Human papillomavirus (HPV) is the main cause of genital cancer. In the Netherlands number of cervical cancer cases is low due to a programmatic and opportunistic screening program for women aged 30 years or older. Unfortunately, not all women get screened or malignancies develop despite screening, which led to 699 new cases in 2008 (Dutch cancer registration). To reduce cervical malignancies, vaccination against HPV types 16/18 was introduced in 2009 in the Netherlands for girls aged 12 years, accompanied by a catch-up campaign for girls aged 13 to 16 years.
The prevalence of HPV is high, especially among adolescents and young adult women.1–4 For women participating in opportunistic screening,1 women included in an American population-based sample,5 female health clinic attendees,6 and women recruited through the internet,7 HPV prevalence rates ranged from 19% to 68%. For American male university students,8,9 men recruited through advertisements,10 male sexually transmitted infections (STI) clinic attendees,11 and male soldiers,12 penile HPV prevalence rates ranged from 26% to 63%.
To determine HPV type-specific differences between male and female heterosexuals, one needs a study population where both genders are equally represented within age groups and sampled at the same time. Most studies including both genders were based on heterosexual couples and may therefore not have resulted in a representative comparison of prevalence rates and risk factors between genders.
This study focuses on a high-risk population selected from visitors at STI-centers: young1–4 and highly sexually active5,13,14 of both men and women. A major goal was to determine type-specific prevalence rates and associated risk factors within a high-risk population of heterosexual males and females aged 16 to 24 years and to assess possible differences between genders before the introduction of HPV vaccination for girls. Our prevalence results serve as baseline data. In combination with future cross-sectional HPV prevalence analyses in the same high-risk population and using a very sensitive detection method, it will give insight in the impact of HPV vaccination of girls on prevalence and genotype-distribution of HPV within both genders.
MATERIALS AND METHODS
Study Population and Design
Between February and April 2009, a cross-sectional study was performed in 12 STI clinics throughout the Netherlands. All heterosexual STI clinic attendees in the age group 16 to 24 years were given written information and asked for consent. Participation in the study included genital sampling (vaginal or penile), use of serum already collected during routine consultation or an extra tube if routine serum was not available (data not shown), filling in a questionnaire, and permission to link the anonymous standardized data collected during the intake of the routine consultation with STI test results (e.g., chlamydia, gonorrhoea, syphilis, HIV). The questionnaire covered sexual habits like condom use, number of sex partners, and history of STI. Demographic data were also collected. Because of the anonymous testing and the very sensitive polymerase chain reaction (PCR) (with as of yet unknown clinical relevance), participants did not receive feedback about their HPV test results.
The study was approved by the Medical Ethical Committee of the University of Utrecht, the Netherlands.
Women were requested to self-collect a vaginal swab. They were asked to insert a swab (Copan Diagnostics, Italy) about 4 cm into the vagina until resistance was felt and to turn it around along the walls of the vagina for 15 seconds. Men had to self-collect a penile swab with the instruction to firmly move the swab up and down the entire shaft of the penis, the glans, the coronal sulcus, and under the foreskin of the penis. Swabs were put into 1-mL universal transport medium (Copan Diagnostics, Italy). In some cases, for example among high-risk patients, a physical examination was performed by a nurse or physician, who then also collected the swab(s) instead of the participant for practical reasons.
All swabs were stored at −20°C until processing. After thawing, swabs were vortexed and 0.2-mL universal transport medium was spiked with phocine herpes virus-1, and DNA was subsequently extracted using the MagnaPure platform (Total Nucleic Acid Isolation Kit, Roche) and eluted in 100-μL elution buffer. HPV-DNA was amplified using the SPF10 primer set according to the manufacturer's instructions (DDL Diagnostic Laboratory, the Netherlands15). HPV-specific amplicons were detected using the DNA enzyme-linked immunoassay (HPV-DEIA, DDL Diagnostic Laboratory, the Netherlands). Amplicons of HPV-positive samples were subsequently analyzed in Line probe assay (HPV-LiPA, DDL Diagnostic Laboratory, the Netherlands), containing all 12 high-risk (HR)-HPV genotypes (i.e., HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59) and 12 other HPV types with limited evidence for cervical cancer in humans (i.e., HPV-6, -11, -34, -40, -42, -43, -44, -53, -54, -66, -70, -74) (classification based on the last International Agency for Research on Cancer report16). In addition, HPV types 68, 73, and 97 can be detected on the membrane, although no distinction between them can be made, and they were classified as other HPV types. In this report, all non-HR-HPV genotypes were referred as low-risk (LR) HPV. Samples found positive in the HPV-DEIA but yielding no HPV genotype-specific hybridization pattern on a LiPA membrane were scored as HPV-negative, because no confirmation was available that the DEIA-positive results were due to a HPV-amplicon representing a genital specimen.
