Population-Based Human Papillomavirus Prevalence in Lampang and Songkla, Thailand

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1 MAJOR ARTICLE Population-Based Human Papillomavirus Prevalence in Lampang and Songkla, Thailand Sukhon Sukvirach, 1 Jennifer S. Smith, 6 Sirirat Tunsakul, 1 Nubia Muñoz, 6 Vitaya Kesararat, 2 Oranuj Opasatian, 3 Saibua Chichareon, 4 Vichien Kaenploy, 5 Rhoda Ashley, 8 Chris J. L. M. Meijer, 9 Peter J. F. Snijders, 9 Pierre Coursaget, 7 Silvia Franceschi, 6 and Rolando Herrero 6,10 1 Research Division, National Cancer Institute, Bangkok, 2 Lampang Hospital and 3 Provincial Health Office, Lampang, and 4 Prince of Songkla University and 5 Provincial Health Office, Songkla, Thailand; 6 International Agency for Research on Cancer, Lyon, and 7 Molecular Virology Laboratory, INSERM, Tours, France; 8 University of Washington, Seattle; 9 Vrije Universiteit Medical Center, Amsterdam, The Netherlands; 10 Proyecto Epidemiologico Guanacaste, San Jose, Costa Rica To investigate the prevalence and determinants of human papillomavirus (HPV) infection, the primary cause of cervical cancer, we studied 1741 women 15 years of age from Lampang and Songkla, Thailand. Exfoliated cervical cells were collected for Papanicolaou smear screening and DNA detection of 36 different HPV types. Serum immunoglobulin G antibodies against L1 virus-like particles (anti-vlps) of HPV-16, -18, -31, -33, and -58 were evaluated using enzyme-linked immunosorbent assay. Overall, 110 women (6.3%) were HPV DNA positive; the most common types were HPV-16, -52, and -72. The age-standardized prevalence of HPV DNA was higher among the 1035 women from Lampang (9.1%; 95% confidence interval [CI], ) than among the 706 women from Songkla (3.9%; 95% CI, 2.3% 5.6%). Anti-VLPs were found in 21.8% of all women and were more frequent among women from Lampang (29.2%) than among women from Songkla (10.9%). Major risk factors for cervical HPV DNA were age!35 years, HSV-2 seropositivity, and having a husband with extramarital sexual partners. Invasive cervical cancer (ICC) is the second most common cancer among women worldwide and the leading cancer in Thailand [1]. The estimated age-standardized incidence rate of ICC in Thailand is 20.7/100,000 population [1]. In 1993, 5500 new cases were reported, and the age-standardized incidence rates of ICC were highest in Chiang Mai (25.7/100,000) and Lampang Received 15 July 2002; accepted 10 December 2002; electronically published 26 March Presented in part: 19th International Papillomavirus Conference, Florianopolis, Brazil, 1 7 September 2001 (abstract P 43). Written informed consent was obtained from patients, and the study protocols were approved by the International Agency for Research on Cancer (IARC) Ethical Review Committee and the National Research Council in Thailand. Financial support: Ministry of Public Health and Cancer Research Foundation, National Cancer Institute, Thailand; Terry Fox Run Fund; IARC. Reprints or correspondence: Dr. Silvia Franceschi, International Agency for Research on Cancer, Unit of Field and Intervention Studies, 150 cours Albert Thomas, F Lyon Cedex 08, France (franceschi@iarc.fr). The Journal of Infectious Diseases 2003; 187: by the Infectious Diseases Society of America. All rights reserved /2003/ $15.00 (23.1/100,000), in the north, and lowest in Songkla (15.8/100,000), in the south [2]. Human papillomavirus (HPV) infection is the primary cause of ICC [3, 4]. Genital HPV infection is one of the most prevalent sexually transmitted infections worldwide, and the prevalence is consistently higher among women with higher-risk sexual behavior [5, 6]. More than 35 different HPV types may infect the female genital tract, and at least 14 HPV types are considered to be oncogenic or high-risk (HR) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) [7]. To prevent HPV-associated cervical cancer in humans, candidate prophylactic HPV vaccines are being actively developed [8]. These vaccines are chiefly based on virus-like particles (VLPs) of HPV-16, alone or in conjunction with other HPV types. Vaccines against HPV-16 were shown to be safe and highly immunogenic [9] and efficacious in preventing persistent HPV- 16 infections [10]. Epidemiological data on the ageand type-specific prevalence of genital HPV infection 1246 JID 2003:187 (15 April) Sukvirach et al.

