Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-10-02T19:41:10Z No. of bitstreams: 2 Dissertação - Liliam Borges Fernades - 2013.pdf: 1021436 bytes, checksum: ed17067b456cf27093e83d9cf999cd72 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-02T20:11:13Z (GMT) No. of bitstreams: 2 Dissertação - Liliam Borges Fernades - 2013.pdf: 1021436 bytes, checksum: ed17067b456cf27093e83d9cf999cd72 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-02T20:11:13Z (GMT). No. of bitstreams: 2 Dissertação - Liliam Borges Fernades - 2013.pdf: 1021436 bytes, checksum: ed17067b456cf27093e83d9cf999cd72 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-06-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / It is estimated that between 60 and 80 million people worldwide enfretem difficulties to reproduce at some point in their lives and it is estimated that this ratio reaches approximately 20% of couples of reproductive age. Gonorrhea is a common bacterial infection caused by Neisseria gonorrhoeae (NG), transmitted almost exclusively by sexual or perianal contact. In women genital infection can be asymptomatic and when it discovers there may be serious complications such as pelvic inflammatory disease, and ectopic pregnancy, a leading cause of female infertility. The molecular diagnostic techniques are powerful allies in the tracking of NG and other sexually transmitted diseases (STD). Therefore, an investigation was conducted in the scientific literature in the form of a Systematic Literature Review (SLR) on the prevalence of NG, molecular diagnostic techniques and the samples used in the detection of this bacterium. Simultaneously, we developed a chart review in the last 4 years to detect the prevalence of gonococcal infection among other diagnoses as the cause of infertility in women seen at the Laboratory of Human Reproduction - Clinical Hospital, Goiânia, Goiás. SLR was found in 720 articles of which 694 were excluded due to previously established criteria and 26 were included in this review. The population was predominantly young 7 (27%) and female sex workers 6 (23%). The age ranged from 12 to 59 years. The more frequent biological material was endocervical swab 10 (38.46%), vaginal swabs 8 (30.76%), urine 4 (15.38%) and the association of these 3 (11.53%). The in-house PCR was the choice of 12 trials for the diagnosis of NG. In the other 14 studies different commercial diagnostic kits were used. Gradually more it is observed the use of biological samples collected from a less invasive diagnosis of STD. Molecular techniques found in this study are the most used in asymptomatic patients, since they have greater efficiency in the detection of NG, whose gold standard applies only in symptomatic patients. In reviewing the records among the 340 women studied, the prevalence of NG 0.6% and co-infection with Chlamydia trachomatis (CT) was 0.6%. DIP was present in 2.1% of cases and 2 cases of coinfection of NG and CT and the mean age was 33 years. The CT and NG infections had low prevalence in the studied sample and studies are needed in other breeding centers in the country to confirm the prevalence of infections in this particular group of women with infertility problems. / Estima-se que entre 60 e 80 milhões de pessoas em todo o mundo em algum momento de suas vidas enfrentem dificuldades para se reproduzirem e calcula-se que este índice atinja aproximadamente 20% dos casais em idade reprodutiva. A gonorréia é uma infecção bacteriana frequente, causada por Neisseria gonorrhoeae (NG), transmitida quase que exclusivamente por contato sexual ou perianal. Nas mulheres a infecção genital pode ser assintomática e quando se descobre pode haver complicações sérias como a doença inflamatória pélvica (DIP), uma das principais causas da infertilidade feminina e da gravidez ectópica. As técnicas diagnósticas moleculares são grandes aliadas no rastreamento de NG e outras doenças sexualmente transmissíveis (DST). Objetivos: realizar uma investigação na literatura científica no formato de uma Revisão Sistemática de Literatura (RSL) sobre a prevalência de NG em mulheres assintomáticas, observando a faixa etária, as técnicas moleculares diagnósticas e as amostras utilizadas na detecção dessa bactéria; e, uma revisão de prontuários dos últimos 4 anos para detectar a prevalência da infecção gonocócica entre outros diagnósticos como fator causal da infertilidade em mulheres atendidas no Laboratório de Reprodução Humana – Hospital das Clínicas/UFG, Goiânia, Goiás. Na RSL foram encontrados 720 artigos dentre os quais 694 foram excluídos por critérios previamente estabelecidos e 26 incluídos nesta revisão. A população predominante foi de jovens 7 (27%) e mulheres profissionais do sexo 6 (23%). A idade variou de 12 a 59 anos. O material biológico mais frequente foi esfregaço endocervical 10 (38,46%), esfregaço vaginal 8 (30,76%), urina 4(15,38%) e a associação dos mesmos 3(11,53%). A técnica PCR convencional foi a técnica de escolha em 12 estudos para diagnóstico de NG. Nos outros 14 estudos foram utilizados distintos kits diagnósticos comerciais. Cada vez mais se observa o uso de amostras biológicas coletadas de forma menos invasiva para o diagnóstico de DST. As técnicas moleculares encontradas no presente estudo são as mais empregadas em pacientes assintomáticos, pois apresentam maior eficiência na detecção de NG, cujo padrão ouro só se aplica em pacientes sintomáticos. Na revisão de prontuários entre as 340 mulheres estudadas, a prevalência de NG de 0,6% e coinfecção com Chlamydia trachomatis (CT) foi 0,6%. DIP foi presente em 2,1% dos casos estudados sendo 2 casos por coinfecção de NG e CT e a média de idade foi 33 anos. As infecções por CT e NG tiveram baixa prevalência na amostra estudada e são necessários estudos em outros centros de reprodução do país para confirmar a prevalência de infecções nesse grupo particular de mulheres com problemas de infertilidade.

A gonorréia permanece até os dias atuais como um problema de saúde pública. A resistência plasmidial à penicilina, em cepas de Neisseria gonorrhoeae, constitui um problema crescente, preocupando autoridades sanitárias em todo o mundo. O conhecimento das características epidemiológicas e da dinâmica destas cepas na população é imperativo, para o sucesso de intervenções de combate e controle da gonorréia no Brasil e no mundo. Os fenômenos de instalação, manutenção e propagação da Neisseria gonorrhoeae produtora de penicilinase são ainda pouco conhecidos. Este trabalho estudou as características fenotípicas (comportamento de sensibilidade às drogas antimicrobianas, características do plasmídio de resistência, auxotipo e sorotipo), da Neisseria gonorrhoeae produtora de penicilinase (NGPP), objetivando fornecer conhecimentos quanto aos fenômenos de instalação, manutenção e propagação destas cepas, de forma a contribuir com o programa de combate e controle da gonorréia na cidade de São Paulo e no Brasil. Foram estudadas 15 cepas de Neisseria gonorrhoeae produtora de penicilinase (NGPP) e 50 de Neisseria gonorrhoeae não