Prevalence of cervicovaginal human papillomavirus infection and genotype distribution in Shanghai, China

The evaluation of human papillomavirus (HPV) prevalence rate dynamics and genotype distribution could support the adoption of more targeted prevention and treatment of cervical cancer. We aimed to assess the infection status and genotype characteristics of HPV among gynecological outpatients in Shanghai, China. Clinical specimens were collected from patients attending gynaecological department of the Putuo Hospital, Shanghai University of Traditional Chinese Medicine, between January 2015 and December 2019. The cervicovaginal infection of 17 high-risk genotypes and 10 low-risk genotypes were analyzed by Luminex-based multiple assays. The overall HPV infection rate was 18.81% (95% CI 18.31–19.30%) in Shanghai city, with high-risk, low-risk and mixed high- and low-risk HPV prevalence being 11.65% (95% CI 11.24–12.06%), 4.19% (95% CI 3.94–4.44%) and 2.96% (95% CI 2.74–3.17%), respectively. The five most prevalent high-risk genotypes were HPV-52 (2.95%), HPV-16 (2.34%), HPV-58 (2.07%), HPV-53 (1.67%) and HPV-39 (1.36%). The most common low-risk genotype was HPV-61 (1.52%), followed by HPV-6 (1.29%) and HPV-81 (1.19%). Moreover, the coverage of HPV genotype by nonavalent vaccine was 10.42%, and non-vaccine-covered high-risk genotype was 7.70%. The 15–24 years age group demonstrated the highest HPV prevalence (43.14%), and significant differences were observed among different age groups (P < 0.001). This study revealed the HPV prevalence and genotype distribution among women in Shanghai city, which could serve as guidance for HPV vaccination and preventative strategies against cervical cancer in this area.


Introduction
Human papillomavirus (HPV) is one of most commonly encountered sexually transmitted infection, which mainly causes cervical cancer and other cancers (vaginal, vulvar, anal, penile and oropharyngeal cancer) [1]. More than 200 HPV genotypes have been identified, and they can be classified into high-risk (HR) and low-risk HPV (LR) genotypes based on their carcinogenicity. It has been demonstrated that persistent infection with HR HPV genotype including HPV16/18/31/33/35/39/45/52/58/ 59 is a major cause of cervical precancerous lesions and cervical cancer [2,3], while LR-HPVgenotypes such as HPV6/11 are associated with condyloma acuminatum or hyperplastic lesions [4]. Approximately 99% cervical cancers were associated with HPV infection worldwide [5], and there are over 130,000 women suffered from cervical cancer per year in China [6]. Therefore, HPV detection Open Access screening is of great importance to reduce the burden of cervical cancer and other HPV-related diseases.
Currently, three licensed HPV vaccines are available, including bivalent (HPV- 16 and -18), quadrivalent (HPV- 6, -11, -16 and -18) and nonavalent vaccines (HPV-6, -11,-16, -18, -31, -33, -45, -52 and -58) in mainland China. They were launched and approved for use by China Food and Drug Administration (CFDA) in 2016, 2017 and 2018, respectively [7]. However, all these commercially vaccines only provide protection against a few genotypes, which were based on epidemiological data from western countries [8]. Furthermore, the HPV infection rate and genotype distribution vary by countries and regions [9,10]. For instance, HPV-31 and HPV-33 are more frequent in Europe and America, whereas HPV-52 and -58 are more prevalent in Asia and HPV-35 and -45 in Africa [4]. Recent a meta-analysis indicated the most prevalent genotypes were HPV-16, -52 and -58, followed by HPV-18, -31, -33 and -35 in women with normal uterine cervix in different regions of China [11]. Thus, acquiring updates on prevalence and distribution of HPV genotypes among different areas will provide crucial information for decision on HPV vaccination program and development of new vaccine in China.
Shanghai have 16 districts with over 20 million population in its area. Previous studies have investigated the prevalence and genotype distribution of HPV in Zhoupu [12], Minghang [13] and Songjiang district [14] of Shanghai China. However, there is still limited information on the distribution of HPV infection in Shanghai, China. The primary objective of this study was to investigate the prevalence and genotype distribution of HPV infection among women attending gynecology clinics in Putuo district of Shanghai and to further evaluate the infection patterns in terms of age groups and geographical areas.

Study participants
From Janauary 2015 to December 2019, women who attended at the Shanghai Putuo Hospital and Liqun Hospital and received HPV DNA genotyping test were included in this restrospective and cross-sectional study. Inclusion criteria for individual were as follows: (1) was ≥ 15 years old; (2) was living in Shanghai city; (3) was first time to receive the test and did not have any treatment; (4) not pregnant and had sexual activity. Finally, a total of 23,866 women with results of genotype-specific HPV were enrolled in this study. This study was approved by the ethics committees of Putuo Hospital, Shanghai University of Traditional Chinese Medicine (PTEC-A-2020-24-1), and the written informed consent was obtained from all the participants at each clinic visit.