2 among women in different areas of the world are needed to define appropriate strategies for the introduction of an HPV vaccine. Little is currently known about the prevalence of HPV infection in population-based samples of women from areas throughout the world, particularly in Asia. In the framework of a multicenter study coordinated by the International Agency for Research on Cancer (IARC) of the prevalence of HPV infection in different countries worldwide, the National Cancer Institute in Bangkok, Thailand, performed 2 prevalence surveys of HPV infection and cervical intraepithelial neoplasia, in northern and in southern Thailand. We report the results of those studies here. SUBJECTS, MATERIALS, AND METHODS Study population and enrollment. A random sample of women who resided in the Koaka District, Lampang province, northern Thailand, was selected in each of eleven 5-year age groups: 15 19, 20 24, 25 29, 30 34, 35 39, 40 44, 45 49, 50 54, 55 59, 60 64, and 65 years. Complete population lists of all residents, obtained from community health centers, were used for this purpose. Of a total of 1893 selected women, 521 did not live at the address given on the population list or refused to participate. Eighty women were ineligible because they (1) were currently pregnant, (2) had a history of hysterectomy or conization, or (3) were mentally incompetent. A total of 1292 women (71.3% of eligible women) were, therefore, interviewed at home and then transported to Lampang Hospital between November 1997 and October Of these, 221 women (10 married and 211 single women) consented to provide a blood sample but refused to have a pelvic examination. The majority of the women who refused a pelvic examination were!25 years of age (83.7%) and reported being virgins (94.1%). Thus, 1071 women provided a cervical specimen for HPV DNA testing. In Songkla province, southern Thailand, sampling was restricted to women who had been married at some point, because of the large number of unmarried women who refused pelvic examination in Lampang. Women were chosen among residents from 3 districts: Ranod, Ratapoom, and Natawee. The type of population lists, age-stratified sampling methods, and exclusion criteria for participation in the study were the same as in Lampang. A total of 2288 women were randomly selected, but 1094 were not found at the address given on the population list or refused to join the study. An additional 197 women were ineligible due to exclusion criteria. Thus, 997 women (47.7% of eligible women) were interviewed and examined in 1 of 3 district clinics between September 1999 and June Seven women consented to provide a blood sample but refused to have a pelvic examination. Thus, 990 women provided a cervical specimen for HPV testing. At both sites, a standardized questionnaire on sociodemographic characteristics, sexual behavior, reproductive and contraceptive history, tobacco smoking and tobacco and betel chewing history, Papanicolaou smear screening history, and the woman s report of the sexual behavior of her husband was administered to study participants by trained female interviewers. Clinical examination and specimen collection. Participating women underwent a pelvic examination. Cervical exfoliated cells were collected for Pap smear screening and HPV DNA detection by sampling of the ectocervix with 2 wooden spatulas and the endocervix with a cytobrush (Cervibrush; CellPath). After Pap smear preparation, the remaining cervical cells were placed in tubes with PBS and stored in a cool box on ice. A 10-mL sample of blood was also collected from each consenting participant. All samples were shipped daily to central laboratories at the Lampang Hospital in Lampang and at the Songklanakarind Hospital in Songkla for processing. Exfoliated cervical cell specimens were processed by centrifugation at high speed (maximum, 3000 g) for 10 min. Excess PBS was discarded using a Pasteur pipette, and the remaining cell pellet was left in a small quantity of PBS ( ml), resuspended in the same volume of PBS, and shaken. The diluted cell pellet was then poured into 2 tubes, using a Pasteur pipette, and frozen at 80 C. Blood samples were centrifuged at 1500 g; aliquoted into plasma, buffy coats, and red blood cells; and stored at 20 C. All samples were sent on dry ice to the IARC (Lyon, France) for storage. All Pap smears from Lampang and Songkla were read at a central laboratory in the National Cancer Institute in Bangkok. Cytological smears were classified according to the Bethesda system. Participants with abnormal cytological findings were asked to undergo a colposcopic examination for biopsy and treatment, as appropriate. HPV DNA testing. HPV DNA testing was performed on exfoliated cervical cell samples in the pathology laboratory of Vrije University, Amsterdam. To analyze the quality of target DNA, b-globin gene-specific primers were used, and only b- globin positive samples were included in analyses. In Lampang, 35 (3.3%) of 1071 samples were b-globin negative, and 1 woman had no result of cytological examination, leaving a total of 1035 women with adequate HPV DNA and cytological findings. In Songkla, 272 (28%) of 990 samples were b-globin negative, and 12 women had no result of cytological examination, leaving a total of 706 women with adequate HPV DNA and cytological findings. HPV DNA positivity was assessed using a general primermediated GP5 /6 polymerase chain reaction (PCR). PCR positivity was assessed by hybridization of PCR products in an EIA using 2 HPV oligoprobe cocktails that together detect the following 36 HPV types: 6, 11, 16, 18, 26, 31, 33 35, 39, 40, 42 45, 51 59, 61, 66, 68, 70, 71 (equivalent to CP8061), 72, 73, 81 (equivalent to CP8304), 82 (IS39 and MM4 subtypes), HPV in Lampang and Songkla, Thailand JID 2003:187 (15 April) 1247

