Epidemiology and genotypes analysis of human papillomavirus infection in Beijing, China

Background This study aimed to investigate the epidemiology of high-risk human papillomavirus (HPV) in the female population in Beijing, China, and identify the relationship between HPV genotypes and host factors. Methods HPV testing was performed on women aged 15–89 (mean age 38.0 ± 10.9 years) from Beijing in 2020. High-risk HPV genotyping real-time polymerase chain reaction was used to determine HPV genotypes. The overall prevalence, age-specific prevalence, genotype distribution, and the correlation between HPV genotypes and cervical cytology were analyzed. Results Among the 25,344 study participants, the single and double infection rates were 18.8% (4,777/25,344) and 4.2% (1,072/25,344), respectively. A total of 6,119 HPV-positive individuals were found to have 91.6% negative results for intraepithelial lesion or malignancy (NILM), 5.8% atypical squamous cells of undetermined significance (ASC-US), 0.9% low-grade squamous intraepithelial lesion (LSIL), and 1.7% high-grade squamous intraepithelial lesion (HSIL). In single HPV infections, the HPV16 genotype was highly associated with cervical cytology severity (χ2 trend = 172.487, P < 0.001). Additionally, HPV infection rates increased gradually with age, and statistical differences were observed across age groups (χ2 = 180.575; P < 0.001). High-risk HPV genotypes were highly prevalent in women below 25 years of age and those aged 55–59 years. Cluster analysis revealed that the 13 HPV genotypes could be roughly divided into two groups in a single infection; however, patterns of infection consistent with biological characteristics were not observed. Conclusion High-risk HPV was found in 24.1% of outpatients, with HPV52, HPV58, HPV16, HPV39, and HPV51 being the most common high-risk genotypes. Single high-risk HPV infection was predominant. HPV16, HPV39, HPV51, and HPV52 were associated with cervical lesion progression. HPV16 infection was especially worrying since it aggravates cervical lesions. Because the infection rates of the 13 HPV genotypes differed by age, the peak HPV infection rate should not guide vaccination, screening, and prevention programs. Instead, these initiatives should be tailored based on the regional HPV distribution characteristics. Moreover, it was determined that Beijing’s populace needed to receive treatment for HPV39 infection.


Introduction
Cervical cancer is one of the most common cancers in women worldwide.In 2020, there were about 342,000 deaths and about 604,000 new cases identified worldwide, with a large share of these deaths taking place in low-and middle-income nations [1].The persistence of human papillomavirus (HPV) infection is widely recognized as the most important causative factor in the development of cervical cancer [2].
The predominant HPV genotypes may cause different outcomes at different stages of the disease [13].Negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) are the four categories used to categorize precancerous phases [14].Among cases of HSIL, the prevalence of HPV16/18 is 52%.However, HPV 16,18, and 45 are significantly under-represented, and other high-risk HPV types are significantly over-represented in HSIL compared with invasive cervical cancer, suggesting differences in typespecific risks for progression [15].The most common HPV types in HSIL samples from China are HPV16, 58, 52, 18, and 33 [16].
Furthermore, age has a significant role in determining the risk of HPV infection [17].In Western countries, HPV prevalence peaks only in women in their mid-twenties and then steadily declines as age increases [18].However, the prevalence of high-risk HPV has two peaks in China: one at age 15-24 years and the other at age 35-49 years [19].
To investigate the state of high-risk HPV infection, the correlation between high-risk HPV genotypes and cervical lesion severity, and the genotype distribution in the area, 25,344 samples from female outpatients in Beijing, China, were gathered for this study in 2020.We aimed to investigate cervical cancer epidemiology, diagnosis, and vaccination in Beijing, China.

Study population
The study's data came from patients who visited Beijing Obstetrics and Gynecology Hospital, Capital Medical University's gynecological outpatient department in 2020.After the exclusion of unqualified samples, the quantitative measurement and biopsy of high-risk HPV DNA from a total of 25,344 women was performed.The median age was 38.0 ± 10.9 years, and the age range was 15-89 years (Table 1).The study was approved by the ethics committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University, and written informed consent was acquired from the study participants.

Cervical specimen collection and high-risk HPV genotyping
Following instructions, women's cervical exfoliated cell samples were obtained using cytobrushes, and the samples were utilized to extract genomic DNA.DNA was isolated using a nucleic acid extraction reagent (Shanghai ZJ Bio-Tech Co., Ltd., Shanghai, China).Then, a commercial HPV genotyping kit (Shanghai ZJ Bio-Tech Co., Ltd.) was used to detect 13 high-risk HPV types (HPV16, 18,31,33,35,39,45,51,52,56,58,59, and 68) with use of TaqMan real-time fluorescent quantitative polymerase chain reaction.The kit's instructions were strictly followed for every procedure.

