Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V.cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V.cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V.cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease. / Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V.cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V.cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.

No description available.

A dengue é uma doença infecciosa de transmitida pela picada de mosquitos do gênero Aedes. O vírus da dengue (DENV), pertencente ao gênero Flavivirus, família Flaviviridae, é atualmente um importante problema de saúde pública em todo o mundo. São reconhecidos quatro sorotipos antigenicamente distintos (DENV-1, -2, -3 e -4). A doença causada por qualquer um dos sorotipos cursa de forma assintomática ou com quadro clínico que varia desde uma febre indiferenciada e autolimitada, passando pela febre clássica da dengue (FD) até quadros graves de febre hemorrágica da dengue (DHF). O diagnóstico clínico é difícil de ser realizado principalmente na fase aguda da doença em que os sintomas são muito similares aos de outras infecções febris agudas, ficando a cargo do laboratório o diagnóstico definitivo. Os métodos sorológicos para detecção de anticorpos IgM/IgG são os mais amplamente utilizados, mas inadequados para diagnóstico precoce uma vez que detectam anticorpos a partir do sexto dia do início dos sintomas. Os métodos moleculares estão sendo cada vez mais utilizados para o diagnóstico precoce por serem mais rápidos e sensíveis que a sorologia e o isolamento viral. Neste estudo comparamos a sensibilidade de uma RT-PCR em tempo real gênero específica com um ELISA para detecção da proteína NS1 comercialmente disponível analisando amostras de soro de pacientes com dengue. Também foram desenvolvidos dois protocolos de RT-PCR em tempo real para identificação do sorotipo viral, uma contendo primers para extremidade 5 do genoma viral e outra contendo primers para a região codificadora da proteína NS5. A RT-PCR em tempo real gênero específica mostrou-se mais sensível que o ELISA, principalmente nas amostras que apresentavam baixa carga viral. A RT-PCR em tempo real contendo os primers para a extremidade 5 apresentou uma sensibilidade baixa quando comparada com a RT-PCR genérica, porém foi mais sensível que aquela contendo os primers para a região codificadora da proteína NS5. Considerando os resultados obtidos, sugerimos uma estratégia de triagem dos casos suspeitos de dengue utilizando a RT-PCR genérica para posteriormente identificar o sorotipo viral com o protocolo que utiliza os primers da extremidade 5. Embora este último protocolo tenha sido pouco sensível, a identificação do sorotipo infectante em algumas amostras é suficiente para definir qual o sorotipo circulante durante uma epidemia. / Dengue is an infectious disease transmitted by the biting of mosquitoes of Aedes genus. Dengue virus (DENV), belonging to the Flavivirus genus, Flaviviridae family, is an important public health problem worldwide. Four antigenically distinct viruses are recognized (DENV-1, -2, -3, e -4). Infection with any of the virus serotypes causes a spectrum of the illness ranging from inapparent or mid viral syndrome to classic dengue fever (DF) and severe hemorrhagic disease (DHF). The clinical diagnosis is difficult especially in the acute phase of the disease when the symptoms are very similar to other febrile illness, corresponding to the laboratory the definitive diagnosis. Serological methods detecting antibodies IgM/IgG are the more widely used; however, they are inappropriate for early diagnosis since these methods detect antibodies after six day of the onset of symptoms. The molecular methods are more frequently used for the early diagnosis because they are faster and more sensitive than serological methods and virus isolation. In this study, we have compared the sensitivity of a generic real time RT-PCR with a commercial ELISA for the NS1 protein analyzing serum samples from patients with dengue virus infection. We have also developed two protocols of real time RT-PCR to identify dengue serotype, one of them containing primers to the 5 end and the other, primers to the NS5 coding region. The generic real time RT-PCR showed to be more sensitive than the ELISA, principally, between the samples with low viral load. The real time RT-PCR containing primers to the 5 end showed a lower sensitivity than the generic real time RT-PCR; however, it was more sensitive than that containing primers to the NS5 coding region. Considering these results, we suggest the use of the generic real time RT-PCR to screen the dengue suspected cases and then to identify the serotype using the protocol that include the primers to the 5 end. Although the last protocol has shown a low sensitive, the identification of the infecting serotype in some of the samples is enough to define which serotype is circulating during the epidemic period.