produtora penicilinase (não NGPP), isolados no serviço de Doenças Sexualmente Transmissíveis (DST) da Faculdade de Saúde Pública da Universidade de São Paulo, no período de abril de 1985 a abril de 1990. Com uma incidência de 3,11 por cento de cepas de NGPP, os resultados permitiram colaborar para formação de perfil epidemiológico das cepas produtoras de penicilinase na cidade de São Paulo. Quanto as características de sensibilidade aos antimicrobianos, foram observados altos níveis de resistência a tetraciclina. As demais características fenotípicas, plasmídios, auxotipo e sorotipo, demonstram que a cidade de São Paulo é uma região geográfica \"aberta\" ao fenômeno de \"importação\", tendo sido observadas características epidemiológicas descritas em várias outras localidades no mundo. Os resultados obtidos neste trabalho reforçam a necessidade da instalação de um programa de vigilância epidemiológica, visando manter sob controle a dinâmica da Neisseria gonorrhoeae, colaborando com o programa de combate e controle da gonorréia na cidade de São Paulo e no Brasil. / Gonorrhoea has been considered as a public health problem up to these days. The plasmid penicilin resistance, in Neisseria gonorrhoeae strains, is an increasing problem which concerns authorities from the whole world. The Knowledge on the epidemiological characteristics and on the dynamics of these strains in the populations is extremely important for the success of interventions and gonorrhoea contrai programmes in Brazil and in the world. The onset phenomena, maintenance and proliferation of the penicilin producing Neisseria gonorrhoeae (PPNG) are not well known yet. This study presents the phenotype characteristics (sensibility to antimicrobial drugs, plasmid resistance characteristics, auxotype and serotype) of the PPNG. The objectives of the study are to contribute to the knowledge related to the onset phenomena, maintenance and proliferation of these strains, so that we can contribute to the gonorrhoea contrai programme in the city of São Paulo and in Brazil. We studied 15 strains of penicilin producing Neisseria gonorrhoeae (PPNG) and 50 of non penicilin producing Neisseria gonorrhoeae (non PPNG), which were isolated in the Sexually Transmitted Disease Service of the Faculty of Public Health of the University of São Paulo, from April 1985 to April 1990. With an incidence rate of 3.11 per cent of Neisseria gonorrhoeae, the results allowed us to design an epidemiological profile of the penicilin producing strains in São Paulo. Regarding the antimicrobial sensibility characteristics, we observed high leveis of resistance to tetracycline. The other phenotype characteristics (plasmid, auxotype, and serotype) show that the city of São Paulo is a geographic region open to \"importation\" phenomenon, since we have observed epidemiologic charactéristics described in several places in the world. The results of this reinforce the necessity of setting up an epidemiological surveillance programme with the objective of controlling the Neisseria gonorrhoeae dynamics and, in this way, to colaborate with the gonorrhoea control programme in the city of São Paulo and in Brazil.

