|Home | About | Journals | Submit | Contact Us | Français|
Uncircumcised HIV-negative men aged 15-49 years were randomized to immediate circumcision (n=441) or delayed circumcision (n=399). HPV was detected by Roche HPV Linear Array at enrollment, 6, 12 and 24 months. Incident HR-HPV was estimated in men who acquired a new HR-HPV genotype. HR-HPV clearance was determined in men with prior genotype-specific HR-HPV infections. Rate ratios (RR) and 95% confidence intervals (95%CI) of HR-HPV acquisition were estimated by Poisson multiple regression
Enrollment characteristics were comparable between groups. HR-HPV incidence was 19.7/100 py in the intervention (70/355.8 py) and 29.4/100 py (125/424.8 py) in the control arm (RR=0.67, 95%CI 0.51-0.89, p = 0.006.) The incidence of multiple HR-HPV infections was 6.7/100 py in the intervention and 14.8/100 py in control arm (RR = 0.45, 95%CI 0.28-0.73), but there was no significant effect on single infections (RR=0.89, 95%CI 0.60-1.30). HR-HPV incidence was lower in the intervention arm for all genotypes and demographic/behavioral subgroups. The clearance of pre-existing HR-HPV infections was 215.8/100py (205/95 py) in intervention and 159.1/100py (255/160.25 py) in control arm men (adjRR=1.39, 95%CI 1.17-1.64).
Male circumcision reduces the incidence of multiple HR-HPV infections and increases clearance of HR-HPV infections in HIV-uninfected men.
The trial was registered with ClinicalTrials.gov numbers NCT00425984
Two trials have shown that male circumcision reduces the prevalence of high risk penile human papillomavirus (HR-HPV) infection by approximately 35% in HIV-negative men [1, 2], and several cross-sectional studies have found a lower rate of prevalent penile HPV infection in circumcised HIV-uninfected men [3-7]. However, it is unclear whether circumcision reduces the incidence of HPV acquisition or increases the clearance of preexisting HPV infection. One longitudinal observational study of 285 U.S. men, 25 of whom were circumcised, found no statistically significant difference in the hazards ratio (HR) of acquisition of either high risk HPV (HR-HPV) infections (adj HR = 1.7, 95% 0.6-4.9) or any HPV infection (adj HR = 0.8, 95%CI 0.4-1.9) in circumcised versus uncircumcised men . However, the authors reported a significantly faster rate of HR-HPV clearance (adj HR = 6.5, 95%CI 2.1-19.7). 
We used data from a randomized trial of male circumcision conducted in Uganda to assess whether circumcision affected the acquisition of new HR-HPV infections and clearance of pre-existing infections.
We conducted a trial of male circumcision for prevention of HIV acquisition in initially HIV-uninfected men in Rakai District, Uganda between 2003-06 [2, 8]. The effect of circumcision on HPV was a secondary trial endpoint. The trial design has been previously described.[2, 8] In brief, we enrolled uncircumcised HIV-negative men aged 15-49 years who were randomized to receive immediate circumcision (intervention arm) or circumcision delayed for 24 months (control arm). At enrollment, and at 6, 12 and 24 months follow-up, trial participants provided information on sociodemographic characteristics, sexual risk behaviors and symptoms suggestive of sexually transmitted infections (STIs).
At each study visit penile swabs were collected for HPV detection. Moistened Dacron swabs were taken from the from the coronal sulcus/glans of circumcised men using a standard protocol, placed in Digene specimen transport medium, and stored at −80° C. HPV genotyping was performed using the Roche HPV Linear Array (Roche Diagnostics, Indianapolis, IN) as previously described [9-11]. 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), or 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. Penile swabs without amplifiable cellular or viral DNA were considered to be insufficient for HR-HPV detection. [12, 13] Informative samples with amplifiable DNA were defined as swabs with detectable beta-globin indicating the presence of cellular DNA, or detectable HPV indicating sufficient viral DNA was present even in the absence of detectable beta-globin.
