Contribution for the knowledge on curable sexually transmitted infections with special emphasis on Chlamydia trachomatis

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2017.Introdução e objetivos: As infeções sexualmente transmissíveis (ISTs) abrangem um amplo conjunto de síndromes com quadros clínicos variados que vão desde a ausência de sintomas até grave morbilidade, incluindo a própria morte. Existem mais de 30 agentes patogénicos de origem bacteriana, viral ou parasitária, que podem ser transmitidos de uma pessoa para outra durante o ato sexual. As ISTs estão entre as condições agudas mais comuns no mundo, sendo Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis e Mycoplasma genitalium as quatro principais causas de ISTs curáveis, sendo responsáveis por uretrite, cervicite, vaginite e proctite. Em Portugal, não há dados sobre a prevalência destas ISTs, desconhecendo-se o seu respetivo impacto para saúde pública. Deste modo, a presente dissertação teve por objetivo: a) o estudo da prevalência dos quatro microrganismos acima referidos no contexto de uma consulta de ISTs (Unidade de consulta de DST/CAD da Lapa); b) a análise da variabilidade genética do gene usado para genotipagem de C. trachomatis, ompA, o qual codifica a principal proteína da membrana externa de C. trachomatis, com vista a caracterizar as diferenças genéticas entre estirpes, as quais podem estar subjacentes a um processo contínuo de evolução e adaptação desta bactéria. Foram usadas as estirpes reunidas pelo laboratório de acolhimento ao longo de um período de 27 anos; c) o estudo da variabilidade intra-estirpes de C. trachomatis, de alvos potencialmente associados à variação de fase e à adaptação bacteriana no contexto da infeção in vivo. A variação de fase, que pode ser causada por mutações reversíveis que influenciam a expressão génica e a sua função, é conhecida por ser essencial para a adaptação e virulência de algumas bactérias, mas pouco se sabe sobre o seu eventual papel na biologia e na patogenicidade de C. trachomatis. Materiais e Métodos: a) Foram avaliadas 1034 amostras biológicas colhidas na consulta DST/CAD Lapa, durante o período de tempo compreendido entre Setembro de 2016 e Setembro de 2017, relativamente à presença de C. trachomatis, N. gonorrhoeae, T. vaginalis e M. genitalium, utilizando dois sistemas de PCR em tempo real, Cobas® 4800 CT/NG (Roche Sistemas de Diagnóstico) e SDiaMGTVTM (Diagenode); b) Foi efetuada genotipagem-ompA de 370 amostras utilizando uma técnica de nested-PCR, seguida de sequenciação pelo método de Sanger e a análise de similaridade das sequências genéticas obtidas relativamente à sequência ompA de estirpes protótipo de C. trachomatis. Posteriormente, essas amostras foram incluídas na base de dados do laboratório de acolhimento, num total de 2579 estirpes de C. trachomatis reunidas entre 1990 e 2017, tendo sido analisada toda a colecção relativamente ao genótipo ompA, ao género, à localização anatómica da infeção e à distribuição das variantes genotípicas; c) Foram analisadas 167 amostras (96 amostras de DNA selecionadas de entre as constantes na base de dados de C. trachomatis do laboratório, juntamente com um conjunto de 71 amostras para as quais os dados de sequenciação total do genoma tinham sido recentemente disponibilizados) relativamente a 12 tratos homopoliméricos potencialmente variáveis no genoma desta bactéria, através de uma técnica com base na sequenciação de nova geração (NGS), de produtos de amplificação (amplicões). Resultados: a) Um quinto da população estudada estava infetada por pelo menos uma IST, tendo sido C. trachomatis a mais frequentemente detetada, seguida por N. gonorrhoeae, M. genitalium e T. vaginalis. Os indivíduos do sexo masculino revelaram-se mais frequentemente infetados que as mulheres, com especial ênfase para os indivíduos com idades compreendidas entre os 25 e os 34 anos, e para os indivíduos com mais de um parceiro sexual. No nosso estudo, N. gonorrhoeae foi a segunda IST mais frequente, sendo que os homens contribuíram com mais de dois terços dos casos e que cerca de metade deles eram homens que faziam sexo com homens (HSH); b) A genotipagem-ompA de estirpes clínicas de C. trachomatis evidenciou 12 genótipos-ompA, de entre os quais os genótipos E e F foram os mais frequentes, enquanto os genótipos B e o C foram os mais raros. Os genótipos G e L2 foram muito mais comuns nos homens do que nas mulheres, tendo sido o endocervix/uretra o local anatómico onde