ThinPrep cytologic test
Cervical cells were detected using the ThinPrep cytology test (TCT).Senior physicians assessed cytological pathology results according to the Bethesda System of Cervical Cytology, which classifies precancerous phases as NILM, ASC-US, LSIL, or HSIL.Histopathological diagnoses were made by a pathologist who was unaware of the HPV detection results.

Cluster analysis
We investigated the similarity of infection for 13 highrisk HPV types in abnormal cytology across age groups using cluster analysis.We considered that the classical K-means algorithm was suitable for this study.K-means used Manhattan distance to measure the distance between two observations.Hierarchical cluster analysis was conducted using a "hclust" function in R-Studio (Version 4.2.1).

Statistical analysis
All statistical analyses performed in this study were with R software version 4.

Prevalence of high-risk HPV genotypes in the cervical samples
The high-risk HPV infection rate among the 25,344 patients was 24.1% (6,119/25,344), with 6,119 participants showing positive findings from high-risk HPV tests.Among the women who were HPV-positive, 4,777 were positive for a single HPV type (4,777/6,119 = 78.1%,4,777/25,344 = 18.8%).

The relationship between high-risk HPV genotypes and abnormal cervical cytology
We examined the connection between TCT outcomes and high-risk HPV genotypes in HPV-positive people.
The infection rate of HPV16 showed a gradual increase with disease progression, whereas the infection rates of HPV31, 33, and 45 were slightly higher in HSIL than in NILM samples.The infection rates of HPV18, 35, 39, 51, 52, 56, 58, 59, and 68 showed a decreasing trend.In the case of abnormal TCT, the infection rate of HPV16 (χ 2 trend = 172.487,P < 0.001) increased with increasing cervical cytological severity, whereas the infection rates of HPV39 (χ 2 trend = 8.569, P = 0.003), 51 (χ 2 trend = 7.708, P = 0.005), and 52 (χ 2 trend = 16.949,P < 0.001) showed the opposite trend (Table 4).An examination of the cervical cytological state and HPV genotype was not conducted due to the lack of data on multiple infections.Two categories of HPV genotypes may be identified by cluster analysis based on the trend of high-risk HPV genotypes by cervical cytological status [20].HPV16, 18, 31, 33, 35, 45, and 59 had similar infection trends, whereas HPV39, 51, 52, 56, 58, and 68 had similar infection trends (Fig. 1).The result is associated with the risk estimates of high-risk HPV genotypes in tumors.

Age-specific prevalence of HPV infection
The participants were divided into ten groups by age category: <20, 20-24, 25-29, 30-34, 35-39, 40-44, 45-49, 50-54, 55-59, and ≥ 60 years.The prevalence of highrisk HPV infection was significantly different across age groups (χ 2 = 180.575;P < 0.001).In this study, the "two peaks" pattern was observed for the prevalence of   5).We compiled the infection status of 13 high-risk HPV genotypes across 10 age groups in this investigation (Table 6).The age of the second peak varied among highrisk HPV genotypes; for HPV16 and HPV18, the second infection peak occurred after age 35 years, and for HPV31 and HPV51, it occurred at age 50-54 years.Despite this, the prevalence of all HPV genotypes increased once more to form a second peak at age 55-59 years.
In addition, we observed differing trends for HPV genotypes and age groups for single and multiple infections.We concentrated on the distribution of each HPV genotype in single infections solely because numerous factors affect multiple infections.Among the single infections, the first peak was observed in young women (15-25 years old), whereas the second peak was observed at different ages.HPV31, 33, 45, 51, 52, 56, and 68 had  2).HPV genotypes may be separated into two groups depending on the age group infection rate, according to cluster analysis based on the trend of the percentage change of HPV genotypes among patients who were HPV-positive in each age group.HPV16, 18, 35, 45, 52, and 68 had similar infection trends, whereas HPV31, 33, 39, 51, 56, 58, and 59 had similar infection trends (Fig. 3).However, the infection trend of HPV genotypes in age groups was not significantly associated with biological characteristics.The correlates of infection trends were unclear.

