Epidemiology of Oral Human Papillomavirus Infection among Diverse Chinese Adults in Typical Areas of China: Findings from the DLCC Study

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide with an annual 705,781 newly diagnosed cases and 358,108 deaths (1). Interestingly, the incidence rate of newly diagnosed HNSCC cases has been abruptly increasing among young adults, even those who have minimal exposure to classical risk factors like smoking and alcohol in recent times (2, 3). It has been found that this rise in cases is linked to human papillomavirus (HPV) infection, particularly in oropharyngeal squamous cell carcinoma, which is believed to be acquired through increased oral exposure to infected anogenital sites due to changes in sexual behaviors (4). Notably, these patients with HPV-related head and neck cancer are more frequently observed in younger patients and tend to have better prognoses than those exposed to alcohol and tobacco for many years (5).

The increasing significance of oral HPV infection in the progression of HNSCC makes it crucial to comprehend the prevalence of oral HPV in the general population and the pertinent relevant risk factors. This understanding is necessary to develop efficient and targeted prevention or intervention strategies. Previously reports have documented a prevalence of HPV infection in normal oral cavity mucosa ranging from 2% to 20%, with most studies being conducted in Europe or the United States (6, 7). However, this variation can be attributed to disparities in the study population, sample size, specimen collection, or detection procedures.

Although China has a vast territory and a large population, there are limited data on the prevalence of oral HPV infection in Chinese adults. Therefore, we aimed to assess the type-specific prevalence and risk factors of oral HPV infection in Chinese adults from typical geographical areas with diverse sociocultures in northern and southern China, using data from a large prospective study, the diverse life-course cohort (DLCC; ref. 8).

The DLCC is a population-based prospective cohort study conducted by the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) in 2017. It covers the entire life course from prenatal life to aging in China (8). This study is based on the second visit (Visit 2) of the DLCC, which began in 2021 and recruited participants from two areas: Shantou and Meizhou cities in Guangdong Province (including three different kinds of culture: “Chaoshan,” “Hakka,” and island culture in South China), as well as Baoding city in Hebei Province. These two newly enrolled areas have unique characteristics in terms of dietary patterns, significant differences in environmental risk factors, and prevalence of noncommunicable diseases (NCD). One of the newly enrolled study sites, Nan'ao Island in Shantou city, was selected as a representation of island culture. It is a relatively isolated place with native residents who have maintained unchanged customs. This site provides an ideal population for studying environmental risk factors and their interaction with genetic backgrounds in relation to health. In addition, it allows for migrant epidemiology research, as individuals may relocate to developed urban areas due to increased job opportunities.

To ensure the representativeness of the target population, a multistage stratified sampling method was utilized to select subjects. In the initial stage of sampling, provinces were chosen from various geographic regions. In the second stage, cities and counties were selected from each province. In the subsequent stage, districts were chosen from cities and rural townships were chosen from counties. In the fourth stage, communities were selected from districts in urban areas, while villages were selected from townships in rural areas. The sampling process was stratified on the basis of geographic regions (north, south), degree of urbanization (provincial capitals, midsize cities, county seats, and rural townships), and economic development status evaluated through gross domestic product for each province. Participants from five areas in Guangdong Province (South China) and two areas in Hebei Province (North China) were tested for oral HPV infection status. The study design, sampling method, and inclusion and exclusion criteria have been described previously (8, 9). Briefly, individuals of at least 20 years of age and who had been living in designated areas for at least 1 year were invited to participate. Pregnant individuals, those with severe mental or physical illness, or active service soldiers were excluded.

This study was conducted in accordance with the Declaration of Helsinki and study protocols were approved by the Institutional Review Board of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (no. 055-2020). A signed informed consent was obtained from each participant before participating in the survey.

Trained interviewers conducted face-to-face questionnaire interviews. The information gathered included items about (i) demographic and socioeconomic characteristics, such as date of birth, sex, marital status, urban or rural living, and educational level, etc.; and (ii) health-related lifestyle factors, such as alcohol consumption, smoking habits, and tea consumption. The definitions of alcohol intake and cigarette smoking were in line with those used in our previous publications (8, 10). Furthermore, the interviewers inquired about the interviewees’ individual history of HPV vaccination.