The study was approved by four Institutional Review Boards (IRBs): the Science and Ethics Committee of the Uganda Virus Research Institute, the Uganda National Council for Science and Technology, the Committee for Human Research at Johns Hopkins University, Bloomberg School of Public Health, and the Western Institutional Review Board. The trial was registered with ClinicalTrials.gov numbers NCT00425984
The trial profile is given in Figure 1. The population assessed in this analysis of HR-HPV incidence/clearance was restricted to HIV-negative married men because we planned to assess HPV infections in their female partners, and given assay costs and limited resources, this was the most efficient sampling design to address effects in men and women. We also excluded men with HIV-infected wives because of high rates of HPV infection in HIV-positive women.[14, 15] In addition, we excluded men whose wives were not concurrently enrolled with their husbands, because we would not be able to rigorously study the efficacy of circumcision for prevention of HPV infection in women, unless these female partners’ enrollment status was known. We then randomly selected married men with concurrently enrolled HIV-negative wives who provided samples obtained at enrollment and at 24 months follow-up to determine the effects of circumcision on prevalent HPV infection as previously reported.  There were 835 HIV-negative intervention arm men with HIV-uninfected concurrently enrolled wives, and due to assay cost and resource limitation, we randomly selected 441 (52.8%) of these male participants who provided a penile swab at enrollment. Of these men, seven failed to accept circumcision by the time of the six month visit and, per protocol, were classified as crossovers at that time. In the control arm, there were 803 HIV-negative men with concurrently enrolled HIV-negative wives, of whom we randomly selected 399 (49.7%) who provided a penile swab at enrollment. Three controls became circumcised between the 1 and 2 year visits and were classified as crossovers at that interval. We conducted HPV assays on randomly selected penile samples at the 6 and 12 month follow up visits to further reduce assay costs. There were 330 randomly selected penile samples assayed in intervention arm men at 6 and 12 months. In controls, we assayed 321 randomly selected samples at 6 months and 314 samples at 12 months. Because our original objective was to determine HPV prevalence at 24 months we assayed all available samples at that time point; 440 in the intervention arm and 399 in the control arm.
We assessed participant characteristics at enrollment by study arm. We then determined the proportion of penile swabs with amplifiable cellular or viral DNA by study visit to assess whether detection varied over time between study arms. An intention-to-treat analysis was then used to assess the efficacy of male circumcision for prevention of new HR-HPV infections among participants with amplifiable cellular or viral DNA detected at sequential study visits. An as treated analysis classified the 7 intervention arm crossovers as uncircumcised if they failed to receive surgery by the six month visit, and three control subjects who received circumcision from non-project sources between the 12 and 24 month visits were classified as circumcised for the last follow up interval. Acquisition of HR-HPV was defined as a new infection identified in men who were initially negative for any HR-HPV and who acquired one or two or more new HR-HPV infection(s) during the next follow up, or men who were initially positive for a specific HR-HPV genotype but acquired one or more new HR-HPV genotype(s) during the next follow up. HR-HPV incidence rates per 100 person years (py) were estimated assuming that the new HR-HPV infection was acquired at the mid-point of the sequential follow up interval at which the new infection was detected. Similar analyses were conducted for the low risk HPV (LR-HPV) genotypes. The unit of observation was an individual participant, and each man with an incident HR-HPV infection was counted only once per follow up interval irrespective of whether he acquired a single HR-HPV genotype or multiple HR-HPV genotypes. We separately assessed the incidence of single and multiple (two or more) new HR-HPV genotype-specific infections, again using the individual participant as the unit of observation. We also assessed the incidence of each HR-HPV genotype; for each genotype, the population at-risk was men without that genotype at a prior study visit, irrespective of the co-infection with other HR-HPV genotypes. Acquisition of HR-HPV infection was examined by study arm, stratified by sociodemographic and behavioral covariates at enrollment. We estimated the incidence rate ratio (IRR) and 95% confidence intervals (95%CI) of HR-HPV acquisition in intervention versus control arm cumulatively over 12 and 24 months using a Poisson log linear regression model with offset. Multivariate Poisson models were used to adjust for covariates found to be associated with incident HR-HPV at p<0.20 in univariate analyses.
Clearance of HR-HPV was estimated among men with pre-existing HR-HPV infections who had sequential samples with amplifiable viral or cellular DNA. Clearance was expressed as the proportion of men with pre-existing HR-HPV who were negative for that genotype at a subsequent sequential study visit, and as the rate of clearance per 100 py observation subsequent to detection of a HR-HPV infection. Clearance was assessed for each HR-HPV genotype, irrespective of the number of HR-HPV infections per individual, and genotype-specific clearance rates were summed to provide global estimates. The rate ratio of clearance was estimated using a log-linear model based on an underdispersed Poisson distribution to account for multiple clearance events within individuals. Potential confounders were examined in univariate analyses, and covariates found to be associated at α <0.20, or suspected confounders based on biological reasoning or prior studies were included in multivariate analyses of men with consecutive follow-up samples.
Analyses were performed using Stata™ Version 8.0 (College Station, Texas) and SAS 9.2 (Carry NC).
The intervention and control arm men were comparable with respect to sociodemographic and behavioral characteristics at enrollment (Table 1). The prevalence of HR-HPV at enrollment was 39.1% in the intervention arm and 38.6% in the control arm (p = 0.17).