mais frequentemente se efetuou a pesquisa e, consequentemente, se detetou a infeção. Aliás, o endocervix/uretra foi o único local anatómico onde os 12 genótipos foram detetados; c) Os resultados deste estudo revelaram diferentes perfis de variação intra- e inter-paciente para os poli (Ns) estudados, os quais inequivocamente sublinham o papel de um homopolímero em desencadear mecanismos reversíveis de "ON/OFF" da citotoxina de C. trachomatis (CT166) in vivo. Foram ainda identificados outros novos potenciais mediadores de mecanismos de variação de fase em C. trachomatis. Discussão: a) De acordo com os dados de prevalência de ISTs nos EUA e na Europa, nos quais C. trachomatis é descrita como a IST bacteriana mais frequente, e com o aumento do número de casos que têm sido descritos nos últimos anos, era esperado que C. trachomatis fosse a IST mais frequentemente detetada na população estudada. No entanto, as taxas de prevalência podem subestimar a verdadeira dimensão desta infeção dado o seu caráter assintomático. Por outro lado, a sua maior frequência pode também ser apenas um reflexo da melhoria dos sistemas de vigilância das ISTs, em muitos países, muitas vezes suportados por estudos de rastreio em populações assintomáticas; tais estudos são facilitados pela disponibilidade de melhores ferramentas de diagnóstico laboratorial (sensibilidade e especificidade próximas dos 100%). O número de casos positivos de ISTs na região anorretal e na orofaringe, testadas apenas em HSH, justificam a necessidade de alargar o diagnóstico a esses locais anatómicos em homens heterossexuais e em mulheres. Deste modo, e considerando que este estudo envolveu pacientes de apenas uma consulta IST, é de salientar a necessidade de uma avaliação a nível nacional, uma vez que até à data a prevalência destas ISTs na população portuguesa é desconhecida; b) A elevada frequência dos genótipos-ompA E e F, e a menor ocorrência de estirpes variantes nas estirpes destes genótipos, sugere uma melhor adaptação desses genótipos; tal situação favorecerá que não sejam reconhecidos pelos mecanismos de defesa do hospedeiro e que não sejam eliminados pelos sistemas de defesa do hospedeiro, facilitando a sua disseminação e justificando o seu predomínio em termos epidemiológicos, em detrimento doutros genótipos-ompA. No entanto, são necessários estudos envolvendo o genoma total de estirpes clínicas de C. trachomatis, que deverão ser acompanhados pela análise dos respetivos dados clínicos, para uma mais completa compreensão das características genéticas de C. trachomatis e a sua relação com o desenvolvimento de patologia; c) Este estudo enriquece o conhecimento sobre a variabilidade intra-paciente de tratos homopoliméricos potencialmente mediadores de mecanismos de variação de fase, consolidando a hipótese de que a funcionalidade da conhecida citotoxina de C. trachomatis (CT166), durante o processo infecioso, será regulada por mecanismos de variação de fase, o que ficou evidenciado pela elevada variabilidade inter e intra-paciente. Por outro lado, foi também possível demonstrar, pela primeira vez, que existem outros tratos homopoliméricos com variabilidade intra-estirpe para além do referido acima. É de destacar a necessidade de se alargar esta abordagem, utilizando-se um conjunto mais homogéneo e amplo de amostras de C. trachomatis, considerando também os respetivos dados clínicos e os genes relacionados putativamente com mecanismos de variação de fase, de modo a consolidar algumas das hipóteses suscitadas no decurso da presente dissertação de mestrado.Introduction and objectives: Sexually transmitted infections (STIs) refer to a wide variety of clinical syndromes and infections, which are among the most common acute conditions in the world, causing serious morbidity, and even death. Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium are the four main causes of curable STIs, being responsible for urethritis, cervicitis, vaginoses and proctitis. In Portugal, there is no data on their prevalence, being unknown the impact of these STIs. This dissertation aimed at a) studying the prevalence of these four microorganisms in the context of a sexually transmitted diseases (STDs) consultation; b) analyzing the variability of C. trachomatis genotyping gene, ompA – which codes to the major outer membrane protein –, throughout a timeline of 27 years. The genetic differences detected in this gene may provide important clues about the continuous process of evolution and