3 83 (equivalent to MM7), 84 (equivalent to MM8), and CP6108 [11]. Probes and procedures used for EIA detection are described elsewhere [11]. The sensitivity and specificity of EIA detection were previously determined using dilution lines of cloned HPVs or cervical smears in which these types were identified in earlier studies [12]. In addition, HPV positivity was assessed by low-stringency Southern blot analysis of PCR products with a cocktail probe of HPV-specific DNA fragments [13]. Subsequently, GP5 /6 PCR was repeated on positive samples in triplicate to generate sufficient products for further typing. After pooling these PCR products, typing was performed using EIA and HPV type specific oligoprobes for the HPV types listed above [14]. Samples that were GP5 /6 positive by lowstringency Southern blot analyses but could not be identified by EIA were considered to be uncharacterized HPV types. Special precautions were taken to minimize false-positive results of PCR, as has been described in detail elsewhere [13]. HPV types considered to be HR for this analysis included HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68. The group of low-risk (LR) types included all other HPV types tested. Some of the LR HPV types are not associated with cancer (e.g., HPV-6, -11, -40, -42, -43, and -44), whereas others have undetermined oncogenic potential (e.g., HPV-26, -34, -53, -54, -70, -72, and -73). Infection with 11 HPV type was considered to be HR if any HPV type detected was an HR type. HPV serological testing. Serological testing for the detection of antibodies against HPV VLPs was conducted using an ELISA, as described elsewhere [15]. In brief, HPV VLPs were produced in Sf21 insect cells using recombinant baculoviruses encoding the L1 gene of HPV-16, -18, -31, -33, and -58 [15 17] and were purified by isopyonic binding in cesium chloride. Flat-bottomed wells were coated overnight at 4 C with ng of VLPs (test well) or 200 ng of bovine serum albumin (BSA; control well) in PBS (ph 7.4). Each plasma sample was tested at a dilution of 1:10 against each of the 5 VLP types and BSA. The background reactivity in the BSA-coated wells was subtracted from the optical density in each of the HPV VLP coated wells. Negative values were adjusted to 0. The cutoff values for positivity were set at 0.4. For comparison with data on HPV DNA prevalence, data on the prevalence of anti-vlps against HPV-16, -18, -31, -33, and -58 are presented for 1033 women from Lampang and 706 women from Songkla who had valid HPV DNA and Pap smear results. Serological testing for herpes simplex virus type 2 (HSV- 2). Serological testing that was type specific for HSV-2 antibodies was conducted blindly in the virology laboratory of Children s Hospital and Regional Medical Center, Seattle, using an HSV-2 ELISA developed by Focus Technology [18]. HSV-2 positivity was confirmed by retesting of positive serum samples with another Focus Technology HSV-2 ELISA. Statistical analysis. The age-standardized prevalence of HPV DNA detection was computed using, as a standard, the population of Thailand in 1993 [2] or the world standard population [1]. Odds ratios (ORs) for HPV DNA detection and corresponding 95% confidence intervals (CIs) were calculated by means of unconditional multiple logistic regression, adjusted for place of residence and age (categorized into 6 groups:!25, 25 34, 35 44, 45 54, 55 64, and 65 years of age). Because of the similarity in the distribution of age, education, and sexual behavior variables and in the association with various risk factors for HPV DNA in Lampang and Songkla, data from the 2 sites were combined. Analyses were performed on data from 1741 women, including 14 women who reported themselves to be virgins. The statistical significance of trends for ORs was assessed by considering the categorical variable to be a continuous variable in the logistic model. In the final multivariate model, all variables that showed statistically significant (P!.05) associations with HPV DNA detection in the age- and center-adjusted analyses were included. RESULTS HPV prevalence and type-specific distribution. Overall, 6.3% of women were HPV DNA positive (table 1), and 28 different HPV types were detected. The majority of the HPVpositive women had infections with HR types (69.1%). HPV infections with multiple types were found in 1.7% of women (26.4% of HPV-positive women). When the presence of an HPV type in either a single or multiple infection was considered, the 3 most common types were 72, 52, and 16, each of which was found in 15 women (0.9% of all women and 13.6% of HPV-positive women). HPV-11, -33, -34, -35, -40, -43, -45, -66, -68, -MM7, -MM8, -CP6108, and -CP8061 were found only in women with multiple infections. The HPV prevalence was higher among 13 women with a diagnosis of low-grade squamous intraepithelial lesion (LSIL; 61.5%) and 11 women with high-grade squamous intraepithelial lesions (HSIL) or ICC (100%) than among 1673 women with normal cytological findings (4.8%). The HPV prevalence was 25.0% among women with cytological abnormalities that were not histologically confirmed. Among women with either LSIL or HSIL/ICC, HPV-52 was the most common type (found in 3 women with LSIL and 4 with HSIL/ICC). Among 80 women with normal cytological findings who were HPV DNA positive, the most common types found in either single or multiple infections were HPV-72, -16, -70, and -33. Age-specific prevalence of HPV infection. As shown in table 2, HPV DNA positivity overall was highest among women!35 years of age (9.8%) and then formed a plateau among 1248 JID 2003:187 (15 April) Sukvirach et al.

