Prevalence and Genotype Distribution of HPV Infection Among People Living With HIV in Colombia

This was a single-centre, cross-sectional observational study involving one group of adults living with HIV, selected through simple random sampling from the HIV clinic at Hospital Universitario San Ignacio (HUSI), a tertiary-care university hospital in Bogotá, Colombia. Study procedures were conducted between March 2023 and September 2025.

Eligibility Eligible participants were PLWH aged 18 years or older receiving ART for at least three months. Individuals with obstructive anal, cervical, or oral tumours, contraindications to study procedures, cognitive impairment limiting questionnaire comprehension, active cancer treatment, pregnancy with threatened abortion or preterm labour, or refusal to consent were excluded.

Sample size and sampling strategy Participants were selected by simple random sampling from eligible adults at the HUSI HIV clinic. The sample size was calculated to estimate anal high-risk HPV prevalence, assuming a 95% confidence level, 5% absolute precision, a design effect of 1.0, and a source population of 2,200 PLWH. Based on an expected prevalence of 80%, the required minimum was 222 participants; allowing for non-participation and non-evaluable samples, 319 individuals were invited.

Study variables and procedures

Baseline

At baseline, the investigators collected sociodemographic, clinical, behavioural, mental health, and HIV-related information through a structured interview and review of medical records. A stable partnership was defined by marital status and sexual behaviour: participants were classified as having a stable partner if they were married or cohabiting and reported only one sexual partner in the previous year. Condom use referred to condom use at the most recent sexual intercourse, and recreational drug use to self-reported use of psychoactive substances other than alcohol or tobacco. Sexual-practice variables included self-reported anal and oral intercourse. Socioeconomic status was classified as low or middle/high according to the Colombian socioeconomic stratification system. Other variables included previous HPV-related screening, HPV vaccination status, sexually transmitted infections in the previous year, the most recent CD4+ T-cell count, and HIV viral load. All participants were receiving ART, recorded as a nucleoside backbone plus a third or fourth agent when applicable; for the analysis, the regimen was classified by whether it included an integrase strand transfer inhibitor.

Physical examination After eligibility was confirmed and informed consent obtained, participants underwent a directed physical examination of the anal, genital (when applicable), and oral/oropharyngeal regions. Anal, cervical (when applicable), and oropharyngeal samples were assessed by cytology and extended HPV DNA genotyping.

Procedures Site-specific samples were collected by trained personnel according to the internal laboratory protocol; when an anal sample was unsatisfactory for cytology or genotyping, repeat sampling was offered. Before anal sampling, participants were advised to avoid anal intercourse and enemas during the preceding 24 hours. Cervical samples were obtained with a Rovers® Cervex-Brush®, and anal and oropharyngeal samples with a cytobrush suitable for epithelial cell sampling. All specimens were transferred to SurePath™ liquid-based cytology medium (BD, Franklin Lakes, NJ, USA) and processed according to the internal laboratory protocol.

Cytology Cytological evaluation was performed on all specimens, and aliquots from the same samples were used for high-risk HPV DNA detection and extended genotyping. Cytological findings from all sites, including anal sample adequacy, were classified according to the Bethesda System. Results were categorised as negative when no epithelial abnormality was reported, and as abnormal when any epithelial abnormality was present (ASC-US, ASC-H, LSIL, HSIL, HSIL suspicious for invasion, or squamous cell carcinoma). As the Bethesda System is standardised for cervical cytology, criteria for sample representativeness and adequacy at the oropharyngeal site were adapted following previous work by the research group.

HPV genotyping Extended high-risk HPV genotyping was performed using the BD Onclarity™ HPV Assay, which individually detects genotypes 16, 18, 31, 45, 51, and 52 and reports the remaining high-risk genotypes in three groups: 33/58, 56/59/66, and 35/39/68. Although the assay is validated and FDA-approved for cervical specimens, its use in anal and oropharyngeal specimens is off-label. Before study implementation, the laboratory performed an internal analytical validation of SurePath-preserved anal and oropharyngeal specimens on this platform. For anal specimens, the assay has been evaluated in a recent prospective study using histologically confirmed anal intraepithelial neoplasia grade 2 or worse (AIN2+) as the clinical endpoint, reporting a sensitivity of 87.7% (95% CI 76.3-94.9) for extended high-risk HPV detection.

