O câncer cervical acomete anualmente cerca de 470.000 mulheres em todo o mundo e mais de 16.000 mulheres no Brasil. O desenvolvimento do câncer cervical e sua associação ao Papilomavírus Humano (HPV) estão bem documentados, sendo este o fator principal para o desenvolvimento do câncer cervical. A infecção genital por Chlamydia trachomatis é estudada como um co-fator no desenvolvimento de neoplasias intraepiteliais cervicais (NICs) e outras alterações celulares significativas em mulheres com histórico de infecção por HPV. Este estudo tem como objetivo conhecer a prevalência de infecção por HPV e Chlamydia trachomatis em uma amostra de mulheres assintomáticas de uma área geográfica localizada na zona norte de Porto Alegre, bem como verificar as características associadas à presença desta co-infecção e sua relação com lesões cervicais. Trata-se de um estudo transversal cujo desfecho é a positividade ao HPV e à Chlamydia trachomatis em uma amostra de mulheres assintomáticas de Porto Alegre. Um total de 1217 amostras de material do colo do útero foi coletado para realização do exame citopatológico e para a identificação do DNA-HPV e DNA-CT através da técnica da Reação em Cadeia da Polimerase (PCR). Colposcopia e biópsia foram realizadas sempre que a citologia estivesse alterada e/ou a PCR para o HPV-DNA fosse positiva. A prevalência de HPV e Chlamydia trachomatis e sua distribuição por faixa etária são descritas, bem como a sua associação com as variáveis estudadas através das Razões de Chances (RC) estimadas por regressão logística múltipla. Observou-se uma prevalência de HPV-DNA de 28,4% (n=346/1217), de CT-DNA de 12,6% (n=152/1208) e de co-infecção por HPV e CT de 6,5% (n=78/1208). Mulheres não brancas (Razão de Chance (RC) =1,60; Intervalo de Confiança (IC) de 95%:1,10-2,38),assalariadas (RC=1,74; IC95%:1,17-2,60) e com parceiro apresentando história de condiloma genital (RC=2,35; IC95%:1,17-4,72) mostraram-se associadas com a positividade para HPV. A infecção por CT mostrou uma associação positiva com mulheres que iniciaram a vida sexual antes dos vinte anos (RC=1,82; IC95%:1,05-3,15) e assalariadas (RC=1,93; IC95%:1,15-3,25). Quanto à co-infecção por HPV e CT, mulheres com mais de três de parceiros sexuais na vida (RC=2,02; IC 95%:1,12-3,65) apresentaram uma associação positiva com o desfecho. Com relação à citologia, tanto a infecção por HPV quanto a co-infecção apresentaram associação significativa com anormalidades citológicas (p≤0.001). Os resultados mostraram uma elevada prevalência de HPV, de CT e de co-infecção em uma amostra de mulheres assintomáticas reforçando dados relatados na literatura. A associação destas infecções com variáveis sócioeconômicas, de comportamento sexual e com lesões do colo uterino, indicam a importância de medidas para a promoção e prevenção de saúde com este alvo específico dentro da rotina de serviços de atenção primária. Desta forma, acredita-se que estes dados possam ser muito úteis no planejamento de programas, incluindo o controle de Doenças Sexualmente Transmissíveis e a utilização de vacinas para o HPV.