adaptation of this human pathogen; c) studying C. trachomatis intra-strain variability in vivo, as a means to identify potentially phase-variable targets associated with bacterial adaptation in the context of infection. Materials and Methods: a) We evaluated 1034 samples collected from patients of the major Portuguese STD clinic (Unidade de consulta de DST/CAD da Lapa) for the microorganism’s presence using two real-time PCR systems, Cobas® 4800 CT/NG (Roche Sistemas de Diagnóstico) and SDiaMGTVTM kit (Diagenode); b) ompA-genotyping of 370 samples was performed using a nested-PCR technique, Sanger sequencing and ompA-sequence similarity analysis regarding C. trachomatis prototype strains. Then, we added these samples to the Instituto Nacional de Saúde Doutor Ricardo Jorge database collection, composed by 2579 C. trachomatis specimens, gathered from 1990 to 2017, and analyzed the whole database regarding ompA-genotype, gender, anatomical site of infection and genotypic variants distribution; c) We analyzed 167 samples (96 selected C. trachomatis-positive DNA samples from the laboratory database, along with a set of 71 samples for which whole-genome sequencing data had been recently released) with an amplicon-based next generation sequencing (NGS) technique for 12 selected potentially variable poly (Ns). Results: a) We found that almost a fifth of our study population was infected by at least one STI, with C. trachomatis being the most commonly detected, followed by N. gonorrhoeae, M. genitalium and, finally, T. vaginalis. Men were, in general, more infected than women, with especial evidence for individuals aged 25 to 34 years, and for people who had more than one sexual partner; b) ompAgenotyping of C. trachomatis clinical strains evidenced 12 ompA-genotypes, among which E and F were the most represented and B and C were the least frequent. Genotypes G and L2 were much more common among men than among women and the endocervix/urethra was the anatomical site of infection more represented, and the only site where all genotypes could be detected; c) The scrutiny of the selected potentially variable poly (Ns) revealed distinct trends of inter- and intra-patient variation that, not only unequivocally reinforce the role of a reversible poly(N) in the ON/OFF switching of C. trachomatis cytotoxin (CT166) in vivo, but also launch other poly(N)s as potential mediators of phase variation mechanisms in C. trachomatis. Discussion: a) According to its prevalence in the USA and Europe, and because the number of detected cases have been rising the past few years, C. trachomatis was expected to be the most frequently detected STI. However, reported prevalence rates underestimate the true burden of this STI due to its asymptomatic character. On the other hand, its frequency may also reflect the expansion of screening, namely among asymptomatic because of the development of better and more sensitive diagnostic tools, and also due to the improvement of reporting systems. The rectum and the oropharynx were only tested in MSM, and the number of positive STIs cases in these anatomical locations highlight the need for their investigation among heterosexual men and women; b) The high frequency of E and F together with the low occurrence of variant strains suggest a better fitness of these ompA-genotypes that would favor them to go undetected by the host defense mechanisms, facilitating their dissemination, and consequently leading to their predominant epidemiological rates. However, genomic studies involving the whole genome sequence together with clinical data are required for a more complete understanding of the genetic features of C. trachomatis; c) This study constitutes an unequivocal turning point on our knowledge of the intra- and inter-patient heterogeneity affecting C. trachomatis poly (Ns), by identifying novel potential targets of phase variation in the context of C. trachomatis infection. In particular, it consolidates the hypothesis that the functionality of the well-known C. trachomatis cytotoxin (CT166) is regulated by an ON/OFF mechanism of phase variation during infection. A future scale-up of the strategy applied in the present study approach, using an homogeneous and wider range of C. trachomatis-positive DNA samples together with their clinical background, along with other putative phase variation related genes, should be performed in order to consolidate some of the hypothesis raised through the present master’s thesis