Objetivos: Avaliar o impacto global, direto, indireto e a tendência da duração de proteção da vacinação contra o Haemophilus influenzae tipo b (Hib), no estado de São Paulo (ESP) e no município de São Paulo (MSP), na população de 0 - 59 meses, comparando os períodos pré-vacinal (1996 - 1998) e pós-vacinal (2001 - 2009). Métodos: estudo com componente descritivo e de cunho analítico, retrospectivo. A população de estudo incluiu os menores de cinco anos residentes no ESP e no MSP. Adotou-se como definição de caso confirmado o menor de cinco anos identificado como positivo para o Hib em cultura e/ou contraimunoeletroforese e/ou látex e/ou RT-PCR, em amostra de LCR e sangue, e/ou vínculo epidemiológico. Os dados foram obtidos a partir do SINAN, SIGH-Web Instituto Adolfo Lutz e Fundação IBGE. As variáveis de estudo incluíram as demográficas, clínicas e relativas ao agente, apresentadas em séries temporais e períodos estabelecidos para parametrização e comparabilidade. O parâmetro das avaliações de impacto foi a magnitude da variação da incidência de meningite causada pelo Hib. Para cada estimativa de impacto construiu-se um Intervalo de Confiança (IC) de 95 por cento a partir do cálculo de Risco Relativo (RR). As estimativas do risco relativo (RR) e os respectivos intervalos de 95 por cento de confiança foram analisados utilizando-se o software R. Resultados: nos períodos considerados, foram descritos 1.561 casos confirmados de meningites por Hib no ESP, sendo 27,16 por cento (424/1.561) no MSP, e 80,78 por cento (1.261/1.561) dos casos foram registrados em menores de cinco anos. A maioria dos casos foi confirmada por cultura, com percentual médio de 65 por cento no ESP e 66 por cento no MSP. As taxas médias de incidência de meningites por Hib mais significativas no período pré-vacinal verificaram-se nos menores de um ano (30,56/105- ESP; 32,06/105 - MSP), considerada a faixa etária de maior risco de adoecimento. Após a introdução da vacina contra o Hib em 1999 (menores de dois anos), as taxas de incidência de meningites por Hib declinaram de forma sustentável nos períodos subsequentes analisados. A incidência de meningite por Hib durante o período pós-vacinal variou de 4,02/105- 1,68/105 nos menores de um ano, no ESP e MSP respectivamente e, de forma similar, de 1,43/105 1,01/105 nos menores de cinco anos. Nos menores de 7 - 23 meses (impacto direto), o percentual de redução foi de 95,11 por cento [66,43 - 99,29] no ESP e 95,91 por cento [70,63 - 99,43] no MSP. O impacto global observado nos menores de cinco anos foi 88,19 por cento [26,58 - 98,10] no ESP e 91,06 por cento [33,99 - 98,79] no MSP. Os dados de vigilância mostram que os casos de meningites por Hib continuam ocorrendo, porém em níveis baixos, ao longo de 10 anos após a introdução do esquema de três doses primárias da vacina conjugada específica. Conclusão: a partir deste racional pode-se inferir a utilidade prática e econômica a favor desta modalidade programática adotada no território paulista, com a evidência de redução relativa de meningites por Hib. / Objectives: To evaluate global impact, direct and indirect, as well as the tendency of the duration of vaccine protection against Haemophilus influenzae type b (Hib) in the state of São Paulo (ESP) and in the city of São Paulo (MSP), amongst the population between 0-59 months of age during the periods pre-vaccine (1996-1998) and post vaccine (2001-2009). Methods: a retrospective study with a descriptive component and with analytic venue. Studied population included children under five years old, dwelling in ESP and MSP. Criteria adopted as definition of confirmed case was child under five years of age identified as positive for Hib in culture and/or counterimmunelectroforesis and/or latex and/or RT/PCR, in LCR sample and blood. and/or epidemiologic link. Data were obtained from the SINAN, SIGH-Web Instituto Adolfo Lutz and IBGE Foundation. Variables under study included socio-demographic and clinical ones, and those related to the agent; they were presented in temporal series and periods established in order to allow parametric and comparison. Impact evaluation was established upon the variation of incidence magnitude of meningitis caused by Hib. For each impact estimate a Confidence Interval (IC) of 95 per cent from the calculus of Relative Risk (RR). Estimates of relative risk (RR) and the respective intervals of 95 per cent confidence were analyzed employing the R software. Results: During the analyzed periods 1561 confirmed cases of meningitis caused by Hib were described in the state of São Paulo, 27.16 per cent of which (424/1561) in MSP; 80.78 per cent (1261/1561) of the registered cases occurred