O câncer cervical acomete anualmente cerca de 470.000 mulheres em todo o mundo e mais de 16.000 mulheres no Brasil. O desenvolvimento do câncer cervical e sua associação ao Papilomavírus Humano (HPV) estão bem documentados, sendo este o fator principal para o desenvolvimento do câncer cervical. A infecção genital por Chlamydia trachomatis é estudada como um co-fator no desenvolvimento de neoplasias intraepiteliais cervicais (NICs) e outras alterações celulares significativas em mulheres com histórico de infecção por HPV. Este estudo tem como objetivo conhecer a prevalência de infecção por HPV e Chlamydia trachomatis em uma amostra de mulheres assintomáticas de uma área geográfica localizada na zona norte de Porto Alegre, bem como verificar as características associadas à presença desta co-infecção e sua relação com lesões cervicais. Trata-se de um estudo transversal cujo desfecho é a positividade ao HPV e à Chlamydia trachomatis em uma amostra de mulheres assintomáticas de Porto Alegre. Um total de 1217 amostras de material do colo do útero foi coletado para realização do exame citopatológico e para a identificação do DNA-HPV e DNA-CT através da técnica da Reação em Cadeia da Polimerase (PCR). Colposcopia e biópsia foram realizadas sempre que a citologia estivesse alterada e/ou a PCR para o HPV-DNA fosse positiva. A prevalência de HPV e Chlamydia trachomatis e sua distribuição por faixa etária são descritas, bem como a sua associação com as variáveis estudadas através das Razões de Chances (RC) estimadas por regressão logística múltipla. Observou-se uma prevalência de HPV-DNA de 28,4% (n=346/1217), de CT-DNA de 12,6% (n=152/1208) e de co-infecção por HPV e CT de 6,5% (n=78/1208). Mulheres não brancas (Razão de Chance (RC) =1,60; Intervalo de Confiança (IC) de 95%:1,10-2,38),assalariadas (RC=1,74; IC95%:1,17-2,60) e com parceiro apresentando história de condiloma genital (RC=2,35; IC95%:1,17-4,72) mostraram-se associadas com a positividade para HPV. A infecção por CT mostrou uma associação positiva com mulheres que iniciaram a vida sexual antes dos vinte anos (RC=1,82; IC95%:1,05-3,15) e assalariadas (RC=1,93; IC95%:1,15-3,25). Quanto à co-infecção por HPV e CT, mulheres com mais de três de parceiros sexuais na vida (RC=2,02; IC 95%:1,12-3,65) apresentaram uma associação positiva com o desfecho. Com relação à citologia, tanto a infecção por HPV quanto a co-infecção apresentaram associação significativa com anormalidades citológicas (p≤0.001). Os resultados mostraram uma elevada prevalência de HPV, de CT e de co-infecção em uma amostra de mulheres assintomáticas reforçando dados relatados na literatura. A associação destas infecções com variáveis sócioeconômicas, de comportamento sexual e com lesões do colo uterino, indicam a importância de medidas para a promoção e prevenção de saúde com este alvo específico dentro da rotina de serviços de atenção primária. Desta forma, acredita-se que estes dados possam ser muito úteis no planejamento de programas, incluindo o controle de Doenças Sexualmente Transmissíveis e a utilização de vacinas para o HPV.

O câncer cervical acomete anualmente cerca de 470.000 mulheres em todo o mundo e mais de 16.000 mulheres no Brasil. O desenvolvimento do câncer cervical e sua associação ao Papilomavírus Humano (HPV) estão bem documentados, sendo este o fator principal para o desenvolvimento do câncer cervical. A infecção genital por Chlamydia trachomatis é estudada como um co-fator no desenvolvimento de neoplasias intraepiteliais cervicais (NICs) e outras alterações celulares significativas em mulheres com histórico de infecção por HPV. Este estudo tem como objetivo conhecer a prevalência de infecção por HPV e Chlamydia trachomatis em uma amostra de mulheres assintomáticas de uma área geográfica localizada na zona norte de Porto Alegre, bem como verificar as características associadas à presença desta co-infecção e sua relação com lesões cervicais. Trata-se de um estudo transversal cujo desfecho é a positividade ao HPV e à Chlamydia trachomatis em uma amostra de mulheres assintomáticas de Porto Alegre. Um total de 1217 amostras de material do colo do útero foi coletado para realização do exame citopatológico e para a identificação do DNA-HPV e DNA-CT através da técnica da Reação em Cadeia da Polimerase (PCR). Colposcopia e biópsia foram realizadas sempre que a citologia estivesse alterada e/ou a PCR para o HPV-DNA fosse positiva. A prevalência de HPV e Chlamydia trachomatis e sua distribuição por faixa etária são descritas, bem como a sua associação com as variáveis estudadas através das Razões de Chances (RC) estimadas por regressão logística múltipla. Observou-se uma prevalência de HPV-DNA de 28,4% (n=346/1217), de CT-DNA de 12,6% (n=152/1208) e