Human Papillomavirus in Women With and Without HIV-1 Infection Attending an STI Clinic in Vitoria, Brazil

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1 Human Papillomavirus in Women With and Without HIV-1 Infection Attending an STI Clinic in Vitoria, Brazil Journal of the International Association of Physicians in AIDS Care 8(5) ª The Author(s) 2009 Reprints and permission: sagepub.com/journalspermissions.nav DOI: / Bettina Moulin Coelho Lima, MD, 1 Jonathan E. Golub, PhD, 2 Adriana Tonani Mattos 1 Luciana Bueno de Freitas, 1 Liliana Cruz Spano, PhD, 1 and Angélica Espinosa Miranda, MD, PhD 1 Abstract We conducted a cross-sectional study in Vitória, Brazil, to assess the prevalence of human papillomavirus (HPV) infection in HIVpositive and HIV-negative women attending a sexually transmitted infection (STI)/AIDS clinic. We also investigated the presence of HPV genotypes and assessed covariates for HIV infection. Enrolled patients received a gynecological evaluation, and cervical scrape samples were collected for cytological analysis and HPV-DNA polymerase chain reaction (PCR). A blood sample was obtained to determine HIV status. HPV infection and squamous intraepithelial lesions were studied in 284 women, 112 (39.4%) HIV-positive women and 172 (60.5%) HIV-negative women. HPV-DNA was detected in 133 (46.8%). HIV-infected women were almost twice as likely to be concurrently infected with HPV than HIV-negative women (OR ¼ % CI: ). The high proportion of HPV detected among women attending an STI/AIDS clinic, particularly among HIV-infected women, proves the importance of screening this high-risk group in the hope of earlier detection and treatment of cervical intraepithelial neoplasia (CIN). Keywords human papillomavirus (HPV), HIV, women, sexually transmitted infection (STI) clinic, cervical intraepitial neoplasia (CIN), Brazil Introduction Human papillomavirus (HPV)-associated malignancies are more frequent in HIV-infected persons, and HPV DNA is commonly detected in the genital mucosa of HIV-infected women. The prevalence of infection is generally much higher than in HIV-negative women of similar sociodemographic characteristics. 1-3 The status of the immune system in HPV-infected women may determine the development of persistence after primary infection, which has emerged in several studies as an important risk factor for cervical neoplasia. 4,5 Thus, an HIVimpaired immune system, which permits a high HPV viral load and persistent HPV infection, may lead to an increased risk for the development of cervical intraepithelial neoplasia (CIN). 6,7 Recent studies on the correlation between HIV and HPV infections indicate higher frequencies of high-risk HPV types in HIV-positive individuals as opposed to the low-risk genotypic frequencies observed in HIV-negative populations. 8,9 Impaired cell-mediated immunity could be a likely explanation for the advancement of high-risk HPV in HIV-positive individuals. Several studies have shown a strong and consistent association between HIV and HPV coinfection and the development of CIN and genital cancer. 7,10 There is evidence to show that HIV-positive women have a significantly higher rate of CIN than their counterparts and are more likely to progress to invasive carcinoma than HIV-negative women. 8,11-13 Brazilian studies also have reported a very high proportion of HPV among HIV-infected women and detection of multiple HPV genotypes in individuals. 9,14,15 The aims of the current study were to (1) assess the prevalence rate of HPV infection in HIV-positive and HIVnegative women attending a sexually transmitted infection (STI)/AIDS clinic; (2) investigate the presence of specific HPV genotypes; and (3) assess covariates for HIV infection. Methods Women aged 18 to 49 years attending an STI/AIDS clinic in Vitoria, Brazil, between March and December 2006 were 1 Nucleo de Doencas Infecciosas, Universidade Federal do Espirito Santo, Brazil 2 School of Medicine, Johns Hopkins University, Baltimore, Maryland Corresponding author: Angélica Espinosa Miranda, Núcleo de Doenças infecciosas, Universidade Federal do Espírito Santo, Av. Marechal Campos 1468, Maruípe, Vitória, ES, Brazil; espinosa@ndi.ufes.br 286

