Herpes simples virus (HSV) type 1 and type 2 have significant impact at both the individual and public health levels.1,2 HSV-2 is the predominant cause of genital ulcer disease worldwide3 and although HSV-1 predominantly causes orolabial disease, it also contributes significantly to HSV-related genital ulcer disease.4–7 In the United States, the prevalence of HSV-1 and HSV-2 are 58% and 17%, respectively.8 Despite the high prevalence of HSV infection, most individuals are unaware that they are infected or at risk for HSV infection. This is because a significant proportion of infected persons remain asymptomatic. In those with symptomatic disease, the cutaneous manifestations of HSV are often not recognized as genital herpes. 9 Finally, asymptomatic shedding of HSV from the oral cavity and genitalia is well established,9–13 and the majority of cases of HSV transmission occur as a consequence of asymptomatic HSV shedding.14 Because herpes is a life-long infection that is associated with physical and psychological distress, and a risk factor for acquisition of other STIs including HIV, it is important to identify measures that prevent primary genital herpes infection.
Circumcision is protective against several STIs, including HIV. However, the role of male circumcision in protection against genital herpes infection remains controversial. To determine whether circumcision is associated with lower HSV prevalence of herpes infection and/or herpes disease, we evaluated HSV-1 and HSV-2 seroprevalence, clinical disease, and asymptomatic HSV shedding in a group of black heterosexual men.
MATERIALS AND METHODS
A retrospective analysis was performed on prospectively collected data from a sample of men attending the Jefferson County Department of Health Sexually Transmitted Diseases (STD) Clinic, Birmingham, AL. Study enrollment and data collection from this sample have been reported previously.15 Briefly, self-identified heterosexual men, presenting to clinic for perceived STI symptoms, as a sexual contact to someone with a diagnosed STI, or for STI screening were approached for study enrollment. Human experimentation guidelines from the US Department of Health and Human Services and the University of Alabama at Birmingham were followed in the conduct of this study. The study was supported in part by a grant from GlaxoSmithKline, however, GlaxoSmithKline did not participate in the study design, analyses, or manuscript preparation. Participants were given a description of the study goals and procedures then asked to give written informed consent. Each consenting participant completed a standardized questionnaire gathering demographic, STI, and sexual partner history. Circumcision status was established by physical examination. If present, genital lesions were identified and characterized (i.e., ulcer, scale, vesicle). Serum was collected for HSV-1 and HSV-2 antibodies. Individual swab specimens were collected for herpes culture and HSV PCR from the urethra, shaft, coronal sulcus, glans, and foreskin (or foreskin remnant), and, if present, genital lesion(s). Participants were included in the current analysis only if they identified themselves as black. All Caucasian men and men of other minorities were excluded from the study. Participants were excluded if circumcision status, HSV-1 or HSV-2 serologies were not reported.
PCR for HSV was performed on pooled aliquots of all specimens collected from each participant. Because of small numbers of positive samples, data are presented for pooled samples only. Methods of DNA extraction, DNA amplification, and identification of amplified products have been described elsewhere.15 Briefly, after DNA amplification PCR primers to the polymerase (pol) marker were initially used.16 Samples positive for HSV DNA using the pol primer were confirmed using a second primer to glycoprotein B. When samples were sequentially positive, viral type was identified by exposing the pol PCR product to the Cfo restriction enzyme obtained from Clostridium formicoaceticum. This restriction enzyme recognizes a 4 base sequence, GCGC, which appears in the polymerase amplicon of HSV-1 once and twice in the amplicon of HSV-2. When run on an agarose gel the pol product yields 2 bands for HSV-1 and 3 bands for HSV-2, allowing identification of specific virus type.
HerpeSelect 1 ELISA IgG and HerpeSelect 2 ELISA IgG assays (Focus Diagnostics, Cypress, CA) were used to determine HSV-1 and HSV-2 status, respectively. As per the manufacturer's protocol, optical density (OD) values ≥1.1 were considered positive. OD values <0.9 were considered to be negative. Indeterminate samples (0.9–1.1) were repeated. If still indeterminate, the specimen was considered negative. If the OD value on repeat did not fall in the indeterminate range, the second value was used to assign serological status.
