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Studies in women indicate that some sexually transmitted infections promote human papillomavirus (HPV) persistence and carcinogenesis. Little is known about this association in men, therefore we assessed whether Chlamydia trachomatis (CT) infection and herpes simplex virus type 2 (HSV-2) serostatus are associated with genital HPV prevalence, an early event in HPV related pathogenesis.
Genital exfoliated cells, first-void urine and blood from 3,971 men recruited in the USA, Mexico, and Brazil, were tested for HPV, CT, and HSV-2 antibodies, respectively. Multivariable logistic regression was used to assess the association of CT infection and HSV-2 serostatus with four HPV outcomes (any, oncogenic, non-oncogenic only, and multiple infections).
A total of 64 (1.6%) men were CT positive and 811 (20.4%) men were HSV-2 seropositive. After adjustment for potential confounders, CT was associated with any HPV (aOR 2.19, 95%CI: 1.13–4.24), oncogenic HPV (aOR 3.10, 95%CI: 1.53–6.28), and multiple HPV (aOR 3.43, 95%CI: 1.69-6.95) prevalence. HSV-2 serostatus was associated with any HPV (aOR 1.25, 95%CI: 1.02-1.52), non-oncogenic HPV only (aOR 1.38, 95%CI: 1.08-1.75), and multiple HPV (aOR 1.33, 95%CI: 1.06-1.68) prevalence. In analyses stratified by sexual behaviour, CT infection was significantly associated with HPV detection among men reporting ≥2 recent sexual partners, while HSV-2 serostatus was significantly associated with HPV detection in men reporting 0-5 lifetime sexual partners.
In this population, CT infection and HSV-2 serostatus were associated with prevalent genital HPV infection. Future prospective studies should investigate whether these infections influence HPV acquisition and/or persistence.
Human papillomavirus (HPV) is a common sexually transmitted infection (STI) and the primary etiologic agent in the development of anogenital cancers and condylomas (1). Factors shown to enhance progression of infection to cancer in women are smoking, long-term oral contraceptive use, and high parity (2). STIs also have been investigated as possible cofactors because they may directly interact with HPV, or damage the epithelial barrier that helps protect against HPV infection (1-3). Studies in women indicate that Chlamydia trachomatis (CT), assessed by the presence of DNA (4;5), may increase the risk of HPV persistence and that CT (assessed by the presence of DNA (6) and antibodies (7-9)) increase the risk of cervical cancer, although some uncertainty remains (10-12). The association between herpes simplex virus type 2 (HSV-2) (detected by DNA (13;14) or antibodies (15;16)) and cervical cancer remains equivocal. Little is known about the influence of STIs on HPV natural history in men. One prospective study found current CT infection to increase the risk of acquiring an additional HPV type in HPV-positive men (17). Four cross-sectional studies showed that STIs were associated with current HPV infection (18-21), but not all studies adjusted fully for sexual behavior.
In a study among men (22), we examined the association of CT infection and HSV-2 serostatus with four outcomes: any HPV, oncogenic HPV, non-oncogenic HPV only, and multiple HPV infections, after adjustment for confounding factors. Furthermore, we assessed whether such associations remained after stratifying by sexual behavior.
The HPV in Men (HIM) study is an ongoing prospective study of the natural history of HPV infection in men. Participants were recruited from Sao Paulo (Brazil), Cuernavaca (Mexico), and Tampa (United States), from March 2005 through September 2009. Men were eligible for participation if they were 18 to 70 years of age, reported no history of anogenital cancer or anogenital warts, had not participated in an HPV vaccine study, reported no current penile discharge or dysuria, and were not being treated for an STI. A full description of the study methods and procedures has been published previously (22).
