Analysis of Male Human Papillomavirus Infection and Genotyping in the Heyuan Region

Summary

This study analyzed male patients who visited the outpatient department of Heyuan People's Hospital and underwent human papillomavirus (HPV) nucleic acid testing, providing preliminary insights into the epidemiological characteristics and genotype distribution of HPV infection in the region. These findings provide support for the prevention and control of HPV-related diseases.

Abstract

This study aimed to investigate the prevalence and genotype distribution of human papillomavirus (HPV) among male outpatients in Heyuan and to compare infection rates across age groups, to inform local prevention and treatment strategies. A total of 1,559 men who underwent HPV testing at Heyuan People's Hospital between 2021 and 2024 were enrolled, and only results from initial visits were included. Twenty-three HPV genotypes were identified by polymerase chain reaction (PCR)- reverse dot blot hybridization. A retrospective cross-sectional analysis was conducted to evaluate overall HPV prevalence, age-specific infection rates, infection patterns, subtype distribution, and vaccine-relevant genotype coverage. Statistical analyses were performed using SPSS version 23.0. The count data were summarized as numbers and percentages, and group differences were assessed using the chi-square test, with statistical significance defined as p < 0.05. The overall HPV infection rate was 45.3% (707/1,559). Infection rates across age groups (≤24, 25–34, 35–44, 45–54, 55–64, and ≥65 years) were 42.8%, 43.1%, 45.8%, 48.2%, 68.3%, and 46.4%, respectively. Single-type infections were the most common pattern, accounting for 57.0% of positive cases. The five most frequently detected high-risk genotypes were HPV52 (9.05%), 16 (5.90%), 53 (5.09%), 51 (5.01%), and 59 (4.93%), while the predominant low-risk genotypes were HPV6 (21.24%), 11 (9.85%), and 43 (5.17%). The theoretical coverage rates for the bivalent, quadrivalent, and nine-valent vaccines were 8.65%, 39.74%, and 55.41%, respectively. The prevalence of HPV infection among male outpatients in Heyuan was relatively high, with single-type infections being the dominant pattern. The most common high-risk genotypes were HPV52, 16, 53, 51, and 59, and the leading low-risk genotypes were HPV6, 11, and 43. The highest infection rate was observed in the 55–64 age group. These findings provide region-specific data that may support the development of tailored HPV prevention and vaccination strategies for males in this population.

Introduction

Human papillomavirus (HPV) is a DNA virus that is transmitted primarily through sexual contact, with humans as its sole host. The prevalence of HPV is similar among males and females worldwide, ranging from 3.5% to 45% in males and 2% to 44% in females¹. Nearly 80% of sexually active men and women will contract the virus at least once during their lifetime, as transmission routes include not only sexual contact but also friction or contact with skin surfaces and mucous membranes, maternal-infant vertical transmission, etc². To date, more than 200 different HPV genotypes have been described, classified as high-risk based on their association with the onset of cervical, vaginal, vulvar, anal, head and neck, and penile cancers, and low-risk associated with genital warts or condyloma acuminata³. In most cases, the infection can be completely cleared by the immune system within 12 to 24 months without clinical complications. However, if the immune system fails to clear the virus, a persistent infection develops, laying the foundation for the onset of neoplastic hyperplastic lesions⁴. Specifically, persistent high-risk human papillomavirus (HR-HPV) infection is associated with the development of squamous intraepithelial lesions (SILs) as well as penile intraepithelial neoplasia (PeiN); Both of these lesions are precancerous conditions for cervical cancer in women and penile cancer in men^5,6. Additionally, lifestyle factors such as long-term use of oral contraceptives, smoking, immunodeficiency, or concurrent sexually transmitted infections may also promote neoplastic proliferation⁷.

At present, there are many studies on the prevalence, risk factors, and prevention strategies of HPV infection in women in China, and the awareness rate and vaccination rate of the HPV vaccine in female groups have also increased significantly. HPV spreads easily between sexual partners, and in many cases, multiple undetected transmission events may occur between spouses⁸. The latest clinical guidelines in China prioritize HPV vaccination for females aged 9–26 years, with a particular focus on girls aged 9–14 years. Additionally, vaccination is recommended for females aged 27–45 years and males aged 9–26 years to advocate for joint prevention efforts between genders⁹.

HPV infection in males may be the cause of HPV infection and repeated infection in female sexual partners¹⁰. In addition, HPV infection can cause diseases such as male common warts, genital warts, genital cancers, oropharyngeal and anal cancers¹¹, and may also be associated with male infertility¹². However, there are relatively few studies on HPV infection in men in China. Furthermore, China has a vast territory and a large number of ethnic groups. The concepts of marriage and living habits differ across regions. HPV infection also has regional, racial, and diversity¹³. Other studies have demonstrated that HPV vaccination in men exhibits significant preventive potential against cervical lesions among sexual partners¹⁴. A comprehensive understanding of male HPV infection patterns and genotyping profiles can enhance public health systems and reduce the costs associated with managing and treating both male and female patients. Reports indicate that HPV-related diseases in males impose a more substantial economic burden on overall healthcare expenditures in Italy¹⁵.

Therefore, this study aims to understand the prevalence, age-stratified positivity rate, genotype frequency, infection pattern distribution, and vaccine type coverage of male outpatient patients who underwent testing in the Heyuan region from 2021 to 2024, providing an important basis for early screening, diagnosis, prevention, and treatment of male HPV infection in the region.