in children under five years of age. The majority of the cases were confirmed by culture, with an average percentage of 65 per cent in ESP and 66 per cent in MSP. More significant average rates of meningitis per Hib during the pre-vaccine period were registered in children under one year of age (30.56/105- ESP; 32.06/105 - MSP), considered the bracket under higher risk of disease. After the introduction of the vaccine against Hib, in 1999 (for children under two years of age), the rates of meningitis incidence per Hib decreased in sustainable fashion for the subsequent periods analyzed. The incidence of meningitis per Hib during the post vaccine period varied from 4.02/105 - 1.68/105 for children under one year old, in ESP and MSP, respectively and, in similar way, from 1.43/105 to 1.01/105 for those under five years old. For children between 7-23 months old (direct impact), the percentage of reduction was of 95.11 per cent [66.43 - 99.29] in ESP and 95.91 per cent [70.63 - 99.43] in MSP, whereas the global impact observed in children under five years old was 88.19 per cent [26.58 - 98.10] in ESP and 91.06 per cent [33.99 98.79] in MSP. Surveillance data show that cases of meningitis by Hib continue to occur but in low levels along 10 years after the introduction of the three doses primary scheme of administration of the specific conjugated vaccine. Conclusion: from this rationale, it is possible to infer the practical and economic utility favoring this programmatic modality adopted in São Paulo, with the evidence of the relative reduction of meningitis caused by Hib.

Made available in DSpace on 2014-07-29T15:26:20Z (GMT). No. of bitstreams: 1 TeseCristyaneBenicio2010.pdf: 531193 bytes, checksum: 9a3d68036fe340ca60db84cb20f7066b (MD5) Previous issue date: 2010-02-18 / Objectives (i) Present review article focusing on pneumococcal vaccines and carriage; (ii) to validate sequential multiplex PCR for identifying pneumococcal capsular serotypes from children attending day-care centers; (iii) determine the multilocus sequence typing; (iv) to identify the capsular types of multiple colonies of S. pneumoniae isolates from a single sample of nasopharyngeal secretions of children attending day-care centers in Goiânia. Materials and Methods S. pneumoniae was obtained from health children less than 5 years old attending 62 day care centers of Goiânia. The laboratory procedures were performed according to WHO recommendations. Were selected 217 isolates (penicillin resistant and sensitive) for capsular typing by multiplex PCR technique. MLST was performed for 55 isolates representing the serotypes detected and the different susceptibility patterns for penicillin. Quellung reaction was used for typing isolates serotypes 6A, 6B, 18C and the isolates not typed by multiplex PCR. For 28 presumptive pneumococcal positive NP swabs, 3 colonies were picked to acess possible serotype diversity. Eighty four pneumococci were identified by conventionally procedure and multiplex PCR was performed. Results Serotypes were deduced for 177/217 (81.6%) of the pneumococcal. The most frequent serogroups/serotypes were 14, 6, 23F, 19F and 18. Multiple serotypes were detected in 13 specimens. Were found 19 MLST types and two new ST. Forty (18.4%) were not serotyped by the multiplex PCR and quellung reaction. The analysis of three colonies from the same NP permitted the detection of differente serotypes in 7/28 (25%) NP samples. Conclusion (i) The multiplex PCR is simple and cost-effective method for detecting multiple serotypes in nasopharyngeal isolates; (ii) and thus might be useful for the monitoring of pneumococcal colonization over time; (iii) the use of multiplex PCR can further broaden our understanding of the dynamics of pneumococcal carriage, including multiple serotypes, the effect of vaccination on carriage, and transmission, as well as surveillance of IPD and co-colonization. / Objetivos: (i) Apresentar uma revisão focando as vacinas pneumocócicas e o portador de S. pneumoniae na nasofaringe; (ii) realizar a tipagem capsular de pneumococos colonizadores de nasofaringe de crianças de creches pela técnica de multiplex PCR; (iii) identificar o perfil MLST dos pneumococos isolados na nasofaringe; (iv) identificar os tipos capsulares de múltiplas colônias de S. pneumoniae isolados de uma única amostra de secreção da nasofaringe de crianças que frequentam creches do município de Goiânia pela técnica de multiplex PCR. Material e Métodos: Um estudo de prevalência de portador de pneumococo foi conduzido de agosto a dezembro de 2005, em crianças de dois a 59 meses de idade, atendidas em 62 creches em Goiânia. Os procedimentos laboratoriais