Bacterial transformation is defined as a mechanism of genetic exchange whereby a population of bacteria can obtain genetic informa-tion as a result of cellular uptake and integration of extracellular deoxyribonucleic acid released from other bacteria by natural or in-duced lysis. In order for a transformable strain of bacteria to take up DNA and to undergo transformation, the cells must be in a physiolog-ical state called competence. A cell is referred to as competent if it has the ability to bind extracellular DNA irreversibly and subsequently to integrate and express the DNA.

Die Inzidenz invasiver H. influenzae-Infektionen in Deutschland steigt seit Jahren an. Die akkurate Identifizierung und Resistenztestung dieses Erregers sind von großer klinischer und epidemiologischer Bedeutung. Daher wurden im Rahmen der vorliegenden Promotionsarbeit umfangreiche Untersuchungen zur Diagnostik und zur Epidemiologie von Antibiotikaresistenzen bei H. influenzae durchgeführt. Es konnte gezeigt werden, dass die in der Routinediagnostik mittlerweile weit verbreitete MALDI-TOF-MS-Diagnostik durch das VITEK MS IVD nur eingeschränkt zur sicheren Unterscheidung von H. influenzae und H. haemolyticus einsetzbar ist. H. influenzae-Isolate erkannte das System mit einer Genauigkeit von 100 %. Bei H. haemolyticus-Isolaten wurden dagegen 42 % der untersuchten Stämme fälschlicherweise als H. influenzae erkannt. Dieser Fragestellung wurde mit der bisher umfangreichsten molekularbiologisch charakterisierten Studienpopulation beider Bakterienspezies nachgegangen. Die kalkulierte antibiotische Therapie einer Sepsis oder Meningitis erfolgt häufig mit Carbapenemen, die leitliniengerechte Therapie invasiver H. influenzae-Infektionen mit Drittgenerations-Cephalosporinen. Imipenem und Cefotaxim gehören zu den Hauptvertretern dieser Gruppen. Bezüglich der Antibiotikaresistenztestung wurde erstmalig für H. influenzae herausgefunden, dass die routinemäßig verwendete Gradientenagardiffusion (GAD) bei der Testung von Cefotaxim im Vergleich zum Goldstandard Bouillon-Mikrodilution gleichwertig und bei Imipenem sogar sensitiver in der Detektion von Heteroresistenzen ist. Die Epidemiologie dieser Resistenzen wurde in dieser Arbeit erstmalig für Deutschland systematisch erfasst, indem alle verfügbaren invasiven Isolate gemeldeter H. influenzae-Infektionen der Jahre 2016 (Imipenem) beziehungsweise 2016-2019 (Cefotaxim) untersucht wurden. Es wurde eine hohe Prävalenz einer Imipenem-Resistenz von 13,5 % festgestellt. Die Prävalenz einer Cefotaxim-Resistenz lag bei 0,9 %. Zur molekularen Typisierung wurde bei den Imipenem-resistenten Isolaten eine Multilocus-Sequenztypisierung, bei den Cefotaxim-resistenten Stämmen eine Sequenzierung des vollständigen Genoms durchgeführt. Hierbei wurde eine hohe genetische Diversität der Stämme festgestellt, was die Schlussfolgerung zulässt, dass resistente Mutanten sporadisch entstehen. Die Untersuchung möglicher spatio-temporaler Cluster führte zum Nachweis einer sehr selten vorkommenden Übertragung eines Imipenem-resistenten Stamms. Durch die Sequenzierung von Resistenzgenen wurde die Epidemiologie und Relevanz bekannter Aminosäuresubstitutionen beleuchtet. Unter anderem wurde für die PBP3-Substitutionen L389F und Y557H eine hochsignifikante Korrelation mit dem Auftreten von Cefotaxim-Resistenzen nachgewiesen. Die gewonnenen Genomdaten bieten die Grundlage für die Forschung an weiteren Antibiotikaresistenzdeterminanten von H. influenzae. / Haemophilus influenzae is a fastidious, facultative anaerobic, Gram-negative bacillus that colonizes the respiratory tract and can cause respiratory and invasive infection such as meningitis and sepsis. Invasive H. influenzae infections are potentially life-threatening and incidence rates have been increasing for years. Therefore, fast and accurate diagnostics, reliable testing of antibiotic resistance and a successful antibiotic treatment is of great importance. Therefore, the objective of the first part of this thesis was to evaluate the diagnostic and discriminative potential of the