Male papillomavirus infection and genotyping in the Qingyuan area

This study was a retrospective trial. From January 2014 to September 2018, male patients with high-risk for HPV infection who were hospitalized and received a physical examination in Qingyuan People's Hospital were recruited in this study. These participants were hospitalized due to their disease. HPV infection can be divided into low-risk type and high-risk type according to the pathogenicity or carcinogenic risk. High-risk populations mainly include the following: (1) subjects with symptoms of urethritis, wrapping balanitis, etc.; (2) subjects with suspected condyloma acuminata, papules, and ulcers; and (3) subjects who are conscious of the symptoms of discomfort or have a physical examination. This study was conducted in accordance with the declaration of Helsinki and approved by the Ethics Committee of the Sixth Affiliated Hospital of Guangzhou Medical University and Qingyuan People’s Hospital. Informed consent was obtained from the patient or his guardian.

Inclusion criteria: (1) male patients; (2) patients over 14 years old; (3) high-risk populations for HPV infection. Exclusion criteria: (1) patients with incomplete clinical data; (2) patients with non-conforming specimen collection.

Urethral epithelium or scraped penile epidermis were collected. Urethral swab was used to sample the urethral epithelium [4]. The swab was inserted ~ 2 cm into the urethra and rotated 360 degrees while removing it. Subjects with verrucous vegetations were scraped with aseptic blades.Samples were placed in separate tubes containing Eagle Minimum Essential Medium, and immediately sent for examination. Specimens that could not be sent in time for examination were stored in a refrigerator at 4 °C or − 20 °C. All specimens were taken from patients in the hospital, and the hospital arranged special personnel to transport them, without any commercial organization to assist in transportation.

The HPV-DNA genotyping (23 types) detection reagent kit was purchased from Shenzhen Yaneng Biological Technology Co., Ltd, which includes 18 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 83, and MM4) and five low-risk types (HPV6, 11, 42, 43, 81, and 82). The main inspection instruments included the following: Life express PCR expander, which was purchased from Hangzhou Bioer Technology Co. Ltd.; YN-H18 semi-automatic nucleic acid molecular hybridometer, which was purchased from Yaneng BioSciences (Shenzhen).

One ml of sample containing medium was transferred to a 1.5-ml centrifuge tube and centrifuged for 10 min at a speed of 13,000 rpm. The liquid supernatant was discarded to retain the cell precipitate at the bottom of the tube. Then, 50 μl of lytic solution was added to the suspended precipitate, centrifuged for 10 min at 13,000 rpm, and heated for 10 min to retain the liquid supernatant containing DNA sample for future use.

Five μl of the extracted DNA was taken for the PCR reaction under the following conditions: 50 °C for 15 min; 95 °C for 10 min; 94 °C for 10 s, 42 °C for 90 s, 72 °C for 30 s, 10 cycles; 94 °C for 10 s, 46 °C for 60 s, 72 °C for 20 s, 30 cycles; 72 °C for five minutes. After amplification, the membrane strip was marked with the patient number and 2 mL of liquid A and all the corresponding PCR products were lightly shaken and hybridized at 51 °C for 30 min. Liquid A was discarded, 2 ml of liquid B was added and shaken gently for 10 min. Liquid B was then discarded, an incubation solution was prepared according to liquid A:POD = 2000:1, 2 ml of incubation solution was added to each film strip and it was gently soaked at room temperature for 15 min. The incubation liquid was discarded, and each film strip was washed at room temperature with liquid A and liquid C. The film was soaked in 2 ml color-developing liquid for 15 min. Then, 2 ml of deionized water was added into the discoloration solution, it was shaken gently for 3 min, and the results were observed. Result interpretation: (1) the IC site on the membrane strip had a signal interpreted as genotype positive in the corresponding position; (2) blue spots were found at one or more HPV genotypic sites, which were judged to be positive for the genotypic markers at the corresponding position; (3) there was no signal at the IC site on the membrane strip, or at the HPV genotyping site. The human ACTB (β-actin) gene was used as an internal reference. The positive control was the HPV-16 type plasmid, while the negative control was H₂O. The controls (both internal reference β-globin and HPV-plasmids) were included in the kit. The testing reagent was provided by Shenzhen Yaneng biological company.

