Background
Human papillomavirus (HPV) is the leading cause of cervical cancer and responsible for about 91% of anal cancers, 75% of vaginal cancers and 70% oropharynx cancer [
1]. It is estimated that there were over 528,000 new cervical cancer patients and 266,000 death worldwide recorded in 2012, among which 85% occurred in developing countries [
2]. In China, 130,000 new cases of cervical cancer are diagnosed annually, especially in young women within the first few years after sexual debuts [
3,
4]. The morbidity and mortality of cervical cancer in developed countries has fallen over the last two decades because of an enhanced awareness of cervical cancer prevention and effective screening [
5]. However, infection rate of HPV has fluctuated and not shown a downward trend, indicating a relatively heavy burden of HPV infection in China [
6,
7].
HPV genotypes are linked to the degree of cervical lesions [
6]. Infections with low-risk HPV (LR-HPV) types, such as HPV6 and − 11, cause benign or low-grade changes in cervix cells, genital warts, and recurrent respiratory papillomatosis. Contrarily, high-risk HPV (HR-HPV) type cause cervical, anal, and other genital cancers, which could be detected in 99% of cervical cancers—the second most common cancer in women worldwide. HPV16 and − 18 have been found to be the most pathogenic HR-HPV types, causing about 70% of cervical cancers worldwide [
7‐
10]. Of note, most HPV infections are completely asymptomatic, resulting in a delay in diagnosis and follow-up treatment with disastrous consequences [
11]. Moreover, HPV-infected individuals without early intervention will develop long-lasting HPV infections that put them at risk for cervical cancer, and increase the risk of sexual transmission to their partners, resulting in approximately estimated $1.7 billion in direct medical costs annually in the United States [
12].
The prevalence of HPV varies geographically widely, ranging from 6% in southeastern Asia to 32% in eastern Africa, and from 6.7 to 44.5% in China [
13]. HPV screening and vaccine are considered being the most effective measures for preventing HPV infection. HPV vaccine has been clinically applied for more than 10 years [
14], however, officially launched on July 2017 in mainland China and available mainly in the developed regions in China currently, such as Beijing, Shanghai, and Guangzhou. Besides, the HPV vaccine made in China was approved in 2020. Representative data on type-specific prevalence of HPV infection in China could provide a baseline estimation to the burden of HPV infection and could help guiding programs on HPV-based cervical cancer screening and strategies on vaccine-based HPV prevention. To date, the existing data about epidemiological characteristics of HPV infection in Southern China are still scantly, especially lacking large sample study on the genotype prevalence of HPV. Therefore, this paper was conducted to determine the prevalence and type distribution of HPV among 214,715 females in Southern China, including its trends from 2012 to 2018 and age-specific prevalence. Such data provide a baseline pre-vaccine population-based prevalence of HPV in Southern China, and help guiding models evaluating impact and cost-effectiveness by comparison between future vaccinated populations, and implementation of the prophylactic HPV vaccine according to the specific types’ distribution in the region.
Methods
Study population
From January 2012 to December 2018, 214,715 females (age range from 19 to 83 years) were enrolled in this study. All participants were from gynecology department and physical examination center of Chenzhou NO.1 People’s Hospital. They have got HPV tests for various reasons, including physical examination, vaginitis, urethritis, irregular vaginal bleeding, cervicitis, undiagnosed abdominal pain and genital warts. In addition, repeated samples from the same women were excluded.
Cervical specimen collection and management
All of the cervical samples were taken by the physician, but not self-sampling, using a 200 mm polyethylene Cervix brush device (Hybribio Corp, Guangdong) following the regular procedures for speculum examination. The samples were transferred to a sampling tube containing a transport medium (Hybribio Corp, Guangdong) and stored at 2–8 °C until HPV DNA extraction within a week. Cervical samples can be kept in a transport medium for 2 weeks at 4 °C according to the manufacturer’s manual.