MALDI-TOF-MS system VITEK® MS regarding H. influenzae and H. haemolyticus. H. influenzae can cause invasive infections, whereas H. haemolyticus is mostly apathogenic. The system showed excellent accuracy for the identification of H. influenzae isolates, as 100 % of the 236 isolates were correctly identified. When testing 50 H. haemolyticus strains, however, the system showed significant limitations, since 42 % of these strains were misidentified as H. influenzae. According to the current German guidelines for the treatment of sepsis and meningitis, treatment of invasive H. influenzae infections is carried out using carbapenems, such as imipenem, or third-generation cephalosporins, such as cefotaxime. Therefore, the prevalence of antibiotic resistances to these substances was investigated and possible resistance mechanisms were examined. The two antibiotic susceptibility testing methods Gradient agar diffusion (GAD) and broth microdilution (BMD) were compared. As a result, for the determination of the cefotaxime MIC, the two methods showed an excellent correlation, whereas for imipenem there were significant differences in the measured MIC values. Since strains tested by GAD often showed double or fuzzy inhibition zones, heteroresistances may be more apparent using this method and GAD may be more sensitive at detecting imipenem resistance. The prevalence of imipenem resistance was determined for the year 2016. The analysis of 474 different invasive isolates showed a high prevalence of 5.5 %. If including all resistant isolates according to GAD, the prevalence would be even as high as 13.5 %. MLST was performed on all isolates to investigate the genetic relationship. As a result, however, some sequence types were observed more frequently, it revealed a significant diversity. Both the analysis of ftsI and acrR showed previously described amino acid substitutions. Cefotaxime resistance was investigated for all 2432 invasive H. influenzae for the years 2016-2019. The low prevalence of 0.9 % shows that cefotaxime is still well suited for the treatment of invasive H. influenzae infections. For the investigation of the genetic relationship and possible causes of cefotaxime resistance whole genome sequencing (WGS) was performed on all resistant isolates. The strains showed high genetic diversity and the geographic analysis also showed that the resistant strains were evenly spread throughout the population in Germany. This led to the conclusion that cefotaxime resistance is more likely caused by sporadic mutation events rather than by specific clones spreading in certain areas. The analysis of the ftsI gene showed that the amino acid substitutions L389F and Y557H are significantly associated with elevated cefotaxime MICs. This dissertation provides comprehensive data regarding diagnostics, antimicrobial susceptibility testing and the epidemiology of antibiotic resistances of invasive H. influenzae. It could be shown that the VITEK MS IVD, although established in the routine diagnostics, can only be used to a limited extent for reliably differentiating H. influenzae and H. haemolyticus isolates. Regarding antibiotic susceptibility testing, it was found that GAD showed similar results compared to the gold standard BMD when testing cefotaxime. When testing imipenem, this method was even more sensitive in detecting heteroresistances compared to the gold standard. For the first time, the epidemiology of antibiotic resistance against cefotaxime and imipenem was carried out using a large set of precisely defined invasive H. influenzae isolates to obtain representative data for the prevalence of imipenem and cefotaxime resistance in Germany. By investigating amino acid substitutions in the ftsI and acrR gene the epidemiology and relevance of these substitutions could be shown. A well-founded statement on the relationship of the resistant strains could be made using the state-of-the-art typing methods MLST and whole genome sequencing. The genome data also offers the possibility of examining other genes of these strains in more detail.