2 Lima et al 287 invited to participate in the study. Participants were interviewed after providing informed consent. Enrolled participants underwent a gynecological evaluation and cervical scrape samples were collected for cytological analysis and HPV- DNA polymerase chain reaction (PCR). A blood sample was obtained to determine HIV status. Patient interviews included demographic, behavior, and clinical data using a questionnaire validated during a pilot study. A second interview took place approximately 2 weeks later where laboratory test results and appropriate counseling were provided to the patient and biopsies for cases with positive pap smears were conducted after colposcopy. The Ethical Committee on Research of the Federal University of Espírito Santo approved this study. Laboratory Specimens A gynecological examination was performed to collect cervical samples of HPV-DNA and to conduct a pap smear. HPV-DNA was investigated by PCR, using the protocol previously established which detects a fragment of 450 base pair (bp) of L1 gene region using consensus primers MY09/11. All positive samples were screened for HPV genotype 16 using a PCR reaction to detect the most common types: 6, 11, 16, 18, 31, 33, and 35. A blood sample was collected for HIV testing: enzymelinked immunosorbent serologic assay (ELISA; HIV-1 Microelisa, Organon Laboratories, Boxtel, Holland) and confirmed by indirect immunofluorescent assay (Fiocruz, Rio de Janeiro). CD4 count and viral load were determined for all HIV-positive women. Statistical Analyses Standard descriptive statistical analyses were performed, including frequency distributions for categorical data and calculation of medians and interquartile ranges (IRQs) for continuous variables. HPV prevalence was calculated to reflect the cumulative frequency of this outcome, with corresponding 95% confidence intervals (CI) in the 2 primary groups (HIV infected and HIV negative). Associations among demographic and behavior variables with HIV infection were tested using the w 2 test, with Yates correction or Fisher exact test, as appropriate. Odds ratios and 95% CI were calculated in bivariate analyses to estimate the strength of the associations between HIV infection and each covariate. Independent risk factors for HIV-infected individuals were assessed through multiple logistic regression using a forward stepwise approach, with 15% as the critical P value for variable entry and 10% as the criterion for variable elimination. Results HPV infection and squamous intraepithelial lesions (SILs) were studied in 284 women, 112 (39.4%) HIV-positive women and 172 (60.6%) HIV-negative women. One woman refused to participate. The frequency of HPV infection was 133 (46.8%; 95% CI ), 63 (56.3%) in HIV-positive women and 70 (40.7%) in HIV-negative women. HPV infection was significantly more frequent in the HIV-positive group (OR ¼ 1.87; 95% CI ), P ¼.001. The median age among all patients was 30 (interquartile range [IQR]: 23-36) years and the median years of education was 8 (IQR: 5-11). Median age of first sexual intercourse was 16 (IQR: 15-18) years. There was no statistical difference between the 2 groups regarding age, education, age at first sexual intercourse, age of partner at first sexual intercourse, and number of sexual partners. Cervical screening revealed 222 (78.2%) patients with normal cytology, 39 (13.7%) with inflammatory disorders, 10 (3.5%) with atypical squamous