The applied DEIA/LiPA detection method was able to detect 99% of the genotype-specific HPV DNAs (n = 72) in the 2010 WHO HPV LabNet Proficiency panel (data not shown). To test for accuracy among the different participating laboratories 5% of both the HPV-positive and -negative DNA-extracts were retested at the central laboratory (RIVM). Retesting of the negative samples yielded 91% concordant results (21/23). In 2 initially negative samples, a single LR-HPV was found (i.e., HPV-42 and HPV-66). On retesting, all initial HPV-positive samples (n = 61) were found HPV-positive.
Type-specific vaginal or penile prevalence rates were estimated per gender. Factors associated with the presence of HPV were assessed using univariable logistic regression analysis. Odds ratios (OR) with 95% confidence intervals (CI) were computed for any HPV, any HR-HPV, and any LR-HPV. HR-HPV also included multiple infections with HR-HPV and LR-HPV. The group of LR-HPV included no HR-HPV. An additional analysis was done for single and multiple HR-HPV infections. For all analyses, the same control group was used, that is, genital HPV-negative persons (including all persons positive with an untypable HPV infection, as we assumed that with most genital types included in the test these untypable HPV types were most likely skin types). Factors with a P value of <0.05 were considered significant and included in a multivariable logistic regression model, with exception of genital warts because of its clinical relation with certain type-specific LR-HPV infections. Missing groups were included in univariable regression analyses to test for selection bias. No significant relation was present and therefore all missing group were excluded in multivariable analyses.
The Pearson χ2 test was used to compare HPV prevalence rates between males and females. All analyses were performed using SAS statistical package version 9.2.
Description Study Population
A total of 1566 young heterosexuals from 12 STI clinics in the Netherlands were tested, 430 men and 1136 women. Overall, the response rate per STI clinic ranged from about 60% to 90%. A questionnaire was filled in by 97.3% (n = 1524) of them and routine STI and risk data collected during consultation were available for 99.6% (n = 1559) of the study population. The median age was 22 years for men and 21 years for women. The main group was Dutch with a high educational level (n = 1135, 66%). Median age of first sexual intercourse was 16 years (range, 10–24 years), median number of sex partners in the past 6 months was 2, and 21% reported an STI in the past. Females started to be sexually active at older age than boys and reported less sex partners, nonetheless they more often reported a history of STI (Table 1).
Genital HPV Prevalence
Males less often tested HPV positive than females: 53.7% versus 71.8% (Table 2). Not included were the male (n = 36) and female (n = 61) samples which tested positive with the DEIA, but could not be genotyped with the LiPA. Of all genital HPV-infected males and females, 73.6% and 81.0% had a HR-HPV type, respectively. These differences between males and females were statistically significant (P < 0.01). HPV-infected men were also more often detected with genital LR-HPV types than women (P = 0.01), while women were more often detected with multiple HR-HPV types (P = 0.05). The 3 most prevalent HPV types in females were HPV-51, HPV-16, and HPV-52. In males, HPV-51 was also the most prevalent HPV type, followed by HPV-6, HPV-16, and HPV-66 (Fig. 1). The HR-HPV vaccine type 16/18 was present in 16.3% of men and in 22.5% of women tested, accounting for 30.3% and 31.4%, respectively, of the HPV positive men and women. Only 1.4% of the tested men and 1.8% of women were positive for both types.