Haemophilus influenzae, as well as other members of the genus which require V-factor, display a unique growth requirement for intact NAD. This organism, the primary cause of bacterial meningitis, is incapable of synthesis of pyridine nucleotides from the usual precursors. An externally directed nucleotide pyrophosphatase was extracted from the organism and purified 700-fold using ammonium sulfate precipitation, ion-exchange and affinity chromatography. The enzyme was determined to be a periplasmic glycoprotein consisting of a single polypeptide of M_r= 65,000. The enzyme had a pH optimum over the range .pH 8.0-9.0 and was not activated by the addition of mono or divalent cations, nor was it inhibited by EDTA. The enzyme was observed to have a broad substrate specificity and functioned in a manner indicative of negative cooperativity with all substrates except several modified in the adenine ring. The most effective inducer of negative cooperativity was NAD as indicated by its Hill coefficient of 0.26. The enzyme was inhibited by adenine nucleotides _ and 5'-AMP, at 20 μM, abolished the negative cooperativity of the enzyme. The enzyme was determined to possess excitation and emission maxima at 286 and 337 nm, respectively, indicative of the presence of tryptophan. The fluorescence of the enzyme was quenched by addition of aliquots of adenine nucleotides. The quenching occurred in a biphasic manner. The enzyme was inactivated by 2,3- butanedione and by Woodward's Reagent K. Studies of the ability of compounds to serve as V-factor revealed that nicotinamide mononucleotide (NM), NAD, as well as analogs of NAD, served as V-factor. The ability of compounds to inhibit growth was also accessed, and the growth of the organism was seen to be inhibited by adenine nucleotides as well as other compounds. The inhibition of growth of Haemophilus influenzae has important clinical implications which are discussed, as well as a model of the NAD metabolism of the organism which is presented. / Ph. D.

Haemophilus influenzae, the primary causative factor in bacterial meningitis, displays a unique growth requirement for intact NAD. Selective inhibitors of the pyridine nucleotide-requiring enzymes from H. influenzae could have a pronounced effect on growth of the organism. Haemophilus malate dehydrogenase was purified 109-fold with a 26% recovery through a 4-step procedure involving salt fractionation, and hydrophobic and dye affinity chromatography. The purified enzyme was demonstrated to be a dimer of M,= 61,000. Initial velocity, product, and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme. Several NAD analogs structurally altered in either the pyridine or purine moiety functioned as coenzymes in the reaction catalyzed. Selective interactions occurring at the coenzyme binding sites were investigated. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate moiety of the coenzyme. Positive chain length effects in the coenzyme-competitive inhibition by aliphatic carboxylic acids indicated the presence of a hydrophobic region close to the pyrophosphate region at the coenzyme binding site. Several structural analogs of NAD and malate were evaluated as selective inhibitors of the enzyme. The enzyme was inactivated by incubation with diethylpyrocarbonate whereas no inactivation was observed with sulfhydryl reagents. Haemophilus influenzae 6-phosphogluconate dehydrogenase was purified 308-fold with a 16% recovery through a 4-step chromatographic procedure involving a PhenylSepharose hydrophobic column, and affinity chromatography on Matrex gel Green A, Matrex gel Red A, and 2',5’ADP-Sepharose resin. The purified enzyme was demonstrated to be a dimer of M,= 70,000. Initial velocity studies of 6-phosphogluconate oxidation indicated a sequential reaction mechanism. Although certain product and dead-end inhibition studies were consistent with an ordered mechanism, the direct binding of 6-phosphogluconate in protection experiments did not support a strictly ordered reaction sequence. Inhibition by adenosine derivatives indicated that the 2’-phosphate is important in binding to the coenzyme binding site of the enzyme. The 3-acetylpyridine analogs of NAD and NADP which support growth of H. influenzae were demonstrated to function as coenzymes with the two dehydrogenases studied. The most effective inhibitors of the purified malate dehydrogenase and 6-phosphogluconate dehydrogenase were observed to inhibit the growth of Haemophilus influenzae. However, the most potent inhibition of growth by 3-aminopyridine analogs of NAD and NADP could not be explained on the basis of interactions of these analogs with the two dehydrogenases studied. / Ph. D.

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.

