Prevalence and genotype distribution of human papillomavirus infection among women aged 30–65 years in Xi’an, China: a population-based study of 14,655 women

ABSTRACT

Cervical cancer is the fourth most common cancer among women worldwide in terms of both incidence and mortality. Persistent infection with high-risk human papillomavirus (HPV) has been identified as a cause of cervical intraepithelial neoplasia and invasive cervical cancer. The distribution of human papillomavirus genotypes varies regionally. To acquire baseline data on the population-based prevalence and genotype distribution of HPV infection, we investigated the molecular epidemiology of HPV infection among women in Xi’an, China. The study was conducted from September 2018 to December 2020. A total of 14,655 women aged 30–65 years were screened. The overall prevalence of HPV infection was 13.5% (95% confidence interval [CI]: 13.0–14.1%), with 10.4% of participants being positive for a single HPV type and 3.1% being positive for multiple HPV types. The prevalence of high-risk HPV (HR-HPV), low-risk HPV (LR-HPV) and mixed HPV infection was 10.1% (95% CI: 9.6–10.5%), 2.2% (95% CI: 2.0–2.4%), and 1.3% (95% CI: 1.1–1.5%), respectively. The five most frequently detected HR-HPV types were types 52 (2.6%), 16 (1.9%), 53 (1.8%), 58 (1.4%), and 51 (0.9%). The most frequently detected LR-HPV type was HPV-42 (1.1%). The prevalence and HPV genotype distribution varied by region and age. Age-specific HPV prevalence peaked in the over 60 years age group (18.8%), and Beilin District had the highest HPV prevalence (18.1%). The results of this first population-based study provide a reference for HPV-based cervical cancer screening and HPV vaccination programs in Xi’an.

KEYWORDS:

• Human papillomavirus infection
• cervical cancer screening
• prevalence survey
• genotype distribution
• general population
• China

Introduction

Cervical cancer is the fourth most common cancer among women in terms of both incidence and mortality. It is estimated to result in 604,000 new cases and 342,000 deaths worldwide in 2020. The incidence and mortality are disproportionately high in transitioning countries relative to transitioned countries.¹ There is geographical variation in the incidence of cervical cancer according to socioeconomic conditions.² Cervical cancer is a considerable burden in China, which is a large populous country with geographical and socioeconomic inequalities,³ with an estimated 106,000 new cases and 48,000 deaths in 2018.⁴ Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. Persistent infection with high-risk HPV (HR-HPV) is a necessary risk factor for precancerous lesions and cervical cancer.^5,6 Effective implementation of cervical cancer screening programs significantly lowered cervical cancer incidence and mortality in some developed countries. As HPV is prevalent worldwide and genotype distribution vary by region, acquiring population-based data and the region-specific molecular epidemiological characteristics of HPV infection are valuable to the development of new screening strategies and HPV prevention by vaccination. Although studies have been conducted in some regions of China, the HPV prevalence varies by region, and the available data are outdated and inadequate. The limited number of studies available do not provide an accurate picture of the prevalence and genotype distribution of HPV infection among the general population in Xi’an. HPV vaccines have been available since 2017 in Xi’an,⁷ but the effect of HPV vaccination on the prevalence and genotype distribution of HPV infection is unknown. Therefore, we conducted a retrospective analysis of the cervical cancer screening database initiated in 2018. The overall prevalence, age, and district-specific prevalence and genotype distribution were calculated and analyzed.

Materials and methods

Study participants

The inclusion criteria were as follows: (1) female sex; (2) aged between 30 and 65 years; (3) history of sexual experience; (4) permanent resident of Xi’an. The exclusion criteria were as follows: (1) age <30 years or >65 years; (2) pregnancy; (3) presence of acute genital inflammation; (4) history of cervical resection or hysterectomy. The screened participants were divided into seven age groups: 30–34 years, 35–39 years, 40–44 years, 45–49 years, 50–54 years, 55–59 years, and ≥ 60 years. All the participants enrolled in this study lived in one of the six districts of Xi’an (Lianhu, Yanta, Baqiao, Changan, Weiyang, and Beilin), the capital city of Shaanxi Province in China. This study and the consent procedure were approved by the Ethics Committee of the Maternal and Child Health Hospital of Xi’an and were performed in accordance with the approved guidelines and the principles adopted in the Declaration of Helsinki and its later amendments or comparable ethical standards. All participants provided written informed consent. Women attending cervical cancer screening program from September 2018 to December 2020 for HPV DNA testing were recruited for the study if they met the study criteria.

