Predictors of human papillomavirus infection in women undergoing routine cervical cancer screening in Spain: the CLEOPATRE study

Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide. It is well established that HPV is the necessary cause of cervical cancer and its precancerous lesions. In addition, HPV has been associated with a wide range of other diseases including: genital warts; vulvar, vaginal, penile and anal cancers; head and neck cancers; and recurrent respiratory papillomatosis [1, 2]. Most sexually active women will be infected with HPV during their lifetime [3]. While the majority of HPV infections are cleared within 2 years, persistent infections and the presence of risk factors that promote persistence may lead to the development of precancerous and cancerous lesions in a small fraction of women. HPV genotypes are classified as high-risk (HR) or low-risk (LR) based on their association with cervical cancer, HPV 16 being the type most commonly identified in this cancer [4].

Epidemiological studies conducted among women over the past decade have shown consistently that the prevalence of cervical HPV infection, particularly HR HPV types, is greatest among women below the age of 25–30 years and lower in older women [5, 6]. The increased infection rates among young women are attributable to higher exposure to HPV through multiple sexual partners. In contrast, older women tend to have fewer new partners and they may have developed some degree of immunity against HPV [7]. Other potential risk factors for cervical HPV infection include tobacco smoking [8], nulliparity [9], use of oral contraceptives [9], and recent new sexual partners [10]. Use of condoms may protect against cervical HPV infection [11].

Data on cervical HPV prevalence and determinants of HPV infection are sparse and limited in Spain. In a previous analysis of the CLEOPATRE Spain study, we reported the prevalence and type-specific distribution of cervical HPV infection among women aged 18–65 years attending cervical cancer screening in Spain [12]. In the current analysis we report on the association between socio-demographic, lifestyle and medical factors and the prevalence of cervical HPV infection. Only one previous epidemiological study has been carried out in the general female population of Spain to assess these risk factors [13]. Thus, the current analysis represents the first large-scale, country-wide epidemiological study exploring risk factors for cervical HPV infection in Spain.

This is the first large-scale, country-wide study in Spain to evaluate potential risk factors for cervical HPV infection. In addition to young age, a lifetime number of two or more sexual partners, a history of genital warts, and being unmarried were the strongest risk factors for cervical HPV infection among women aged 18–65 years attending routine cervical cancer screening across Spain. Living in an urban community, country of birth other than Spain, low level of education, and current smoking were also identified as independent risk factors for cervical HPV infection. Condom use provided a borderline protective effect against HPV infection.

Our results agree with those of a previous epidemiological study in the general female population of Spain (N = 973) conducted in 2003 by de Sanjose et al., which found the following independent risk factors of HPV infection: overseas birth, divorce, more than one sexual partner, and smoking marijuana or related products. Use of condoms with a regular partner was found to be protective against HPV, but with a borderline effect [13].

As in other studies [7, 10, 11, 13], an increased lifetime number of sexual partners was the most consistent risk factor for cervical HPV infection identified in our study. In contrast, age at first sexual intercourse was not identified as an independent risk factor in the multivariate analysis, in accordance with some previous studies [7, 10, 13]. In our study, young age at first intercourse increased the risk of HPV infection in the unadjusted model but in the adjusted one the effect was reversed. The inclusion of number of sexual partners in the adjusted model explained this change, indicating reverse co-linearity between the two variables. Our further stratified analyses, as shown in Figure 1, strongly suggested that the key determinant of cervical HPV infection is the number of sexual partners the woman had had and not the age at first sexual intercourse. These findings are in agreement with other studies suggesting that age at sexual debut is a likely predictor of lifetime number of sexual partners and not a factor independently associated with cervical HPV infection [15, 16]. A similar effect was observed when time since first sexual intercourse was assessed rather than age at first sexual intercourse.

Country of birth other than Spain was identified as an independent risk factor for cervical HPV infection, a finding that was reported in a previous, smaller study conducted in Spain [13]. In our study, most women born outside Spain were immigrants from Latin America. The HPV prevalence in these countries is higher than that in Spain [17, 18]. The increased prevalence of HPV among immigrant women compared with women born in Spain may be related to well-known differences in sexual behavior in both men and women.