Data Management and Statistical Analysis
Data were entered into a Microsoft Access database as previously described.15 Analyses were performed using Stata (Stata Corp, Release 8.0, College Station, TX). The relationship of circumcision status to HSV seroprevalence and to demographical and behavioral variables was assessed univariately using the Fisher exact, chi square, or Mann-Whitney U tests as appropriate. Then, this relationship was assessed by multivariate modeling (logistic regression) with the outcome being circumcision status and the independent variables being HSV seroprevalence and age (both included a priori) and other variables with P < 0.1 on univariate analysis. To further address the influence of age on the relationship of circumcision status to HSV seroprevalence, separate multivariate models were performed stratified by age groups (18–25 vs. 26–35 vs. 36–45 years of age in the initial stratified model, then 18–25 vs. 26–45 years of age as described in the results). Descriptive statistics of the relationship of circumcision status to clinical disease and asymptomatic shedding are presented, but smaller numbers of observations precluded meaningful statistical analyses.
Characteristics of Study Participants
Of 529 men enrolled, we excluded 69 (13%) from further analysis 51 (10%) self-identified as white or other and 18 (3%) lacked HSV serologies or report of circumcision status by physical examination. A total of 460 black men were included in the final analysis of which 335 (73%) were circumcised.
Table 1 presents participant characteristics by circumcision status. Uncircumcised men were older than circumcised men (median age: 30 vs. 24 years; P < 0.001). In addition, uncircumcised men were further from sexual debut (median: 15 vs. 9 years; P < 0.001). They reported more lifetime sexual partners (median: 20 vs. 15 partners; P = 0.058). Uncircumcised black men were also more likely to report a history of a sexually transmitted disease (P = 0.02). They reported more genital ulcer disease overall (P = 0.031), more syphilis (P = 0.047), but no more genital HSV than circumcised men (P = 0.45). After adjusting for age, there were no significant differences between circumcised and uncircumcised black men about the fore-mentioned variables, suggesting that age rather than circumcision status may account for the difference observed between groups.
Circumcision, Age, and HSV Seroprevalence
Over 80% of men aged 18 to 25 years were circumcised. Circumcision frequency declined in each successive age group with <35% of men >45 years of age being circumcised (Fig. 1A). Overall, HSV-1 and HSV-2 seroprevalence increased with each successive age group. For HSV-1, 53% of men 18 to 25 years of age were seropositive and seroprevalence increased with age, peaking in those 36 to 45 years of age (75%). HSV-2 prevalence followed a similar pattern with 32% of the youngest age group being seropositive and those 36 to 45 years of age demonstrating the highest seroprevalence (71%) (Fig. 1B).
Thus, in this sample of black men, circumcision status is closely linked to age and herpes prevalence is higher in older participants. To address age as a potential confounder, we evaluated the relationship between HSV infection and circumcision between men in the same age groups (18–25, 26–35, and 36–45 years of age: men over 45 years of age were excluded from further analyses because of the limited number of participants in this age group). In the 18 to 25 year age group, HSV-1 seroprevalence was higher in the uncircumcised versus circumcised men (73% vs. 48%) (Fig. 2A). In contrast, in the 26 to 35 and 36 to 45 year age groups, there was no real difference in the HSV-1 seroprevalence by circumcision status: the seroprevalence already high (about 70%) in both age groups irrespective of circumcision. For HSV-2, there was no difference in seroprevalence by circumcision status in the 18 to 25 year age group (30% and 33% in uncircumcised and circumcised men, respectively). In the older age groups, uncircumcised men consistently demonstrated a slightly higher HSV-2 seroprevalence than circumcised men although differences were not significant (68% and 80% vs. 57% and 63% in men aged 26–35 and 36–45 years, respectively) (Fig. 2B).