Specimen collection
Cervicovaginal cell samples were collected from each participant by professional gynecologists using plastic brushes (Tellgen Life Science, Shanghai, China). The brushes were placed into sterile tubes containing 3 ml of cell preservation solution (Tellgen Life Science, Shanghai, China) and stored at 4 °C, and finally trasported to our clinical lab within one week for HPV DNA genotype testing.

DNA extraction and HPV genotyping
HPV DNA extraction was performed using a domestic commercial available viral DNA extraction kit (Tellgen Life Science, Shanghai, China) according to manufacturers, procedure. HPV detection and genotyping were conducted using Tellgenplex ™ HPV DNA Test (Tellgen Life Science, Shanghai, China). The Test is a suspension bead array method that involves PCR, hybridization onto a bead using amplified PCR products and digital singnal processing [8]. In brief, 5 μL of the extracted DNA was used in the 15 μL PCR master mix reaction solution, followed by hybridization with oligonucleotide probes at 95 °C for 5 min, and 48 °C for 30 min.The hybridization product was stained with streptavidin-R-phycoerythrin, and analyzed

HPV genotype categories
Beides overall HPV, type-specific, defined HR, LR and mixed HPV prevalence, we also analyzed the prevalence of several different groupings of HPV genotypes including: (1)  , and the prevalence of HPV infection in those age groups was calculated. Moreover, the potential impact of current vaccines in different age groups was also evaluated.

Statistical analysis
Excel (version 2010), SPSS software (version 22.0) were used for data processing and analysis. Bubble plots were created with ggplot2 and reshape2 packges, and heatmap plot was conducted with pheatmap package in R (version 4.1.2). The 95% confidence interval (CI) for HPV prevalence was estimated. Considering the impact of 4v and 9v vaccines, McNemar exact test and multiple chi-square test were used to compare the significant difference between two paired percentages and prevalence among different groups, respectively. The prevalence of 17 HR-HPV infection was summarized according geographical division of China based on epidemiological studies published from Janauary 2015 to August 2021. Difference in 17 HR-HPV genotypes distribution were visualized by Non-metric Multi-Dimensional Scaling (NMDS) using PAST software [15]. The linear-by linear association and gamma value were used to evaluate the trend in HPV prevalence over the five years, and P value < 0.05 was considered statistically significant for all analyses.

Age-specific Prevalence of HPV infection
The age-specific prevalence of HPV infection is shown in Fig. 1. There were two peaks in the prevalence of overall HPV infection. The first peak found in women aged ≤ 24 years (43.14%), decreased sharply after the first peak, and maintained a plateau at middle age. The   (Fig. 1A). Single HPV infection also peaked at ≤ 24 years, then dropped drastically with age, and stabilized in women aged 35-64 years without significant variation, and then decreased sharply again among ≥ 65 years group. Interestingly, the trend of the dual and multiple infection was similar to that overall HPV infection (Fig. 1B).

Distribution of HPV genotypes
The distribution of HR-HPV and LR-HPV genotypes in different age groups are shown in Fig. 2 Table S1). Hierarchical clustering analysis showed that relative young-age group (≤ 34 years) and middle/older group (35-64 years and ≥ 65 years) shared similar distribution of HPV types (Fig. 3).

Prevalence of HPV according to vaccine types
The prevalence of detected genotypes targeted by 2v, 4v, and 9v vaccines were 3.25%, 5.08%, and 10.42%, respectively, whereas the prevalence of non-vaccine HR-HPV genotypes was 7.70% as shown in Table 1. The prevalence Fig. 1 The prevalence of HPV infection by age groups. A any, high-risk, low-risk and low and high-risk HPV infection. B single, dual and multiple infection. C bivalent, quadrivalent, nonavalent vaccine targeting genotypes and non-vaccine high-risk genotypes. Error bars represent 95% confidence intervals Fig. 2 Bubble plots showing the relative prevalence of detected A 17 high-risk HPV genotypes and B 10 low-risk HPV genotypes across age groups of HPV genotypes grouped by age was shown in Fig. 1C. Moreover, we also compared the prevalence of patients with type included in 4v and 9v vaccines among age groups. A significant relationship was observed between vaccines (4v and 9v) and groups (P < 0.001), and a significantly higher coverage in 9v vaccine than in 4v vaccine for each age group (P < 0.001, Additional file 1: Table S2).

