Oral human Papillomavirus DNA detection in HIV-positive men: prevalence, predictors, and co-occurrence at anal site

This cross-sectional, clinic-based study was designed and conducted at the National Institute for Infectious for Disease, Rome, Italy, according to the Helsinki declaration and approved by the Institutional Review Board (n.42–18/6/2013).

All HIV-positive men, aged 18 years or older, consecutively observed within an anal cancer screening program between February 2015 and June 2016 provided written informed consent. The indication for anal screening was given based on current European AIDS Clinical Society (EACS) guidelines [20] by the HIV physician during routine clinic visit. Patients with history of anal warts or of pre-cancerous lesions were not excluded.

A questionnaire on health behaviours was created ad hoc and interviewer-administered. Questions focused on the following information: demographics, tobacco and alcohol use (more than 3 units per day), previous history of anal/genital condylomatosis, number of sexual partners in lifetime and in the previous 12 months, oral and anal sexual intercourse in lifetime and in the previous 12 months.

Clinical charts were used to abstract presence and type of current cART, as well as viroimmunological parameters (CD4 cell count and HIV RNA needed to be determined within 3 months of samples collection).

At the same time of anal cancer screening, participants provided a sample of oral rinse obtained by swishing and gargling in the oral cavity 10–15 ml of bottle mineral water (Fonte Tullia) for 20–30 s and spitting into a sterile specimen cup. One aliquot (5–7 ml) was sent to the Laboratory of Pathology for cytological evaluation, while the second was centrifuged for 15 min at 3000 g. Cell pellet was washed with sterile phosphate buffered saline and re-suspended in RPMI medium. Nucleic acids were extracted by automated QIASYMPHONY purification system (QIAGEN, Hilden, Germany). QuantiFast Pathogen Internal Control (QIAGEN, Hilden, Germany), was used to check presence of inhibiting factors. Furthermore, to ensure specimen adequacy, i.e. the presence of DNA, the samples were tested by amplification of a 110 bp fragment of the human β-globin gene using PCO3/PCO4 primers [21] followed by analysis in 2% agarose gel. Samples positive for β-globin were considered evaluable.

For HPV DNA testing, a PCR for the L1 region was used. Products were analysed by electrophoresis in 1.7% agarose gel and stained with ethidium bromide, as previously described [22]. HPV-positive samples by MY09/11 PCR were typed using Genomica CLART assay. Samples, that could not be typed with the CLART assay, were directly sequenced using a Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Ca). Sequence alignments were obtained using returned results from GeneBank’s on-line BLAST server (http//https://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi, nih.​gov/​BLAST) [23]. A similarity above 90% was used to identify all known HPV genotypes.

Anal swabs were collected in a standardized manner with a flocked swab according to the manufacturer’s instructions (Eswab, Copan Italia Spa, Brescia, Italy) [24]. The swab was then placed in a sterile tube containing 1 ml of phosphate buffer saline and processed as described above.

Subjects with positive HPV DNA were classified in having a high-risk (HR) HPV, if at least one HR HPV-type was found, or otherwise classified having a low-risk (LR) HPV, if harbouring only low-risk HPV-types. Carcinogenic risk classification was done according to IARC monograph (2007) [25]. Detection was classified in single or multiple HPV if only one or more than one types were characterized. Number of HPV types targeted by nonavalent HPV vaccine were counted in oral and anal samples.

All assays were performed by trained laboratory technicians blinded to clinical information of the patient and to the assay results.

Fixed cytological smears were stained according to the Papanicolau method [26] and adequacy evaluated based on the cellularity, as well as on the presence of acceptable fixation and staining.

Two pathologists, blinded to clinical and HPV information, independently evaluated cytological specimens. A cytological assessment of the quality of the obtained cells from each oral sample was undertaken, using standard parameters that include quality of preparation, cellularity, and types of cells present, in addition, data on micro-organisms, leucocytes/inflammatory cells and artefacts were also obtained. Cytological findings were reported using the following categories of the Bethesda 2001 guideline [27] modified as appropriate for the site: NILM (negative for intraepithelial lesion or malignancy), ASC-US (atypical squamous cells of undetermined significance), L-SIL (low grade squamous intraepithelial lesion), or H-SIL (high grade squamous intraepithelial lesion).

