Multiple-Type Human Papillomavirus Infection in Male Anogenital Sites: Prevalence and Associated Factors

Human papillomavirus (HPV) is the necessary cause of cervical cancer and is the most common sexually transmitted infection. Although HPV infections may clear spontaneously, persistent infection with oncogenic HPV is the strongest risk factor for cervical neoplasia (1). With >40 different HPV types known to infect the anogenital tract, concurrent infection with multiple HPV types is common. A large population-based study found that 20% to 30% of HPV-positive women had multiple HPV types (2), and 12.3% of all women in another cohort were infected with multiple types over a period of 1 year (3). A recent study of women showed a greater prevalence of multiple high-risk types among women ages 20 to 29 years (10.5%) than among women ages 40 to 59 years (0.9%; ref. 4). We reported previously that 27.4% of participants in the HPV Detection in Men study had more than one HPV type detected across multiple sites sampled (5).

Reports of the effects of HPV-type coinfection among women have been inconsistent but have included a greater risk of persistent infection, acquisition of additional HPV types, and cytologic abnormalities (2, 3, 6-8). Among men, multiple HPV infection at baseline has been associated with HPV persistence (9, 10). Therefore, coinfection with multiple HPV types may be an important risk factor for HPV-related cancers.

In the current study, we examined the prevalence of multiple oncogenic and nononcogenic HPV types among men in the cross-sectional HPV Detection in Men study within each anogenital site sampled and the associations between multiple HPV and potential risk factors.

Study design, clinical sampling, and HPV testing have been described previously in detail (5). Briefly, a cross-sectional study of HPV in 463 men was completed. Men were eligible if they (a) were between ages 18 and 40 years, (b) had had sexual intercourse with a woman within the past year, (c) acknowledged no previous diagnosis of genital warts or penile or anal cancer, (d) had no current penile discharge or pain during urination, and (e) had no current diagnosis of a sexually transmitted disease (STD). Primary methods of recruitment were through advertisements in city and university newspapers, flyers in public places, and in-person recruitment at the local air force base and the county health department STD clinic.

All participants gave written informed consent, and all procedures were approved by the University of Arizona Human Subjects Protection Program, Centers for Disease Control and Prevention Institutional Review Board, U.S. Department of Defense, and the University of South Florida Institutional Review Board.

Men collected a semen sample by masturbation 12 to 36 h before the clinical visit. The clinician used a calcium alginate or Dacron urethral swab to sample the first 2 cm of the urethral epithelium. She sampled the following sites by rubbing separate saline-wetted Dacron swabs to sample the entire surface of the (a) glans penis/coronal sulcus, (b) penile shaft (including prepuce, if present), (c) scrotum, and (d) perianal area. The anal canal was sampled with another saline-wetted Dacron swab. Participants completed a self-administered questionnaire that included questions on demographic factors (race, ethnicity, age, income, occupation, education, country of origin, and length of U.S. residency), alcohol and tobacco use, age at first sexual intercourse, lifetime number of female sex partners, frequency of sexual intercourse, ever having had sex with a man or having been diagnosed with a STD, and condom use during the past 3 months.

HPV testing of swabbed cellular material and semen was conducted using PCR for amplification of a fragment of the L1 gene (11). HPV genotyping was conducted using the reverse line blot method on all samples regardless of HPV PCR result (12). This detection method uses the HPV L1 consensus PCR products labeled with biotin to detect 37 HPV types. HPV detection assays were run on each sampling site and in the semen specimen separately.

The oncogenic HPV types associated with cervical dysplasia and cancer include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 66. The nononcogenic types detected with the line blot methodology are 6, 11, 26, 40, 42, 53 to 55, 61, 62, 64, 67 to 73, 81 to 84, IS39, and CP6108. The absence of HPV DNA was defined as a negative result by genotyping in a sample positive for human β-globin. Samples without detection of β-globin or HPV were deemed inadequate for evaluation and treated as missing. Between 93.7% and 95.7% of the external genital site samples and anal samples were adequate, whereas 83.7% of urethral samples and 99.7% of semen were adequate. A “multiple-type” variable was created such that, at each sampling site, results were categorized as having 0, 1, or ≥2 oncogenic types and 0, 1, or ≥2 nononcogenic types. A participant was considered to have “multiple-type” oncogenic infection if ≥2 oncogenic types were detected at any of the sites that were sampled. Multiple nononcogenic HPV infection was similarly defined. Multiple “any type” infection was defined as having ≥2 types (whether oncogenic or nononcogenic) detected at any site. “Unclassified” HPV types were not included in these analyses.

The frequencies and means of the responses to the questionnaire items and the results from the clinical examination were calculated. These calculations were compared between men who had no HPV infection at any site or specimen to those who had one type of HPV detected and those who had more than one HPV type detected. ANOVA was used to compare continuous measures and a χ² test for categorical variables, with a P for trend calculated for those categories that were ordinal. The frequencies of men who had the HPV types included in current prophylactic vaccines (6, 11, 16, or 18) alone or in combination with other types were also calculated.