Participants provided written informed consent and underwent a clinical examination two weeks (baseline) before the enrollment visit. Participants completed a computer-assisted self-interview questionnaire to determine sociodemographic characteristics, sexual history, condom use practices, tobacco use, self and partner history of STI, and self and partner recent diagnosis of an STI or warts. Using three saline prewetted Dacron swabs, study clinicians collected exfoliated cells from the coronal sulcus, penile shaft, and scrotum to test for HPV infection. Samples were combined into one genital external specimen and stored at -70°C until analysis. Blood samples were collected to test for HSV-2 serostatus and men provided a first-void urine specimen for CT infection testing. Baseline (visit 1) samples and study characteristics were used for analyses.
Detection of HPV DNA was done by means of PCR amplification and genotyping. DNA was extracted with the QIAamp Media MDx (QIAgen, Valencia, CA, USA) by a robotic system according to the manufacturer's instructions. DNA was stored at -20°C until use. 30 ng of DNA was amplified with the PGMY09/11 L1 consensus primer system (23). HPV DNA was detected by running the sample on a 2% agarose gel to visualize a band at 450 bp. The PCR product was used for HPV genotyping and analyzed by using the Linear Array method (Roche Molecular Diagnostics city, CA, USA) (24). HPV genotyping was conducted on all samples regardless of HPV PCR results.
Serum for HSV-2 testing was stored at 4°C and analyzed within 24 hours. Detection of HSV-2 IgG antibodies was conducted with the HerpeSelect2 ELISA IgG kit (Focus Diagnostics Cypress, CA, USA). Samples were considered positive if the OD was >1.1 times above the cut-off calibrator.
Participants' first-void urine samples were collected into urine collection cups free of preservatives. Samples were transferred within 24 hours after collection into the GenProbe specimen transport tube. CT was tested using the Chlamydia LCx assay (Abbott Laboratories, Abbott Park, Chicago, IL, USA). Samples were concentrated (target capture), amplified (Transcription-Mediated Amplification) and detected (Dual Kinetic Assay) according to manufacturer's procedures.
We considered four HPV categories in our analyses: any HPV, oncogenic HPV, non-oncogenic HPV only, and multiple HPV infections. A participant was considered HPV positive if he tested positive by PCR or tested positive for at least one genotype. The oncogenic HPV category included men who were positive for at least 1 of 13 oncogenic types (HVP types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) regardless of co-infection with non-oncogenic types. Non-oncogenic HPV only included single or multiple infections with non-oncogenic HPV types (HPV types 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 66, 67, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39 and CP6108) in the absence of oncogenic infection (25). A participant was considered positive for multiple HPV types if he tested HPV positive for >1 genotype, regardless of type. Samples were considered valid if they were HPV positive by PCR or by genotyping, or β-globin positive, regardless of HPV result. In total, 98% (3,971/4,074) of samples were considered valid. In a sensitivity analysis, similar results were obtained for the full cohort (n=4,074) as for the group with valid HPV samples only (n=3,971).
Pearson's Chi-squared test was used to compare HPV prevalence with CT prevalence and HSV-2 seroprevalence. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using bivariable and multivariable logistic regression analyses. We constructed four separate models, one for each HPV outcome: any HPV, oncogenic HPV, non-oncogenic HPV only, and multiple HPV infections. Men who had no detectable HPV (HPV negative on both PCR and genotyping assays) served as the reference group for all analyses.
Multivariable logistic regression analyses were performed using three different approaches. In approach 1, we used the same set of variables for all models; these were variables that had previously been associated with genital HPV prevalence or acquisition (22;26). This resulted in a list of six possible confounders for the CT model: (1) age (four categories); (2) country of residence; (3) smoking status (never, former, current); (4) education, (5) number of female partners during the past three to six months; and (6) number of male anal sex partners during the past three months. For the HSV-2 model, we also selected possible confounders (1) through (4) and additionally adjusted for (5) number of lifetime female sex partners and (6) number of lifetime male anal sex partners. Recent number of sex partners was used with CT, and lifetime number of sex partners was used with HSV-2, as CT diagnosis indicates current infection, while HSV-2 antibody status reflects cumulative lifetime exposure.