para isolamento e identificação dos pneumococos foram realizados de acordo com as técnicas recomendadas pela Organização Mundial de Saúde. Foram selecionados 217 isolados (resistentes e sensíveis à penicilina) para a tipagem capsular pela técnica de multiplex PCR. O perfil MLST foi realizado para 55 isolados, representando os sorotipos detectados e os diferentes perfis de suscetibilidade à penicilina. A reação de Quellung foi usada para tipar os sorotipos 6A, 6B e 18C e os isolados não tipados pelo multiplex PCR. Para a análise de múltiplas colônias de S. pneumoniae, utilizou-se 28 amostras positivas para pneumococo, das quais se recuperou 3 colônias de cada placa de ágar sangue, totalizando 84 colônias, que foram submetidas aos testes de tipagem fenotípica e caracterização capsular pela técnica de multiplex PCR. Resultados: Cento e setenta e sete sorotipos em duzentos e dezessete (177/217), totalizando 81,6% dos pneumococos foram tipados. Os sorotipos mais freqüentes foram 14, 6, 23F, 19F e 18. Foram identificadas múltiplas colônias em 13 amostras de nasofaringe. Foram observados 19 tipos MLST e dois novos tipos de seqüência (ST). Quarenta (18,4%) dos isolados não foram tipados pelo multiplex PCR e todos não tipados pela reação de Quellung. A análise de múltiplas colônias de S. pneumoniae pela técnica de multiplex PCR permitiu a detecção de mais de um tipo em 25% (7/28) das amostras. Conclusões: (i) O método de multiplex PCR mostrou-se seguro e simples na detecção de diferentes tipos capsulares incluídos na reação, além de mais barato; (ii) representou uma valiosa ferramenta em investigações de vigilância de pneumococos; (iii) Aplicação da técnica multiplex PCR permitiu o conhecimento da diversidade genética de pneumococos colonizadores da nasofaringe, detectando a dinâmica da colonização desta bactéria na população, incluindo a colonização por múltiplos sorotipos; a substituição ou mudança de sorotipo como resultado da vacinação; como também vigilância das doenças pneumocócicas invasivas.

A dengue é uma doença infecciosa de transmitida pela picada de mosquitos do gênero Aedes. O vírus da dengue (DENV), pertencente ao gênero Flavivirus, família Flaviviridae, é atualmente um importante problema de saúde pública em todo o mundo. São reconhecidos quatro sorotipos antigenicamente distintos (DENV-1, -2, -3 e -4). A doença causada por qualquer um dos sorotipos cursa de forma assintomática ou com quadro clínico que varia desde uma febre indiferenciada e autolimitada, passando pela febre clássica da dengue (FD) até quadros graves de febre hemorrágica da dengue (DHF). O diagnóstico clínico é difícil de ser realizado principalmente na fase aguda da doença em que os sintomas são muito similares aos de outras infecções febris agudas, ficando a cargo do laboratório o diagnóstico definitivo. Os métodos sorológicos para detecção de anticorpos IgM/IgG são os mais amplamente utilizados, mas inadequados para diagnóstico precoce uma vez que detectam anticorpos a partir do sexto dia do início dos sintomas. Os métodos moleculares estão sendo cada vez mais utilizados para o diagnóstico precoce por serem mais rápidos e sensíveis que a sorologia e o isolamento viral. Neste estudo comparamos a sensibilidade de uma RT-PCR em tempo real gênero específica com um ELISA para detecção da proteína NS1 comercialmente disponível analisando amostras de soro de pacientes com dengue. Também foram desenvolvidos dois protocolos de RT-PCR em tempo real para identificação do sorotipo viral, uma contendo primers para extremidade 5 do genoma viral e outra contendo primers para a região codificadora da proteína NS5. A RT-PCR em tempo real gênero específica mostrou-se mais sensível que o ELISA, principalmente nas amostras que apresentavam baixa carga viral. A RT-PCR em tempo real contendo os primers para a extremidade 5 apresentou uma sensibilidade baixa quando comparada com a RT-PCR genérica, porém foi mais sensível que aquela contendo os primers para a região codificadora da proteína NS5. Considerando os resultados obtidos, sugerimos uma estratégia de triagem dos casos suspeitos de dengue utilizando a RT-PCR genérica para posteriormente identificar o sorotipo viral com o protocolo que utiliza os primers da extremidade 5. Embora este último protocolo tenha sido pouco sensível, a identificação do sorotipo infectante em algumas amostras é suficiente para definir qual o sorotipo circulante durante uma epidemia. / Dengue is an infectious disease transmitted by the biting of mosquitoes of Aedes genus. Dengue virus (DENV), belonging to the Flavivirus genus, Flaviviridae family, is an important public health problem worldwide. Four antigenically distinct