Zugl.: Berlin, Humboldt-Univ., Diss., 2009

Background. Keratinocytes are the first target of infection for lymphogranuloma venereum (LGV) Chlamydia trachomatis, yet they have been omitted from pathogenesis studies. We infect keratinocytes and cervical cells with C. trachomatis and hypothesize different growth and cytotoxicity profiles among the strains. Methods. HaCaT human keratinocytes and ME-180 cervical cells were infected with C. trachomatis (multiplicity of infection (MOI) 0.025) serovars L1, L2, L3, 3 LGV clinical isolates or serovar E and incubated at 37 or 33°C for 5 days. Cytotoxicity was quantified daily using the CytoTox96® Non-Radioactive Cytotoxicity Assay, cells stained with the MicroTrak C. trachomatis Culture Confirmation kit and growth quantified by area of 100X photographs covered by Chlamydia. HaCaT and ME-180 cervical cells were infected with C. trachomatis (MOI 0.25) serovar L2 or E, incubated at 37 or 33°C for 48 hours and viewed with a transmission electron microscope (TEM). Mitochondrial activity was quantified using the MTT assay. The DeadEndTM Colorimetric TUNEL System with C. trachomatis Culture Confirmation kit as a counter-stain was used to assess cell death in infected versus uninfected cells. The BioVisionTM CaspGLOW Fluorescein Caspase Staining Kit and Transwell® Permeable Supports was used to differentiate between apoptosis mediated by cell-to-cell contact or a secreted molecule. Results. Growth in ME-180 versus HaCaT cells at 37°C was similar, but slower at 33 versus 37°C in HaCaT cells (p < 0.05). By day 5 L2 had grown faster than other strains in HaCaT cells at 37°C (p < 0.05), faster than clinical isolates in ME180 cells (p < 0.01), and faster than serovar E, and 2 clinical isolates at 33°C (p < 0.01). After 5 days L2 induced cytotoxicty was 11% in ME180 cells, which was higher than the clinical isolates (p < 0.01). In HaCaT cells at 33°C L2 EB were identified in a non-membrane state in the cytoplasm but not in the inclusion at 48 hours post infection. Serovar E but not L2 caused mitochondrial swelling at 1 h post infection in HaCaT cells at 37°C. This corresponded with a 16% reduction in mitochondrial activity (p < 0.001). TUNEL assay analyses demonstrated numerous dead cells adjacent to chlamydial inclusions for strains L2 and L3 but not L1 and E. An elevated number of caspase positive cells was detected in uninfected cell monolayers exposed to both L2 and E at 37°C but not 33°C. Conclusions. 1. C. trachomatis infects human keratinocytes in vitro. 2. Fresh clinical isolates behaved differently to the L2 reference strain. This demonstrates the need for fresh clinical isolates in pathogenesis studies of LGV. 3. In HaCaT cells at 33°C serovar L2 EB leave the intact inclusion and migrate through the cytoplasm in a non-membrane bound state 4. C. trachomatis induces apoptosis in uninfected cells exposed to infected cells via a secreted molecule at 37°C. This is more marked with serovar L2 exposure than serovar E exposure. / Thesis (Ph.D)-University of KwaZulu-Natal, Durban, 2010.