cells of undetermined significance (ASCUS/HPV), and 11 (3.9%) squamous intraepithelial lesions (SILs; low grade and high grade). High-grade SILs were identified in 2 (0.7%) women. A prior STI was found in 157 patients (55.5%): 50 (31.8%) genital warts, 34 (21.8%) genital herpes, 25 (15.9%) syphilis, 18 (11.6%) Chlamydia infection, 15 (9.6%) trichomoniasis, and 13 (8.3%) gonorrhea. HIV-positive women were less educated than HIV-negative women (P ¼.003; Table 1). HIV-positive women (42.0%) reported a lower history of STIs over the previous 3 years than HIV-negative women (64.3%; P <.001). HIV-positive women used condoms more frequently (82.1% vs. 57.3%, P <.001) and were more likely to have been raped (39.1% vs. 23.5%, P ¼.007). No difference in drug abuse or prostitution was reported (Table 2). High viral load and low CD4 counts were not associated with HPV in the HIV-infected group. In the final logistic regression model, independent covariates associated with HIV-infection were prevalent HPV infection (OR ¼ 2.29; 95% CI: ), more than 1 sexual partner over the past 12 months (OR ¼ 1.93; 95% CI: ), lower education (OR ¼ 1.70; 95% CI: ), and no history of a prior STI (OR ¼ 2.56; 95% CI ; Table 3). HPV genotypes were determined in 73 (54.9%) of the PCRpositive samples (Table 4). Genotype 16 (12%) was the most frequent, followed respectively by 6 (10.5%), 33 (8.5%), 31 (7.5%), 35 (6.8%), 18 (5.3%), and 11 (4.5%). Coinfection with 2 or 3 types was observed in 16 cases, half in HIV-positive women. In HIV-positive women, genotypes 16, 6, and 35 were the most frequent and in HIV-negative women, 6, 11, and 16 were most frequent. High-risk genotypes (16, 18) of HPV were present in 72% (13/18) of HIV-positive women and in 50% (12/ 24) of HIV-negative women. Regarding low-risk HPV genotypes (6, 11), they were found in 28% (5/18) of HIV-positive women and in 50% (12/24) of HIV-negative women (Table 4). Discussion We found an HPV prevalence of 56.3% in HIV-positive women and 40.7% among HIV-negative women attending an STI clinic in Vitória, Brazil. Levi et al reported 64.5% of HIV-positive women to be coinfected with HPV and Grinsztejn et al found 48.0% in different cities in Brazil. 9,14 A high frequency of cervical abnormalities (21.8%) was detected in our study population, with no difference between

3 288 Journal of the International Association of Physicians in AIDS Care 8(5) Table 1. Demographic Variables by HIV Status in Women Attending the Reference Center for STI/AIDS in Vitória, Brazil, 2006 Variables Total N (%) HIVþ N(%) HIV N (%) OR (95% CI) Age (years) 18 to (47.9) 38 (33.9) 98 (57.0) 0.62 ( ) 30 to (33.8) 54 (48.2) 42 (24.4) 2.06 ( ) 40 to (18.3) 20 (17.9) 32 (18.6) Reference Schooling 1 to 4 years 54 (19.9) 33 (29.5) 25 (14.5) 3.02 ( ) 5 to 8 years 99 (36.4) 44 (39.3) 59 (34.3) 1.92 ( ) 9 years 119 (43.8) 35 (31.2) 88 (51.2) Reference Marital status Single 101 (35.6) 27 (24.1) 74 (43.0) 0.18 ( ) Married 131 (46.1) 59 (52.7) 72 (41.9) 0.41 ( ) Divorced 37 (13.0) 16 (14.3) 21 (12.2) 0.38 ( ) Widow 15 (5.3) 10 (8.9) 5 (2.9) Reference Monthly income a Up 1 73 (25.7) 37 (33.0) 36 (20.9) 1.29 ( ) 1.1 to (53.9) 56 (50.0) 97 (56.4) 0.72 ( ) 3.1 to 5 47 (16.5) 15 (13.4) 32 (18.6) 0.59 ( ) >5 11 (3.9) 4 (3.6) 7 (4.1) Reference # Sexual partners in last 12 months 1 25 (8.8) 8 (7.1) 17 (9.9) 0.68 ( ) 2 to (47.5) 52 (46.4) 83 (48.3) 0.97 ( ) 6 to 7 51 (18.0) 23 (20.5) 28 (16.3) 1.27 ( ) >10 73 (25.7) 29 (26.0) 44 (25.5) Reference Abbreviation: STI, sexually transmitted infection. a Brazilian monthly income was approximately US$180 at that time. HIV-positive and -negative women, consistent with previous studies. 