4 Table 1. Prevalence of type-specific human papillomavirus (HPV) infection of the cervix among 1741 women in Lampang and Songkla, Thailand. Variable Total population, no. (%) Women with abnormal cytology, no. (%) No histological confirmation a LSIL b HSIL/ICC c Women with normal cytology, no. (%) HPV DNA negative 1631 (93.7) 33 (75.0) 5 (38.5) 0 (0) 1593 (95.2) HPV DNA positive 110 (6.3) 11 (25.0) 8 (61.5) 11 (100) 80 (4.8) High risk type 76 (4.4) 9 (20.5) 7 (53.8) 11 (100) 49 (2.9) Low risk type 33 (1.9) 2 (4.5) 1 (7.7) 0 (0) 30 (1.8) 16 9 (0.52) (0.48) 18 7 (0.40) (0.24) 31 4 (0.23) d 2 (0.12) 39 4 (0.23) (0.18) 42 3 (0.17) (0.18) 44 1 (0.06) (0.06) 51 5 (0.29) (0.24) (0.69) (0.18) 54 1 (0.06) (0) 56 5 (0.29) (0.24) 58 3 (0.17) (0.12) 59 1 (0.06) (0.06) 70 7 (0.40) (0.36) (0.75) (0.78) CP (0.29) (0.30) X e 1 (0.06) (0.06) Single-type subtotal 81 (4.7) (3.6) 11, 33, 39, 66 1 (0.06) (0) 16, 31 1 (0.06) (0) 16, 18, 31, 35 1 (0.06) (0.06) 16, 33, 35 1 (0.06) (0.06) 16, 33, 42, 58 1 (0.06) d 0(0) 16, 34, 68 1 (0.06) (0.06) 16, 52 1 (0.06) (0.06) 18, 31 1 (0.06) (0.06) 18, 58 1 (0.06) (0.06) 31, 35 1 (0.06) (0) 31, 33, 39 1 (0.06) (0.06) 33, 35 3 (0.17) (0.12) 33, 58 4 (0.23) (0.18) 33, 58, 72 1 (0.06) (0.06) 33, 58, CP6108, CP (0.06) (0) 33, 70 1 (0.06) (0.06) 34, 68 1 (0.06) (0.06) 39, 68 1 (0.06) (0.06) 39, 52 1 (0.06) (0) 40, MM8, CP (0.06) (0.06) 43, 45 1 (0.06) (0.06) 52, 70 1 (0.06) (0.06) 72, CP (0.06) (0.06) MM7, MM8, CP (0.06) (0) Multiple-type subtotal 29 (1.7) (1.2) Total NOTE. High-risk HPV types are shown in bold. ASCUS, atypical squamous cells of undetermined significance; AGUS, atypical glandular cells of undetermined significance; CIN, cervical intraepithelial neoplasia; HSIL, high-grade squamous intraepithelial lesion; ICC, invasive cervical cancer; LSIL, low-grade squamous intraepithelial lesion. a Includes 20 women with ASCUS, 10 with AGUS, and 14 with abnormal cytology and normal histological findings. b Includes histological confirmation in 8 women with CIN grade I. c Includes 4 women with CIN grade II, 3 with CIN grade III, and 2 with ICC. d Includes 1 woman with ICC. e Uncharacterized type; not classified as low or high risk.