Psychiatric evaluation Anxiety and depressive symptoms were assessed using the GAD-7 and PHQ-9. Based on previously reported evidence, at a cut-off of ≥10 the GAD-7 has a sensitivity and specificity of 89% and 82% for generalised anxiety disorder, and the PHQ-9 of 88% for major depression. Participants scoring ≥10 on either scale were referred for mental health evaluation. For the analysis, clinically relevant depressive symptoms were defined as a PHQ-9 score ≥5 (at least mild symptoms). Mental health symptoms will be completely addressed in a separate analysis.

Outcomes The main outcomes were overall and site-specific HPV detection, high-risk HPV genotype detection, and cytological abnormalities. High-risk genotypes were defined as types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, consistent with genotypes commonly reported as high-risk.

Exploratory secondary outcomes included associations of sociodemographic, clinical, behavioural, mental health, and immunovirological factors with anal high-risk HPV detection, together with agreement between anal cytology and anal high-risk HPV genotyping.

Data management Clinical information was recorded in the institutional electronic medical record, and GAD-7 and PHQ-9 questionnaires on paper. All data were entered into REDCap by the study coordinator, with a sequential anonymous identifier per participant. Cytology and genotyping reports were entered into the same database, and a 100% audit verified completeness and concordance with the primary source. Database access was restricted to authorised personnel, and identifiable information was excluded from the analysis dataset, following institutional confidentiality procedures and Colombian data protection regulations.

Statistical analysis Descriptive analysis Sociodemographic, clinical, behavioural, and HIV-related characteristics were summarised using absolute and relative frequencies. Baseline characteristics were presented for the overall sample and stratified by detection of anal HPV-16/18 and HPV-16/18/31/45.

Bivariate and multivariable analyses An exploratory analysis assessed associations between anal high-risk HPV genotype detection and variables previously reported as potentially associated with a positive result, including age, condom use, recreational drug use, stable partnership, antiretroviral regimen, CD4+ T-cell count, and HIV viral load; a positive depression screen was also explored. This analysis was restricted to participants with adequate/evaluable anal cytology and valid anal HPV genotyping. Associations were assessed for two outcomes (anal HPV-16/18 and HPV-16/18/31/45). These genotypes were selected because of their well-established oncogenic relevance. Crude associations were estimated by entering each variable separately into a log-binomial model to obtain crude prevalence ratios (PRs) with 95% confidence intervals (CIs). All prespecified covariates were then entered simultaneously; as the multivariable log-binomial models did not converge, Poisson regression with a log link and robust variance estimator (HC0) was used to estimate adjusted prevalence ratios (aPRs) with 95% CIs. Diagnostics included convergence and multicollinearity (variance inflation factors); no substantial multicollinearity was found.

Missing data Missing data were assessed within the analytical population (participants with adequate/evaluable anal cytology and valid anal HPV genotyping; N=255). Missingness was below 10% for all variables and was handled using multiple imputation by chained equations (MICE), assuming data missing at random. Imputation was applied only to the secondary analysis of factors associated with high-risk genotype detection. Twenty imputed datasets were generated with 20 iterations each; convergence was assessed with trace plots and imputed values compared with observed distributions. Estimates and 95% CIs were pooled using Rubin's rules.

Concordance analysis between HPV genotyping and cytology findings Agreement between anal HPV genotyping categories (HPV-16 alone, HPV-16/18, HPV-16/18/31/45, and any high-risk genotype) and anal cytology was assessed using row percentages and Cohen's kappa (κ) with 95% CIs. Associations between the same categories and anal epithelial abnormalities were assessed using row percentages and Cramér's V with 95% CIs, with significance tested by the χ² test. Given their exploratory nature, findings were interpreted according to clinical relevance and precision rather than statistical significance alone.

Software Data cleaning, descriptive analyses, and statistical modelling were performed using R version 4.5.1 in RStudio (version 2026.07.0+139).

Ethical considerations The study protocol was approved by the Research and Ethics Committee of Hospital Universitario San Ignacio and Pontificia Universidad Javeriana (FM-CIE-1162-22). Written informed consent was obtained from all participants before enrolment. The study was conducted in accordance with the Declaration of Helsinki and Colombian regulations, and participant confidentiality was protected according to institutional data protection procedures.