de co-infecção por HPV e CT de 6,5% (n=78/1208). Mulheres não brancas (Razão de Chance (RC) =1,60; Intervalo de Confiança (IC) de 95%:1,10-2,38),assalariadas (RC=1,74; IC95%:1,17-2,60) e com parceiro apresentando história de condiloma genital (RC=2,35; IC95%:1,17-4,72) mostraram-se associadas com a positividade para HPV. A infecção por CT mostrou uma associação positiva com mulheres que iniciaram a vida sexual antes dos vinte anos (RC=1,82; IC95%:1,05-3,15) e assalariadas (RC=1,93; IC95%:1,15-3,25). Quanto à co-infecção por HPV e CT, mulheres com mais de três de parceiros sexuais na vida (RC=2,02; IC 95%:1,12-3,65) apresentaram uma associação positiva com o desfecho. Com relação à citologia, tanto a infecção por HPV quanto a co-infecção apresentaram associação significativa com anormalidades citológicas (p≤0.001). Os resultados mostraram uma elevada prevalência de HPV, de CT e de co-infecção em uma amostra de mulheres assintomáticas reforçando dados relatados na literatura. A associação destas infecções com variáveis sócioeconômicas, de comportamento sexual e com lesões do colo uterino, indicam a importância de medidas para a promoção e prevenção de saúde com este alvo específico dentro da rotina de serviços de atenção primária. Desta forma, acredita-se que estes dados possam ser muito úteis no planejamento de programas, incluindo o controle de Doenças Sexualmente Transmissíveis e a utilização de vacinas para o HPV.

Chlamydia trachomatis infections are associated with a spectrum of clinical diseases including urethritis, prostatitis and epididymitis among men and cervicitis and pelvic inflammatory disease (PID), with an increased risk of infertility and ectopic pregnancy (EP), among women. In the search for other pathogens causing urethritis, Mycoplasma genitalium was isolated from urethral specimens from two men with acute urethritis (1980). Mycoplasma bacteria are extremely difficult to isolate by culture, and clinical studies have been possible only after the advent of the first PCR-based detection method. M. genitalium has been found to be associated with lower genital tract infections in both men and women. Finding evidence for a connection between M. genitalium and upper genital tract infections in women is still of major importance. The aim in papers I and II was to develop a PCR method for genetic characterization of clinical C. trachomatis isolates by sequence analysis of the omp1 gene, and to study the distribution of genotypes within sexual networks and determine if genotyping would improve partner notification. The method was used to determine the genotypes of C. trachomatis in 237 positive urogenital and/or urine specimens from men and women attending the STDClinic in Örebro during one year. Sequence analysis of the omp1 gene revealed that the most prevalent genotypes corresponded to C. trachomatis serovar E (47%), followed by F (17%), and K (9%). There were 161 networks found and specimens were sequenced from at least two patients in 47 networks. In seven of these 47 networks there were discrepant genotypes. In the largest network comprising 26 individuals two different C. trachomatis genotypes were found, and one partner had urethritis due to a Mycoplasma genitalium infection but was C. trachomatis negative. The need for a new method for M. genitalium DNA detection was one reason for study III. An existing conventional PCR protocol for detection of M. genitalium DNA was further developed into a real-time PCR (RT-PCR) with hybridisation probes. In order to evaluate the RT-PCR assay with clinical material, specimens from 398 men and 301 women attending the STD Clinic in Örebro were analysed, using the RT-PCR assay, and also by the well established conventional PCR in Copenhagen. Using the conventional PCR method as “gold standard”, the sensitivity for the RT-PCR assay was 72.2% and 68.2% and the specificity was 99.7% and 98.6%, respectively, in urogenital specimens from men and women. The aim in paper IV was to adapt a Triton X-114 extracted Lipid-Associated Membrane Protein (LAMP) Enzyme Immuno Assays (EIA) method to detect antibodies against M. genitalium and to evaluate the association between M. genitalium and PID and EP, using sera sampled in Örebro during the 1980s, and also to compare the number of sera having M. genitalium antibodies against those having C. trachomatis antibodies, using a commercial anti- Chlamydia trachomatis EIA assay. No statistical significant association could be demonstrated between M. genitalium antibodies and PID or EP in our serum material. However, a slight trend toward association was found when focusing on younger individuals. Antibodies against C. trachomatis were found to be significantly associated with PID and EP.

