Chlamydial infections have been associated with several chronic human diseases, including trachoma, pelvic inflammatory disease, chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease. In Chlamydia-associated disease, the organisms are believed to exist in an atypical, persistent phase that is not well understood at the genetic level. The research presented in this thesis investigated chlamydial gene expression in in vitro cell culture models of persistence. The first set of studies analysed a continuous-infection model of persistence that has been recently developed for two C. pneumoniae isolates (TW-183 and CM-1). The spontaneous establishment and unique cyclical nature of continuous infections could be particularly relevant to in vivo events. An initial analysis using a semi-quantitative reverse transcriptase PCR (sqRT-PCR) approach provided evidence of differential gene expression in C. pneumoniae TW-183 continuous infections relative to acute control infections. Using a subsequently established fully quantitative real-time reverse transcriptase PCR (rtRT-PCR) assay, up-regulated expression profiles were confirmed for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in the continuous C. pneumoniae TW-183 infections. The omcB, pmp1 and porB genes, all of which encode membrane proteins, showed similar patterns of expression over both the acute and continuous time courses tested. Gene expression data for a second C. pneumoniae isolate, CM-1, revealed similar overall expression trends to those seen for C. pneumoniae TW-183 but also supported previous observations of different growth characteristics between the two isolates in the continuous-infection model. The rtRT-PCR assay was further optimised for use in gene expression studies of the gamma interferon (IFN-γ)-mediated model of C. pneumoniae A-03 persistence, in which altered growth and morphological traits typical of chlamydial persistence have been well characterised. Meanwhile, chlamydial genes such as euo, ftsK and hctB were emerging from the literature as reliable genetic markers of persistence. Therefore, a preliminary rtRT-PCR analysis of marker gene expression was used to assess the likely extent of persistence in individual IFN-γ-treated C. pneumoniae A-03 infections from a series of experiments that had been prepared for this persistence model. In this way, an appropriate pair of duplicate experiments was selected for further studies based on strong genetic evidence of persistence in IFN-γ-treated samples at 48 h post-infection (PI) in those experiments. Using rtRT-PCR, 14 genes of interest from the related peptidoglycan, aminosugars and lipopolysaccharide (LPS) biosynthetic pathways were analysed in the validated experiments of the IFN-γ-mediated C. pneumoniae A-03 persistence model. Selective up- and down-regulated expression trends were associated with IFN-γ-treatment at 48 h PI for genes encoding products that are located at specific enzymatic points in these pathways. Most strikingly, the expression of glmU, the product of which controls the amount of an essential precursor metabolite that enters both peptidoglycan and LPS biosynthesis, was strongly and reproducibly down-regulated in the 48-h PI IFN-γ-treated samples. This expression profile may contribute to a reduced rate of peptidoglycan biosynthesis in this persistence model and may therefore be related to the inhibited cell division and RB-to-EB differentiation that characterise chlamydial persistence. While most other genes in these pathways showed unchanged expression associated with IFN-γ treatment, murA and kdsB (from peptidoglycan and LPS biosynthesis, respectively) were selectively up-regulated in the 48-h PI IFN-γ-treated samples. Taken together, these data supported the concept of a persistence stimulon in C. pneumoniae that is regulated at key points in various metabolic pathways. In addition to the analysis of biosynthetic genes, the up-regulated gene set from continuous C. pneumoniae TW-183 infections was also analysed in the validated IFN-γ-mediated C. pneumoniae A-03 persistence experiments. The data revealed similarities and differences in gene expression patterns between these two in vitro persistence models. Furthermore, the profiles obtained for genes such as pmp1 and porB provided insights into the widely predicted phenomenon of late developmental gene shut-down during chlamydial persistence. A final investigation into an analogous IFN-γ-mediated persistence system for C. trachomatis serovar L2 focussed on one up-regulated (murA) and one down-regulated (glmU) gene from the validated IFN-γ-mediated persistent C. pneumoniae A-03 data set. Both genes were significantly down-regulated in persistent C. trachomatis, adding to a growing body of evidence for key differences among chlamydial species in their persistent gene expression patterns. This project has contributed significantly to our understanding of the molecular basis of the important persistent phase of chlamydial development.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 5 uppsatser.

The bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection worldwide. When left untreated, chlamydial infections can lead to severe complications, such as infertility. Lack in current prevention and management due to its asymptomatic course of infection highlight the need for an effective vaccine against chlamydia. There is no vaccine at present to protect against chlamydia, but research is ongoing. A research group at Örebro University has developed a protein antigen candidate. This project focused on the production of the candidate, here called Protein X, for preclinical trials. This included optimising production in Escherichia coli to maximise formation of soluble protein, optimising purification, buffer exchange and removal of His-tag. It was found that formation of soluble protein was favoured in lower expression temperatures. Furthermore, purification was performed on soluble and insoluble protein fractions using immobilised metal affinity chromatography. However, issues with inefficient binding to the resin and purity could not be solved and further optimisation is needed. Buffers were tested to find a suitable buffer for preclinical experiments, but the protein precipitated in all buffers. It was however found that protein from the insoluble fraction dissolved in pure water. Lastly, removal of the His-tag was performed with a non-enzymatic method that utilises nickel ions instead of expensive proteases. Efficient removal was however not achieved and enzymatic methods may be considered instead. In conclusion, this project highlighted issues in the production of Protein X and may guide the research group towards improving this process for efficient preclinical preparations.

Indiana University-Purdue University Indianapolis (IUPUI) / Progression of Chlamydia into upper reproductive tract epithelium and the induction of subsequent immune responses to infection are major contributors to Chlamydia-induced pathogenesis of the genital tract. We reported that C. muridarum infection of the oviduct epithelial cells (OEs) secrete IFN-β in a TLR3 dependent manner. However, we showed that the C. muridarum infected TLR3-deficient OEs were still able to secrete minimal amounts of IFN-β into the supernatants, which is suggestive that there are other signaling pathways that contribute to Chlamydia-induced IFN-β synthesis in these cells. Previous studies describing the activation of the JAK/STAT signaling pathway during Chlamydia infection of cervical epithelial cells proposes a putative role for STAT1 in the synthesis of type I IFNs during Chlamydia infection. The present study investigated the role of STAT1 in Chlamydia-induced IFN-β production in OEs. OEs were infected with Chlamydia muridarum and analyzed at 24 hours by RT-PCR and western blot to determine STAT1 expression. STAT (-/-) OEs were infected and IFN-β production measured by ELISA. Quantitative real-time PCR analyses were performed at 6 and 16 hour post-infection to elucidate the mechanisms involved in IFN-β production during infection. Fluorescent microscopy was used to observe changes in Chlamydia replication. STAT1 activation and expression were significantly increased in wild-type (WT) OEs upon infection. TLR3 (-/-) OEs showed diminished STAT1 protein activation and expression. Augmented STAT1 protein expression corresponded to STAT1 mRNA levels. ELISA analyses revealed significantly less IFN-β production in infected STAT1 (-/-) OEs compared to WT OEs. Quantitative real-time PCR data showed that gene expression of IFN-β and of type I IFN signaling components were significantly increased during late stage Chlamydia infection, dependent on STAT1. Temporal regulation and increases in expression of IFN-α subtypes during infection were STAT1-dependent. Our results implicate STAT1-mediated signaling as a contributor to the C. muridarum-induced synthesis of IFN-β and other type I IFNs in OEs. We previously described a major role for TLR3 in the early-stage Chlamydia-induced synthesis of IFN-β in OEs; the results from this study suggest a role for STAT1 in the synthesis of type I IFNs that occurs during early and late stages of infection.

Yes / With the aim of targeting high-risk hidden heterosexual young people for Chlamydia trachomatis (CT) testing, an innovative web-based screening strategy using Respondent Driven Sampling (RDS) and home-based CT testing, was developed, piloted and evaluated. Two STI clinic nurses encouraged 37 CT positive heterosexual young people (aged 16-25 years), called index clients, to recruit peers from their social and sexual networks using the web-based screening strategy. Eligible peers (young, living in the study area) could request a home-based CT test and recruit other peers. Twelve (40%) index clients recruited 35 peers. Two of these peers recruited other peers (n = 7). In total, 35 recruited peers were eligible for participation; ten of them (29%) requested a test and eight tested. Seven tested for the first time and one (13%) was positive. Most peers were female friends (80%). Nurses were positive about using the strategy. The screening strategy is feasible for targeting the hidden social network. However, uptake among men and recruitment of sex-partners is low and RDS stopped early. Future studies are needed to explore the sustainability, cost-effectiveness, and impact of strategies that target people at risk who are not effectively reached by regular health care.