viruses are recognized (DENV-1, -2, -3, e -4). Infection with any of the virus serotypes causes a spectrum of the illness ranging from inapparent or mid viral syndrome to classic dengue fever (DF) and severe hemorrhagic disease (DHF). The clinical diagnosis is difficult especially in the acute phase of the disease when the symptoms are very similar to other febrile illness, corresponding to the laboratory the definitive diagnosis. Serological methods detecting antibodies IgM/IgG are the more widely used; however, they are inappropriate for early diagnosis since these methods detect antibodies after six day of the onset of symptoms. The molecular methods are more frequently used for the early diagnosis because they are faster and more sensitive than serological methods and virus isolation. In this study, we have compared the sensitivity of a generic real time RT-PCR with a commercial ELISA for the NS1 protein analyzing serum samples from patients with dengue virus infection. We have also developed two protocols of real time RT-PCR to identify dengue serotype, one of them containing primers to the 5 end and the other, primers to the NS5 coding region. The generic real time RT-PCR showed to be more sensitive than the ELISA, principally, between the samples with low viral load. The real time RT-PCR containing primers to the 5 end showed a lower sensitivity than the generic real time RT-PCR; however, it was more sensitive than that containing primers to the NS5 coding region. Considering these results, we suggest the use of the generic real time RT-PCR to screen the dengue suspected cases and then to identify the serotype using the protocol that include the primers to the 5 end. Although the last protocol has shown a low sensitive, the identification of the infecting serotype in some of the samples is enough to define which serotype is circulating during the epidemic period.

<p>Streptococcus agalactiae, also designated group B streptococcus (GBS), is a Gram-positive coccus, and it is an important pathogen that causes invasive disease in neonates, pregnant adults, and non-pregnant adults with predisposing conditions. The group II intron GBSi1 is one of the major mobile genetic elements identified in S. agalactiae. The aim of this thesis was to characterize the GBSi1 distribution pattern, the population structure, and the influence of serotype- and clone-specific properties on the invasive capacity among clinical invasive and non-invasive isolates of S. agalactiae.</p><p>Two additional copies of GBSi1 were identified at sites different from the primarily identified scpB-lmb locus. The distribution of GBSi1 was uneven among different serotypes. Three intron copies were only found in isolates of serotype III, and these targeted all the three identified gene loci. In contrast, a single copy of GBSi1 was found in isolates of serotype II and V and only located at the scpB-lmb locus. Furthermore, at the 5′ flanking region of the scpB-lmb gene locus, a novel 2.1 kb DNA fragment with plasmid features was identified only in intron carrying isolates. This may suggest that GBSi1 once was brought into the S. agalactiae genome by an integrated plasmid.</p><p>Multilocus sequence typing was used to characterize totally 314 invasive and non-invasive S. agalactiae isolates collected in Northern and Western Sweden from the years 1988 to 2004. Five major genetic lineages (clonal complexes) were identified among both invasive and non-invasive isolates, including serotype Ia, Ib, and II to V, indicating a clonal population structure of S. agalactiae isolates. A number of genetically highly related isolates were found to express different capsular types, suggesting that capsule switching occurs rather frequently between isolates. Furthermore, non-invasive isolates belonging to the same clonal complexes displayed more heterogeneity in capsule expression as well as in the distribution patterns of mobile genetic elements than invasive isolates. This indicates that less variability is allowed in a highly selective environment such as the blood. All major clonal complexes and serotypes caused invasive disease, although their ability to do so varied greatly. CC17 was significantly associated with neonatal invasive disease; whereas CC19 was equally common among isolates from adult and neonatal disease, despite that both CC17 and CC19 expressed capsular type III. This striking difference seen between CC17 and CC19 suggests that clonal complex associated properties, in addition to capsular type, play important roles in the virulence of S. agalactiae. CC1, a new emerging clone since early 1990s, has caused substantial amount of disease among adults. In addition, mutually exclusive distribution of mobile elements GBSi1 and IS1548 was seen, and they were shown to constitute genetic markers for serotype III CC17 and CC19 isolates, respectively.</p>

