The prevalence of human papillomavirus among women in northern Guangdong Province of China

Globally, cervical cancer, whose etiologic factor is Human papillomavirus (HPV), is the third most common cancer among women. In cervical cancer screening, HPV testing is important. However, the prevalence of HPV in northern Guangdong Province has not been conclusively determined. A total of 100,994 women attending Yuebei People's Hospital Affiliated to Shantou University Medical College between 2012 and 2020 were recruited. HPV was tested by a polymerase chain reaction (PCR)-based hybridization gene chip assay. The prevalence of HPV among these women was established to be19.04%. Peak prevalence was observed in women aged 40–49 (7.29%). Besides, the prevalence of single-type HPV infection (14.46%) was significantly high, compared to multiple-type infection (4.58%) (p < 0.01), while the prevalence of high-risk HPV infection (19.97%) was significantly higher than that of low-risk genotypes (5.48%) (p < 0.01). The most prevalent high-risk genotypes were HPV52 (4.16%), HPV16 (2.98%), HPV58 (2.15%), HPV53 (1.58%) and HPV68 (1.34%). HPV co-infection with up to 10 genotypes was reported for the first time. Our findings suggested a high burden of HPV infections among women in northern Guangdong. Establishing the prevalence and genotype distribution characteristics of HPV infections in the region can contribute to cervical cancer prevention through HPV vaccination.

HPV DNA testing. HPV DNA PCR amplification and genotyping were performed using the HPV genotyping Kit (Yaneng Biotechnology, Ltd., Shenzhen, China) according to the manufacturer's instructions. The test kit was approved by the National Medical Products Administration (NMPA, No.20193401918). The test included 23 type-specific oligonucleotides, designed to detect 17 HR-HPV genotypes (HPV 16,18,31,33,35,39,45,51,52,53,56,58,59,66,68,73 and 82) together with 6 LR-HPV genotypes (HPV6, 11, 42, 43, 81 and 83). Amplification was performed using the BIOER Genetouch PCR amplification instrument. The amplification system consisted of 5 μl HPV-DNA and 20 μl reaction system. Reaction conditions were: 50 °C for 15 min, denaturation at 95 °C for 10 min, denaturation at 94 °C for 30 s, annealing at 42 °C for 90 s, elongation at 72 °C for 30 s, 40 cycles, elongation at 72 °C for 5 min, and storage at 4 °C. HPV genotyping was performed by hybridization in a YN-H16 thermostatic hybridization instrument. The final result was determined by directly observing color reactions on the chip. The negative control showed no color reaction at all sites except the blue dot on the internal control (IC) site. The positive control must be color reaction of the positive HPV genotype and IC site (shown in blue), and the genotype is read according to the site of the blue spot on the chip. Statistical analysis. Data analysis was performed using SPSS22.0 software (IBM, USA). Data were presented as mean ± standard deviation or frequency and percentage for numerical or discrete variables, respectively. The Chi-square test was used to determine significant differences between groups, and differences were considered statistically significant at p < 0.05. Ethics statement. This research was approved by the Ethics Committees of Yuebei People's Hospital.

Results
Age-specific HPV prevalence. A total of 100,994 women visited the Yuebei People's Hospital and subjected to HPV DNA testing. The basic characteristics of study participants are shown in Table 1. It was established that 19,233 (19.04%) of the women had HPV infections, with most of the infections occurring in women of the 40-49 age group (7.29%). Single-type HPV infections were common among women aged 40-49 years (6.44%), while multiple-type HPV infections were common among women aged 50-59 years (1.71%). Singletype HPV infections (14.46%) were more common than multiple-type infections (4.58%), (p < 0.01; Table 2 and Fig. 1).
Distribution of single and multiple HPV infections. Single, double, and multiple-type HPV infections accounted for 75.94%, 17.52% and 4.47%, respectively (Table 5). Single-type infection was more common than multiple-type infection (p < 0.01). HPV co-infection with up to 10 subtypes was discovered for the first time (Fig. 5).

Discussion
In this study, we established that the HPV infection rate among women in northern Guangdong was 19.04%, with the infection rate being highest among women aged 40-49 (7.29%). Single-type and HR-HPV infections were the most prevalent form. The most prevalent HR-HPV infections were HPV52, 16 www.nature.com/scientificreports/ 56 while LR-HPV infections were mainly HPV81 and 43. We also report, for the first time, HPV co-infection with up to 10 genotypes in two cases. Studies have reported differences in HPV prevalence and genotype distribution in different countries 13 . According to a comprehensive meta-analysis involving over 1 million women, the global HPV prevalence is estimated to be 11.7%. Sub-Saharan Africa (24.0%), Eastern Europe (21.4%), and Latin America (16.1%) have the highest prevalence 14 . In Japan, the prevalence of HPV is 25%, with HPV52, 16, and 58 being the most common genotypes 15 . In Korea, the prevalence of HPV is 16.71%, with HPV 53, 58, and 52 being the most common genotypes 16 . A previous meta-analysis reported that HPV infection rate among women in mainland China is 19.0%, with HPV16, 52, and58 being the most common genotypes 17 . These types are also the most common types of CIN in China 18 . This contrasts with the situation in Western countries, where HPV16 and 18 infections are prevalent 19,20 . www.nature.com/scientificreports/ In China, there are regional differences in HPV prevalence and genotype distribution. Population-based screening revealed that the HPV infection rate in China ranges from 9.9% to 31.94% [21][22][23][24] . Among the sampled regions, Haikou (31.94%), Beijing (21.06%), and Guangdong (20.02%) have the highest HPV incidences. Our results are consistent with HPV distribution characteristics previously reported in China [25][26][27] . The overall HPV infection rate is high and single-type as well as high-risk infections are more common. However, the top five genotypes are slightly different. For instance, the main genotypes in Meizhou 28 , were HPV 16, 52, 58, 18, and 81, while in northern Guangdong, they were HPV 52, 16, 58, 53 and 68. In short, the top three in most regions of China were HPV 52, 16, and 58.
HPV screening and vaccination are the most effective measures for preventing cervical cancer. China's cervical cancer screening program adopts a combined method for HPV and cytology (TCT) screening, which can maximize screening Sensitivity and specificity to improve the detection rate. The program is led by the government, with the participation of social groups, medical institutions, third-party testing institutions, etc. It is completely free for the subjects, voluntary, and covers women aged 35-64. In short, cervical cancer screening has a long way to go, and various efforts are being made to explore solutions with Chinese characteristics. Most developed countries have adopted vaccination as their main preventive strategy. Therefore, if these prevention strategies     29 .
China approved the importation of bivalent vaccines in 2016. Nationally, the HPV vaccination rate for women is only 11.0% 30 , and the vaccination situation is not optimistic. HPV vaccination is expensive, and uncertainties regarding the side effects of the vaccine are the main reason for reluctance. In this study, we found that the HPV infection rate remained stable without any downward trend from 2012 to 2020. Therefore, China needs to develop HPV vaccines that are suitable for the Chinese population, while putting into consideration the characteristics of vaccine genotypes and the age of vaccination, further localization, reducing costs, and including government free vaccination programs to reduce the incidences of HPV infections and cervical cancer.

Conclusions
For the first time, we report on prevalence and subtype distribution of women HPV in northern Guangdong, which is of great significance for guiding the formulation of local vaccination and other prevention strategies. However, epidemiological investigation of HPV requires multi-center and long-term monitoring, especially evaluation of the effects after vaccination. Besides, persistent HR-HPV and multiple-type infections as well as cancer outcomes should be investigated further.