Study design and participants
Between September, 2003, and September, 2005, we recruited sexually active (an average of at least four sex acts per month) women aged 18–49 years to join the MIRA trial in Durban and Johannesburg, South Africa, and Harare, Zimbabwe. Participants who met protocol inclusion and exclusion criteria (see panel 1) were then randomised into one of two groups: the intervention group, who received a clinician-fitted diaphragm (All-Flex Arcing Spring diaphragm; Ortho-McNeil Pharmaceutical, Raritan, NJ, USA), a supply of lubricant gel (Replens, Lil’ Drug Store Products, Cedar Rapids, IA, USA) and male condoms; and the control group, who received male condoms only. All participants received a comprehensive HIV prevention package consisting of HIV/STI pre-test and post-test counselling, treatment of curable, laboratory-diagnosed STIs, and intensive risk reduction counselling, which emphasised condom negotiation, and was tailored to each participant’s individual circumstances. Pre-test and post-test counselling was individual and confidential, and was offered by qualified research counsellors at the research clinic sites, in accord with guidelines of the US Centers for Disease Control and Prevention (CDC).
Panel 1: MIRA protocol inclusion and exclusion criteria
- Aged 18–49 years*
- Sexually active (defined as an average of four acts per month)
- C trachomatis and N gonorrhoeae negative or treated† at enrolment
- Healthy cervix
- Intending to live in study area for 24 months
- Willing to be randomised
- Willing to provide informed consent
- Known sensitivity or allergy to latex
- History of toxic shock syndrome
- Currently pregnant or wanting to be pregnant in the next 24 months
- Refused treatment for STI/RTI diagnosed at enrolment
- No cervix (total hysterectomy)
- Epithelial disruptions or lesions
- Recent pelvic surgery or pregnancy outcome (within 6 weeks)
- Unable (within five attempts) or unwilling to insert diaphragm correctly
- Illicit drug use or recent blood transfusions (within past 12 months)
- Co-enrolment in other trials
- Unable to speak the study languages (English, Shona, Sotho, Zulu)
- Condition that, in the opinion of the investigator, would constitute contraindications to participation in the study or would compromise ability to comply with the study protocol, such as a major psychiatric disorder
- Did not meet eligibility criteria within five screening or five enrolment visits
RTI = reproductive tract infection. *In November, 2003, 3 months after the beginning of screening, the upper age limit was set at 49 years; previously, women aged 50 years or older had been enrolled into the study. †Single-dose treatment taken at enrolment qualified women as eligible participants.
The randomisation scheme was developed by Ibis Reproductive Health, and used randomly permuted blocks of sizes eight, ten, and 12, stratified by site. Sealed, opaque randomisation envelopes were stored in a secure, central location at each clinic. At the point of randomisation, the designated staff member retrieved the next sequential envelope, and both the staff member and the participant signed the envelope to verify that it was intact. The participant then opened the envelope for their assignment. The study was open label, since no placebo could be used for a diaphragm. Data were managed at the Center for International Data Evaluation and Analysis at the University of California at San Francisco. Data were electronically faxed from the clinic sites directly into a database system. The investigators and co-ordinators were able to access individual records for quality control purposes, but all information identifying the study arm was coded. Investigators and study statisticians were not allowed to examine any data by study arm until unblinding in early February, 2007, when the final analysis began.
Participants were followed quarterly from September, 2003, to December, 2006. The follow-up period for the study was designed to be staggered, with the first cohort of enrolled participants followed up for 24 months, the last enrolled followed for 12 months, and an overall average of 18 woman-months of follow-up per participant.
Women were recruited from family planning, well-baby, and general health clinics, and from community-based organisations, through printed media and radio. In Zimbabwe, the study was done in two clinics within 30 km of Harare: Chitungwiza, a peri-urban municipality; and Epworth, a slightly poorer and less developed suburb. In Durban, the study was undertaken from a peri-urban clinic in Umkomaas and a less urbanised clinic in Botha’s Hill. In Johannesburg, the study clinic was located on the campus of the Perinatal HIV Research Unit of the Chris Hani Barangwanath Hospital in Soweto, a large urban township area.
