Expedited partner therapy (EPT) is a strategy for treating the partners of chlamydia index cases by which a health care provider gives an index patient drugs or a prescription for treatment of chlamydia to deliver to their sex partner without an intervening medical evaluation of the partner. Despite routine offer of EPT in New York City Department of Health and Mental Hygiene (DOHMH) sexual health clinics, the majority of patients who are eligible for EPT do not receive it, largely because EPT eligibility requires lab confirmation of chlamydial infection, which is lacking in situations where patients are treated for chlamydia on the same day they are tested for chlamydia (i.e., presumptive treatment). These patients become eligible for EPT after they leave the clinic and often do not return for EPT. This dissertation includes three papers: one systematic review and two original analyses. The objective of the systematic review was to synthesize existing estimates of EPT effectiveness to better understand the impact of biases on these estimates; a meta-analysis provided an aggregate estimate of the effectiveness of EPT for preventing index patient reinfection with chlamydia and/or gonorrhea. We found 6 studies that included some measure of EPT effectiveness. Meta-analysis revealed that EPT significantly reduced the risk of reinfection from chlamydia and/or gonorrhea, but it also revealed a substantial amount of heterogeneity. Systematic review revealed that inclusion of patients whose sex partners were at the clinic or already treated for infection was a common source of bias among existing estimates of EPT effectiveness. The two original analyses used data from NYC DOHMH sexual health clinics where EPT is routinely offered as patient delivered partner therapy (PDPT), a form of EPT where medication is given directly to the index patient. The objective of the first analysis was to identify predictors of presumptive treatment and predictors of being offered PDPT among patients eligible for PDPT in the NYC clinics. This analysis demonstrated that patient diagnosis as a contact to a sexually transmitted infection (STI) that would warrant treatment with azithromycin or doxycycline (termed STI contacts) was the best predictor of presumptive treatment in NYC DOHMH sexual health clinics. Patients who were not contacts to such STIs or who were STI contacts with more than one sex partner were more likely to be offered PDPT compared to patients who were STI contacts and reported ≤ 1 sex partner. Males not diagnosed as STI contacts were identified as a target population for increasing rates of PDPT offer. The objective of the last analysis was to provide an estimate of PDPT effectiveness for preventing index patient reinfection with chlamydia. This analysis was novel compared to existing estimates of EPT effectiveness in that we excluded patients whose sex partners were at the clinics at the time of chlamydia testing, treatment, or PDPT offer. We found that PDPT significantly reduced the risk of repeat chlamydial infection at both 6 months and 1 year after initial infection. This result was unchanged by multiple sensitivity analyses that assessed the validity of our estimate. In this dissertation, we were able to fill gaps in the literature regarding EPT implementation. The results may help to decrease missed opportunities for offering patients EPT and to support the continued scale-up and optimization of EPT.

The obligate human pathogen Neisseria gonorrhoeae is responsible for the widespread sexually transmitted disease gonorrhoea, which in rare cases also leads to the development of disseminated gonococcal infection (DGI). DGI is mediated by PorBIA-expressing bacteria that invade host cells under low phosphate condition by interaction with the scavenger receptor-1 (SREC-I) expressed on the surface of endothelial cells. The interaction of PorBIA and SREC-I was analysed using different in vitro approaches, including surface plasmon resonance experiments that revealed a direct phosphate-independent high affinity interaction of SREC-I to PorBIA. However, the same binding affinity was also found for the other allele PorBIB, which indicates unspecific binding and suggests that the applied methods were unsuitable for this interaction analysis. Since N. gonorrhoeae was recently classified as a “super-bug” due to a rising number of antibiotic-resistant strains, this study aimed to discover inhibitors against the PorBIA-mediated invasion of N. gonorrhoeae. Additionally, inhibitors were searched against the human pathogen Chlamydia trachomatis, which causes sexually transmitted infections as well as infections of the upper inner eyelid. 