Specimen collection

According to the currently accepted protocols of practice, cervicovaginal samples were collected by a gynecologist. Cervical cell specimens were collected using the special cytobrush included in the kit (Jiangsu Jianyou Co., Ltd., ORE00704). The cervical samples were kept in a sample transport medium according to the manufacturer’s instructions. All samples were shipped to the laboratory at the HPV Testing Center of Xi’an immediately at 4°C, and HPV genotyping tests were performed within 24 hours.

DNA extraction and HPV genotype testing

DNA extraction, polymerase chain reaction (PCR) amplification and genotype detection by reverse dot blot hybridization (RDB), were performed according to the manufacturer’s instructions. HPV DNA genotype detection was performed using a commercial HPV genotyping kit (HEAS BioTech Co., Ltd., DQE00501). PCR amplification was performed using an ABI7500 Thermo Cycler (Applied Biosystems, USA). The amplification parameters were: 50°C for 3 min; 95°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, for 40 cycles; and then 72°C for 10 min. PCR reverse dot membrane hybridization was used to detect 28 HPV genotypes (types 6, 11, 40, 42, 43, 44, 54, 61, 81, 83, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 82, 26, 53 and 73). The HPV types were determined according to the distribution of positive points on the membrane strips. Negative and positive controls were used throughout the process. All the HPV DNA testing was performed at the HPV Testing Center of Xi’an.

Statistical analysis

All statistical analyses were performed using SPSS version 19.0 (IBM Corp, Armonk, NY, USA). The overall prevalence, type-specific prevalence, genotype distribution, and prevalence of single and multiple HPV infections were calculated and analyzed. The prevalence of HPV infection among the different districts and age groups was compared. Chi-squared (χ²) tests were used to evaluate the significance of differences between groups. For all analyses, P-values ≤ 0.05 were considered statistically significant.

Results

A total of 14,889 women were screened during the study period, of whom 234 were excluded due to unknown age.

Overall prevalence of HPV infection in Xi’an

The age of participants ranged from 30 to 65 years, and the mean age was 44.2 ± 8.9 years. The overall HPV prevalence was 13.5% (1985/14655, 95% CI: 13.0–14.1%). The prevalence of HR-HPV, LR-HPV and mixed HR-HPV and LR-HPV infection was 10.1% (1474/14655, 95% CI: 9.6–10.5%), 2.2% (324/14655, 95% CI: 2.0–2.4%), and 1.3% (187/14655, 95% CI: 1.1–1.5%), respectively. Thus, 74.3% of the HPV infections were caused by HR-HPV.

HPV prevalence by region

The prevalence of HPV infection varied by region (χ² = 14.439, p = .013). The prevalence of HPV infection in Lianhu, Yanta, Baqiao, Changan, Weiyang, and Beilin Districts was 11.9%, 12.7%, 12.8%, 14.4%, 14.6%, and 18.1%, respectively. The prevalence of single HR-HPV, single LR-HPV, multiple HR-HPV, multiple LR-HPV, and mixed HR-HPV and LR-HPV infections according to the region is shown in Table 1. Single HPV infections were dominant in all districts. The highest prevalence of single HPV infection occurred in Beilin District (14.7%), and the lowest prevalence occurred in Lianhu District (10.1%). Among the women positive for HPV DNA, single HR-HPV infection was the most common pattern. The highest prevalence of a single HR-HPV infection occurred in the Chang’an District (9.2%). Wei’yang District had the highest prevalence of multiple HR-HPV infections (2.4%). Beilin District had the highest prevalence of single LR-HPV (5.9%), multiple LR-HPV (0.8%), and mixed HR-HPV and LR-HPV (1.7%) infections.

Table 1. Prevalence of human papillomavirus infection in women in Xi’an, China according to district (p = .013)

HPV types	Lianhu		Yanta		Baqiao		Changan		Weiyang		Beilin		Unknown
	n = 3082		n = 3187		n = 2683		n = 2821		n = 296		n = 238		n = 2348
	Single	Multiple**	Single	Multiple	Single	Multiple	Single	Multiple	Single	Multiple	Single	Multiple	Single	Multiple
HR-HPV types only	241	45	244	41	218	46	260	44	23	7	18	5	223	59
	(7.8%)	(1.5%)	(7.7%)	(1.3%)	(8.1%)	(1.7%)	(9.2%)	(1.6%)	(7.8%)	(2.4%)	(7.6%)	(2.1%)	(9.5%)	(2.5%)
LR-HPV types only	46	4	68	5	50	3	64	5	9	0	14	2	50	4
	(1.5%)	(0.1%)	(2.1%)	(0.2%)	(1.9%)	(0.1%)	(2.3%)	(0.2%)	(3.0%)	0	(5.9%)	(0.8%)	(2.1%)	(0.2%)
Mixed HR and LR HPV types	23*	8	27	20	15	14	20	14	4	0	3	1	18	20
	(0.7%)	(0.3%)	(0.8%)	(0.6%)	(0.6%)	(0.5%)	(0.7%)	(0.5%)	(1.4%)	0	(1.3%)	(0.4%)	(0.8%)	(0.9%)
Total	310	57	339	66	283	63	344	63	36	7	35	8	291	83
	(10.1%)	(1.8%)	(10.6%)	(2.1%)	(10.5%)	(2.3%)	(12.2%)	(2.2%)	(12.2%)	(2.4%)	(14.7%)	(3.4%)	(12.4%)	(3.5%)

*indicating single HR-HPV and single LR-HPV mixed infection.

