Human papillomavirus (HPV) is one of the most common pathogens of sexually transmitted disease (STD).1 There are more than 100 different HPV subtypes, over 40 of which are sexually transmitted. Although there is variability by region, approximately 70% of cervical cancers are associated with HPV 16 and 18. These are followed by subtypes HPV 45, 31, 33, 35, 52, and 58; these variants account for 10% of total cases.2–5
Data on the epidemiology of HPV infection are needed to develop optimal vaccination strategy and to assess the efficacy of HPV vaccination. The HPV DNA test not only identifies women with cervical disease, but also those at risk for developing disease within a few years. However, HPV prevalence in genital organs can only provide data on current infection, not on cumulative exposure.2,6 In addition, the HPV DNA test is also limited by the reluctance and discomfort of unmarried women regarding gynecologic examination.7–9 In contrast, HPV seroprevalence is a useful epidemiologic marker that reflects cumulative exposure to HPV. However, only 54-69% of HPV DNA-positive women are seroconverted 6-18 months after HPV DNA detection and the persistence of HPV antibodies may vary.6,10
Because there are little epidemiological data on HPV infection in the general population of Korea,8,11,12 we estimated the prevalence and seroprevalence of high-risk HPV in Korean girls and women aged 9-59 years. Because the age of sexual debut is relatively late in Korean conservative culture,13 we hypothesized that the prevalence and seroprevalence of high-risk HPV might be lower in Korea than in Western countries. These data are valuable for evaluating the guidelines regarding optimal age for vaccination depending on the characteristics of Korean women. We also establish baseline data for estimating vaccine efficacy.
MATERIALS AND METHODS
The current study was conducted in 1,143 girls and women aged 9-59 years who visited our medical institution for a regular medical checkup from July 2008 to May 2009. All study samples were selected according to equal case distributions in five geographic areas and predefined strata of five age groups. The sample was obtained using a complex, stratified, multistage probability cluster design to select a nationally representative sample.
Girls and women aged 9-19 years were pediatric patients in whom cancer and immunologic diseases were ruled out. Considering that the age of sexual debut is relatively late in Korea and Korea's comparatively conservative culture, for girls and women aged 9-19 years, the HPV antibody test was performed without a questionnaire study or a HPV DNA test requiring cytology of the uterine cervix. Women aged 20 years or older were asked to complete a self-reporting questionnaire that included questions on sociodemographic information and daily habits associated with HPV infections such as sexual behavior, history of STD, tobacco use, and oral contraceptive use. Of a total of 1,143 girls and women, the prevalence of high-risk HPV was analyzed in a total of 902 women aged 20-59 years, and the seroprevalence of high-risk HPV was analyzed in a total of 1,094 girls and women aged 9-59 years (Table 1). All participants signed an informed consent form and the study protocol was approved by the Institute of Review Boards of Gangnam Severance Hospital.
In the 902 women aged 20-59 years, exfoliated cervical cells were collected with a cytobrush (Cervibrush; Cell-Path). These cells were collected in an Eppendorf tube containing 20 mL phosphate-buffered saline. In 1,094 girls and women aged 9-59 years, 10-mL blood sampling was done with the use of the Vacutainer system. Blood samples were centrifuged at 1,500×g for 10 minutes and the serum was isolated. All samples were sent daily to a central laboratory and stored in prelabeled tubes at −70°C until processing.
High-risk HPV DNA testing was performed using a liquid hybridization assay (Hybrid Capture II, Digene) and polymerase chain reaction (PCR) assays. To confirm the presence of high-risk HPV DNA (HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and viral load, cells from the uterine cervix were collected with the use of a female swab specimen collection kit (Dacron swab, Digene). The cells were placed in a liquid hybridization assay collection kit and then preserved at −20°C for further analysis. RNA probes of high-risk HPV were hybridized with denatured, single-stranded DNA. This reaction mixture was transferred to a tube of which the surface was coated with anti-DNA-RNA hybrid antibodies. The immobilized hybrids were then reacted with an alkaline phosphatase-conjugated antihybrid monoclonal antibody. After rinsing, these reactants were treated with Lumi-Phospho 530 (Lumigen), which reacts to alkaline phosphatase, a dioxetane-based chemiluminescent substrate. The light emitted from the reaction was measured using a luminometer. The intensity was expressed using units that were relative to the reactions. The solutions containing 10 pg/mL HPV 16 DNA served as positive controls for high-risk HPV. The relative unit for the light emitted from all the samples was defined as the relative degree of illumination to the positive control group. A positive cutoff value was set at 1 pg HPV DNA per milliliter in each specimen.