A profilaxia pré-exposição (PrEP) ao HIV se consolidou como uma importante estratégia de combate ao avanço da epidemia. Ainda assim, a incidência de casos da infecção vem aumentando na população jovem, assim como a de outras infecções sexualmente transmissíveis (ISTs), que atuam como importante fator de risco para transmissão do HIV-1. Entre as infecções mais frequentemente diagnosticadas estão Chlamydia trachomatis (CT) e Neisseria gonorrhoeae (NG). A presença de lesões na mucosa genital e anal são fatores de risco estabelecidos para a transmissão do HIV-1, porém o impacto das infecções assintomáticas ainda é pouco conhecido. Dados recentes mostram que a ativação sistêmica de linfócitos T é um fator de risco para a aquisição da infecção pelo HIV-1. Nesse estudo, estudamos a ativação de linfócitos T na presença de infecção anorretal assintomática por CT e/ou NG. Células mononucleares do sangue periférico de voluntários do PrEP Brasil, um estudo clínico demonstrativo de PrEP ao HIV em homens que fazem sexo com homens, foram descongeladas para análise da ativação de linfócitos T. Trinta e quatro participantes com swab anorretal positivo para CT e/ou NG foram selecionados, enquanto assintomáticos e negativos para outras ISTs. Trinta e cinco controles foram selecionados randomicamente. Encontramos uma maior frequência de linfócitos T CD8+ HLA-DR+CD38+ (1,5 vs. 0,9% p < 0,005) no grupo com infecção assintomática. Os linfócitos T CD8+ de memória também apresentaram uma maior expressão dos marcadores de ativação. Os marcadores de exaustão e senescência foram significantemente mais expressos no grupo com a infecção. Não foi observado aumento ou diferença nos níveis de CD14 solúvel no plasma. Nossos achados demonstram que as infecções anorretais assintomáticas por CT e NG induzem a ativação sistêmica de linfócitos T CD8+. Considerando a alta prevalência dessas infecções e o risco associado de aquisição da infecção pelo HIV-1, o rastreamento periódico e o tratamento sistemático devem sem explorados em conjunto com as estratégias de prevenção ao HIV / Oral antiretroviral pre-exposure prophylaxis (PrEP) has been established as a pivotal strategy in the prevention against HIV epidemic. However, the incidence of HIV-1 infections has been rising among the youth, as well as other sexually transmitted infections (STIs), acting as an important risk factor for HIV-1 acquisition. Infection by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most diagnosed. Although the presence of mucosal lesions is a known risk factor for HIV-1 acquisition, the potential increase in risk associated with asymptomatic STIs is not completely understood. Recent data defined higher T cell activation as a single risk factor for sexually acquired HIV-1 infection. We examined the effect of asymptomatic CT and/or NG anorectal infection on immune activation. Peripheral blood mononuclear cells from participants of PrEP Brasil, a study of daily oral PrEP among healthy men who have sex with men, were analyzed for T cell activation by flow cytometry. Thirty-four participants with positive anorectal swab for CT and/or NG were selected, while negative for other STIs and without any reported symptoms. Thirty-five controls were randomly selected. We found a higher frequency of CD8+ HLA-DR+CD38+ T cells (1.5 vs. 0.9% p < 0.005) in the group with CT and/or NG infection and a greater median proportions of activation markers expression in CD8+ T cells with memory phenotype. Exhaustion and senescence markers were also significant higher in the infected group. No difference was observed in the soluble CD14 levels. Our findings suggest that asymptomatic CT and NG anorectal infection lead to a systemic activation of the T cell compartment. Considering the high prevalence of asymptomatic infection and the risk of HIV-1 acquisition associated, regular screening and treatment should be explored as an adjuvant tool for HIV prevention

A profilaxia pré-exposição (PrEP) ao HIV se consolidou como uma importante estratégia de combate ao avanço da epidemia. Ainda assim, a incidência de casos da infecção vem aumentando na população jovem, assim como a de outras infecções sexualmente transmissíveis (ISTs), que atuam como importante fator de risco para transmissão do HIV-1. Entre as infecções mais frequentemente diagnosticadas estão Chlamydia trachomatis (CT) e Neisseria gonorrhoeae (NG). A presença de lesões na mucosa genital e anal são fatores de risco estabelecidos para a transmissão do HIV-1, porém o impacto das infecções assintomáticas ainda é pouco conhecido. Dados recentes mostram que a ativação sistêmica de linfócitos T é um fator de risco para a aquisição da infecção pelo HIV-1. Nesse estudo, estudamos a ativação de linfócitos T na presença de infecção anorretal assintomática por CT e/ou NG. Células mononucleares do sangue periférico de voluntários do PrEP Brasil, um estudo clínico demonstrativo de PrEP ao HIV em homens que fazem