In agreement with the pooled analysis of the International Agency for Research on Cancer (IARC) on smoking and HPV prevalence [8], we identified current smoking status as an independent risk factor for cervical HPV infection. A large population-based study conducted in women from Costa Rica found a positive association between smoking status and HPV 16, an association that was also found to be statistically significant in our study [10]. Intensity of smoking was identified as another predictor of HPV infection in the IARC analysis, but these data were not statistically significant in our adjusted model. An association between smoking and cervical HPV infection is difficult to assess because of the strong confounding effect of sexual behavior. To address this we performed further logistic regression analyses to formally test for the interaction between smoking and number of sexual partners, and found a statistically significant interaction between the two risk factors (Table 3). Smoking was associated with a two-fold increased prevalence of cervical HPV in women with multiple sexual partners but not among monogamous women. In contrast, in the IARC study a significant relationship of smoking intensity was seen only among women who reported one sexual partner, but not among women reporting more partners. A possible explanation for this interaction is that among women with a high exposure to HPV (those with multiple partners) the risk of infection is much increased if they are concomitantly exposed to smoking. Conversely, smoking in the absence of high exposure to HPV is not as relevant in determining the risk of HPV infection. Some studies have documented that in the presence of HPV, tobacco products may reduce local immunological responses, which might facilitate HPV acquisition, reduce HPV clearance, or increase HPV persistence, thus increasing the overall HPV prevalence [19, 20].

Condom use was found to be inversely but weakly associated with cervical HPV infection. The protective effect of condom use against HPV infection is controversial in the literature. Whereas some previous studies have reported a weak association with HPV infection [10, 11, 13], others have not [7]. Taking into account the weak magnitude of the associations found overall and in the subgroup analyses performed in our study, the beneficial effect of condom use on HPV prevalence is probably small.

Although male circumcision was not identified as a predictor of cervical HPV infection in our study, interaction analyses showed a protective effect among women at increased risk of HPV infection (i.e. women who had more sexual partners). Previous studies have shown that circumcised men are less likely to have penile HPV infection than uncircumcised men and that male circumcision infers a protective effect in female sexual partners for cervical HPV infections as well as for cervical cancer [21, 22].

A strength of this study is that the age-stratified sampling strategy used within each of the Autonomous Communities of Spain (except Ceuta and Melilla) provided a good demographic representation of the age-specific female population attending cervical cancer screening across the country. However, a potential limitation is that women were selected from opportunistic cervical cancer screening programs, which may have resulted in the preferential recruitment of health-conscious or healthier women. This potential over-representation of “healthy” women could underestimate the true prevalence of risk factors related to cervical HPV infection, such as tobacco smoking, high number of sexual partners or having never used a condom. The potential effect of this would be an underestimation of the true effects related to these risk factors. In order to assess potential selection bias, the distribution of key socio-demographic and lifestyle characteristics of women included in the CLEOPATRE study was compared with that of the general female population of Spain using public data such as marital status, parity, use of contraceptive methods, and number of sexual partners among others. We ascertained that the study population was indeed representative of the general female population in Spain (data not shown). As with any other cross-sectional design, there is a risk of reporting bias, particularly in relation to variables that address aspects of sexual behavior, such as “age at first sexual intercourse,” “lifetime number of sexual partners,” and “partner’s circumcision status.” Other studies have noted also the possibility of this non-differential misclassification related to sexual behavior [7]. It is well known that non-differential misclassification cannot inflate true effects; instead, it would shift the observed association towards the null value.