Cells exfoliated from the oral cavity were collected from all individuals by a trained otolaryngologist using a swab. Oral specimens were collected by rubbing the swab five times at each site: the buccal mucosa, palate, tonsils, top and bottom of the tongue, inner upper and lower lips, and gingival surfaces. Each patient's swab was then stored in a sterile tube (Boke, Shandong) containing a cell preservation solution which consisted of Tris-HCL, sodium chloride (NaCl), potassium chloride (KCl), guanidine isothiocyanate (GITC), ethylene diamine tetraacetic acid (EDTA), Na2, Tween-20, and phenol red. The tubes were frozen at −80°C until analysis.

DNA extraction was performed using the E.Z.N.A. Mag-Bind Tissue DNA Kit (M6223; Omega Bio-Tek, Inc.) on a NAE-96/24 automated workstation. The quality of the DNA was assessed by amplifying the β-globin gene using the PC03 and PC04 primers. HPV DNA in samples that tested positive for β-globin was tested using a highly sensitive and specific nested PCR approach. This approach involved using the MY09/11 primer set for the primary PCR and the GP5+/6+ primer set (11). The amplified target bands were gel-purified using the AxyPrep DNA Gel Extraction kit (AP-GX-250; Axygen). For TA cloning, purified DNA for each sample was ligated with TOPO new vector (RuiBiotech), and then transformed into competent DH5α Escherichia coli cells. The colonies were randomly selected for DNA sequencing. The obtained results were subsequently analyzed using Basic Local Alignment Search Tool (BLAST) database (http://blast.ncbi.nlm.nih.gov).

Rigorous quality control procedures were applied to eliminate false-positive and false-negative results, as described elsewhere (12, 13). In brief, separate work areas were established for each PCR reaction step. Separate sets of pipettes and pipette tips with aerosol filters, lab coats, glove boxes, and waste baskets were used and changed regularly. Reagents should be prepared and aliquoted in either location based on their use in pre-PCR or post-PCR applications. In each 96-well PCR reaction plate, the following negative and positive controls were included: four controls without any DNA template; one set of CaSki cells genome DNA (HPV 16 positive), each containing 1 and 0.1 ng genome DNA (14). Test results were used only when controls met the following criteria: (i) all four negative controls were negative and (ii) two positive controls (1 and 0.1 ng CaSki cell genome DNA) are both positive, and the signal intensity and concentration correspond to each other. Because of our rigorous quality control procedures, there were no negative controls which were positive in this study.

A flow chart depicting the study sample selection can be found in Supplementary Fig. S1. The final sample consisted of 9,867 participants.

Categorical variables were described using frequency and percentage. The age- and sex-standardized prevalence of oral HPV was calculated using the direct standardization method, with the reference population being the sum of participants in the two typical areas. Because sociodemographic and health-related lifestyle risk factors vary significantly between sexes, logistic regressions were conducted separately for males and females. In addition, in this study, HPV types were categorized as high-risk types (including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) or low risk (include 6, 11, 26, 40, 42, 43, 44, 54, 57, 61, 62, 64, 72, and 81; ref. 15). Understanding the prevalence of high-risk HPV infection and exploring its associated factors are crucial in terms of public health and disease prevention. Therefore, we examined the prevalence of oral HPV based on risk stratification. Multivariable logistic regression models were used to identify potential factors associated with oral HPV infection. 95% confidence intervals (CI) were calculated for all standardized prevalence and OR estimates. All statistical analyses were performed using SAS version 9.4 (SAS Institute).

The datasets generated or analyzed for this study are available from the corresponding author upon reasonable request.

The basic characteristics of the study population are summarized in Table 1. A total of 9,867 individuals were ultimately included in the sample. The mean age of the participants was 56.62 ± 12.88, and 30.9% were men. Of the participants, 53.1% living in urban areas and the majority were married (86.8%). Significant disparities were observed between sexes in sociodemographic characteristics, health-related lifestyle factors, and HPV vaccination (all P values <0.001), with the exception of urban and/or rural residence (P = 0.301).

