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Background: There is an urgent need to improve our understanding of the mucosal immuno-pathogenesis of HIV acquisition in the female genital tract, particularly in high-risk women such as female sex workers (FSWs). Cervical biopsy samples offer technical advantages over cytobrush sampling, but there are concerns that this might increase HIV acquisition, particularly if healing is slow and/or women do not abstain from sex during healing. Methodology/Principal Findings: Cervical biopsy samples and cervico-vaginal swabs for co-infection diagnostics, prostate specific antigen (PSA) and immune studies were collected from 59 women, including HIV seropositive and HIV-exposed seronegative (HESN) FSWs as well as lower risk women from Nairobi, Kenya. A clinical-demographic questionnaire was administered and women were instructed to avoid sexual intercourse, douching and the insertion of tampons for 14 days. All participants underwent a repeat exam to assess healing within the 14 days, and had HIV diagnostics at six months. Cervical sampling was well tolerated, and 82% of participants had healed macroscopically by 5 days. Both self-report and PSA screening suggested high levels of compliance with pre- and post-procedure abstinence. Delayed healing was associated with vulvovaginal candidiasis (VVC) and HESN status. At six-month follow up all low-risk and HESN participants remained HIV seronegative. Conclusion: Cervical biopsy sampling is a safe and well-tolerated method to obtain cervical biopsies in this context, particularly if participants with VVC are excluded. As healing could be delayed up to 11 days, it is important to support (both financially and with rigorous counseling) a period of post-procedure abstinence to minimize HIV risk. © 2012 Hasselrot et al.
The study was reviewed and approved by the regional ethical boards at Kenyatta National Hospital, Nairobi, Kenya; the Karolinska Institutet, Stockholm, Sweden; and the University of Manitoba, Winnipeg, Canada. Written informed consent was obtained from all study participants. All ethical committees approved the consent procedure. HIV-uninfected and infected FSW participants were recruited at the Majengo Sex Worker Clinic [3] and HIV-uninfected lower risk controls were recruited at a Maternal Health Clinic based at the Pumwani Maternity Hospital [11]. Inclusion criteria were: (1) age >18 years; (2) uterus and cervix present; (3) willingness to undergo pelvic exams and ectocervical biopsies; (4) willingness to abstain from vaginal sex for 15 days as part of the study; (5) antiretroviral treatment (ART) naïve and (6) general good health. Exclusion criteria were: (1) pregnancy; and (2) active menstruation. All HIV-uninfected female sex workers were currently active in sex work and had been enrolled in the Majengo Clinic for at least three years; thereby meeting previously published epidemiologic criteria for relative HIV resistance [3]. All lower risk individuals enrolled reported no history of sex work and only one sexual partner for the last 6 months. The detected HIV viral load in the HIV-infected women was 20–64800 copies/mL (median: 11735 copies/mL) and the CD4 count ranged 121–1737 cells/µL (median: 493 cells/µL). Cervical biopsies were collected using a protocol previously shown to be safe and well-tolerated in Swedish participants at low risk of HIV exposure [12]. An external and internal genital exam was performed and cervicovaginal secretions (CVS) were collected from all women by rotating one cotton-tipped swab 360° in the cervical os, and one swab to collect secretions from the posterior vaginal fornix. Both swabs were transferred into a vial containing 5 mL of phosphate-buffered saline (PBS).Next, cervical cells were collected by rotating one cytobrush 360° in the cervical os and two ectocervical biopsies (3 mm2) from the superior portion of the ectocervix were collected with Schubert biopsy forceps (B. Braun Aesculap AG, Tuttlingen, Germany). A polycresulin gel was applied after sampling to induce vasoconstriction and subsequent homeostasis and all participants were observed for up to one hour to ensure that no active bleeding occurred prior to clinic discharge. Data regarding demographical, reproductive, sexual and clinical characteristics were collected using a questionnaire. All participants were provided with both written and verbal information about the potential for cervical biopsy to increase the risk of HIV acquisition and/or transmission, this information was repeated at two separate clinic visits prior to enrolment. Participants were asked to abstain from unprotected vaginal sex for a period of one day prior to the procedure and to refrain from any vaginal sex, vaginal douching or tampon insertion for fourteen days after the procedure. Since the study protocol precluded income from sex work, FSW participants received monetary compensation equivalent to the expected lost income; lower risk controls received the same compensation. Genital infection diagnostics included: HIV (Chiron, Emeryville, CA, USA) and Herpes Simplex Virus type 2 (HSV2) serology (HerpeSelect® 1 and 2 Immunoblot IgG, Focus Diagnostics, CA, USA); urine for Chlamydia trachomatis and Neisseria gonorrhoeae (Amplicor PCR Diagnostics, Roche Diagnostics, Quebec, Canada); syphilis serology (Macro-Vue Rapid Plasma Reagin test, Becton Dickinson, Franklin Lakes, NJ, USA); Gram stain for bacterial vaginosis (BV; defined as a Nugent score of 7 to 10) and lactobacillus colonization and vulvovaginal candidiasis (VVC) (defined as the Gram stain finding of any lactobacilli or yeast, respectively). All participants were provided with HIV/STI prevention counseling, male and female condoms, family planning services, treatment of STIs, medical care for acute and chronic illnesses, access to adequate diagnostic testing and referral for specialist consultant and/or hospitalization at Kenyatta National Hospital if needed. Study participants were asked to return for clinical follow-up 3–5 days post-procedure; those who were actively menstruating were requested to come back as soon as menses had ceased. Participants were clinically evaluated and asked about any bleeding or discomfort, and whether they had had vaginal sex since the procedure. A gynecological exam was performed, including clinical assessment of biopsy healing. The biopsy site was considered as healed when no bleeding from biopsy site, no hyperemia and no abnormalities were detected during the macroscopic evaluation. Vaginal lavages (VagL) were collected by gently aspirating 2 mL of PBS without getting in contact with the cervix. Participants were again informed of the importance of sexual abstinence for a full two weeks post-procedure. PSA levels in enrolment CVS and follow up VagL were assayed using a Chemiluminescent microparticle immunoassay (ARCHITECT Instrument, Abbott Laboratories, IL, USA) as a marker of recent unprotected sex [13]. Univariate analyses were performed using Fisher’s exact test when comparing categorical variables, and non-parametric Mann-Whitney test when comparing continuous variables. Forward conditional binary logistic regression analyses were performed with healing status of biopsy site as the dependent variable and all variables associated with non-healing of the biopsy site in univariate analysis as covariates. P-value of <0.05 was considered significant. Software products used were Prism 5.00 (GraphPad Software Inc, CA, USA) for Windows and PASW Statistics 18 (SPSS/IBM Corporation, NY, USA).
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