Prevalence of four urogenital sexually transmitted infections in a dedicated clinic from Lisbon

Background/Objectives: To determine the prevalence of urogenital Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) among attendees of an open and freely available sexually transmitted infections (STI) dedicated clinic in Lisbon, at Centro de Saúde da Lapa, during 1-year. Methods: Molecular testing for CT, NG, MG, and TV was performed on 1,062 urogenital specimens (one specimen per person). A descriptive, cross- sectional, observational study was conducted to evaluate the characteristics of infected persons. Statistical analysis was performed. Results: Around 237 infections were detected in 214 patients. CT was the most prevalent (11.6%), with a similar infection rate between men and women. NG was the second most frequently detected (7.3%), followed by MG and TV (2.9 and 0.5%, respectively). Statistically significant associations were found: 1) between younger age and CT and NG prevalence, where being < 25 years old constituted an increased risk factor; 2) between CT and NG prevalence and sexual orientation, where heterosexuals presented an increased risk for CT infections while men who have sex with men (MSM) had a higher risk for NG infections; and 3) between “having symptoms” and gonococcal infection. Conclusions: This study highlights the rising of CT and NG in contrast to a low rate of MG and to the scarceness of TV.info:eu-repo/semantics/publishedVersio

Chlamydia trachomatis: when the virulence-associated genome backbone imports a prevalence-associated major antigen signature

Chlamydia trachomatis is the most prevalent sexually transmitted bacterium worldwide and the causative agent of trachoma. Its strains are classified according to their ompA genotypes, which are strongly linked to differential tissue tropism and disease outcomes [ocular disease, urogenital disease and lymphogranuloma venereum (LGV)]. While the genome-based species phylogenetic tree presents four main clades correlating with tropism/prevalence, namely ocular, LGV, urogenital T1 (more prevalent genotypes) and urogenital T2 (less prevalent genotypes), inter-clade exchange of ompA is considered a rare phenomenon probably mediating marked tropism alterations. An LGV epidemic, associated with the clonal expansion of the L2b genotype, has emerged in the last few decades, raising concerns particularly due to its atypical clinical presentation (ulcerative proctitis) and circulation among men who have sex with men (MSM). Here, we report an LGV outbreak, mostly affecting human immunodeficiency virus-positive MSM engaging in high-risk sexual practices, caused by an L2b strain with a rather unique non-LGV ompA signature that precluded the laboratory notification of this outbreak as LGV. C. trachomatis whole-genome capture and sequencing directly from clinical samples was applied to deeply characterize the genomic backbone of this novel LGV outbreak-causing clone. It revealed a chimeric genome structure due to the genetic transfer of ompA and four neighbouring genes from a serovar D/Da strain, likely possessing the genomic backbone associated with the more prevalent urogenital genotypes (T1 clade), to an LGV (L2b) strain. The hybrid L2b/D-Da strain presents the adhesin and immunodominant antigen MOMP (major outer membrane protein) (encoded by ompA) with an epitope repertoire typical of non-invasive genital strains, while keeping the genome-dispersed virulence fingerprint of a classical LGV strain. As previously reported for inter-clade ompA exchange among non-LGV clades, this novel C. trachomatis genomic mosaic involving a contemporary epidemiologically and clinically relevant LGV strain may have implications on its transmission, tissue tropism and pathogenic capabilities. The emergence of variants with epidemic and pathogenic potential highlights the need for more focused surveillance strategies to capture C. trachomatis evolution in action.info:eu-repo/semantics/publishedVersio