Microbial interactions represent an understudied facet of human health and disease. In this study, the interactions that occur between Chlamydia trachomatis and the opportunistic fungal pathogen, Candida albicans were investigated. Candida albicans is a common component of the oral and vaginal microbiota responsible for thrush and vaginal yeast infections. Normally, Candida exist in the body as yeast. However, disruptions to the microbiota create conditions that allow expanded growth of Candida, conversion to the hyphal form, and tissue invasion. Previous studies have shown that a myriad of outcomes can occur when Candida albicans interacts with pathogenic bacteria. To determine if C. trachomatis physically interacts with C. albicans, we incubated chlamydial elementary bodies (EB) in medium alone or with C. albicans yeast or hyphal forms for 1 h. Following incubation, the samples were formaldehyde-fixed and processed for immunofluorescence assays using anti-chlamydial MOMP or anti- chlamydial LPS antibodies. Replicate samples were replenished with culture medium and incubated at 35°C for 0-120 h prior to fixation for immunofluorescence analysis or collection for EB infectivity assays. Data from this study indicates that both C. trachomatis serovar E and C. muridarum EB bind to C. albicans yeast and hyphal forms. This interaction was not blocked by pre-incubation of EB with the Candida cell wall components, mannan or β-glucans, suggesting that EB interact with a Candida cell wall protein or other structure. Bound EB remained attached to C. albicans for a minimum of 5 days (120 h). Infectivity assays demonstrated that EB bound to C. albicans are infectious immediately following binding (0h). However, once bound to C. albicans, EB infectivity decreased at a faster rate than EB in medium alone. At 6h post binding, 40% of EB incubated in medium alone remained infectious compared to only 16% of EB bound to C. albicans. Likewise, pre-incubation of EB with laminarin, a soluble preparation of β-glucan, alone or in combination with other fungal cell wall components significantly decreases chlamydial infectivity in HeLa cells. These data indicate that interactions between EB and C. albicans inhibit chlamydial infectivity, possibly by physically blocking EB interactions with host cell receptors.

Studies indicate that estrogen enhances Chlamydia trachomatis serovar E infection in genital epithelial cells. Hormones have direct and indirect effects on endometrial epithelial cells. Estrogen and progesterone exposure induces endometrial stromal cells to release effectors that subsequently regulate growth and maturation of uterine epithelial cells. Estrogen enhances C. trachomatis infection by aiding entry and intracellular development in endometrial epithelial cell (Ishikawa, IK)/SHT-290 stromal cell co-culture. Enhanced chlamydial infection was mediated by direct estrogen-stimulated signaling events in epithelial cells and indirectly via estrogen-induced stromal cell effectors. The current study investigates the effects of hormones on chlamydial development using culture conditions representative of the menstrual cycle. Chlamydia trachomatis-infected IK or IK/SHT-290 cultures were exposed to 10(-8) M estrogen (E2), 10(-7) M progesterone (P4) or a combination of both hormones (10(-8) M E2 followed by 10(-9) M E2/10(-7) M P4). Chlamydial infectivity and progeny production were significantly decreased (30-66%) in cultures exposed to progesterone or estrogen/progesterone combination compared to estrogen alone. Thus, progesterone antagonized the positive effects of estrogen on chlamydial infection. These data indicate the susceptibility of endometrial epithelial cells to C. trachomatis infection during the menstrual cycle is altered by phase specific actions of sex hormones in the genital tract.