**indicating HR-HPV types only: two and more HR-HPV types infections; LR-HPV types only: two and more LR-HPV types infections; Mixed HR and LR HPV types: two or more HR-HPV types and two or more LR-HPV types mixed infection.

HPV infection characteristics by age group

The prevalence of HPV infection varied with age in Xi’an (χ² = 59.649, p < .001). The prevalence of HPV infection was 12.3%, 11.6%, 11.5%, 14.3%, 14.7%, 17.1%, and 18.7% for women aged 30–34 years, 35–39 years, 40–44 years, 45–49 years, 50–54 years, 55–59 years, and ≥60 years respectively. The prevalence of single HR-HPV, single LR-HPV, multiple HR-HPV, multiple LR-HPV, and mixed HR- and LR-HPV infection according to different age groups is shown in Table 2. The prevalence of single HR-HPV infection (χ² = 18.611, p = .005), single LR-HPV infection (χ² = 15.314, p = .018), multiple HR-HPV infections (χ² = 38.694, p < .001), and mixed HR-HPV and LR-HPV infections (χ² = 32.743, p < .001) differed significantly by age. Single HPV infection was significantly more prevalent than multiple HPV infections in all age groups. The highest prevalence of single HR-HPV (10.4%), multiple HR-HPV (3.2%), and mixed HR-HPV and LR-HPV infections (3.2%) occurred in women aged ≥60 years. The lowest prevalence of single HR-HPV (7.3%) occurred in women aged 40–44 years.

Table 2. Prevalence of human papillomavirus infection in women in Xi’an, China according to age (p < .001)

HPV Types	30–34y		35–39y		40–44y		45–49y		50–54y		55–59y		≥60y
	n = 2537		n = 2680		n = 2599		n = 2510		n = 1982		n = 1605		n = 742
	Single	Multiple**	Single	Multiple	Single	Multiple	Single	Multiple	Single	Multiple	Single	Multiple	Single	Multiple
HR-HPV types only	209	37	196	31	189	35	232	33	167	38	157	49	77	24
	(8.2%)^a	(1.5%)^c	(7.3%)	(1.2%)	(7.3%)	(1.3%)	(9.2%)	(1.3%)	(8.4%)	(1.9%)	(9.8%)	(3.1%)	(10.4%)	(3.2%)
LR-HPV types only	32	5	57	4	48	3	60	2	55	3	35	6	14	0
	(1.3%)^b	(0.2%)	(2.1%)	(0.1%)	(1.8%)	(0.1%)	(2.4%)	(0.1%)	(2.8%)	(0.2%)	(2.2%)	(0.4%)	(1.9%)	0
Mixed HR and LR HPV types	18*	10	16	7	11	12	24	9	16	13	13	14	12	12
	(0.7%)	(0.4%)^d	(0.6%)	(0.3%)	(0.4%)	(0.5%)	(1.0%)	(0.4%)	(0.8%)	(0.7%)	(0.8)	(0.9%)	(1.6%)	(1.6%)
Total	259	52	269	42	248	50	316	44	238	54	205	69	103	36
	(10.2%)	(2.0%)	(10.0%)	(1.6%)	(9.5%)	(1.9%)	(12.6%)	(1.8%)	(12.0%)	(2.7%)	(12.8%)	(4.3%)	(13.9%)	(4.9%)

*indicating single HR-HPV and single LR-HPV mixed infection.

**indicating HR-HPV types only: two and more HR-HPV types infections; LR-HPV types only: two and more LR-HPV types infections; Mixed HR and LR HPV types: two or more HR-HPV types and two or more LR-HPV types mixed infection.

^aThe prevalence of single HR-HPV infection differed by age (P = .005).

^bThe prevalence of single LR-HPV infection differed by age (P = .018).

^cThe prevalence of multiple HR-HPV infections differed by age (P < .001).

^dThe prevalence of mixed HR-HPV and LR-HPV infections differed by age (P < .001).