Urogenital chlamydia infection, caused by the bacterium Chlamydia trachomatis (CT), is the most common sexually transmitted bacterial infection in Sweden. In 2008 it was estimated by WHO that there were 105.7 million new cases of CT worldwide, an increase by 4.2 million cases (4.1%) compared to 2005. If untreated, CT infections can progress to serious reproductive health problems, especially in women. These complications include subfertility/infertility, ectopic pregnancy and chronic pain. The CT infection is often asymptomatic and reliable diagnostic methods and contact tracing are important tools for identifying infected individuals. CT infection is classified in the Swedish Communicable Diseases Act as a serious disease; consequently, written reporting and contact tracing are compulsory. Previous or ongoing CT infection is not uncommon in infertile couples, especially in women with tubal factor infertility (TFI). We have tested 244 infertile couples for CT antibodies, and CT IgG positive couples were tested for CT DNA in urine. The prevalence of CT antibodies was higher in infertile men and women, and ongoing CT infection was common. Our results support a role of CT in infertility and underscore the importance of prevention of CT infection. Contact tracing was studied during using questionnaires. A total of 544 questionnaires was sent to tracers in a Swedish county and 534 (98%) were completed. Centralized contact tracing performed by experienced tracers is effective; on average 65% of sexual contacts found by contact tracing are CT-infected. Our data show that it is worthwhile to extend the tracing period beyond 6 months as 30% of reported sexual contacts between months 7-12 were CT-infected. Contact tracing may be performed face-to-face at the clinic or by telephone. Because of the severe consequences of CT infection there is a need for useful methods for both primary and secondary prevention of CT and other sexually transmitted infections (STIs). An important sub-population for CT/STI-prevention is the “core group”, i.e. a subpopulation with high incidence of STIs combined with risky sexual behaviours. This subpopulation contributes particularly to the spread of STIs in the population. Therefore, we have developed and evaluated a brief standardised but flexible manual-based single-session intervention based on motivational interviewing (MI) for the reduction of high risk sexual behaviour. Women (n=105) and men (n=119) at high risk of contracting CT infection were randomly eighter offered brief MI counselling or standard care. Our findings support the effectiveness of brief MI-based counselling in reducing high-risk sexual behaviour and incident CT infection in women (p&lt;0.01) but not in men. Our results suggest that gender aspects need to be considered and that men and women should be treated differently for achieving maximal risk-reduction. Whereas it might be sufficient to include information and motivation when performing risk-reducing counselling on women, counsellors may also add other components, such as behavioural skills and booster sessions, when counselling is performed on men. / Klamydiainfektion orsakas av Chlamydia trachomatis och är den vanligaste sexuellt överförda bakterieinfektionen. WHO har uppskattat att det år 2008 var 105,7 miljoner nya fall av klamydia i världen, en ökning med 4,2 miljoner fall (4,1 %) jämfört med år 2005. Klamydiainfektion är ett folkhälsoproblem och klassificeras i den svenska smittskyddslagen som en allmänfarlig sjukdom varför det är obligatoriskt att smittspåra och göra en skriftlig anmälan till smittskyddsläkaren och Smittskyddsinstitutet. Klamydiainfektionen ger oftast inga symtom och tillförlitliga diagnostiska metoder och smittspårning är viktiga ”redskap” för att hitta smittade personer. Om klamydiainfektionen inte behandlas kan den leda till allvarliga hälsoproblem, speciellt hos kvinnor. Bland komplikationer efter klamydiainfektion ingår ofrivillig barnlöshet, utomkvedshavandeskap och kronisk buksmärta. Tecken på tidigare eller pågående klamydiainfektion är vanliga hos ofrivilligt barnlösa par, speciellt hos kvinnor med skadade äggledare som orsak till barnlösheten. Våra resultat ger stöd för betydelsen av klamydia vid ofrivillig barnlöshet och understryker vikten av förebyggande åtgärder mot klamydia samt klamydiaprovtagning av både män och kvinnor vid utredning av ofrivillig barnlöshet. Centraliserad klamydiasmittspårning utförd av erfarna smittspårare är effektiv och i genomsnitt är 65 % av spårade sexuella kontakter klamydiasmittade. Våra data visar att det lönar sig att förlänga smittspårningsperioden från 6 till 12 månader eftersom betydligt fler klamydiasmittade kontakter då hittas. Den så kallade ”Västerbottensmodellen” med en smittspårningsperiod på 12 månader rekommenderas nu av Socialstyrelsen. Kontaktspårning kan utföras antingen på mottagningen eller per telefon. På grund av risk för allvarliga konsekvenser av klamydia finns det behov av metoder för att förebygga klamydiasmitta. En viktig grupp för prevention är den så kallade ”kärngruppen", alltså de personer som har en hög förekomst av klamydia och andra sexuellt överförda infektioner i kombination med sexuellt riskbeteende. Denna grupp bidrar särskilt till spridningen av sexuellt överförda infektioner bland befolkningen. Därför har vi utvecklat och utvärderat en kort samtalsmetod som bygger på metoden motiverande samtal (MI, motivational interviewing) för att minska sexuellt risktagande. Våra fynd visar att kort MI-baserad rådgivning för att minska sexuellt riskbeteende och klamydiainfektion fungerar bra på kvinnor men inte lika bra på män. Resultaten tyder på att genusaspekter måste beaktas och att kvinnor och män ska behandlas på olika sätt för att uppnå maximal riskminskning. Det kan vara tillräckligt att fokusera på information och motivation vid rådgivning av kvinnor men för rådgivning av män kan man behöva komplettera med beteendemässiga färdigheter och/eller upprepad MI-baserad rådgivning för att nå god effekt.