The study protocol was reviewed and approved by the University of California at San Francisco Institutional Review Board Committee on Human Research, and by the ethics review committees at all local institutions and collaborating organisations, including the Medical Research Council of Zimbabwe, the Medicines Control Authority of Zimbabwe, Biomedical Research Ethics Committee of the University of KwaZulu-Natal, Human Research Ethics Committee of the University of the Witwatersrand, and Western Institutional Review Board. An independent external audit, sponsored by Ibis Reproductive Health, was done by the Quintiles Corporation in November 2005, after study accrual was completed.
At screening, we obtained verbal consent to assess initial eligibility, followed by written informed consent for screening procedures, including diagnostic testing and answering an interviewer-administered questionnaire on demographics and sexual behaviour. Participants received counselling before tests for HIV and STIs, and provided a urine specimen for PCR testing for N gonorrhoeae, C trachomatis and Trichomonas vaginalis (Roche Pharmaceuticals, Branchburg, NJ, USA). Two HIV rapid tests were done on whole blood samples from finger-prick or venipuncture by use of Determine HIV-1/2 (Abbott Laboratories, Tokyo, Japan) and Oraquick (OraSure Technologies, Bethlehem, PA, USA). All participants were notified of their HIV test results and received post-test counselling, condoms, and referral to supporting services, as needed. Discordant results of rapid tests were confirmed on serum samples by ELISA (Vironostika, Biomerieux, Durham, NC, USA; BioRad, Redmond, WA, USA; or AxSYM HIV Ag/Ab Combo assay, Abbott Laboratories, Abbott Park, IL, USA). The enrolment visit was scheduled within 2 weeks for participants who met the initial eligibility criteria.
At their enrolment visit, participants provided written informed consent, had a pelvic examination, provided a blood sample to be used in tests for syphilis (rapid plasma reagin [RPR] and Treponema pallidum haemagglutinin [TPHA], Randox Laboratories, Crumlin, UK) and herpes simplex virus 2 (HSV2; ELISA, FOCUS Diagnostics, Cypress, CA, USA), and urine for pregnancy testing. Women who tested positive for syphilis at enrolment were treated and kept in the study. Because of the high prevalence (>50%) of HSV2 in study areas, and the logistical challenges of identifying individuals who were negative for both HSV2 and HIV, we did not screen out individuals with prevalent HSV2 infection. On the day of enrolment, if women had not had an HIV test done within the past 14 days or tests for N gonorrhoeae, C trachomatis, and T vaginalis done within the past 30 days, they were required to be re-tested before being eligible for randomisation.
To ensure that women in the intervention group were not inherently better at using the diaphragm than those in the control group, all women were fitted for a diaphragm before randomisation, received a demonstration of diaphragm placement on a pelvic model with instructions on use, practised diaphragm insertion on themselves, and were assessed for correct insertion and removal of the device. Women enrolled in the trial completed an Audio Computer Assisted Self-Interviewing (ACASI) baseline questionnaire on demographics, sexual behaviour, and product use.
After randomisation and ACASI, women in the intervention group were counselled to insert the diaphragm into their vagina at any time that was convenient to them before coitus, and to leave it in place for at least 6 h after sex. They were given detailed instructions on maintenance, cleaning, and storage of their diaphragm. Women were asked to empty an applicator of gel (about 2.5 g) into the dome of the diaphragm at the time of insertion, to spread gel onto the rim to facilitate insertion, and to insert another applicator of gel into the vagina before each act of vaginal sex. At each visit, women received a 3-month supply of gel, and were counselled that the effectiveness of the diaphragm and lubricant gel for the prevention of HIV infection was not known. To prevent HIV, they were asked to use condoms regardless of whether or not they used the diaphragm and lubricant gel. Participants were also told that they should not use the diaphragm and lubricant alone as a method of contraception. The diaphragm is approved for contraception when used with a spermicide (typical effectiveness 84%), but its effectiveness without nonoxynol-9 has not been fully established28,29
(nonoxynol-9 is not indicated for use in settings of high HIV prevalence, and therefore was not used in this study). Women were advised to use other contraceptive methods and were provided with hormonal contraceptives through the clinic. They were encouraged to return to the clinic if they experienced any problems or needed more study products.