Distribution of single and multiple HPV infections

Among all HPV-positive women, 1528 women were positive for single HPV infection, accounting for 10.4% of all participants and 77.0% of HPV-positive cases. A total of 457 women were positive for multiple HPV infections, accounting for 3.1% of all participants and 23.0% of HPV-positive cases (Table 3). In terms of multiple HPV infections, double HPV infection was the most common infection pattern, with an overall prevalence of 2.2%. Three HPV genotype infections were found in 104 women, accounting for 0.7% of all participants and 5.2% of HPV-positive participants. Only 0.2% of the participants were infected with four or more HPV genotypes.

Table 3. Distribution of single and multiple human papillomavirus infections in women in Xi’an, China

HPV infections	Positive case	Percentage (x/1985)	Prevalence (x/14655)
Single type	1528	77.0%	10.4%
Two types	320	16.1%	2.2%
Three types	104	5.2%	0.7%
Four types	26	1.3%	0.2%
≥Five types	7	0.4%	0%
Total	1985	100%	13.5%

Genotype distribution of HPV infection in Xi’an

The distribution of HPV genotypes is shown in Table 4. Among all HPV-positive samples, the six most prevalent HR-HPV genotypes were HPV-52 (2.6%), HPV-16 (1.9%), HPV-53 (1.8%), HPV-58 (1.4%), HPV-51 (0.9%), and HPV-56 (0.9%). Furthermore, the three most prevalent LR-HPV types were HPV-42 (1.1%), HPV-81 (0.8%), and HPV-43 (0.7%). In terms of the region-specific distribution of HPV genotypes, the most prevalent HR-HPV genotype was HPV-52. However, HPV-16 (2.9%) was the most common genotype in Beilin District. HPV-42 was the most prevalent LR-HPV genotype in most districts.

Table 4. Prevalence of human papillomavirus in women in Xi’an, China according to genotype and district

Overall prevalence			Prevalence by different districts of Xi’an
	All population	Lianhu	Yanta	Baqiao	Changan	Weiyang	Beilin	Unknown
	n = 14655	n = 3082	n = 3187	n = 2683	n = 2821	n = 296	n = 238	n = 2348
HPV subtype	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n(%)
16	278 (1.9)	59 (1.9)	52 (1.6)	39 (1.5)	58 (2.1)	4 (1.4)	7 (2.9)	59 (2.5)
18	99 (0.7)	16 (0.5)	18 (0.6)	14 (0.5)	28 (1.0)	0 (0)	1 (0.4)	22 (0.9)
31	66 (0.5)	14 (0.5)	10 (0.3)	9 (0.3)	16 (0.6)	2 (0.7)	0 (0)	15 (0.6)
33	76 (0.5)	15 (0.5)	14 (0.4)	17 (0.6)	13 (0.5)	1 (0.3)	2 (0.8)	14 (0.6)
35	38 (0.3)	6 (0.2)	8 (0.3)	6 (0.2)	12 (0.4)	0 (0)	0 (0)	6 (0.3)
39	71 (0.5)	13 (0.4)	18 (0.6)	11 (0.4)	14 (0.5)	1 (0.3)	1 (0.4)	13 (0.6)
45	29 (0.2)	5 (0.2)	8 (0.3)	5 (0.2)	6 (0.2)	0 (0)	1 (0.4)	4 (0.2)
51	136 (0.9)	19 (0.6)	25 (0.8)	22 (0.8)	25 (0.9)	3 (1.0)	4 (1.7)	38 (1.6)
52	374 (2.6)	77 (2.5)	81 (2.5)	63 (2.3)	68 (2.4)	8 (2.7)	3 (1.3)	74 (3.2)
56	136 (0.9)	18 (0.6)	31 (1.0)	34 (1.3)	26 (0.9)	1 (0.3)	2 (0.8)	24 (1.0)
58	200 (1.4)	26 (0.8)	39 (1.2)	36 (1.3)	44 (1.6)	7 (2.4)	3 (1.3)	45 (1.9)
59	70 (0.5)	10 (0.3)	11 (0.3)	10 (0.4)	14 (0.5)	4 (1.4)	1 (0.4)	20 (0.9)
66	106 (0.7)	22 (0.7)	20 (0.6)	28 (1.0)	17 (0.6)	3 (1.0)	3 (1.3)	13 (0.6)
68	85 (0.6.)	17 (0.6)	13 (0.4)	15 (0.6)	17 (0.6)	1 (0.3)	5 (2.1)	17 (0.7)
82	17 (0.1)	4 (0.1)	2 (0.1)	2 (0.1)	1 (0)	1 (0.3)	0 (0)	7 (0.3)
26	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
53	258 (1.8)	53 (1.7)	54 (1.7)	49 (1.8)	54 (1.9)	6 (2.0)	5 (2.1)	37 (1.6)
73	15 (0.1)	2 (0.1)	2 (0.1)	2 (0.1)	4 (0.1)	1 (0.3)	0 (0)	4 (0.2)
6	39 (0.3)	5 (0.2)	7 (0.2)	6 (0.2)	11 (0.4)	1 (0.3)	0 (0)	9 (0.4)
11	11 (0.1)	0 (0)	3 (0.1)	2 (0.1)	4 (0.1)	0 (0)	1 (0.4)	1 (0)
40	28 (0.2)	6 (0.2)	10 (0.3)	2 (0.1)	8 (0.3)	0 (0)	0 (0)	2 (0.1)
42	155 (1.1)	23 (0.7)	29 (0.9)	25 (0.9)	35 (1.2)	2 (0.7)	9 (3.8)	32 (1.4)
43	99 (0.7)	16 (0.5)	20 (0.6)	12 (0.4)	21 (0.7)	3 (1.0)	2 (0.8)	25 (1.1)
44	54 (0.4)	10 (0.3)	22 (0.7)	12 (0.4)	4 (0.1)	0 (0)	0 (0)	6 (0.3)
54	98 (0.7)	16 (0.5)	38 (1.2)	24 (0.9)	12 (0.4)	0 (0)	0 (0)	8 (0.3)
61	94 (0.6)	26 (0.8)	28 (0.9)	14 (0.5)	18 (0.6)	0 (0)	0 (0)	8 (0.3)
81	120 (0.8)	14 (0.5)	23 (0.7)	15 (0.6)	24 (0.9)	7 (2.4)	8 (3.4)	29 (1.2)
83	11 (0.1)	1 (0)	4 (0.1)	3 (0.1)	1 (0)	0 (0)	2 (0.8)	0 (0)