Multinomial logistic regression was used to model the association of potential risk factors on multiple-type HPV detection. These factors included current smoking status and smoking history, lifetime number of female sex partners, different female partners in the past 3 months, frequency of sexual intercourse in the past 3 months and in the past 1 month, frequency of condom use with vaginal sex in the past 3 months, circumcision status, history of various STD diagnoses, and detection of genital warts during the clinical sampling visit. Odds ratios (OR) were first calculated using multinomial logistic regression for each potential risk factor adjusting for date of laboratory analysis. Next, independent factors were determined by construction of a multivariable model using backward selection logistic regression modeling, starting with the factors that had statistically significant (P < 0.10) ORs with either level of outcome (multiple or single HPV-type infection) in the previous step. Finally, factors that were independently associated with either multiple oncogenic or nononcogenic HPV detection were included in three multivariable multinomial models with (a) oncogenic, (b) nononcogenic, and (c) any type outcome. Age and date of laboratory analysis were included in all multivariable models. Analyses were conducted using Intercooled Stata 9.1 for Windows (StataCorp).

A complete description of the study population and the HPV types detected by anatomic site have been reported (5). Men were ages between 18 and 40 years, with 49.9% ages <25 years. Most (77.5%) were recruited from Tucson and 22.5% were recruited from Tampa. Most participants were White in both Tucson (72.7%) and Tampa (60.6%). Most participants (70.6%) were single, never married, and the majority (61.4%) of men reported more than five female sexual partners in their lifetime. Sixty-five percent of men had at least one type of HPV detected (including “unclassified” types), with the penile shaft, glans/corona, and scrotum having the greatest proportion of positive results (5).

Approximately 23% of men had multiple HPV types detected in at least one sampling site/specimen, whereas 42.6% had a single HPV type detected (Table 1). Multiple infection with nononcogenic types occurred in a greater proportion of men (13.4%) than did multiple oncogenic infection (8.6%). Each of the four quadrivalent vaccine types was detected approximately equally as often as a single type as they were with other types (Table 1). For example, HPV-16 was the only type found for 6.2% of men and was detected with other types in 4.8% of men. At each site, the majority of samples with multiple types had two or three types detected, although as many as seven types were detected in one shaft sample. For example, in the glans/corona sample, 57 (12.8%) men had multiple types: 35 (61.4% of 57) of these contained two types and 10 (17.5% of 57) contained three HPV types (data not shown). Fewer than 10 samples (2%) at each sampling site had more than three HPV types detected.

Multiple HPV was found more often at the glans/corona, shaft, and scrotum than at the other sampling sites. With 18.7% of shaft samples positive for more than one type, this site had the greatest proportion of multiple types (6.5% of shaft samples were positive for multiple oncogenic types and 10.2% were positive for multiple nononcogenic types). The glans/corona had a similar proportion of samples with multiple types (12.8%), whereas 7.3% of scrotum samples had multiple types (Table 2). Perianal and anal canal samples had lower proportions of multiple types (2.8% and 2.1%, respectively) and <1% of urethra and semen samples had multiple HPV types.

HPV-16 and -18 co-occurred in only one man (at the penile shaft). No participant had HPV-6 and -11 detected simultaneously. At least one HPV type included in the quadrivalent HPV vaccine was detected in 17.3% of men overall. Among men with multiple infections, 45.2% had infection with a vaccine HPV type. Of men with HPV-6, -11, -16, or -18 infections, 7 (58.3%), 2 (100%), 36 (70.6%), and 6 (66.7%), respectively, had at least one other type detected. However, only two men had two HPV vaccine types detected, one had three types, and no one had all four quadrivalent vaccine types detected.

In bivariate analyses, with “no oncogenic HPV” as the base outcome, factors associated with single-type HPV infection were current smoking, lifetime number of female sexual partners, condom use, and ever having had a partner with an abnormal Papanicolaou (Pap) smear (Table 3A). The latter three factors were also associated with multiple-type oncogenic HPV as were the number of partners in the previous 3 months, frequency of sexual intercourse, and concurrent detection of genital warts (Table 3A). Factors pertaining to numbers of sexual partner, frequency of intercourse, and abnormal Pap smear appeared to be more strongly associated with multiple-type oncogenic infection than with single-type oncogenic infection.

In bivariate analyses, with “no nononcogenic HPV” as the base outcome, factors associated with single-type nononcogenic HPV infection were Hispanic ethnicity, currently smoking, number of female partners in the lifetime and in the previous 3 months, and concurrent detection of genital warts (Table 3B). Factors associated with multiple-type nononcogenic HPV infection were current smoking, number of female partners in the lifetime and in the previous 3 months, frequency of sexual intercourse, concurrent detection of genital warts, previous diagnosis of a STD, and ever having had a partner with an abnormal Pap smear. For sexual partner and frequency of intercourse behavior variables and for concurrent detection of genital warts, associations tended to be stronger for multiple nononcogenic HPV than for single-type nononcogenic infection (Table 3B).

