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Prevalence and determinants of HPV infection among Colombian women with normal cytology

Prevalence and determinants of HPV infection among Colombian women with normal cytology Molecular and Cellular Pathology British Journal of Cancer (2002) 87, 324 – 333 ã 2002 Cancer Research UK All rights reserved 0007 – 0920/02 $25.00 www.bjcancer.com Prevalence and determinants of HPV infection among Colombian women with normal cytology 1,2 2 3 ,1 2 3 1 M Molano , H Posso , E Weiderpass , AJC van den Brule* , M Ronderos , S Franceschi , CJLM Meijer , 3 3 4 A Arslan , N Munoz and the HPV Study Group Department of Pathology, Unit of Molecular Pathology, Vrije Universiteit Medical Centre, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands; 2 3 Division de Investigacion, Instituto Nacional de Cancerologia, Bogota, Colombia; Unit of Field and Intervention Studies, International Agency for Research on Cancer, Lyon, France Human papillomavirus is the principal risk factor associated with cervical cancer, the most common malignancy among women in Colombia. We conducted a survey, aiming to report type specific prevalence and determinants of human papillomavirus infection in women with normal cytology. A total of 1859 women from Bogota, Colombia were interviewed and tested for human papillomavirus using a general primer GP5+/GP6+ mediated PCR – EIA. The overall HPV DNA prevalence was 14.8%; 9% of the women were infected by high risk types, 3.1% by low risk types, 2.3% by both high risk/low risk types and 0.4% by uncharacterized types (human papillomavirus X). Thirty-two different human papillomavirus types were detected, being human papillomavirus 16, 58, 56, 81(CP8304) and 18 the most common types. The human papillomavirus prevalence was 26.1% among women younger than 20 years, 2.3% in women aged 45 – 54 years, and 13.2% in women aged 55 years or more. For low risk types the highest peak of prevalence was observed in women aged 55 years or more. Compared to women aged 35 – 44 years, women aged less than 20 years had a 10-fold increased risk of having multiple infections. Besides age, there was a positive association between the risk of human papillomavirus infection and number of regular sexual partners and oral contraceptive use. In women aged below 25 years, high educational level and having had casual sexual partners predicted infection risk. In conclusion, there was a broad diversity of human papillomavirus infections with high risk types being the most common types detected. In this population multiplicity of sexual partners and, among young women, high educational level and casual sexual partners seem to determine risk. British Journal of Cancer (2002) 87, 324 – 333. doi:10.1038/sj.bjc.6600442 www.bjcancer.com ã 2002 Cancer Research UK Keywords: HPV; Colombia; epidemiology; cervix uteri Cervical cancer is the most common cancer among women in On the basis of their main association with benign or (pre-) developing countries, and the sixth most common cancer in malignant lesions, HPVs can be subdivided into low risk (LR) women in developed countries (Parkin et al, 1999). In Colombia, and high-risk (HR) oncogenic types. Women with normal cervical where the annual standardised incidence rate of cancer of the cytology who are infected with HR HPV have an approximately cervix is high (23 per 100 000), it is also the principal cause of 100-fold increased risk of developing CIN III compared to unin- cancer mortality among women (Globocan, 1998). fected women (Rozendaal et al, 1996). Therefore, it has been Infection with certain types of human papillomavirus (HPV) is suggested that HR HPV detection might be used as a tool to iden- the main cause of cervical cancer and its precursor lesions (Van tify women at high risk of cervical cancer, in addition to – or as den Brule et al, 1991; Munoz et al, 1992; Chichareon et al, 1998; an alternative to – Pap smears (Rozendaal et al, 1996, 2000; Meijer Ngelangel et al, 1998; Rolon et al, 2000). HPV infection is now et al, 1998). The marked geographic differences in incidence of cervical considered as virtually necessary but not sufficient to cause this malignancy (Walboomers et al, 1999). Other host factors such as cancer are not only the result of differences in screening patterns, genetic alterations, endogenous female hormones and immune but also of differences in exposure to risk factors. For example, the status or environmental factors (e.g. smoking and use of exogenous very low incidence rates (2 – 4 per 100 000) reported from some hormones) are also likely to be involved in cervical carcinogenesis areas in China and Spain (Parkin et al, 1997) where population- (Bosch et al, 1992; Ngelangel et al, 1998; Gostout et al, 1998; based screening programs do not exist, are probably the result of Palefsky et al, 1999). low exposure to HPV resulting from the conservative sexual beha- viour prevalent in the previous decades. To investigate geographic differences in the prevalence of type- *Correspondence: AJC van den Brule; E-mail: vandenbrule@vumc.nl specific HPV infection and of other risk factors for cervical cancer, HPV Study Group: M Gonzalez, J Luna, G Martinez, E Mora, G Perez, the International Agency for Research on Cancer is co-ordinating a JM Fuentes, C Gomez, E Klaus, C Camargo, C Tobon, T Palacio, C Suarez, series of surveys of prevalence of HPV infection and CIN lesions in C Molina countries with high and low incidence of cervical cancer, this study Received 22 January 2002; revised 17 April 2002; accepted 8 May 2002 being one of them. HPV in Colombia M Molano et al Table 1 Characteristics of the study population of HPV-DNA detection among women with normal cytology in Bogota, Colombia Proportion of women positive for HPV- DNA b a a No Overall % % any HPV-DNA %HR %LR Total 1845 100 14.9 11.4 3.2 Age (years) 520 230 12.4 26.1 20.4 5.2 20 – 24 220 11.8 22.7 18.2 4.1 25 – 29 337 18.1 12.7 10.4 1.8 30 – 34 404 21.7 16.6 11.9 4.2 35 – 39 284 15.3 8.1 6.0 2.1 40 – 44 192 10.3 8.1 7.8 0.5 45 – 54 86 4.6 2.3 2.3 0.0 555 106 5.7 13.2 5.7 7.6 Education Low 659 35.7 11.7 8.3 3.2 Intermediate 740 40.1 15.5 12.3 2.9 High 445 24.1 18.9 14.4 3.6 Missing 1 0.1 0.0 0.0 0.0 Age at 1st sexual intercourse 520 628 34.0 11.9 9.1 2.5 17 – 19 585 31.7 14.7 10.9 3.4 416 632 34.3 18.2 14.1 3.6 Age at 1st regular sexual intercourse 520 759 41.1 11.7 8.9 2.5 17 – 19 556 30.1 15.6 11.7 3.6 416 478 26.0 18.6 14.4 3.5 Missing 52 2.8 21.1 15.3 5.7 Number of regular sexual partners 1 1370 74.3 14.0 10.9 2.7 2 299 16.2 17.4 12.7 3.7 3 59 3.2 22.0 15.2 6.8 Missing 117 6.3 16.2 11.1 5.1 Casual sexual partners No 1307 70.8 13.8 10.5 2.8 Yes 445 24.1 18.2 13.9 4.0 Missing 93 5.0 15.0 10.7 4.3 Parity 0 – 1 644 34.9 29.2 22.0 6.2 2 – 3 845 45.8 14.4 11.3 2.5 54 356 19.3 11.4 8.3 3.2 Age at first birth 521 843 45.7 10.4 7.8 2.2 18 – 20 493 26.7 16.2 12.4 3.4 417 293 15.9 15.7 12.6 3.1 Missing 216 11.7 28.7 21.3 6.5 Oral contraceptives Never 957 51.9 13.8 10.3 3.0 Ever 845 46.3 16.3 12.4 3.5 Former user 721 39.1 15.4 12.0 2.9 Current user 133 7.2 21.0 14.3 6.7 Missing 34 1.8 14.7 14.7 0.0 IUD (Intrauterine Device) Never 779 42.2 15.8 12.2 3.1 Ever 1032 56.0 14.2 10.7 3.3 Former user 524 28.4 14.1 10.5 3.6 Current user 508 27.6 14.4 10.8 2.9 Missing 34 1.8 17.6 14.7 2.9 Condom Never 1203 65.2 13.7 10.2 3.1 Ever 618 33.5 17.5 13.7 3.4 Missing 24 1.3 12.5 8.3 4.1 Continued ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology Molecular and Cellular Pathology HPV in Colombia M Molano et al Table 1 (Continued ) Proportion of women positive for HPV- DNA b a a No Overall % % any HPV-DNA %HR %LR Smoking Never 1343 72.8 14.7 11.3 3.0 Ever 502 27.2 15.7 11.6 3.6 Former smoker 205 11.1 14.1 9.7 3.9 Current smoker 297 16.1 16.8 12.8 3.4 High-risk (HR) types: 16, 18, 26, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 ISO39. Low-risk (LR) types: 6, 11, 40, 42, 43, 44, 82 (MM4), 83 (MM7), 84 (MM8), 71 (CP8061), CP6108, 81 (CP8304), 54, 55, 57, 61, 70, 72, 73. Proportion of women infected with any HPV types, HR and LR types, respectively, in each category of age, education, etc. When women were infected by both HR and LR simultaneously they were counted in the HR group only. Information on age was available for 1859 individuals. Include nulliparous women. The prevalence of HPV, as detected by HPV DNA, characteris- Biological specimens tically peaks after initiation of sexual activity and decreases with age. A second peak in peri-menopausal women has been recently During the gynaecological examination, cervical scrapes were described in some populations (Cuzick et al, 1999; Herrero et al, collected from each woman using two Ayre spatulas and two endo- 2000; Lazcano et al, 2001). It is currently unknown whether cervical brushes. The first spatula and brush were used for a different age-specific curves of HPV prevalence are attributable routine Pap smear, which was classified according to the Bethesda to an effect of age per se or to differences in risk between birth system terminology. The second spatula and brush and the remain- cohorts. ing cells of the first spatula and brush, were placed in a tube A prospective cohort study in Bogota, Colombia, was initiated in containing 5 ml of phosphate-buffered saline (PBS 16)+0.05% the early 1990s by the Colombian National Institute of Cancer and thiomersal. Cells were detached from the spatula and endocervical the International Agency for Research on Cancer (IARC). The aim brush through vortex, and subsequently centrifuged at 3000 g for of this study was to investigate the natural history of HPV infection 10 min. The cell