Interaction of Herpes Simplex Virus (HSV) glycoprotein D (gD) with the host cell surface during Chlamydia trachomatis/HSV co-infection stimulates chlamydiae to become persistent. During viral entry, gD interacts with one of 4 host co-receptors: HVEM (herpes virus entry mediator), nectin-1, nectin-2 and 3-O-sulfated heparan sulfate. HVEM and nectin-1 are high-affinity entry receptors for both HSV-1 and HSV-2. Nectin-2 mediates HSV-2 entry but is inactive for HSV-1, while 3-O-sulfated heparan sulfate facilitates HSV-1, but not HSV-2, entry. Western blot and RT-PCR analyses demonstrate that HeLa and HEC-1B cells express nectin-1 and nectin-2, but not HVEM. Because both HSV-1 and HSV-2 trigger persistence, these data suggest that nectin-1 is the most likely co-receptor involved. Co-infections with nectin-1 specific HSV-1 mutants stimulate chlamydial persistence, as evidenced by aberrant body (AB) formation and decreased production of elementary bodies (EBs). These data indicate that nectin-1 is involved in viral-induced chlamydial persistence. However, inhibition of signal transduction molecules associated with HSV attachment and entry does not rescue EB production during C. trachomatis/HSV-2 co-infection. HSV attachment also does not activate Cdc42 in HeLa cells, as would be expected with viral stimulated activation of nectin-1 signaling. Additionally, immunofluorescence assays confirm that HSV infection decreases nectin-1 expression. Together, these observations suggest that gD binding-induced loss of nectin-1 signaling negatively influences chlamydial growth. Chlamydial infection studies in nectin-1 knockdown (NKD) HeLa cell lines support this hypothesis. In NKD cells, chlamydial inclusions are smaller in size, contain ABs, and produce significantly fewer infectious EBs compared to C. trachomatis infection in control HeLa cells. Overall, the current study indicates that the actions of host molecule, nectin-1, are required for successful C. trachomatis development.

Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/β-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester β-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures (p < 0.005) and chlamydial infectivity (p ≤ 0.01) in both IK and Hec-1B cells. Confocal and electron microscopy analysis of chlamydial inclusions revealed that Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis.

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial STD agent in the US. This bacterium has a unique biphasic developmental cycle in which the infectious elementary body (EB) infects a host mucosal epithelial cell and differentiates into the replicative form (the reticulate body or RB) within a modified vacuole called an inclusion. The RB later divides and develops back into an EB and is released, perpetuating the infectious cycle. When developing chlamydiae are exposed to unfavorable environmental conditions, they deviate from the normal developmental cycle into a non-infectious but viable state termed persistence. Previous data from our laboratory indicate that i) during C. trachomatis/HSV co-infection, the chlamydiae become persistent and ii) HSV gD interaction with host cell surface is sufficient to induce this response. During viral entry, HSV gD interacts with one of four host co-receptors, one of which is the host adhesion molecule nectin-1. Interestingly, Western blotting demonstrated that nectin-1 is significantly decreased in C. trachomatis-infected HeLa cells. Additional studies indicated that active C. trachomatis replication is required for nectin-1 down-regulation and nectin-1 is likely down-regulated post-translationally. CPAF, a chlamydia-secreted protease, is responsible for degrading several host proteins. Both in vivo experiments using CPAF-specific chemical inhibitors and cell-free cleavage assays using recombinant CPAF indicate that nectin-1 is degraded by CPAF in C. trachomatis-infected cells. Further studies suggest that nectin-1 is the most likely candidate involved in triggering HSV-induced chlamydial persistence. Co-infection experiments using nectin-1-specific HSV-1 mutants suggest that nectin-1 is, indeed, required for persistence induction. Additional studies in single co-receptor-expressing CHO cells demonstrate that, despite the fact that HSV-1 enters both HVEM- and nectin-1-expressing cells, viral co-infection reduces chlamydial infectivity only in the CHO-nectin-1 cell line. These data confirm that HSV/nectin-1 interaction is sufficient for chlamydial persistence induction. Although nectin-1 ligation is known to activate Cdc42, pull-down assays indicate that Cdc42 is not activated in co-infected HeLa cells. Taken together, these data suggest that: i) HSV gD-nectin-1 binding activates a novel host epithelial cell pathway that restricts chlamydial development and ii) the chlamydiae may degrade nectin-1 to evade this inhibitory host response.