Descriptive analysis was conducted to characterize the subjects included in the study. Median values and interquartile ranges (IQR) were used to describe numerical variables, while counts and percentages were employed for qualitative variables. The number of patients with at least one nonavalent vaccine-targeted HPV type over the total number of subjects found to be HPV DNA-positive at each anatomical site were employed to calculate the proportion of vaccine-targeted infections separately for each anatomical site. The association between variables was assessed using chi-squared test or Fisher’s exact test, as appropriate. Two-tailed p-values were calculated and a value <0.05 was considered statistically significant. A univariate logistic regression was used to analyse the strength of association between positive oral HPV detection and other variables, calculating odds ratios (OR) and their 95% confidence intervals (CI). A multivariable regression analysis, adjusting for variables associated with the outcome at univariate analysis with a p < 0.10 and forcing age, was carried out. Data management and analysis were performed using SPSS version 23 [28].

The study population included 305 HIV-infected males and general characteristics are shown in Table 1. Briefly, patients were predominantly Caucasians (92.8%), with a median age of 44.7 years (IQR: 37.4–51.0). Overall, 262 (85.9%) were MSM. The remaining patients reported HIV acquisition by heterosexual intercourse in 27 cases (8.9%) and by intravenous drug use in 4 cases (1.3%). Mother-to-child transmission and blood transfusion were registered in 1 case each. In the remaining 10 subjects (3.3%) HIV transmission modality remained unknown. At anal cancer screening, 289 (94.8%) patients were on cART since a median of 4.4 years (IQR: 1.9–9.3). Viroimmunological exams showed HIV RNA below 40 copies/ml in 268 patients (87.9%) and a median CD4 cell count of 699/μL (IQR: 529–878). Sixty-two patients (20.3%) had a CD4 cell count <500 cells/μL and 8 < 200 cells/μL. Self-reported previous or current anal/genital condylomatosis was recorded in 112 (36.7%) men.

Health behaviours assessed in the interview showed that 64.9% of patients stated cigarette smoke-exposure (17.4% previously and 47.5% currently), 26.5% reported previous or current recreational drug use, and 3.6% declared current alcohol intake. Sexual history in the patient-reported questionnaire showed at that 152 men (49.8%) counted more than 100 sexual partners in lifetime. In the previous 12 months, 21 men (6.9%) declared no sexual activity, while 97 (31.8%) recalled 10 or more partners. The 284 patients who were sexually-active in the preceding 12 months, recalled the following type of sexual partners: only heterosexual in 31 (10.9%), both sexes in 44 (15.5%), and only homosexual in 209 (73.6%) cases. Median number of sexual partners in the previous 12 months were: 1 for heterosexuals (IQR: 1–2), 7 for bisexuals (IQR: 3.0–18.8), and 4 for homosexuals (IQR: 1–10). Overall, 275 (90.2%) patients reported receptive oral sex. None of the participating patients had been vaccinated for HPV.

HPV DNA was found in oral rinses of 64 out of 305 HIV-positive men (20.9%). HPV genotype was detected by MY09/11 primers in all but 12 cases, in which direct sequencing identified: HPV13 n = 2; HPV22 n = 3; HPV32 n = 8, HPV97 n = 2; HPV107 n = 1; HPV145 n = 1.

The upper panel of Fig. 1 shows the different HPV types detected in the oral cavity, according to multiplicity of infection and risk types. Multiple HPV infections were detected in oral rinses in 18 cases (28.1%): two types in 16 patients, and three to four types in one case each. In HPV DNA-positive oral rinses, alpha-3 species were found in 23 (HPV61 n = 5, HPV62 n = 4, HPV72 n = 6, HPV81 and HPV83 in 2 cases each, HPV84 n = 4), alpha-7 in 14 (HPV18, HPV39, HPV45 and HPV59 in 1 case each, HPV70 and HPV85 in 4 cases each, HPV97 n = 2), and alpha-9 in 12 cases (HPV16 n = 3, HPV33 n = 5, HPV35 and HPV58 in 2 cases each). HPV16 and HPV18 were found in three and one oral samples, respectively. Beta-2 species were detected in 5 samples (HPV22 n = 3, HPV107 and HPV145 in 1 case each). HR types were found in 50% oral rinses with detectable HPV DNA (n = 32), and were more frequently observed in presence of multiple than of single HPV types (77.8% versus 39.1%; p = 0.005).