pellet was resuspended in 1 ml buffer Tris-HCl and CIN lesions among low-income Colombian women. We report 10 mM pH 8.3 and stored at 7708C until their use. For analysis, here the results of a cross-sectional analysis of the cohort at enrol- 100 ml aliquots were boiled for 10 min at 1008C, cooled on ice ment, with focus on determinants of HPV infection among women and centrifuged for 1 min at 30006g. 10 ml of these pre-treated with normal cervical cytology. crude cell suspensions were used for PCR analysis (Van den Brule et al, 1990; De Roda Husman et al, 1994). To assess the quality of the target DNA, all pellets were pre-screened using a 209 base pair MATERIALS AND METHODS amplifying b-globin PCR using the primer combination BPCO3 and BPCO5 as described by De Roda Husman et al (1995b). Study population Between November 1993 and November 1995 the Colombian HPV detection by PCR National Cervical Cancer Control Program conducted a census in four health districts in Bogota, which had no previously HPV-DNA detection was performed by a standard GP5+/GP6+ implemented population-based cervical screening program. The PCR based assay, as described by De Roda Husman et al first 2000 women aged 18 – 85 years identified in the census (1995a). Briefly, the GP – PCR reaction was carried out using were invited to participate in the study. Additionally, 200 50 ml of PCR solution containing 10 mM Tris HCL pH 8.3, adolescents aged 13 – 17 years were invited to participate. They 50 mM KCl, 200 mM of each deoxynucleotide, 3.5 mM of MgCl , were identified consecutively from an adolescent clinic giving 1 U of DNA polymerase (AmpliTaq; Perkin-Elmer, USA) and contraceptive counselling in the study area. At recruitment, all 25 pmol of each of the GP5+ and biotinylated GP6+ primers women were interviewed face-to-face by specially trained inter- (Eurogentec, Belgium): 40 cycles of amplification were carried viewers. They answered to a structured questionnaire on out using a Perkin-Elmer 9600,USA thermocycler. Each cycle socio-demographic characteristics, lifelong sexual behaviour, included a denaturation step at 958C for 1 min, one annealing step reproductive and menstrual history, and smoking and dietary at 408C for 1 min, and a chain elongation step at 728C for 1.5 min. habits. After interview all women underwent a pelvic examina- The first step was preceded by a denaturation step of 4 min and tion. the last step was followed by an elongation step of 10 min. From the 2200 invited women, 53 refused to participate, eight Three dilutions of the cell line SiHa containing 1 – 10 copies of were considered ineligible (mental illness, hysterectomy, history HPV16 (100 pg, 1 ng and 10 ng) were used as positive control. As of cervical cancer) and 29 did not provide cell specimens for negative PCR controls, distilled water and processing blanks were HPV DNA detection, so that 2110 women had cell specimens to used every 10 samples. HPV positivity was assessed by Southern be analysed. Of them 150 had abnormal cytology (according to blot hybridization of GP5+/GP6+ PCR products with a cocktail the Bethesda system classification) and 101 of the women with probe of specific [a- P]dCTP labelled DNA fragments from cloned normal cytology were beta-globin PCR negative (which is consid- DNA of HPV6, 11, 16, 18, 31 and 33 under low stringent condi- ered an indicator of poor DNA quality). After these exclusions, tions (Van den Brule et al, 1990; De Roda Husman et al, 1995a,b). 1859 women remained in the analysis presented here. In the multi- variate analysis (see below) we further excluded 14 women who did Type specific HPV detection not answer the questionnaire completely, resulting in 1845 women with normal cytology and risk factors analysis. Informed consent HPV positive samples were first subjected to HPV group-specific was obtained from all participants included in the study. The local analysis using cocktail probes for HR and LR HPVs (Jacobs et al, ethical committee and the ethical committee at IARC approved the 1997, 2000). The HR HPV cocktail probe consisted of oligoprobes study protocol. for HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 British Journal of Cancer (2002) 87(3), 324 – 333 ã 2002 Cancer Research UK HPV in Colombia M Molano et al HR% LR% HPVX% 45 –54 55&+ <20 20 –24 25 –29 30–34 35 – 39 40–44 (n =230) (n =220) (n = 337) (n = 404) (n =284) (n =192) (n =86) (n =106) Age Figure 1 Prevalence of high and low risk of human papillomavirus among women with normal cytology from Bogota, Colombia Table 2 Prevalence of type-specific HPV single infection by age-group, in women with normal cytology Age 525 years Age 25 – 34 years Age 535 years All ages (n=450) (n=741) (n=668) (n=1859) HPV type No No No No % 6 010 1 0.4 11 1 2 1 4 1.4 16 12 20 13 45 16.3 18 4 4 0 8 2.9 31 2 2 1 5 1.9 33 1 2 1 4 1.4 34 0 1 0 1 0.4 39 2 3 1 6 2.2 40 3 1 1 5 1.9 42 2 2 3 7 2.5 45 2 1 0 3 1.1 51 2 3 3 8 2.9 52 1 3 2 6 2.2 53 0 2 0 2 0.7 54 2 0 2 4 1.4 56 5 3 2 10 3.6 58 9 5 3 17 6.2 59 0 1 0 1 0.4 61 0 1 0 1 0.4 66 2 2 0 4 1.4 68 0 2 0 2 0.7 70 2 1 1 4 1.4 72 1 1 0 2 0.7 81 (CP8304) 3 5 2 10 3.6 Iso 39 0 1 0 1 0.4 82 (MM4) 0 1 0 1 0.4 83 (MM7) 2 0 0 2 0.7 84 (MM8) 0 2 2 4 1.4 HR (cocktail) 3 3 3 9 3.3 LR (cocktail) 4 5 1 10 3.6 HPV X 2 4 1 7 2.5 Total 67 84 43 194 70.3 % relative to 276 HPV-positive women. and the LR HPV consisted of oligoprobes for HPV 6, 11, 40, 42, considered in the results as HR types, based on both the align- 43, 44, HPV 82 (MM4), HPV83 (MM7), HPV84 (MM8), Iso39, ment analysis of the E6 gene (modified from Myers et al, 1996) HPV71 (CP8061), CP6108, HPV 81 (CP8304), HPV 26, 34, 53, and the risk estimates obtained for the various HPV types within 54, 55, 57, 61, 70, 72, 73. However HPV types with unknown a multi-center case – control study of cervical cancer conducted by oncogenic potential – namely 26, 53, 73, 34 and Iso 39 – were the IARC (Mun ˜ oz et al, 1992, Mun ˜ oz, 2000; Bosch et al, 1995; ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology Molecular and Cellular Pathology HPV in Colombia M Molano et al Table 3 Risk factors for HPV-DNA detection among women with normal cytology Positive for HPV-DNA Positive for HPV-DNA Positive for HPV-DNA a b detection HR types LR types a a a No OR No OR No OR Age (years) 520 60 4.2 (2.1 – 8.3) 47 3.7 (1.8 – 7.9) 12 9.6 (2.2 – 42.0) 20 – 24 50 3.7 (2.2 – 6.4) 40 3.5 (2.0 – 6.4) 9 6.0 (1.8 – 19.6) 25 – 29 43 1.6 (1 – 2.7) 35 1.7 (1.0 – 2.8) 6 3.8 (1.4 – 4.8) 30 – 34 67 2.1 (1.4 – 3.3) 48 1.8 (1.1 – 3.0) 17 3.8 (1.4 – 10.0) 35 – 44 40 1 (reference) 32 1 (reference) 7 1 (reference) 545 16 1.2 (0.6 – 2.3) 8 0.8 (0.3 – 1.8) 8 3.2 (1.0 – 10.3) Education Low 77 1 55 1 21 1 Intermediate 115 1 (0.7 – 1.5) 91 1.0 (0.7 – 1.6) 22 0.9 (0.4 – 2.0) High 84 1.4 (0.9 – 2.1) 64 1.4 (0.9 – 2.3) 16 1.3 (0.5 – 3.0) No of regular sexual partners 1 192 1 150 1 38 1 2 52 1.4 (1.0 – 2.0) 38 1.3 (0.9 – 2) 11 1.5 (0.7 – 3.1) 3 13 2.1 (1.1 – 4.2) 9 2 (0.9 – 4.5) 4 3.0 (0.9 – 9.7) Casual sexual partners No 181 1 138 1 37 1 Yes 81 1.1 (0.8 – 1.5) 62 1.1 (0.8 – 1.6) 18 1.3 (0.7 – 2.5) Parity 061 1 46 1 13 1 1 – 2 136 0.7 (0.4 – 1.1) 107 0.7 (0.4 – 1.1) 24 0.7 (0.3 – 1.8) 53 79 0.7 (0.4 – 1.3) 57 0.7 (0.4 – 1.4) 22 1.0 (0.3 – 3.0) Age at first sexual intercourse 520 75 1 57 1 16 1 17 – 19 86 1.0 (0.7 – 1.4) 64 1.0 (0.7 – 1.5) 20 1.0 (0.5 – 2.1) 416 115 1.0 (0.6 – 1.5) 89 1.0 (0.6 – 1.6) 23 0.7 (0.3 – 1.8) Oral contraceptives Never 132 1 99 1 29 1 Ever 139 1.4 (1.1 – 1.9) 106 1.4 (1.0 – 1.9) 30 1.6 (0.9 – 2.9) Former user 111 1.4 (1 – 1.9) 87 1.4 (1.0 – 2.0) 21 1.3 (0.7 – 2.5) Current user 28 1.5 (0.9 – 2.4) 19 1.2 (0.7 – 2.2) 9 3.1 (1.3 – 7.2) IUD Never 123 1 95 1 24 1 Ever 147 1.1 (0.8 – 1.5) 110 1.0 (0.7 – 1.4) 34 1.6 (0.8 – 3.0) Former user 74 1.4 (0.9 – 1.8) 55 1.2 (0.8 – 1.7) 19 1.8 (0.9 – 3.8) Current user 73 1.5 (0.7 – 1.4) 55 0.9 (0.6 – 1.3) 15 1.3 (0.6 – 2.9) Condom Never 165 1 123 1 37 1 Ever 108 1.1 (0.7 – 1.3) 85 1.0 (0.7 – 1.4) 21 0.8 (0.4 – 1.4) Smoking Never 197 1 152 1 41 1 Ever 79 1.1 (0.8 – 1.5) 58 1.1 (0.8 – 1.5) 18 1.2 (0.6 – 2.3) Former smokers 29 1.0 (06 – 1.6) 20 0.9 (0.5 – 1.6) 8 1.2 (0.5 – 3.0) Current smoker 50 1.2 (0.8 – 1.7) 38 1.2 (0.8 – 1.8) 10 1.2 (0.5 – 2.6) a a OR =odds ratio adjusted for age, education, number of regular partners, parity, age at first sexual intercourse, oral contraceptives, condom use and smoking habit. High-risk (HR) types: 16, 18, 26, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 ISO39. Low-risk (LR) types: 6, 11, 40, 42, 43, 44, 82 (MM4), 83 (MM7), 84 (MM8), 71 (CP8061), CP6108, 81 (CP8304), 54, 55, 57, 61, 70, 72, 73. Chaouki et al, 1998; Chichareon et al, 1998; Ngelangel et al, 1998; our assay a cut-off point was defined using three times mean Rolon et al, 2000). OD of the negative controls. Briefly, in the enzyme immune assay (EIA), 5 ml of the biotiny- The HPV positive samples were analysed successively in other lated PCR products were captured in streptavidine-coated specific EIA probe sub-cocktails and finally specific oligoprobes microtitle plates (Roche, Mannheim, Germany). Subsequently, were used to identify each individual HPV type. Samples which the wells were washed three times with 16SSC; the captured were positive by Southern blot analyses and negative by HR and DNA was denatured by alkaline treatment with 0.1 M NaOH LR EIA, were considered as positive for HPV of undetermined type and hybridised to digoxigenin-labelled type-specific oligoprobes. (HPV X). After several washings the hybrids were detected using anti-digox- igenin (Fab fragments) labelled with alkaline phosphatase (Roche, Data analysis Mannheim, Germany) and paranitrophenyl phosphate (Sigma, USA) was used as substrate. The optical density (OD) was We computed odds ratios (ORs) and 95% confidence intervals (CI) measured at 405 nm using a Labsystem Multiscan reader. In using unconditional logistic regression models, considering HPV British Journal of Cancer (2002) 87(3), 324 – 333 ã 2002 Cancer Research UK HPV in Colombia M Molano et al Table 4 Risk factors for HPV-DNA detection in women with normal cytology by age groups Age 525 years 25 – 34 years 535 years n=450 n=741 n=668 a a a HPV+ (%) OR HPV+ (%) OR HPV+ (%) OR Education Low 15.5 1 14.8 1 9.1 1 Intermediate 23.6 1.6 13.7 1.0 6.0 0.7 (0.8 – 3.5) (0.6 – 1.7) (0.3 – 1.4) High 35.0 2.6 16.5 1.0 10.1 1.1 (1.1 – 6.1) (0.5 – 2.0) (0.5 – 2.6) Number of regular partners 1 22.6 1 12.4 1 9.4 1 52 38.8 2.1 23.0 1.9 7.0 0.6 (1.1 – 4.1) (1.2 – 3.0) (0.3 – 1.3) Casual partners No 21.9 1 13.7 1 9.1 1 5Yes 33.3 1.7 17.2 1.1 7.2 0.7 (1.0 – 2.9) (0.6 – 1.7) (0.3 – 1.6) Parity 0 31.5 1 30.2 1 10.0 1 1 – 2 21.1 0.9 13.4 0.5 8.0 0.6 (0.5 – 1.5) (0.2 – 1.2) (0.1 – 3.0) 3+ 22.2 1.2 15.0 0.5 8.7 0.6 (0.4 – 3.7) (0.2 – 1.2) (0.1 – 2.9) Oral contraceptives Never 23.0 1 11.8 1 7.9 1 Ever 27.0 1.4 18.0 1.5 9.4 1.4 (0.9 – 2.4) (0.9 – 2.3) (0.5 – 2.1) Former user 23.0 1.2 19.0 1.6 9.3 1.3 (0.7 – 2.1) (1.0 – 2.5) (0.7 – 2.5) Current user 34.6 2.0 12.7 0.9 11.1 1.7 (0.6 – 1.5) (0.4 – 2.2) (03 – 8.2) OR =odds ratio adjusted for age, education, number of regular partners, parity, age at first sexual intercourse, oral contraceptives, condom use and smoking habit. infections as dependent variables and several known or hypothe- HPV-DNA prevalence sised risk factors for cervical cancer as independent variables (STATA; Stata Press, College Station, TX, USA). We performed The overall HPV-DNA prevalence rate was 14.9% (Table 1), and 32 both age adjusted (grouped as 520, 20 – 24, 25 – 29, 30 – 34, 35 – different HPV types were detected. Of the HPV-DNA positive 39, 40 – 44, 45 – 54, 55 or more years) and multivariate analyses. women, 9% were infected with HR types only, 3.2% with LR types The following variables were included in the models: age (as cate- only, and 0.4% with HPV X. 2.3% of women were infected with gorised above), educational level (low, intermediate and high), both HR/LR types, which were grouped together with HR types number of regular (1, 2, 3 or more) sexual partners, parity (0, in all analysis. Thus the total HR type prevalence was 11.4% (Table 1 – 2, 3 or more children), age at first sexual intercourse (less 17, 1). HPV-DNA age-specific prevalence was highest among women 17 – 19, 20 or more years), use of oral contraceptives (ever or never below age 20 (26%), and lowest among women aged 45 – 54 years use), condoms (ever or never use), and smoking habits. The differ- (2.3%). Women aged 55 or more years had a prevalence rate of ent age-adjusted and multivariate models did not differ materially 13.2% (Table 1). from each other. Therefore we present in this report only results of HR HPV types were at least three times more prevalent than LR the fully adjusted multivariate models. types in all age groups, except among women aged 55 or more years (Table 1). Similarly to the overall HPV prevalence, the HR age-speci- fic prevalence rate curve presented a U-shape (Figure 1), the highest RESULTS prevalence being among women aged less than 20 years (20.4%), the lowest among women aged 45 – 54 years (2.3%), and an intermediate Characteristics of the study population among women aged 55 years or more (5.7%). For LR types, the age The characteristics of the study population are summarised in Table specific pattern was rather different: while no women aged 45 – 54 1. Most of the participating women were aged 25 – 34 years years were positive, those aged 55 years or more had the highest (median age 32), had low (36%) or intermediate (40%) educational prevalence rate (7.6%) of all age groups. Thus, among older women, level, had their first sexual intercourse or first regular sexual partner LR types were more common than HR types (Figure 1). before the age of 20 (median 18 and 19 years, respectively), Single infections (infections with only one HPV type) were reported a single regular lifelong sexual partner (74%), and had detected in 194 women (10.4% of the entire study population; at least two full term pregnancies (median=2). Intrauterine device 70.3% of the HPV positive women). Among these women, the (IUD) was the most common contraceptive method ever used most common HPV HR types were 16 (16.3% of the women with (56%), followed by oral contraceptives (46.3%) and condoms single infections), 58 (6.2%), 56 (3.6%) and 18 and 51 (2.9%). The (33.5%). Only one third of the women had ever smoked regularly. most frequent HPV LR types were HPV 81 (CP8304) (3.6%), 42 ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology Molecular and Cellular Pathology HPV in Colombia M Molano et al Table 5 Risk-factors for HPV-DNA multiple detection in women from Bogota, Colombia HPV single detection HPV multiple detection a a Number OR Number OR Age (years) 520 33 2.8 (1.3 – 6.3) 27 10.8 (3.0 – 38.4) 20 – 24 34 3.1 (1.7 – 5.7) 16 6.8 (2.3 – 20.2) 25 – 29 28 1.2 (0.7 – 2.2) 15 3.7 (1.3 – 10.3) 30 – 34 56 2.1 (1.3 – 3.4) 11 2.1 (0.7 – 6.0) 35 – 44 33 1 (reference) 7 1 (reference) 545 10 0.8 (0.4 – 1.8) 6 3.2 (1.0 – 10.7) Education Low 57 1 20 1 Intermediate 81 1.0 (0.7 – 1.5) 34 1.1 (0.5 – 2.2) High 56 1.2 (0.7 – 1.9) 28 2.1 (1.0 – 4.5) Number of regular sexual partners 1 138 1 54 1 2 36 1.3 (0.9 – 2.0) 16 1.7 (0.9 – 3.2) 3 9 2.0 (0.9 – 4.3) 4 2.9 (0.9 – 9.4) Parity 0 35 1 26 1 1 – 2 97 0.7 (0.4 – 1.3) 39 0.6 (0.3 – 1.2) 53 62 0.9 (0.4 – 1.7) 17 0.5 (0.2 – 1.3) Oral contraceptives Never 92 1 40 1 Ever 101 1.3 (1.0 – 1.8) 38 1.6 (1.0 – 2.7) Former user 83 1.3 (0.9 – 1.9) 28 1.6 (0.9 – 2.7) Current user 18 1.3 (0.7 – 2.4) 10 1.8 (0.8 – 3.9) Condom Never 110 1 55 1 Ever 83 1.2 (0.9 – 1.7) 25 0.5 (0.3 – 0.9) OR =odds ratio adjusted for age, education, number of regular partners, parity, age at first sexual intercourse, oral contracep- tives, condom use and smoking habit. (2.5%), 40 (1.9%) and 70 (1.4%). The prevalence of single HPV In women below the age of 25 years, high educational level (OR infection decreased with age, the same HPV types being the 2.6, 95% CI 1.1 – 6.1), and more than a single regular (OR 2.1, commonest ones in all age groups (Table 2). 95% CI 1.1 – 4.1) or casual (OR 1.7, 95% CI 1.0 – 2.9) sexual partner Multiple infections (infections with two or more HPV types) predicted infection risk (Table 4). There was also a suggestion of an were detected in 82 women (4.4% of the entire study population; increased risk with current use of oral contraceptives (OR 2.0, 95% 29.7% of the HPV positives). Women aged less than 25 years had CI 0.6 – 1.5 – Table 4). The addition of women attending an adoles- an almost five times higher prevalence rates of multiple infections cent clinic (age 13 – 17 years) did not generate a bias in the study (9.6%) than women aged 35 years or more (1.9%). Most of the results, since no statistically significant differences were found when multiple infections were caused by HR/LR types (53.7%) or HR/ they were excluded from the analysis. The association with number HR types (42.7%) and less (3.6%) by LR/LR types. Among women of partners persisted with a slightly lower odds ratio (OR 1.7, 95% with multiple infections, HR types were present in 97.6% of those CI 0.7 – 4.2). On the other hand, the association with education women aged less than 25 years, 100% among those aged 25 – 34 was stronger (OR 7.4, 95% CI 1.9 – 29.0 for high level of education). years, and 85.7% among those aged 35 – 54 years, and 83.3% among In the age group 25 – 34 years, having had more than one regu- those aged 55 or more years. HPV 35, 43, 44 and CP6108 were lar sexual partner increased HPV infection risk (OR 1.9, 95% CI detected only in multiple infections (data not shown). 1.3 – 3.0 – Table 4). There was no clear association between the existence of casual sexual partners, age at first sexual intercourse or first birth, use of IUDs or condoms and risk. Former use of oral Risk factors for HPV infections contraceptives increased the infection risk slightly (OR 1.6, 95% CI Besides age, number of regular sexual partners and OC use, no 1.0 – 2.5) and parity slightly decreases the risk (OR 0.5, 95% CI other risk factors were clearly associated with overall risk for 0.2 – 1.2). No clear pattern of association was observed among HPV infections in our study population (Tables 1 and 3). For women aged 35 or more years. The study had insufficient women HR HPV infections, there was a trend of a decreased risk among of the age of 55 years or more to allow further subgroup analysis women with parities (OR 0.7), and of an increased risk among with meaningful statistical power. users of oral contraceptives (OR 1.4, 95% CI 1.0 – 1.9). For LR Young age and high level of attained education were better types, current use of oral contraceptives increased the infection risk predictors of multiple infections than of single ones (Table 5). significantly (OR 3.1, 95% CI 1.3 – 7.2) and use of IUD also tended For multiple infections, there was a trend for a protective effect to increase the risk of infection (OR 1.6, 95% CI 0.8 – 3.0 – Table of parity (for three or more children compared to nulliparous, 3). Although there was no statistically significant interaction OR 0.5, 95% CI 0.2 – 1.3) and use of condoms (OR 0.5, 95% CI between the effects of age and other potential risk factors for 0.3 – 0.9) (Table 5). In most instances, however, examined charac- HPV infections, some interesting age-specific