Incidence, persistence, and clearance of cervical human papillomavirus infection among gynecological outpatients in Kunming, Yunnan, China, 2019–2023: a retrospective cohort study

Study population

Data were collected from women who visited the gynecological clinic of the First People’s Hospital of Yunnan Province between October 2019 and August 2023 and were advised to undergo routine HPV testing. Women were eligible if they met the following criteria: (i) attended the hospital’s gynecological clinic; (ii) provided cervical brush samples for HPV DNA testing; and (iii) were aged 15 years or older. Patients were excluded if they met any of the following criteria: (i) male sex; (ii) missing relevant clinical data; and (iii) age younger than 15 years. This study was approved by the ethics committee of the First People’s Hospital of Yunnan Province (No. KHLL2023-KY068). All study procedures complied with the Declaration of Helsinki, and written informed consent was obtained from all participants prior to study initiation, in accordance with the requirements of the research ethics committee. In this study, the terms 2vHPV, 4vHPV, and 9vHPV refer to HPV genotype groups covered by the bivalent, quadrivalent, and nonavalent HPV vaccines, respectively (as licensed in China). These terms are used solely for categorizing and analyzing the prevalence and distribution of HPV types according to their vaccine coverage, rather than to indicate participants’ vaccination status or to classify patients into groups. The following variables were collected: patient ID, date of HPV testing, age at HPV testing, and HPV type results. All data were de-identified prior to analysis. Details are presented in Fig. 1. Portions of this text were previously published as part of a preprint (https://www.researchsquare.com/article/rs-4606669/v1).

Cervical sample collection

Vaginal administration and irrigation were prohibited for three days prior to sampling, and vaginal intercourse was prohibited for twenty-four hours. Using a cervical brush, exfoliated cervical cells were collected and stored at 4 °C in sterile sample tubes containing cell preservation solution (HEALTH Gene Technologies Co., Ltd., Ningbo, China).

DNA extraction

DNA was extracted from the sample within 24 h of collection using a nucleic acid extraction kit (HEALTH Gene Technologies Co., Ltd., Ningbo, China) according to the manufacturer’s instructions. Samples containing cervical exfoliated cells in preservation solution were vortex-mixed, transferred to a 1.5 mL sterile tube, and centrifuged at 12,000 g for 5 min. The supernatant was discarded, and the cell pellet was washed with 500 µL of PBS to remove residual contaminants such as blood or mucus. Finally, the pellet was lysed for 15 min at 100 °C in 200 µL of 5% Chelex-100 chelating resin, followed by centrifugation at 12,000 g for 5 min.

HPV testing and genotyping

HPV genotyping was performed using an HPV Genotyping Kit (HEALTH Gene Technologies Co., Ltd., Ningbo, China) according to the manufacturer’s instructions. Specific primers targeting the human β-globin locus, plasmid pcDNA 3.1(+) and the early HPV genes E6, E7, and E1 were designed. Polymerase chain reaction (PCR) amplification was performed in a Veriti heat cycler (Applied Biosystems, Foster City, CA, USA) using the following program: 55 min at 42 °C, pre-denaturation for 8 min at 94 °C, 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 70 °C for 1 min, followed by a final 1-minute extension at 70 °C. The reaction mixture contained DNA template (9 µL), PCR Master Mix (9 µL), and Taq DNA polymerase (2 µL). PCR amplicon was subsequently analyzed using a 3500DX Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), which identifies 25 HPV subtypes by capillary electrophoresis based on product length in a single run. These subtypes include 15 HR-HPVs (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82), three probable HR-HPVs (26, 53, and 66), and seven LR-HPVs (6, 11, 42, 43, 44, 81, and 83). To exclude false-negative results due to insufficient sample, amplification of pcDNA and human β-globin was performed as internal controls to monitor the PCR process.

Statistical analysis

Participants who underwent HPV retesting within 24 months of their initial test were included to compute the incidence, persistence, and clearance rates. The 24-months follow-up was divided into four intervals: 0–6, 6–12, 12–18, and 18–24 months after the initial test.

For incident infection, the denominator was the number of individuals who tested negative for HPV at baseline and had a follow-up test within two years, and the numerator was the number who tested positive for any HPV subtype at the second test.

Persistence rates were calculated as the number of individuals who tested positive for the same HPV subtype at both baseline and the follow-up test (numerator) divided by the total number who were positive at baseline and retested within two years (denominator).

Clearance rates were calculated as the number of individuals who tested negative for the same HPV type at follow-up (numerator) divided by the number who tested positive for that type at baseline and were retested within 24 months (denominator). The statistical software SPSS, version 26.0 (SPSS, Inc., Chicago, IL, USA), was used for all statistical analyses, and a P < 0.05 was considered statistically significant.

Overall characteristics of women with multiple HPV results among gynecological outpatients

A total of 45,149 individuals who met the inclusion criteria were analyzed to examine the prevalence of HPV infection in the region. Of these, 10.01% (n = 4,521) underwent two or more HPV tests within a 24-month period (Fig. 1). Among those who underwent multiple HPV tests, 76.97% (n = 3,272) had exactly two tests, with the number of tests per woman ranging from one to eight. The mean age was 40.09 ± 10.30 years. Participants were stratified into the following age categories: <30, 30–39, 40–49, 50–59, and ≥60 years, which accounted for 15.86%, 34.97%, 30.55%, 15.00%, and 3.63% of the cohort, respectively. Among those who underwent HPV retesting, 39.50% had a single infection and 16.55% had multiple infections (Table 1). Among individuals who retested positive for HPV, 42.00% tested positive for HR-HPV, and 14.05% for LR-HPV (Table 1).