In approach 2, a list of potentially confounding variables that were associated (P<0.25) with each of the four HPV outcomes in bivariable analyses was added to the above named model (e.g. marital status, circumcision status, age of sexual debut, ever diagnosed with an STI, condom use during vaginal or anal sex in the past 3-6 months, and partner diagnosed with genital warts or with an STI during the past 6 months).
In approach 3, all variables associated (P<0.25) with each of the four HPV outcomes in bivariable analyses were added to the main model and dropped one by one based on the likelihood ratio test at (p<0.05), until a parsimonious model was obtained.
To explore whether sexual behavior modified the association of CT infection and HSV-2 serostatus with the HPV outcomes, we performed a stratified analysis in which we divided the study population into three equal-size groups by number of recent sexual partners (RSP) during the past 3-6 months, and by lifetime number of sex partners (LSP), respectively. Stratification was performed by number of partners, regardless of the gender of the partner. Men who refused to answer the number of male or female partners were excluded from the stratified analyses. Additionally, stratified analyses by country were performed.
All statistical tests were two-sided and all analyses were performed using Stata software version 11.2 (Stata Intercooled, College Station, TX, USA).
Participating men had a median age of 31 years (interquartile range: 23-40) and were recruited from Brazil (35%), Mexico (33%), and the United States (32%). Overall, 2,650 (67%) men were positive for any HPV, 1,183 (30%) positive for oncogenic HPV, 898 (23%) positive for non-oncogenic HPV only, and 1,146 (29%) positive for multiple HPV infections.
Population characteristics are presented by CT infection and HSV-2 serostatus in Table 1. In total, 64 (1.6%) men were infected with CT at baseline. CT infection was most common (2.4% CT-infected) in the youngest age group (18-30 years) (P<0.001). CT prevalence was highest in men recruited from Brazil (2.3%) followed by Mexico (1.3%) and the United States (1.2%) (P=0.038). A higher number of recent female sexual partners during the past three to six months (P=0.026), and having a partner diagnosed with genital warts during the past six months (P=0.028) were significantly associated with CT infection.
In total, 811 (20.4%) men had antibodies against HSV-2. HSV-2 seroprevalence increased with age (P<0.001), with men from Brazil having the highest HSV-2 seroprevalence (38.4%). HSV-2 seroprevalence was associated with marital status (P<0.001), education (P<0.001), smoking (P=0.045), and circumcision (P<0.001). HSV-2 seroprevalence was highest in men who reported ≥20 lifetime female sexual partners (P<0.001) and increased with lifetime number of male anal sexual partners (P<0.001). All other sexual behavior variables were associated with HSV-2 serostatus, except condom use during vaginal or anal sex during the past 3-6 months and number of recent female sexual partners.
Table 2 shows the prevalence of HPV infection by CT infection status and HSV-2 serostatus. HPV prevalence was higher in CT-infected men compared to CT-negative men for the following HPV outcomes: any HPV; oncogenic HPV; ≥1 HPV genotype; multiple HPV genotypes; HPV vaccine types; HPV 11, and HPV 18 (P≤0.05). HPV prevalence was higher in HSV-2 seropositive men than in HSV-2 seronegative men for the following HPV outcomes: any HPV; oncogenic HPV; non-oncogenic HPV only; ≥1 HPV genotype; and multiple HPV genotypes (P≤0.05).