patterns emerged in teristics shared similar associations with single and multiple the analysis, of which some are presented in Table 4. infections. British Journal of Cancer (2002) 87(3), 324 – 333 ã 2002 Cancer Research UK HPV in Colombia M Molano et al with cervical cancer and the alignment analysis of the E6 gene DISCUSSION (modified from Myers et al, 1996). However the percentage of To date, few studies evaluated age-specific HPV prevalence patterns these HPVs is very low and for absolute HR or LR classification, and determinants of HPV infection among women with normal transformation studies in combination with follow up epidemiolo- cytology in countries with high incidence of cervical cancer. We gical studies are necessary. report here the first investigation of this type done in Colombia. The presence of multiple infections (29.7%) was higher than An overall HPV DNA prevalence of 14.9% was found, which is previously observed among control subjects in the IARC studies done similar to that reported in other high-risk populations in Mexico in Brazil (0%) (Eluf-Neto et al, 1994), the Philippines (14.3%) (14.5%) (Lazcano et al, 2001) and Costa Rica (16%) (Herrero et (Ngelangel et al, 1998) Thailand (9.8%) (Chichareon et al, 1998), al, 2000). In addition, it is similar to the HPV-DNA prevalence Morocco (5.3%) (Chaouki et al, 1998) and Paraguay (16.7%) (Rolon among control women (13%) in a concurrent case – control study et al, 2000), lower than in a population-based study from Costa Rica in Cali, Colombia (Mun ˜ oz et al, 1996). (39%) (Herrero et al, 2000) and similar to the results from a study Several studies have suggested that the prevalence of HPV infec- done in The Netherlands (28%) (Jacobs et al, 2000), where 3305 cyto- tion decreases with age, HPV being uncommon in cytological logical normal cervical scrapes were analysed using the same normal women over age 35 years (Melkert et al, 1993; Morrison, laboratory technique as in the present study. These differences in 1994). However, a few recent large population-based surveys from prevalence of multiple infections could be due to differences in the Costa Rica and Mexico also presented some increase in HPV infec- technique used (sensitivity, specificity and types identified), or real tion among women in peri- and post-menopausal ages. While in differences in the prevalence of the HPV types searched for and iden- Costa Rica the peak was observed in women older than 55 years, tified in the populations studied. In addition, these populations with a predominance of LR HPV types, in Mexico the second peak differed in age-composition: mean age 32 years in this study, whereas started earlier, after 45 years of age, with predominance of HR in Thailand it was 49.7 years. Taking into account the age-dependent HPV types and with an increase in detection of LR HPV types prevalence of multiple infections, this fact may also explain the compared to younger ages. discrepancy in results. In our study, 97% of the women with multiple In our study the HPV prevalence in the age group 45 – 54 years was infections presented a HR HPV type. The long-term follow up of low, but there was an increased prevalence of HPV infection in these infections will help to clarify the role of HR multiple infections women aged 55 or more years, with a predominance of LR types in the development of cervical lesions. and multiple HPV infections, but still 5.7% were HR HPV positive. Besides age, number of regular sexual partners and OC use If confirmed, the risk of HPV infection – most notably LR types (specially in women below age 35 years) which were risk factors – among post-menopausal women, it may be explained in different for HPV positivity, other reproductive and sexual behaviour factors ways: (1) reactivation of latent HPV infections by decreased immune considered by us were only weakly and inconsistently related to response. Some immunological studies show a decrease in circulating HPV infection. It might be explained by the relatively high mature T cells in older people as a result of a decrease in CD8+ frequency of HPV infections in the studied women and possibly lymphocytes, a decline in the frequency of CD4+ T cells producing the predominance of ‘male role’ in the transmission of HPV infec- IL-2 and/or a decreased expression in IL-2 receptors (Lesourd and tions to women (particularly over age 35 years). Unfortunately, no Meaume, 1994; Gostout et al, 1998; Ginaldi et al, 1999). (2) Reactiva- information was available on the sexual behaviour of the partners tion of latent HPV infections by hormonal changes related to the in our study population. Our results suggest an age-dependent gradual decline of ovarian function around menopause (Lazcano et association between HPV infection and some risk factors. A possi- al, 2001). (3) A cohort effect (Herrero et al, 2000), where older ble explication of the highest prevalence of HPV infections among women were exposed to the virus early in life and belonged to genera- young, highly educated women and with more than one regular or tions more heavily exposed to HPV infections. casual sexual partner could reflect changes in lifestyle and sexual An alternative explanation for this second peak lies in the fact behaviour in younger generations (possibly reduced influence of that only women with normal cervical cytology were included in religion and greater freedom). this study. Persistent infections with HR HPV types will lead to We observed an increase of HPV infections and particularly of LR cervical intraepithelial lesions with subsequent treatment. It may types in women older than 55 years and we tried to analyse if these result in an under-representation of HR HPV types and the women were different in terms of exposure to risk factors. Due to proportion of them in the population will diminish at older age. the low number of women in this group we could not find a clear In contrast, LR HPV infections that rarely give rise to cervical pattern of association. We found that use of oral contraceptives dysplasia will remain (Jacobs et al, 2000). may be a risk factor for both HR and LR HPV infections. Use of We identified 32 different HPV types in our study population. exogenous hormones – as contraceptives or hormone replacement This broad diversity of infections is consistent with previous – could influence HPV infections in different ways. Results from studies showing a greater HPV heterogeneity in mild cervical laboratory work have indicated that the HPV genome contains a dysplasias than in severe dysplasias (De Roda Husman et al, hormone-recognition segment, which might show an interactive 1994; Liaw et al, 1999; Jacobs et al, 2000; Lazcano et al, 2001). It effect of oral contraceptives and HPV (Monsonego et al, 1991). Addi- has been estimated that at least 50% of sexually active adults have tionally, steroid hormones do interfere with cellular gene function ever had a genital HPV infection, most of these, however, being involved in cell cycle regulation and apoptosis, and they also might transient and resolving spontaneously (Koutsky, 1997). inhibit the immunologically mediated resolution of minor HPV- In our study HPV 16, 58, 56, 52, HPV 81 (CP8304), 51 and 18 induced cervical lesions (Von Knebel Doeboritz et al, 1997). were the most prevalent HPV types detected as single infections in With respect to parity, in our study there was a trend of all age groups. Although HPV16 is the predominant type detected decreased risk of HR and multiple HPV infections with increase in the majority of studies (Jacobs et al, 2000; Lazcano et al, 2001; of number of births. Previous studies have presented conflicting Rolon et al, 2000), remarkable differences were noted in our study results: an increase in risk with number of births (Hildesheim et population with a high prevalence of other HPV types such as al, 1993) or decrease or no association with parity at all (Lazcano HPV 56, 58 and the LR type HPV 81 (CP8304). This information et al, 2001). There is still insufficient data to give final conclusions should be taken into account when developing HPV vaccines about the effect of number of births on the risk of HPV infections. tailored to this population. In conclusion, our results showed a broad diversity of HPV In this study HPV 26, 53, 73, 34 and Iso 39 were analysed as infections in women with normal cytology, with HR types being types with oncogenic potential according their presence in patients at least three times more common than LR types. Whereas young ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology Molecular and Cellular Pathology HPV in Colombia M Molano et al women were particularly at risk of HR and multiple HPV infec- are indebted to all the study participants, gynaecologists, nurses tions, post-menopausal women showed an increased frequency of and social workers who collaborated in the fieldwork, to L Rozen- LR HPV types. In this population, high educational level and daal for helpful comments, R van Andel, R Pol, N Fransen- multiplicity of sexual partners seem to determine risk among Daalmeijer and H Schrijnemakers for technical support. M Molano young women. No other hypothesized risk factor was clearly asso- obtained a fellowship from Colciencias of the Colombian Govern- ciated with the risk of HPV infection. ment. 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Int J Cancer 68: 766 – 769 ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

Prevalence and determinants of HPV infection among Colombian women with normal cytology

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Springer Journals
