This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Neisseria meningitidis, also referred to as meningococcus, is one of the leading causes of epidemic meningitis and septicaemia worldwide. Despite modern treatment, meningococcal disease remains associated with a high mortality (about 10%). Meningococcal disease is mainly restricted to specific hypervirulent lineages and specific capsular groups (serogroups), which have a changing global distribution over time. At the end of the 2000s, the previously unusual serogroup Y emerged, corresponding to half of all of the invasive meningococcal disease (IMD) cases in Sweden by the beginning of the 2010s. The aim of this thesis is to describe the emergence of serogroup Y meningococci genetically in an effort to understand some of the factors involved in the successful spread of this group throughout Sweden. In addition, genetic typing schemes were evaluated for surveillance and outbreak investigation. Our results indicate that the currently recommended typing for surveillance of meningococci could be altered to include the factor H-binding protein (fHbp). A highly variable multilocus variable number tandem repeat analysis (HV-MLVA) was able to confirm connected cases in a suspected small outbreak. In addition, a strain type sharing the same porA, fetA, porB, fHbp, penA and multilocus sequence type was found to be the principal cause of the increase in serogroup Y disease. However, a deeper resolution obtained from the core genomes revealed a subtype of this strain, which was mainly responsible for the increase. Finally, when the Swedish serogroup Y genomes were compared internationally, different strains seemed to dominate in different regions. This indicates that the increase was probably not due to one or more point introductions of a strain previously known internationally but more probably multifactorial.

Neisseria meningitidis (N. meningitidis) is a Gram-negative commensal bacterium colonizing nasopharynx of about 10 % of healthy individuals, which can cause invasive diseases, such sepsis and meningitis, upon occasional penetration into bloodstream. Pathogenesis of N. meningitidis appears to be directly related to conditions of limited iron availability. Under these conditions two proteins of unknown function: FrpC and FrpD, are synthesized. FrpD is a highly conserved lipoprotein of N. meningitidis anchored to the bacterial outer membrane. It is known that FrpD tightly binds the FrpC protein, which belongs to the Repeat-in-Toxin (RTX) protein family and may act as bacterial exotoxin. However, the mechanism of FrpD-FrpC interaction and the exact function of this complex are unknown due to the absence of structural information on these proteins. Therefore, we set out to determine the structure of FrpD and provide insights into its interaction mechanism with FrpC and structure-functional relationships of these two proteins. We determined the first crystal and solution structures of the FrpD protein. We found that atomic structures of FrpD reveal a novel protein fold. We uncovered the structure-function relationships underlying the mechanism of interaction between the FrpD and FrpC proteins and tested the putative function of the FrpD-FrpC1-414 complex in vitro. Finally, we proposed the putative function of the FrpD-FrpC1-414 complex as a new minor adhesin of N. meningitidis, which mediates the bacterial adhesion to the host epithelial cells and facilitate the colonization. Our work constitutes the first step in clarifying the molecular basis of the FrpD-FrpC interaction and sets the base for further investigation of the role of FrpD and FrpC in the virulence mechanism of N. meningitidis.

