Design of the FemCure study: prospective multicentre study on the transmission of genital and extra-genital Chlamydia trachomatis infections in women receiving routine care

BACKGROUND: In 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. METHODS: A 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. DISCUSSION: The 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 REGISTRATION: ClinicalTrials.gov Identifier: NCT02694497

Design of the FemCure study: Prospective multicentre study on the transmission of genital and extra-genital Chlamydia trachomatis infections in women receiving routine care

Background: In 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. Methods: A 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. Discussion: The 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

Viability-PCR Shows That NAAT Detects a High Proportion of DNA from Non-Viable Chlamydia trachomatis

OBJECTIVES: According 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. METHODS: Technical 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. RESULTS: Technical 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. CONCLUSIONS: V-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

Evaluation of clinical samples by viability-PCR.

<p>(A) Δlog CT/ml of the clinical samples are presented as scatter dot plots displaying the median with the interquartile ranges. (B) Clinical samples categorized according to the amount of DNA originating from viable CT.</p

Effect of PMA treatment on defined viability ratios of CT.

<p>representing a untreated viable CT culture proportion of 0%, 0.1%, 1%, 10%, 50%, and 100%, respectively. qPCR was performed using primers specific for the chlamydial single-copy <i>ompA</i> gene. Error bars represent standard deviations from three independent replicates. (A) Log CT/ml obtained for each viability ratio treated with (w PMA; white bars) and without (w/o PMA; grey bars) PMA. (B) Bars present the change in log CT/ml observed as a result of treatment with PMA prior to DNA purification.</p

Treatment effectiveness of azithromycin and doxycycline in uncomplicated rectal and vaginal Chlamydia trachomatis infections in women: a multicentre observational study (FemCure).

Rectal infections with Chlamydia trachomatis (CT) are prevalent in women visiting a sexually transmitted infection (STI) outpatient clinic, but it remains unclear what the most effective treatment is. We assessed the effectiveness of doxycycline and azithromycin for the treatment of rectal and vaginal chlamydia in women. This study is part of a prospective multicentre cohort study (FemCure). Treatment consisted of doxycycline (100 mg twice daily for 7 days) in rectal CT positive women, and of azithromycin (1 g single dose) in vaginal positive women who were rectally untested or rectally negative. Participants self-collected rectal and vaginal samples at enrolment (treatment timepoint) and during 4 weeks of follow-up. The endpoint was microbiological cure by a negative nucleic acid amplification test at 4 weeks. Differences between cure proportions and 95% confidence intervals (CIs) were calculated. We analysed 416 patients of whom 319 had both rectal and vaginal chlamydia at enrolment, 22 had rectal chlamydia only, and 75 had vaginal chlamydia only. In 341 rectal infections, microbiological cure in azithromycin treated women was 78.5% (95%CI: 72.6 to 83.7, n=164/209) and 95.5% (95%CI: 91.0 to 98.2, n=126/132) in doxycycline treated women (difference 17.0%, 95%CI: 9.6 to 24.7, p<0.001). In 394 vaginal infections, cure was 93.5% (95%CI: 90.1 to 96.1,n=246/263) in azithromycin treated women and 95.4% (95%CI: 90.9 to 98.2,n=125/131) in doxycycline treated women (difference 1.9%, 95%CI: -3.6 to 6.7,p=0.504). The effectiveness of doxycycline is high and exceeds that of azithromycin for the treatment of rectal CT infections in women