Risk factors for oral HPV infection among a high prevalence population of HIV-positive and at-risk HIV-negative adults

Study Population and Study Design

This cross-sectional study was based within two multi-site U.S. cohort studies of HIV-positive and at-risk HIV-negative individuals: one cohort of women, the Women Interagency HIV Study (WIHS)(28, 29) and one cohort of men who have sex with men (MSM), the Multicenter AIDS Cohort Study (MACS).(30–32) These cohorts were designed to be methodologically similar and include HIV-negative individuals at risk for HIV infection based on their sexual or injection drug use behaviors. The current study is the initial phase of a larger natural history study of oral HPV infection in these two cohorts.

Briefly, the MACS and WIHS began in 1984 and 1994 respectively, with additional enrollment in 1987–91 (MACS- only), and 2001–03. MACS and WIHS participants undergo a semi-annual interview which includes medical history, demographic, and behavioral questions. There is also a physical examination and venipuncture, where the blood is tested for CD4 T cell count and HIV viral load. Between April 2009 and March 2010, we enrolled a convenience sample of 365 men and 280 women.

The study design stratified enrollment by cohort (365 MACS and 280 WIHS), HIV status (379 HIV-positive and 266 HIV-negative), and among HIV-positive individuals by ever/never HAART use (312 HAART experienced and 67 never HAART users). To enrich for HAART-naïve participants; all interested HAART naïve participants at these sites were enrolled. We enrolled 192 HIV-positive and 173 HIV-negative men from the Baltimore, Chicago and Pittsburgh MACS sites, and 187 HIV-positive, 93 HIV-negative women from the Brooklyn, Bronx, and Chicago WIHS sites. This sample represents 25% of current MACS and 26% of current WIHS participants at the sites enrolled. The study protocol was approved by the MACS and WIHS executive committees as well as the Institutional Review Boards at each site. Written informed consent was received from each participant.

An oral rinse sample was collected as part of this sub-study using a 30 second swish and gargle of 10 mL of Scope™ mouthwash, as previously described.(33) Relevant behavioral measures routinely collected by the MACS and WIHS were also evaluated in this study. Self reported lifetime use and past six months use (called recent use hereafter) of recreational and therapeutic drugs, alcohol, cigarettes, and sexual behavior were collected by interview administered questionnaire in the WIHS and computer assisted self interview (CASI) in the MACS. Fellatio (performing oral sex on a man) was defined as “he put his penis in your mouth” in the MACS and “a blow job or putting his penis in your mouth” in the WIHS. Cunnilingus (performing oral sex on a woman) was defined as “you used your tongue to touch or lick her genitals (vagina, clitoris)” in the MACS and any reported sexual activity with a female in the WIHS. The number of male or female partners an individual performed fellatio or cunnilingus on is referred to as the number of oral sex partners hereafter. Data on rimming, collected only in the MACS, was defined as “you used your tongue to touch or lick his or her anus/butt”. Updated information on lifetime oral sex history was acquired for most participants, but the complete history was not acquired for a proportion of MACS (44%) and WIHS (21%) participants. The updated number of lifetime oral sex partners for these individuals was imputed (see statistical methods below). Recent cigarette smoking was defined as using at least one cigarette per day for the past six months. Recent alcohol use was defined as drinking at least once in the past month.

The definition of HAART was guided by the DHHS/Kaiser Panel(34) and is defined as: the reported use of three or more antiretroviral medications, one of which has to be a protease inhibitor (PI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), one of the NRTIs abacavir or tenofovir, an integrase inhibitor, or an entry inhibitor. Nadir CD4 was the lowest CD4 T cell count observed while in the MACS/WIHS study.

Laboratory Analyses

After collection, oral rinse samples were stored at 4°C and shipped to a laboratory run by Dr. Maura Gillison at the Ohio State University for processing and testing. DNA was isolated from Scope™ oral rinse samples by centrifugation, re-suspended in Phosphate buffered saline (PBS), and purified using a magnetic bead-based automated platform (QIAsymphony SP, Qiagen), as previously described.(35) The purified DNA was then analyzed for 36 different HPV types using PGMY09/11 PCR primer pools and reverse line blot hybridization. Samples were considered evaluable if β-globin positive, and considered insufficient and excluded from the analysis if β-globin negative. HPV types detected were classified into non-oncogenic (low-risk) and oncogenic (high-risk) type categories as previously established by the World Health Organization’s International Agency for Research on Cancer (IARC).(36–38) Oncogenic HPV types included HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73. All other types detected were considered non-oncogenic including HPV 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 66, 67, 69, 70, 71, 72, 81, 82, 83, 84, and 89.