Chlamydia trachomatis (Ct), the most common sexually transmitted bacterium, has been associated with adverse pregnancy outcomes including controversial data on miscarriage, intrauterine growth restriction and low birth weight, however the causative mechanisms are unknown. A successful pregnancy requires normal endometrial stromal cell (ESC) decidualisation and trophoblast invasion, processes that involve chemokine action and lead to successful implantation. My objectives were to determine whether Ct infection impacts upon ESC decidualisation and chemokine secretion on human primary ESC invitro, to investigate the role of Ct infection in pregnancy in-vivo using a murine model of pregnancy and to investigate the role of Ct in miscarriage in a statistically powered case control study. A novel finding is that Ct can infect and proliferate in ESC, resulting in suboptimal decidualisation as measured by decidualisation marker prolactin’s reduced mRNA and protein levels in infected ESC. Furthermore, the altered secreted chemokine profile of decidualised ESC suggests an attenuated innate immune response from infected ESC. Focusing on chemokines C-X-C motif chemokine 12 (CXCL12) and CXCL16, important for trophoblast invasion, decreased mRNA and protein concentrations were detected in infected decidualised cells. From the in-vivo mouse model of past Ct infection in pregnancy, it was demonstrated that Ct infection did neither affect the fertility of the mice, pregnancy or resorption numbers in C3H mice nor alter embryonic and placental weight on e12 embryos. However, Ct infection caused reduction of embryo and placenta weight on e14 embryos. Finally, preliminary data from the case control study indicate that past Ct infection is not associated with miscarriage. Our in house PGP3 ELISA that detects past Ct infection was more sensitive than a commercially available MOMP ELISA. My data suggests that Ct infection affects pregnancy during the implantation stage by impairing decidualisation and altering chemokine secretion predisposing for adverse pregnancy outcomes that include growth restriction during later gestation.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, 2008. / Submitted by Jaqueline Oliveira (jaqueoliveiram@gmail.com) on 2008-12-08T18:17:18Z No. of bitstreams: 1 DISSERTACAO_2008_JoseAugustodeOliveiraBotelho.pdf: 2980105 bytes, checksum: f5b5a96a5b7e8a0bd74ff1e7bbaafbd7 (MD5) / Approved for entry into archive by Georgia Fernandes(georgia@bce.unb.br) on 2009-02-17T18:04:51Z (GMT) No. of bitstreams: 1 DISSERTACAO_2008_JoseAugustodeOliveiraBotelho.pdf: 2980105 bytes, checksum: f5b5a96a5b7e8a0bd74ff1e7bbaafbd7 (MD5) / Made available in DSpace on 2009-02-17T18:04:52Z (GMT). No. of bitstreams: 1 DISSERTACAO_2008_JoseAugustodeOliveiraBotelho.pdf: 2980105 bytes, checksum: f5b5a96a5b7e8a0bd74ff1e7bbaafbd7 (MD5) / Introdução: A infecção em gestantes por Chlamydia trachomatis é um grande problema de saúde pública e que não raramente tem como conseqüência a ocorrência de abortos. Objetivos: Conhecer a prevalência de infecção por Chlamydia trachomatis em gestantes e abortos, por faixa etária e município de residência no Mato Grosso do Sul, de 2005 a 2007. Método: Realizou-se estudo epidemiológico descritivo. Examinaram-se 74.701 cartões do Programa de Proteção à Gestante, juntamente com dados dos resultados de testes laboratoriais (Enzimaimunoensaio) em papel de filtro; nos resultados positivos realizaram-se testes sorológicos confirmatórios. Empregou-se o teste Qui-quadrado (X2), com 95% confiabilidade na análise dos dados. Resultados: Encontrou-se uma prevalência de infecção por C. trachomatis de 6,64% e taxa de infecção na faixa etária de 20 a 35 anos de 6,8% e OR 1,07 (p=0,002). A freqüência de infecção e abortos relatados anteriormente foi de 6,77% com OR 1,2 (p<0,0001). Conclusões: A prevalência de infecção por C. trachomatis nas gestantes pesquisadas no Mato Grosso do Sul está em conformidade com as taxas brasileiras. Verificou-se que existem diferenças na ocorrência de abortos entre os municípios localizados