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Copyright © 2002 by The Author(s)
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Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
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0007-0920
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1532-1827
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10.1038/sj.bjc.6600442
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Abstract

Molecular and Cellular Pathology British Journal of Cancer (2002) 87, 324 – 333 ã 2002 Cancer Research UK All rights reserved 0007 – 0920/02 $25.00 www.bjcancer.com Prevalence and determinants of HPV infection among Colombian women with normal cytology 1,2 2 3 ,1 2 3 1 M Molano , H Posso , E Weiderpass , AJC van den Brule* , M Ronderos , S Franceschi , CJLM Meijer , 3 3 4 A Arslan , N Munoz and the HPV Study Group Department of Pathology, Unit of Molecular Pathology, Vrije Universiteit Medical Centre, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands; 2 3 Division de Investigacion, Instituto Nacional de Cancerologia, Bogota, Colombia; Unit of Field and Intervention Studies, International Agency for Research on Cancer, Lyon, France Human papillomavirus is the principal risk factor associated with cervical cancer, the most common malignancy among women in Colombia. We conducted a survey, aiming to report type specific prevalence and determinants of human papillomavirus infection in women with normal cytology. A total of 1859 women from Bogota, Colombia were interviewed and tested for human papillomavirus using a general primer GP5+/GP6+ mediated PCR – EIA. The overall HPV DNA prevalence was 14.8%; 9% of the women were infected by high risk types, 3.1% by low risk types, 2.3% by both high risk/low risk types and 0.4% by uncharacterized types (human papillomavirus X). Thirty-two different human papillomavirus types were detected, being human papillomavirus 16, 58, 56, 81(CP8304) and 18 the most common types. The human papillomavirus prevalence was 26.1% among women younger than 20 years, 2.3% in women aged 45 – 54 years, and 13.2% in women aged 55 years or more. For low risk types the highest peak of prevalence was observed in women aged 55 years or more. Compared to women aged 35 – 44 years, women aged less than 20 years had a 10-fold increased risk of having multiple infections. Besides age, there was a positive association between the risk of human papillomavirus infection and number of regular sexual partners and oral contraceptive use. In women aged below 25 years, high educational level and having had casual sexual partners predicted infection risk. In conclusion, there was a broad diversity of human papillomavirus infections with high risk types being the most common types detected. In this population multiplicity of sexual partners and, among young women, high educational level and casual sexual partners seem to determine risk. British Journal of Cancer (2002) 87, 324 – 333. doi:10.1038/sj.bjc.6600442 www.bjcancer.com ã 2002 Cancer Research UK Keywords: HPV; Colombia; epidemiology; cervix uteri Cervical cancer is the most common cancer among women in On the basis of their main association with benign or (pre-) developing countries, and the sixth most common cancer in malignant lesions, HPVs can be subdivided into low risk (LR) women in developed countries (Parkin et al, 1999). In Colombia, and high-risk (HR) oncogenic types. Women with normal cervical where the annual standardised incidence rate of cancer of the cytology who are infected with HR HPV have an approximately cervix is high (23 per 100 000), it is also the principal cause of 100-fold increased risk of developing CIN III compared to unin- cancer mortality among women (Globocan, 1998). fected women (Rozendaal et al, 1996). Therefore, it has been Infection with certain types of human papillomavirus (HPV) is suggested that HR HPV detection might be used as a tool to iden- the main cause of cervical cancer and its precursor lesions (Van tify women at high risk of cervical cancer, in addition to – or as den Brule et al, 1991; Munoz et al, 1992; Chichareon et al, 1998; an alternative to – Pap smears (Rozendaal et al, 1996, 2000; Meijer Ngelangel et al, 1998; Rolon et al, 2000). HPV infection is now et al, 1998). The marked geographic differences in incidence of cervical considered as virtually necessary but not sufficient to cause this malignancy (Walboomers et al, 1999). Other host factors such as cancer are not only the result of differences in screening patterns, genetic alterations, endogenous female hormones and immune but also of differences in exposure to risk factors. For example, the status or environmental factors (e.g. smoking and use of exogenous very low incidence rates (2 – 4 per 100 000) reported from some hormones) are also likely to be involved in cervical carcinogenesis areas in China and Spain (Parkin et al, 1997) where population- (Bosch et al, 1992; Ngelangel et al, 1998; Gostout et al, 1998; based screening programs do not exist, are probably the result of Palefsky et al, 1999). low exposure to HPV resulting from the conservative sexual beha- viour prevalent in the previous decades. To investigate geographic differences in the prevalence of type- *Correspondence: AJC van den Brule; E-mail: vandenbrule@vumc.nl specific HPV infection and of other risk factors for cervical cancer, HPV Study Group: M Gonzalez, J Luna, G Martinez, E Mora, G Perez, the International Agency for Research on Cancer is co-ordinating a JM Fuentes, C Gomez, E Klaus, C Camargo, C Tobon, T Palacio, C Suarez, series of surveys of prevalence of HPV infection and CIN lesions in C Molina countries with high and low incidence of cervical cancer, this study Received 22 January 2002; revised 17 April 2002; accepted 8 May 2002 being one of them. HPV in Colombia M Molano et al Table 1 Characteristics of the study population of HPV-DNA detection among women with normal cytology in Bogota, Colombia Proportion of women positive for HPV- DNA b a a No Overall % % any HPV-DNA %HR %LR Total 1845 100 14.9 11.4 3.2 Age (years) 520 230 12.4 26.1 20.4 5.2 20 – 24 220 11.8 22.7 18.2 4.1 25 – 29 337 18.1 12.7 10.4 1.8 30 – 34 404 21.7 16.6 11.9 4.2 35 – 39 284 15.3 8.1 6.0 2.1 40 – 44 192 10.3 8.1 7.8 0.5 45 – 54 86 4.6 2.3 2.3 0.0 555 106 5.7 13.2 5.7 7.6 Education Low 659 35.7 11.7 8.3 3.2 Intermediate 740 40.1 15.5 12.3 2.9 High 445 24.1 18.9 14.4 3.6 Missing 1 0.1 0.0 0.0 0.0 Age at 1st sexual intercourse 520 628 34.0 11.9 9.1 2.5 17 – 19 585 31.7 14.7 10.9 3.4 416 632 34.3 18.2 14.1 3.6 Age at 1st regular sexual intercourse 520 759 41.1 11.7 8.9 2.5 17 – 19 556 30.1 15.6 11.7 3.6 416 478 26.0 18.6 14.4 3.5 Missing 52 2.8 21.1 15.3 5.7 Number of regular sexual partners 1 1370 74.3 14.0 10.9 2.7 2 299 16.2 17.4 12.7 3.7 3 59 3.2 22.0 15.2 6.8 Missing 117 6.3 16.2 11.1 5.1 Casual sexual partners No 1307 70.8 13.8 10.5 2.8 Yes 445 24.1 18.2 13.9 4.0 Missing 93 5.0 15.0 10.7 4.3 Parity 0 – 1 644 34.9 29.2 22.0 6.2 2 – 3 845 45.8 14.4 11.3 2.5 54 356 19.3 11.4 8.3 3.2 Age at first birth 521 843 45.7 10.4 7.8 2.2 18 – 20 493 26.7 16.2 12.4 3.4 417 293 15.9 15.7 12.6 3.1 Missing 216 11.7 28.7 21.3 6.5 Oral contraceptives Never 957 51.9 13.8 10.3 3.0 Ever 845 46.3 16.3 12.4 3.5 Former user 721 39.1 15.4 12.0 2.9 Current user 133 7.2 21.0 14.3 6.7 Missing 34 1.8 14.7 14.7 0.0 IUD (Intrauterine Device) Never 779 42.2 15.8 12.2 3.1 Ever 1032 56.0 14.2 10.7 3.3 Former user 524 28.4 14.1 10.5 3.6 Current user 508 27.6 14.4 10.8 2.9 Missing 34 1.8 17.6 14.7 2.9 Condom Never 1203 65.2 13.7 10.2 3.1 Ever 618 33.5 17.5 13.7 3.4 Missing 24 1.3 12.5 8.3 4.1 Continued ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology Molecular and Cellular Pathology HPV in Colombia M Molano et al Table 1 (Continued ) Proportion of women positive for HPV- DNA b a a No Overall % % any HPV-DNA %HR %LR Smoking Never 1343 72.8 14.7 11.3 3.0 Ever 502 27.2 15.7 11.6 3.6 Former smoker 205 11.1 14.1 9.7 3.9 Current smoker 297 16.1 16.8 12.8 3.4 High-risk (HR) types: 16, 18, 26, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 ISO39. Low-risk (LR) types: 6, 11, 40, 42, 43, 44, 82 (MM4), 83 (MM7), 84 (MM8), 71 (CP8061), CP6108, 81 (CP8304), 54, 55, 57, 61, 70, 72, 73. Proportion of women infected with any HPV types, HR and LR types, respectively, in each category of age, education, etc. When women were infected by both HR and LR simultaneously they were counted in the HR group only. Information on age was available for 1859 individuals. Include nulliparous women. The prevalence of HPV, as detected by HPV DNA, characteris- Biological specimens tically peaks after initiation of sexual activity and decreases with age. A second peak in peri-menopausal women has been recently During the gynaecological examination, cervical scrapes were described in some populations (Cuzick et al, 1999; Herrero et al, collected from each woman using two Ayre spatulas and two endo- 2000; Lazcano et al, 2001). It is currently unknown whether cervical brushes. The first spatula and brush were used for a different age-specific curves of HPV prevalence are attributable routine Pap smear, which was classified according to the Bethesda to an effect of age per se or to differences in risk between birth system terminology. The second spatula and brush and the remain- cohorts. ing cells of the first spatula and brush, were placed in a tube A prospective cohort study in Bogota, Colombia, was initiated in containing 5 ml of phosphate-buffered saline (PBS 16)+0.05% the early 1990s by the Colombian National Institute of Cancer and thiomersal. Cells were detached from the spatula and endocervical the International