Statistical Analyses

Characteristics of HIV-positive and HIV-negative individuals were compared using chi-square tests for categorical data, and t-tests for continuous variables. Overall oral HPV status was defined as positive for anyone who had any of the 36 HPV types detected, while all others were considered HPV-negative. Additionally, type-specific HPV prevalence was estimated where participants were categorized as positive or negative for each of the 36 different types of HPV tested.

While the number of lifetime sexual partners at MACS and WIHS study baseline was collected for all participants, 34% of the 645 participants were not asked about their updated number of lifetime number of oral sex partners. Therefore, an updated number of lifetime oral sex partners were imputed for these individuals by calculating the average increase in lifetime oral sexual partners from baseline to current visit among the 425 participants with complete sexual behavioral data. These increases were averaged within subgroups defined by gender, years of follow-up, and number of oral sex partners in the past six months and then imputed to individuals without updated lifetime sexual data within each strata. Results were similar when the 220 individuals without an updated measure of lifetime number of oral sex partners were excluded from the analysis.

Risk factors for oral HPV infection were assessed using logistic regression models with robust variance and generalized estimating equations (GEE) to adjust for within-individual correlations related to multiple observations of the same person (i.e., multiple HPV types) using previously described methods.(39, 40) Crude and adjusted odds ratios (OR & aOR), and 95% confidence intervals (CI) were reported. Type-specific risk factors for oral HPV16 infection and multiple HPV infection were calculated using standard logistic regression models. Factors statistically significant (p<0.05) in the univariate analysis and those considered relevant based on previous literature (age, sexual behavior, gender) were included in multivariate models. Models were stratified by HIV-status, per the study design. The final multivariate model for HIV-negative subjects included age, gender, recent cigarette use, number of recent (last six months) and lifetime oral sex partners, and education level. The final multivariate model for HIV-positive subjects included these variables as well as current CD4 T cell count (called current CD4 hereafter), study site, and recent alcohol use. Analyses that stratified by cohort (MACS vs. WIHS) study were also performed. Fourteen individuals had incomplete covariate information. Therefore, our multivariate analyses were based upon 631 individuals.

Trends in the odds ratios were tested using continuous data when available (age, current CD4, HIV viral load, number of recent and lifetime oral sexual partners) and considering ordinal values as a single continuous variable when data was collected ordinally (education, recent cigarette and alcohol use). Correlation between variables was tested using Pearson’s correlation coefficient. All statistical tests were two-sided and considered significant at α=0.05 level. STATA software (version 10.0; Stata Corp., College Station, TX) was used for all statistical analyses.

Participant characteristics

Characteristics of the 645 participants enrolled are shown in Table 1, stratified by HIV-status and gender. Participants in this study sample were similar to those in the MACS and WIHS cohorts. Participants in the MACS sample were similar to the larger MACS population with regards to median current CD4 (550 vs. 557 cells//µL), median age (52 vs. 54 years), proportion White (66% vs. 66%) and proportion sexually active (71% vs 66%, each p>0.05). Participants in the WIHS samples were similar to the larger WIHS population with regards to median age (45 vs. 46 years) and the proportion White (11% vs. 15%), but those in the WIHS study sample had lower median current CD4 (458 vs. 510 cells/µL) and were more likely to be sexually active in the past six months (71 vs. 63%, each p<0.05) when compared to the entire WIHS cohort. By design, the proportion of HIV-infected participants which were HAART naïve was larger in our sample (19%) than the current MACS (6%) or WIHS (7%) study populations.

In this study, most HIV-positive participants were receiving HAART, with median nadir and current CD4 of 266 and 524 cells/µL, respectively. HIV-positive individuals were less educated, and less likely to have recently performed oral sex or to drink alcohol than HIV-negative individuals (each p<0.05, Table 1). HIV-positive individuals were also more likely to be from the WIHS, to be African American, and to smoke cigarettes than HIV-negative individuals (each p<0.05, Table 1).