Another limitation of our study is that only 80% of the HC2-positive samples were positive by LiPA, a much more sensitive assay than HC2. This apparently low performance of the LiPA assay may be explained by the inherent differences between the two assays. Although the HC2 assay is designed to detect 18 HPV types (including the low-risk types), several studies have noted that the HC2 HR probe mix may cross-react with other HPV types that are not represented in the mix [23‐25]. Data on the cross-reactivity of the low-risk probe are not available, but it can be assumed that cross-reactivity also takes place with that probe. In contrast, in the LiPA test (which is designed to detect 27 HPV genotypes by identification of highly specific HPV DNA sequences), cross-reactivity with other HPV types has not been observed. It is thus conceivable that HPV types detected by HC2 through cross-hybridization are not detectable with the LiPA assay. The HR probe and the LR probe, with an unknown extent of cross-reactivity, were both used in the present study, and this may explain the observed relatively low sensitivity of the LiPA assay. When comparing the two methods, a Kappa value of 0.67 (95% CI, 0.64–0.70) was found, which could be interpreted as being acceptable.

This analysis of the CLEOPATRE Spain study has identified several socio-demographic, lifestyle, and medical factors associated with cervical HPV infection. Cervical HPV is a highly prevalent sexually transmitted infection in women in Spain, particularly among those under 30 years of age in whom the prevalence approaches 30%. In addition to young age, increasing number of lifetime sexual partners and a history of genital warts were identified as key risk factors for cervical HPV infection, particularly for HR HPV, reinforcing knowledge that cervical HPV infection is acquired early in a woman’s sexual life. Living in an urban community, country of birth other than Spain, low level of education, and current smoking were also identified as independent risk factors for HPV infection. A weak inverse association between condom use and HPV infection was also observed. In non-monogamous women, circumcision of the partner was associated with a reduced risk of HPV infection and smoking with an increased risk of HPV infection. Our results emphasize the importance of HPV vaccination programs for young women before they acquire sexual partners to prevent HR and LR HPV infections. Surveys of cervical HPV prevalence and its related risk factors increase our understanding of the natural history of this infection and establish the basis for future assessments of the impact of HPV immunization programs and the dynamics of risk factors for HPV infection in the population of Spain.

The 77 study investigators:

Rafael Comino, Hospital Universitario de Puerto Real, Puerto Real, Spain

José Antonio Borrego, Hospital Materno Infantil Reina Sofia, Córdoba, Spain

Mónica García, Centro de Especialidades “La Bola Azul”, Almería, Spain

Rogelio Garrido, Consulta Privada, Sevilla, Spain

Francisco Montoya, Hospital Materno Infantil Virgen de las Nieves, Granada, Spain

José Antonio Vargas, Consulta Privada, Sevilla, Spain

José Luis Cuadros, Hospital Clínico Universitario San Cecilio, Granada, Spain

Blas Hervías, Hospital Universitario Puerta del Mar, Cádiz, Spain

Miguel Ochando, Complejo Hospitalario Cuidad de Jaén, Jaén, Spain

Antonio de Toro, Hospital San Juan de Dios, Bormujos, Spain

Antonino Parrilla, Centro de Especialidades Esperanza Macarena, Sevilla, Spain

Tomás Carrascal, Consulta Privada, Huelva, Spain

Pedro Abad, Hospital Provincial de Almería, Almería, Spain

José Manuel Ramón, Hospital de San Jorge, Huesca, Spain

María Isabel Morollón, Hospital Universitario Miguel Servet, Zaragoza, Spain

Luis González, Consulta Privada, Oviedo, Spain

Concepción Solares, Centro de Salud del Quirinal, Avilés, Spain

Javier Barrés, Policlínica Miramar, Palma de Mallorca, Spain

Gabriel Ferret, Clínica Miramar, Palma de Mallorca, Spain

Orlando Falcón, Instituto Falcón Vizcaíno, Las Palmas, Spain

Lucía Almeida, Hospital Ntra Sra. De la Candelaria, Santa Cruz de Tenerife, Spain

Juan Miguel Falcón, Clínica de obstetricia y ginecología Dr. Falcón, Telde, Spain

José Bretones, Residencia Hospital Marqués de Valdecilla, Santander, Spain

José Antonio Mínguez, Consulta Privada, Valladolid, Spain

Guadalupe Bombín, Hospital Río Carrión, Palencia, Spain

Jaime Moreno, Hospital Vírgen de la Concha, Zamora, Spain

Diana Fernández, Hospital General de Segovia, Segovia, Spain

Gaspar González, Complejo Hospitalario Universitario de Albacete, Albacete, Spain