To further evaluate our observation that younger, uncircumcised men are more likely than circumcised men to be HSV-1 infected, both circumcised and uncircumcised men were stratified into 2 age groups, those ≤25 years and those ≥26 years. On univariate analyses, uncircumcised men ≤25 years were more likely to be HSV-1 seropositive than circumcised men in this age group (OR: 2.83, CI: 1.38–5.83; data not shown), but were no more likely than circumcised men to be HSV-2 seropositive. In contrast, in men ≥26 years, uncircumcised men were no more likely than circumcised men to be HSV-1 seropositive. Although not significant, uncircumcised men ≥26 years of age were more likely to be HSV-2 seropositive when compared to circumcised men in the same age group (OR: 1.71, CI: 0.94–3.41; data not shown). When adjusting for age, years since sexual debut, number of lifetime sex partners, and history of genital ulcer disease, uncircumcised men ≤25 years of age had a significantly higher HSV-1 seroprevalence (OR: 2.69, CI: −1.27–5.71) (Table 2).
Circumcision and HSV Shedding
Forty-two (9%) of participants were found to have HSV shedding by PCR: 30 (33%) of 92 men with observed genital lesions and 12 (4%) of 341 participants without lesions. The number of participants with HSV shedding was too small to permit meaningful statistical analyses. The frequency of genital lesions was similar in both circumcised and uncircumcised men as was HSV shedding from their lesions. Interestingly, asymptomatic shedding was observed in 6% of circumcised men and in none of the 81 uncircumcised participants (Table 3).
Removal of foreskin tissue from the male penis results in epithelial changes and facilitates drying of penile skin surfaces.17 These changes have been suggested to modify the efficiency of STI transmission. Recent studies have clearly shown that male circumcision is protective against HIV. Three randomized controlled trials in Africa have demonstrated that male circumcision leads to a 53% to 60% risk reduction of female-to-male transmission of HIV.18–20 Similar observations have been reported in the US.21 The WHO now recommends male circumcision as an important strategy to reduce the risk of HIV acquisition in heterosexual men in HIV endemic regions of the world.22
Male circumcision has also been shown to modify the natural history and complications of human papillomavirus (HPV) infection. Observational studies report that male circumcision reduces HPV prevalence and is associated with a lower prevalence of penile cancer.23–25 Importantly, male circumcision has also been associated with reduced rates of cervical cancer among circumcised males' female partners.26 Uncircumcised men are more likely to harbor HPV DNA than circumcised men at specific genital sites (i.e., glans, shaft, and foreskin/foreskin remnant),27,28 and in a recent randomized controlled trial, a reduction in the prevalence of high risk HPV infection was reported after circumcision.29 This reduction in HPV is thought to be related to a reduction in clearance of HPV virus from the uncircumcised penis.30
The relationship between circumcision status and genital herpes is less clear. Although male circumcision has been suggested to be protective against HSV infection, the validity of these findings has been called into question. In older studies, herpes diagnosis was established only by patient report, physical examination, or viral culture.31–33 More recent studies, using serological testing to define HSV infection, suggest little or no association between circumcision and herpes infection in the United States.34 Interestingly, however, a recent prospective study looking at incidence of herpes infection based on circumcision status did show a higher incidence of herpes infection in uncircumcised men.35 As well, a randomized-controlled trial in Uganda recently showed that male circumcision did significantly reduce HSV-2 infection with a cumulative probability of HSV-2 infection of 7.8% versus 10.3% in circumcised versus uncircumcised men, respectively.36 In our study, we found that age is a potentially important confounder in evaluation of the relationship between genital herpes and circumcision. Circumcision rates were lower among black older men, a group who had higher HSV prevalence, more lifetime sexual partners, and more years at risk for infection. While we did not see a statistically significant difference in HSV-2 seroprevalence when participating men were stratified by circumcision status, we did note, after additionally stratifying into age groups that there was an age-related trend towards higher HSV-2 seroprevalence in uncircumcised versus circumcised men in the older age groups. Interestingly, we did observe a significantly higher HSV-1 prevalence in uncircumcised men, specifically men ≤25 years of age.