5 Table 2. Sociodemographic characteristics, chewing betel or tobacco, and smoking tobacco as risk factors for cervical human papillomavirus (HPV) infection in a population-based sample of women in Lampang and Songkla, Thailand. Variable HPV infection status Uninfected, no. Infected, no. (%) OR a (95% CI) Center Songkla (3.8) 1 b Lampang (8.0) 2.1 ( ) Age, years! (10.6) 2.5 ( ) (9.2) 2.1 ( ) (5.0) 1.1 ( ) (6.0) 1.4 ( ) (4.5) 1.0 ( ) (4.5) 1 b P for trend.001 Religion Buddhist (6.6) 1 b Muslim (1.0) 0.2 ( ) Christian 8 1 (11.1) 2.1 (0.2 18) Education None or some primary (3.6) 1 b Completed primary (6.2) 1.6 ( ) Secondary or higher (8.9) 2.0 ( ) P for trend.097 Marital status Married (6.7) 1 b Divorced 74 5 (6.3) 0.9 ( ) Widowed (4.7) 0.9 ( ) Single 15 0 (0) 0.0 Chewing betel or tobacco Never (6.9) 1 b Ever (3.2) 0.8 ( ) Smoking Never (7.0) 1 b Ever (3.5) 0.4 ( ) NOTE. CI, confidence interval; OR, odds ratio. a Adjusted for age and center. b Reference category. women 35 years of age (4.5% 6.0%). The prevalence of HR HPV types increased from 5.6% among women!25 years of age to 6.5% among those aged years and then gradually decreased with age to 2.9% among those aged 65 years (figure 1). For LR types, the highest prevalence was found among women!25 years of age (5.1%) and the lowest among women aged years (0.3%). Among HPV-positive women, the proportion of LR HPV infection was highest among women!25 years of age (48%). The majority of HPV-positive women had single-type infections in all age groups (data not shown). Figure 2 shows a comparison of the age-specific prevalence of HPV DNA of any type and the prevalence of DNA of and anti-vlps against HPV-16, -18, -31, -33, and -58 among study participants. Seropositivity for anti-vlps was 17.2% among women!25 years of age, increased to 26.9% among women years of age, and decreased thereafter. The age curves for HPV DNA of the same types as anti-vlps were notably lower than the anti-vlp age curves. The prevalence of anti- VLPs against 5 HPV types was higher in Lampang (29.2%) than in Songkla (10.9%) (data not shown). Risk factors for HPV infection. The prevalence of cervical HPV DNA was significantly higher in Lampang (8.0%; 95% CI, 6.4% 9.8%) than in Songkla (3.8%; 95% CI, 2.5% 5.5%) (table 2). Using the Thailand population in 1993 [2] as the standard, the age-standardized HPV prevalence was found to be 9.1% (95% CI, 7.1% 11.1%) in Lampang and 3.9% (95% CI, 2.3% 5.6%) in Songkla, respectively (prevalences were 9.1% and 3.7%, respectively, using the world standard population). The prevalence of HR HPV types was higher in Lampang (5.8%) than in Songkla (2.3%) ( P!.001), but the prevalence of LR types was similar (2.1% in Lampang and 1.6% in Songkla). Abnormal cytological findings were more common among women from Lampang (4.9%) than among those from Songkla (2.4%) ( P p.008; data not shown). HPV DNA detection was not statistically significantly associated with religion or marital status, but it was marginally more frequent among the most educated women. No association was found with chewing betel or tobacco, but women who reported a history of tobacco smoking (19.8%) appeared to have a lower risk of HPV DNA detection than did women who did not smoke (table 2). HPV positivity was not related to a younger age at menarche, first marriage, or intercourse (table 3). Most women (85.0%) reported having only 1 sexual partner in their lifetimes. Women who reported having 2 sexual partners in their lifetimes appeared to be at an increased risk of HPV DNA detection, but the risk trend was of borderline statistical significance. No HPV infection was detected among 14 women who reported being virgins. HPV DNA was significantly associated with a reported history of sexually transmitted infections (OR, 3.2) and HSV- 2 seropositivity (OR, 2.1). HPV DNA detection was also higher among women who reported that their husband had other sexual partners (OR, 1.9) or contact with prostitutes (OR, 1.6) (table 3). HPV DNA detection was not significantly associated with reproductive characteristics, including age at first pregnancy, number of pregnancies, and parity (table 4). Women who reported a history of abortion were at an increased risk (OR, 1.7). Use of contraceptives was unrelated to HPV detection, with the exception of condom use (OR, 2.0). Women who reported condom use, however, were significantly younger and more likely to be HSV-2 seropositive than were women who did not use condoms ( P!.01). The association between HPV DNA detection and living in 1250 JID 2003:187 (15 April) Sukvirach et al.