Chan, Lap Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 131-147). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / LIST OF ABBREVIATIONS --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- Prevalence of antimicrobial reisitance in gonococcal strains --- p.4 / Chapter 1.2.1 --- Prevalence of penicillin resistant gonococcal strains --- p.5 / Chapter 1.2.2 --- Prevalence of tetracycline resistant gonococcal strains --- p.6 / Chapter 1.2.3 --- Prevalence of quinolone resistant gonococcal strains. --- p.7 / Chapter 1.2.4 --- Emergence of generation cephalosporin reduced susceptible gonococcal strains --- p.8 / Chapter 1.2.5 --- Monitoring the prevalence of gonorrhea --- p.9 / Chapter 1.3 --- Gonococcal Antimicrobial Surveillance Program --- p.10 / Chapter 1.4 --- Antimicrobial Resistance Mechanisms in N. gonorrhoeae --- p.11 / Chapter 1.4.1 --- "Innate Resistance Mechanisms in N, gonorrhoeae" --- p.11 / Chapter 1.4.1.1 --- Natural mechanisms in N. gonorrhoeae against toxic substance --- p.11 / Chapter 1.4.1.2 --- Efflux pump inhibitors --- p.13 / Chapter 1.4.2 --- Development of acquired antimicrobial resistance in N. gonorrhoeae --- p.14 / Chapter 1.4.2.1 --- Penicillin --- p.14 / Chapter 1.4.2.1.1 --- Chromosomal-mediated resistance --- p.14 / Chapter 1.4.2.1.2 --- Plasmid-mediated resistance --- p.16 / Chapter 1.4.2.2 --- Tetracycline --- p.16 / Chapter 1.4.2.2.1 --- Plasmid-mediated resistance --- p.17 / Chapter 1.4.2.2.2 --- Chromosomal mediated resistance --- p.18 / Chapter 1.4.2.3 --- Fluroquinolone --- p.18 / Chapter 1.4.2.3.1 --- Resistant mechanism in quinolone resistant gonococcal strains --- p.19 / Chapter 1.4.2.3.1.1 --- gyrA andparC --- p.19 / Chapter 1.4.2.3.1.2 --- NorM efflux system --- p.21 / Chapter 1.4.2.4 --- 3rd generation cephalosporins --- p.22 / Chapter 1.4.2.4.1 --- Mosaic penA structure in reduced susceptible gonococcal strains --- p.22 / Chapter 1.4.2.4.2 --- Other mechanisms related to reduced susceptibility in gonococcal strains --- p.24 / Chapter 1.5 --- Application of molecular typing methods to study the epidemiology of N. gonorrhoeae --- p.24 / Chapter 1.5.1 --- Opa typing --- p.25 / Chapter 1.5.2 --- K gonorrhoeae Multi-Antigen Sequence Typing --- p.25 / Chapter 1.6 --- Project Objectives --- p.27 / Chapter CHAPTER 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Collecting gonococcal strains --- p.29 / Chapter 2.2 --- Culturing of N. gonorrhoeae --- p.29 / Chapter 2.3 --- Identification --- p.29 / Chapter 2.3.1 --- Gram staining test --- p.30 / Chapter 2.3.2 --- Oxidase test --- p.31 / Chapter 2.3.3 --- Cabohydrate utilization test --- p.31 / Chapter 2.4 --- In-vitro antimicrobial susceptibility testing --- p.32 / Chapter 2.4.1 --- Preparation of cell cultures for MIC tests --- p.32 / Chapter 2.4.2 --- Preparation of antimicrobial agents for MIC tests --- p.32 / Chapter 2.4.3 --- Inoculum preparation and delivering --- p.33 / Chapter 2.5 --- Preparation of genomic DNA for detection of mutations --- p.34 / Chapter 2.6 --- Study of Resistant Mechanism against fluoroquinolone --- p.34 / Chapter 2.6.1 --- PGR detection of mutations in gyrA and parC genes --- p.35 / Chapter 2.6.2 --- Optimization of gyrA and parC genes PGR --- p.35 / Chapter 2.6.3 --- Detection of PGR products for gyrA and parC genes --- p.37 / Chapter 2.6.4 --- Purification of Amplified DNA products --- p.37 / Chapter 2.7 --- Tests of efflux inhibitor on N. gonorrhoeae --- p.38 / Chapter 2.7.1 --- Effect ofCCCP --- p.39 / Chapter 2.7.2 --- Effect of Reserpine --- p.39 / Chapter 2.8 --- Study of Resistant mechanism against β-lactams --- p.40 / Chapter 2.8.1. --- Detection for the presence of β-lactamase --- p.40 / Chapter 2.8.2 --- Mosaic penA patterns --- p.40 / Chapter 2.8.2.1 --- Detection of mutations in penA gene --- p.40 / Chapter 2.8.2.2 --- Optimization of penA gene PGR --- p.41 / Chapter 2.8.2.3 --- Detection