For typing of high-risk HPV DNA (HPV 16, 18, 31, 33, 45, 52, 56, 58, and 59), DNA was extracted using the Qiagen DNA extraction kit (Qiagen). For the DNA quality check, β-globin PCR was performed. Only samples showing positive results to β-globin were used to confirm whether they were positive for HPV using GP5+/6+ primer. To identify HPV subtypes, samples testing positive for high-risk HPV were subjected to PCR with the use of each HPV primer.
For the high-risk HPV serologic test, samples were tested for specific neutralizing antibodies to high-risk HPV subtypes 16 and 18 by Merck and Co. Inc. using a multiplexed competitive assay (Luminex). The measurement of antibody levels was done through methods for measuring antibodies against HPV as described by Opalka et al in 200314 and reported in milli-Merck units per milliliter. The monoclonal antibodies used in the HPV multiplexed competitive assay included H16.V5 and H18.J4 for HPV subtypes 16 and 18, respectively. Seropositivity for HPV was defined as having anti-HPV antibodies titers of at least 20 and at least 24 milli-Merck units per milliliter for HPV subtypes 16 and 18, respectively.15
Demographic characteristics and sexual behaviors between the two groups were analyzed using the Pearson χ2, t test, and Fisher exact test. Unconditional multiple logistic regression was used to calculate odds ratios and corresponding 95% confidence intervals (CIs) to assess the associations between HPV DNA positivity and seropositivity and selected characteristics. For all analyses, a significance level of P<.05 was chosen.
The demographic characteristics and sexual behaviors of the study patients are shown in Table 2. There were significant differences between high-risk HPV DNA-positive women and high-risk HPV DNA-negative women with respect to age, marital status, education level, tobacco use, age at sexual debut, child birth, oral contraceptives use, lifetime number of sexual partners, new sexual partners in recent 6 months, and history of STD. When compared with high-risk HPV-seronegative women, there were significant differences in marital status, child birth, lifetime number of sexual partners, new sexual partners in recent 6 months, and history of STD in high-risk HPV-seropositive women.
Of 902 Korean women aged 20-59 years, the prevalence of high-risk HPV was 12.6% (95% CI 10.5-14.8%). To calculate the age-standardized prevalence and seroprevalence, age-specific proportions were standardized to female population figures from the National Statistics Office for Korea in 2008. Thus, HPV prevalence standardized by age on the basis of the female population of Korea was 12.6% (95% CI 10.4-14.8%). Prevalence of high-risk HPV reached its highest peak at age 20-29 years (23.2%, 95% CI 17.4-29.0%) and appeared to decline with increasing age. However, there appeared to be another increase in the prevalence of high-risk HPV in women aged 50-59 years (Table 3).
In regard to the type-specific distribution of high-risk HPV, of the 106 samples that were positive to liquid hybridization assay, the number of samples whose subtypes were confirmed by PCR was found to be 74 (69.8%). In the remaining 32 samples, no subtypes were confirmed. Of the 74 samples whose subtypes were confirmed, 50 samples (67.6%) were found to be of a single infection where a single subtype of HPV was confirmed to be present. In the remaining 24 samples (32.4%), there were multiple infections where more than two subtypes of HPV were confirmed. The most common high-risk HPV subtype was HPV 56 (1.9%). This was followed by HPV 18 (1.8%), HPV 52 (1.7%), HPV 16 (1.3%), HPV 31 (1.3%), HPV 33 (1.3%), HPV 58 (0.8%), HPV 45 (0.7%), and HPV 59 (0.7%) (Fig. 1).
Of 1,094 Korean girls and women aged 9-59 years, the seroprevalence of either high-risk HPV types 16 or 18 was 8.7% (95% CI 7.2-10.6%) and seroprevalence standardized by age on the basis of the female population was 9.0% (95% CI 7.3-10.8%). Seroprevalence increased through 25-29 years of age (13.4%, 95% CI 8.2-20.1%), decreased to 7.6% in women aged 30-39 years, and plateaued to approximately 10% after age 40 years. The type-specific seroprevalence for HPV 16 and HPV 18 were 7.4% (95% CI 5.9-9.1%) and 2.7% (95% CI 1.9-3.9%), respectively. Peak seroprevalence occurred in women aged 20-24 years for HPV 16 and in women aged 25-29 years for HPV 18. The seroprevalence of antibodies to both high-risk HPV 16 and 18 was rare (1.4%, 95% CI 0.8-2.2%) and peaked among women aged 25-29 years (Table 4).