sexo com homens, foram descongeladas para análise da ativação de linfócitos T. Trinta e quatro participantes com swab anorretal positivo para CT e/ou NG foram selecionados, enquanto assintomáticos e negativos para outras ISTs. Trinta e cinco controles foram selecionados randomicamente. Encontramos uma maior frequência de linfócitos T CD8+ HLA-DR+CD38+ (1,5 vs. 0,9% p < 0,005) no grupo com infecção assintomática. Os linfócitos T CD8+ de memória também apresentaram uma maior expressão dos marcadores de ativação. Os marcadores de exaustão e senescência foram significantemente mais expressos no grupo com a infecção. Não foi observado aumento ou diferença nos níveis de CD14 solúvel no plasma. Nossos achados demonstram que as infecções anorretais assintomáticas por CT e NG induzem a ativação sistêmica de linfócitos T CD8+. Considerando a alta prevalência dessas infecções e o risco associado de aquisição da infecção pelo HIV-1, o rastreamento periódico e o tratamento sistemático devem sem explorados em conjunto com as estratégias de prevenção ao HIV / Oral antiretroviral pre-exposure prophylaxis (PrEP) has been established as a pivotal strategy in the prevention against HIV epidemic. However, the incidence of HIV-1 infections has been rising among the youth, as well as other sexually transmitted infections (STIs), acting as an important risk factor for HIV-1 acquisition. Infection by Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are among the most diagnosed. Although the presence of mucosal lesions is a known risk factor for HIV-1 acquisition, the potential increase in risk associated with asymptomatic STIs is not completely understood. Recent data defined higher T cell activation as a single risk factor for sexually acquired HIV-1 infection. We examined the effect of asymptomatic CT and/or NG anorectal infection on immune activation. Peripheral blood mononuclear cells from participants of PrEP Brasil, a study of daily oral PrEP among healthy men who have sex with men, were analyzed for T cell activation by flow cytometry. Thirty-four participants with positive anorectal swab for CT and/or NG were selected, while negative for other STIs and without any reported symptoms. Thirty-five controls were randomly selected. We found a higher frequency of CD8+ HLA-DR+CD38+ T cells (1.5 vs. 0.9% p < 0.005) in the group with CT and/or NG infection and a greater median proportions of activation markers expression in CD8+ T cells with memory phenotype. Exhaustion and senescence markers were also significant higher in the infected group. No difference was observed in the soluble CD14 levels. Our findings suggest that asymptomatic CT and NG anorectal infection lead to a systemic activation of the T cell compartment. Considering the high prevalence of asymptomatic infection and the risk of HIV-1 acquisition associated, regular screening and treatment should be explored as an adjuvant tool for HIV prevention

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-18T15:08:54Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PrevalenciaVariabilidadeGenotipica.pdf: 1636799 bytes, checksum: 6bc83b2eaa373bb9dc5c14d10a80cbc0 (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-11-08T14:41:08Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PrevalenciaVariabilidadeGenotipica.pdf: 1636799 bytes, checksum: 6bc83b2eaa373bb9dc5c14d10a80cbc0 (MD5) / Made available in DSpace on 2017-11-08T14:41:08Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_PrevalenciaVariabilidadeGenotipica.pdf: 1636799 bytes, checksum: 6bc83b2eaa373bb9dc5c14d10a80cbc0 (MD5) Previous issue date: 2015 / A infecção por Chlamydia trachomatis é a Infecção Sexualmente Transmissível (IST) bacteriana mais prevalente no mundo, podendo ser assintomática em até 80% dos casos, e associa-se às complicações tardias. As jovens universitárias fazem parte de uma demanda diferenciada da população por apresentarem alto grau de escolaridade. Objetivo foi verificar a prevalência e a variabilidade dos genótipos de C. trachomatis em infecção cervical das estudantes de universidade pública do estado do Pará, Brasil, e avaliar a associação deste às respectivas características socio-comportamentais e de queixas ginecológicas. Foram incluídas 438 estudantes universitárias entre setembro de 2012 a outubro de 2014 e as amostras endocervicais foram obtidas durante exame ginecológico. Realizou-se a técnica de fenol-clorofórmio para a extração de DNA total da amostra de secreção cervical, e para a detecção de C. trachomatis, utilizou-se a Reação em Cadeia da Polimerase (PCR) do tipo seminested-PCR para amplificação de 224pb do gene omp1. Para a identificação dos genótipos, realizou-se uma Nested-PCR, para a amplificação de 990pb do gene omp1, no qual, foi purificada e submetida ao sequenciador ABI3130, posteriormente as sequencias nucleotídicas foram comparadas com as depositadas no GenBank. A prevalência da infecção cervical por C. trachomatis