The distribution of HPV genotypes is shown according to age group in Table 5. HPV-52, −53, −16, and −58 were the most common HR-HPV genotypes among all age groups. HPV-52 was the most common HR-HPV genotype among all age groups, except for the age group of 30–34 years, among which HPV-16 (2.4%) was the most prevalent genotype and HPV-52 was the second most prevalent genotype detected (2.3%). HPV-42 was the most common LR-HPV genotype among all age groups, except for the age group of 50–54 years, in which HPV-81 was the most prevalent genotype.

Table 5. Prevalence of human papillomavirus infection in women in Xi’an, China according to genotype and age group

	Prevalence by age group
	30–34y	35–39y	40–44y	45–49y	50–54y	55–59y	≥60y
	n = 2537	n = 2680	n = 2599	n = 2510	n = 1982	n = 1605	n = 742
HPV subtype	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n (%)	Positive, n (%)
16	61 (2.4)	41 (1.5)	39 (1.5)	43 (1.7)	39 (2.0)	31 (1.9)	24 (3.2)
18	19 (0.7)	13 (0.5)	11 (0.4)	21 (0.8)	17 (0.9)	12 (0.7)	6 (0.8)
31	6 (0.2)	12 (0.4)	12 (0.5)	9 (0.4)	17 (0.9)	4 (0.2)	6 (0.8)
33	9 (0.4)	10 (0.4)	7 (0.3)	13 (0.5)	14 (0.7)	14 (0.9)	9 (1.2)
35	3 (0.1)	3 (0.1)	7 (0.3)	9 (0.4)	4 (0.2)	9 (0.6)	3 (0.4)
39	12 (0.5)	15 (0.6)	14 (0.5)	13 (0.5)	5 (0.3)	6 (0.4)	6 (0.8)
45	2 (0.1)	4 (0.1)	3 (0.1)	3 (0.1)	5 (0.3)	8 (0.5)	4 (0.5)
51	28 (1.1)	15 (0.6)	17 (0.7)	23 (0.9)	20 (1.0)	23 (1.4)	10 (1.3)
52	58 (2.3)	55 (2.1)	44 (1.7)	73 (2.9)	49 (2.5)	56 (3.5)	39 (5.3)
56	17 (0.7)	19 (0.7)	22 (0.8)	21 (0.8)	15 (0.8)	28 (1.7)	14 (1.9)
58	25 (1.0)	31 (1.2)	30 (1.2)	28 (1.1)	32 (1.6)	38 (2.4)	16 (2.2)
59	11 (0.4)	11 (0.4)	10 (0.4)	16 (0.6)	10 (0.5)	10 (0.6)	2 (0.3)
66	25 (1.0)	15 (0.6)	14 (0.5)	17 (0.7)	16 (0.8)	15 (0.9)	4 (0.5)
68	19 (0.7)	13 (0.5)	17 (0.7)	10 (0.4)	12 (0.6)	11 (0.7)	3 (0.4)
82	1 (0)	5 (0.2)	7 (0.3)	1 (0)	2 (0.1)	0 (0)	1 (0.1)
26	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
53	31 (1.2)	27 (1.0)	45 (1.7)	45 (1.8)	32 (1.6)	43 (2.7)	35 (4.7)
73	0 (0)	3 (0.1)	1 (0)	4 (0.2)	5 (0.3)	2 (0.1)	0 (0)
6	6 (0.2)	5 (0.2)	6 (0.2)	9 (0.4)	7 (0.4)	3 (0.2)	3 (0.4)
11	3 (0.1)	1 (0)	2 (0.1)	3 (0.1)	1 (0.1)	1 (0.1)	0 (0)
40	4 (0.2)	4 (0.1)	4 (0.2)	8 (0.3)	2 (0.1)	6 (0.4)	0 (0)
42	16 (0.6)	23 (0.9)	26 (1.0)	31 (1.2)	23 (1.2)	23 (1.4)	13 (1.8)
43	15 (0.6)	21 (0.8)	15 (0.6)	14 (0.6)	18 (0.9)	11 (0.7)	5 (0.7)
44	10 (0.4)	8 (0.3)	12 (0.5)	8 (0.3)	6 (0.3)	6 (0.4)	4 (0.5)
54	16 (0.6)	18 (0.7)	12 (0.5)	14 (0.6)	14 (0.7)	20 (1.2)	4 (0.5)
61	14 (0.6)	16 (0.6)	14 (0.5)	6 (0.2)	16 (0.8)	20 (1.2)	8 (1.1)
81	14 (0.6)	18 (0.7)	13 (0.5)	23 (0.9)	24 (1.2)	16 (1.0)	12 (1.6)
83	0 (0)	1 (0)	1 (0)	5 (0.2)	1 (0.1)	2 (0.1)	1 (0.1)