Haemophilus influenzae (Hi) é uma bactéria Gram negativa dependente dos fatores de crescimento NAD e hemina. O sorotipo b (Hib) é causador de doenças invasivas de importância clínica. É uma bactéria anaeróbia facultativa, e se adapta em devido à versatilidade do seu arsenal metabólico e assim, sendo capaz de causar infecção em nichos com e sem oxigênio no hospedeiro. O objetivo deste trabalho foi estudar o comportamento cinético de Hib em anaerobiose, aerobiose e a interface entre as duas condições, utilizando diferentes aceptores de elétrons (fumarato, DMSO, nitrato e oxigênio). Nitrato possui o maior potencial de redução padrão, porém o nitrito gerado de sua oxidação inviabilizou o crescimento de Hib. DMSO apresentou o melhor crescimento celular em frascos agitados, frente a fumarato de sódio. Ensaios em biorreator demonstraram que em anaerobiose, com fumarato de sódio como aceptor, succinato é o metabólito principal formado. Acetato foi produzido com todos os aceptores testados, seja em anaerobiose ou aerobiose, majoritário na última condição. Lactato de sódio e glicerol foram totalmente consumidas em aerobiose e lentamente em anaerobiose, sem esgotar nenhuma das fontes de carbono. / Haemophilus influenzae (Hi) is a Gram-negative bacterium and require growth factors for its development (hemin and NAD). Serotype b (Hib) is an important human pathogen for public health. It is a facultative anaerobic bacterium, which is allowed to adapt in several host niches due to its metabolic versatility with oxygen or its absence. The aim of this work is to study the growth kinetic of Hibs in anaerobiosis and aerobiosis, in the presence of different final electron acceptors (fumarate, DMSO, nitrate and oxygen). Nitrate has the major reduction potential among the electron acceptors tested, however Hib did not growth. In the presence of DMSO, Hib has the better growth. Anaerobic essay carried out in bioreactor showed that in the presence of fumarate as electron acceptor, succinate was the major metabolite produced. Acetate was produced in the presence of all electron acceptors tested, in both conditions (anaerobiosis and aerobiosis. Lactate and glycerol were entirely consumed in aerobiosis as carbon source and in anaerobiosis, the consumption of both carbon sources were not expressive.

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 2014-07-29T15:30:37Z (GMT). No. of bitstreams: 1 Camila Xavier de Carvalho_dissertacao.pdf: 504724 bytes, checksum: a41997b1490987005878aa95bc646e53 (MD5) Previous issue date: 2010-03-19 / Haemophilus influenzae (Hi) causes infection in children, and is presented in two ways: with six encapsulated serotypes a-f and non-encapsulated or nontypeable (NTHi). Capsulated strains are responsible for a variety of invasive diseases, with meningitis being the most frequent. Nontypeable strains are responsible for respiratory tract infections and acute otitis media in children under 24 months. Children who attend day care centers have increased risk of developing otitis media when colonized with NTHi. Our goal was to describe the prevalence of colonization by Hi and risk factors associated with carrier status in children attending day care centers. Nasopharyngeal swabs collected from 1192 healthy children under five years of age who attended one of 62 daycare centers in Goiânia - Goiás, Brazil were analyzed. The samples were placed on chocolate agar plates and incubated in an atmosphere containing 5% CO2 at 37 ° C overnight. Hi were identified according with colony morphology in culture, Gram staining, and their requirement for V (hemin) and X (NAD) factors. Capsular typing and the presence of the genes TEM1 and ROB1 for resistance to β-lactams were evaluated by PCR. Differences between proportions and means were tested using Chi-square and Student's t test, respectively. Estimates of relative risk (odds ratio) were evaluated by univariate and multivariate logistic regression, p values less than 5% were considered statistically significant. The prevalence of colonization among the 1192 children was 32.1% and 23.3% for HiNT and 8.8% for encapsulated strains. The prevalence of strains carrying the gene TEM1 was 38.4%. Among HiNT strains the prevalence of TEM1 gene was 43.2%. Previous hospitalization of children in the last 6 months was independently associated with the risk carrier by H. influenzae typeable. The data described in this study will aid investigation on the impact of the 10-valent pneumococcal vaccine (PHiD-CV) introduction. / Haemophilus influenzae (Hi) é uma das espécies de bactéria que causa infecção em crianças, e se apresenta sob duas formas: capsulados com seis sorotipos de a-f e não capsulados ou não tipáveis (HiNT). Cepas capsuladas são responsáveis por uma variedade de doenças invasivas, sendo a meningite