Insights on the intra-patient genetic heterogeneity of Chlamydia trachomatis

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Prevalence of four urogenital sexually transmitted infections in a dedicated clinic from Lisbon

Background/Objectives: To determine the prevalence of urogenital chlamydia trachomatis (CT), neisseria gonorrhoeae (NG), trichomonas vaginalis (TV), and mycoplasma genitalium (MG) among attendees of an open and freely available sexually transmitted infections (STI) dedicated clinic in Lisbon, at Centro de Saúde da Lapa, during 1-year. Methods: Molecular testing for CT, NG, MG, and TV was performed on 1,062 urogenital specimens (one specimen per person). A descriptive, crosssectional, observational study was conducted to evaluate the characteristics of infected persons. Statistical analysis was performed. Results: Around 237 infections were detected in 214 patients. CT was the most prevalent (11.6%), with a similar infection rate between men and women. NG was the second most frequently detected (7.3%), followed by MG and TV (2.9 and 0.5%, respectively). Statistically significant associations were found: 1) between younger age and CT and NG prevalence, where being < 25 years old constituted an increased risk factor; 2) between CT and NG prevalence and sexual orientation, where heterosexuals presented an increased risk for CT infections while men who have sex with men (MSM) had a higher risk for NG infections; and 3) between “having symptoms” and gonococcal infection. Conclusions: This study highlights the rising of CT and NG in contrast to a low rate of MG and to the scarceness of TV

Clinical and Epidemiological Characterization of Lymphogranuloma Venereum in a Sexually Transmitted Diseases Clinic in Lisbon, 2001 to 2020

Between 2001 and 2020, 54 LGV cases were diagnosed in a sexually transmitted disease clinic in Lisbon, most in men who have sex with men (87%), HIV negative (63%), from the anorectal mucosa (72.2%). Cases among heterosexuals were also identified (13%). Surveillance programs irrespective of sexual orientation and HIV status are needed to avoid the morbidity associated with LGV.A study of all patients with lymphogranuloma venereum in a sexually transmitted disease clinic in Lisbon, Portugal, found that most cases occurred in HIV-negative men who have sex with men in the anorectal mucosa.info:eu-repo/semantics/publishedVersio

Chlamydia trachomatis outbreak: when the virulence-associated genome backbone imports a prevalence-associated major antigen signature

Chlamydia trachomatis is the most prevalent sexually transmitted bacterium worldwide and the causative agent of trachoma. Its strains are classified according to their ompA genotypes, which are strongly linked to differential tissue tropism and disease outcomes [ocular disease, urogenital disease and lymphogranuloma venereum (LGV)]. While the genome-based species phylogenetic tree presents four main clades correlating with tropism/prevalence, namely ocular, LGV, urogenital T1 (more prevalent genotypes) and urogenital T2 (less prevalent genotypes), inter-clade exchange of ompA is considered a rare phenomenon probably mediating marked tropism alterations. An LGV epidemic, associated with the clonal expansion of the L2b genotype, has emerged in the last few decades, raising concerns particularly due to its atypical clinical presentation (ulcerative proctitis) and circulation among men who have sex with men (MSM).N/

Transcontinental dissemination of the L2b/D-Da recombinant Chlamydia trachomatis Lymphogranuloma venereum (LGV) strain: need of broad multi-country molecular surveillance

Previously, we identified a Chlamydia trachomatis Lymphogranuloma venereum (LGV) recombinant strain possessing a unique non-LGV ompA genotype. Here, culture-independent genome sequencing confirms its circulation in Europe, Middle East and North America, and unveils genetic evidence of emergence of antibiotic resistance. Multi-country and systematic molecular surveillance is needed.This work results in part from the GenomePT project (POCI-01–0145-FEDER-022184), supported by COMPETE 2020— Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). This work was also supported by Fundos FEDER through the Programa Operacional Factores de Competitividade—COMPETE and by Fundos Nacionais through the Fundação para a Ciência e a Tecnologia within the scope of the project UID/BIM/00009/2019 (Centre for Toxicogenomics and Human Health -ToxOmics).info:eu-repo/semantics/publishedVersio