Discussion

Human papillomaviruses are double-stranded DNA viruses that belong to the papillomavirus family. More than 200 genotypes have been identified in the human papillomavirus family, which preferentially infect mucosa and epithelial cells.^8,9 According to their carcinogenic potential, HPVs are categorized as HR-HPV types, which are closely associated with carcinomas, and LR-HPV types, which mainly cause benign warts. Persistent infection with HR-HPV has been identified as a causal and necessary risk factor for cervical cancer.⁵ HPV-16 and HPV-18 are recognized as the most common cervical cancer-associated HR-HPV genotypes and are responsible for approximately 70% of cervical cancers worldwide.¹⁰ In addition to HPV-16 and HPV-18, HPV-31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are also defined as HR-HPV genotypes.¹¹

Cervical cancer causes more deaths in less-developed countries than that in developed countries.¹² As the major risk factors for cervical cancer, HPV vaccination and cervical cancer primary screening are the two most effective approaches to prevent cervical cancer. The global goal to eliminate cervical cancer as a public health problem included a target of screening 70% of women aged 35 and 45 years.¹³ Currently, HPV DNA testing is regarded as an effective primary screening method.¹⁴ In order to lower the incidence and mortality of cervical cancer, a cervical cancer screening program was initiated in 2009 in China.¹⁵ Xi’an initiated an HPV screening program in 2018. The characteristics of HPV infection vary considerably according to region and age. Differences in HPV genotype distribution among the general population should be considered when developing screening strategies and the next-generation of vaccines. However, there has been limited research on the molecular epidemiology of HPV infection in Xi’an. This study described a cross-sectional and large-scale investigation of the molecular epidemiology of HPV infection in women attending cervical cancer screening program in Xi’an, the central city of northwest China.

With the development of laboratory medicine and sensitive testing technology being clinically available, multiple infection caused by different HPV genotypes can be detected accurately, making mass population HPV screening possible. As a sensitive technique for molecular hybridization, PCR reverse dot blot assay has been recognized as an appropriate method for HPV genotyping.¹⁶ This method is particularly suitable for detecting infections with multiple HPV types. The kit used in our study can simultaneously detect 28 different HPV genotypes, including 18 HR-HPV types and 10 LR-HPV types.