Neisseria meningitidis é o agente mais prevalente entre os casos de meningite bacteriana, acompanhados de altas taxas de letalidade, causando também outras doenças invasivas. Os objetivos deste estudo foram: caracterizar as cepas de N. meningitidis de pacientes com doença meningocócica no Rio Grande do Sul (RS), de 2003 a 2005, assim como determinar a diversidade de tipos de PorA e avaliar uma técnica de PCR em tempo real para detectar o DNA de N. meningitidis em amostras clínicas. Alguns isolados e amostras clínicas foram caracterizados por MLST e tipagem de PorA, e amostras clínicas foram amplificados por uma técnica de PCR em tempo real utilizando como alvo o gene bexA. Este estudo demonstrou alta prevalência de algumas linhagens hipervirulentas e emergência de algumas delas, incluindo as linhagens W135:P1.5,2:complexo ST-11, e C:P1.22,14-6:complexo ST-103. Estas linhagens são provavelmente responsáveis pelos aumentos de incidência dos sorogrupos W135 e C observados no período. Os complexos clonais mais prevalentes foram os complexos ST-32, ST-103, ST-11, ST-41/44. Os tipos de PorA mais encontrados para o sorogrupo B foram P1.19,15, P1.7,16, e P1.18-1,3, representando uma distribuição de tipos de PorA diferente de outros estados do Brasil, o que tem implicações na escolha e na eficácia de vacinas baseadas em OMPs. A caracterização detalhada e precisa das cepas de meningococo é um elemento importante nos estudos da epidemiologia, biologia populacional e evolução do meningococo, e fornece informações para o desenvolvimento de estratégias de controle da doença. Os resultados de sensibilidade (96%) e especificidade (97%) alcançados pela PCR para N. meningitidis, testando 186 amostras clínicas, foram adequadas para a sua utilização como método de confirmação de casos de meningite por meningococo, excluindo a etapa de extração de DNA das amostras clínicas, o que diminui o custo da reação. / Neisseria meningitidis is the most prevalent agent among bacterial meningitis cases, with high fatality rates and also causing other invasive diseases. The aims of this study were: to characterize N. meningitidis strains causing invasive disease in Rio Grande do Sul (RS), during 2003 to 2005, as well as to determine the diversity of PorA VR types; and to evaluate a real time PCR assay to detect N. meningitidis DNA in clinical specimens. To achieve that, isolates and clinical specimens were characterized by MLST and PorA VR typing, and clinical specimens were amplified by real time PCR using target sequence from gene bexA. This study demonstrated high prevalence of some hypervirulent lineages and emergence of new ones, including the lineages W135:P1.5,2:ST-11 complex, and C: P1.22,14-6:ST-103 complex. These lineages are probably responsible for the increasing incidence of serogroups C and W135 observed. The most prevalent clonal complexes were ST-32, ST-103, ST-11 e ST-41/44 complexes. The most prevalent PorA VR types found for serogroup B were P1.19,15, P1.7,16, and P1.18-1,3, representing a different distribution of PorA types if compared to other states of Brazil. The different distribution of PorA VR types in RS has implications in vaccine design and efficacy. Detailed and accurate meningococcal characterization is an important element in studies of meningococcal epidemiology, population biology, and evolution and provides information for the design of control strategies. Good sensitivity (96%) and specificity (97%) were achieved for N. meningitidis PCR, testing 186 specimens. The method is appropriate to be used for confirmation of cases of meningococcal meningitis, and eliminating the DNA purification step reduces reaction costs.

Infecções por Neisseria meningitidis são uma importante causa de morbidade e mortalidade em todo o mundo devido à habilidade do patógeno em provocar epidemias e até pandemias. Por ser capaz de se recombinar geneticamente existe uma grande variabilidade de tipos circulantes que podem diferir de acordo com a população atingida e localização geográfica. Por isso os objetivos deste estudo foram: avaliar a epidemiologia da doença meningocócica (DM) em pacientes do Rio Grande do Sul (RS), caracterizar fenotípica e genotipicamente isolados de N. meningitidis utilizando métodos convencionais e propor um novo método de tipagem para o estudo de surtos, bem como avaliar sua susceptibilidade à penicilina. Para isso foi realizado um estudo retrospectivo de coorte em 2.215 casos de DM notificados entre 1995 a 2003 no RS. Foi observada uma redução de quase 50% dos casos durante o período e a taxa de letalidade foi de 22%. Ainda assim, uma incidência marcante continua sendo observada mesmo depois do final do período epidêmico, em 1999, onde as