Carlos Zorzo, Hospital Gral. De Guadalajara, Guadalajara, Spain

Manuel Sánchez, Consulta Privada, Toledo, Spain

Montserrat Cararach, USP Institut Universitari Dexeus, Barcelona, Spain

Jordi Antoni, Hospital Teknogin, Barcelona, Spain

Pere Cavallé, Hospital Universitario Sant Joan de Reus, Tarragona, Spain

Pere Fusté, A.G. GINOCOLEGS, Barcelona, Spain

Jordi Xercavins, Hospital Universitari Vall d’Hebrón, Barcelona, Spain

Cristina Centeno, Hospital Universitari Vall d’Hebrón, Barcelona, Spain

María Eulalia Fernández, Centro de Atención Primaria Ramona Vía, El Prat de Llobregat, Spain

Aureli Torné, Hospital Clinic i Provincial, Barcelona, Spain

Luis Puig, Consulta Privada, Barcelona, Spain

Ramón Carreras, Hospital Mare de Deu del Mar, Barcelona, Spain

Joan Meléndez, Meléndez Ginecológica SC, Blanes, Spain

Juan Carlos Martínez, Centro de Salud S. Vicente del Raspeig, Alicante, Spain

Ana de Gonzalo, Centro de Especialidades Monteolivete, Valencia, Spain

Ana Boldo, Hospital La Plana, Villareal, Spain

Alberto Romeu, Centro de Salud de Godella, Godella, Spain

José Antonio López, Consulta Ginecológica, Alicante, Spain

Mar Sanz, Centro de Planificación Familiar “El Carmen”, Manises, Spain

José María Galán, Hospital Gral. San Pedro de Alcántara, Cáceres, Spain

Francisco Solano, Hospital Materno-Infantil Perpetuo Socorro, Badajoz, Spain

Alejandro Novo, H. C. U. de Santiago de Compostela, Santiago de Compostela, Spain

José Luis Gómez, H. Materno-Infantil Teresa Herrera, La Coruña, Spain

Concepción Moreno, Centro de Orientación Familiar del Ferrol, Ferrol, Spain

Angel de la Orden, Hospital Do Meixoeiro, Vigo, Spain

Francisco Vázquez, Clínica Ginecológica CEOGA, Lugo, Spain

Pilar Miranda, Hospital de Fuenlabrada, Fuenlabrada, Spain

Pilar Benavides, Hospital Universitario Santa Cristina, Madrid, Spain

Emilia de Dios, Centro de Salud Dos de Mayo, Móstoles, Spain

José Manuel Hernández, Hospital 12 de Octubre, Madrid, Spain

José Miguel Seoane, Hospital 12 de Octubre, Madrid, Spain

Miguel Angel Huertas, Hospital Universitario de Getafe, Getafe, Spain

Mónica García, Fundación Hospital de Alcorcón, Alcorcón, Spain

Juan Ordás, Centro de Especialidades José Marvá, Madrid, Spain

Dolores Rubio, Hospital Ramón y Cajal, Madrid, Spain

Esperanza Díaz, Hospital Ramón y Cajal, Madrid, Spain

Juan Carlos Recio, Centro de Salud Andrés Mellado, Madrid, Spain

Álvaro Zapico, OBS-GYN S.L., Murcia, Spain

José Ramón Rodriguez, Consulta Privada, Murcia, Spain

José Ramón Rodriguez, Hospital Los Arcos, Santiago de la Ribera, Spain

Juan Carlos Muruzábal, Instituto Navarro de Ginecología, Pamplona, Spain

Daniel Andía, Hospital de Basurto, Bilbao, Spain

Enrique Izaguirre, Hospital Txagorritxu, Vitoria, Spain

Borja Rivero, Consulta Privada, Zarauz, Spain

José Ramón Serrano, Clínica San Martín Osasunaegía, Berbara, Spain

Esteban Campeny, Consulta Privada, Logroño, Spain