This study aimed to characterize the HPV infection status in adolescents and young university women in Portugal. The distribution of HPV genotypes was evaluated by PCR DNA genotyping after self-sampling collection from 435 women of exfoliated cervical cells using a commercial kit. We observed an overall frequency of HPV infection of 11.5%. Furthermore, HPV DNA prevalence was 16.6% in those young women that self-declared as sexually active. The more frequently detected HPV types were 31, 16, 53, and 61. Statistical analysis identified median age (
Genital Human papillomavirus (HPV) is highly prevalent in sexually active women. This infection has been established as the etiological agents of genital warts and squamous intraepithelial lesions of the cervix, and nevertheless, only certain types of HPVs are able to induce cervical cancer development [
Cervical cancer is one of the leading causes of mortality by cancer with approximately 495 000 women newly diagnosed each year [
Adolescents and young-adult women are more vulnerable to HPV infection. These observations are based on biological/physiological differences in the cervical epithelium. While in adults the predominant cell type is squamous, in adolescents the predominant cell type is columnar and metaplastic [
HPV molecular detection has been implemented in several countries and it was already proposed as the primary test for screening by replacing the Pap smear test [
Nevertheless, HPV genotyping may be considered a more important methodology than detection only, since it helps to identify the persistent infections which are predictive of HPV infection outcome (development of intraepithelial lesions or cervical cancer). Several
Despite the incidence and mortality rates of cervical cancer in Portugal, until now no national screening program was implemented and there is no sufficient epidemiological data for HPV infection, particularly in young women. Screening programs in young women are essential to understand not only the epidemiology of HPV but also behaviours and community practices and consequently prevent the propagation of infection. Adolescents should be educated regarding HPV and associated risk factors of infection. Hence, they should also be encouraged to obtain suitable gynecological cares after initiating sexual activity [
Several studies have reported lately that self-obtained samples from the anogenital tract were accurate and suitable for HPV-DNA testing and have shown a similar correlation to clinically obtained samples [
The present study is the first to characterize the presence of HPV in adolescents and university women from northern region of Portugal using the self-sampling method.
A population-based study was developed to obtain a profile of HPV infection status in the Northern region of Portugal. Between March and May 2010, female students aged from 14 to 30 years old, from eight high schools and two university institutions from northern region of Portugal, were invited to participate in the study. Ethics approval was obtained from the Ethics Committee of
This study was undertaken in 435 women (median age 17.0 ± 2.46 years old), who have answered an anonymous questionnaire for epidemiological characterization. The study population was characterized according to age, age of menarche (median age 12.0 ± 1.34), education level, HPV vaccination, sexual activity (
Characteristics of the study participants.
Age and sexual intercourse | |
Age (median ± sda) | 17.0 ± 2.46 (range 14 to 30) |
Age of menarche (median ± sda) | 12.0 ± 1.34 (range 8 to 16) |
Age of first sexual intercourse (median ± sda) | 16.0 ± 1.67 (range 13 to 22) |
Number of years after first sexual intercourse (median ± sda) | 2.0 ± 2.06 (range 0 to 12) |
Education level ( | |
Elementary (≤9th grade) | 27 (6.2) |
High school (10th–12th grade) | 296 (68.2) |
University | 111 (25.6) |
HPV vaccination ( | |
Yes | 161 (37.0) |
No | 231 (53.1) |
NRb | 43 (9.9) |
Sexual activity ( | |
Yes | 277 (63.7) |
No | 38 (8.7) |
NRb | 120 (27.6) |
No. of Sexual partners ( | |
1 | 174 (62.8) |
2–5 | 94 (33.9) |
NRb | 9 (3.3) |
aStandard deviation; bNonresponders.
All participants were volunteers and informed of the study through workshops developed specifically to explain the study and self-sampling procedure. Briefly, exfoliated cervical cells were collected by self-sampling using the commercial kit
Sample processing included the concentration of cells by centrifugation, followed by DNA extraction using QiAmp DNA Blood mini Kit (QIAGEN, Hilden, Germany), according to manufacturer’s instructions. DNA quality was assessed by measuring the absorbance at 260 nm using a UV/Visible spectrophotometer and its purity assessed by the ratio of the values of absorbance at 260/280 nm.
To assess the validation of extraction method, the presence of genomic DNA was tested with a PCR protocol for amplification of beta-globin gene with the PCO3 primer 5′-ACA CAA CTG TGT TCA TAG C-3′ and BGII primer 5′-GTC TCC TTA AAC CTG TCT TG-3′.