Bivariable and multivariable analyses (approach 1) examining associations between the four HPV outcomes and the two main exposures (CT infection and HSV-2 serostatus) are presented in Table 3. In bivariable and multivariable analyses, CT infection was independently associated with any HPV (aOR 2.19, 95%CI: 1.13-4.24), oncogenic HPV (aOR 3.10, 95%CI: 1.53-6.28), and multiple HPV infections (aOR 3.43, 95%CI: 1.69-6.95). In the case of non-oncogenic HPV only, the association was marginally significant in the bivariable analysis (crude OR 2.16, 95%CI: 1.00-4.68, P=0.050) and was not significant in the multivariable analysis (aOR 1.95, 95%CI: 0.88-4.32, P=0.101). HSV-2 seropositivity was significantly associated with any HPV (aOR 1.25, 95%CI: 1.02-1.52), non-oncogenic HPV only (aOR 1.38, 95%CI: 1.08-1.75), and multiple HPV infections (aOR 1.33, 95%CI: 1.06-1.68). However, in the case of oncogenic HPV, HSV-2 serostatus was significantly associated in the bivariable analysis (crude OR 1.52, 95%CI: 1.24-1.85) and marginally significant in multivariable analyses (aOR 1.26, 95%CI: 1.00-1.59, P=0.051).
Multivariable logistic regression analyses were performed in two additional ways (see methods). In approach 2, addition of the covariates mentioned above did not improve the model based on the likelihood ratio test, and did not significantly alter the association between either CT infection and HSV-2 serostatus with the four HPV outcomes by >5%. Using approach 3, the association between multiple HPV infections and HSV-2 seropositivity was not significant (aOR 1.25; 95%CI 0.99-1.57; P=0.056); all other results were comparable.
Stratified analyses for the association between CT infection and the four HPV outcomes are presented in Table 4a. A significant association for all outcomes was found in the group that reported ≥2 RSP, except for the association between CT infection and non-oncogenic HPV only (aOR 3.22, 95%CI: 0.86 - 12.06). Stratified analyses by LSP for the association between HSV-2 serostatus and the four HPV outcomes are shown in Table 4b. HSV-2 serostatus was significantly associated with the four HPV outcomes among men reporting 0-5 LSP, but this association was weaker among men reporting 6-14 LSP, and absent among men reporting ≥15 LSP. The same trend was observed for the three other HPV outcomes.
In additional stratified analyses by country CT was positively associated with HPV in each country; however, the strength of these associations was highest in the US, followed by Mexico, and weakest in Brazil. HSV-2 was positively associated with HPV in each country (data not shown).
This is one of the first studies investigating the association of genital HPV infection with CT infection and HSV-2 serostatus in men. After adjusting for confounding factors, CT infection was positively associated with any HPV, oncogenic HPV, and multiple HPV infections; HSV-2 was positively associated with any HPV, non-oncogenic HPV, and multiple HPV infections. In expanded models adjusting for additional variables generally focused on sexual behavior, ORs for CT and HSV-2 were unchanged and remained significant. Thus, additional correction for sexual behavior did not alter our conclusion, suggesting that in this cross-sectional analysis confounding due to sexual behavior was well controlled.
We measured prevalent genital HPV infection, while HSV-2 seropositivity represented cumulative sexual exposure. The association between two markers reflecting different stages of infection may be questioned. However, as HSV-2 after a primary infection leads to clinical recurrences, HSV-2 seropositive individuals may have frequent reactivations of genital HSV-2 eruptions (54% of the men with genital HSV-2 infection or HSV-2 antibodies had 6 or more recurrences per year) (27).
The association we observed between CT infection and HPV infection may raise some questions as these infections affect different anatomical sites: CT infection is located in the urethra, whereas HPV infection is mostly located at the coronal sulcus, penile shaft, and scrotum. The biological mechanism responsible for an association between these two STIs therefore remains to be elucidated.
Men who reported a current penile discharge or dysuria and men who were being treated for an STI were excluded from participation in this study. Therefore we are only measuring associations between asymptomatic CT and HPV infections. Although this is a limitation, greater than 50% of CT infections in males are asymptomatic (28). This explains why, despite the exclusion criteria, we observed 1.6% of men had a CT infection.
A limitation of the current study is the inability to address temporality. It is unclear whether the STI was transmitted concurrently with HPV, or preceded or followed HPV. In the case of HSV-2, temporality may not be an issue as HSV-2 seroprevalence indicates past infections that may have influenced the current HPV infection. Although HSV-2 assessed with antibodies does not distinguish between oral, anal or genital HSV-2 infection, HSV-2 seropositivity indicates genital herpes in almost all patients (29). Since both CT and HPV infection are current infections, it is challenging to draw conclusions on temporality of these two infections. Another limitation is the relatively low prevalence of each HPV type, therefore it was not possible to investigate CT/HSV-2 associations with individual HPV types.