Oral rinses samples were not adequate for cytological examination in 27 patients because of low cellularity. Among 278 cases, cytology was normal in 228 (82.0%) cases, while cellular atypia (ASC-US or more) was found in 50 patients: ASC-US n = 49 (17.6%) and H-SIL n = 1. In Table 2, the distribution of cytological findings according to HPV risk group is shown. The risk of cellular atypia (ASC-US or more) was higher in presence of HR types when compared to subjects harbouring only LR types (OR 2.31; 95% CI 0.83–6.69; p = 0.050).

HPV DNA was detected at the anal site in 260 out of 305 (85.2%) male patients. Thus, the proportion of subjects with positive HPV DNA at the oral cavity was significantly lower when compared to that of persons with HPV DNA at the anal site (21.0% versus 85.2%; p < 0.001). The finding of multiple HPV infection was more frequent in anal (206/260) than in oral (18/64) samples (79.2% versus 28.1%; p < 0.001). Out of 265 HPV DNA-positive patients regardless the anatomic site, only in 59 cases (22.3%) HPV DNA was recovered in oral rinses and in anal samples at the same time (19.3% of the overall cohort). Detection of HPV at the oral cavity tended to be more frequent in men with HPV DNA detected at anal site when compared to those with negative results (22.7% versus 11.1%, p = 0.078). Concerning specific HPV types, among the 59 patients with detectable HPV DNA at both anatomical sites, 51 (86.4%) had completely different HPV types. Out of the remaining eight, seven cases shared only one HPV type (HPV11 n = 2; HPV61, HPV81 and HPV83 in 1 case each among LR HPV types; HPV51 and HPV66 in 1 case each among HR HPV types), while one patient harboured the same double infection by HR HPV39 and HPV59 types at both sites. In Fig. 1, the distribution of HPV types detected at the anal site are shown by multiplicity and oncogenicity of HPV types.

Table 3 shows the univariate analysis that found significantly higher odds for oral HPV DNA detection in men who have had more than 5 sexual partners in the previous 12 months (OR 2.72; 95% CI 1.39–5.34; p = 0.004) or more than 100 partners in lifetime (OR 5.77; 95% CI 1.31–25.35; p = 0.020). Further, a significant association between oral HPV DNA and self-reported history of anal/genital condylomatosis (OR 1.86; 95% CI 1.06–3.24; p = 0.030), use of recreational drugs (OR 2.0; 95% CI 1.01–3.98, p = 0.048), and current CD4 cell count below 200/μL when compared to higher values (OR 12.36; 95% CI 2.43–62.83; p = 0.002) was found.

At multivariable analysis, after adjusting for age as well as all variables found to be associated with positive oral HPV DNA at univariate analysis (p < 0.01) and choosing to include “number of sexual partner in the previous 12 months” instead of “number of lifetime partners” to avoid collinearity, a significant independent association between detectable oral HPV DNA and the following conditions was confirmed: CD4 cell count ≤200/μL versus > 200/μL (OR 12.58; 95% CI 2.19–72.30; p = 0.005) and to report >5 sexual partners in the previous 12 months versus 0–1 partner (OR 2.78; 95% CI 1.30–5.93; p = 0.008).

A minimum of one nonavalent vaccine-targeted HPV type was found in 17 out of 64 patients (26.6%) with positive oral HPV DNA, and in 199 out of 260 of patients (76.5%) with HPV positivity at the anal site. The nonavalent vaccine-targeted vaccine would have covered all identified HPV types in 11/17 (64.7%) of patients with HPV at the oral cavity and in 33/199 (16.6%) of patients with HPV at the anal site (Fig. 1).