Made available in DSpace on 2015-07-08T12:28:20Z (GMT). No. of bitstreams: 2 5.pdf: 1093384 bytes, checksum: 9432b61bcb4cfac37f05a1600b8f2fae (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Haemophilus influenzae pode ser encontrado, normalmente, na microbiota do trato respiratório, do trato gênito-urinário e da cavidade oral. Porém, essa espécie inclui um dos mais importantes patógenos bacterianos em infecções principalmente pediátricas. As cepas de Hi podem ser capsuladas, variando de a-f, ou não capsuladas (não tipáveis - NT). O tipo capsular b foi o mais frequente em infecções graves infantis até a utilização da vacina conjugada contra Hib, sendo ainda considerado patogênico. No Brasil, essa vacina foi introduzida no Programa Nacional de Imunização do Ministério da Saúde em agosto de 1999, e como em outros países, promoveu uma acentuada diminuição em doenças causadas por esse agente. No entanto, estudos realizados na era pós-vacinal têm mostrado que a incidência de doenças invasivas causadas por H. influenzae não b e NT têm aumentado, inclusive no Brasil. O objetivo desse trabalho foi obter informações sobre as cepas de Hi circulantes no município do Rio de Janeiro. Utilizou-se 96 amostras de quadros infeciosos (46 invasivas e 50 não invasivas), isoladas no período pós-vacinal (2000-2012). Em relação à idade, dos 54 pacientes que tiveram esse dado, 32 tiveram doenças invasivas e 22 não invasivas. Em doenças invasivas, houve o predomínio de crianças < 5 anos. Enquanto que nas não invasivas os adultos > 70 anos predominaram. Entre as cepas obtidas, 15 foram capsuladas e 81 não capsuladas. A maioria das cepas capsuladas foi proveniente de sítios invasivos, cuja faixa etária predominante foi de < 5 anos. O tipo capsular mais isolado foi o b, seguido do a e f. As cepas Hib, predominaram no início do período de estudo, enquanto os outros sorotipos predominaram no final. No presente trabalho, não encontramos cepas mutantes deficientes de cápsula tanto Hib-, quanto Hia- / Haemophilus influenzae can be found usually in the microbiota of the respiratory tract, genitourinary tract and oral cavity. However, this species includes one of the most important bacterial pathogens mainly in pediatric infections. The strains can be capsulated Hi, (serotypes a-f), or not capsulated (nontypeable - NT). The type b capsular was the most frequent serious infection in children until the use of conjugate vaccine against Hib and is still considered pathogenic. In Brazil, this vaccine was introduced in the National Immunization Program of the Ministry of Health in August 1999, and as in other countries, promoted a significant decrease of disease caused by this agent. However, studies during the post-vaccine period have shown that the incidence of invasive disease caused by H. influenzae b and NT have increased, including in Brazil. The aim of this study was to obtain information on the Hi strains circulating in the municipality of Rio de Janeiro. We used 96 samples (46 invasive and 50 noninvasive), isolated during the post-vaccination period (2000-2012). Concerning the age of the 54 patients who had this data, 32 had invasive and 22 noninvasive disease. In invasive disease, there was a predominance of children <5 years. While in the noninvasive group, adults > 70 years predominated. Among the strains obtained, 15 were capsulated and 81 non-capsulated. Most capsulated strains originated from invasive sites whose predominant age group was <5 years. The most frequent capsular type was b, followed by a and f. Hib strains predominated at the beginning of the study period, while the other serotypes prevailed in the end. In this study, we did not found mutant strains deficient in both capsule Hib- and Hia-. NT strains accounted for the vast majority of isolates in this study, 32 strains isolated from invasive sites and 49 sites noninvasive, and were obtained from patients of all age groups. The capsulated strains were predominantly biotype I and II, while non-typeable strains were most II and III. In this study, only the NT strains were resistant to two drugs: ampicillin and trimethoprim - sulfamethoxazole. These were mostly non-invasive. Thus, none of the capsulated isolates were resistant. The PFGE patterns for the 96 strains were quite different, however eight NT strains belonged to the same genotype. Capsulated strains of the same serotype were similar, getting most isolates grouped in the same cluster. We therefore conclude that it is necessary to monitor the Hi strains circulating in Rio de Janeiro, because of the geographic and economic importance of this municipality. Such conduct should be extended to the whole country in order to understand the possible changes of serotypes today, which will certainly guide for the design of new vaccines, improvement of existing ones and the use of antibiotics, resulting in a public health impact.