Our study concerns a well-characterized international cohort of men with a wide age range, a large sample size, with extensive data regarding demographic and sexual behavior characteristics, high rate of specimen adequacy (98%), and comprehensive laboratory ascertainment. As such, the study provides a good starting point to investigate the association of CT infection and HSV-2 serostatus with HPV in more detail.
Our results are in good agreement with previous studies in women (10;30). A prospective study among men from Denmark found CT infection to be a risk factor for acquiring an additional HPV type in HPV-positive men (17). A recent study investigating heterosexual couples in Rwanda found a moderate association between high-risk HPV infection and HSV-2 seropositivity (OR=2.4, 95%CI: 1.2-5.0) in men (19). A Japanese study in men found CT not to be associated with HPV in a bivariable analysis, although in a multivariable analysis STIs were significantly associated with HPV infection (OR=2.41, 95%CI: 1.04-5.61) (20). In a study among male attendees of the STI clinic in the Netherlands, an association between history of Chlamydia and gonorrhea with HPV infection was observed (18). Among Kenyan men, CT infection was significantly independently associated with HPV presence in the glans (aOR=1.87, 95%CI 1.24-2.81) but not the shaft, and HSV-2 serostatus was only marginally independently associated with HPV presence in the glans (aOR=1.25, 95%CI 1.01-2.81) (21).
In stratified analyses regarding CT and HPV infection, the strength of the association increased with increasing number of RSP. Men with ≥2 RSP may be acquiring both STIs simultaneously from their high-risk partners. As the association among men with ≥2 RSP was stronger than in the whole study population this may indicate residual confounding due to sexual behavior. However, it is also possible that CT increases risk of HPV acquisition or persistence through a biological interaction. In an analogy of the hypothesized interaction between CT and HPV at the cervix, CT induces an inflammatory reaction that indirectly may increase HPV persistence through decreases in cell-mediated immunity (3). When stratifying by LSP, the strength of the HSV-2 and HPV association increased with decreasing number of LSP. In the case of HSV-2, the biological interaction in men reporting ≥6 LSP may be obscured by riskier sexual behavior that overwhelms the added risk conferred by HSV-2.
Further prospective studies in men are needed to clarify the role of STIs as a cofactor in HPV natural history, specifically HPV acquisition and persistence. There may be concern that STIs are surrogates for higher-risk behaviors that increase the exposure to HPV. Our stratified analyses for HSV-2 indicate the contrary. Prospective studies in men are needed in which multiple measures of HPV infections and STIs would help differentiate between STIs as true HPV cofactors or as indicators of sexual behavior. Overall, these results show that after correcting for sexual behavior, men infected with CT and men seropositive for HSV-2 are at higher risk for HPV infection.
The authors would like to thank all participants who provided personal information and biological samples for the study, and the HIM Study Teams in the USA (Tampa), Brazil (São Paulo), and Mexico (Cuernavaca). We would also like to thank Susan T. Landry for editorial review. The HIM Study was supported through a grant from the National Cancer Institute, US National Institutes of Health (CA R01CA098803).
This manuscript is in partial fulfillment of C.J. Alberts PhD thesis.
Conflict of Interest: M.F. Schim van der Loeff receives research funding from Merck and Sanofi-Pasteur MSD, A.G. Nyitray receives research funding from Merck, A.R. Giuliano receives research funding from Merck and GSK and is also a consultant to Merck, a member of the Merck HPV Advisory Board and is on the speakers' bureau, L.L. Villa is on the speakers' bureau of Merck and is also a member of its advisory board; other authors of this manuscript declare no commercial or other association that might pose a conflict of interest for the work submitted.