In the multivariable model for oncogenic HPV, Hispanic ethnicity [adjusted OR (AOR), 2.71; 95% CI, 1.17-6.32], lifetime number of female partners (e.g., AOR for ≥21 partners versus 1-5, 4.62; 95% CI, 1.59-13.4), less frequent condom use (AOR, 2.52; 95% CI, 1.12-5.71), and detection of genital warts (AOR, 5.54; 95% CI, 1.26-24.3) were significantly associated with multiple oncogenic HPV infection. Significant associations were observed for multiple nononcogenic HPV with Hispanic ethnicity (AOR, 2.44; 95% CI, 1.03-5.79), lifetime number of partners (e.g., AOR for ≥21 partners versus 1-5, 22.89; 95% CI, 7.43-70.5), and concurrent detection of genital warts (AOR, 11.94; 95% CI, 1.84-77.7; Table 4). In the multivariable model of multiple infection with any HPV types, significant associations were observed for Hispanic ethnicity, currently smoking, lifetime number of partners, condom use, and concurrent detection of warts (Table 5). All associations were stronger for multiple infection and than for single-type infection.

In this cross-sectional study of primarily asymptomatic, heterosexual men, 22.9% of participants had more than one HPV type detected. Relatively few (8.6%) had multiple oncogenic types only and 13.4% had multiple nononcogenic types. The prevalence of multiple-type HPV has been reported in men as between 2.1% and 34.8% (10, 13-15). The use of a HPV detection method for 37 genotypes may partially explain the relatively high proportion of multiple types detected in this study compared with those previously published.

Regarding HPV types found in the quadrivalent vaccine, HPV-16, -18, and -6 were detected approximately as frequently as single HPV types as they were with other HPV types. However, no participant in this study had infection with all four vaccine types concurrently. Among men with multiple infections, 45.2% had infection with a vaccine HPV type.

Statistically significant, independent associations were seen between multiple HPV infection and the detection of warts, currently smoking ≥10 cigarettes/d, increasing lifetime number of female partners, and less frequent use of condoms during vaginal sex in the past 3 months. These estimates of association were stronger for multiple-type infection than for single-type infection. We have reported previously on the risk factors for HPV detection in this sample of men (16). That analysis found somewhat different risk factors for oncogenic and nononcogenic types. In the current report, few differences in associations between oncogenic and nononcogenic type infections were observed, and 95% CIs were overlapping. Apparently stronger associations between multiple oncogenic HPV and both Hispanic ethnicity and condom use might be of particular interest for evaluation in future, larger studies of multiple-type HPV detection in men.

The effect of multiple-type HPV infection on male health remains to be determined. However, an increased risk of persistent HPV infection and anogenital cancer might be hypothesized. Of two female cohort studies that published factors associated with multiple-type HPV infection, one found no association between number of HPV types and presence of cytologic abnormalities (2), whereas another found strong associations between high-grade squamous intraepithelial lesions and number of HPV types (3). In a case-control study, women with cervical intraepithelial neoplasia 1 or worse were more likely to have multiple HPV types than women with normal cytology (7).

Infection with multiple HPV types has been reported as a risk factor for “persistent” HPV infection (17) and has been associated with longer duration of infection (8). However, in a large cohort study, persistence was independent of coinfection with other HPV types, but acquisition of a HPV infection was more likely among women who had a HPV infection at baseline (18). In another cohort study, women were more likely to acquire multiple HPV types than was expected by chance, after adjustment for common risk factors, such as recent and lifetime number of sex partners (19).

Limitations of this study include the difficulty of detecting multiple types (assays are not optimized for variably amplifying types); simultaneous measurement of both exposure and outcome, which precludes causal inferences; and small numbers of HPV-positive results in each category when results are categorized by single/multiple and oncogenic/nononcogenic HPV types. Generalizability of these results is limited by the self-selection of men into the study. However, similarities to the base population and other cohorts can be observed. For example, the proportion of men reporting a sexual partner with a history of abnormal Pap smear in this study (23%) is similar to that reported by women ages ≥21 years who responded to the National Health Interview Survey in the United States, in which 20% reported ever having had an abnormal Pap smear (20). The majority (61.4%) of men in this study reported having had more than five female sex partners in their lifetime. In comparison, approximately half (48.5%) of the men in another study in the United States reported six or fewer partners (21) and 50.7% of the men in a Mexican study reported fewer than three partners (10). Although the men in this study who were recruited from STD clinics may have been more likely to have behaviors related to HPV acquisition and may have contributed to a higher HPV prevalence estimate, we believe that our exclusion criteria limit the number of “high-risk” men who participated. In future studies of HPV among men, the effect of multiple HPV types on persistence of infection and acquisition of new types should be examined. A strength of this study is the ability to examine number of HPV types within each sampling site separately. This information may aid in future studies of transmission to partners and development of anogenital warts or lesions or anal cytologic abnormalities among men themselves.

A.R. Giuliano: commercial research grant with Merck & Co. Inc.; speakers' bureau/honoraria for Merck & Co., Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the staff members of the University of Arizona Health Research Clinic, the Men's Research Clinic at Lifetime Cancer Screening and Prevention Center, H. Lee Moffitt Cancer Center, and the Pima County Theresa Lee Health Center; Danelle Smith, Steven McAnany, Shannon McCarthy, and Sireesha Banduvula for work in the laboratory; and Digene for donating reagents and supplies.