of PGR products --- p.43 / Chapter 2.8.2.4 --- Purification of Amplified DNA products --- p.44 / Chapter 2.9 --- Detection of the presence of tetM determinant --- p.45 / Chapter 2.9.1 --- Optimization of tetM determinant PCRs --- p.46 / Chapter 2.9.2 --- Detection of PGR products --- p.47 / Chapter 2.10 --- Detection of different allele types in tbpB andpor genes --- p.48 / Chapter 2.10.1 --- Optimization of PGR for NG-MAST --- p.48 / Chapter 2.10.2 --- Detection of PCR products --- p.49 / Chapter 2.10.3 --- PCR product purification --- p.50 / Chapter 2.11 --- Sequencing of the PCR products --- p.51 / Chapter 2.12 --- Data Analysis --- p.52 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1 --- Gonococcal strains collected --- p.55 / Chapter 3.2 --- Identification of gonococcal strains --- p.55 / Chapter 3.3 --- MIC of Antimicrobial agents --- p.56 / Chapter 3.3.1 --- Interpretive Criteria --- p.56 / Chapter 3.3.2 --- Ciprofloxacin --- p.56 / Chapter 3.3.3 --- Penicillin --- p.57 / Chapter 3.3.4 --- Tetracycline --- p.57 / Chapter 3.3.5 --- Ceftriaxone --- p.58 / Chapter 3.3.6 --- Cefixime --- p.58 / Chapter 3.3.7 --- Cefotaxime --- p.58 / Chapter 3.3.8 --- Spectinomycin --- p.58 / Chapter 3.3.9 --- Levofloxacin --- p.59 / Chapter 3.3.10 --- Ceftibuten --- p.59 / Chapter 3.4 --- Result of PGR --- p.60 / Chapter 3.4.1 --- gyrA andparC genes --- p.60 / Chapter 3.4.2 --- penA gene --- p.61 / Chapter 3.4.3 --- tbpB and por genes --- p.62 / Chapter 3.4.4 --- tetM determinant --- p.62 / Chapter 3.5 --- β-lactamase --- p.63 / Chapter 3.6 --- Efflux pump inhibitor --- p.63 / Chapter 3.6.1 --- CCCP --- p.64 / Chapter 3.6.2 --- Reserpine --- p.64 / Chapter 3.7 --- Detection of Mutations --- p.66 / Chapter 3.7.1 --- gyrA and parC genes --- p.66 / Chapter 3.7.2 --- penA gene --- p.68 / Chapter 3.8 --- NG-MAST --- p.70 / Chapter 3.8.1 --- tbpB and por gene --- p.71 / Chapter 3.9 --- Porin mutation --- p.72 / Chapter CHAPTER 4. --- DISCUSSION / Chapter 4.1 --- Sampling --- p.74 / Chapter 4.2 --- Methodology --- p.75 / Chapter 4.3 --- MIC distribution of different antimicrobial agents --- p.76 / Chapter 4.4 --- Mechanisms of quinolone resistance --- p.78 / Chapter 4.4.1 --- Mutations at QRDRs --- p.78 / Chapter 4.4.2 --- Association of the number of mutations at parC gene with MIC levels against fluroquinolones --- p.80 / Chapter 4.5 --- Penicillin and Tetracycline Resistant Mechanisms --- p.81 / Chapter 4.6 --- Efflux system --- p.85 / Chapter 4.7 --- NG-MAST --- p.88 / Chapter 4.8 --- Mosaic penA pattern --- p.89 / Chapter 4.9 --- Management of gonorrhea --- p.90 / Chapter CHAPTER 5. --- CONCLUSIONS / REFERENCES

A gonorréia permanece até os dias atuais como um problema de saúde pública. A resistência plasmidial à penicilina, em cepas de Neisseria gonorrhoeae, constitui um problema crescente, preocupando autoridades sanitárias em todo o mundo. O conhecimento das características epidemiológicas e da dinâmica destas cepas na população é imperativo, para o sucesso de intervenções de combate e controle da gonorréia no Brasil e no mundo. Os fenômenos de instalação, manutenção e propagação da Neisseria gonorrhoeae produtora de penicilinase são ainda pouco conhecidos. Este trabalho estudou as características fenotípicas (comportamento de sensibilidade às drogas antimicrobianas, características do plasmídio de resistência, auxotipo e sorotipo), da Neisseria gonorrhoeae produtora de penicilinase (NGPP), objetivando fornecer conhecimentos quanto aos fenômenos de instalação, manutenção e