Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-18T18:53:21Z No. of bitstreams: 1 Vivian Galvão. Caracterizaçao de cepas...2012.pdf: 1132531 bytes, checksum: c5a7374a74f6f74d8bd7cc7573c224f5 (MD5) / Made available in DSpace on 2013-10-18T18:53:21Z (GMT). No. of bitstreams: 1 Vivian Galvão. Caracterizaçao de cepas...2012.pdf: 1132531 bytes, checksum: c5a7374a74f6f74d8bd7cc7573c224f5 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A doença pneumocócica invasiva (DPI) continua sendo uma das principais causas de morbidade e mortalidade no mundo, mesmo com a disponibilidade atual de terapias antimicrobianas e vacinas conjugadas. O objetivo desse estudo foi caracterizar o perfil fenotípico e genotípico das cepas invasivas de Streptococcus pneumoniae isoladas de diferentes sítos de infecção que circulam em hospitais públicos e privados da cidade de Salvador-Brasil no período de janeiro de 2008 a julho de 2011. Os isolados de S. pneumoniae de doença invasiva foram identificados por métodos microbiológicos clássicos e submetidos à determinação capsular através da técnica de Multiplex-PCR. A sensibilidade aos antimicrobianos foi determinada pela técnica de microdiluição em caldo. A caracterização genotípica foi realizada por PFGE e MLST. No período do estudo foram identificados 75 casos de DPI com cultura positiva, sendo 82,7% provenientes de hemocultura, 9,3% de líquido pleural e 8,0% de líquor. As crianças representaram 37,9% e os idosos 24,0% da população em estudo. Os sorotipos mais prevalentes foram o 14 (14,7%), 19F (13,3%), 6B (10,7%), 23F (9,3%), 3 (9,3%) e 19A (6,7%). Um total de 57,3% dos sorotipos identificados estão representados na vacina PCV10. Não-susceptibilidade à penicilina (CIM ≥ 4μg/mL) foi observada em 5,3% dos isolados. Para o SMX-TMP, tetraciclina e eritromicina, os índices de não-susceptibilidade foram de 55%, 15% e 11%, respectivamente. A tipagem por PFGE classificou 61,3% dos isolados de DPI como não-clonais e 29 (38,7%) em 10 perfis clonais. Quando comparados aos isolados de meningite isolados no Hospital Couto Maia, 22,7% apresentaram perfis semelhantes, que foram distribuídos em seis grupos clonais (quatro grupos clonais com isolados não-susceptíveis à penicilina e dois sensíveis). Foram encontrados 22 STs diferentes entre as 26 amostras caracterizadas por MLST. Quando comparado aos clones já caracterizados pelo PMEN, verificou-se que na cidade de Salvador circulam clones já identificados em outros países, a exemplo dos clones: Colombia23F-26 (SLV 338), Portugal19F-21 (ST 177), Spain9V-3 (SLV 156) e Netherlands3-31 (ST 180). Os isolados de pneumococos deste estudo apresentam maior taxa de resistência, incluindo resistência a múltiplas drogas quando comparados aos dos casos de meningite identificados em casos de meningite, com exceção da penicilina. Embora os clones mais frequentemente associados aos casos de meningite pneumocócia em Salvador tenham sido identificados nesta casuistica, os isolados de pneumococos provenientes de outras formas de doença invasiva apresentaram uma maior diversidade fenotípica e genotípica, ressaltando a importância do monitoramento contínuo das cepas invasivas nas diferentes manifestações da doença pneumocócica no tempo das vacinas conjugadas. / Invasive pneumococcal disease (IPD) remains one of the major causes of morbidity and mortality worldwide, even with the current availability of antimicrobial therapies and conjugate vaccines. The objective of the present study was to characterize the phenotypic and genotypic profiles of invasive pneumococcal isolates obtained from patients admitted to public and private hospitals in Salvador, from the period of Jan/2008 to Jul/2011. The pneumococcal isolates from invasive disease were identified by classical microbiological methods and submitted to capsular deduction by multiplex-PCR. The antimicrobial susceptibility was performed by broth microdilution method. The