The prevalence of oral HPV infection [HPV (+)] is presented in Tables 2 and 3, broken down by sex. Figure 1 illustrates the prevalence and proportions of specific HPV types in the overall population, categorized as high or low risk. In the high-risk HPV (+) group, type 16 was the most prevalent at 37.0%. In the low-risk HPV (+) group, type 81 accounted for 71.0% of the cases.

Overall, the prevalence of oral HPV infection was 3.0% in the population as a whole, with males having a higher prevalence at 3.7% compared with females at 2.7%. Within the study population, males also had a higher prevalence of both high-risk (1.7% vs. 1.2%) and low-risk (2.0% vs. 1.6%) HPV oral infections. Generally, individuals residing in the typical areas of South China (Guangdong province) had a lower prevalence compared with those in the North (Hebei province). The age- and sex-standardized prevalence were 2.2% (1.8–2.6) in Guangdong and 4.10% (3.5–4.7) in Hebei. The prevalence of the sex- and age-standardized high-risk HPV infection in Guangdong and Hebei provinces was 0.9% (0.7–1.1) and 1.89% (1.5–2.3), respectively.

To analyze the geographic variations in oral HPV infection, we additionally calculated the prevalence adjusted for age and sex among the seven study locations in Guangdong and Hebei. It is worth noting that although the overall prevalence of oral HPV infection in Guangdong is low, there are significant differences depending on the location. For instance, Jiaoling, situated in the mountainous region of Guangdong, had the highest HPV (+) prevalence at 5.3% (3.9–6.7). In contrast, Nan'ao Island exhibited the lowest HPV (+) prevalence of 0.2% (0–0.4), with no infections found among male participants.

The age-specific prevalence of oral HPV infection among both sexes and different geographic areas can be found in Supplementary Table S1. Out of 134 HPV-vaccinated women, 7 were HPV positive (3 were high-risk and 4 were low-risk), there was no difference between the vaccination group and their counterparts in the oral HPV prevalence.

The factors associated with oral HPV infection are presented in Table 4. According to multivariable logistic regression models, individuals in Hebei and were male, had a higher likelihood of being HPV positive, with an OR and 95% CI of 2.01 (1.53–2.65) and 1.42 (1.09–1.85), respectively. In addition, we conducted a regression analysis stratified by HPV types. The findings demonstrated that individuals in Hebei (OR and 95% CI: 2.41, 1.61–3.63) and those residing in urban areas (OR and 95% CI: 2.15, 1.43–3.26) were more susceptible to high-risk HPV infection. Conversely, individuals who had resided in urban areas had a lower risk of low-risk HPV infection (OR: 0.50, 95% CI: 0.36–0.71). Participants who smoked also had a reduced risk of low-risk HPV infection (OR and 95% CI: 0.56, 0.34–0.93). Because sex may modify the relationship between these factors and HPV infection, we conducted sex-stratified logistic regressions (Table 5). In both sexes, individuals in Hebei and those residing in urban areas had an increased likelihood of being infected with high-risk HPV. However, discrepancies between sexes were only observed in terms of low-risk HPV infections, and no geographic disparities were noted in males. Educational level and health-related lifestyle factors were not found to be significantly associated with any types of HPV infection.

To the best of our knowledge, this study is the first to report on the prevalence of oral HPV infection among a general Chinese adult population from diverse geographical areas with a relatively large sample size in China. Our findings indicate a generally low prevalence of oral HPV infection, with significant variations exist between sex and geographic locations. In addition, our study highlights that nearly half of all oral HPV infections are high-risk types, with HPV 16 being the most frequently identified high-risk oncogenic strain.

Recently, the incidence of HPV-related oropharyngeal cancers has increased drastically, prompting an urgent need for a deeper understanding of the infections that cause these cancers (16). As a result, it is crucial to monitor the prevalence of oral HPV infection in healthy populations. This will help in identifying high-risk populations, assessing the burden of HPV-related disease, and planning effective prevention programs. Our study contributes to our knowledge of the natural history of oral HPV infection among adults in China.