At enrollment, and prior to circumcision, the proportions of samples with amplifiable cellular DNA were 78.0%% (344/441) in the intervention and 76.7% (306/399) in the control arm. Among the intervention arm men, the proportion of samples with amplifiable cellular DNA declined over time to 63.0% (208/330) at twelve months and 60.8% (240/395) at 24 months (p <0.001). However, there was no decline in the proportion of samples with amplifiable cellular DNA among uncircumcised controls at 12 months (76.8%, 241/314) or 24 months (75.3%, 272/361), and this difference between study arms at 24 months was statistically significant (p<0.001).
The incidence of new HR-HPV infections or genotypes was determined for samples with amplifiable cellular or viral DNA at sequential study visits (Table 2). In an intention to treat analysis over the 24 months follow up, 70 intervention arm men acquired one or more new HR-HPV infections (incidence 19.7/100 py), whereas among controls 125 men acquired one or more HR-HPV infections with an incidence rate of 29.4/100 py (IRR = 0.67, 95%CI 0.50-0.90). During the first year of follow up, the incidence rate was significantly lower in the intervention group (IRR= 0.61, %95CI 0.44-0.85), while during the second year of follow up, the effect was of borderline statistical significance (IRR=0.64, 95%CI 0.38-1.07). HR-HPV incidence declined over time in both study arms, partly because participants with higher risk sexual behaviors acquired infections in the first follow up year, and thus could not contribute to incident infections with the same genotype during the second follow up year. There were no significant differences in HR-HPV incidence between study arms among men who only acquired a single HR-HPV infection (IRR=0.89, 95%CI 0.60-1.30). However, the acquisition of multiple HR-HPV infections over 24 months was markedly lower among men in the intervention arm (6.7/100 py) than in the control arm (14.8/100 py) with an IRR = 0.45 (95%CI 0.28-0.73). In an as treated analysis, the incidence of any HR-HPV infection was 20.6/100 py (72/350) in the intervention arm and 28.6/100 py (123/430.5 py) in the control with an IRR of 0.72 (95%CI 0.54-0.96). The as treated incidence of multiple (two or more) HR_HPV infections was 7.1/100 py (25/350 py) in the circumcised men and 14.4/100 py (62/430.5 py) in the controls with an IRR = 0.50, 95%CI 0.31-0.79).
The incidence of type-specific HR-HPV over 24 months was lower in intervention than control arm men for all high risk genotypes examined (Table 3), and these differences were statistically significant for HR-HPV genotypes 18 (IRR = 0.30, 95%CI 0.10-0.75) and 33 (IRR = 0.17, 95%CI 0.19-0.0.74).
The rates of HR-HPV acquisition were lower in the intervention than the control arm in all sociodemographic and behavioral subgroups (Table 4). Among controls, HR-HPV incidence was higher in younger men and declined with age, but no age-related trend was observed among intervention arm men. The effect of circumcision on HR-HPV acquisition was statistically significant among men reporting high risk sexual behaviors such as non-use of condoms (IRR=0.67, 95%CI 0.46-0.98), and multiple sex partners (IRR=0.56, 95% 0.36-0.87). After adjustment for covariates associated with HR-HPV at enrollment (age, education, condom use, alcohol consumption with sex, and number of sex partners), the adjusted incidence rate ratio of HR-HPV acquisition in circumcised relative uncircumcised men was 0.67 (95%CI 0.50-0.91, p = 0.008.)
We also assessed acquisition of low risk HPV genotypes (LR-HPV). The 24 month incidence of any LR-HPV genotype was 29.9/100 py (102/341.5 py) in the intervention arm and 35.0/100 py (144/411 py) in the control arm (IRR 0.84, 95%CI 0.66-1.10). For single LR-HPV infections, the incidence was 19.3/100py (36/41.5py) in the intervention arm and 17.8/100 py (73/411 py) in the controls (IRR = 1.09, 95%CI 0.78-1.52). However, the incidence of multiple LR-HPV infections was 10.5/100 py (36/341.5py) in the intervention and 17.3/100 py (71/411 py) in the controls (IRR = 0.61, 95%CI 0.41-0.91).
Clearance of HR-HPV infections is shown in Table 5. Clearance rates per 100 py observation were higher in the intervention (215.8/100 py) than the control arm (159.1/100 py), with a RR=1.36 (95%CI 1.13-1.63). The rates of clearance per 100 py were increased for most genotypes and this was statistically significant for types 39, 51 and 58 (Table 7). After adjustment for age, education, number of sex partners and condom use, the ratio of clearance rates in the intervention relative to the control arm was 1.39 (95%CI 1.17-1.64). Among men in the intervention arm, 77.7% (205/264) of pre-existing HR-HPV infections were cleared over the 24 months observation period, whereas clearance was 66.9% (255/381) among the controls (RR = 1.16, 95%CI 1.05-1.28). In multivariate analyses, the adjusted RR of percent clearance was 1.17 (95%CI 1.05-1.31).