Risk Factors for a Prevalent Genital HPV Infection
Overall, all univariable odds ratios were stronger for HR-HPV than for LR-HPV (data not shown). In women, the univariable risk for an HR- or LR-HPV infection increased with increasing number of lifetime sex partners, increasing number of sex partners in the last 6 months, and when having had a chlamydia and/or gonorrhea infection in the past. Condom use in combination with having a steady and/or casual partner in the last 6 months was negatively associated. In addition to these 4 factors, the risk for an HR-HPV infection also increased with increasing age, increasing number of sexually active years, and the presence of an urogenital chlamydia infection at time of sampling. Similar factors were observed in men, although number of sex partners in the last 6 months was not significantly related to LR-HPV, and the negative association of condom use in combination with having a steady and/or casual partner in the last 6 months with LR-HPV was borderline significant (P = 0.06). Having genital warts at time of sampling was only significantly associated with LR-HPV genotypes and not with HR-HPV: in men (OR = 3.6, 95% CI: 1.0–13.0) and in women (OR = 7.3, 95% CI: 2.0–26.8).
Multivariable analyses showed that women had a significantly higher risk of having an HPV infection compared with men (any HPV: adjusted odds ratio [AOR] = 3.0, 95% CI: 2.3–4.0; HR-HPV: AOR = 3.2, 95% CI: 2.4–4.2; LR-HPV: AOR = 1.9, 95% CI: 1.3–2.8). The multivariable models for any genital HPV, HR-HPV, and LR-HPV per gender are shown in Table 3. Based on overlapping confidence intervals, no statistically significant differences were seen between men and women for any of the outcome variables. One exception is condom use in combination with having a steady and/or casual partner in the last 6 months which was significantly associated with lower risk for an HR-HPV infection in women, but not in men. Furthermore, HR-HPV was more strongly associated with high-risk sexual behavior than LR-HPV.
Risk Factors for Single or Multiple Genital HR-HPV Infections
Univariable analyses of multiple HR-HPV infections showed stronger relations with sexual risk behavior in comparison with a single HR-HPV infection (data not shown). In the multivariable analysis, this relationship remained (Table 4). Number of lifetime sex partners and having had a chlamydia or gonorrhea infection in the past were independently associated with multiple HR-HPV infections in both genders, whereas condom use in combination with having a steady or casual partner in the last 6 months was only negatively associated with multiple HR-HPV in women. Overall, women had a significantly higher risk of having a single HR-HPV infection (AOR = 2.9, 95% CI: 2.1–4.0) or multiple HR-HPV infection (AOR = 3.8, 95% CI: 2.6–5.6) than men.
Before the introduction of HPV-16/-18 vaccination for girls, HR-HPV prevalence among young high-risk heterosexuals attending Dutch STI clinics was 58% in females and 40% in males. The majority of these were HPV-16 and HPV-51 infections. Furthermore, females were more often infected with multiple HR-HPV types and vaccine types HPV-16/-18 in comparison with males. Repeated cross-sectional studies in this population will allow early assessment of the impact of vaccination on changes in the prevalence and distribution of type-specific HPV infections in young females as well as males.