For evaluation, 868 women aged 20-59 years were tested with both liquid hybridization assay for the presence of high-risk HPV DNA and the HPV serologic test for seropositivity of high-risk HPV subtypes 16 and 18. Overall, seropositivity was identified in 91 women (10.5%). Thirteen of the 110 women (11.8%) who were HPV DNA-positive for any of the 13 high-risk HPV subtypes considered were seropositive, whereas 13 of the 91 (14.3%) women with seropositivity were HPV DNA-positive. Thus, concordance between HPV DNA and antibodies in individual women was poor (κ=0.016, 95% CI 0.053-0.086) (Table 5).
We analyzed the association between sociodemographic and behavioral risk factors and HPV infections to identify determinants of prevalence and seroprevalence of high-risk HPV. In multivariable analysis with the variables of significant P value in Table 2, the prevalence of high-risk HPV was correlated only with the number of lifetime sexual partners, reaching 8.7% among women reporting just one partner and increasing to 26.6% among those with at least three partners (P=.006). The effects of the remaining risk factors were not statistically significant (Table 6). For the seroprevalence of high-risk HPV subtypes 16 and 18, the correlation to an increased number of lifetime sexual partners was similar to that of prevalence (Table 7). Seroprevalence increased from 8.6% among women with one lifetime sexual partner to 17.6% among those with at least three partners (P=.037). Taken together, these data support the role of sexual behavior as the key determinant of HPV infection.
The prevalence of high-risk HPV was 12.6% (age-standardized prevalence 12.6%) among women aged 20-59 years with the highest prevalence at 20-29 years of age. This result is consistent with the 10.4-15.5% prevalence of HPV infection found in previous studies conducted with Korean women16,17 and similar to the 15.2% prevalence of high-risk HPV in the United States.18 The prevalence of high-risk HPV in this study showed a decreasing tendency in age after its highest peak at 20-29 years and thereafter reached a second peak at 50-59 years. This tendency was also common in many studies,19–21 which can be explained by the following: First, there is the reactivation of latent HPV infections that patients acquired in their 20s as a result of impaired immune response as a result of hormone deficiency in the perimenopausal or postmenopausal years. Second, this age-specific phenomenon can be attributed to increased sexual contact with new sexual partners in middle age. Finally, there could be a cohort effect. Although infection might have occurred earlier, during their 20s, it was not tested and thus only detected later.19,20,22
In the current study, HPV 56 was the most common subtype followed by HPV 18, 52, 16, 31, and 33. This is different from patients with cervical cancer, in whom the most commonly observed subtypes are HPV 16 and 18. According to several other studies, the distribution pattern of high-risk HPV types in the general population showed that in addition to HPV 16 and 18, HPV 33, 58, 56, and 52 have also been frequently found,3–5 possibly as a result of geographic variation in the relative distribution of different high-risk HPV types in the general population. Moreover, HPV 16 and HPV 18 are found more commonly in women with cervical neoplasia compared with women with normal cytology. Therefore, the screening of a vaccinated population needs to focus on detecting cervical lesions caused by nonvaccine-targeted HPV types. Ultimately, there is a growing need of broad-spectrum HPV vaccines against all oncogenic HPV types.
In the present study, the seroprevalence of either high-risk HPV subtypes 16 or 18 was 8.7% (age-standardized seroprevalence 9.0%) among girls and women aged 9-59 years. The prevalence of HPV DNA cannot fully indicate the persistent exposure to HPV because most HPV infections appear to be transient. On the other hand, HPV seroprevalence can be a useful marker, although it is an imperfect marker of cumulative HPV exposure. High-risk HPV seroprevalence in the current study was lower than that found in Western countries.23–27 This can be attributed to the fact that the frequency of exposure to sex for Korean women is low in the conservative sexual culture. According to previous studies, the seroprevalence of high-risk HPV overall increased or formed a plateau with increasing age.23,24,27–29 In our study, however, the seroprevalence of high-risk HPV reached its highest peak at 20-29 years of age and decreased thereafter. It then reached a second peak after age 40, similar to the age-specific pattern of the prevalence of high-risk HPV. Considering that the prevalence of HPV infection reaches a peak within 5-10 years of sexual debut and that seroconversion occurs 6-18 months after HPV detection,10,30 it is possible that the age of HPV exposure was accelerated in Korean women and that there was reactivation of latent infection as well as new HPV infection resulting from the active sexual life of middle-aged women.