Oral HPV prevalence

Oral HPV prevalence (34%), including infection with multiple HPV types (14%) was high in this study population (Table 2). The overall prevalence of oncogenic oral HPV infections was lower than that of non-oncogenic infections (18% vs. 23%, p=0.03). However, HPV16 was the most common HPV type detected in the study (prevalence of 5.7%), detected in 2.2% and 5.4% of HIV-negative and HIV-positive women (p=0.21), respectively and in 6.9% and 6.8% of HIV-negative and HIV-positive MSM (p=0.95), respectively. HPV16 constituted a larger proportion of infections detected among HIV-negative than HIV-positive participants (16% vs. 8% p=0.04). The distribution of all oral HPV types detected by HIV status are displayed in Supplemental Table 2, including the most commonly detected oncogenic (HPV16, 59 and 33) and non-oncogenic (HPV55, 62 and 53) types.

Risk factors for prevalent oral HPV infection

HIV-positive individuals were significantly more likely than HIV-negative participants to have a prevalent oral HPV infection detected (adjusted odds ratio [aOR]=2.1, 95%CI=1.6–2.8). This was true for both oncogenic (aOR=1.8, 95%CI=1.3–2.7) and non-oncogenic (aOR=2.3, 95%CI=1.6–3.4) types of oral HPV. Oral HPV16 infection, however, was similarly common among HIV-positive and HIV-negative individuals (aOR=1.3, 95%CI=0.62–2.7).

Recent cigarette smoking was associated with significantly increased odds of oral HPV among both HIV-positive and HIV-negative individuals. There were, however, notable differences between HIV-positive and HIV-negative participants in other risk factors for oral HPV infection (Table 3). The number of recent oral sex partners was the strongest risk factor for prevalent oral HPV among HIV-negative (p-trend=0.003) but not HIV-positive (p-trend=0.88) participants. In the MACS, where data on rimming (oral-anal contact) was collected, number of recent rimming partners was also significantly associated with oral HPV among HIV-negative MSM. However, the number of recent oral sex and rimming partners were highly correlated (r=0.65). In contrast to that observed for recent sex behavior, lifetime oral sex behavior was associated with oral HPV prevalence in HIV-positive (p-trend=0.03), but not HIV-negative (p-trend=0.11) individuals. Odds of oral HPV also increased significantly with older age among HIV-negative individuals (Table 3, p-trend=0.01). Always using a condom during oral sex, race/ethnicity, cigarette smoking intensity and duration, and recent marijuana use were not associated with odds of oral HPV in HIV-positive or HIV-negative individuals (data not shown).

Severity of immunosuppression was the strongest risk factor for prevalent oral HPV among those with HIV (p-trend<0.001, Figure 1 and Table 3, 4) including an association with lower current and nadir CD4, and higher HIV viral load (Table 3). Current CD4 was moderately correlated with current HIV viral load (r=0.48) and with nadir CD4 (r=0.28). However, when modeled together current CD4 was a stronger predictor than either nadir CD4 or current HIV-viral load (Supplemental Table 1). HIV-positive individuals with current CD4>500 remained at significantly increased odds of oral HPV (aOR=1.6, 95%CI=1.1–2.3) but had similar odds of HPV16 (aOR=0.47, 95%CI=0.16–1.4) compared to HIV-negative participants. Oral HPV prevalence was similar among HIV-positive HAART ever users and never users (42% vs. 34%, p=0.24) and current HAART users and non-users (43% vs. 34%, p=0.11).

A strong dose response with lower current CD4 was observed among both HAART-naïve (p-trend=0.03) and HAART-experienced (p-trend=0.002) individuals. This trend was especially strong for HPV16 infection where the median current CD4 was 238 cells/uL among those with HPV16 infection compared to 481 cells/uL among those infected with other HPV types (p=0.002). The association between current CD4 and oral HPV16 was significantly stronger than the association of current CD4 with all other HPV types (p=0.02). Contrasting individuals with current CD4<200 to HIV-positive individuals with current CD4≥500, the OR for HPV16 was over four fold greater than the OR for all other HPV types (aOR=4.5, 95%CI=1.3–16).

When the study population was stratified by MACS/WIHS cohort, current CD4 remained influential among HIV-positive individuals in both cohorts, but there were differences by cohort in other risk factors. The associations of cigarette smoking and lifetime number of oral sexual partners with oral HPV prevalence were more notable among WIHS participants, while recent number oral sex partners was more notable among MACS participants (Table 4).