Circumcision was associated with a significant reduction in the acquisition of HR-HPV infection with an estimated efficacy of approximately 33% over two years. Moreover, the efficacy was 55% for prevention of multiple HR-HPV infections (Table 2). The protective effect of circumcision was observed for all HR-HPV genotypes (Table 4) and was consistently observed in all sociodemographic and behavioral subgroups (Table 4). These findings are consistent with the observed reduction in the prevalence of HR-HPV in two randomized trials [1, 2] and in several observational studies [3-7]. Therefore, it would appear that circumcision protects men from acquiring new HR-HPV infections. Similar reductions were observed in the acquisition of LR-HPV genotypes in circumcised men.
The rate of clearance of HR-HPV per 100 py was greater among circumcised than uncircumcised men (Table 5) which is consistent with one observational study, but the magnitude of the effect of circumcision in this trial was less than that reported in the observational study (adj HR = 6.5, 95%CI 2.1-19.7). 
The implications of these findings for male-to-female transmission of HR-HPV are unknown but observational studies suggest lower HR-HPV carriage in female partners of circumcised men. [3, 16] Recent studies have also shown a high degree of type-specific concordance between sexual partners within couples, so it is likely that reduced male HR-HPV carriage following circumcision, resulting from lower acquisition and faster clearance, will reduce female HR-HPV infections. We are currently evaluating the efficacy of circumcision for reducing female infections among sexual partners of men enrolled in this trial.
It is plausible that circumcision might reduce HR-HPV acquisition. In uncircumcised men, the foreskin is retracted over the shaft during intercourse and the inner preputial mucosa is exposed to vaginal and cervical fluids. In addition, the intact foreskin is vulnerable to microtears during intercourse which could facilitate viral entry, and the moist subpreputial cavity may provide a favorable environment for HR-HPV survival . Thus, by removing the foreskin, circumcision may both reduce exposure to female genital HR-HPV and reduce access of HPV to epidermal basal cells. Additionally, keratinization of the circumcision scar may reduce the risk of HR-HPV infection. We observed a significant reduction in detection of beta-globin in penile samples from circumcised men, but there was no change in detection of cellular DNA among uncircumcised controls. This suggests that progressive keratinization of surgical scar may reduce the number of basal cells vulnerable to HPV infections over time.
This study has limitations. The analysis was confined to a random sample of married men so we cannot assess efficacy among single men. The six month intervals between visits in the first follow up year and the 12 month interval between visits in the second year were long and we likely missed incident infections which cleared prior to the subsequent follow up visit. In addition, the long follow up intervals did not allow precise estimation of time to clearance of pre-existing infections. We were conservative in our estimates of incidence and clearance which were restricted to sequential samples with amplifiable cellular or viral DNA to ensure the adequacy of sample collection. Since swabs with amplifiable DNA decreased in the intervention relative to control arm, this led to omission of more follow up intervals for circumcised men (11.0%) than for controls (8.0%), which differentially reduced person time for the intervention arm and thus possibly inflated rates of acquisition and clearance for this group.
We collected swabs from the coronal sulcus/glans and the shaft, but only had resources to assay the corona sulcus/glans samples. Therefore, we do not know whether HR-HPV carriage at other penile sites is affected by circumcision. However, the South African trial sampled the urethral meatus and found that circumcision reduced HR-HPV prevalence, suggesting that the effect may not be site specific . Some studies to determine the optimal site for penile HPV detection report a lower prevalence of HPV from swabs of the coronal sulcus/glans compared to the shaft,[12, 13] whereas others found no difference in detection between these two sites. Collection of samples from one site may be a limitation to this study, but since swab procedures were identical in both study arms, this should not bias the estimates of efficacy.
In summary, circumcision significantly reduced the incidence of multiple penile HR-HPV infections, and increased clearance of pre-existing HR-HPV in married HIV-negative men. Thus, male circumcision may potentially reduce exposure of female partners to HR-HPV infection.
The study was primarily supported by a grant (UO1 AI11171-01-02) from the National Institutes of Allergy and Infectious Disease (NIAD), Division of AIDS, National Institutes of Health (NIH), and in part by the Division of Intramural Research, NIAID, NIH. This publication was supported, in part, by a fellowship/grant from the Fogarty International Center/USNIH: Grant # 2 D 43 TW000010-19-AITRP. We also are grateful for the advice provided by the Rakai Community Advisory Board. Finally, we wish to express our gratitude to study participants whose commitment and cooperation made the study possible.
All authors declare that there is no conflict of interest
The authors have no commercial or other associations that might pose a conflict of interest