The potential for age-related changes in circumcision status is not unique to our study. Nationally, circumcision rates have changed over time with higher rates in younger men. While the difference in circumcision status by age is modest overall, among black men, circumcision prevalence has increased from 50% to 90% during the last 50 years.34 A product of age, the uncircumcised men will report more years of sexual activity and more lifetime sexual partners. This translates into more exposures to HSV-1 and HSV-2 and a higher HSV seroprevalence.
A further potential obstacle in understanding the association between male circumcision and herpes infection is changes in sexual practices over time. Oral-genital exposure has become more prevalent among adolescent and young adults than in the past.37 Furthermore, while traditionally most HSV-1 infections were acquired relatively early in childhood, more recently age-related HSV-1 acquisition rates have declined. The intersection of these 2 points creates a circumstance in which HSV-1 now causes an increasing proportion of first episode genital herpes disease.3 Although the majority of participants in the current study were HSV-1 seropositive, with a large proportion expected to have acquired HSV-1 during childhood, there were differences in prevalence in young circumcised versus uncircumcised men, the group more likely to engage in oral-genital sex. Because seroprevalence of HSV-1 was significantly lower in circumcised men, male circumcision may be protective against or delay acquisition of HSV-1 infection.
A major limitation of the current study is the small sample size of uncircumcised men. This may have interfered with our ability to identify an association between HSV-2 infection and male circumcision. As fore-mentioned, Tobian et al, recently demonstrated a modest but significant reduction in HSV-2 incidence after male circumcision.36 In the current study, older uncircumcised men had a higher HSV-2 seroprevalence than circumcised men of similar age. With a larger population, this “trend” may have reached statistical significance, supporting these recent findings. The current study is also limited by other factors. Data regarding sexual behavior and STI history were collected from patient interview, and thus are subject to the inherent errors associated with recall. HerpeSelect was used to make the diagnosis of herpes infection with on OD of 1.1 considered positive. Although this OD was the manufacturer-recommended cut-off for positive results at the time this study was performed, this OD may be too low, increasing the likelihood of false positives.38,39 Although this could bias results in populations where HSV seroprevalence is low, in high seroprevalence populations, such as ours, the effect, although present, is likely reduced.
Although it is well-established that nearly all participants who are serologically HSV-2 positive were infected through sexual contact, HSV-1 infection is more complex with both oral and genital exposures resulting in infection. The proportion of the study population with a history of herpes labialis is unknown. We did no ascertain the proportion of participants receiving oral sex. The association between circumcision status and HSV-1 seroprevalence may therefore be related to higher levels of herpes labialis acquisition and/or higher rates of receipt of fellatio in uncircumcised men. It seems unlikely that circumcision status would affect either of these variables but further work is needed to address this.
Although our study was too small to satisfactorily evaluate the relationship between circumcision status and HSV shedding, it provides observations worthy of further study. While genital ulcers were observed slightly more often in uncircumcised men, the proportion of ulcers in which HSV was detected was similar. Asymptomatic HSV shedding from the glans penis and foreskin were only detected in circumcised men. As recently shown by Mark et al, HSV reactivation with viral shedding occurs often and is rapidly cleared.40 By sampling only a single time point, we may have missed a significant proportion of episodes of HSV shedding in both the circumcised and uncircumcised population. It is unclear whether uncircumcised men are less likely to shed HSV or are more difficult to collect sufficient genital specimens from. Alternatively, asymptomatic shedding may simply have been missed more of the time in this population by collecting samples from only 1 time point. Further research is warranted to understand how male circumcision influences symptomatic HSV genital ulcer disease and asymptomatic HSV shedding.
Despite these limitations, the current study does suggest an age-related relationship between HSV-1 and HSV-2 seroprevalence and circumcision supporting the need to account for age carefully in future studies of this relationship. It also suggests an important and previously unrecognized association with HSV-1 seroprevalence and circumcision. Future work is necessary to verify these findings and to assess whether male circumcision offers a clinical benefit for HSV-1 and HSV-2 genital disease, represented by a reduction in genital ulcer disease or asymptomatic shedding.
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