In the present study, a total of 1044 male patients were involved in the detection of HPV genotyping. The age of these subjects ranged from 15 years old to 83 years old and the infection rate of HPV in each age group was above 50%. The overall trend of the HPV infection rate increased with age. Among these positive infections, the majority of infected subjects were within 21–40 years old, accounting for more than 70% of the infected population. The specific results are presented in Table 1.

Among the 1044 male high-risk subjects, 567 infected subjects were detected and the infection rate was 54.31%. The HPV infection rate for high-risk types, low-risk types, and high- and low-risk types co-infections was 32.09%, 38.80%, and 29.10% respectively.

There were 23 genotypes in the 567 HPV-infected subjects. Among these, HPV52 was the most high-risk subtype with the highest detection rate (8.81%), and this was followed by the HPV16 (6.90%), HPV51 (5.08%), HPV68 (4.12%), and HPV58 (3.26%) subtypes. The low-risk subtypes with high detection rates were HPV6 (18.87%), HPV11 (10.25%), HPV42 (5.36%), and HPV43 (4.78%) (Table 2).

Among the 567 HPV-infected subjects, the constituent ratio for single HPV infection was 56.61%, which were mainly low-risk HPV6 and HPV11, followed by HPV52, HPV16, HPV42, HPV51, etc. The ratio for double HPV co-infection was 26.63%, and the ratio for high- and low-risk mixed co-infection was 16.40%. The ratio for high- and low-risk mixed infection in multiple infections was 12.70%, and the ratio for low-risk co-infection subjects was very low, only 0.88% (Table 3).

151 people were infected with 2 types of HPV. 59 patients were screened by physical examination, among whom 25 (43.86%) were aged 21–30 years. HPV6, HP81 and HP11 were the most common types, with 20 patients (35.09%), 9 patients (15.79%) and 8 patients (14.04%), respectively. 54 patients were diagnosed with warts, 21 of them were the ages of 31–40 years (38.89%). HPV6, HPV11 and HPV16 were the three common types, account for 25 patients (46.3%), 15 patients (27.78%) and 8 (14.81%) patients, respectively. Compared with the physical examination population, patients with clinical diagnosis of warts had higher detection frequency of HPV16. The other diagnostic types included foreskin glanthitis, skin infection, dermatitis, urethritis in a total of 38 people (Table 4).

There were 95 people with three or more types of HPV infection. About half of them (46 patients) were clinically diagnosed with warts, of whom 28 patients (60.87%) were aged 21–30 years. Among them, 25 cases (54.35%) contained HPV6, 16 cases (34.78%) contained HPV11, and 7 cases (15.22%) combined with HPV6 and HP11. Physical examination screened 26 patients with 3 or mor types of HPV co-infections. Nine of them were aged 21–30 years, accounting for 39.13%. There were 14 patients (60.87%) contained HPV52 and 9 patients (39.13%) with HPV6. The remaining 23 cases were clinically diagnosed with skin infection, urethritis, paraphernalitis, skin rash, prostatitis and other related diseases, accounting for 24.21% (Table 4).

In this study, we found that the clinical signs of HPV infection included warts, dermatitis, acroposthitis, urethritis, rash, and prostatitis. Warts are skin-surface vegetations caused by the human papillomavirus. Dermatitis is a general term for inflammatory skin disorders caused by a variety of internal, external, or non-infectious factors. Acroposthitis is the inflammation of the foreskin’s inside surface and the penis head. A rash is a skin lesion that can take many forms, from simple skin-color changes to skin-surface bulges or blisters. Urethritis is a common disease that refers to inflammation of the urethral mucosa. Prostatitis (prostatitis) refers to prostate disease caused by a variety of complex causes, with urethral stimulation symptoms and chronic pelvic pain as the main clinical manifestations. The positive detection rate of patients with clinical manifestations was the highest, reaching 66.47% (Table 5).