a mais freqüente. As cepas não capsuladas ou não tipáveis são responsáveis por infecções do trato respiratório e otite média aguda em menores de 24 meses. As crianças que frequentam creches têm risco aumentado de desenvolverem otite média quando colonizadas por HiNT. Nosso objetivo foi descrever a prevalência de colonização por Hi e fatores de risco associados ao estado de portador em crianças atendidas em creches. Foram analisados swabs de nasofaringe coletados de 1192 crianças saudáveis menores de cinco anos de idade que frequentavam uma das 62 creches de Goiânia - Goiás, Brasil. As amostras foram semeadas em placas de ágar chocolate e incubadas em atmosfera contendo 5% de CO2 a 37°C durante a noite. Os Hi foram identifiados de acordo com a morfologia da colônia em meio de cultura, coloração de Gram e a exigência dos fatores V (hemina) e X (NAD). A tipagem capsular assim como a presença dos genes TEM1 e ROB1 para resistência a β-lactâmicos foi avaliada pela PCR. Diferenças de proporção e de média foram avaliadas pelo teste do Chi-quadrado e teste t de student, respectivamente. Estimativas de risco relativo (odds ratio) foram avaliadas por regressão logística uni e multivariada, valores de p menores que 5%, foram considerados estatisticamente significantes. A prevalência de colonização entre as 1192 crianças foi de 32,1% sendo 23,3% para HiNT e 8,8% para cepas capsuladas. A prevalência de cepas portadoras do gene TEM1 foi de 38,4%. Dentre os HiNT a prevalência de cepas portadoras do gene TEM1 foi de 43,2%. Internação prévia da criança nos últimos 6 meses esteve independentemente associado ao risco de portador por H. influenzae tipável. Os dados obtidos neste estudo poderão subsidiar o impacto da introdução da vacina pneumocócica decavalente PHiD-CV.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-08T17:19:50Z No. of bitstreams: 1 01d.pdf: 1587828 bytes, checksum: 1a8694d38c20b5c410d6e957fd6a2575 (MD5) / Made available in DSpace on 2012-11-08T17:19:50Z (GMT). No. of bitstreams: 1 01d.pdf: 1587828 bytes, checksum: 1a8694d38c20b5c410d6e957fd6a2575 (MD5) Previous issue date: 2005-06 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O objetivo deste trabalho foi aprimorar a qualidadeanalítica do laboratório de Controle Físico-Químico, LAFIQ, de Bio - Manguinhosatravés da implantação de ferramentas que permitam avaliar o desempenho dos resultados do laboratório e criar um modelo de controle. Foram estudados dois métodosde análise: determinação do conteúdo de polissacarídeo na vacina contra Haemophillus influenzaetipo B – Hib que corresponde a categoria de método tipo I, conforme Farmacopéia Americana e da condutividade em água para injetáveis correspondente a categoria de método tipo III. Os parâmetros de validação e adequação dos dois métodos foram estudados e cartas controle foram construídas utilizando os dados das análises de amostras referência realizadas por diversos analistas em dias diferentes. Com parâmetros calculados a partir destas cartas foi possível avaliar quão correto é um resultado comparado ao valor desejado, representado pelo valor da grandeza que está sendo medido. O parâmetro desvio-padrão, e o correspondente intervalo de confiança foram utilizados para medir a dispersão dos resultados através do cálculo das incertezas das medições. Para o método tipo I as estimativas das incertezas padrão combinada e expandida foram u = 0,50 µg / mL e U = 1,00 µg / mL, respectivamente. Foram considerados os parâmetros Reprodutibilidade, a concentração da solução de trabalho, do volume, da pureza do reagente, e da massa, como os que mais contribuírampara a incerteza desta categoria de método. Para o método tipo III foram encontradasas incertezas u = 0,03 µS / cm e U = 0,06 µS / cm, como incertezas padrão combinada e padrão expandida. Os fatores que mais contribuíram na incerteza deste método foram os relacionados a qualificação / calibração do equipamento, a estabilidade da amostra e a precisão do procedimento dos analistas. O uso destes recursos estatísticos desenvolvidos e estudados na presente dissertação levarão ao aprimoramento do controle da qualidade dos imunobiológicos garantindo a qualidade do trabalho dos analistas e a maior confiabilidade dos resultados fornecidos pelo LAFIQ. / The objective of this work was to improve the quality of the Bio-Manguinhos’ Laboratory of Physico-Chemical Quality Control using tools that allow to evaluate the analytical performance. The work was carried out studying two analytical methodologies: determination of the content of polysaccharide in Haemophillus influenzaetype b vaccine, that is type I according to American Pharmacopeia’s and conductivity measurement in injectable waters, type III. In thisstudy it was suggested a model that allows to guarantee the quality of results emitted by