foi de 12,5% (IC: 95% ±5,89) e os genótipos identificados foram o genótipo J(36,3%), seguido dos genótipos D (18,2%), E (18,2%), F (18,2%) e Ia (9,1%). Não houve associação significativa para a idade, início da vida sexual, número de parceiros, se usam preservativo camisinha, presença de queixas ginecológicas e de genótipos encontrados na população de estudo. Embora a prevalência encontrada apresentar-se alta entre as estudantes universitárias, a falta de significância estatística pode ser devido ao número amostral pequeno e/ou consequência de respostas socialmente aceitáveis. Esforços sejam feitos para que a ampliação do rastreio da infecção por C. trachomatis em populações restritas. / Infection with Chlamydia trachomatis is a Sexually Transmitted Infection (STI) more prevalent bacterial in the world and can be asymptomatic in up to 80% of cases and is associated with late complications. The university students are part of a differentiated demand of the population for their high level of education. Objective was to assess the prevalence and variability of the genotypes of C. trachomatis in cervical infection of public university students in the state of Pará, Brazil, and to evaluate the association of the respective socio-behavioral characteristics and gynecological complaints. They included 438 college students from September 2012 to October 2014 and cervical samples were obtained during gynecological examination. It was performed phenol-chloroform technique for extracting total DNA from the sample cervical secretion, and for the detection of C. trachomatis used the Polymerase Chain Reaction (PCR) PCR-seminested the type for amplification 224pb gene omp1. For the identification of genotypes, we carried out a nested PCR for the amplification of the gene omp1 990pb, which was purified and subjected to ABI3130 sequencer, then the nucleotide sequences were compared with GenBank. The prevalence of cervical infection by C. trachomatis was 12.5% (CI: 95% ± 5.89) and genotypes were identified J genotype (36.3%), followed by D genotypes (18.2%) E (18.2%), F (18.2%) and la (9.1%). There was no significant association for age, first sexual intercourse, number of partners, and condoms are used, the presence of gynecological complaints and genotypes found in the study population. Although the prevalence found present itself high among university students, the lack of statistical significance may be due to small sample size and/or result in socially acceptable answers. Efforts are made to trace the expansion of C. trachomatis infection in restricted populations.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-05-27T22:34:44Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DeteccaoLaboratorialChlamydia.pdf: 3759603 bytes, checksum: 3decacb6f231b37120ea7adb1f579960 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-06-03T15:35:10Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DeteccaoLaboratorialChlamydia.pdf: 3759603 bytes, checksum: 3decacb6f231b37120ea7adb1f579960 (MD5) / Made available in DSpace on 2013-06-03T15:35:10Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DeteccaoLaboratorialChlamydia.pdf: 3759603 bytes, checksum: 3decacb6f231b37120ea7adb1f579960 (MD5) Previous issue date: 2012 / O tracoma como principal causa de cegueira prevenível no mundo, é uma doença negligenciada relacionada a baixas condições socioeconômicas e locais sem saneamento básico. Presente principalmente nos países subdesenvolvidos traz grandes prejuízos aos cofres públicos com a perda de produtividade e a deficiência visual. Com a criação da Aliança para Eliminação Global de Tracoma em 1997 (GET2020), o Estado do Pará, com apoio do Ministério da Saúde do Brasil, realizaram em 2006 o inquérito epidemiológico do tracoma em escolares de 1ª a 4ª série da rede oficial de ensino, nos municípios com índice de desenvolvimento humano inferior a média nacional, para conhecer a prevalência da doença. Os dados obtidos no inquérito comprovaram que a doença não foi erradicada, revelando 35 municípios paraenses prioritários e prevalências acima de 5%. Uma sub-amostra da conjuntiva de escolares clinicamente positivos foi coletada para a confirmação diagnóstica por Imunofluorescência direta (IFD). O presente estudo utilizou 52 amostras crio conservadas obtidas durante o inquérito, para serem analisadas pelos métodos de IFD e de biologia molecular, na identificação laboratorial da Chlamydia trachomatis. Foram encontradas as frequências de 26,92% (14/52) e 49% (24/49) de resultados positivos pelas técnicas de IFD e reação em cadeia da polimerase (nested-PCR), respectivamente. Considerando as 49 amostras analisadas pelas duas metodologias, as sensibilidades para a detecção do agente etiológico, por IFD e PCR foram de 28,57% (14/49) e 48,98% (24/49), respectivamente (p = 0,0127). As duas técnicas juntas confirmaram a infecção em 57,14% (n=28) das amostras, onde 50% (n=14) foram positivas apenas pela