HPV genotyping
DNA was extracted by Cell Lysis Kit (Hybribio Corp, Guangdong) accompanying with the negative and positive quality control products throughout the whole process. HPV genotyping have been performed using HPV assay kit (Hybribio Corp, Guangdong) since 2012 with no modifications. This kit detected 21 HPV genotype via gene amplification technology and diversion hybridization. The low limit of detection (LOD) for HPV DNA is 500 copies/ml according to manufacturer’s protocol. This assay uses HPV L1 consensus PCR primers (MY09/11) for the amplification and a human housekeeping gene β-globin as an endogenous internal control to ensure appropriate DNA purification, PCR reaction and specimen quality. PCR was carried out in 25 μl reaction mixture in a thermal cycler, the cycling parameters of which were as follows: 20 °C for 10 min, 95 °C for 9 min, followed by 40 cycles (20s at 95 °C, 30 s at 55 °C and 30 s at 72 °C), with a final extension at 72 °C for 5 min. HPV types were then classified via a nylon membrane immobilized with 21 different type-specific probes. Quality controls of HPV genotyping including External Quality Assessment and Internal Quality Control were implemented throughout the study.
Statistical analysis
Analyses were conducted with SPSS version 19.0. Descriptive statistical analysis was performed on the distribution of HPV genotypes using indicators such as frequency and prevalence. The Chi-square test was used to test the differences between prevalence, genotypes and number of co-infections of HPV in different age groups and time groups.
Ethical consideration
The study was approved by the ethics Committee of Chenzhou NO.1 People’s Hospital and conducted strictly in accordance with the Declaration of Helsinki, including the confidentiality and anonymity. No informed consent or other action on the part of the patients was required due to anonymous analyses of the data. All experiments were carried out in the lab certified by the National Center for Clinical Laboratories following the laboratory biosafety guidelines.
Discussion
Here we measured the prevalence of 21 distinct HPV types and found the overall prevalence of HPV in this representative sample of women was 18.71% and HR-HPV of 15.17%, amongst 214,715 women prior to HPV vaccination. The high HR-HPV prevalence indicated the inadequacy of routine cervical screening in the region. Additionally, the large sample size allows for precise estimation of both increases and decreases in HPV type specific prevalence, which could be used as a baseline of comparison to future sampling of the entire population.
Global HPV prevalence estimates are known to vary by the region, study design, target population and calendar time [
16]. According to the previous reports, HPV positive rates range from 6.70 to 44.50% in China [
11]. Notably, Western Asia (1.70%) and North America (4.70%) had a low rates of HPV infection, while East Africa (33.6%) and the Caribbean (35.4%) had a high rates of HPV infection [
17‐
19]. HR-HPV genotype distribution was also heterogeneous, ranging from 9.90–27.50% in China, which is 15.17% in current study, as with that in Guangdong (20.02%), Guiyang (20.45%) and Nanning (22.28%) [
20]. The variation was expected to be explained by cultural diversity, the sampling strategy, methods and devices, as well as the sensitivity and specificity of the HPV detection assays.
In present study, the sampling methods and devices, and Hybribio test were enrolled for HPV detection assays unaltered for over 7 years, which could monitor the annual changes of HPV prevalence sufficiently. We found HPV prevalence showed a significant downward trend from 2012 to 2016. Analogous results of the HR-HPV positive rates were also found to decline from 25.3% in 2007 to 18.4% in 2014, in Guangzhou [
21]. This decline may be due to the following reasons: 1) emphasis on cervical cancer has led to increase participation in screening, including those without cervical abnormalities; 2) in the last decade, some women tested positive for HPV became negative, due to immunization and treatment; and 3) individuals were more likely to be aware of HPV and HPV vaccination following the continuous improvements of living conditions and public health awareness. Several studies have demonstrated the association of HPV with economic development. Women from impoverished countries and areas suffered a high prevalence of HPV, e.g., 66.7% among young females in South Africa and 44.5% in Henan province of China [
19,
22]. On the contrary, a low rate of 6.7% was found among women in Beijing—the capital of China, which remained the most flourishing cultural and economic center in China, and also had the excellent healthcare system, indicating a strong correlation of HPV with socioeconomic development [
23].