The results showed that the overall prevalence of HPV infection was 13.5%, which is higher than the estimated global HPV prevalence among women without cervical abnormalities (10.4%)¹⁷ and is comparable to the prevalence in Southeast Asia (14%).¹⁸ Due to the large population and wide territory, HPV prevalence varies considerably in China, from 8.92% in Yangqu¹⁹ to 44.5% in Henan.²⁰ Compared with other region-specific data in China, the overall HPV prevalence in Xi’an is higher than that reported in Yunnan in southwest China (12.9%);²¹ lower than that reported in Shandong (north China, 28.4%),²² Jiangsu (East China, 26.92%),²³ Shenzhen (South China, 17.83%),²⁴ Wuhan (central China, 17.68%),²⁵ and Jilin (northeast China, 34.4%);²⁶ and similar to the prevalence reported in Xinjiang (14.02%), another city in north-western China.²⁷ A meta-analysis found that the prevalence of HR-HPV in Chinese women was 19.0%,²⁸ which is higher than the prevalence of HR-HPV in Xi’an. The difference may be associated with the study setting (clinic-based or population-based), population composition, sampling periods, examination techniques, and geographic region.^17,29,30 As the central city of northwest China, Xi’an has unique advantages in economy, culture, education, and medical resources, which may contribute to the relatively low prevalence of infection. Previous studies showed that the overall HPV prevalence was 30.21%³¹ and 26.3%⁷ in Xi’an. The possible difference from our study may be that hospital-based studies are opportunistic screenings with selection bias. The participants in the previous studies were mainly women visiting hospital gynecology departments, and most participants had genital lesions. This study had a much larger sample size and was based on a screening population in which participants were recruited from the general population and screened for cervical cancer. Moreover, participants in previous studies included women aged 16–30 years, who were more susceptible to acquiring HPV infection because younger women tend to be more sexually active than older women. However, this study did not include participants <30 years. The increasing awareness of cervical cancer screening and the application of HPV vaccines have contributed to a decrease in the number of asymptomatic HPV-positive individuals. A previous population-based investigation focused on the prevalence and genotype distribution of women with normal cytology results,¹⁷ so the estimated prevalence was low, given that the prevalence of HPV infection was higher in women with abnormal cytology results than that of women with normal cytology results. This study investigated the prevalence and genotype distribution of women among the general population. Therefore, the prevalence of HPV determined in this study is likely to be a more accurate estimate of the true prevalence of HPV infection in women in Xi’an.

The prevalence of HPV infection varies by age.³² The prevalence and genotype distribution of HPV infection exhibited age-related differences in our study. The highest prevalence was in the over-60-years age group, followed by the 55–59-years age group, which is consistent with a trend toward a high prevalence in older women. In other age groups, the HPV prevalence was higher in women younger than 35 years than in those in the 35–39-years and 40–44-years age groups. Women younger than 30 years were excluded from our study, so it is impossible to determine whether there is a peak in HPV prevalence in women younger than 30 years in Xi’an. Age-specific analyses showed a bimodal peak in HPV prevalence worldwide. The global prevalence of HPV infection is higher in younger women. In Africa, Americas, and Europe, HPV prevalence presented a second peak in women aged 45 years or older.¹⁷ A possible reason for this pattern is that younger women tend to be more sexually active, and older women are more susceptible to HPV infection due to reduced immune functions. The notable increase in total infection, HR-HPV infection, and mixed HPV infection in older women might result from an impaired immune response and hormonal changes at menopause, which may lead to persistent HPV infection. Older women should pay more attention to the prevention and treatment of cervical precancerous lesions and require more frequent follow-ups because of the increasing risk of persistent infection.

The prevalence of HPV infection also showed regional differences. Our study found that women from Beilin District had the highest HPV prevalence, which was noticeably higher than the overall prevalence in Xi’an. Single HPV infection, single LR-HPV infection, multiple LR-HPV infections, and mixed high-low-risk HPV infection in Beilin District were consistently higher than in other districts in Xi’an. This suggests that the regional characteristics of HPV infection are heterogeneous. However, there may have been selection bias due to the limited number of participants from the Beilin District.The participants were selected from women who participated in the HPV screening program during the study. Women volunteering to participate in cervical cancer screening programs were less in Beilin and Weiyang than other districts. Correspondingly, participants meeting the inclusion criteria in the districts were less than in other districts. This is associated with the fact that the total population of Beilin and Weiyang Districts is relatively smaller than that of other districts in Xi’an, and the population composition may differ in different districts. A lack of cervical cancer screening awareness can contribute to the low acceptability of HPV screening in these districts. Moreover, socioeconomic disparities and differences in health care service may account for the differences in the prevalence of HPV infection between different districts in Xi’an. The differences may also be attributable to susceptibility factors in women exposed to HPV infection. Studies have shown that HPV infection is associated with factors such as sexual behavior, the number of childbirths and abortions, smoking, oral contraception, and personal hygiene.^30,33–35 Therefore, it is necessary to further investigate the cause of the higher HPV prevalence among women in Beilin District.

In this study, single HPV infection predominated in HPV-positive women. Double HPV infection was the most common pattern of multiple infections. HPV16 + 52 and HPV52 + 58 have been reported to be the most common combinations of HPV genotypes in women with multiple infections in China,²⁹ while the most common genotype combinations in our study were HPV53 + 58 and HPV53 + 52.