6,11,13,17,18 In our study, HIV-infected women were almost twice as likely to have HPV infection, less likely to have reported more than 1 partner in the last year, and less educated. Surprisingly, a history of drug abuse or commercial sex work was not correlated with HIV infection, and history of an STI in the last 3 years was actually strongly associated with HIV-negative status. The literature overwhelmingly states that a history of other STIs facilitates HIV transmission through direct, biological mechanisms and that early STI treatment should be part of a high-quality, comprehensive HIV prevention strategy. 19 In our study, HIV-infected women reported less STIs over the past 3 years, but they also reported considerably more frequent condom use than HIV-negative women. Because many of our HIV-infected women had been infected for longer than 3 years, and our STI question was limited to this time period, it is quite possible that HIV-positive women were more likely to have had an STI prior to HIV infection than that estimated here. Moreover, at each routine follow-up clinic visit, HIVinfected women receive counseling for reducing high-risk behaviors and are provided with free condoms, potentially resulting in lower STI risk since HIV infection in this group. Finally, our overall prevalence of STIs (55.5%) is lower than what would have been expected at an STI clinic. However, this clinic is a reference center for HIV/AIDS care and treatment, thus many women may have been seeking care here for an HIV test and not only for STI symptoms. These are important factors for consideration when implementing prevention strategies for women attending STI clinics. Although HPV infection was frequent in this sample, the prevalence of high-grade SILs was very low. One possible explanation is that these women have earlier access to health assistance and are detected before developing high-grade SILs. Even if SILs were not frequent in this group, these lesions need to be monitored. The literature suggests that both HPV infection and SILs are common in women with HIV infection. 13,17,18 HIV disease may be asymptomatic, however, and therefore women with rapidly progressive SILs should be tested for HIV. Recurrence rates are substantial with standard local ablative therapy for SILs; thus, frequent and regular cervical smears are recommended. Women with any degree of abnormal cytology must be referred for colposcopy. Invasive cervical carcinoma in HIV-positive woman is usually advanced and more likely to relapse following treatment. 13,18 HIV-infected women with cervical cancer have a worse prognosis than HIV-negative women with cervical cancer; higher recurrence rates and highly active antiretroviral therapy (HAART) do not appear to decrease the risk of cervical cancer in HIV-infected women. 13,17 Medical management for cervical cancer is the traditional treatment, regardless of HIV status. HPV is regarded as the most significant risk factor for cervical carcinoma. 20,21 Cancer of the cervix was identified as an STI in 1834, and the hypothesis that HPV is involved was suggested in the mid-1970s The risk varies according to the infecting HPV subtype, for example, high-risk types (16 and

4 Lima et al 289 Table 2. Behavior Variables by HIV Status in Women Attending the Reference Center for STI/AIDS in Vitória, Brazil, 2006 Variables Total N (%) HIVþ N(%) HIV N (%) OR (95% IC) Smoking Yes 66 (23.2) 26 (23.2) 40 (23.3) 1.02 ( ) No 218 (76.8) 86 (76.8) 132 (76.7) Reference Drug abuse (no IDU) Yes 62 (21.8) 28 (25.0) 34 (19.8) 1.38 ( ) No 222 (78.2) 84 (75.0) 138 (80.2) Reference IDU Yes 6 (2.1) 3 (2.7) 3 (1.8) 1.54 ( ) No 278 (97.9) 109 (97.3) 169 (98.2) Reference Previous rape Yes 85 (29.9) 44 (39.3) 41 (23.3) 2.09 ( ) No 199 (70.1) 68 (60.7) 131 (76.2) Reference 1 intercourse 15 Yes 112 (39.4) 50 (44.6) 62 (36.0) 1.43 ( ) No 172 (60.6) 62 (55.4) 110 (64.0) Reference Partner in life More than one 23 (8.1) 7 (6.3) 16 (9.3) 1.54 ( ) Only one 261 (91.9) 105 (93.7) 