We found HPV52 was the most commonly detected genotype, in agreement with that stated in Japan, Taiwan, and eastern Africa [
24]. However, it was inconsistent with the data reported that HPV16 was the predominant genotype in other studies [
3] and HPV35 in sub-Saharan Africa [
25]. Moreover, prevalence of both HPV52 (5.12%) and − 58 (2.51%) was higher than that reported (2.3 and 1.0% respectively) in the United States [
3], and a nationwide population-based investigation in 37 cities in China [
20]. HPV 16 is the strain most likely to cause cancer. Thus, clinic-based studies usually found higher prevalence of HPV16 than population-based studies [
24,
26]. We found a relatively low prevalence of HPV16, which was in accordance with an estimated HPV16 prevalence of 3.2% from a meta-analysis of 1 million women with normal cytology [
27]. It is possible that most women seemingly healthy from the physical examination center enrolled in our study were more likely to detect HPV types not related to cervical infection. Our data also indicated that HPV52, − 16, and − 58 were consistently the top three HR-HPV genotypes from 2012 to 2018, suggesting the HPV vaccine covering these HR-HPV types is routinely recommended, especially for those females at young ages exposed to HPV in this region.
Evaluations in the United State showed that young women had the highest HPV prevalence [
3], consistent with our the findings of the highest HPV prevalence (33.11%) among women aged less than 21 years. Young women often have a high infection rate, mainly because they are sexually more active before their immune systems become less sensitive [
17]. Although they were known to have high risks for HPV infection, however it is temporary and supposed to disappear within a year or two, and thus its prevalence declined gradually with respect to ages [
18]. Predictably, the prevalence of HPV slightly declined in middle ages, yet significantly increased among the oldest people, which are consistent with those in most developed countries and the data from Bruni and colleagues [
27]. The mechanism of this increase in infection rates is unclear at present. Other than persistent infections that seem to be more prominent among females at older ages [
19], this increase could be also explained by re-marriage, reactivation of latent HPV in menopausal women and the cohort variation [
16]. In addition, women aged 31–40 years were the most common population for screening, and those aged 41–50 years showed an increase trend in this study, thus routine screening is strongly recommended for women over 30.
In an unweighted analysis of women with multiple HPV genotype infections, we found HR-HPV genotypes accounted for over 80% of multiple infections, which mainly occurred at ages either younger than 21 or older than 60. Some have suggested HPV infection with multiple genotypes may prolong the duration of infection and increase the risk of cervical cancer and cervical precancerous lesions [
28], leading to a complication of multiple infections among older women. Multiple infections were believed to have competitive and/or cooperative interactions between HPV genotypes [
29]. Although the mechanisms and potential oncogenic effects of multiple genotype infections still require further investigation, this study could still be beneficial to the development of HPV prophylactic vaccines.
The overall strengths of this study include the large sample size, unaltered HPV genotyping methods over 7 years, decade-long study period measuring the trends of HPV infection on a yearly basis, and the use of PCR testing rather than serologic tests, which allows for accurate determination of simultaneous co-infection, all of that provide a good baseline for epidemiological surveillance after the introduction of HPV vaccine at population level. However, several limitations exist in our study. Firstly, HPV DNA testing does not reflect the previous and cumulative incidence of HPV infections, but only the current infection status. Secondly, our study included specimens from women, without pathological data, such as, cervical cytology and histology results, which was unable to explain the relationship between HPV infection and pathology. Thirdly, no male samples were analyzed, so our study would not be able to represent the infection of HPV in general population in the region. Lastly, the detailed information about the patients, such as education level, economic status and background related to HPV infection, were not documented in this study, hindering a more comprehensive evaluation of the effects of these factors on the prevalence of HPV infection.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.