We conducted two parallel trials of male circumcision in HIV-negative and HIV-positive men in Rakai district of southwestern Uganda from 2003–2007. The design and conduct of the trials has been described previously.[3
] In brief, both HIV-infected and uninfected men were informed of the trial objectives and procedures, and consenting men were then screened for eligibility. Criteria for enrollment included being uncircumcised, aged 15–49 years, and having no indications for or contraindication to surgery. Men found to have genital infections or a hemoglobin level ≥ 8 grams/dL were treated and rescreened. All screened participants were offered voluntary HIV counseling and testing, health education on HIV/STI prevention and free condoms.
HIV status at enrollment was assessed by two enzyme immunoassays: Vironostika HIV-1 (Organon Teknika, Charlotte, North Carolina, USA] and Cambridge Biotech [Worcester, Massachusetts, USA). Discordant EIA results were confirmed by Western blot (Calypte Biomedcial Corparation, Rockville, MD, USA).
Because the safety of surgery in HIV-infected men was unknown, we excluded HIV-positive men from enrollment if they had evidence of immunosuppression as indicated by a CD4 cell count below 350 cells/mm3 or WHO clinical stage 3 or 4 disease. All HIV-infected men were referred for management of their HIV disease by the Rakai Health Sciences Program’s HIV care services supported by the President’s Emergency Fund for AIDS Relief. All HIV-infected men received a basic care package of cotrimoxazole prophylaxis, insecticide impregnanted bed nets and hypochlorite water disinfection packets. Men with a CD4 cell count below 250 cells/mm3 or WHO stage 4 disease were advised to initiate antiretroviral therapy which was available free of charge through the Rakai Program.
Eligible participants provided written informed consent for enrollment which described study procedures, potential risks and benefits and the voluntary nature of participation. Men who were randomized to the intervention arm received circumcision within two weeks of enrollment using the sleeve procedure under local anesthesia and were followed postoperatively at 1–2 and 7–9 days and 4–6 weeks. All intervention and control participants were followed at 6, 12 and 24 months. At enrollment and at each follow up visit, participants completed an interview to ascertain sociodemographic characteristics, sexual risk behaviors and symptoms suggestive of STIs or AIDS.
At each visit, clinical officers examined the men’s genitalia recording any abnormality and took a penile swab for HPV detection. Moistened Dacron swabs were taken from the subpreputial cavity of uncircumcised men and from the coronal sulcus/glans of circumcised men, placed in Digene specimen transport medium (STM), and stored at −80° C until assay. HPV genotyping was performed using the Roche HPV Linear Array (Roche Diagnostics, Indianapolis, IN) as previously described [16
]. HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were considered the primary high risk HPV (HR-HPV) carcinogenic viral genotypes. Genotypes 6, 11, 26, 40, 42, 43,53, 54, 55, 61, 67, 70, 71 72, 73, 81 82, 83, 84, and 108 were considered low risk HPV (LR HPV) genotypes. To ensure the adequacy of penile swabs for PCR detection we restricted analyses to samples with amplifiable DNA, defined as swabs with the detection of any high or low risk HPV DNA and/or detectable human beta-globin control amplification. Penile swabs negative for both HPV and the beta-globin internal control were considered to be insufficient for HR-HPV detection and were excluded from HPV analyses. [19
The trial profile is shown in . There were 1151 HIV-infected men identified at screening of whom 922 (80.1%) were enrolled and randomized to the intervention (n = 474) or control (n = 448) arms. The lack of balance between study arms was due to the fact that enrollment and randomization was decentralized in these rural communities and took place at 10 sites. Also, all men (HIV-infected and uninfected) were randomized at time of enrollment men. Thus, imbalances due to chance occurred in the numbers allocated to intervention and control arms. Due to financial constraints and assay costs, we only tested a random sample of HIV-positive participants who provided penile swabs at enrollment and at 24 months follow up. Resources were insufficient to assay samples at the 6 and 12 month visits. There were 103 enrollment samples tested in the intervention arm (21.7% of men enrolled) and 107 in the control arm (23.9% of men enrolled). At 24 months follow up, assay results were available for 93 intervention arm men (90.3% of those tested at enrollment). Ten intervention arm samples were excluded due to insufficient volume or suspected contamination. There were 8 crossovers in the intervention arm, defined as men who failed to accept circumcision by six months post-randomization. Follow up assays were conducted for all 107 control arm men at follow up and there were no control crossovers.
The trials were approved by two IRBs in Uganda (The Scientific and Ethics Committee of the Uganda Virus Research Institute and the Uganda National Council of Research and Technology), and in the United States (The Johns Hopkins University, Bloomberg School of Public Health, Committee for Human Research, and Western Institutional Review Board, Olympia, WA). The trial in HIV-infected men was registered with ClinicalTrials.gov, number NCT00124878
Characteristics, behaviors and HR-HPV infections were assessed by arm of assignment at enrollment. We assessed the efficacy of circumcision for reduction of prevalent HR-HPV using an intention-to-treat analysis, based on two classifications of HR-HPV outcomes: 1) the prevalence of any (one or more HR-HPV infections), and 2) the prevalence of single or multiple (more than one) HR-HPV infections at 24 months follow up. Similar analyses were conducted for low risk HPV infections (LR-HPV). An as treated analysis classified intervention arm crossovers as uncircumcised if they failed to accept surgery by the six month visit. There were no control arm crossovers in this analytic sample. The units of observation were individual study participants. The prevalence risk ratio (PRR) of HR-HPV in the intervention relative to the control arm was estimated by comparing the two binomial proportions.
The incidence of new HR-HPV infections at 24 months was determined among men who were either HR-HPV negative at enrollment, or who were HR-HPV positive for a given genotype but acquired a new HR-HPV genotype at follow up. The denominator for estimation of incidence consisted of men with valid samples at both enrollment and 24 months follow up. Incidence was estimated as the proportion of men with a new HR-HPV infection at the 24 months follow up. We assessed acquisition of any (ie., one or more new HR-HPV infection), as well as single and multiple infections per person. The proportion of new genotype-specific infections was estimated from the number of new genotypes detected at 24 month follow up among men with samples negative for the newly acquired genotypes at enrollment. The incidence rate ratio (IRR) of new HR-HPV infections acquired over 24 months was estimated by Poisson log linear regression. Adjusted IRRs were estimated by multivariable Poisson regression and included covariates for age, marital status, number of sex partners, condom use and alcohol consumption with sex.
The clearance of pre-existing genotype-specific HR-HPV infections over 24 months was estimated from the proportions of prevalent genotype-specific infections detected at enrollment which were not detected at follow up, among samples with amplifiable viral or cellular DNA at both time points. The clearance ratio of any HR-HPV genotype infection was estimated using a log binomial model with robust variance estimates, assuming an exchangeable correlation structure between the multiple clearances observed on the same individual.
Analyses were performed in R 2.8.1 (R Foundation for Statistical Computing: Vienna, Austria, 2007.).