faixas etárias de crianças entre 1 e 4 anos e menores de 1 ano representam juntas quase 55% dos casos notificados, com uma média de incidência de 11,3/100.000 hab e 31.3/100.000 hab, respectivamente. A maioria dos casos foi atribuída ao sorogrupo B (69,8%), que aumentou significativamente. Também foi observada uma diminuição significativa dos casos pelo sorogrupo C. Os fenótipos B:4,7:P1.19,15, B:15:P1.7,16 e B:NT:P1.3 representam quase 50% de todos os isolados sorotipados. Cinqüenta e seis isolados obtidos de pacientes do RS, durante o primeiro ano não-epidêmico, somados a 20 isolados dos estados de Santa Catarina e Paraná, Dinamarca e França foram genotipados por Multilocus Sequence Typing (MLST). Foram identificados 20 Sequence Types (STs) distintos, dos quais 8 foram caracterizados como tipos novos, encontrados somente no RS. ST-33 (27%) e ST-259 (18%) foram os tipos mais freqüentes, ambos pertencentes ao complexo clonal ST-32/ET-5. Os casos ST-259 do RS demonstraram uma maior tendência de desfecho de casos fatais. Não foram encontrados isolados ST-259 entre os controles geográficos ou em outros estudos previamente realizados no Brasil. Nossos resultados sugerem que estes dois clones observados durante todo o período de estudo, somados à emergência do ST-103 contribuem para a manutenção da alta incidência da DM no RS. Quase 18% dos isolados apresentaram suscetibilidade reduzida à penicilina. A presença de suscetibilidade reduzida ou plena à penicilina associada ao clone ST-461 foi estatisticamente significativa (P=0,017) quando comparada à resistência nos demais clones. Nenhum isolado demonstrou atividade para a ß-lactamase. Também foi padronizado um novo método de genotipagem, chamado de Variable Site Specific-PCR (VSS-PCR) que apresentou um excelente poder discriminatório, de baixo custo operacional e de fácil execução e interpretação, que poderá ser utilizado em estudos de surtos da DM, assim que seja validado. / Neisseria meningitidis infections are an important cause of morbidity and mortality worldwide because of the pathogen ability to cause epidemics and pandemics. The recombination ability results in a large variability of circulating types that can differ depending on the infected population and geographic localization. Considering these aspects, the aims of this study were: to evaluate meningococcal disease epidemiology affecting patients from the state of Rio Grande do Sul (RS), phenotypic and genotypic characterization of N. meningitidis isolates, using conventional methods and standardizing a new method for outbreak study; and evaluation of penicillin susceptibility. A retrospective cohort study was carried out among 2,215 meningococcal disease (MD) cases reported from 1995 to 2003 in RS, Brazil. A reduction of almost 50% in the overall incidence was observed, and the case-fatality rate during this period was 22%. Even so, high incidence of MD continued to be observed after the epidemic period which ended in 1999. The age group of 1-4 years old and infants under 1 year of age represent together almost 55% of the reported cases with a mean incidence of 11.3/100,000 pop and 31.3/100,000 pop, respectively. The majority of cases were caused by N. meningitidis serogroup B (69.8%), which increased significantly. There was a significant decrease in serogroup C cases. The phenotypes B:4,7:P1.19,15, B:15:P1.7,16 and B:NT:P1.3 represented almost 50% of all serotyped cases. Fifty-six isolates obtained from RS patients during the first non-epidemic year 2000 plus 20 isolates from other southern Brazilian states (Santa Catarina and Paraná), Denmark and France were typed by Multilocus Sequence Typing (MLST). Twenty different Sequence Types (STs) were identified, 8 of them found only in RS. ST-33 (27%) and ST-259 (18%) were the most frequent, both belonging to the ST-32/ET-5 complex. The ST-259 cases showed a trend towards higher risk of fatal outcome. ST-259 isolates were not detected among geographic controls or in other studies carried out in Brazil. Our data suggest that these two clones wich occurred over the entire study period and the emergence of the ST-103 isolates contributed to the continued high incidence of MD in RS. Almost 18% of isolates presented moderate resistance to penicillin. The decreased susceptibility or complete resistance to penicillin associated to ST-461 clone was statistically significant (P=0,017) when compared to the other clones. None of the isolates have showed ß-lactamase activity. We standardized a new genotyping method, called Variable Site Specific-PCR (VSS-PCR) that shows high discriminatory power, lower costs, easy performance and interpretation, which may be used in MD outbreaks, after its validation.