Streptococcus agalactiae, also designated group B streptococcus (GBS), is a Gram-positive coccus, and it is an important pathogen that causes invasive disease in neonates, pregnant adults, and non-pregnant adults with predisposing conditions. The group II intron GBSi1 is one of the major mobile genetic elements identified in S. agalactiae. The aim of this thesis was to characterize the GBSi1 distribution pattern, the population structure, and the influence of serotype- and clone-specific properties on the invasive capacity among clinical invasive and non-invasive isolates of S. agalactiae. Two additional copies of GBSi1 were identified at sites different from the primarily identified scpB-lmb locus. The distribution of GBSi1 was uneven among different serotypes. Three intron copies were only found in isolates of serotype III, and these targeted all the three identified gene loci. In contrast, a single copy of GBSi1 was found in isolates of serotype II and V and only located at the scpB-lmb locus. Furthermore, at the 5′ flanking region of the scpB-lmb gene locus, a novel 2.1 kb DNA fragment with plasmid features was identified only in intron carrying isolates. This may suggest that GBSi1 once was brought into the S. agalactiae genome by an integrated plasmid. Multilocus sequence typing was used to characterize totally 314 invasive and non-invasive S. agalactiae isolates collected in Northern and Western Sweden from the years 1988 to 2004. Five major genetic lineages (clonal complexes) were identified among both invasive and non-invasive isolates, including serotype Ia, Ib, and II to V, indicating a clonal population structure of S. agalactiae isolates. A number of genetically highly related isolates were found to express different capsular types, suggesting that capsule switching occurs rather frequently between isolates. Furthermore, non-invasive isolates belonging to the same clonal complexes displayed more heterogeneity in capsule expression as well as in the distribution patterns of mobile genetic elements than invasive isolates. This indicates that less variability is allowed in a highly selective environment such as the blood. All major clonal complexes and serotypes caused invasive disease, although their ability to do so varied greatly. CC17 was significantly associated with neonatal invasive disease; whereas CC19 was equally common among isolates from adult and neonatal disease, despite that both CC17 and CC19 expressed capsular type III. This striking difference seen between CC17 and CC19 suggests that clonal complex associated properties, in addition to capsular type, play important roles in the virulence of S. agalactiae. CC1, a new emerging clone since early 1990s, has caused substantial amount of disease among adults. In addition, mutually exclusive distribution of mobile elements GBSi1 and IS1548 was seen, and they were shown to constitute genetic markers for serotype III CC17 and CC19 isolates, respectively.

Atlantic cod (Gadus morhua L.) is one of the most promising new fish species introduced to cold water aquaculture due to the large established market in Europe and the USA and the decline in wild stock. So far, the production of farmed cod has been relatively low, with the main hindrance due to diseases. Vibrio anguillarum has been recognised as the biggest disease problem of farmed cod and has slowed the development of a successful cod aquaculture industry. When the first incidences of V. anguillarum occurred in cod aquaculture, vaccines designed for vibriosis in Atlantic salmon (Salmo salar L.) were used in an attempt to combat the disease. However, these vaccines did not provide sufficient protection, possibly because they lacked serotype O2b, which is known to affect cod and to a lesser extent salmonids. Recently, vibriosis vaccines specifically designed to protect Atlantic cod have been formulated, but outbreaks of vibriosis in vaccinated fish are still being reported, suggesting that these formulations are inadequate. The aim of this project was to develop a whole cell inactivated vaccine formulation specifically tailored to protect Atlantic cod against Vibrio anguillarum. The serological classification of V. anguillarum was first investigated by producing a set of monoclonal antibodies (mAbs). Using lipopolysaccharides (LPS) extracted with butan-1-ol, 4 mAbs were selected and shown to react specifically with V. anguillarum serotypes O1, O2a and O2b. A collection of over 150 V. anguillarum