The high prevalence rates in this population suggest that this is indeed a group at high risk for (multiple) HPV infections. The composition of the study population (young1–4 and highly sexually active5,13,14) in combination with a very sensitive HPV detection method17,18 will have contributed to these high prevalence rates. The higher HPV prevalence in women compared with men could be related to a higher incidence, and/or a longer duration of infection. Indeed, Trottier et al observed a median duration of 6.3 to 9.7 months to clear an incident HPV infection in women,19 whereas Lu et al reported a median duration of 5.9 months in men.20 To unravel the determinants of this higher prevalence in women, a prospective study is needed in which men and women are followed to establish incidence and to assess time to clearance once infected. Another possible explanation could be the use of self-collected samples. Self-collecting vaginal swabs is already applied in STI testing in the Netherlands, therefore implementation was simple. Self-collecting a penile swab was never applied in the STI clinics. However, other studies showed good agreement between samples taken by a physician and self-collected samples.21,22
Type distribution between genders was not equal. The three most common HPV types among women were HPV-51, -16, and -52, all HR types. This corresponds with types seen in literature where almost all studies rank these three among the 5 most frequent types.1,2,6,23,24 In men, the most common types were HPV-51 and -16, but also LR types HPV-6 and -66, suggesting a type-specific difference in susceptibility between genders. In addition to these types, other studies also frequently found HPV-53 and HPV-59.8,9,12,25 Although HPV-53 prevalence was also high in our study, HPV-59 was very low. The differences between HPV type-specific prevalences we found and those observed in other studies could have been influenced by different sensitivities of the applied detection methods.26
We have used a sensitive HPV detection method to provide baseline data for monitoring HPV vaccination effects on HPV dynamics. There are some limitations to such a sensitive test. First of all, using this test within a high-risk population prevents us to extrapolate the HPV prevalence to the general Dutch young population and compare the prevalence rates found in this study with other studies using other test methods. Also, the clinical relevance of this PCR is as of yet unknown and type-specific prevalence rates found can, therefore, not be related to cancer risks. Finally, discrepancies in genotypes detection will become more prominent when a sensitive test is used on samples with multiple HPV genotypes with different viral loads. The discrepancies observed in about 15% of the results of the initial study and retesting were indeed profoundly associated with presence of multiple HPV genotypes (average = 3.8 HPV genotypes [data not shown]). Furthermore, testing in as many as 7 laboratories could have negatively influenced the validity of the test results. To prevent this, several quality control methods were implemented as mentioned in the section “Laboratory methods” so we could establish that internal validity was assured.
We have chosen to include all DEIA positive samples that could not be genotyped with the LiPA in the HPV negative group, because we assume that most untypable samples contain HPV skin genotypes. However, it is possible that other genital HPV genotypes, which are not represented on the used LiPA membranes, are present in these samples. Therefore, we repeated the univariate and multivariate analyses with exclusion of all samples containing untypable HPV genotypes. Multivariate results showed only 2 significant differences with regard to Tables 3 and 4: (1) Having a current urogenital chlamydia infection became borderline significant in women for any HPV, HR-HPV, and single HR-HPV where it was not significant for these specific HPV groups when the untypable HPV types were included in the HPV negative group; (2) Condom use in combination with having a steady and/or casual partner in the last 6 months was significant associated in men with a HR-HPV infection and with a single HR-HPV infection, where it was not significant when the untypable HPV types were included in the HPV negative group.
Reported condom use in combination with having a steady and/or casual partner was a protective factor for an (HR-)HPV infection in men as well as in women. Men who consistently used condoms regardless of having a steady or casual partner had a decreasing risk for a prevalent (HR-)HPV infection, in women also not having a casual partner was protective. Data about the effect of condom use against HPV infection are inconsistent. In men, including areas not covered by a condom in the analysis could dilute any effect.27 Nielson et al observed a protective effect in men, taking into account the covered and uncovered areas,27 Baldwin et al also found a protective effect including only covered areas.28 For women data are also inconsistent, but suggests that condom use is not protective against HPV infection2,6,24; however, some studies did find a strong protective effect.14,29
The fact that HPV is a sexually transmitted disease and is transmitted by skin-to-skin as well as by mucosa-to-mucosa contact30 suggests that not only women will be protected against HPV through vaccination, but that also a decrease in HPV prevalence in men is to be expected. To monitor these possible changes in the HPV dynamics over time, we will perform follow-up studies in this young high-risk population every 2 years. In future studies, we will also explore whether reducing the prevalence of certain HPV types can have an effect on the prevalence of other prevalent STI, in particular chlamydia. But there are still questions which remain unanswered, for example, what explains the differences in type-specific prevalence between men and women? And what is the mode of influence of the associated factors of HPV prevalence: increasing HPV transmission or slowing down the clearance of the virus?
Using a very sensitive assay, we found a high HPV prevalence in genital specimen of a high-risk population of young males and females. Females had higher HPV prevalence rates than men, and were more often infected with a HR-HPV type. Also HPV-16/-18 was more commonly detected in females than in males. HR-HPV infection was especially related with high sexual risk behavior in contrast to LR-HPV types.
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