The overall concordance between HPV DNA positivity and seropositivity was poor. Approximately 11% of HPV DNA-positive women were seropositive for high-risk HPV. This discrepancy can be explained by the effect of a long lag time required for seroconversion, waning of detectable antibodies, and variable persistence of type-specific antibodies.
Correlation between the general characteristics of study patients and the prevalence and seroprevalence of high-risk HPV was evaluated. We found that an increase in the number of lifetime sex partners was significantly associated with the prevalence and seroprevalence of high-risk HPV and that the effects of the remaining risk factors were not statistically significant.
Seroconversion within 12 months after HPV infection is seen in approximately 50% of all cases. It is known that only 54-69% of women with HPV infection present detectable antibodies.6,10 A low seroconversion rate and the short-term persistence of antibodies may also attribute to underestimation of HPV seroprevalence. Under the assumption that the antibody is formed in 60% of women with HPV infection, our data suggest that 14.5% of Korean girls and women aged 9-59 years and 22.3% of women aged 25-29 years have been exposed to high-risk HPV. This result is similar to the 23.2% of HPV prevalence found among women aged 20-29 years.
It is possible that sexually active women could be infected with multiple types of HPV. However, according to previous studies, there were few cases in which even sexually active women were infected with all four types of HPV vaccine. Accordingly, even in women previously infected with more than one subtype of HPV, vaccination for HPV may provide protection against other HPV subtypes.
In conclusion, our results confirm that the prevalence and seroprevalence of high-risk HPV begins to increase noticeably after the teenage years. Therefore, it is clear that girls and women aged 10-19 years who have not yet been exposed to HPV through sexual activity should be vaccinated. We also found that a high percentage of middle-aged women have been exposed to high-risk HPV infection. Therefore, it is necessary to raise the recommended age of catchup vaccination. Moreover, it is thought that the existing stipulated optimum age of routine vaccination should be lowered considering the rapid change in sexual behavior among teenagers in Korea. Our findings have important implications for the development of HPV vaccine policy. Our data also provide baseline information for assessing current vaccination strategies and monitoring the effect of HPV vaccination.
1.Koutsky L. Epidemiology of genital human papillomavirus infection. Am J Med 1997;102:3–8.
2.HPV and cervical cancer in the world: 2007 report. Vaccine 2007;25(suppl 3):C1–230.
3.Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer 2003;88:63–73.
4.Munoz N, Bosch FX, Castellsague X, Diaz M, de Sanjose S, Hammouda D, et al. Against which human papillomavirus types shall we vaccinate and screen? The international perspective. Int J Cancer 2004;111:278–85.
5.Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–27.
6.Ho GY, Studentsov YY, Bierman R, Burk RD. Natural history of human papillomavirus type 16 virus-like particle antibodies in young women. Cancer Epidemiol Biomarkers Prev 2004;13:110–6.
7.Dunne EF, Nielson CM, Stone KM, Markowitz LE, Giuliano AR. Prevalence of HPV infection among men: a systematic review of the literature. J Infect Dis 2006;194:1044–57.
8.Shin HR, Franceschi S, Vaccarella S, Roh JW, Ju YH, Oh JK, et al. Prevalence and determinants of genital infection with papillomavirus, in female and male university students in Busan, South Korea. J Infect Dis 2004;190:468–76.
9.Sukvirach S, Smith JS, Tunsakul S, Munoz N, Kesararat V, Opasatian O, et al. Population-based human papillomavirus prevalence in Lampang and Songkla, Thailand. J Infect Dis 2003;187:1246–56.
10.Carter JJ, Koutsky LA, Hughes JP, Lee SK, Kuypers J, Kiviat N, et al. Comparison of human papillomavirus types 16, 18, and 6 capsid antibody responses following incident infection. J Infect Dis 2000;181:1911–9.
11.Clifford GM, Shin HR, Oh JK, Waterboer T, Ju YH, Vaccarella S, et al. Serologic response to oncogenic human papillomavirus types in male and female university students in Busan, South Korea. Cancer Epidemiol Biomarkers Prev 2007;16:1874–9.
12.Shin HR, Lee DH, Herrero R, Smith JS, Vaccarella S, Hong SH, et al. Prevalence of human papillomavirus infection in women in Busan, South Korea. Int J Cancer 2003;103:413–21.
13.Kim CJ, Kim BG, Kim SC, Kim YT, Kim YM, Park SY, et al. Sexual behavior of Korean young women: preliminary study for the introducing of HPV prophylactic vaccine. Korean J Gynecol Oncol 2007;18:209–18.