Overall characteristics of HPV incidence

Among women who underwent repeat HPV testing within a 24-month period, the overall incidence rate of any HPV, 2vHPV, 4vHPV, and 9vHPV were 36.84% (95% confidential interval (CI) [34.72%–38.96%]), 4.43% (95% CI [3.52%–5.33%]), 5.64% (95% CI [4.62%–6.65%]), and 18.42% (95% CI [16.71%–20.13%]), respectively (Table 2). The majority of incident infections occurred in the 30–39 and 40–49 age groups. The five most frequently detected incident HPV subtypes were HPV 52, 51, 81, 58, and 16 (Fig. 2A). Among HR-HPV, the subtypes with the highest incidence rates were HPV 52 (5.59%), HPV 51 (3.72%), and HPV 58 (3.37%). Among LR-HPVs, HPV 81 (3.47%), HPV 43 (2.67%), and HPV 42 (2.52%) were the most prevalent (Fig. 2A). The incidence rates of any HPV infection were 13.14%, 15.60%, 8.76%, and 11.83% among women retested at 0–6, 6–12, 12–18, and 18–24 months, respectively (Fig. 3). HPV 52, 51, 58, and 53 were the most frequently detected subtypes during the 0–6 and 6–12 month intervals. Longer screening intervals were associated with higher incidence rates of HPV 16 and 39. Across all follow-up intervals, the most prevalent LR-HPV subtypes were HPV 81, 42, 43, and 44. A progressive increase in the incidence of HPV 42 was observed with longer retesting intervals, suggesting that HPV 42 may require more time for natural clearance.

Overall characteristics of HPV persistence

Among women who underwent repeat HPV testing within a 24-month period, the overall persistent rates of any HPV, 2vHPV, 4vHPV, and 9vHPV were 55.56% (95% CI [53.62%–57.50%]), 7.10% (95% CI [6.10%–8.10%]), 7.93% (95% CI [6.88%–8.98%]), and 28.57% (95% CI [26.81%–30.33%]), respectively (Table 3). The persistence rates in the <30, 30–39, 40–49, 50–59, and ≥60 years age groups were 56.60%, 49.94%, 49.43%, 69.23%, and 78.99%, respectively, demonstrating a pronounced age-related increase in persistent HPV infection. The five most frequently persistent HPV subtypes were HPV 42, 52, 58, 81, and 56 (Fig. 1B). Among HR-HPVs, the subtypes with the highest persistence rates were HPV 52 (47.83%), 58 (46.88%), and 56 (41.18%). Among LR-HPVs, HPV 42 (51.82%), 81 (45.65%), and 44 (32.93%) had the highest persistence rates (Fig. 1B). The persistent rates of any type of HPV were 12.57%, 13.26%, 6.62%, and 6.37% among women retested at 0–6, 6–12, 12–18, and 18–24 months, respectively (Fig. 4). Across the different time intervals, persistent infections were more prolonged for HPV 58 and 52 among HR-HPVs, and for HPV 42 and 81 among LR-HPVs. Among patients who tested positive for HPV at the first screening and were found to carry different HPV genotypes at the second screening (233, 9.19%), dynamic changes in genotype profiles were frequently observed (Table S1). Overall, 136 (58.37%) showed co-infection with new genotypes while retaining at least one of their baseline types, whereas 97 (41.63%) experienced complete genotype replacement, with all baseline types cleared and only new genotypes detected at follow-up. Within the co-infection group, 90 (38.63%) transitioned from single-type infection at baseline to multiple-type infection at follow-up. HR-HPV accounted for the majority of newly detected genotypes, particularly genotypes HPV 52 and 58 (Fig. S1).

Overall characteristics of HPV clearance

Among women who underwent repeat HPV testing within a 24-month period, the overall clearance rates for any HPV, 2vHPV, 4vHPV, and 9vHPV were 74.43% (95% CI [72.73%–76.13%]), 13.46% (95% CI [12.13%–14.79]), 17.68% (95% CI [16.19%–19.17%]), and 44.44% (95% CI [42.50%–46.37%]), respectively (Table 4). The clearance rates among HPV-positive individuals in the <30, 30–39, 40–49, 50–59, and ≥60 age groups were 77.87%, 76.66%, 75.64%, 67.55%, and 62.18%, respectively. HPV clearance rates showed a progressive decline with increasing age. The five HPV subtypes most likely to be cleared were HPV 26, 83, 11, 82, and 44 (Fig. 1B). Among HR-HPVs, the subtypes with the lowest clearance rates were HPV 58 (53.66%), 52 (53.99%), and 35 (62.50%), while for LR-HPVs, the lowest clearance rates were observed for HPV 42 (56.93%), 81 (62.50%), and 43 (85.15%). The majority of HPV infections were cleared rapidly, with approximately two-thirds resolving within the first year (Fig. 5).