em diferentes áreas do Estado, microrregião de influência de Campo Grande, assentamentos e áreas indígenas. _________________________________________________________________________________________ ABSTRACT / Introduction: The infection in pregnant women for Chlamydia trachomatis is a major public health problem and not rarely has to occur as a result of abortions. Objectives: To know the prevalence of C. trachomatis infection in pregnant women and abortions, by age and city of residence in Mato Grosso do Sul, between 2005 and 2007. Method: There was descriptive epidemiological study. Were examined to 74,701 collection cards of the Pregnancy Protection Program, together with data of the results of laboratory tests (Enzyme immunoassay) in tissue dried blood filter paper; the positive results were confirmed by serological tests. We applied the Chisquare (X2), with 95% confidence in the analysis of data. Results: We found a prevalence of infection by C. trachomatis of 6.64% and rate of infection in age from 20 to 35 years of 6.8%, OR 1.07 (p = 0002). The frequency of infection and abortions reported previously was 6.77% with OR 1.2 (p <0.0001). Conclusions: The prevalence of infection by C. trachomatis in pregnant women searched in Mato Grosso do Sul is in accordance with the Brazilian rates. It was found that there are differences in the occurrence of abortions among cities in different areas of the state, micro regions by influence of Campo Grande, settlements and indigenous areas.

Infecções por Chlamydia trachomatis estão entre as mais freqüentes doenças sexualmente transmissíveis (DST) em todo o mundo, apresentando grande importância epidemiológica. A identificação deste patógeno pode ser difícil e um método de detecção baseado na amplificação de ácidos nucleicos é altamente desejável, por sua acurácia e rapidez. O presente estudo avaliou a acurácia, sensibilidade e especificidade de uma reação em cadeia da polimerase (PCR) in “house” em amostras de urina de homens com e sem sintomas de DST comparados a um teste comercial, o COBAS Amplicor CT/NG (Roche, Suiça). Foram utilizados primers específicos para amplificar um segmento do plasmídio críptico de C. trachomatis gerando um fragmento de 201 pb, cuja seqüência foi confirmada por clivagem enzimática e seqüenciamento automático. A especificidade analítica dos primers foi confirmada frente ao DNA de diferentes microrganismos patogênicos e não patogênicos da microbiota urogenital masculina. A detecção limite do DNA clamidial pela PCR in house após hibridização (Southern blot), foi de 1 pg. O COBAS Amplicor CT/NG foi considerado o teste de referência por ser automatizado e conter um programa de controle interno da reação para identificar inibição da DNA polimerase. Entre as 37 amostras testadas positivas para C. trachomatis pelo COBAS Amplicor, 33 foram confirmadas positivas pela PCR in house. Foram detectados inibidores da reação nas 4 amostras que apresentaram resultados falso-negativos, utilizando-se a técnica de contaminação da reação com o DNA clamidial (spiked) e um controle interno da reação com primers que amplificam a β-globina do DNA humano. Após congelamento e descongelamento para eliminar prováveis substâncias inibidoras lábeis, as 4 amostras foram re-testadas, resultando na positividade de mais 2 amostras, completando 35 amostras positivas pela PCR in house. Entre as 74 amostras testadas negativas para C. trachomatis pelo COBAS Amplicor, 72 foram confirmadas negativas pela PCR in house. Das 2 amostras prováveis falso-positivas, apenas 1 permaneceu positiva, após re-teste. Dos 111 pacientes analisados, 108 apresentaram resultados idênticos nos dois testes, o equivalente a uma acurácia = 97,3%, sensibilidade = 94,6% e especificidade = 98,6%. A alta sensibilidade e especificidade apresentada pela PCR in house, demonstra a possibilidade de triar e diagnosticar C. trachomatis em urina de homens, importante reservatório da infecção clamidial para as mulheres. / Chlamydia trachomatis infections are among the most commum sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid amplification based detection method. The present study was designe to evaluate the accuracy, sensitivity and specificity of a in house plasmid-based polymerase chain reaction (PCR) and hybridization on first void urine specimens from symptomatic and asymptomatic men, compared with a commercial test the PCR COBAS Amplicor CT/NG (Roche, Switzerland), for detection of C. trachomatis (CT). Specific primers for the cryptic plasmid of C. trachomatis were designed to amplify a 201 bp fragment confirmed by enzymatic cleavage and automatic sequencing. The analytic specificity was determined by submitting to the intended protocol, the DNA of normal and pathogenic urogenital microbiota, the specific primers anneling was confirmed. The detection limit of the PCR and the Southern blot hibridization, was mesured in 1 pg of chlamydial DNA. The COBAS Amplicor CT/NG was considered the reference test, it contains a internal control (IC) programme to identify DNA polymerase inhibition. Among 37 positive specimens tested by COBAS Amplicor, 33 were confirmed positive by in house PCR and inhibitors of PCR were demonstrated in the 4 possibly false-negative samples performing DNA chlamydial spiking experiments and using a positive internal control of human β-globin DNA. The specimens were freezedthowed and re-tested to remove labile inhibitors, but 2 samples remained negative. Among 74 negative specimens by COBAS Amplicor, 72 were confirmed negative by in house PCR. The 2 problaby false-positive samples were re-tested and just one remained positive. The final results of the two tests were identical for 108 of the 111 pacients, accuracy = 97,3% ; sensitivity = 94,6% and specificity = 98,6%. This study shows that the in house plasmid-based PCR is fasible for detection of Chlamydia trachomatis in male urine specimens. This test presented high accuracy, sensitivity and specificity, offering great potencial for the screening of men, an important reservoir of infection chlamydial for women.

Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2015-04-08T21:24:34Z No. of bitstreams: 1 Márcia Sacramento Cunha Machado.pdf: 2145514 bytes, checksum: 5c4ac4862de6cc6c8841617df08590b4 (MD5) / Made available in DSpace on 2015-04-08T21:24:34Z (GMT). No. of bitstreams: 1 Márcia Sacramento Cunha Machado.pdf: 2145514 bytes, checksum: 5c4ac4862de6cc6c8841617df08590b4 (MD5) Previous issue date: 2011 / A incidência de doenças sexualmente transmissíveis (DST) vem aumentando em todo o mundo, especialmente entre adolescentes. Entretanto, poucos estudos foram realizados no Brasil para abordar este tema. Com o objetivo de estimar a prevalência, identificar a etiologia e possíveis fatores associados para DST na adolescência, foi realizado um estudo transversal. Cem adolescentes sexualmente ativas do sexo feminino foram avaliadas em Salvador, Bahia entre 2008 e 2010. Foram realizados exames citológicos, microbiológicos, PCR da secreção cervico-vaginal para Chlamydia trachomatis e HPV, testes sorológicos para sífilis, Herpes simplex vírus I/II (IgG e IgM), AgHBs, anti-HBc e anti-HBs, para hepatite B; anti-HCV, para hepatite C; HTLV I/II e HIV. A idade média das adolescentes foi de 16,6 + 1,6 anos. A prevalência de DST foi de 90%. O HPV representou principal etiologia (88%), seguido de Chlamydia trachomatis (31%). Quatro pacientes (4%) apresentaram positividade para herpes simples vírus IgM e uma adolescente apresentou Trichomonas vaginalis. Co-infecção foi observada em 30% dos casos. Nenhuma paciente foi encontrada com infecção por hepatite B ou C, HIV, HTLV e sífilis. Conclusão: A prevalência de doenças sexualmente transmissíveis foi elevada entre as adolescentes avaliadas. Os resultados reforçam a situação de risco e a necessidade de políticas públicas voltadas para esta população específica. Além disso, técnicas para o diagnóstico molecular de DST, especialmente para HPV e Chlamydia trachomatis, devem ser disponibilizadas no SUS, sobretudo na adolescência, possibilitando a identificação do risco para câncer de colo uterino.