14.Opalka D, Lachman CE, MacMullen SA, Jansen KU, Smith JF, Chirmule N, et al. Simultaneous quantitation of antibodies to neutralizing epitopes on virus-like particles for human papillomavirus types 6, 11, 16, and 18 by a multiplexed luminex assay. Clin Diagn Lab Immunol 2003;10:108–15.
15.Dias D, Van Doren J, Schlottmann S, Kelly S, Puchalski D, Ruiz W, et al. Optimization and validation of a multiplexed luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18. Clin Diagn Lab Immunol 2005;12:959–69.
16.Joo WD, Kim SH, Kim DY, Suh DS, Kim JH, Kim YM, et al. Prevalence of human papillomavirus infection in Korean women: risks of abnormal Pap smear and cervical neoplasia. Korean J Gynecol Oncol Colposc 2004;15:309–16.
17.Oh JK, Franceschi S, Kim BK, Kim JY, Ju YH, Hong EK, et al. Prevalence of human papillomavirus and Chlamydia trachomatis infection among women attending cervical cancer screening in the Republic of Korea. Eur J Cancer Prev 2009;18:56–61.
18.Dunne EF, Unger ER, Sternberg M, McQuillan G, Swan DC, Patel SS, et al. Prevalence of HPV infection among females in the United States. JAMA 2007;297:813–9.
19.Herrero R, Hildesheim A, Bratti C, Sherman ME, Hutchinson M, Morales J, et al. Population-based study of human papillomavirus infection and cervical neoplasia in rural Costa Rica. J Natl Cancer Inst 2000;92:464–74.
20.Lazcano-Ponce E, Herrero R, Muñoz N, Cruz A, Shah KV, Alonso P, et al. Epidemiology of HPV infection among Mexican women with normal cervical cytology. Int J Cancer 2001;91:412–20.
21.Castle PE, Schiffman M, Herrero R, Hildesheim A, Rodriguez AC, Bratti MC, et al. A prospective study of age trends in cervical human papillomavirus acquisition and persistence in Guanacaste, Costa Rica. J Infect Dis 2005;191:1808–16.
22.Trottier H, Franco EL. The epidemiology of genital human papillomavirus infection. Vaccine 2006;24(suppl 1):S1–15.
23.Wang SS, Schiffman M, Shields TS, Herrero R, Hildesheim A, Bratti MC, et al. Seroprevalence of human papillomavirus-16, -18, -31, and -45 in a population-based cohort of 10000 women in Costa Rica. Br J Cancer 2003;89:1248–54.
24.Newall AT, Brotherton JM, Quinn HE, McIntyre PB, Backhouse J, Gilbert L, et al. Population seroprevalence of human papillomavirus types 6, 11, 16, and 18 in men, women, and children in Australia. Clin Infect Dis 2008;46:1647–55.
25.Markowitz LE, Sternberg M, Dunne EF, McQuillan G, Unger ER. Seroprevalence of human papillomavirus types 6, 11, 16, and 18 in the United States: National Health and Nutrition Examination Survey 2003-2004. J Infect Dis 2009;200:1059–67.
26.Skjeldestad FE, Mehta V, Sings HL, Ovreness T, Turpin J, Su L, et al. Seroprevalence and genital DNA prevalence of HPV types 6, 11, 16 and 18 in a cohort of young Norwegian women: study design and cohort characteristics. Acta Obstet Gynecol Scand 2008;87:81–8.
27.Jit M, Vyse A, Borrow R, Pebody R, Soldan K, Miller E. Prevalence of human papillomavirus antibodies in young female subjects in England. Br J Cancer 2007;97:989–91.
28.Chen CJ, Viscidi RP, Chuang CH, Huang YC, Chiu CH, Lin TY. Seroprevalence of human papillomavirus types 16 and 18 in the general population in Taiwan: implication for optimal age of human papillomavirus vaccination. J Clin Virol 2007;38:126–30.
29.Dondog B, Clifford GM, Vaccarella S, Waterboer T, Unurjargal D, Avirmed D, et al. Human papillomavirus infection in Ulaanbaatar, Mongolia: a population-based study. Cancer Epidemiol Biomarkers Prev 2008;17:1731–8.
30.Munoz N, Manalastas R Jr, Pitisuttithum P, Tresukosol D, Monsonego J, Ault K, et al. Safety, immunogenicity, and efficacy of quadrivalent human papillomavirus (types 6, 11, 16, 18) recombinant vaccine in women aged 24-45 years: a randomised, double-blind trial. Lancet 2009;373:1949–57.
© 2010 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.
Figure. No caption available.