isolates were screened using these, which revealed that most of the isolates had been previously correctly classified. A new sub-serotype of V. anguillarum O2 was identified from isolates recovered from outbreaks of vibriosis in Norway as well as Scotland. This new sub-serotype was referred to as O2d since the subserotype O2c has been recently identified in vibriosis cases from Atlantic cod. However, it was shown that the O2c sub-serotype might not belong to the O2 serotype, but in fact belongs to another serotype. To protect Atlantic cod against all the V. anguillarum serotypes (and subserotypes) which they are susceptible to, it is recommended that isolates from serotypes O1, O2a, O2b, O2c and O2d should all be included in a bacterin vaccine for cod. In order to determine which isolates from each of the serotypes to include in the vaccine, a variety of virulence factors of V. anguillarum were investigated in vitro. The interaction of some candidate isolates from O1, O2a and O2b serotypes (O2c and O2d were not identified at the time this part of the study took place) with cod phagocytic cells were studied using flow cytometry. Phagocytosis and respiratory burst of cod macrophages and neutrophils as well as cod serum killing of V. anguillarum were quantified. It was found that isolates within the same serotype displayed varying degrees of resistance to phagocytosis and the subsequent respiratory burst activity as well as that all the V. anguillarum strains tested were resistant to Atlantic cod serum killing. These in vitro assays were found to be very useful in assessing the virulence of V. anguillarum. The isolate within each serotype eliciting the highest percentage of positive phagocytic cells was selected in order to increase the antigen presentation pathway, thus theoretically enhancing the protection elicited by the vaccine. A multivalent formalin-inactivated non-adjuvanted vaccine was prepared which included all the serotypes previously described and was injected intraperitoneally into Atlantic cod. A bath challenge was performed on vaccinated and mock-vaccinated fish, 6 weeks post immunisation, using V. anguillarum isolates from the serotypes O2b, O2c and O2d that were not included in the vaccine. An excellent level of protection was obtained against O2b and O2d (relative percentage survival 100% and 96.4%, respectively), but the challenge with the sub-serotype O2c isolate did not produce any mortality in the control group and needs to be repeated. The vaccine formulation was very efficient at protecting Atlantic cod against vibriosis but further challenges need to be performed with other serotypes included in the vaccine (O1 and O2a), as well as with more isolates from the O2b, O2c and O2d sub-serotype. To conclude, Atlantic cod is a species which will certainly have a major influence in marine aquaculture, but many areas have to be improved. The development of an effective and broad range vaccine to protect cod against Vibrio anguillarum offers another advance which should help Atlantic cod aquaculture to reach its full potential.

Objetivos: Avaliar o impacto global, direto, indireto e a tendência da duração de proteção da vacinação contra o Haemophilus influenzae tipo b (Hib), no estado de São Paulo (ESP) e no município de São Paulo (MSP), na população de 0 - 59 meses, comparando os períodos pré-vacinal (1996 - 1998) e pós-vacinal (2001 - 2009). Métodos: estudo com componente descritivo e de cunho analítico, retrospectivo. A população de estudo incluiu os menores de cinco anos residentes no ESP e no MSP. Adotou-se como definição de caso confirmado o menor de cinco anos identificado como positivo para o Hib em cultura e/ou contraimunoeletroforese e/ou látex e/ou RT-PCR, em amostra de LCR e sangue, e/ou vínculo epidemiológico. Os dados foram obtidos a partir do SINAN, SIGH-Web Instituto Adolfo Lutz e Fundação IBGE. As variáveis de estudo incluíram as demográficas, clínicas e relativas ao agente, apresentadas em séries temporais e períodos estabelecidos para parametrização e comparabilidade. O parâmetro das avaliações de impacto foi a magnitude da variação da incidência de meningite causada pelo Hib. Para cada estimativa de impacto construiu-se um Intervalo de Confiança (IC) de 95 por cento a partir do cálculo de Risco Relativo (RR). As estimativas do risco relativo (RR) e os respectivos intervalos de 95 por cento de confiança foram analisados utilizando-se o software R. Resultados: nos períodos considerados, foram descritos 1.561 casos confirmados de meningites por Hib no ESP, sendo 27,16 por cento (424/1.561) no MSP, e 80,78 por cento (1.261/1.561) dos casos foram registrados em menores de cinco