Agency for Research on Cancer (IARC). The aim brush through vortex, and subsequently centrifuged at 3000 g for of this study was to investigate the natural history of HPV infection 10 min. The cell pellet was resuspended in 1 ml buffer Tris-HCl and CIN lesions among low-income Colombian women. We report 10 mM pH 8.3 and stored at 7708C until their use. For analysis, here the results of a cross-sectional analysis of the cohort at enrol- 100 ml aliquots were boiled for 10 min at 1008C, cooled on ice ment, with focus on determinants of HPV infection among women and centrifuged for 1 min at 30006g. 10 ml of these pre-treated with normal cervical cytology. crude cell suspensions were used for PCR analysis (Van den Brule et al, 1990; De Roda Husman et al, 1994). To assess the quality of the target DNA, all pellets were pre-screened using a 209 base pair MATERIALS AND METHODS amplifying b-globin PCR using the primer combination BPCO3 and BPCO5 as described by De Roda Husman et al (1995b). Study population Between November 1993 and November 1995 the Colombian HPV detection by PCR National Cervical Cancer Control Program conducted a census in four health districts in Bogota, which had no previously HPV-DNA detection was performed by a standard GP5+/GP6+ implemented population-based cervical screening program. The PCR based assay, as described by De Roda Husman et al first 2000 women aged 18 – 85 years identified in the census (1995a). Briefly, the GP – PCR reaction was carried out using were invited to participate in the study. Additionally, 200 50 ml of PCR solution containing 10 mM Tris HCL pH 8.3, adolescents aged 13 – 17 years were invited to participate. They 50 mM KCl, 200 mM of each deoxynucleotide, 3.5 mM of MgCl , were identified consecutively from an adolescent clinic giving 1 U of DNA polymerase (AmpliTaq; Perkin-Elmer, USA) and contraceptive counselling in the study area. At recruitment, all 25 pmol of each of the GP5+ and biotinylated GP6+ primers women were interviewed face-to-face by specially trained inter- (Eurogentec, Belgium): 40 cycles of amplification were carried viewers. They answered to a structured questionnaire on out using a Perkin-Elmer 9600,USA thermocycler. Each cycle socio-demographic characteristics, lifelong sexual behaviour, included a denaturation step at 958C for 1 min, one annealing step reproductive and menstrual history, and smoking and dietary at 408C for 1 min, and a chain elongation step at 728C for 1.5 min. habits. After interview all women underwent a pelvic examina- The first step was preceded by a denaturation step of 4 min and tion. the last step was followed by an elongation step of 10 min. From the 2200 invited women, 53 refused to participate, eight Three dilutions of the cell line SiHa containing 1 – 10 copies of were considered ineligible (mental illness, hysterectomy, history HPV16 (100 pg, 1 ng and 10 ng) were used as positive control. As of cervical cancer) and 29 did not provide cell specimens for negative PCR controls, distilled water and processing blanks were HPV DNA detection, so that 2110 women had cell specimens to used every 10 samples. HPV positivity was assessed by Southern be analysed. Of them 150 had abnormal cytology (according to blot hybridization of GP5+/GP6+ PCR products with a cocktail the Bethesda system classification) and 101 of the women with probe of specific [a- P]dCTP labelled DNA fragments from cloned normal cytology were beta-globin PCR negative (which is consid- DNA of HPV6, 11, 16, 18, 31 and 33 under low stringent condi- ered an indicator of poor DNA quality). After these exclusions, tions (Van den Brule et al, 1990; De Roda Husman et al, 1995a,b). 1859 women remained in the analysis presented here. In the multi- variate analysis (see below) we further excluded 14 women who did Type specific HPV detection not answer the questionnaire completely, resulting in 1845 women with normal cytology and risk factors analysis. Informed consent HPV positive samples were first subjected to HPV group-specific was obtained from all participants included in the study. The local analysis using cocktail probes for HR and LR HPVs (Jacobs et al, ethical committee and the ethical committee at IARC approved the 1997, 2000). The HR HPV cocktail probe consisted of oligoprobes study protocol. for HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 British Journal of Cancer (2002) 87(3), 324 – 333 ã 2002 Cancer Research UK HPV in Colombia M Molano et al HR% LR% HPVX% 45 –54 55&+ <20 20 –24 25 –29 30–34 35 – 39 40–44 (n =230) (n =220) (n = 337) (n = 404) (n =284) (n =192) (n =86) (n =106) Age Figure 1 Prevalence of high and low risk of human papillomavirus among women with normal cytology from Bogota, Colombia Table 2 Prevalence of type-specific HPV single infection by age-group, in women with normal cytology Age 525 years Age 25 – 34 years Age 535 years All ages (n=450) (n=741) (n=668) (n=1859) HPV type No No No No % 6 010 1 0.4 11 1 2 1 4 1.4 16 12 20 13 45 16.3 18 4 4 0 8 2.9 31 2 2 1 5 1.9 33 1 2 1 4 1.4 34 0 1 0 1 0.4 39 2 3 1 6 2.2 40 3 1 1 5 1.9 42 2 2 3 7 2.5 45 2 1 0 3 1.1 51 2 3 3 8 2.9 52 1 3 2 6 2.2 53 0 2 0 2 0.7 54 2 0 2 4 1.4 56 5 3 2 10 3.6 58 9 5 3 17 6.2 59 0 1 0 1 0.4 61 0 1 0 1 0.4 66 2 2 0 4 1.4 68 0 2 0 2 0.7 70 2 1 1 4 1.4 72 1 1 0 2 0.7 81 (CP8304) 3 5 2 10 3.6 Iso 39 0 1 0 1 0.4 82 (MM4) 0 1 0 1 0.4 83 (MM7) 2 0 0 2 0.7 84 (MM8) 0 2 2 4 1.4 HR (cocktail) 3 3 3 9 3.3 LR (cocktail) 4 5 1 10 3.6 HPV X 2 4 1 7 2.5 Total 67 84 43 194 70.3 % relative to 276 HPV-positive women. and the LR HPV consisted of oligoprobes for HPV 6, 11, 40, 42, considered in the results as HR types, based on both the align- 43, 44, HPV 82 (MM4), HPV83 (MM7), HPV84 (MM8), Iso39, ment analysis of the E6 gene (modified from Myers et al, 1996) HPV71 (CP8061), CP6108, HPV 81 (CP8304), HPV 26, 34, 53, and the risk estimates obtained for the various HPV types within 54, 55, 57, 61, 70, 72, 73. However HPV types with unknown a multi-center case – control study of cervical cancer conducted by oncogenic potential – namely 26, 53, 73, 34 and Iso 39 – were the IARC (Mun ˜ oz et al, 1992, Mun ˜ oz, 2000; Bosch et al, 1995; ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology Molecular and Cellular Pathology HPV in Colombia M Molano et al Table 3 Risk factors for HPV-DNA detection among women with normal cytology Positive for HPV-DNA Positive for HPV-DNA Positive for HPV-DNA a b detection HR types LR types a a a No OR No OR No OR Age (years) 520 60 4.2 (2.1 – 8.3) 47 3.7 (1.8 – 7.9) 12 9.6 (2.2 – 42.0) 20 – 24 50 3.7 (2.2 – 6.4) 40 3.5 (2.0 – 6.4) 9 6.0 (1.8 – 19.6) 25 – 29 43 1.6 (1 – 2.7) 35 1.7 (1.0 – 2.8) 6 3.8 (1.4 – 4.8) 30 – 34 67 2.1 (1.4 – 3.3) 48 1.8 (1.1 – 3.0) 17 3.8 (1.4 – 10.0) 35 – 44 40 1 (reference) 32 1 (reference) 7 1 (reference) 545 16 1.2 (0.6 – 2.3) 8 0.8 (0.3 – 1.8) 8 3.2 (1.0 – 10.3) Education Low 77 1 55 1 21 1 Intermediate 115 1 (0.7 – 1.5) 91 1.0 (0.7 – 1.6) 22 0.9 (0.4 – 2.0) High 84 1.4 (0.9 – 2.1) 64 1.4 (0.9 – 2.3) 16 1.3 (0.5 – 3.0) No of regular sexual partners 1 192 1 150 1 38 1 2 52 1.4 (1.0 – 2.0) 38 1.3 (0.9 – 2) 11 1.5 (0.7 – 3.1) 3 13 2.1 (1.1 – 4.2) 9 2 (0.9 – 4.5) 4 3.0 (0.9 – 9.7) Casual sexual partners No 181 1 138 1 37 1 Yes 81 1.1 (0.8 – 1.5) 62 1.1 (0.8 – 1.6) 18 1.3 (0.7 – 2.5) Parity 061 1 46 1 13 1 1 – 2 136 0.7 (0.4 – 1.1) 107 0.7 (0.4 – 1.1) 24 0.7 (0.3 – 1.8) 53 79 0.7 (0.4 – 1.3) 57 0.7 (0.4 – 1.4) 22 1.0 (0.3 – 3.0) Age at first sexual intercourse 520 75 1 57 1 16 1 17 – 19 86 1.0 (0.7 – 1.4) 64 1.0 (0.7 – 1.5) 20 1.0 (0.5 – 2.1) 416 115 1.0 (0.6 – 1.5) 89 1.0 (0.6 – 1.6) 23 0.7 (0.3 – 1.8) Oral contraceptives Never 132 1 99 1 29 1 Ever 139 1.4 (1.1 – 1.9) 106 1.4 (1.0 – 1.9) 30 1.6 (0.9 – 2.9) Former user 111 1.4 (1 – 1.9) 87 1.4 (1.0 – 2.0) 21 1.3 (0.7 – 2.5) Current user 28 1.5 (0.9 – 2.4) 19 1.2 (0.7 – 2.2) 9 3.1 (1.3 – 7.2) IUD Never 123 1 95 1 24 1 Ever 147 1.1 (0.8 – 1.5) 110 1.0 (0.7 – 1.4) 34 1.6 (0.8 – 3.0) Former user 74 1.4 (0.9 – 1.8) 55 1.2 (0.8 – 1.7) 19 1.8 (0.9 – 3.8) Current user 73 1.5 (0.7 – 1.4) 55 0.9 (0.6 – 1.3) 15 1.3 (0.6 – 2.9) Condom Never 165 1 123 1 37 1 Ever 108 1.1 (0.7 – 1.3) 85 1.0 (0.7 – 1.4) 21 0.8 (0.4 – 1.4) Smoking Never 197 1 152 1 41 1 Ever 79 1.1 (0.8 – 1.5) 58 1.1 (0.8 – 1.5) 18 1.2 (0.6 – 2.3) Former smokers 29 1.0 (06 – 1.6) 20 0.9 (0.5 – 1.6) 8 1.2 (0.5 – 3.0) Current smoker 50 1.2 (0.8 – 1.7) 38 1.2 (0.8 – 1.8) 10 1.2 (0.5 – 2.6) a a OR =odds ratio adjusted for age, education, number of regular partners, parity, age at first sexual intercourse, oral contraceptives, condom use and smoking habit. High-risk (HR) types: 16, 18, 26, 31, 33, 34, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 ISO39. Low-risk (LR) types: 6, 11, 40, 42, 43, 44, 82 (MM4), 83 (MM7), 84 (MM8), 71 (CP8061), CP6108, 81 (CP8304), 54, 55, 57, 61, 70, 72, 73. Chaouki et al, 1998; Chichareon et al, 1998; Ngelangel et al, 1998; our assay a cut-off point was defined using three times mean Rolon et al, 2000). OD of the negative controls. Briefly, in the enzyme immune assay (EIA), 5 ml of the biotiny- The HPV positive samples were analysed successively in other lated PCR products were captured in streptavidine-coated specific EIA probe sub-cocktails and finally specific oligoprobes microtitle plates (Roche, Mannheim, Germany). Subsequently, were used to identify each individual HPV type. Samples which the wells were washed three times with 16SSC; the captured were positive by Southern blot analyses and negative by HR and DNA was denatured by alkaline treatment with 0.1 M NaOH LR EIA, were considered as positive for HPV of undetermined type and hybridised to digoxigenin-labelled type-specific