Ältere Menschen sind gegenüber invasiven Infektionen und Sepsis besonders vulnerabel mit ungünstiger Prognose. Staphylococcus aureus und Haemophilus influenzae können beide invasive Infektionen verursachen. Oft geht eine asymptomatische Besiedelung einer Infektion voraus und ist ein Risikofaktor für eine invasive Infektion. Daher wurde eine bizentrische Querschnittstudie in den Regionen Aachen und Würzburg durchgeführt, um die Prävalenz von H. influenzae, S. aureus und MRSA (Methicillin resistenter S. aureus) bei asymptomatischen Senioren zu bestimmen, wie auch Risikofaktoren für eine Besiedelung. Von Oktober 2012 bis Mai 2013 wurden 677 Erwachsenen im Alter von 65 Jahren oder älter eingeschlossen, die zu Hause oder in Seniorenheimen lebten. Die Prävalenz von H. influenzae bei älteren Menschen war mit einer Trägerrate von nur 1,9% ([95% CI: 1,0 - 3,3%]; 13/677) sehr niedrig. Trägerisolate waren überwiegend nicht typisierbare H. influenzae, zeigten eine hohe clonale Diversität und waren alle Ampicillin-sensibel. Die Prävalenz von S. aureus war mit 28,5% ([95% CI: 25,1 - 32,1%]; 193/677) hoch, wie für die deutsche Allgemeinbevölkerung bekannt, während MRSA bei weniger als 1% der Teilnehmer gefunden wurde (0,7% [95% CI: 0,2 - 1,7%]; 5/677). Die Prävalenz von H. influenzae, S. aureus und MRSA unterschied sich nicht signifikant zwischen selbständig zu Hause lebenden Senioren und Pflegeheimbewohnern. Ältere, selbständig lebende Menschen mit höherem Bildungsniveau hatten signifikant höhere Kolonisierungsraten mit S. aureus (adjusted OR: 1,905 [95% CI: 1,248 - 2,908]; p = 0,003). Bei Pflegeheimbewohnern war eine Kolonisierung signifikant mit Verheiratet sein assoziiert (adjusted OR: 3,367 [95% CI: 1,502 - 7,546]; p = 0,003). Diese Ergebnisse unterstreichen die Bedeutung von sozio-demographischen Faktoren für eine Kolonisierung mit S. aureus und schließen eine Lücke bei epidemiologischen Daten zu H. influenzae. / Elderly people are especially vulnerable to invasive infections and sepsis with often poor outcome. Staphyloccus aureus and Haemophilus influenzae both can cause invasive infections. Asymptomatic colonization often precedes infection and poses a risk for invasive infection. Therefore, a bi-centric cross-sectional carrier study was conducted in the regions of Aachen and Wuerzburg, Germany, to determine the prevalence of H. influenzae, S. aureus and MRSA (methicillin resistant S. aureus) in asymptomatic elderly people and to identify risk factors for colonization. From October 2012 to May 2013 677 adults aged 65 years and older were included, living at home or in nursing homes. In contrast to children and younger adults the prevalence of H. influenzae was very low among elderly people with a carriage rate of only 1.9% ([95% CI: 1.0 - 3.3%]; 13/677). Carrier isolates were predominantly non typeable H. influenzae, showed a high clonal diversity and were all susceptible to ampicillin. The prevalence of S. aureus was expectedly high as known for the German general population (28.5% [95% CI: 25.1 - 32.1%]; 193/677), while MRSA was found in less than 1% of the individuals (0.7% [95% CI: 0.2 - 1.7%]; 5/677). The prevalence of H. influenzae, S. aureus und MRSA did not differ significantly between community dwellers and nursing home residents. Elderly community-dwellers with higher education level had significantly higher colonization rates with S. aureus (adjusted OR: 1.905 [95% CI: 1.248 - 2.908]; p = 0.003). Among nursing home residents, colonization was significantly associated with being married (adjusted OR: 3.367 [1.502 - 7.546]; p = 0.003). These results underline the importance of socio-demographic factors for colonization with S. aureus and close a gap in epidemiological data on H. influenzae.

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 &#946;-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 &#946;-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.