We demonstrated that in 416 women attending a sexually transmitted infection clinic, microbiological cure was low in azithromycin-treated (78.5%) compared with doxycycline-treated (95.5%) rectal chlamydia; microbiological cure in vaginal chlamydia was high for both treatment types (93.5% and 95.4%).

ObjectivesAccording to the current guidelines for laboratory diagnosis of sexually transmitted infections (STIs), nucleic acid amplification tests (NAATs) are the preferred diagnostic method for Chlamydia trachomatis (CT) infections. However, NAATs amplify the available target DNA without discriminating between DNA originating from viable or non-viable CT. Assessing CT viability will provide more insights in the clinical and public health relevance of a CT positive test result. The aim of this study was to technically validate and implement viability-PCR (V-PCR) to asses CT viability.MethodsTechnical validation of V-PCR was performed by the assessment of predefined viability ratios of CT. Samples were subjected to V-PCR which consisted of propidium monoazide (PMA) treatment prior to DNA extraction followed by quantitative PCR (qPCR) targeting the ompA gene for the detection of CT DNA. Finally, V-PCR was applied to vaginal swabs of 50 CT positive patients, as indicated by routine NAAT, collected at our outpatient STD clinics before antimicrobial treatment.ResultsTechnical validation of V-PCR showed that PMA treatment of heat-inactivated CT culture resulted in an almost complete loss of qPCR signal. PMA treated samples of the fresh viable CT culture showed no marked reduction of PCR signal, indicating that all DNA from viable CT could be detected. Applying V-PCR to clinical samples showed that in 36% of samples (18/50) less than 1% of CT DNA originated from viable bacteria.ConclusionsV-PCR showed to be a fast and easy method to assess CT viability in clinical samples, without the need of traditional challenging cell culture methods. Furthermore, V-PCR results of clinical samples have indicated that a substantial amount of the amplified CT DNA originated from non-viable cells. Although results might be influenced by cell death during transport, this study suggests that there is a potential overestimation of quantitative CT positivity by currently used NAATs.

BackgroundIn women, anorectal infections with Chlamydia trachomatis (CT) are about as common as genital CT, yet the anorectal site remains largely untested in routine care. Anorectal CT frequently co-occurs with genital CT and may thus often be treated co-incidentally. Nevertheless, post-treatment detection of CT at both anatomic sites has been demonstrated. It is unknown whether anorectal CT may play a role in post-treatment transmission. This study, called FemCure, in women who receive routine treatment (either azithromycin or doxycycline) aims to understand the post-treatment transmission of anorectal CT infections, i.e., from their male sexual partner(s) and from and to the genital region of the same woman. The secondary objective is to evaluate other reasons for CT detection by nucleic acid amplification techniques (NAAT) such as treatment failure, in order to inform guidelines to optimize CT control.MethodsA multicentre prospective cohort study (FemCure) is set up in which genital and/or anorectal CT positive women (n = 400) will be recruited at three large Dutch STI clinics located in South Limburg, Amsterdam and Rotterdam. The women self-collect anorectal and vaginal swabs before treatment, and at the end of weeks 1, 2, 4, 6, 8, 10, and 12. Samples are tested for presence of CT-DNA (by NAAT), load (by quantitative polymerase chain reaction -PCR), viability (by culture and viability PCR) and CT type (by multilocus sequence typing). Sexual exposure is assessed by online self-administered questionnaires and by testing samples for Y chromosomal DNA. Using logistic regression models, the impact of two key factors (i.e., sexual exposure and alternate anatomic site of infection) on detection of anorectal and genital CT will be assessed.DiscussionThe FemCure study will provide insight in the role of anorectal chlamydia infection in maintaining the CT burden in the context of treatment, and it will provide practical recommendations to reduce avoidable transmission. Implications will improve care strategies that take account of anorectal CT.Trial registrationClinicalTrials.gov Identifier: NCT02694497.

IntroductionIn prior studies it is demonstrated that, in women, the prevalence of anorectal infections with Chlamydia trachomatis (CT) is comparable to genital CT. Yet, the clinical relevance and the role in overall transmission of anorectal CT in women is still under debate. The assessment of CT viability will gain new insight in current knowledge gaps. Recently, we validated the viability-PCR (V-PCR) method to assess CT viability in genital CT positive samples. In this study, V-PCR was utilised to assess CT viability in anorectal samples from CT positive women. MethodsCOBAS 4800 CT/NG routine testing was used for CT diagnosis. Women positive for genital and/or anorectal CT (n=66), collected self-taken vaginal and anal swabs at our outpatient STI clinic (South Limburg Public Health Service) prior to treatment at the initial screening and at treatment consultation. V-PCR and culture were used to assess CT viability. ResultsV-PCR results showed that in up to 31% (8/26) of anorectal positive samples less than 1% of the detected CT DNA originated from viable bacteria. However, in 62% (16/26) of anorectal positive samples more than 10% of the detected CT DNA originated from viable CT. In this category, routine COBAS results also showed a stable bacterial load between initial screening and treatment consultation, further supporting the presence of large amounts of viable CT. Finally, culture results confirmed results of V-PCR and showed a direct relation to the proportion of viable CT in clinical samples.ConclusionAlthough the cohort was relatively small, results in this study showed that a substantial amount of anorectal CT positive samples contained viable CT. Overall, these results provide further evidence that anorectal CT infections in women are clinically relevant. In a currently ongoing larger cohort study, clinical samples from CT positive women (n=400) will be assessed for viability before and after treatment (FemCure Study).

IntroductionLongitudinal cohort studies provide unique insights but preventing drop-out and missing values is challenging. The aim of this study is to describe how to recruit and maximise retention of 400 Chlamydia trachomatis (CT) positive women in FemCure, an ongoing longitudinal multicenter cohort study aiming to recruit (extra)genital CT positive women.MethodsRecruitment took place at 3 STI-clinics (South Limburg, Amsterdam and Rotterdam). Of the invited women 29% joined. They collected vaginal, anorectal and (nurse-taken) oral swabs and completed online questionnaires pre-treatment, 1, 2, 4, 6, 8, 10 and 12 weeks post-treatment. To minimise loss to follow up (ltfu) various reminders were sent, small incentives were given and no show at the 2nd visit were replaced. Logistic regression analyses were used to assess predictors for ltfu (i.e. age, education level, steady partner, ethnicity, previous CT diagnosis, recruitment clinic, and anatomic site of infection). The results include the first 7 months of recruitment.ResultsBy 11-dec-16 143 participants completed fu. Of them, 81% had complete data, 17% (n=24) was ltfu, of which 18 before the 2nd visit. Nine cases had missing data. Of all questionnaires and swabs (n=1144), 12% was completed after a reminder. Multivariate logistic regression showed that education and age were associated with ltfu. Low education was related to more ltfu than higher education (OR=2.8, 95% CI 1.1–6.8). Those aged 23=> were related to more ltfu than those <23 (OR=2.5, 95% CI 1.0–6.4). No other variables were statistically significantly associated, this may change with ongoing recruitment. At the last visit 86% reported to participate again in a similar study (reasons given include helping others/friendliness of the staff) while the others mention time constraints as a barrier. ConclusionThese preliminary results show a retention rate of 81% without missing data. Approaches described, especially combined with committed healthcare providers can be used to motivate participants to complete fu in future longitudinal multicenter studies.