Infecções por Chlamydia trachomatis estão entre as mais freqüentes doenças sexualmente transmissíveis (DST) em todo o mundo, apresentando grande importância epidemiológica. A identificação deste patógeno pode ser difícil e um método de detecção baseado na amplificação de ácidos nucleicos é altamente desejável, por sua acurácia e rapidez. O presente estudo avaliou a acurácia, sensibilidade e especificidade de uma reação em cadeia da polimerase (PCR) in “house” em amostras de urina de homens com e sem sintomas de DST comparados a um teste comercial, o COBAS Amplicor CT/NG (Roche, Suiça). Foram utilizados primers específicos para amplificar um segmento do plasmídio críptico de C. trachomatis gerando um fragmento de 201 pb, cuja seqüência foi confirmada por clivagem enzimática e seqüenciamento automático. A especificidade analítica dos primers foi confirmada frente ao DNA de diferentes microrganismos patogênicos e não patogênicos da microbiota urogenital masculina. A detecção limite do DNA clamidial pela PCR in house após hibridização (Southern blot), foi de 1 pg. O COBAS Amplicor CT/NG foi considerado o teste de referência por ser automatizado e conter um programa de controle interno da reação para identificar inibição da DNA polimerase. Entre as 37 amostras testadas positivas para C. trachomatis pelo COBAS Amplicor, 33 foram confirmadas positivas pela PCR in house. Foram detectados inibidores da reação nas 4 amostras que apresentaram resultados falso-negativos, utilizando-se a técnica de contaminação da reação com o DNA clamidial (spiked) e um controle interno da reação com primers que amplificam a β-globina do DNA humano. Após congelamento e descongelamento para eliminar prováveis substâncias inibidoras lábeis, as 4 amostras foram re-testadas, resultando na positividade de mais 2 amostras, completando 35 amostras positivas pela PCR in house. Entre as 74 amostras testadas negativas para C. trachomatis pelo COBAS Amplicor, 72 foram confirmadas negativas pela PCR in house. Das 2 amostras prováveis falso-positivas, apenas 1 permaneceu positiva, após re-teste. Dos 111 pacientes analisados, 108 apresentaram resultados idênticos nos dois testes, o equivalente a uma acurácia = 97,3%, sensibilidade = 94,6% e especificidade = 98,6%. A alta sensibilidade e especificidade apresentada pela PCR in house, demonstra a possibilidade de triar e diagnosticar C. trachomatis em urina de homens, importante reservatório da infecção clamidial para as mulheres. / Chlamydia trachomatis infections are among the most commum sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid amplification based detection method. The present study was designe to evaluate the accuracy, sensitivity and specificity of a in house plasmid-based polymerase chain reaction (PCR) and hybridization on first void urine specimens from symptomatic and asymptomatic men, compared with a commercial test the PCR COBAS Amplicor CT/NG (Roche, Switzerland), for detection of C. trachomatis (CT). Specific primers for the cryptic plasmid of C. trachomatis were designed to amplify a 201 bp fragment confirmed by enzymatic cleavage and automatic sequencing. The analytic specificity was determined by submitting to the intended protocol, the DNA of normal and pathogenic urogenital microbiota, the specific primers anneling was confirmed. The detection limit of the PCR and the Southern blot hibridization, was mesured in 1 pg of chlamydial DNA. The COBAS Amplicor CT/NG was considered the reference test, it contains a internal control (IC) programme to identify DNA polymerase inhibition. Among 37 positive specimens tested by COBAS Amplicor, 33 were confirmed positive by in house PCR and inhibitors of PCR were demonstrated in the 4 possibly false-negative samples performing DNA chlamydial spiking experiments and using a positive internal control of human β-globin DNA. The specimens were freezedthowed and re-tested to remove labile inhibitors, but 2 samples remained negative. Among 74 negative specimens by COBAS Amplicor, 72 were confirmed negative by in house PCR. The 2 problaby false-positive samples were re-tested and just one remained positive. The final results of the two tests were identical for 108 of the 111 pacients, accuracy = 97,3% ; sensitivity = 94,6% and specificity = 98,6%. This study shows that the in house plasmid-based PCR is fasible for detection of Chlamydia trachomatis in male urine specimens. This test presented high accuracy, sensitivity and specificity, offering great potencial for the screening of men, an important reservoir of infection chlamydial for women.