genotypic profile was accessing by PFGE and MLST. During the study period, 75 consecutive culture-positive cases of IPD were characterized, being 82.7% from blood, 9.3% from pleural fluid and 8.0% from CSF. The study population comprised of 37.9% of children (under 5 years old) and 24.0% of elderly (upper 64 years old). The most prevalent serotypes were: 14 (14.7%), 19F (13.3%), 6B (10.7%), 23F (9.3%), 3 (9.3%) and 19A (6.7%). A total of 57.3% of the serotypes identified are represented in the vaccine PCV10. Non-susceptibility to penicillin (MIC ≥ 4μg/mL) was observed in 5.3% of the isolates. The non-susceptibility rate for SMX-TMP, tetracycline and erythromycin was found in 55%, 15% and 11% of the isolates, respectively. The PFGE pattern analysis classified 61.3% of IPD isolates as non-clonal and 29 (38.7%) isolates were clustered in ten PFGE profiles. When the clonal strains were compared with pneumococcal meningitis isolates from Hospital Couto Maia, 22.7% showed similar profiles by PFGE, being distributed into six clonal groups (four clonal groups of penicillin non-susceptible and two of penicillin susceptible). Twenty-two STs were observed among the 26 samples characterized by MLST. When compared to already characterized PMEN clones, it was found that some circulating clones in the city of Salvador have been identified in other countries, such as: Colombia23F-26 (SLV 338), Portugal19F-21 (ST 177), Spain9V-3 (SLV 156) and Netherlands3-31 (ST 180). The pneumococcal strains in the study have a higher rate of resistance, including multidrug resistance when compared to cases of meningitis identified in the HCM, except for penicillin. Although the clones most frequently associated with cases of meningitis in Salvador have been identified in this study, isolates of pneumococci from other forms of IPD had a higher phenotypic and genotypic diversity, highlighting the importance of continuous monitoring of invasive strains in different manifestations of pneumococcal infection in the time of conjugate vaccines.

Meningitis is an infection of the meninges, a layer of tissue surrounding the brain. In cases of pneumococcal meningitis (where the bacterium Streptococcus pneumoniae is the causat- ive agent) this causes severe inflammation, requiring intensive care and rapid antibiotic treatment. The contribution of variation in host and pathogen genetics to pneumococcal meningitis is unknown. In this thesis I develop and apply statistical genetics techniques to identify genomic variation associated with the various stages of pneumococcal meningitis, including colonisation, invasion and severity. I start by describing the development of a method to perform genome-wide association studies (GWAS) in bacteria, which can find variation in bacterial genomes associated with bacterial traits such as antibiotic resistance and virulence. I then applied this method to longitudinal samples from asymptomatic carriage, and found lineages and specific variants associated with altered duration of carriage. To assess meningitis versus carriage samples I applied similar analysis techniques, and found that the bacterial genome is crucial in determining invasive potential. As well as bacterial serotype, which I found to be the main effect, I discovered many independent sequence variants associated with disease. Separately, I analysed within host-diversity during the invasive phase of disease and found it to be of less relevance to disease progression. Finally, I analysed host genotype data from four independent studies using GWAS and heritability estimates to determine the contribution of human sequence variation to pneumococcal meningitis. Host sequence accounted for some variation in susceptibility to and severity of meningitis. The work concludes with a combined analysis of pairs of bacterial and human sequences from meningitis cases, and finds variation correlated between the two.