Discussion
Cervical cancer is one of the most preventable cancers.The primary cause of HSIL in the cervix and cervical cancer is high-risk HPV persistent infection.In 2018, the World Health Organization called for global action to eliminate cervical cancer and proposed that 90% of girls aged 9-14 years receive the HPV vaccine [21].In most nations, a complete strategy that includes HPV vaccination and HPV-based screening is economical [22].However, regional disparities in cervical cancer screening have been caused by the varying strengths of local governments and differing patient participation rates.China launched a national public health program to curb cervical cancer in 2009 [23].However, immunization and cervical cancer screening rates are still low [24].
The frequency of high-risk HPV genotypes among Beijing Obstetrics and Gynecology Hospital outpatients was 24.1% in this study, which is in line with earlier studies [25,26].The results supported that HPV52, 58, and 16 are the most prevalent HPV genotypes among women in Beijing.Previous studies have demonstrated that HPV52 and 58 are the two dominant HPV genotypes in East Asia and China [27].This study's high-risk HPV-type prevalence is consistent with earlier studies [9,28,29].A single genotype typically causes high-risk HPV infection; however, multiple infections have gradually gained attention in recent years.Infection with multiple HPV types is reported in 20-45% of women worldwide who are infected with HPV [30,31].Regarding the distinction between the effects of a single high-risk HPV infection and numerous high-risk infections on cervical precancer and cancer, there is no conclusive evidence [9,32,33].In this study, high-risk HPV infection was mainly caused by a single genotype (18.8%), whereas multiple infections   Recent studies indicate that HSIL, a significant stage of cervical precancerous lesions, and cervical cancer may also be caused by other high-risk HPV strains.This study provides an in-depth analysis of the relationship between cervical cytology and HPV infection.We found that the relationship between cervical cytological state and HPV type varied depending on whether the infection was single or multiple.In single infection, HPV16 was the most common type among women with HSIL, followed by HPV58, 33, 31, 52, and 18.Furthermore, the prevalence of HPV16, 39, 51, and 52 infections was strongly correlated with the severity of cervical cytology.The proportion of HPV16 gradually increased with disease progression.On the other hand, as the illness advanced, the proportions of HPV39, 51, and 52 steadily decreased.These results were consistent with previous findings [10,34].In multiple infections, high-risk HPV was less likely to progress to HSIL than in single infections.This study observed no effect of multiple infections on abnormal cervical cytology.
In addition, the prevalence of overall high-risk HPV infections displayed a bimodal age distribution, with one peak at ≤ 25 years, a decline with age, and a second peak at 55-59 years of age.However, the infection rates of different HPV genotypes differed across age groups.For instance, HPV-16 and 18 peaked again between the ages of 35 and 39.Consequently, we suggest that women over 35 be mandated to undergo an annual HPV test.Despite similar infection trends for single and multiple infections, we only analyzed the shifts in infection curves for single HPV infections because the etiologies of multiple infections are more complex.We found that the peak age of each HPV strain varied.
Furthermore, the 13 high-risk HPV types classified as oncogenic based on epidemiologic and/or phylogenetic evidence are members of four species within the Alphapapillomavirus genus.HPV 16, 31, 33, 35, 52, and 58 are the prototypes of the A9 species; HPV 18, 39, 45, 68, and 59 are the prototypes of the A7 species; HPV51 is the Fig. 2 The prevalence by age of each high-risk HPV genotype in a single HPV infection prototype of the A5 species; and HPV56 is the prototype of the A6 species.Our study categorized the 13 HPV types by cluster analysis and finally divided them into two groups: (1) HPV16, 18, 35, 45, 52, and 68, which had a similar age distribution, and (2) HPV31, 33, 39, 51, 56, 58, and 59, which had similar infection trends.Nevertheless, no noteworthy correlation was detected with epidemiology and systems biology classification techniques.
Ultimately, immunization is one of the most important strategies for lowering cervical cancer incidence.Vaccines should be effectively and rationally distributed by region according to HPV epidemiological characteristics.Cervical cancer vaccination is considered an important measure for the effective prevention of cervical precancer, cervical cancer, and acromegaly.Moreover, variations in HPV subtype infection are noted between regions and ethnic groups.Therefore, a thorough big sample survey may offer useful therapeutic value for vaccine development and vaccination to prevent cervical cancer in pertinent places.The sample in this study reflects the high-risk HPV infection status of the Beijing population.The imported nine-valent HPV vaccine has prevented Fig. 3 Cluster analysis of HPV genotypes in single infection across age groups the HPV subtypes, including HPV6, 11, 16, 18, 31, 33, 45, 52, and 58 in the Beijing population.Notably, however, HPV39 was not covered by the vaccine.As a result, the vaccine's ability to prevent high-risk HPV in Beijing is still limited.

Conclusion
In conclusion, this study examined the frequency of highrisk HPV infection in females, the correlation between high-risk HPV genotypes and cervical lesion severity, and the association between high-risk HPV infection and age distribution features in Beijing, China in 2020.Our study will provide helpful information for screening and vaccinating cervical cancer in Beijing, China.

Table 1
Information about women with high-risk human papillomavirus infection

Table 2
Distribution of HPV genotypes in 25,344 women with high-risk human papillomavirus infection

Table 3
Distribution of cervical cytological status by age group

Table 4
The relationship between high-risk genotypes and TCT results in single HPV-positive specimens

Table 5
Distribution of HPV infection by age group in 25,344 women with high-risk HPV infection Fig. 1 Cluster analysis of high-risk HPV genotypes and cervical cytological status

Table 6
Age distribution of 13 high-risk HPV types