Although several studies have reported oral HPV infection in otherwise healthy individuals, most of these studies were conducted on European and American populations or primarily focusing on Chinese adults in rural areas. There is still a lack of data regarding a general Chinese population.

Berenson and colleagues used data from the National Health and Nutrition Examination Survey (NHANES) in the United States between 2011 and 2016 and reported that the prevalence of oral HPV infection, regardless of subtype variation, is 6.9%. They also found that males may be 3.5 times more susceptible to the disease than females (17). In addition, the Michigan HPV and oropharyngeal cancer cohort study revealed that the baseline oral HPV prevalence is 10.0% for any detected genotype (out of the 338 valid oral tests at baseline) and 6.5% for high-risk subtypes (18). Similarly, the multinational HPV infection in men study was conducted in the United States, Mexico, and Brazil. During the first 12 months of follow-up, 4.4% of men acquired an incident oral HPV infection, while 1.7% acquired oncogenic HPV. The study found that oral HPV infection was significantly higher in men living in Mexico (19). Furthermore, in a Finnish cohort, approximately 10% of men and women had oral infections after 24 months of follow-up, and 14.3% of men and women had oral infections after 7 years (20).

Notably, incidences of oral HPV infection vary significantly in normal oral cavity mucosa, ranging from 0.0% (95% CI: 0.0–3.7) to 24.1% (95% CI: 18.2–31.1; ref. 6). This wide range of variability might be attributed to differences in geographical regions, ethnic groups, genetic susceptibilities, or specimen collection, processing, and detection procedures. Importantly, our findings indicate a much higher incidence rate of oral HPV infections in the Chinese population compared with the previously reported numbers. For example, Ke and colleagues have reported mucosal HPV infection prevalence of 0.67% (95% CI: 0.47–0.93) in young Chinese adult males living in rural areas during 2009–2011 (21).

Differences among studies can be attributed to various factors, such as the heterogeneity of the study populations and varying time course of oral HPV infections due to a rapid socioeconomic development (5), during which sexual attitudes and behavior might have experienced substantial changes in Chinese adults (22). For instance, only 3.94% of participants reported oral sexual practice in Ke and colleagues’ study, compared with 86.9% in the United States (17, 21).

The prevalence of overall oral HPV infection among male participants was significantly higher than that in females in this study, which is consistent with the results of the U.S. population-based NHANES (15, 23). Therefore, this study represents the most reliable estimate of oral HPV prevalence in the Chinese population up to now. Several European studies, however, have suggested no significant differences in oral HPV prevalence rates between males and females (24, 25, 26). Of all the participants in this cohort, 30% were male and only 3% of these males were under the age of 30. Because the young male participants showed the highest prevalence of oral HPV infections (15), it is likely that the actual rate of HPV infection was still underestimated.

Previous studies on oral HPV infection also suggest significant differences across geographic regions (6, 7). China, being a vast country with diverse geographical areas and cultures, provides an interesting context for studying the prevalence of oral HPV infection. In the study, we focused on the second wave of DLCC from urban communities and rural villages from Baoding city of Hebei Province, Shantou and Meizhou cities (including three different kinds of culture: “Chaoshan,” “Hakka,” and island culture in South China) in Guangdong province. The two areas have some unique characteristics in terms of living patterns, substantial differences in environmental risk factors (such as the concentration of ambient air pollutants), and NCD prevalence (8).

Chosen to represent island culture, Nan'ao is a fairly secluded area where native residents lead conservative lifestyles. This may partially explain the low prevalence of HPV (+). The differences in prevalence according to location may be indicative of demographic disparities. Likewise, a multinational study including men from the United States, Mexico, and Brazil found variations in oral HPV prevalence, with Mexico having the highest prevalence (27). The specific reasons behind these geographic disparities in HPV infection prevalence are still unclear. Conducting additional multinational studies with standardized sample collection and detection methods may provide a deeper insight.