propagação destas cepas, de forma a contribuir com o programa de combate e controle da gonorréia na cidade de São Paulo e no Brasil. Foram estudadas 15 cepas de Neisseria gonorrhoeae produtora de penicilinase (NGPP) e 50 de Neisseria gonorrhoeae não produtora penicilinase (não NGPP), isolados no serviço de Doenças Sexualmente Transmissíveis (DST) da Faculdade de Saúde Pública da Universidade de São Paulo, no período de abril de 1985 a abril de 1990. Com uma incidência de 3,11 por cento de cepas de NGPP, os resultados permitiram colaborar para formação de perfil epidemiológico das cepas produtoras de penicilinase na cidade de São Paulo. Quanto as características de sensibilidade aos antimicrobianos, foram observados altos níveis de resistência a tetraciclina. As demais características fenotípicas, plasmídios, auxotipo e sorotipo, demonstram que a cidade de São Paulo é uma região geográfica \"aberta\" ao fenômeno de \"importação\", tendo sido observadas características epidemiológicas descritas em várias outras localidades no mundo. Os resultados obtidos neste trabalho reforçam a necessidade da instalação de um programa de vigilância epidemiológica, visando manter sob controle a dinâmica da Neisseria gonorrhoeae, colaborando com o programa de combate e controle da gonorréia na cidade de São Paulo e no Brasil. / Gonorrhoea has been considered as a public health problem up to these days. The plasmid penicilin resistance, in Neisseria gonorrhoeae strains, is an increasing problem which concerns authorities from the whole world. The Knowledge on the epidemiological characteristics and on the dynamics of these strains in the populations is extremely important for the success of interventions and gonorrhoea contrai programmes in Brazil and in the world. The onset phenomena, maintenance and proliferation of the penicilin producing Neisseria gonorrhoeae (PPNG) are not well known yet. This study presents the phenotype characteristics (sensibility to antimicrobial drugs, plasmid resistance characteristics, auxotype and serotype) of the PPNG. The objectives of the study are to contribute to the knowledge related to the onset phenomena, maintenance and proliferation of these strains, so that we can contribute to the gonorrhoea contrai programme in the city of São Paulo and in Brazil. We studied 15 strains of penicilin producing Neisseria gonorrhoeae (PPNG) and 50 of non penicilin producing Neisseria gonorrhoeae (non PPNG), which were isolated in the Sexually Transmitted Disease Service of the Faculty of Public Health of the University of São Paulo, from April 1985 to April 1990. With an incidence rate of 3.11 per cent of Neisseria gonorrhoeae, the results allowed us to design an epidemiological profile of the penicilin producing strains in São Paulo. Regarding the antimicrobial sensibility characteristics, we observed high leveis of resistance to tetracycline. The other phenotype characteristics (plasmid, auxotype, and serotype) show that the city of São Paulo is a geographic region open to \"importation\" phenomenon, since we have observed epidemiologic charactéristics described in several places in the world. The results of this reinforce the necessity of setting up an epidemiological surveillance programme with the objective of controlling the Neisseria gonorrhoeae dynamics and, in this way, to colaborate with the gonorrhoea control programme in the city of São Paulo and in Brazil.

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.

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.