Protocol

This study has been reviewed and approved by the Medical Research Ethics Committee of Heyuan People's Hospital (Approval Number: YXYJLL-SFKJ202461; Approval Date: May 15, 2024). The ethical certificate has been uploaded as a supplementary file. The patient, parent, or guardian of a minor has signed a statement during registration stating “I agree that my clinical biological samples and data will be used for scientific research, technological innovation, and clinical research approved by the Medical Research Ethics Committee after removing identifiable personal information”, and this analysis will not pose any additional risks to the patient and complies with relevant regulations. All the materials used in this study are listed in the Table of Materials.

Subjects

A total of 1,559 male patients who visited the outpatient department of Heyuan People's Hospital and underwent HPV testing between January 2021 and December 2024 were included in the study. The subjects were aged from 2 to 80 years old (The study population consists of all male patients with valid HPV nucleic acid test results from outpatient visits at this medical institution between 2021 and 2024; The primary objective is to provide a comprehensive overview of HPV testing and infection patterns among male patients in the region; Furthermore, the routes of HPV infection not only include sexual activity, but also friction or contact with the skin surface and mucous membranes, as well as vertical transmission from mother to child. Therefore, no age restrictions were imposed, with an average age of (34.2 ± 10.7) years old.

Methods

This study employed a retrospective cross‑sectional design to analyze all male patients who visited our hospital's outpatient department and underwent HPV testing; only the initial sampling results were included for those who underwent repeated testing.

Specimen collection

The exfoliated cell samples were obtained by trained clinicians using sterile, specialized sampling brushes. Collection sites included the dorsal and ventral surfaces of the penis, the internal and external coronal sulci, the glans penis, the distal urethra, and any visible verrucous lesions, with site selection determined based on individual patient findings; after collection, the brush heads were broken in a dedicated sample preservation solution. Specimen preservation conditions: After sampling, the specimens were immediately stored in a refrigerator at 2–8 °C, and the test was completed within 3 days.

DNA extraction

After each sample was oscillated and mixed, 200 µL were sequentially added to the nucleic acid extraction or purification reagent. DNA extraction was performed using magnetic bead technology with the extraction reagent in the SP96 fully automated nucleic acid extraction instrument.

HPV detection

HPV-DNA testing was performed using the human papillomavirus genotyping (type 23) detection kit, which employs a combination of PCR-based in vitro amplification and DNA reverse dot blot hybridization for HPV genotyping. Specific primers were designed based on HPV genetic characteristics, and target fragments from 23 HPV genotypes were amplified. The amplified products were hybridized with the typing probes fixed on the membrane, including 17 HR-HPV (HPV16,51,52,53,18,58,59,31,33,45,56,66,35,39,68,73,82) and 6 LR-HPV (HPV6,11,42,43,81,83). Based on the presence or absence of hybridization signals, the infection status of these HPV genotypes was determined. The specific laboratory procedure involved using a filtered pipette tip to add 5 µL of extracted DNA to the PCR reaction mixture, bringing the total volume to 25 µL. Subsequently, PCR amplification was performed. After amplification, all amplified products were added to the isothermal hybridizer containing membrane strips for colorimetric hybridization. Upon completion of color development, results can be interpreted.

Statistical analysis

SPSS 23.0 software was used for statistical analysis. The count data were expressed as the number of cases (n) and percentage (%). The comparison between groups was performed by χ² test. p < 0.05 was considered statistically significant. Among the 23 HPV genotypes, positivity for any single genotype indicates HPV infection; positivity for only one genotype indicates a single infection; and positivity for two or more genotypes indicates a multiple infection. Multiple infections are further classified based on the number of coexisting genotypes into dual infection, triple infection, quadruple infection, pentuple infection, hexuple infection, and higher." The ratio to total detection frequency” was calculated using the total number of genotype detections (1,238) as the denominator, as patients with multiple infections contributed multiple genotypes.

Results

Overall prevalence of HPV positivity in the study population

Among 1559 male outpatients, 707 cases were positive for HPV DNA, with a positive rate of 45.3%. The annual HPV infection rates were 2021 (44.8%, 100/223), 2022 (45.5%, 231/508), 2023 (45.3%, 182/402), and 2024 (45.5%, 194/426). There was no significant difference in HPV infection rate among different years (χ² = 0.033, p = 0.998) (See Table 1). Except for 2023, when no ...

Discussion

HPV is the most common sexually transmitted virus in men and women. High-risk infection is closely related to cervical, vulvar, and vaginal cancer in women, and penile, anal, and oropharyngeal cancer in men. Persistent low-risk types can lead to most anal genital warts and respiratory papillomatosis¹⁶. An analysis of diseases associated with HPV infection in men showed a high prevalence of HPV in the male penis, penis head / coronary sulcus, semen, and scrotum, perianal and anal regions, ranging f...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Detection of Mycoplasma pneumoniae nucleic acid and drug resistance gene	Guangzhou Da 'an Gene Co., Ltd.	Stream SP96
Filter Tips	Coring Life Science Co., Ltd.	29822001	0–10 µL
Filter Tips	Coring Life Science Co., Ltd.	05623001	5–300 µL
Human papillomavirus genotyping ( type 23 ) detection kit	Shenzhen Ya Neng Biotechnology Co., Ltd.	20193401918
Nuclear acids extracting reagent(magnetic beads method)	Guangzhou Da'an Gene Co., Ltd.	20170583
PCR amplification instrument	Thermo Fisher Scientific	ABI Veriti DX 96
PCR Hybridizer	Shenzhen Ya Neng Biotechnology Co., Ltd.	YN-H16
Sampling brush	Shenzhen Ya Neng Biotechnology Co., Ltd.
Sample preservation solution	Shenzhen Ya Neng Biotechnology Co., Ltd.