6 Figure 1. Type distribution of human papillomavirus infection in population-based samples of women from Lampang and Songkla, Thailand. Lampang, rather than Songkla, remained significant in the multivariate model (table 5). Independent risk factors for HPV infection included a woman s tobacco smoking history, HSV- 2 seropositivity, and report that the woman s husband had had other sexual partners. Associations with factors such as history of abortions, lifetime number of sexual partners, and history of condom use were attenuated and became nonsignificant in the final model. Risk factors for HPV infection did not appear to significantly differ between HR and LR HPV types, although infection with LR types appeared to be more strongly associated with HSV-2 seropositivity than did infection with HR types. The multivariate analyses presented in table 5 were repeated separately for women living in Lampang and those living in Songkla, and risk factors were consistent (data not shown). When women!35 years of age were compared with those 35 years of age, risk factors also were generally similar, although the association with having 2 sexual partners in a lifetime appeared to be stronger among younger women (OR, 2.2; 95% CI, ) than among older women (OR, 1.1; 95% CI, ). [19], although our sample size was too limited for reliable investigation of the type distribution of HPV infection among women with HSIL or ICC. The population-based prevalences of HPV-16 and -18 DNA types among women in our study were 0.9% (13.6% of HPV-positive women) and 0.6% (9.1% of HPV-positive women), respectively. Only a few studies using highly sensitive assays for HPV DNA detection have been conducted in less-developed countries [5, 19, 20, 23 30]. The overall HPV DNA prevalence of 6.3% (4.8% in women with normal cytological findings) in our study is among the lowest reported in the literature to date. It is similar to the prevalence seen in a control group of middleaged women in a case-control study of ICC in Spain (4.9%) [24]; among women with normal cytological findings in 4 studies in Bangkok, where HPV positivity ranged from 5% to 9% [27 30]; and among women with normal cytological findings in a study in Japan (4.5%) [31]. It is lower than the prevalences found among women with normal cytological findings in Costa Rica (11%) [19] and Mexico (14.5%) [5] and among women in the control group (mean age, 43 years) of a case-control study of ICC in Taiwan (9.2%) [25]. The prevalence of HPV DNA positivity that we found in Songkla (3.8%) is lower than the prevalence of 15.7% that was seen among middle-aged women in the control group of a hospital-based case-control study carried out by the IARC in the same province in Thailand [20]. The higher number of sexual partners and fewer refusals among control women in that case-control study [20], compared with the present study, may account for this discrepancy. A highly sensitive PCR-based assay was used in this study to detect a wide range of different HPV types, reducing the likelihood that HPV DNA positivity was underdetected. The specificity of our findings also appears to be high; none of the 14 women who reported themselves to be virgins were HPV DNA positive. Twenty-eight different HPV types were found in women in this study, which confirms that there is great DISCUSSION The age-standardized prevalence of cervical HPV DNA was 2- fold higher in population-based samples of women from Lampang (9.1%) than among women from Songkla (3.9%). When samples from the 2 centers were combined, the HPV DNA prevalence was 6.3%. HPV-52, -72, and -16 were the 3 most common types found by HPV DNA detection for the entire sample of women surveyed, whereas HPV-72, -16, and -70 were the most common types among women with normal cytological findings. HPV-52 was the most common type among the 68 women with abnormal cytological findings (14.7%), which may indicate that HPV-52 is of greater importance in cervical neoplasia in Thailand than elsewhere [7, 19 22]. As expected, our results confirm that the prevalence of HPV positivity increases with increasing severity of cervical neoplasia Figure 2. Prevalence of antibodies against virus-like particles (anti- VLPs) of human papillomavirus (HPV) types 16, 18, 31, 33, and 58 and HPV DNA of these 5 HPV types and of all types. HPV in Lampang and Songkla, Thailand JID 2003:187 (15 April) 1251