Neisseria meningitidis är en bakterie som kan leda till invasiv sjukdom eller endast orsaka bärarskap i nasopharynx. Bakterien delas in i olika serogrupper och klonala komplex. Vissa av dessa grupper och klonala komplex förekommer endast hos invasiva isolat och andra bland bärare, vilket tyder på att det finns genetiska skillnader mellan invasiva och bärarisolat. I denna studie undersöktes genen pilE som kodar för PilE proteinet och ingår i bakteriens pili. Proteinet finns i två klasser, klass 1 och klass 2. Metoden som användes för att studera eventuella skillnader i förekomst och uttryck av pilE genen var digital droplet PCR (ddPCR). Både DNA och RNA kvantifierades med ddPCR för att undersöka antalet kopior av pilE genen (DNA) samt dess uttryck (RNA) mellan invasiva isolat och bärarisolat, mellan klass 1 pilE isolat och klass 2 samt fördelning av klasserna i isolattyperna. Principen för ddPCR är att dela ett prov till tiotusentals nanodroppar där individuella droppar genomgår PCR för en senare avläsning av flourescens från prober. Resultatet visade skillnad mellan bärarisolat och invasiva isolat där de invasiva isolaten visade mindre uttryck av pilE än bärarisolat. Klass 2 isolat hade signifikant färre genuttryck än klass 1 isolat och invasiva isolat visade klass 2 i högre utsträckning än bärarisolat. / Neisseria meningitidis is a bacterium that can cause invasive disease or carriage in the nasopharynx. The bacteria are divided into different serogroups and clonal complexes. Specific serogroups and clonal complexes are more frequent or are only found among invasive isolates or in carriage isolates. This study has investigated pilE, a gene that encodes the PilE protein located in the bacteria´s pilin and the protein is found in either class 1 or class 2. digital droplet PCR was used to investigate differences in presence and expression of the gene pilE. Both DNA and RNA was quantified to study the difference in copy number of the pilE gene (DNA) and its expression (RNA) between invasive isolates and carriage isolates but also between class 1 isolates and class 2 isolates. The distribution of classes between the isolate types was also investigated. The ddPCR method divides a sample into thousands of nanodroplets and a PCR reaction occurs in each droplet followed by droplet reading where fluoroscens from probes is measured. The difference that could be seen was that the invasive isolates expressed pilE in lower copies. Class 2 isolates had a significantly lower gene expression than class 1 isolates and invasive isolates expressed class 2 in a higher frequency.

This thesis is concerned with the level of parents´ awareness and attitudes to above-standard vaccination of children from 0-3 years in Příbram. Among the optional vaccination of children from 0-3 years belong vaccination against pneumococcal disease, rotavirus infections, meningococcal invasive disease (caused by meningococcal of group C, A+C, or A, C, W 135 and Y, now also of group B), tick-borne encephalitis, varicella smallpox, influenza and hepatitis A (or a combination of type A + B). The first part deals with the issue in a theoretical perspective. It foreshadows the basic characteristics of the diseases against which the premium vaccination of children from 0-3 years is offered. Further it is focused on vaccines against these diseases, which can occur in the Czech Republic. The survey is summarized in the research. There were parents of children attending one of the 12 kindergartens in Příbram in the sample of the research. tely 40% of the parents (or mothers) of these children in each kindergarten.The results were summarized in schedules of absolute and relative frequencies, or graphs. It was set three basic goals that were met with five hypotheses. The first of these was to monitor the attitudes of parents to above-standard vaccination of children from 0-3 years in Příbram. To this target relate hypothesis H1: Parents are interested in above-standard vaccination of children from 0-3 years in Příbram, H2: Parents with higher educational level have significantly higher interest in above-standard vaccination of children from 0-3 years and H3: Parents are statistically significantly more interested in vaccination against tick-borne encephalitis than other extra vaccination of children from 0-3 years, due to an endemic area of tick-borne encephalitis in Příbram. These hypotheses were not confirmed statistically. The second aim investigated the main reason for the possible lack of interest in extra vaccination of children from 0-3 years in Příbram. The target was filled with hypothesis H4: The main reason for the possible lack of parents´ interest in extra vaccination of children from 0-3 years is high price of vaccines, which was statistically refuted. The third goal was to explore parents' knowledge about diseases against which the extra vaccination of children from 0-3 years is offered. To this goal was set the hypothesis H5: Parents have sufficient information about the diseases against which the extra vaccination of children from 0-3 years is offered. This hypothesis was confirmed. The hypotheses were verified by ?chi-square? test at a significance level of 5%, which is an instrument of verification or falsification of hypotheses. This work may be used in practice as a preview to the parents´ awareness and interest in extra vaccination of children from 0-3 years. As well as cumulative information materials for professionals and the public about the above-standard vaccination of children from 0-3 years.