Made available in DSpace on 2014-07-29T15:30:37Z (GMT). No. of bitstreams: 1 ZulmireneCardoso2011.pdf: 964377 bytes, checksum: 44d4d3bac7f58b70b63d756a5cde56ae (MD5) Previous issue date: 2011-05-30 / BACKGROUND: Adolescents and youth are the group most vulnerable to sexually transmitted diseases (STDs), which are among the most prevalent infections in the world and represent a serious impact on reproductive health in this age group. OBJECTIVE: To estimate the prevalence and identify risk factors for infection by N. gonorrhoeae and co-positivity with C. trachomatis in sexually active adolescents and young adults living in a midsize city of Goias and describe the socio-demographic and behavioral profile of the population seen at the Family Health Program (PSF). METHODOLOGY: The study was designed as a prevalence study in 651 adolescents and young adults, not pregnant, 15 to 24 years were enrolled in the Family Health Program (FHP) in Inhumas, Goiás were excluded pregnant or postpartum teens in antibiotics or menstruating. The diagnosis of gonococcal infection and infection by C. trachomatis was performed using the Polymerase Chain Reaction (PCR) in endocervical specimens using the Roche Amplicor kit for N. gonorrhoeae and C. trachomatis. The socio-demographic and sexual behavior were obtained through a questionnaire as an interview. Univariate analysis was performed to identify potential risk factors associated with infection. Endocervical swab samples were collected from 322 sexually active patients to dtect the DNA of N. gonorrhoeae and C. trachomatis. RESULTS: The mean age of 428 sexually active adolescents and young adults was 18.6 (SD = 2.7), with 64.3% single. Inconsistent use of condoms was observed in 78.0% of girls and 11.9% never used it. The first sexual intercourse occurred before the age of fifteen in 23.4%. Pregnancy before age 20 occurred in 90% and 23.5% of them before age 15. The prevalence of N. gonorrhoeae was 3.4% (95% CI 1.8 to 6.2) in 322 patients from whom samples were collected endocervical swab. Co-infection with chlamydia was 27.3%. Univariate analysis showed that the risk factor associated with infection by N.gonorrhoeae was not using condoms. CONCLUSIONS: Young women and adolescents in this study exhibited various behaviors considered at risk for STDs and those who reported not using condoms had a higher risk of being infected by the gonococcus. WORDS - KEY: Neisseria gonorrhoeae; Adolescents Prevalence; STD; PCR. / JUSTIFICATIVA: Adolescentes e jovens constituem o grupo mais vulnerável às Doenças Sexualmente Transmissíveis (DST), que estão entre as infecções mais prevalentes no mundo e representam um sério impacto na saúde reprodutiva deste grupo etário. OBJETIVO: Estimar a prevalência, identificar os fatores de risco para a infecção por N. gonorrhoeae e a co-positividade com C. trachomatis em adolescentes e jovens sexualmente ativas residentes em uma cidade de médio porte do estado de Goiás e descrever o perfil sócio demográfico e comportamental da população atendida no Programa de Saúde da Família (PSF). METODOLOGIA: A investigação foi delineada como um estudo de prevalência em 651 adolescentes e jovens, não grávidas, de 15 a 24 anos, cadastradas no Programa de Saúde da Família (PSF) em Inhumas, Goiás. Foram excluídas adolescentes grávidas ou no puerpério, em uso de antibióticos ou menstruadas. O diagnóstico da infecção gonocócica e da infecção por C. trachomatis foi realizado empregando a Reação de Cadeia de Polimerase (PCR) em amostras endocervicais utilizando o kit Amplicor Roche para N. gonorrhoeae e C. trachomatis. Os dados sócio-demográficos e de comportamento sexual foram obtidos através de questionário, na forma de entrevista. Análise univariada foi realizada para identificar potenciais fatores de risco associados à infecção. Amostras de swab endocervical foram colhidas de 322 pacientes sexualmente ativas para a detecção do DNA da N. gonorrhoeae e da C. trachomatis. RESULTADOS: A média de idade das 428 adolescentes e jovens sexualmente ativas foi de 18,6 (dp=2,7), sendo 64,3% solteiras. O uso inconsistente de preservativo foi observado em 78,0% das meninas e 11,9 % nunca o usaram. A sexarca ocorreu antes dos quinze anos em 23,4%. A gravidez ocorreu antes dos 20 anos em 90% delas e em 23,5% antes dos 15 anos. A prevalência de N. gonorrhoeae foi de 3,4% (IC 95%1,8 - 6,2) em 322 pacientes das quais foram obtidas amostras de swab endocervical. A co-infecção com clamídia foi 27,3%. A análise univariada mostrou que o fator de risco associado à infecção por N. gonorrhoeae foi o não uso de preservativos. CONCLUSÕES: As jovens e adolescentes do presente estudo apresentaram vários comportamentos considerados de risco para DST e aquelas que referiram não usar preservativos apresentaram um risco maior de estarem infectados pelo gonococo.