oligoprobes. (HPV X). After several washings the hybrids were detected using anti-digox- igenin (Fab fragments) labelled with alkaline phosphatase (Roche, Data analysis Mannheim, Germany) and paranitrophenyl phosphate (Sigma, USA) was used as substrate. The optical density (OD) was We computed odds ratios (ORs) and 95% confidence intervals (CI) measured at 405 nm using a Labsystem Multiscan reader. In using unconditional logistic regression models, considering HPV British Journal of Cancer (2002) 87(3), 324 – 333 ã 2002 Cancer Research UK HPV in Colombia M Molano et al Table 4 Risk factors for HPV-DNA detection in women with normal cytology by age groups Age 525 years 25 – 34 years 535 years n=450 n=741 n=668 a a a HPV+ (%) OR HPV+ (%) OR HPV+ (%) OR Education Low 15.5 1 14.8 1 9.1 1 Intermediate 23.6 1.6 13.7 1.0 6.0 0.7 (0.8 – 3.5) (0.6 – 1.7) (0.3 – 1.4) High 35.0 2.6 16.5 1.0 10.1 1.1 (1.1 – 6.1) (0.5 – 2.0) (0.5 – 2.6) Number of regular partners 1 22.6 1 12.4 1 9.4 1 52 38.8 2.1 23.0 1.9 7.0 0.6 (1.1 – 4.1) (1.2 – 3.0) (0.3 – 1.3) Casual partners No 21.9 1 13.7 1 9.1 1 5Yes 33.3 1.7 17.2 1.1 7.2 0.7 (1.0 – 2.9) (0.6 – 1.7) (0.3 – 1.6) Parity 0 31.5 1 30.2 1 10.0 1 1 – 2 21.1 0.9 13.4 0.5 8.0 0.6 (0.5 – 1.5) (0.2 – 1.2) (0.1 – 3.0) 3+ 22.2 1.2 15.0 0.5 8.7 0.6 (0.4 – 3.7) (0.2 – 1.2) (0.1 – 2.9) Oral contraceptives Never 23.0 1 11.8 1 7.9 1 Ever 27.0 1.4 18.0 1.5 9.4 1.4 (0.9 – 2.4) (0.9 – 2.3) (0.5 – 2.1) Former user 23.0 1.2 19.0 1.6 9.3 1.3 (0.7 – 2.1) (1.0 – 2.5) (0.7 – 2.5) Current user 34.6 2.0 12.7 0.9 11.1 1.7 (0.6 – 1.5) (0.4 – 2.2) (03 – 8.2) OR =odds ratio adjusted for age, education, number of regular partners, parity, age at first sexual intercourse, oral contraceptives, condom use and smoking habit. infections as dependent variables and several known or hypothe- HPV-DNA prevalence sised risk factors for cervical cancer as independent variables (STATA; Stata Press, College Station, TX, USA). We performed The overall HPV-DNA prevalence rate was 14.9% (Table 1), and 32 both age adjusted (grouped as 520, 20 – 24, 25 – 29, 30 – 34, 35 – different HPV types were detected. Of the HPV-DNA positive 39, 40 – 44, 45 – 54, 55 or more years) and multivariate analyses. women, 9% were infected with HR types only, 3.2% with LR types The following variables were included in the models: age (as cate- only, and 0.4% with HPV X. 2.3% of women were infected with gorised above), educational level (low, intermediate and high), both HR/LR types, which were grouped together with HR types number of regular (1, 2, 3 or more) sexual partners, parity (0, in all analysis. Thus the total HR type prevalence was 11.4% (Table 1 – 2, 3 or more children), age at first sexual intercourse (less 17, 1). HPV-DNA age-specific prevalence was highest among women 17 – 19, 20 or more years), use of oral contraceptives (ever or never below age 20 (26%), and lowest among women aged 45 – 54 years use), condoms (ever or never use), and smoking habits. The differ- (2.3%). Women aged 55 or more years had a prevalence rate of ent age-adjusted and multivariate models did not differ materially 13.2% (Table 1). from each other. Therefore we present in this report only results of HR HPV types were at least three times more prevalent than LR the fully adjusted multivariate models. types in all age groups, except among women aged 55 or more years (Table 1). Similarly to the overall HPV prevalence, the HR age-speci- fic prevalence rate curve presented a U-shape (Figure 1), the highest RESULTS prevalence being among women aged less than 20 years (20.4%), the lowest among women aged 45 – 54 years (2.3%), and an intermediate Characteristics of the study population among women aged 55 years or more (5.7%). For LR types, the age The characteristics of the study population are summarised in Table specific pattern was rather different: while no women aged 45 – 54 1. Most of the participating women were aged 25 – 34 years years were positive, those aged 55 years or more had the highest (median age 32), had low (36%) or intermediate (40%) educational prevalence rate (7.6%) of all age groups. Thus, among older women, level, had their first sexual intercourse or first regular sexual partner LR types were more common than HR types (Figure 1). before the age of 20 (median 18 and 19 years, respectively), Single infections (infections with only one HPV type) were reported a single regular lifelong sexual partner (74%), and had detected in 194 women (10.4% of the entire study population; at least two full term pregnancies (median=2). Intrauterine device 70.3% of the HPV positive women). Among these women, the (IUD) was the most common contraceptive method ever used most common HPV HR types were 16 (16.3% of the women with (56%), followed by oral contraceptives (46.3%) and condoms single infections), 58 (6.2%), 56 (3.6%) and 18 and 51 (2.9%). The (33.5%). Only one third of the women had ever smoked regularly. most frequent HPV LR types were HPV 81 (CP8304) (3.6%), 42 ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology Molecular and Cellular Pathology HPV in Colombia M Molano et al Table 5 Risk-factors for HPV-DNA multiple detection in women from Bogota, Colombia HPV single detection HPV multiple detection a a Number OR Number OR Age (years) 520 33 2.8 (1.3 – 6.3) 27 10.8 (3.0 – 38.4) 20 – 24 34 3.1 (1.7 – 5.7) 16 6.8 (2.3 – 20.2) 25 – 29 28 1.2 (0.7 – 2.2) 15 3.7 (1.3 – 10.3) 30 – 34 56 2.1 (1.3 – 3.4) 11 2.1 (0.7 – 6.0) 35 – 44 33 1 (reference) 7 1 (reference) 545 10 0.8 (0.4 – 1.8) 6 3.2 (1.0 – 10.7) Education Low 57 1 20 1 Intermediate 81 1.0 (0.7 – 1.5) 34 1.1 (0.5 – 2.2) High 56 1.2 (0.7 – 1.9) 28 2.1 (1.0 – 4.5) Number of regular sexual partners 1 138 1 54 1 2 36 1.3 (0.9 – 2.0) 16 1.7 (0.9 – 3.2) 3 9 2.0 (0.9 – 4.3) 4 2.9 (0.9 – 9.4) Parity 0 35 1 26 1 1 – 2 97 0.7 (0.4 – 1.3) 39 0.6 (0.3 – 1.2) 53 62 0.9 (0.4 – 1.7) 17 0.5 (0.2 – 1.3) Oral contraceptives Never 92 1 40 1 Ever 101 1.3 (1.0 – 1.8) 38 1.6 (1.0 – 2.7) Former user 83 1.3 (0.9 – 1.9) 28 1.6 (0.9 – 2.7) Current user 18 1.3 (0.7 – 2.4) 10 1.8 (0.8 – 3.9) Condom Never 110 1 55 1 Ever 83 1.2 (0.9 – 1.7) 25 0.5 (0.3 – 0.9) OR =odds ratio adjusted for age, education, number of regular partners, parity, age at first sexual intercourse, oral contracep- tives, condom use and smoking habit. (2.5%), 40 (1.9%) and 70 (1.4%). The prevalence of single HPV In women below the age of 25 years, high educational level (OR infection decreased with age, the same HPV types being the 2.6, 95% CI 1.1 – 6.1), and more than a single regular (OR 2.1, commonest ones in all age groups (Table 2). 95% CI 1.1 – 4.1) or casual (OR 1.7, 95% CI 1.0 – 2.9) sexual partner Multiple infections (infections with two or more HPV types) predicted infection risk (Table 4). There was also a suggestion of an were detected in 82 women (4.4% of the entire study population; increased risk with current use of oral contraceptives (OR 2.0, 95% 29.7% of the HPV positives). Women aged less than 25 years had CI 0.6 – 1.5 – Table 4). The addition of women attending an adoles- an almost five times higher prevalence rates of multiple infections cent clinic (age 13 – 17 years) did not generate a bias in the study (9.6%) than women aged 35 years or more (1.9%). Most of the results, since no statistically significant differences were found when multiple infections were caused by HR/LR types (53.7%) or HR/ they were excluded from the analysis. The association with number HR types (42.7%) and less (3.6%) by LR/LR types. Among women of partners persisted with a slightly lower odds ratio (OR 1.7, 95% with multiple infections, HR types were present in 97.6% of those CI 0.7 – 4.2). On the other hand, the association with education women aged less than 25 years, 100% among those aged 25 – 34 was stronger (OR 7.4, 95% CI 1.9 – 29.0 for high level of education). years, and 85.7% among those aged 35 – 54 years, and 83.3% among In the age group 25 – 34 years, having had more than one regu- those aged 55 or more years. HPV 35, 43, 44 and CP6108 were lar sexual partner increased HPV infection risk (OR 1.9, 95% CI detected only in multiple infections (data not shown). 1.3 – 3.0 – Table 4). There was no clear association between the existence of casual sexual partners, age at first sexual intercourse or first birth, use of IUDs or condoms and risk. Former use of oral Risk factors for HPV infections contraceptives increased the infection risk slightly (OR 1.6, 95% CI Besides age, number of regular sexual partners and OC use, no 1.0 – 2.5) and parity slightly decreases the risk (OR 0.5, 95% CI other risk factors were clearly associated with overall risk for 0.2 – 1.2). No clear pattern of association was observed among HPV infections in our study population (Tables 1 and 3). For women aged 35 or more years. The study had insufficient women HR HPV infections, there was a trend of a decreased risk among of the age of 55 years or more to allow further subgroup analysis women with parities (OR 0.7), and of an increased risk among with meaningful statistical power. users of oral contraceptives (OR 1.4, 95% CI 1.0 – 1.9). For LR Young age and high level of attained education were better types, current use of oral contraceptives increased the infection risk predictors of multiple infections than of single ones (Table 5). significantly (OR 3.1, 95% CI 1.3 – 7.2) and use of IUD also tended For multiple infections, there was a trend for a protective effect to increase the risk of infection (OR 1.6, 95% CI 0.8 – 3.0 – Table of parity (for three or more children compared to nulliparous, 3). Although there was no statistically significant interaction OR 0.5, 95% CI 0.2 – 