A Chlamydia trachomatis (Ct) à a bactÃria de transmissÃo sexual mais comum em todo o mundo, apesar de existirem poucos dados sobre este agravo na populaÃÃo brasileira. O objetivo da pesquisa foi determinar a prevalÃncia da infecÃÃo por Ct, avaliando os fatores sÃciodemogrÃficos e achados clÃnicos, colposcÃpicos e citopatolÃgicos associados à ocorrÃncia desta infecÃÃo em mulheres jovens na periferia de Fortaleza. Foi realizado um estudo de corte transversal em 200 mulheres sexualmente ativas, com idade entre 12 e 25 anos, atendidas no perÃodo de agosto de 2011 a agosto de 2012, no ambulatÃrio de ginecologia geral do Hospital Distrital Gonzaga Mota â Barra do CearÃ. InformaÃÃes pessoais e dados do exame ginecolÃgico foram anotados em um questionÃrio e as pacientes submeteram-se à coleta de material da endocÃrvice para teste de captura hÃbrida II para Chlamydia trachomatis e para citologia oncÃtica convencional, seguido de colposcopia. Os dados foram analisados utilizando o software Graphpad Prism 5.0, procedendo-se a anÃlise descritiva e analÃtica utilizando o teste t de Student para as variÃveis nominais e o teste exato de Fisher para as variÃveis quantitativas. A prevalÃncia da infecÃÃo por Chlamydia trachomatis foi 15,5% (31/200) em mulheres adolescentes e adultas jovens. NÃo foi encontrada associaÃÃo entre a infecÃÃo estudada e as caracterÃsticas sÃciodemogrÃficas, hÃbitos sexuais, sinais e sintomas questionados, ectopia cilÃndrica e alteraÃÃes colposcÃpicas. Dentre as atipias citolÃgicas, o ASC-US esteve presente em 20,7% dos casos e 4,5% dos controles (p=0,0067, RR=3,452, IC=1,72-6,89), mostrando uma associaÃÃo positiva com a infecÃÃo clamidiana. A G. vaginalis foi encontrada em 54.8% das pacientes infectadas e em 30,7% das pacientes negativas (p=0,0133, RR=2,305. IC=1,21-4,39), mostrando uma relaÃÃo com a infecÃÃo estudada. Concluiu-se que a infecÃÃo por C. trachomatis teve uma prevalÃncia alta na populaÃÃo estudada, que nÃo houve associaÃÃo com fatores de risco sÃcio-demogrÃfico, biolÃgico, com achados clÃnicos e/ou colposcÃpicos. Houve associaÃÃo de ASC-US e G. vaginalis na citologia oncÃtica com a infecÃÃo estudada. / Chlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection worldwide, but there are few published dates about it in Brazil. The aim of this study was to determinate the prevalence of Chlamydia trachomatis infection and to assess the socialdemographic behavioral, clinical and cytopathological factors associated with this infection among adolescents and young women in a low-income area of Fortaleza - Brazil. A cross-sectional study was conducted in 200 sexually active women aged 12 to 25 years, from august 2011 to august 2012, in gynecology outpatient clinic of the General Hospital District Gonzaga Mota - Barra do CearÃ. Personal information and date of gynecogical examination were recorded in a questionnaire and the patients underwent collection of material from the endocervix to hybrid capture test for C. trachomatis and Pap test, followed by colposcopy. Data were analyzed using Graphpad Prism 5.0 software, proceeding to descriptive and analytical analysis using the Student t test for nominal variables and the Fisher exact test for quantitative variables. No association was found between infection and studied the socio-demographic characteristics, sexual habits, signs and symptoms questioned, cylindrical ectropion and colposcopic changes.Among the abnormal cytological atypia, ASC-US was presented in 20,7% of cases and 4,5% of controls (p=0,0067, RR=3,452, IC=1,72-6,89), with a positive association with the infection. G. vaginalis morphotype was identified in 54,8% of infected women and 30,7% of negative patients (p=0,0133, RR=2,305. IC=1,21-4,39), showing a relationship with the infection. It was concluded that infection with C. trachomatis had a high prevalence in the population studied, no association was observed with socio-demographic, biological and clinical findings and colposcopic changes. There was association of ASC-US and G. vaginalis in cytology with the infection studied.