Characteristics of HPV infection by geographical regions
We sorted out the reports according to geographical regions: eastern, central and western China based on previous published studies (Additional file 1: Table S3). The prevalence of those studies and NMDS plots of 17 HR-HPV are shown in Fig. 4. The median HPV prevalence in the eastern area of China (21.66%) was relatively higher than in the western (16.95%) and central (18.19%) regions. In general, the most commonly detected HR-HPV genotypes were HPV-16, HPV-52, HPV-58, HPV-53 and HPV-18.

Comparison of the HPV prevalence in Shanghai with other regions of China
HPV prevalence rate varies greatly in different regions of China as it is influenced by multiple factors such as economic levels, living habits, awareness of prevention and screening, and HPV detection method sensitivity.

Higher prevalence of HPV in younger women
Many studies have shown that HPV infection were significantly age-specific [31][32][33][34]. In the present study, overall HPV infection rate among young women (≤ 24 years, 43.14%) was much higher than that of other age groups, then the rate of HPV infection reduced sharply, which may be associated with their sexual behavior and attitude. However, decreasing trend stopped and rised slowly at 35-55 years, and slightly increased at 55-64 years (18.36%). Notably, the HR-HPV infection demonstrated the similar pattern. A previous study have been showed that the HR-HPV infection rate of Chinese women demonstrated "two-peak" pattern. The first peak presented at youngest age group (15-19 years), and the second peak observed at 50-60 years group [35]. In this study, the highest HR-HPV infection rate was also observed in the youngest age group (≤ 24 years), and followed by a less obvious peak for the 55-64 years group. While LR-HPV infection did not have the similar distribution. Compared with LR-HPV infection, HR-HPV infection was more likely to be prevalent, persistent and less likely to be cleared [36]. We also presented age-specific prevalence of single, dual and multiple HPV infections. The single genotype infection in aged ≤ 24 years group was higher than that of dual and multiple infections in other age groups, which also observed in northern Henan province of China [37]. Young women are thought to have more frequent sexual activities, more than one partner and relatively inadequate immune response, which makes them have a higher probability of exposure to HPV infection.
For menopausal women, immune dysregulation would lead them unable effectively remove and inhibit the virus, may account for viral persistence or reactivation of latent HPV [38]. Therefore, further promotion of vaccination program and preventative screening strategies against cervical cancer for young women susceptible to HPV infection is necessary and urgent for this region. Additionally, cervical cancer screening program like HPV genotyping test is also valuable for perimenopausal women (≥ 55 years).

High frequency of non-vaccine HR-HPV genotypes 53, 39, 56, 51 and 59 in Shanghai women
Knowledge of the genotype distribution of HPV in specific areas will enable the improvement of optimal protective strategies. Previous studies have indicated that HPV-52, -16 and -58 were the most common HPV genotype in many regional of China [16,23,39,40]. In our study, the most common genotype was HPV-52, followed by HPV-16, -58, -53 and -39, which was consistent with the result in Guizhou [41]. HPV-52, -58, -16, -51 and -39 were the five most common HR-HPV genotypes in Yangzhou [42], and HPV-52, -16, -58, -39 and -51 in Wuhan [43]. It has been reported that HPV-52 and -58 were the more prevalent genotypes in Asia, especially in China, and infection with them may have association with the cervical cancer development [38,44]. HPV-16 and HPV-18 were the most commonly encountered genotype worldwide, accounting for up to 70% of cervical cancers [3]. In our study, HPV-16 ranked second, whereas HPV-18 was only 9th common HR-HPV genotype. In Hangzhou, HPV-16 ranked first, and HPV-18 was 5th most common prevalent genotype [45], and 11th in Shanxi [46]. HPV-16 was the most common genotype coinfection with other types [27,33]. HPV-18 was more common in other countries than in China [9]. Persistent infection with one or more high risk genotypes of HPV is one of the leading cause for cervical neoplasia [47]. It has been reported that HR-HPV genotypes can be found in more than 90% cervical cancer specimens [48]. HPV vaccination is an effective strategy for the primary prevention of HPV infection and the potential development of cervical neoplasia. Our study showed that bivalent, quadrivalent and nonavalent vaccines only covered 3.25%, 5.08%, and 10.42% HPV genotypes in this area. It was worth noting that in addition to HPV-52, -16 and -58, there was a high prevalence of HPV-53, -39, -56, -51 and -59 in this region. These HR-HPV genotypes are not included in current available vaccines, and may should be taken into account in the future HPV vaccines to reduce the risk of HPV-related cervical cancer development in Shanghai.