Les hommes ayant des relations sexuelles avec d'autres hommes (HARSAH) ont un risque accru d'infections sexuellement transmissibles (IST). Les données sur les IST chez les HARSAH sont rares au Bénin. Cette étude visait à évaluer la prévalence et les facteurs associés à Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) et Treponema pallidum (TP) chez 204 HARSAH VIH-négatifs de Cotonou au Bénin initiant la prophylaxie préexposition en prévention du VIH. Un questionnaire a été complété et un médecin a réalisé un examen physique des participants. Des échantillons de sang, anaux, pharyngés et urinaires ont été prélevés. Les prévalences des IST ont été présentées avec un intervalle de confiance à 95% et la régression de Poisson a permis d'identifier leurs déterminants. La prévalence était de 18,6%, 15,2%, 9,8% et 27,9% pour CT, NG tous sites confondus, NG anorectal et l'infection par NG ou CT (NG/CT) respectivement. Les IST étaient généralement asymptomatiques (84,2% pour NG/CT) et de localisation extra-génitale (61,4% pour NG/CT). Un seul cas de syphilis non confirmée a été observé. En analyse multivariée, le fait d'avoir moins de 25 ans, de ne pas être célibataire et d'avoir plus de quatre partenaires féminines étaient associés à CT et NG/CT alors qu'un faible revenu mensuel était associé à NG/CT seulement. Le jeune âge, le sexe anal réceptif et ne pas avoir de rapports sexuels payants étaient associés à NG. Seul, le sexe anal réceptif était associé à NG anorectal. Les infections à NG et à CT sont fréquentes chez les HARSAH VIH-négatifs de Cotonou au Bénin. Leur caractère asymptomatique et extra-génital rend la prise en charge syndromique inefficace et le dépistage aux sites anal et pharyngé nécessaire. La syphilis est rare chez ces HARSAH. Les interventions de contrôle et de prévention des IST devraient cibler particulièrement les jeunes HARSAH et leurs partenaires sexuelles féminines. / Men who have sex with men (MSM) are at increased risk of sexually transmitted infections (STIs). Data on STIs among MSM are scarce in Benin. This study aimed to assess the prevalence and factors associated with Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Treponema pallidum (TP) among 204 HIV-negative MSM in Cotonou, Benin initiating pre-exposure prophylaxis (PrEP) for HIV prevention. A questionnaire was completed, and participants underwent a physical examination by a physician. Blood, anal, pharyngeal, and urinary samples were collected. Prevalences are presented with a 95% confidence interval. Poisson regression was used to determine factors associated with STIs. The prevalence was 18.6%, 15.2%, 9.8% and 27.9% for CT, NG-all sites, anorectal NG, and infection by either NG or CT (NG/CT) respectively. STIs were generally asymptomatic (84.2% for NG/CT) and of extra-genital location (61.4% for NG/CT). Only one case of unconfirmed syphilis was observed. In multivariate analysis, younger age, not being single, having more than four female sexual partners were associated with CT and NG/CT while lower monthly income was associated with NG/CT alone. Younger age, receptive anal sex and paid sex were associated with NG. Only receptive anal sex was associated with anorectal NG. NG and CT infections are common among HIV-negative MSM in Cotonou, Benin. Their asymptomatic and extra-genital nature makes syndromic management ineffective and screening at anal and pharyngeal sites necessary. Syphilis is rare among these MSM. STI control and prevention interventions should target young MSM and their female sexual partners.