Infecções por Chlamydia trachomatis estão entre as mais freqüentes doenças sexualmente transmissíveis (DST) em todo o mundo, apresentando grande importância epidemiológica. A identificação deste patógeno pode ser difícil e um método de detecção baseado na amplificação de ácidos nucleicos é altamente desejável, por sua acurácia e rapidez. O presente estudo avaliou a acurácia, sensibilidade e especificidade de uma reação em cadeia da polimerase (PCR) in “house” em amostras de urina de homens com e sem sintomas de DST comparados a um teste comercial, o COBAS Amplicor CT/NG (Roche, Suiça). Foram utilizados primers específicos para amplificar um segmento do plasmídio críptico de C. trachomatis gerando um fragmento de 201 pb, cuja seqüência foi confirmada por clivagem enzimática e seqüenciamento automático. A especificidade analítica dos primers foi confirmada frente ao DNA de diferentes microrganismos patogênicos e não patogênicos da microbiota urogenital masculina. A detecção limite do DNA clamidial pela PCR in house após hibridização (Southern blot), foi de 1 pg. O COBAS Amplicor CT/NG foi considerado o teste de referência por ser automatizado e conter um programa de controle interno da reação para identificar inibição da DNA polimerase. Entre as 37 amostras testadas positivas para C. trachomatis pelo COBAS Amplicor, 33 foram confirmadas positivas pela PCR in house. Foram detectados inibidores da reação nas 4 amostras que apresentaram resultados falso-negativos, utilizando-se a técnica de contaminação da reação com o DNA clamidial (spiked) e um controle interno da reação com primers que amplificam a β-globina do DNA humano. Após congelamento e descongelamento para eliminar prováveis substâncias inibidoras lábeis, as 4 amostras foram re-testadas, resultando na positividade de mais 2 amostras, completando 35 amostras positivas pela PCR in house. Entre as 74 amostras testadas negativas para C. trachomatis pelo COBAS Amplicor, 72 foram confirmadas negativas pela PCR in house. Das 2 amostras prováveis falso-positivas, apenas 1 permaneceu positiva, após re-teste. Dos 111 pacientes analisados, 108 apresentaram resultados idênticos nos dois testes, o equivalente a uma acurácia = 97,3%, sensibilidade = 94,6% e especificidade = 98,6%. A alta sensibilidade e especificidade apresentada pela PCR in house, demonstra a possibilidade de triar e diagnosticar C. trachomatis em urina de homens, importante reservatório da infecção clamidial para as mulheres. / Chlamydia trachomatis infections are among the most commum sexually transmitted diseases and of great epidemiological importance world-wide. Identification of this pathogen can be difficult, and it is highly desirable to have a rapid and accurate nucleic acid amplification based detection method. The present study was designe to evaluate the accuracy, sensitivity and specificity of a in house plasmid-based polymerase chain reaction (PCR) and hybridization on first void urine specimens from symptomatic and asymptomatic men, compared with a commercial test the PCR COBAS Amplicor CT/NG (Roche, Switzerland), for detection of C. trachomatis (CT). Specific primers for the cryptic plasmid of C. trachomatis were designed to amplify a 201 bp fragment confirmed by enzymatic cleavage and automatic sequencing. The analytic specificity was determined by submitting to the intended protocol, the DNA of normal and pathogenic urogenital microbiota, the specific primers anneling was confirmed. The detection limit of the PCR and the Southern blot hibridization, was mesured in 1 pg of chlamydial DNA. The COBAS Amplicor CT/NG was considered the reference test, it contains a internal control (IC) programme to identify DNA polymerase inhibition. Among 37 positive specimens tested by COBAS Amplicor, 33 were confirmed positive by in house PCR and inhibitors of PCR were demonstrated in the 4 possibly false-negative samples performing DNA chlamydial spiking experiments and using a positive internal control of human β-globin DNA. The specimens were freezedthowed and re-tested to remove labile inhibitors, but 2 samples remained negative. Among 74 negative specimens by COBAS Amplicor, 72 were confirmed negative by in house PCR. The 2 problaby false-positive samples were re-tested and just one remained positive. The final results of the two tests were identical for 108 of the 111 pacients, accuracy = 97,3% ; sensitivity = 94,6% and specificity = 98,6%. This study shows that the in house plasmid-based PCR is fasible for detection of Chlamydia trachomatis in male urine specimens. This test presented high accuracy, sensitivity and specificity, offering great potencial for the screening of men, an important reservoir of infection chlamydial for women.