1.3) and use of condoms (OR 0.5, 95% CI between the effects of age and other potential risk factors for 0.3 – 0.9) (Table 5). In most instances, however, examined charac- HPV infections, some interesting age-specific patterns emerged in teristics shared similar associations with single and multiple the analysis, of which some are presented in Table 4. infections. British Journal of Cancer (2002) 87(3), 324 – 333 ã 2002 Cancer Research UK HPV in Colombia M Molano et al with cervical cancer and the alignment analysis of the E6 gene DISCUSSION (modified from Myers et al, 1996). However the percentage of To date, few studies evaluated age-specific HPV prevalence patterns these HPVs is very low and for absolute HR or LR classification, and determinants of HPV infection among women with normal transformation studies in combination with follow up epidemiolo- cytology in countries with high incidence of cervical cancer. We gical studies are necessary. report here the first investigation of this type done in Colombia. The presence of multiple infections (29.7%) was higher than An overall HPV DNA prevalence of 14.9% was found, which is previously observed among control subjects in the IARC studies done similar to that reported in other high-risk populations in Mexico in Brazil (0%) (Eluf-Neto et al, 1994), the Philippines (14.3%) (14.5%) (Lazcano et al, 2001) and Costa Rica (16%) (Herrero et (Ngelangel et al, 1998) Thailand (9.8%) (Chichareon et al, 1998), al, 2000). In addition, it is similar to the HPV-DNA prevalence Morocco (5.3%) (Chaouki et al, 1998) and Paraguay (16.7%) (Rolon among control women (13%) in a concurrent case – control study et al, 2000), lower than in a population-based study from Costa Rica in Cali, Colombia (Mun ˜ oz et al, 1996). (39%) (Herrero et al, 2000) and similar to the results from a study Several studies have suggested that the prevalence of HPV infec- done in The Netherlands (28%) (Jacobs et al, 2000), where 3305 cyto- tion decreases with age, HPV being uncommon in cytological logical normal cervical scrapes were analysed using the same normal women over age 35 years (Melkert et al, 1993; Morrison, laboratory technique as in the present study. These differences in 1994). However, a few recent large population-based surveys from prevalence of multiple infections could be due to differences in the Costa Rica and Mexico also presented some increase in HPV infec- technique used (sensitivity, specificity and types identified), or real tion among women in peri- and post-menopausal ages. While in differences in the prevalence of the HPV types searched for and iden- Costa Rica the peak was observed in women older than 55 years, tified in the populations studied. In addition, these populations with a predominance of LR HPV types, in Mexico the second peak differed in age-composition: mean age 32 years in this study, whereas started earlier, after 45 years of age, with predominance of HR in Thailand it was 49.7 years. Taking into account the age-dependent HPV types and with an increase in detection of LR HPV types prevalence of multiple infections, this fact may also explain the compared to younger ages. discrepancy in results. In our study, 97% of the women with multiple In our study the HPV prevalence in the age group 45 – 54 years was infections presented a HR HPV type. The long-term follow up of low, but there was an increased prevalence of HPV infection in these infections will help to clarify the role of HR multiple infections women aged 55 or more years, with a predominance of LR types in the development of cervical lesions. and multiple HPV infections, but still 5.7% were HR HPV positive. Besides age, number of regular sexual partners and OC use If confirmed, the risk of HPV infection – most notably LR types (specially in women below age 35 years) which were risk factors – among post-menopausal women, it may be explained in different for HPV positivity, other reproductive and sexual behaviour factors ways: (1) reactivation of latent HPV infections by decreased immune considered by us were only weakly and inconsistently related to response. Some immunological studies show a decrease in circulating HPV infection. It might be explained by the relatively high mature T cells in older people as a result of a decrease in CD8+ frequency of HPV infections in the studied women and possibly lymphocytes, a decline in the frequency of CD4+ T cells producing the predominance of ‘male role’ in the transmission of HPV infec- IL-2 and/or a decreased expression in IL-2 receptors (Lesourd and tions to women (particularly over age 35 years). Unfortunately, no Meaume, 1994; Gostout et al, 1998; Ginaldi et al, 1999). (2) Reactiva- information was available on the sexual behaviour of the partners tion of latent HPV infections by hormonal changes related to the in our study population. Our results suggest an age-dependent gradual decline of ovarian function around menopause (Lazcano et association between HPV infection and some risk factors. A possi- al, 2001). (3) A cohort effect (Herrero et al, 2000), where older ble explication of the highest prevalence of HPV infections among women were exposed to the virus early in life and belonged to genera- young, highly educated women and with more than one regular or tions more heavily exposed to HPV infections. casual sexual partner could reflect changes in lifestyle and sexual An alternative explanation for this second peak lies in the fact behaviour in younger generations (possibly reduced influence of that only women with normal cervical cytology were included in religion and greater freedom). this study. Persistent infections with HR HPV types will lead to We observed an increase of HPV infections and particularly of LR cervical intraepithelial lesions with subsequent treatment. It may types in women older than 55 years and we tried to analyse if these result in an under-representation of HR HPV types and the women were different in terms of exposure to risk factors. Due to proportion of them in the population will diminish at older age. the low number of women in this group we could not find a clear In contrast, LR HPV infections that rarely give rise to cervical pattern of association. We found that use of oral contraceptives dysplasia will remain (Jacobs et al, 2000). may be a risk factor for both HR and LR HPV infections. Use of We identified 32 different HPV types in our study population. exogenous hormones – as contraceptives or hormone replacement This broad diversity of infections is consistent with previous – could influence HPV infections in different ways. Results from studies showing a greater HPV heterogeneity in mild cervical laboratory work have indicated that the HPV genome contains a dysplasias than in severe dysplasias (De Roda Husman et al, hormone-recognition segment, which might show an interactive 1994; Liaw et al, 1999; Jacobs et al, 2000; Lazcano et al, 2001). It effect of oral contraceptives and HPV (Monsonego et al, 1991). Addi- has been estimated that at least 50% of sexually active adults have tionally, steroid hormones do interfere with cellular gene function ever had a genital HPV infection, most of these, however, being involved in cell cycle regulation and apoptosis, and they also might transient and resolving spontaneously (Koutsky, 1997). inhibit the immunologically mediated resolution of minor HPV- In our study HPV 16, 58, 56, 52, HPV 81 (CP8304), 51 and 18 induced cervical lesions (Von Knebel Doeboritz et al, 1997). were the most prevalent HPV types detected as single infections in With respect to parity, in our study there was a trend of all age groups. Although HPV16 is the predominant type detected decreased risk of HR and multiple HPV infections with increase in the majority of studies (Jacobs et al, 2000; Lazcano et al, 2001; of number of births. Previous studies have presented conflicting Rolon et al, 2000), remarkable differences were noted in our study results: an increase in risk with number of births (Hildesheim et population with a high prevalence of other HPV types such as al, 1993) or decrease or no association with parity at all (Lazcano HPV 56, 58 and the LR type HPV 81 (CP8304). This information et al, 2001). There is still insufficient data to give final conclusions should be taken into account when developing HPV vaccines about the effect of number of births on the risk of HPV infections. tailored to this population. In conclusion, our results showed a broad diversity of HPV In this study HPV 26, 53, 73, 34 and Iso 39 were analysed as infections in women with normal cytology, with HR types being types with oncogenic potential according their presence in patients at least three times more common than LR types. Whereas young ã 2002 Cancer Research UK British Journal of Cancer (2002) 87(3), 324 – 333 Molecular and Cellular Pathology Molecular and Cellular Pathology HPV in Colombia M Molano et al women were particularly at risk of HR and multiple HPV infec- are indebted to all the study participants, gynaecologists, nurses tions, post-menopausal women showed an increased frequency of and social workers who collaborated in the fieldwork, to L Rozen- LR HPV types. In this population, high educational level and daal for helpful comments, R van Andel, R Pol, N Fransen- multiplicity of sexual partners seem to determine risk among Daalmeijer and H Schrijnemakers for technical support. M Molano young women. No other hypothesized risk factor was clearly asso- obtained a fellowship from Colciencias of the Colombian Govern- ciated with the risk of HPV infection. ment. 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