The PCR reaction was performed in a 50
HPV DNA was detected through PCR using two different pairs of consensus primers: GP5+/6+, which amplify a region of 150 bp [
PCR amplification reaction with GP5+/6+ primers was carried in a 50
PCR amplification reaction with MY09/11 was performed in a 50
HPV-positive cases by amplification with primers MY09/11 were typed by RFLP analysis as described by Nobre et al. in 2008 [
The definition of HPV positivity was based on the result of MY09/11 and/or GP5+/6+ PCR amplification. Nonconcordant results by both primer sets were sent to sequentiate for confirmation of result. HPV genotyping was performed in MY09/11 positive cases and HPV genotypes were divided into four groups based on their oncogenic activity: high-risk types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), probable high-risk types (26, 53, 66, 68, 73, 82), low-risk types (6, 11, 13, 40, 42, 43, 44, 54 and 55, 61, 70, 72, 81, and 89), and types of undetermined risk (30, 32, 34 and 64, 62, 67, 69, 71, 74, 83, 84, 85, 86, 87, 90, 91, 97, 102, 106) [
To ensure the quality of the study, the following quality criteria were defined: (1) blind study; (2) inclusion of negative and positive controls in all PCR reactions: as negative control we have used double distilled water (dd H2O) replacing template DNA in the reaction mix; and as positive control we used an HPV positive sample used in the Virology Service for diagnosis of HPV infection using the commercial kit
Statistical Analysis was performed using the computer software
The presence of HPV was tested in 435 samples obtained by self-collection from young women and 50 positive cases were identified. Positive cases included one case from women who referred no initiation of sexual activity and 3 from which such information was not provided. For data analysis, we have considered only women who referred to have initiated sexual activity (
Distribution of HPV in sexually active women.
HPV positive | ||||||
---|---|---|---|---|---|---|
Total ( | 46 (16.6) | |||||
Age ( | ||||||
≤17 ( | 8 (7.5) | |||||
18–20 ( | 19 (18.3) | |||||
>20 ( | 19 (28.8) | |||||
≤17 ( | 8 (7.5) | |||||
>17 ( | 38 (22.4) | |||||
Age at first menarche ( | ||||||
≤12 ( | 26 (17.2) | |||||
>12 ( | 20 (16.1) | |||||
Age at first sexual intercourse ( | ||||||
≤16 ( | 22 (15.4) | |||||
>16 ( | 24 (18.8) | |||||
No. of Sexual partners ( | ||||||
1 ( | 15 (8.6) | |||||
2–5 ( | 28 (29.8) | |||||
No. of years after first sexual intercourse ( | ||||||
≤2 ( | 23 (12.7) | |||||
>2 ( | 23 (25.6) | |||||
Education level ( | ||||||
Nonuniversity ( | 23 (12.8) | |||||
University ( | 23 (24.0) | |||||
HPV vaccination ( | ||||||
No ( | 34 (22.1) | |||||
Yes ( | 9 (9.3) |
aHigh-risk HPV; bProbable high-risk HPV; cLow-risk HPV; dUndeterminated- risk HPV; eInconclusive. The frequencies represented in the bold values of the table refer only to HPV-positives cases.
HPV infection was analyzed according to age, age of first menarche, age of first sexual intercourse, number of years after first sexual intercourse, number of sexual partners, educational level, and HPV vaccination status (Table
We observed that HPV was more frequent among university students (24.0%) compared with nonuniversity students (12.8%). However, within positive cases, the prevalence of high-risk HPV was higher for nonuniversity students in comparison to university students (47.9% versus 34.8%) (Table
Distribution of different HPV types.
HPV positive cases ( | |
---|---|
HR HPVa ( | |
16 | 6 (13.0) |
18 | 2 (4.3) |
31 | 7 (15.2) |
45 | 1 (2.2) |
56 | 1 (2.2) |
58 | 2 (4.3) |
pHR HPVb ( | |
53 | 4 (8.7) |
66 | 3 (6.5) |
LR HPVc ( | |
6 | 1 (2.2) |
54 | 2 (4.3) |
61 | 4 (8.7) |
89 | 1 (2.2) |
UR HPVd ( | |
30 | 1 (2.2) |
32 | 1 (2.2) |
71 | 1 (2.2) |
84 | 1 (2.2) |
86 | 1 (2.2) |
87 | 1 (2.2) |
97 | 1 (2.2) |
Nonidentified ( |
aHigh-risk HPV; bProbable High-risk HPV; cLow-risk HPV; dUndeterminated- risk HPV.
