Oral HPV infection in a clinic-based sample of Hispanic men

This study consisted of a cross-sectional study with men attending a publicly funded STI clinic in San Juan, Puerto Rico. A convenience sample was drawn from 205 men from within the clinic’s waiting room, coupled with subsequent screening to confirm eligibility (at least 16 years of age) and informed consent. Study participation was voluntary and written informed consent, including Health Insurance Portability and Accountability Act (HIPAA) forms, notifying about the nature of the study, general purpose, the types of research and clinical procedures involved, were provided. All study design, written consent and data collection procedures were approved by the University of Puerto Rico-Medical Sciences Campus (UPR MSC) Institutional Review Board (IRB).

The behavioral interview was completed in approximately 25 minutes by a trained interviewer. Domains included demographic characteristics (e.g. age, place of birth, current residence, employment, and educational attainment), detailed assessment of sexual risk (e.g. including age of onset, current sexual partners and practices), and assessment of alcohol, cigarette and drug use- (e.g. types, frequency, and mode of administration of illicit/illegal drugs such as: marihuana, cocaine, heroin, and crack). Additionally, the assessment included questions about health history (self-reported STI’s and HIV status).

A person was considered oral HPV positive if resulted positive to any of the 27 types tested, whereas all others were considered negative. Behavioral characteristics included patterns of smoking tobacco, smoking marihuana, and drinking. Lifetime and recent cigarette and marihuana smoking, as well as lifetime alcohol use were measured. Lifetime smoking was defined as having smoked at least once during the lifetime. Current smoking was defined as smoking at least one cigarette per day for the past 12 months. Years smoking was defined as the number of years spent smoking regularly. Lifetime smoking of marihuana was measured as “ever smoked marihuana”, which referred to having smoked marihuana at least once during lifetime. “Ever smoked marihuana in the past 12 months” was defined as having smoked marihuana at least once during the past 12 months. Lifetime alcohol use was measured as “ever having an alcoholic beverage”, which referred to drinking at least once during lifetime.

The self-reported sexual characteristics analyzed within the sample dealt with sexual behavior, including lifetime and recent oral sex. Men who had sex with men (MSM) was a self-reported measure from the question: “Regarding your lifetime sexual partners, how many of them have been men?” Those participants that answered “1 or more men”, were categorized as “men who had sex with men” (MSM) in their lifetime. Those that answered zero (0) were categorized as non-MSM. Lifetime oral sex was defined as having had oral sex at least once during lifetime. Recent oral sex was defined as having had oral sex at least once during the past 12 months.

All of the specimen collection and HPV polymerase chain reaction (PCR) procedures used in this study have been extensively tested and refined, and used in multiple previously published studies[28, 29]. After completion of the behavioral interview, participants underwent collection of biological specimens by trained physicians at the study site. An oral mouthwash sample was collected to obtain DNA for HPV genotyping. Oral rinse samples were collected as previously described in the National Health and Nutrition Examination Survey (NHANES)[30]. Briefly, each participant vigorously swished and gargled 10 ml of Scope mouthwash for 30 seconds. This was followed by depositing the Scope and saliva generated from the mouthwash into a specimen collection cup.

DNA was extracted using the DNA purification from buccal cells protocol from Gentra PureGene Kit (Qiagen, Valencia, CA) and following the manufacturer’s instructions. The concentration of each DNA was quantified and qualified spectrophotometrically using a Nanodrop HPV genotyping was determined in all study samples using the INNO-LiPA HPV Genotyping Extra assay (Innogenetics, Belgium) and following the manufacturer’s instructions. The INNO-LiPA HPV assay is a PCR-based hybridization assay that includes a cocktail of biotinylated consensus primers (small primer fragment (SPF) 10) to amplify a portion of the L1 open reading frame (ORF) of 13 established HR HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66), 5 known or putative HR types (26, 53, 68, 73, and 82), 7 LR HPV types (6, 11, 40, 43, 44, 54, and 70), additional non-differentiated HPV types, and types with undefined risk (74 and 69/71). After DNA extraction, all specimens were subjected to PCR amplification (40 cycles) using the Inno-LiPA HPV Genotyping Extra Amp. The PCR product was then denatured, and a 10-μl aliquot was hybridized onto nitrocellulose strips where the HPV type-specific oligonucleotides were already bound. After 60 min at 49°C, the PCR product bound to a specific probe was detected by an alkaline phosphatase-streptavidin conjugate and colorimetric detection. Results were visually interpreted by experienced laboratory personnel using the reference guide provided. Some types are defined by a single positive probe on the genotyping strip (i.e., HPV6, -11, and -16), but others are interpreted as a combination of two to four probes (i.e., HPV18, -33, and -58). While the manufacturer provides interpretation of type detection, including “possible” types, only those that were detected unequivocally were included in the analysis.