anos. A maioria dos casos foi confirmada por cultura, com percentual médio de 65 por cento no ESP e 66 por cento no MSP. As taxas médias de incidência de meningites por Hib mais significativas no período pré-vacinal verificaram-se nos menores de um ano (30,56/105- ESP; 32,06/105 - MSP), considerada a faixa etária de maior risco de adoecimento. Após a introdução da vacina contra o Hib em 1999 (menores de dois anos), as taxas de incidência de meningites por Hib declinaram de forma sustentável nos períodos subsequentes analisados. A incidência de meningite por Hib durante o período pós-vacinal variou de 4,02/105- 1,68/105 nos menores de um ano, no ESP e MSP respectivamente e, de forma similar, de 1,43/105 1,01/105 nos menores de cinco anos. Nos menores de 7 - 23 meses (impacto direto), o percentual de redução foi de 95,11 por cento [66,43 - 99,29] no ESP e 95,91 por cento [70,63 - 99,43] no MSP. O impacto global observado nos menores de cinco anos foi 88,19 por cento [26,58 - 98,10] no ESP e 91,06 por cento [33,99 - 98,79] no MSP. Os dados de vigilância mostram que os casos de meningites por Hib continuam ocorrendo, porém em níveis baixos, ao longo de 10 anos após a introdução do esquema de três doses primárias da vacina conjugada específica. Conclusão: a partir deste racional pode-se inferir a utilidade prática e econômica a favor desta modalidade programática adotada no território paulista, com a evidência de redução relativa de meningites por Hib. / Objectives: To evaluate global impact, direct and indirect, as well as the tendency of the duration of vaccine protection against Haemophilus influenzae type b (Hib) in the state of São Paulo (ESP) and in the city of São Paulo (MSP), amongst the population between 0-59 months of age during the periods pre-vaccine (1996-1998) and post vaccine (2001-2009). Methods: a retrospective study with a descriptive component and with analytic venue. Studied population included children under five years old, dwelling in ESP and MSP. Criteria adopted as definition of confirmed case was child under five years of age identified as positive for Hib in culture and/or counterimmunelectroforesis and/or latex and/or RT/PCR, in LCR sample and blood. and/or epidemiologic link. Data were obtained from the SINAN, SIGH-Web Instituto Adolfo Lutz and IBGE Foundation. Variables under study included socio-demographic and clinical ones, and those related to the agent; they were presented in temporal series and periods established in order to allow parametric and comparison. Impact evaluation was established upon the variation of incidence magnitude of meningitis caused by Hib. For each impact estimate a Confidence Interval (IC) of 95 per cent from the calculus of Relative Risk (RR). Estimates of relative risk (RR) and the respective intervals of 95 per cent confidence were analyzed employing the R software. Results: During the analyzed periods 1561 confirmed cases of meningitis caused by Hib were described in the state of São Paulo, 27.16 per cent of which (424/1561) in MSP; 80.78 per cent (1261/1561) of the registered cases occurred in children under five years of age. The majority of the cases were confirmed by culture, with an average percentage of 65 per cent in ESP and 66 per cent in MSP. More significant average rates of meningitis per Hib during the pre-vaccine period were registered in children under one year of age (30.56/105- ESP; 32.06/105 - MSP), considered the bracket under higher risk of disease. After the introduction of the vaccine against Hib, in 1999 (for children under two years of age), the rates of meningitis incidence per Hib decreased in sustainable fashion for the subsequent periods analyzed. The incidence of meningitis per Hib during the post vaccine period varied from 4.02/105 - 1.68/105 for children under one year old, in ESP and MSP, respectively and, in similar way, from 1.43/105 to 1.01/105 for those under five years old. For children between 7-23 months old (direct impact), the percentage of reduction was of 95.11 per cent [66.43 - 99.29] in ESP and 95.91 per cent [70.63 - 99.43] in MSP, whereas the global impact observed in children under five years old was 88.19 per cent [26.58 - 98.10] in ESP and 91.06 per cent [33.99 98.79] in MSP. Surveillance data show that cases of meningitis by Hib continue to occur but in low levels along 10 years after the introduction of the three doses primary scheme of administration of the specific conjugated vaccine. Conclusion: from this rationale, it is possible to infer the practical and economic utility favoring this programmatic modality adopted in São Paulo, with the evidence of the relative reduction of meningitis caused by Hib.