Statistical significant differences were found when comparing HPV distribution regarding education level, HPV vaccination, mean age, lifetime number of sexual partners, and number of years after first sexual intercourse (Table
Odds Ratio (
95% CIb | |||
---|---|---|---|
Education level | |||
University versus nonuniversity | 0.018 | 2.15 | 1.13–4.08 |
HPV vaccination | |||
Vaccinated versus nonvaccinated | 0.009 | 0.36 | 0.16–0.79 |
Median age | |||
≤17 versus >17 | |||
Median age first menarche | |||
≤12 versus >12 | 0.810 | 0.92 | 0.49–1.75 |
Median age first sexual intercourse | |||
≤16 versus >16 | 0.461 | 1.27 | 0.67–2.40 |
No. of sexual partners | |||
1 versus 2–5 | |||
No. of years after first sexual intercourse ( | |||
≤2 versus >2 |
aOdds Ratio; bConfidence interval.
HPV infection is one of the most common sexually transmitted infections and a major public health concern, especially during adolescence [
Due to the worldwide distribution of HPV infection and to the high-incidence in undeveloped countries, many companies have attempted to developed low-cost tests with high efficiency to detect HPV. Self-sampling has been suggested as a useful tool for testing women for HPV infection mainly in low-resource populations or for people who have difficult access to health care [
Although the number of samples was limited and further large-scale epidemiological studies are needed to assess the significance of less common genotypes, this study provides the first view on the spectrum of the most common mucosal HPV types in young Portuguese women.
Epidemiologic studies refer to a prevalence of HPV infection from 2% to 44% in general population, with the highest prevalence in women aged 20 to 24 years old [
According to several studies, the behavioral risk factors associated with the HPV infection in adolescents and young adult women include early age of first sexual intercourse [
The number of lifetime sexual partners is considered to be one of the major risk factors for HPV infection [
Present results showed differences comparing HPV distribution regarding education level and HPV vaccination. Since the median age of first sexual intercourse was of 16.0 years old, it is expected that university students have more years of sexual life than nonuniversity students. Therefore, they would be exposed more frequently to a HPV infection than nonuniversity students. Regarding HPV vaccination, since the National program of Vaccination in Portugal started to include the HPV vaccine 2 years ago for girls between 13 and 18 years old, it is expected that these girls would be protected from acquiring an HPV infection. Therefore our data is correlated to these evidences and vaccination may reduce efficiently the infection by HPV.
Despite these important epidemiological data, characterization of HPV genotype frequencies among populations is considered to be even more imperative. HPV diagnostic assays such as hybrid capture second-generation (HC2) and PCR-based methods employing the consensus primers, MY09/11 and GP5+/6+, allow the detection of a large number of HPV types, mainly high-risk types [
In our study, we have used the genotyping method described by Nobre et al. in 2008, which allows a good discrimination of mucosal HPV types, including several HPV types that are underdiagnosed by current diagnostic methods. Our data showed that high-risk HPV types are the most frequent, with HPV31 and HPV16 having higher frequency. Surprisingly, HPV31 was found as the most frequent among our study. A recent meta-analysis shows that HPV31 may be the second or third most common type in European women with normal cytology [
Regarding our results, we must consider that after an open invitation to participate in the study, all participants were volunteers and informed of the study through workshops developed specifically to explain the study and self-sampling procedure. We cannot rule out the influence of two factors, that the attending students were more keen to know more about HPV and that subjects at higher risk of HPV infection are fear of being detected positive. However, we do think that the influence of those factors was reduced because knowing HPV results was optional, and only under the supervision of the gynecologist. Furthermore, our rate of participation was not conditioned by the cost of the test (which was free to all participants).
Molecular pathology methodologies have been demonstrated as important in the improvement of screening, diagnostic, or monitoring cancer, and genetic- or viral-associated diseases. Furthermore, they provide valuable tools for the new paradigm in Medicine known as personalized medicine [
It is acceptable to consider that, to achieve the maximum efficacy in health prevention strategies, we must maximize the knowledge of some populations about their epidemiology, costumes, and concerns and that is particularly important in those populations where no screening or vaccination has already been developed [
In conclusion, we have to refer that self-sampling may be a good alternative in substitution of the vaginal speculum examination by a clinician as a tool for primary screening of HPV infection in young asymptomatic women. This study contributes to a better understanding of the epidemiology of HPV infection among young Portuguese women. The exact knowledge of HPV profile in young women may be important to appraise the vaccination strategies within the different population.
There are no conflict of interests for any of the authors.
Funding of this project was made by Portuguese League Against Cancer (
The authors are grateful to the Portuguese League Against Cancer (