156 (90.7) Reference Condom use Frequent/always 191 (67.3) 92 (82.1) 99 (57.6) 3.44 ( ) Rarely/never 93 (32.7) 20 (17.9) 73 (42.4) Reference Previous STI Yes 157 (55.5) 47 (42.0) 110 (64.0) 0.37 ( ) No 127 (44.5) 65 (58.0) 62 (36.0) Reference CSW Yes 60 (21.1) 23 (20.5) 37 (21.5) 1.09 ( ) No 224 (78.9) 89 (79.5) 135 (78.5) Reference Abbreviations: CSW, commercial sex worker; IDU, injecting drug user; STI, sexually transmitted infection. Table 3. Independent Covariates to HIV Infection in Women Attending the Reference Center for STI/AIDS in Vitória, Brazil, 2006 Variables OR 95% CI P Value Schooling (up to 8 years vs. > 8 years) More than one partner at last 12 months No history of a prior STI in last 3 years HPV infection Abbreviation: HPV, human papillomavirus; STI, sexually transmitted infection. 18) and low-risk types (6 and 11). Subsequent studies have shown that almost all cervical cancers are HPV positive and that only certain HPV types are associated with invasive carcinoma (most commonly HPV-16, HPV-18, HPV-31, and HPV- 45). 25 Women participating in our study were tested for the more frequent HPV genotypes, though a high proportion did not have a specific type identified. PCR primers for other HPV genotypes are necessary for further studies. The primary limitation of this study is that all patients were STI clinic patients; thus, they were likely at an increased risk of HPV, regardless of their HIV status. Inferences to Brazilian women in the general population may be limited. Another limitation is the potential reporting bias by the patients regarding questions of a sensitive nature, particularly sexual history. Women may be more likely to provide socially acceptable Table 4. HPV Genotypes Identified by PCR in Women Attending the Reference Center for STI/AIDS in Vitória, Brazil, 2006 HPV genotypes Women HIVþ N(%) HIV- N (%) Total 6 5 (7.9) 9 (12.9) 14 (10.5) 11 1 (1.6) 5 (7.2) 6 (4.5) 16 9 (14.3) 7 (10.0) 16 (12.0) 18 2 (3.2) 5 (7.1) 7 (5.3) 31 6 (9.5) 4 (5.7) 10 (7.5) 33 8 (12.7) 3 (4.3) 11 (8.3) 35 5 (7.9) 4 (5.7) 9 (6.8) No type identified 27 (42.9) 33 (47.1) 60 (45.1) Abbreviations: HPV, human papillomavirus; PCR, polymerase chain reaction; STI, sexually transmitted infection. responses to these questions, which may result in an incorrect interpretation of these data. Misclassification of condom use, age of first intercourse, and number of sexual partners may have occurred. Despite these limitations, the high rate of participation demonstrates that STI and cervical cancer prevention programs can successfully deliver acceptable confidential and private services for women attending an STI clinic. Cancer prevention programs exist and have been shown to be successful at avoiding disease progression. Although this is encouraging, much work remains to identify additional

5 290 Journal of the International Association of Physicians in AIDS Care 8(5) innovative interventions that address the social, cultural, and environmental influences of HPV infection and cervical cancer. There is also a need to find better ways of disseminating evidence-based approaches to HIV prevention, so that effective interventions are more widely used. Sexually active women need access to confidential, low-cost, and friendly services to teach them how to protect themselves from STIs, including HIV infection. Declaration of Conflicting Interests The authors declare that they have no conflict of interest. Funding The authors disclosed receipt of the following financial support for the research and/or authorship of this article: FACITEC (Fundo de Apoio à Ciência e Tecnologia) of Vitória Municipality, NIH grant number 3D43TW S1, National Institutes of Health grant AI06699 (JEG). References 1. Ellerbrock TV, Chiasson MA, Bush TJ, et al. 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