The most common HPV genotypes worldwide are types 16, 18, 31, 52, and 58,¹⁸ and the most common HPV genotypes in Asian countries are types 16, 52, 58, 18, and 56.³⁶ The three most prevalent HPV genotypes in China are types 16, 52, and 58.^28,29 In this study, the six most prevalent HPV genotypes were 52, 16, 53, 58, 51, and 56, in decreasing order, which accounted for 69.6% of all infections and 93.8% of infections with HR-HPV types. The three most prevalent LR-HPV types were types 42, 81, and 43. The genotype distribution pattern was consistent with that of other population-based studies conducted in China.²³ HPV-53 is not common in Asia, but HPV-53 was the third most prevalent type in our study, suggesting a unique distribution of HR-HPV among women in Xi’an. A hospital‐based investigation revealed that the most common HR‐HPV genotypes in Xi’an were types 16, 58, 52, 18, and 31,³¹ and another study found that types 6, 11, 52, and 16 were the four most common HPV types among patients with genital warts in Xi’an.³⁷ This study revealed that the genotype distribution pattern among the general population of Xi’an was significantly different from that among gynecological patients.

HPV vaccination can effectively prevent HPV infection.³⁸ At present, three different prophylactic HPV vaccines, including a bivalent vaccine targeting HPV-16, and −18, quadrivalent vaccine targeting HPV-6, −11, −16, and −18, and nonovalent vaccines targeting HPV-6, −11, −16, −18, −31, −33, −45, −52, and −58, have been used to prevent HPV infection and decrease the risk of cervical cancer. Both bivalent and quadrivalent vaccines protect against HPV-16 and HPV-18, which cause approximately 70% of cases of cervical cancer. The quadrivalent vaccine can also protect against HPV-6 and HPV-11, which cause approximately 90% of cases of genital warts.³⁹ Gardasil 9, a nonovalent HPV vaccine, has been available in China since 2018.¹⁵ Compared with bivalent and quadrivalent HPV vaccines, the nonovalent HPV vaccine offers a broader coverage and is 90% effective at preventing cervical cancer.⁴⁰ Although the distribution of HPV genotypes significantly varies regionally, no HPV vaccines have been developed that are customized to the specific regional distribution of HPV genotypes. Therefore, investigating the spectrum of region-specific HPV genotypes is essential for HPV vaccine development and effective HPV prevention. According to our results, the nonovalent HPV vaccine cannot provide adequate protection for women in Xi’an. Most importantly, HPV-53 was the third most prevalent type in Xi’an, but the nonovalent vaccine does not cover HPV-53. Therefore, the genotype spectrum of the next-generation of prophylactic HPV vaccines should include other common HPV genotypes such as HPV-51, −53, and −56, to efficiently and specifically protect against HR-HPV infection.

This study had several limitations. First, women aged 20–29 years were not included in our study, which may have led to a bias in the prevalence and genotype distribution of HPV infection. HPV infections are extremely common among young women but rarely cause persistent infections because HPV can be easily cleared. Given that the characteristics of HPV infection in the age group <30 years, China has set HPV testing to be carried out in women over 30 years. The initial screening age is set after the peak prevalence of transient HPV infection to effectively avoid an excessively high detection rate of transient HPV infection. As women <30 years are not eligible to attend the screening program for free, we cannot obtain relevant data on HPV infection in the age group. Therefore, this study does not completely represent the overall epidemiology of HPV infection among women in Xi’an. Second, the study duration was insufficient to assess the secular trend in HPV infection. Third, the number of participants in this study was limited. Wider population coverage of cervical cancer screening is essential for preventing cervical cancer. HPV self‐sampling is an effective approach for increasing screening coverage.⁴¹ Providing devices for self-sampling can expand the participation of hard-to-reach women. Finally, detailed information about cervical cytology was not available to evaluate the prognosis of women with HPV infection. In the future, a larger population-based study should be conducted to investigate the secular trend in the HPV prevalence and HPV genotype distribution and the relationship between HPV DNA and cytology.

Conclusions

We conducted a population-based study to investigate the molecular epidemiology of HPV infection in Xi’an. The HPV prevalence showed a significant variation according to age and district. The six most prevalent HR-HPV genotypes were HPV-52, −16, −53, −58, −51, and −56. The results obtained from this study provide a reference for guiding cervical cancer screening and HPV vaccination programs in Xi’an, which are an essential component of public health policies for cervical cancer prevention.

Authors’ contributions

XH and YH conceived and designed the research; XH, GS, YL, ZD, SW, HT, and XW conducted the research; XH, GS, YL, and CL checked and analyzed the data; XH and XY performed statistical analysis; XH wrote the paper; YH had primary responsibility for final content.

Acknowledgments

We would like to thank all the study participants, investigators and the contribution of our coworkers from Xi’an Maternal and Child Health Care Hospital. We also would like to thank Editage for English language editing.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by a grant from Xi’an Health Commission [grant number 2020yb40].