68 compounds, including plant-derived small molecules, extracts or pure compounds of marine sponges or sponge-associated bacteria and pipecolic acid derivatives, were screened using an automated microscopy based approach. No active substances against N. gonorrhoeae could be identified, while seven highly antichlamydial compounds were detected. The pipecolic acid derivatives were synthesized as potential inhibitors of the virulence-associated “macrophage infectivity potentiator” (MIP), which exhibits a peptidyl prolyl cis-trans isomerase (PPIase) enzyme activity. This study investigated the role of C. trachomatis and N. gonorrhoeae MIP during infection. The two inhibitors PipN3 and PipN4 decreased the PPIase activity of recombinant chlamydial and neisserial MIP in a dose-dependent manner. Both compounds affected the chlamydial growth and development in epithelial cells. Furthermore, this work demonstrated the contribution of MIP to a prolonged survival of N. gonorrhoeae in the presence of neutrophils, which was significantly reduced in the presence of PipN3 and PipN4. SF2446A2 was one of the compounds that had a severe effect on the growth and development of C. trachomatis. The analysis of the mode of action of SF2446A2 revealed an inhibitory effect of the compound on the mitochondrial respiration and mitochondrial ATP production of the host cell. However, the chlamydial development was independent of proper functional mitochondria, which excluded the connection of the antichlamydial properties of SF2446A2 with its inhibition of the respiratory chain. Only the depletion of cellular ATP by blocking glycolysis and mitochondrial respiratory chain inhibited the chlamydial growth. A direct effect of SF2446A2 on C. trachomatis was assumed, since the growth of the bacteria N. gonorrhoeae and Staphylococcus aureus was also affected by the compound. In summary, this study identified the severe antichlamydial activity of plant-derived naphthoquinones and the compounds derived from marine sponges or sponge-associated bacteria SF2446A2, ageloline A and gelliusterol E. Furthermore, the work points out the importance of the MIP proteins during infection and presents pipecolic acid derivatives as novel antimicrobials against N. gonorrhoeae and C. trachomatis. / Neisseria gonorrhoeae ist ein obligat humanpathogenes Bakterium, das für die weltweit verbreitete sexuell übertragbare Krankheit Gonorrhoe verantwortlich ist. In seltenen Fällen kann es auch zur Ausbildung der Disseminierten Gonokokken-Infektion (DGI) kommen, die mit der Expression des Gonokokken Oberflächenproteins PorBIA assoziiert ist. PorBIA-exprimierende Bakterien invadieren in die Wirtszelle unter phosphatfreien Bedingungen, was durch eine Interaktion mit dem zellulären Oberflächenrezeptor scavenger receptor-1 (SREC-I) vermittelt wird. Die direkte Interaktion zwischen PorBIA und SREC-I wurde mittels verschiedenster Methoden analysiert, einschließlich einer Oberflächenplasmonresonanz-analyse, die eine direkte Bindung von PorBIA zu SREC-I in einem phosphatunabhängigen Schritt aufzeigte. Allerdings wurde dieselbe Affinität auch zu PorBIB gefunden, was auf eine unspezifische Bindung hindeutet und dafür spricht, dass die verwendeten Methoden für diese Interaktionsanalyse ungeeignet sind. N. gonorrheae wurde vor kurzem wegen der stetig steigenden Anzahl antibiotikaresistenter Stämme als „Superkeim“ bezeichnet. Aufgrund dessen wurden Inhibitoren gegen die PorBIA-vermittelte Invasion von N. gonorrhoeae, aber auch gegen Chlamydia trachomatis, den humanpathogenen Erreger von sexuell übertragbaren Infektionen und chronisch-follikulärer Bindehautentzündung, gesucht. 68 niedermolekulare Substanzen wurden mittels eines automatisierten Fluoreszenzmikroskopieverfahrens auf ihre inhibitorische Wirkung hin analysiert. Zu den getesteten Substanzen zählten pflanzenabstammende Stoffe, Isolate aus marinen Schwämmen oder Schwamm-assoziierten Bakterien, sowie Pipecolinsäure-Derivate. Gegen N. gonorrheae konnten keine Substanzen identifiziert werden, während sieben antichlamydiale Inhibitoren detektiert wurden. Pipecolinsäurederivate wurden synthetisiert als potentielle Inhibitoren des virulenz-assoziierten Proteins “macrophage infectivity potentiator” (MIP), das eine Peptidyl-Prolyl-cis-trans-Isomerase Aktivität besitzt. Diese Arbeit untersuchte die Rolle des MIP Proteins von N. gonorrhoeae und C. trachomatis während einer Infektion. Die zwei Inhibitoren PipN3 und PipN4 senkten die PPIase Aktivität des rekombinanten Chlamydien und Neisserien MIPs. Beide Substanzen beeinträchtigten das chlamydiale Wachstum und die Entwicklung in Epithelzellen. Ebenso konnte eine tragende Rolle des N. gonorrhoeae MIPs für das Überleben der Bakterien in Gegenwart von Neutrophilen aufgezeigt werden, das durch PipN3 und PipN4 inhibiert wurde. SF2446A2 war einer der Inhibitoren, der einen erheblichen Effekt auf das Wachstum und die Entwicklung von C. trachomatis aufgewiesen hat. Während der Analyse des Wirkmechanismus von SF2446A2 konnte eine Hemmung der mitochondrialen Atmungskette und eine Abnahme der mitochondrialen ATP Produktion in der Wirtszelle festgestellt werden. Allerdings war die Entwicklung von C. trachomatis unabhängig von der Funktionsfähigkeit der Mitochondrien. Eine Verbindung zwischen der antichlamydialen Wirkung von SF2446A2 und der Inhibierung der Mitochondrienatmungskette konnte damit ausgeschlossen werden. Nur das Reduzieren von zellulärem ATP durch Blockieren der Glykolyse und mitochondrialen Atmungskette verursachte eine Beeinträchtigung des Chlamydienwachstums. Eine direkte Auswirkung von SF2446A2 auf C. trachomatis wurde angenommen, da die Substanz auch das Wachstum von anderen Bakterien wie N. gonorrhoeae und Staphylococcus aureus inhibierte. Zusammengefasst identifizierte diese Studie die antichlamydiale Aktivität pflanzenabstammender Naphthochinone und der Isolate aus marinen Schwämmen oder Schwamm-assoziierten Bakterien SF2446A2, ageloline A und gelliusterol E. Ebenso verdeutlicht die Arbeit die Bedeutung der MIP Proteine während der Infektion und legt Pipecolinsäurederivate als mögliche neue Antibiotika gegen N. gonorrhoeae und C. trachomatis nahe.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

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.