Infecções por Neisseria meningitidis são uma importante causa de morbidade e mortalidade em todo o mundo devido à habilidade do patógeno em provocar epidemias e até pandemias. Por ser capaz de se recombinar geneticamente existe uma grande variabilidade de tipos circulantes que podem diferir de acordo com a população atingida e localização geográfica. Por isso os objetivos deste estudo foram: avaliar a epidemiologia da doença meningocócica (DM) em pacientes do Rio Grande do Sul (RS), caracterizar fenotípica e genotipicamente isolados de N. meningitidis utilizando métodos convencionais e propor um novo método de tipagem para o estudo de surtos, bem como avaliar sua susceptibilidade à penicilina. Para isso foi realizado um estudo retrospectivo de coorte em 2.215 casos de DM notificados entre 1995 a 2003 no RS. Foi observada uma redução de quase 50% dos casos durante o período e a taxa de letalidade foi de 22%. Ainda assim, uma incidência marcante continua sendo observada mesmo depois do final do período epidêmico, em 1999, onde as faixas etárias de crianças entre 1 e 4 anos e menores de 1 ano representam juntas quase 55% dos casos notificados, com uma média de incidência de 11,3/100.000 hab e 31.3/100.000 hab, respectivamente. A maioria dos casos foi atribuída ao sorogrupo B (69,8%), que aumentou significativamente. Também foi observada uma diminuição significativa dos casos pelo sorogrupo C. Os fenótipos B:4,7:P1.19,15, B:15:P1.7,16 e B:NT:P1.3 representam quase 50% de todos os isolados sorotipados. Cinqüenta e seis isolados obtidos de pacientes do RS, durante o primeiro ano não-epidêmico, somados a 20 isolados dos estados de Santa Catarina e Paraná, Dinamarca e França foram genotipados por Multilocus Sequence Typing (MLST). Foram identificados 20 Sequence Types (STs) distintos, dos quais 8 foram caracterizados como tipos novos, encontrados somente no RS. ST-33 (27%) e ST-259 (18%) foram os tipos mais freqüentes, ambos pertencentes ao complexo clonal ST-32/ET-5. Os casos ST-259 do RS demonstraram uma maior tendência de desfecho de casos fatais. Não foram encontrados isolados ST-259 entre os controles geográficos ou em outros estudos previamente realizados no Brasil. Nossos resultados sugerem que estes dois clones observados durante todo o período de estudo, somados à emergência do ST-103 contribuem para a manutenção da alta incidência da DM no RS. Quase 18% dos isolados apresentaram suscetibilidade reduzida à penicilina. A presença de suscetibilidade reduzida ou plena à penicilina associada ao clone ST-461 foi estatisticamente significativa (P=0,017) quando comparada à resistência nos demais clones. Nenhum isolado demonstrou atividade para a ß-lactamase. Também foi padronizado um novo método de genotipagem, chamado de Variable Site Specific-PCR (VSS-PCR) que apresentou um excelente poder discriminatório, de baixo custo operacional e de fácil execução e interpretação, que poderá ser utilizado em estudos de surtos da DM, assim que seja validado. / Neisseria meningitidis infections are an important cause of morbidity and mortality worldwide because of the pathogen ability to cause epidemics and pandemics. The recombination ability results in a large variability of circulating types that can differ depending on the infected population and geographic localization. Considering these aspects, the aims of this study were: to evaluate meningococcal disease epidemiology affecting patients from the state of Rio Grande do Sul (RS), phenotypic and genotypic characterization of N. meningitidis isolates, using conventional methods and standardizing a new method for outbreak study; and evaluation of penicillin susceptibility. A retrospective cohort study was carried out among 2,215 meningococcal disease (MD) cases reported from 1995 to 2003 in RS, Brazil. A reduction of almost 50% in the overall incidence was observed, and the case-fatality rate during this period was 22%. Even so, high incidence of MD continued to be observed after the epidemic period which ended in 1999. The age group of 1-4 years old and infants under 1 year of age represent together almost 55% of the reported cases with a mean incidence of 11.3/100,000 pop and 31.3/100,000 pop, respectively. The majority of cases were caused by N. meningitidis serogroup B (69.8%), which increased significantly. There was a significant decrease in serogroup C cases. The phenotypes B:4,7:P1.19,15, B:15:P1.7,16 and B:NT:P1.3 represented almost 50% of all serotyped cases. Fifty-six isolates obtained from RS patients during the first non-epidemic year 2000 plus 20 isolates from other southern Brazilian states (Santa Catarina and Paraná), Denmark and France were typed by Multilocus Sequence Typing (MLST). Twenty different Sequence Types (STs) were identified, 8 of them found only in RS. ST-33 (27%) and ST-259 (18%) were the most frequent, both belonging to the ST-32/ET-5 complex. The ST-259 cases showed a trend towards higher risk of fatal outcome. ST-259 isolates were not detected among geographic controls or in other studies carried out in Brazil. Our data suggest that these two clones wich occurred over the entire study period and the emergence of the ST-103 isolates contributed to the continued high incidence of MD in RS. Almost 18% of isolates presented moderate resistance to penicillin. The decreased susceptibility or complete resistance to penicillin associated to ST-461 clone was statistically significant (P=0,017) when compared to the other clones. None of the isolates have showed ß-lactamase activity. We standardized a new genotyping method, called Variable Site Specific-PCR (VSS-PCR) that shows high discriminatory power, lower costs, easy performance and interpretation, which may be used in MD outbreaks, after its validation.