the Control Laboratory. The validation of the chosen methods and the construction of chart controls were made by evaluating the validation parameters, data for reference sample and estimating the uncertainty of the measurements. This last one is considered the quantified attribute that allows estimating if the result is correct comparedto the certified value. The uncertainty corresponds to the range of the established confidence that means the dispersion of the results. All these statistics tools will improve the implementation of a Quality System. For the method type I the uncertainty standard and expanded were u = 0,50 µg / mL and U = 1,00 µg / mL, respectively. The calculations of themeasurement uncertainty were performed considering the follow sources: Reproducibility, concentration of the standard solutions, volume, purity of reagent, and mass. Forthe method type III, the values were u = 0,02 µS/cm and U = 0,06 µS/cm. The parameters that contributed to the uncertainty of this method were: the behavior of the sample, the qualification and performance of equipment used and the stability of water samples. For both studied analytical methods the main contribution for the analytical results uncertainties was the precision of the analytical procedures. The use of these statistic tools willimprove the confidence on the analytical results from the LAFIQ.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-09T17:20:22Z No. of bitstreams: 1 darcy-akemi-hokama.pdf: 626817 bytes, checksum: 700e602e893be0cfb2141252bf9c68b7 (MD5) / Made available in DSpace on 2012-11-09T17:20:22Z (GMT). No. of bitstreams: 1 darcy-akemi-hokama.pdf: 626817 bytes, checksum: 700e602e893be0cfb2141252bf9c68b7 (MD5) Previous issue date: 2005-09 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O objetivo deste trabalho foi a avaliação das melhorias alcançadas no Sistema de Controle de Qualidade de Vacinas produzidas em Bio-Manguinhos, abrangendo o período de 1999 a 2004. Analisamos que uma transferência de tecnologia bem sucedida, como a da vacina contra Haemophilus influenzae(Hib) produzida pela GlaxoSmith-Kline, Bélgica, proporcionou avanços significativos em qualidade. Vários fatoresestudados, como: melhoria no cumprimento às normas de Boas Práticas de Produção e Controle vigentes; o papel do Controle de Qualidade na absorção de tecnologia; investimento em equipamentos analíticos de última geração; captação, capacitação e treinamento de recursos humanos. Os resultados mostraram os altos índices de reprovação no processo produtivo de vacinas durante o ano de 1997, período em que as perdas chegaram a atingir até 23%. As melhorias implementadas nas áreas produtivas e de controle de qualidade, implicaram no aumento significativo do rendimento na produção das vacinas, evidenciado pelas perdas estimadas em apenas 1-3%, durante o período de 1999 a 2004 (exceto em 2000). Avaliamos também que os incrementos no sistema da qualidade não podem ser sustentados, caso não sejam adotadas outras ações essenciais, como a qualificação de fornecedores, uma dificuldade relacionada à complexidade para se adquirir produtos de qualidade, os quais podem estar envolvidos direta ou indiretamente na produção de vacinas. Desta forma, propomos a implementação de um Programa de Qualificação de Fornecedores para Bio-Manguinhos, com o objetivo de se minimizar um fator intrincado na aquisição de materiais e serviços confiáveis e, sobretudo, que garantam a qualidade no processo de fabricação de seus produtos (Vacinas, Biofármacos e Reativos para Diagnóstico). / The current work aimed to evaluate the improvementsoccurred in the Vaccine Quality Control System produced by Bio-Manguinhos from year1999 to 2004. Succeeded technology transfer against Haemophilus influenzatype B (Hib) from GlaxoSmith-Kline, Belgium, has got many improvements on quality system. Various factors were analyzed such as: improvement to meet current Good Manufacturing Practices and Control following procedures; role of Quality Control in technology absorption; investment in new-generation analytical equipments, upgrade and human resources.Results show that after improvement in the system , lost vaccine production that were as high as 23% in 1997, lowered to a range of 1~3% between 1999~2004 (except in 2000), concomitant to enhancement in production mass, what is a very substantial change. Improvements in Quality Control System were not sustainable without observing, for instance, buyingfrom qualified suppliers, one of the actual problems for obtaining proper quality products thatrelate directly or indirectly to vaccine production. From these analyzes we propose to implement Supplier Qualification Program to minimize a serious problem concerning safety material and services (Vaccines, Biopharmaceuticals and Diagnosis Reactives).