Panel 2: MIRA study products
Diaphragm: All-Flex Arcing Spring diaphragm (Ortho-McNeil Pharmaceutical)
Moulded, buff-coloured, natural rubber vaginal diaphragm. Contains a distortion-free, dual spring-within-a-spring that provides arcing action.
Gel: Replens Long-Lasting Vaginal Moisturizer (Lil’ Drug Store Products)
An over-the-counter gel that has been marketed to postmenopausal women for treating symptoms of vaginal dryness and atrophy, and has a positive effect on the maturation of the vaginal epithelium.30
Ingredients are purified water, glycerin, mineral oil, polycarbophil, carbomer 934P, hydrogenated palm oil glyceride, sorbic acid, methylparaben, and sodium hydroxide. Replens is not a known contraceptive. Although it is considered an inactive lubricant, it has weak acid-buffering activity. It has been used as a placebo in microbicide safety and effectiveness trials and has also been shown to provide some protection against herpes simplex virus 2 in a mouse model and to inactivate HIV in vitro.31–36
Both study products (diaphragm and gel) were removed from their original packaging and only study-specific instructions for use were given to participants. Diaphragms were distributed in nine sizes, ranging from 55 mm to 95 mm, in 5 mm increments. The gel was distributed in 35 g tubes (14 applications per tube) with one reusable applicator per tube.
At all visits, participants in both study groups received counselling on risk reduction and as many male condoms as desired. Counsellors emphasised that condoms are the only known method to prevent HIV and STIs, and that condoms should be used for every act of sex. Illustrated instruction sheets on use of diaphragms and male condoms were distributed to all women according to study arm assignment in all study languages. A statement about what is known and unknown about the effectiveness of condoms, diaphragms, and gel was included in the enrolment informed consent form, and women’s comprehension of this information was assessed with a questionnaire at the enrolment and month-12 visits. Study products are described in panel 2.
Participants returned 2 weeks after enrolment for resupply of products, for counselling, and to have any problems assessed. Thereafter, follow-up visits were scheduled quarterly to assess HIV and STI status, recent medical history, use of study products, and sexual behaviour, through a face-to-face clinician-administered questionnaire and ACASI. Women were counted as having attended their quarterly visit if they visited during the period from 14 days before, to 73 days after, the scheduled date. Clinicians addressed any medical problems; a pelvic examination and urinalysis or wet mount were done when clinically indicated, and treatment was provided when appropriate. Syphilis testing was repeated at the closing visit or if clinically indicated. HSV2 testing was repeated at the closing visit for women who were negative for HSV2 at enrolment.
Confirmatory laboratory testing was done for women with double-positive or inconsistent HIV rapid results with HIV ELISA. In cases of weakly reactive ELISA results, Western blot (BioRad Laboratories, Hercules, CA, USA) was used to corroborate ELISA test results. All participants were informed of their HIV status after each test. Women who seroconverted were able to continue participation in the study, and were scheduled for follow-up supportive counselling and referred to local psychosocial and clinical services. The study facilitated access to community-based psychosocial support programmes and to national care and antiretro-viral treatment programmes for all participants who were identified as HIV-positive after enrolment.