7 Table 3. Age and menarche and sexual behavior as risk factors for cervical human papillomavirus (HPV) infection in a populationbased sample of women in Lampang and Songkla, Thailand. Variable HPV infection status Uninfected, no. Infected, no. (%) OR a (95% CI) Age at menarche, years (8.1) 1 b (6.5) 0.9 ( ) (5.1) 0.8 ( ) P for trend.54 Age at first marriage, years (6.3) 1 b (6.9) 1.1 ( ) (6.8) 1.0 ( )! (5.4) 0.7 ( ) P for trend.23 Age at first intercourse, years (6.7) 1 b (6.5) 1.0 ( ) (6.7) 0.9 ( )! (5.6) 0.7 ( ) P for trend.21 Lifetime no. of sexual partners (0) (6.0) 1 b (9.0) 1.7 ( ) (8.9) 1.6 ( ) P for trend.07 Reported history of sexually transmitted infections Never (6.0) 1 b Ever (14.6) 3.2 ( ) Herpes simplex virus 2 serostatus Negative (4.8) 1 b Positive (10.1) 2.1 ( ) Husband has extramarital affairs No (4.9) 1 b Yes (9.0) 1.9 ( ) Husband frequents prostitute(s) No (5.4) 1 b Yes (9.0) 1.6 ( ) NOTE. CI, confidence interval; OR, odds ratio. a Adjusted for age and center. b Reference category. variability in the range of HPV types detected in exfoliated cervical cells at a population level [19]. HPV DNA positivity was highest among women!25 years of age, which is consistent with the high frequency with which HPV infection occurs when women start having sexual intercourse [5, 19]. The representativeness of our estimate of HPV DNA prevalence among women!25 years of age is limited, however, because young single women in Thailand generally were not willing to undergo a pelvic examination. A few studies have suggested that HPV prevalence may vary with age in a U-shaped fashion [5, 19]. In Costa Rica, the second peak of HPV DNA detection in women 165 years of age appeared to result from an increase in the prevalence of LR, rather than HR, HPV types [19]. In Mexico, a second peak among women 145 years of age appears to have resulted from an increase in both LR and HR HPV types. Our data from Thailand show no clear increase in the prevalence of HPV DNA detection among older women. Differences in HPV DNA prevalence found in different studies, overall and by age, may be partly accounted for by differences in cohort effects and PCR methods used for HPV detection (i.e., whether MY09/11, GP5 /6, or PGM09/ 11 primers are used). Results on risk factors for HPV infection in women provide further supportive evidence of the importance of the male role in HPV transmission to female partners [32, 33] in this Thai population, in which the vast majority (85.0%) of married women reported having only 1 sexual partner in their lifetimes. HPV infection was also positively correlated with HSV-2 seropositivity in women, which is considered to be a reliable marker for the woman s sexual behavior [34], as well as that of her male partner [35]. That the prevalence of HPV infection in Lampang was higher than that in Songkla does not appear to be explained by differences in sexual habits or in HPV testing methods. All samples were analyzed in parallel at a centralized laboratory. That the prevalence of anti-vlps against 5 HPV types was 3-fold higher among women from Lampang (29.2%) than among women from Songkla (10.9%) is consistent with HPV DNA findings and suggests that HPV infection varies between the 2 provinces. It is a source of concern, however, that the proportion of refusals to participate to our survey was higher in Songkla than in Lampang. That the prevalence of HPV infection was lower among women with a history of smoking tobacco than among women who had never smoked must be interpreted with caution, because the prevalence of smoking among women in Thailand is low. Although smoking has been consistently associated with an increased risk of ICC [20, 36], the influence of smoking on HPV infection is unclear; some studies have found an increased risk of HPV infection [37], no difference [5], or a reduced risk [33, 38 41] among smokers. Potential limitations of our study include that the survey, most notably in Songkla, had a relatively low participation rate. Because Pap smear screening is not widespread in Thailand, it is not, perhaps, surprising that a substantial proportion of women refused to participate in the study. The elevated prevalence of b-globin negativity among the exfoliated cervical cell samples, notably in Songkla, is another limitation and may be the result of problems in the transport of samples from rural areas to the Songklanakarind Hospital. Our results also showed 1252 JID 2003:187 (15 April) Sukvirach et al.

8 Table 4. Reproductive characteristics and contraceptive use as risk factors for cervical human papillomavirus (HPV) infection in a populationbased sample of women in Lampang and Songkla, Thailand. Variable HPV infection status Uninfected, no. Infected, no. (%) OR a (95% CI) Age at first pregnancy, years (6.7) 1 b (6.3) 0.9 ( )! (6.7) 0.8 ( ) P for trend.55 No. of pregnancies None 75 1 (1.3) 0.1 ( ) (9.1) 1 b (5.0) 0.8 ( ) (4.5) 0.8 ( ) P for trend.45 Parity None 90 1 (1.1) 0.1 ( ) (8.5) 1 b (4.9) 0.9 ( ) (4.4) 0.8 ( ) P for trend.18 Abortion Spontaneous Never (6.1) 1 b Ever (7.4) 1.5 ( ) Induced Never (6.1) 1 b Ever 85 9 (9.6) 1.5 ( ) Any Never (5.8) 1 b Ever (8.2) 1.7 ( ) Contraceptive use Oral Never (4.9) 1 b In past only (7.6) 1.1 ( ) 4 years current use (9.1) 1.1 ( ) 14 years current use 80 7 (8.1) 1.2 ( ) P for trend.66 Injectable or implant Never (5.5) 1 b In past only (6.4) 0.8 ( ) 4 years current use (11.5) 1.4 ( ) 14 years current use 45 4 (8.2) 1.1 ( ) P for trend.50 Condoms Never (6.0) 1 b Ever (10.8) 2.0 ( ) Intrauterine device Never (6.7) 1 b Ever (4.1) 0.6 ( ) NOTE. CI, confidence interval; OR, odds ratio. a Adjusted for age and center. b Reference category.