Frequency distributions and descriptive statistics were first used to characterize the study sample in terms of demographics, behavioral and HIV status. Overall prevalence of oral HPV infection was estimated, along with 95.0% binomial confidence interval (CI). Frequency distributions were used to describe the DNA HPV-types detected in the oral cavity. Chi-square tests, Fisher’s exact tests, and independent t-tests were used to evaluate differences in demographic, behavioral, and HIV status. Statistical significance was set at p-value <0.05. Variables that resulted significantly associated with oral HPV seroprevalence in the bivariate analyses were later evaluated for possible collinearity. A Pearson correlation of 0.5 or greater and a p-value for the two-tailed significance test <0.05 was considered as a significant correlation.

Factors significantly associated with oral HPV infection were included in a logistic regression analysis. Different binary logistic regression models were developed in order to investigate the relationship between the selected risk factors and the occurrence of any type of oral HPV infection. Associations with oral HPV prevalence were reported as crude and adjusted Odds Ratios (ORs), along with 95%CIs and significance assessed through the Wald test p-value < 0.05. Trend tests were conducted across age categories. Goodness of fit for all models was assessed using the log likelihood estimates. In order to choose the best model, the log likelihood estimates were compared by subtracting each model’s log likelihood from the base model’s log likelihood. First-order interactions between the significant factors of the chosen model were also evaluated in the logistic regression analysis.

Statistical analyses were performed using the statistical package SPSS (Version 21.0, Chicago, IL), while 95% confidence intervals (CI) for prevalence estimations were performed using the Binomial CI Calculator from STATA Statistical Software (Release 11, College Station, TX).

The demographic and behavioral characteristics of the 205 participating men are shown in Table 1. The mean age of the study sample was 38.5 years (standard deviation [SD] = 14.2). The majority (65.2%) had a high school education or less and was employed (53.9%). Also, public health insurance was predominant among the sample (58.9%); although 18.3% reported not having any type of medical insurance.

The mean years for smoking was 23.9 years (SD =14.3). More than half of the interviewed men had smoked at least once in their lifetime (73.5%), reported to be current smokers (65.8%), and had at least one alcoholic beverage in their lifetime (95.1%). Though the majority had smoked marihuana at least once in their lifetime (62.3%), they reported not having smoked marihuana in the past 12 months (55.2%).

Half of the sample reported having HIV (50.2%). The mean years of living with HIV was 11.5 years (SD = 8.4 years). Oral HPV infection was observed among 22.3% of the HIV positive men and 17.8% among HIV negative men. Additionally, approximately a third (29.5%) of the participants indicated having had sex with another man (MSM). The vast majority of the MSM’s interviewed in this sample had reported oral sex at least once in their lifetime (91.2%) and at least once in the past 12 months (91.7%). These results were also consistent with those who reported being heterosexual, in which a vast majority (91.9%) reported having had oral sex at least once in their lifetime and 76.0% having had oral sex at least once in the past 12 months.

The prevalence estimates for oral HPV infection by the 27 individual genotypes analyzed among the 205 participating men aged 16–81 years are shown in Table 2. The prevalence of oral HPV infection among men was 20.0% (95% CI = 14.8%, 26.1%). The prevalence of high-risk HPV infection was 10.7% (95% CI = 6.8%, 15.8%) and for low-risk HPV was 7.3% (95% CI = 4.2%, 11.8%). The most prevalent high-risk subtype was HPV-52 (2.9%), while the most prevalent low-risk subtype was HPV-6 (3.9%). HPV types 18, 26, 33, 45, 53, 59, 79/71, and 82 were not detected in this sample.

Table 3 shows the results for the associations between demographic, behavioral, and clinical characteristics with HPV status. Oral HPV prevalence was significantly associated with age, lifetime smoking, years smoking, lifetime marihuana use, and lifetime number of sexual partners. However, bivariate correlation analyses demonstrated that having smoked at least once during lifetime, having smoked marihuana at least once during lifetime, and years smoking were significantly correlated (p-value <0.05). Upon this result, we only included “having smoked at least once during lifetime” for subsequent analyses.

Results for the univariate and multivariate logistic regression models evaluating the association of demographic and behavioral variables with the likelihood of HPV infection are demonstrated in Table 4. In the unadjusted logistic regression model, factors independently associated with increased prevalence odds of HPV infection included: age, lifetime smoking and lifetime number of sexual partners. The prevalence of any type of oral HPV significantly increased over increasing age categories (p-trend = 0.001). Men who had smoked at least once in their lifetime were three times more likely to test positive for any HPV type infection (OR = 3.10; 95% CI = 1.15, 8.36) as compared with those who had never smoked in their lifetime. Also, per every unit increase in lifetime sexual partners, the prevalence of oral HPV infection increased by 2%.

In the complete multivariate model (model 3), lifetime smoking and lifetime sex partners remained independently associated with detection of any type of oral HPV. Odds of detecting any type of oral HPV infection remained three-fold higher among men who had smoked at least once in their lifetime compared to those who had never smoked (OR = 3.00; 95% CI = 0.98, 9.16), after adjusting for differences in age and number of lifetime sexual partners. This association was marginally significant. Odds of detecting any type of oral HPV infection remained significantly higher among men with increased number of lifetime sex partners (OR = 1.02; 95% CI = 1.01, 1.03), after adjusting for differences in age and lifetime smoking. No significant first-order interactions were observed between age, lifetime smoking, and lifetime sex partners.