PCR, 35,72% (n=10) para ambas as técnicas e 14,28% (n=4) somente pela IFD. A análise de sete sequências nucleotídicas demonstrou homologia para isolados de C. trachomatis genótipo L1. Este estudo é pioneiro no Brasil, pois além de confirmar a presença de C. trachomatis em amostras oculares de escolares clínicamente positivos para tracoma, validou protocolo de obtenção de DNA a partir de lâminas de IFD crioconservadas, demonstrou a maior sensibilidade do método molecular frente à IFD e identificou o genótipo L1 presente nas amostras. / Trachoma as a leading cause of preventable blindness in the world is a neglected disease related to low socioeconomic conditions and locations without basic sanitation. Present mainly in developing countries brings great harm to public coffers in lost productivity and visual impairment. With the creation of the Alliance for the Global Elimination of Trachoma in 1997 (GET2020) the State of Pará, with support from the Ministry of Health of Brazil, held in the 2006 epidemiological survey of trachoma in school 1st to 4th grades of the official network of education in municipalities with the human development index of less than national average, to discover the prevalence of the disease. Data from the survey proved that the disease was not eradicated, revealing 35 priority municipalities in Pará and prevalence above 5%. A sub-sample of the conjunctiva of clinically positive students was collected to confirm the diagnosis by direct immunofluorescence (DIF). The present study used 52 cryopreserved samples obtained during the epidemiological survey to be analyzed by the methods of DIF and molecular biology to laboratorial identification of Chlamydia trachomatis. We found frequencies of 26.92% (14/52) and 49% (24/49) of positive results by DIF techniques and polymerase chain reaction (nested-PCR), respectively. Considering the 49 samples analyzed by two methods, the sensitivities for detection of the etiologic agent, by FAT and PCR were 28.57% (14/49) and 48.98% (24/49), respectively (p = 0,0127). The two techniques together confirmed 57.14% (n = 28) of samples with infection, where 50% (n = 14) were positive only by PCR, 35.72% (n = 10) for both technical and 14, 28% (n = 4) only by the IFD. The nucleotide sequence analysis of seven isolates showed homology to C. trachomatis genotype L1. These studies is a pioneer in Brazil, as well as confirm the presence of C. trachomatis in samples from ocular school clinically positive for trachoma, validated protocol for obtaining DNA from cryopreserved sheets DIF, showed the highest sensitivity of the molecular method front DIF and identified the genotype L1 in the sample of the conjunctiva of clinically positive students.

Teenage pregnancies are often not intended, and there is a high risk that unintended pregnancies will lead to abortion. The wide-spread availability of Youth Clinics, the subsidizing of contraceptives and the introduction of new and effective contraceptives have failed to lower the abortion rates. The aim of this thesis was to study possible risk groups and to highlight underlying reasons for contraceptive failure. Methods: Study I and II were quantitative studies with the aims of investigating whether teenagers who sought emergency contraception (Paper I) and teenage mothers (Paper II) were at risk for new unintended pregnancies during a 12-month follow-up period. Study III and IV were qualitative studies. The aim in study III was to see how contraceptive use was documented in medical records (MRs) concerning teenagers who had attended for induced abortion. In study IV the aim was to find out reasons for non- use or inconsistent use of contraceptives among teenagers attending for abortion. Results: In study I and II data were collected from medical and antenatal records. The results showed that both groups, despite contraceptive counselling, were at high risk for new unintended pregnancies leading to abortion. Attendance at the postpartum visit was low and 24% of the teenage mothers did not receive any recommendation about using a particular contraceptive method. Within 12 months 25% had a new pregnancy and of these one third led to legal abortion. In Study III two themes were generated from the analysis of the MRs; ‘Contraceptive methods previously used’ and ‘Plan for future contraceptive use’. All MRs did not contain information about contraceptive use. In study IV one theme was generated from the analysis of the interview text: ‘Struggling with feelings of uncertainty and patterns of behaviour’. Conclusion: Teenagers using emergency contraceptive pills and teenage mothers were at high risk for unintended pregnancies. Contraceptive failure in teenagers who have had an abortion may be due to in part to the absence of contraceptive counselling at abortion visits and in part to problems with contraceptive use due to insufficient knowledge and not knowing what do when side-effects occurs.