9 Table 5. Results of multivariate analysis of risk factors for cervical human papillomavirus (HPV) infection in a population-based sample of women in Lampang and Songkla, Thailand. Variable HPV type, OR a (95% CI) High risk Low risk Any Center Songkla 1 b 1 b 1 b Lampang 3.0 ( ) 1.6 ( ) 2.5 ( ) Age, years! ( ) 2.1 ( ) 1.6 ( ) ( ) 0.8 ( ) 1.2 ( ) ( ) 0.08 ( ) 0.6 ( ) ( ) 0.5 ( ) 0.9 ( ) ( ) 0.6 ( ) 0.8 ( ) 65 1 b 1 b 1 b Smoking Never 1 b 1 b 1 b Ever 0.5 ( ) 0.2 ( ) 0.4 ( ) Lifetime no. of sexual partners 1 1 b 1 b 1 b ( ) 1.8 ( ) 1.4 ( ) Herpes simplex virus 2 serostatus Negative 1 b 1 b 1 b Positive 1.4 ( ) 3.6 ( ) 1.9 ( ) Husband has extramarital affairs No 1 b 1 b 1 b Yes 1.5 ( ) 1.7 ( ) 1.5 ( ) History of abortion Never 1 b 1 b 1 b Ever 1.4 ( ) 1.9 ( ) 1.5 ( ) Condom use No 1 b 1 b 1 b Yes 1.3 ( ) 2.6 ( ) 1.6 ( ) NOTE. CI, confidence interval; OR, odds ratio. a Adjusted for all variables in the table other than the current category. b Reference category. a relatively low prevalence of LSIL and HSIL among women screened in our study, pointing to some potential underdetection of cervical abnormalities. This should not bias, however, the overall detection of HPV DNA in participating women. Because of the relatively low number of HPV-positive women in our study, statistical power to detect meaningful differences in the risk factors for HR and LR HPV types that have been found in some other studies [38, 42] was also limited. In conclusion, the prevalence of HPV infection was relatively low in population-based samples of women from Lampang and Songkla, Thailand, which suggests that the spread of HPV infection in the country is relatively limited. Because the vast majority of the women surveyed reported having only 1 sexual partner in their lifetimes, the sexual behavior of a woman s husband in Thailand appears to be an important determinant of a woman s HPV infection status. That the prevalence of HPV DNA and anti-vlps was higher among women from Lampang than among women from Songkla is consistent with a 50% difference seen in the incidence of ICC between the 2 provinces. Acknowledgments We acknowledge the collaboration of Sujin Wongchusri (Lampang Hospital, Lampang, Thailand), Pipat Yingseri and Boonterm Tunsurat (Lampang Provincial Health Office, Lampang), Sutep Watcharapiyanan (Songkla Provincial Health Office, Songkla, Thailand), Wisit Tunnukij and Apichai Deechaisate (Somdej Hospital, Natawee, Songkla), Teerawat Kornsilp 1254 JID 2003:187 (15 April) Sukvirach et al.

10 (Ranod Hospital, Songkla), and Suwit Kongchuchuy (Ratapoom Hospital, Songkla) and thank them for providing local management and advice. We thank Manop Lertsakornsiri, Busabun Preda-ananthasook, Suwit Pichayanan, and Supavan Hongpatarakeree (Lampang Hospital); Sudjid Kaewsuksri and Supanee Suwan (Ranod Hospital); Sudee Koll and Saniya Mugem (Ratapoom Hospital); and Wannee Janeruksukum and Yuwadee Tanbutr (Somdej Hospital), for conducting the gynecologic examinations and collecting the cervical specimens. We also thank Supanee Nimpoonsawad (Koa-ka Health Office, Lampang) and Sunanta Pongpaiboon (Songkla Provincial Health Office), for their supervision of the health care workers. All health care workers in Koa-ka, Ranod, Ratapoom, and Natawee who were responsible for inviting women in the sampling list to participate in this project are also recognized. 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