Neisseria meningitidis é o agente mais prevalente entre os casos de meningite bacteriana, acompanhados de altas taxas de letalidade, causando também outras doenças invasivas. Os objetivos deste estudo foram: caracterizar as cepas de N. meningitidis de pacientes com doença meningocócica no Rio Grande do Sul (RS), de 2003 a 2005, assim como determinar a diversidade de tipos de PorA e avaliar uma técnica de PCR em tempo real para detectar o DNA de N. meningitidis em amostras clínicas. Alguns isolados e amostras clínicas foram caracterizados por MLST e tipagem de PorA, e amostras clínicas foram amplificados por uma técnica de PCR em tempo real utilizando como alvo o gene bexA. Este estudo demonstrou alta prevalência de algumas linhagens hipervirulentas e emergência de algumas delas, incluindo as linhagens W135:P1.5,2:complexo ST-11, e C:P1.22,14-6:complexo ST-103. Estas linhagens são provavelmente responsáveis pelos aumentos de incidência dos sorogrupos W135 e C observados no período. Os complexos clonais mais prevalentes foram os complexos ST-32, ST-103, ST-11, ST-41/44. Os tipos de PorA mais encontrados para o sorogrupo B foram P1.19,15, P1.7,16, e P1.18-1,3, representando uma distribuição de tipos de PorA diferente de outros estados do Brasil, o que tem implicações na escolha e na eficácia de vacinas baseadas em OMPs. A caracterização detalhada e precisa das cepas de meningococo é um elemento importante nos estudos da epidemiologia, biologia populacional e evolução do meningococo, e fornece informações para o desenvolvimento de estratégias de controle da doença. Os resultados de sensibilidade (96%) e especificidade (97%) alcançados pela PCR para N. meningitidis, testando 186 amostras clínicas, foram adequadas para a sua utilização como método de confirmação de casos de meningite por meningococo, excluindo a etapa de extração de DNA das amostras clínicas, o que diminui o custo da reação. / Neisseria meningitidis is the most prevalent agent among bacterial meningitis cases, with high fatality rates and also causing other invasive diseases. The aims of this study were: to characterize N. meningitidis strains causing invasive disease in Rio Grande do Sul (RS), during 2003 to 2005, as well as to determine the diversity of PorA VR types; and to evaluate a real time PCR assay to detect N. meningitidis DNA in clinical specimens. To achieve that, isolates and clinical specimens were characterized by MLST and PorA VR typing, and clinical specimens were amplified by real time PCR using target sequence from gene bexA. This study demonstrated high prevalence of some hypervirulent lineages and emergence of new ones, including the lineages W135:P1.5,2:ST-11 complex, and C: P1.22,14-6:ST-103 complex. These lineages are probably responsible for the increasing incidence of serogroups C and W135 observed. The most prevalent clonal complexes were ST-32, ST-103, ST-11 e ST-41/44 complexes. The most prevalent PorA VR types found for serogroup B were P1.19,15, P1.7,16, and P1.18-1,3, representing a different distribution of PorA types if compared to other states of Brazil. The different distribution of PorA VR types in RS has implications in vaccine design and efficacy. Detailed and accurate meningococcal characterization is an important element in studies of meningococcal epidemiology, population biology, and evolution and provides information for the design of control strategies. Good sensitivity (96%) and specificity (97%) were achieved for N. meningitidis PCR, testing 186 specimens. The method is appropriate to be used for confirmation of cases of meningococcal meningitis, and eliminating the DNA purification step reduces reaction costs.