We selected a sample size of 4500 to provide 90% power to detect an intervention-related decrease in HIV incidence of at least 33%, significant at the 5% level. We assumed that incidence rates at the three sites ranged between 3.5% and 5% per year, that average adherence to diaphragms would be 80% in the intervention group, that the average yearly rate of loss to follow-up would be 7%, and that the maximum length of follow-up would be 24 months. Midway through the study, in June, 2005, before the first scheduled interim analysis, overall incidence of HIV in the first months of the study was noted to be lower than expected; we therefore increased the sample size to 5000.
An independent Data Safety Monitoring Board (DSMB), chosen and convened by the study investigators, was scheduled to meet once, when a third of the expected 300 HIV seroconversions had occurred. Prespecified stopping rules included lack of safety, futility, or efficacy. Statistical monitoring criteria were based on the Lan-Demets group sequential method with O’Brien-Fleming stopping boundaries to preserve the overall type-1 error rate at 5%.37,38
Based on the results of the first interim analysis, the DSMB requested another interim analysis, which was done when two-thirds of expected seroconversions had been reached, with a recommendation to complete the trial as planned.
The primary outcome was incident HIV infection. To confirm that women with HIV infection identified at their first follow-up visit were incident cases, we tested for viral nucleic acid by HIV DNA PCR (Amplicor HIV-1 DNA v1.5, Roche Molecular Systems, Branchburg, NJ, USA) from stored dried blood spot samples (S&S filter paper). At the South African sites, HIV RNA PCR tests (Amplicor HIV-1 MONITOR, v1.5, Roche Molecular Systems) were also done on stored plasma from the enrolment visit. Participants with a positive for HIV DNA PCR, HIV RNA PCR (>400 copies per μL), or both, were classified as having prevalent HIV infection at enrolment, and therefore were excluded from the intention-to-treat (ITT) analysis.
HIV incident infection was defined as time from enrolment to seroconversion, on the basis of a discrete time scale determined by an individual’s quarterly visit schedule. For participants who seroconverted, the time of seroconversion was defined as the time of first positive HIV test result. For cases in which one or more visits were missed in the intervals between the last negative and first positive tests, the time of seroconversion was assumed to be the visit containing the midpoint between these two time points.
To minimise recall bias, we a priori selected product use at last sex as our primary measures of diaphragm use and of condom use in all analyses.39
For confirmatory purposes, we also assessed a measure of cumulative use of the methods since last study visit (always, sometimes, or never).
Selected baseline characteristics were compared between the groups to examine the effect of randomisation. In addition to safety assessments, we did an ITT and subgroup analyses, and a per-protocol analysis. All analyses were pre-specified in an analytical plan finalised before the group assignments were unblinded.
The primary analysis compared observed HIV incidence between the groups using a stratified Cox model for discrete time outcomes, including a binary indicator of group assignment as the only predictor variable, and with separate baseline hazards for each of the three study sites. This analysis was based on an ITT approach. Results of the primary analysis were summarised by the estimated relative hazard comparing HIV incidence in women in the intervention group to that in controls, with associated 95% CIs. We also did additional subgroup analyses to investigate the consistency of ITT results across categories of baseline characteristics selected a priori. Results were summarised by separate relative hazard estimates and 95% CIs for the effect of the intervention at each level of each of the baseline variables.
The per-protocol analysis repeated the between-group comparison of the primary outcome, excluding follow-up periods where participants in the intervention group did not report use of the diaphragm at last sexual contact. Person-time in the intervention group was included in this analysis if participants reported using the diaphragm without the lubricant gel. Person-time in the control group was included even if no condom use was reported, but was excluded if use of a diaphragm was reported.
For women in the intervention group, we assessed the marginal association between condom and diaphragm use, cumulatively across all follow-up visits, with generalised estimating equation (GEE) logistic regression.
Assessment of safety of the intervention was based on a comparison of the proportions of participants who reported reproductive tract-related, urinary tract-related or pregnancy-related adverse events or serious adverse events by study group.