The Institutional Review Board of University of Washington, Kenya Medical Research Institute, and University of Nairobi approved the protocol. The trial was registered with clinicaltrials.gov, registration number NCT00542074.
Between June 2003-June 2005 at the Nairobi City Council STI Special Treatment Clinic (STC), four satellite City Council clinics, and a faith-based health facility, women with vaginal symptoms, or whose partner had urogenital symptoms, were referred toether with their male sex partners to the study, together or within 24 hours of each other. Study clinic visits took place at the STC.
On day 0, both partners independently gave written informed consent for participation, received HIV counseling and testing, and underwent face-to-face interviews regarding demographic, sexual and behavioral characteristics, and symptoms. Female participants underwent physical examination, including pelvic examination and collection of vaginal swabs for determination of vaginal pH and detection of volatile amines, using the QuickVue Advance pH & Amine test®
(Quidel, San Diego, CA), diagnosis of BV using Nugent’s criteria [38
], detection of Trichomonas vaginalis
by wet mount examination and by culture (InPouch®
, Biomed Diagnostics, Inc., White City, OR); and endocervical specimens for N. gonorrhoeae
and C. trachomatis
DNA detection by polymerase chain reaction amplification (PCR) (Amplicor, Roche Diagnostics Inc., NJ). Men underwent genital examination and urethral swab for T. vaginalis
culture and N. gonorrhoeae
and C. trachomatis
PCR. Participants provided blood for HIV-1 serologic screening (Detect HIV-1, Biochem ImmunoSystems, Inc, Montreal, Canada), with positive tests confirmed by ELISA (Recombigen, Cambridge Biotech LTD, Ireland), and for syphilis serodiagnosis by Rapid Plasma Reagin (RPR, Beckton-Dickinson, Baltimore, MD), confirmed by Treponema pallidum
hemaglutination assay (TPHA, Biotech Laboratories, UK).
BV diagnosis was based upon Nugent score ≥ 7 determined by a laboratory technologist; every fifth smear was reviewed, re-scored and discussed at a weekly meeting of four laboratory technologists, blinded to initial interpretations. Scores were compared (kappa = .90), discussed, and a final consensus score determined. These smears were photographed in at least 8 fields and sent to a blinded microbiologist (CS), who independently scored the slides. The laboratory technologists’ consensus diagnosis of BV had high concordance with that of the microbiologist (kappa = 0.87). All technicians were blinded to randomization assignment for all specimens.
Participants with BV received metronidazole 400 mg TID for 7 days and became eligible for randomization. All couples received STI prevention counseling, free condoms, and STD syndromic management per Kenya Ministry of Health guidelines as indicated. Using a computer-generated random sequence, the study statistician placed single sheets of paper labeled “Intervention” or “Control” into a series of sealed opaque envelopes, kept in a locked cabinet by the study coordinator. Once a couple was deemed willing and eligible, the study nurse consecutively obtained the next envelope from the study coordinator, and provided it to participants, who opened it themselves.
Randomized couples participated in a discussion on improved genital hygiene, and were advised to bathe daily. Men in the intervention arm were requested to use 62% ethanol gel (Purell®, GOJO Healthcare Inc., Akron, OH) at least once daily, and immediately prior to and following intercourse, squeezing a shilling-sized (size of a US quarter) amount of gel on their palm, rubbing it gently onto the palms of both hands, and applying a second similar-sized amount to the genitalia from the tip of the penis towards the base. Uncircumcised men were instructed to retract the foreskin before gel application. We provided gel in pocket-sized 4.25 fluid ounce plastic squeeze bottles and encouraged men to carry it with them at all times. Participants received a brochure concerning STI control and hygienic practices, and (for the intervention arm) information on use of the study product. Participants received diaries to record when (s)he had sex, bathed, and, in the intervention arm, when the male applied gel. Neither participants nor those administering the intervention were blinded to group assignment. The men began the trial immediately following the enrollment visit. Women were advised to abstain for the first week of treatment or to use condoms until they returned to clinic for the one week visit.
Upon completing enrollment visits, community health workers (CHW) escorted couples home to verify locator information, and visited each couple again on days 14 and 45, to encourage continued participation, monitor side effects, and review and replenish study diaries. Female articipants were asked to return one week, one month, and two months after enrollment. Male partners were encouraged to return for follow-up visits but were not required for female participation. Intervention and control participants were seen for follow-up visits on different days to prevent cross-arm communication. Each follow-up included interview and examination; women underwent syndromic management for STI-related symptoms, and vaginal swabbing for determining Nugent’s score. Men also underwent interview, examination, and syndromic management for STI-related symptoms. At the one-week visit, couples received post-test counseling, treatment for additional STI detected, and referral to support groups and/or care for those HIV-infected. After the one-week visit, only the woman was required to return, bringing diaries for herself and her partners. Because men were instructed to wait until the gel had evaporated before starting sex, womens’ motivation to douche to remove gel was expected to be minimal and questionnaires did not include information on douching or other intravaginal practices.
At the two-month follow-up, vaginal swabs were taken for isolation of lactobacilli and transported anaerobically (Anaerobe Systems, San Jose, Calif.), emulsified in pre-reduced 1% yeast extract broth for inoculation onto Brucella medium enriched with vitamin K and hemin, Columbia Agar containing colistin and nalidixic acid (CNA), and Rogosa agar. Cultures were incubated anaerobically for 5 days at 35°C. Growth of each organism type was recorded semi-quantitatively. Each colony type found on any of the three plates was Gram stained, those appearing consistent with lactobacillus underwent additional tests. Lactobacilli were alpha hemolytic, catalase-negative, produced acid in both slant and butt of triple sugar iron agar, and did not grow on bile esculin agar. Strict anaerobes were identified as nonsporeforming Gram-positive rods, negative for indole and nitrate production, and then identified to species level using simple enzymatic tests; (WeeTabs, Key Scientific, Round Rock, TX). For isolates confirmed as lactobacillus, H2O2 production was further determined (Protects, Key Scientific).
Before classification as lost-to-follow-up, participants not returning for study visits were sought three times at home by the CHW. When participants declined further study participation, reasons for discontinuation were elicited.
The estimated target sample size (364 couples), providing 80% power to detect a 33% reduction in recurrence (50% vs. 33.3%) with alpha=0.05 (2-sided), after 20% loss-to-follow-up. However, prolonged transportation strikes in Nairobi limited enrollment to 223.
The primary analysis was comparative time to first BV (Nugent score ≥ 7) by randomization arm, using Wilcoxon’s log rank test. Cox regressions were used to estimate hazard ratios (HRs) for the intervention effect, and to estimate the intervention effect, before and after adjusting for baseline characteristics differing between study arms. Time-varying covariates were used to examine a dose-response relationship between gel use and time to BV, and post-randomization explanatory variables (e.g., reported sexual practices during follow-up).
Because women received syndromic treatment for STI-related symptoms, including vaginal symptoms, at follow-up visits, and because Nugent scores were not concurrently available to clinicians during the visit, some symptomatic women with Nugent score < 7 received metronidazole. Since this could bias comparisons between arms, another analyses compared time to failure defined as either Nugent score ≥ 7 or receipt of treatment with metronidazole during follow-up, by randomization arm. Analyses were intent-to-treat; all couples returning for any follow-up were included, and analyzed according to randomization group. Analyses employed SAS version 9 (Cary, NC) and Stata version 9 (College Station, TX).