Moreover, our data indicated that smoking and alcoholism did not have an independent association with oral HPV infection, which differs from other studies (15). Our previous study reported a prevalence of 67.39% for smoking and 85.22% for alcohol consumption among adult males in China, which is much higher than what was found in this survey (28). It is important to note that in the overall regression analysis without considering sex, ever smoking was identified as a risk factor for HPV infection. However, when we analyzed the data by sex, this positive association did not remain statistically significant. This could be potentially attributed to residual confounding, even after accounting for sex as a covariate in the regression model. Another possible explanation could be the reduction in sample size after stratification, combined with the lower prevalence of ever smoking and alcohol consumption, which may have limited the statistical power to detect such differences.

Taken together, these results showed that HPV vaccination was efficient in preventing HPV infections in the cervix, anal, and oral areas. This finding can serve as a basis for promoting HPV vaccination as a means of preventing HPV infection and cancerization in the anal and oral, particularly in males, who have fewer opportunities and less recognition for receiving the HPV vaccine (29, 30). It is unfortunate that the vaccination rates for women in our investigation were only 2%, which is significantly lower than the rates reported in Western countries (31). In addition to the lack of vaccine availability, the cost of the vaccine and inadequate knowledge about HPV may also contribute to the low HPV vaccination rates in China (32). Although a direct relationship between the HPV vaccine and the prevention of HNSCC is yet to be established, recent years have been a significant decrease of up to 90% in oropharyngeal HPV infections among vaccinated young adults (33). Therefore, there is a pressing need to increase HPV vaccination rates in China.

Comparability has always been emphasized during the whole survey. We took several measures to reach this target. First, we used the same equipment, stringent and consistent criteria for measurements in all the survey sites, which makes the results from different study sites comparable. Second, the core team members of DLCC who took charge in questionnaire interview and physical examination did not change during the survey period. Third, before local staff took part in the survey, standardized training would be initiated for all of them to guarantee their field work capability. Fourth, the definition and classification of each variable was consistent all through the survey, which enable us to integrate the data collected from different period and to do pooled analysis in the future.

This study has several limitations. First, despite having a large sample size, the low prevalence of oral HPV infection may lead to unstable estimation. In addition, when estimating the prevalence stratified by sex or age, some estimates may be further unstable due to the limited sample sizes of specific factors. Moreover, the percentage of men recruited in the study was significantly lower than that of women. However, because the assessment of prevalence and risk factors were adjusted for sex, the main conclusions would remain unchanged. Second, no data on sexual behavior were collected, which restricts our understanding of the risk factors for oral HPV infection. Third, considering the cross-sectional nature of this study, it is not possible to interpret the observed associations as being temporally linked to infection. Large prospective natural history studies with more evaluation cycles are necessary to explore the epidemiology and risk factors of oral HPV infection in China.

In conclusion, our study has revealed a comparatively low prevalence of oral HPV infection within the general population of China. Moreover, we have observed significant regional and sex disparities. Specifically, the prevalence of oral HPV infection is higher in Hebei Province, located in the north of the country, than in Guangdong Province, situated in the south. In addition, men exhibit a higher prevalence of oral HPV infection in comparison with women. These discrepancies in the prevalence of oral HPV prevalence may suggest the existence of a distinct etiology in the progression of the infection, which should be further comprehended.

No disclosures were reported.

Y. Zhu: Conceptualization, software, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. H. He: Conceptualization, software, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. X. Zhu: Validation, investigation, writing–review and editing. Y. Hu: Data curation, validation, investigation, writing–review and editing. S. Yu: Validation, investigation, writing–review and editing. W. Diao: Validation, investigation, writing–review and editing. S. Li: Validation, investigation, writing–review and editing. G. Shan: Conceptualization, resources, supervision, funding acquisition, validation, investigation, project administration, writing–review and editing. X. Chen: Conceptualization, supervision, validation, investigation, writing–review and editing.

G. Shan and X. Chen received a grant from the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-023); X. Chen and Y. Zhu received a grant from the National High Level Hospital Clinical Research Funding (2022-PUMCH-A-032, 2022-PUMCH-B-094) and G. Shan received a grant from the Science & Technology Fundamental Resources Investigation Program (grant no. 2022FY100800). We thank all participants and staff at DLCC for their involvement and efforts.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.