Significance of Genotype-Specific High-Risk Human Papillomavirus Testing in Cervical Cancer Screening: A Hospital-Based Study.

Eligible participants were women who screened positive for hrHPV, had no prior diagnosis of cervical cancer or cervical intraepithelial neoplasia, and were not immunocompromised. Cell samples were collected using a cervical broom, preserved in liquid preparation, and analyzed with the Anyplex II HPV HR detection assay (Seegene Inc., Seoul, Korea). Specimen adequacy was confirmed by a human beta‐globin assay. This simultaneous multiplex real‐time polymerase chain reaction detects 14 hrHPV genotypes, including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68. Colposcopy referral followed the 2019 American Society for Colposcopy and Cervical Pathology recommendations. Women positive for HPV16/18, with or without reflex cytology, were referred, as were those positive for non‐16/18 hrHPV plus any abnormal cytology. Repeat hrHPV positivity with negative cytology (negative for intraepithelial lesion or malignancy [NILM]) was also referred. Colposcopy was performed by gynecologic oncologists or fellows under supervision. Diagnostic procedures, including biopsy, conization, or subsequent hysterectomy, were conducted as indicated. The most severe pathology was used as the reference standard for histopathological diagnosis. All pathology was evaluated and reported by a team of gynecologic pathologists.

All authors contributed to the study design and conceptualization. S.B., N.V., A.P., and S.A. performed participant enrollment. S.B., W.K., and I.R. evaluated all medical records for data retrieval, analysis, and interpretation. S.B. and W.K. drafted the manuscript. I.R. revised the manuscript for critical intellectual content. W.K. and I.R. were essential intellectual contributors. All authors edited and approved the final version of the manuscript.

Clinical characteristics, presenting symptoms, hrHPV testing and cervical cytology results, diagnostic procedures, and histopathological findings are summarized in Table  1 . A total of 1981 medical records were analyzed. The median age was 40 years (IQR 32.0‒49.0), and the median parity was 1 (IQR 0‒2). Most women (77.6%) were premenopausal. A total of 1394 women (70.4%) had no underlying disease. Previous screening tests had been performed in 1223 women (61.7%) before the index visit. Of the entire cohort, 1215 women (61.3%) had prior cytology tests, and 1087 (5.4%) had prior hrHPV tests. Clinical characteristics of the 1981 women included in the study. Note: Data are presented as number and percentage unless otherwise indicated. Abbreviations: AGC‐NOS, atypical glandular cells‐not otherwise specified; AIS, adenocarcinoma in situ; ASC‐H, atypical squamous cells, cannot exclude high‐grade lesion; ASC‐US, atypical squamous cells of undetermined significance; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; HSIL, high‐grade squamous intraepithelial lesion; LSIL, low‐grade squamous intraepithelial lesion; NILM, negative for intraepithelial lesion or malignancy. At index testing, 1480 women (74.7%) harbored a single HPV genotype. Co‐infections involving two genotypes were detected in 371 women, three genotypes in 109 women, four genotypes in 18 women, and five genotypes in three women. The three most common genotypes were HPV52 (23.7%), HPV16 (15.6%), and HPV58 (15.4%). The 9‐valent HPV vaccine would have fully covered the hrHPV genotypes detected in 954 women (48.2%) and partially covered those in 306 women (15.4%). Of the 1981 women in the study population, 1071 were advised to undergo follow‐up, and 910 underwent immediate colposcopy. Some women subsequently underwent loop electrosurgical excision procedure or hysterectomy, as indicated. Hysterectomy was performed for CIN2 + histopathology when free margins could not be achieved through re‐excision, for cervical cancer, or for other benign conditions diagnosed within 3 months of the index test (e.g., uterine fibroids or adenomyosis). CIN2 + lesions were identified in 152 women (7.7%), comprising 130 cases of CIN2/CIN3/AIS and 22 cases of cancer (Table  2 ). Histopathological diagnoses and distribution of high‐risk HPV genotypes in CIN2 + cases. Note: Data are presented as number and percentage. Abbreviations: ADA, adenocarcinoma; AIS, adenocarcinoma in situ; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; SCCA, squamous cell carcinoma; SCNE, small cell neuroendocrine carcinoma. The association between clinical characteristics, HPV genotypes, and histopathological CIN2 + findings is shown in Table  3 . Detection of HPV16 and multiparity were significantly associated with an increased risk of CIN2 + , with odds ratios (OR) of 4.534 (95% CI 3.197‒6.430) and 1.497 (95% CI 1.070‒2.094), respectively. Women with HPV16, HPV35, or HPV18 had an immediate CIN2 + risk exceeding 11%. Those infected with HPV52, HPV51, HPV58, HPV45, HPV33, or HPV59 had an immediate CIN2 + risk ranging from 5% to 9%. The immediate risk of CIN2 + in women infected with HPV31, HPV39, HPV56, HPV66, or HPV68 was below 4%. Association between clinical and virological factors and CIN2 + histopathology. Note: Data are presented as number, percentage, odds ratio, and 95% confidence interval. CIN2 + includes CIN2, CIN3, AIS, and carcinoma. Abbreviations: CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus.

Primary screening using hrHPV testing, whether through clinician‐collected or self‐collected methods, has gained worldwide acceptance [ 5 , 6 , 15 , 16 ]. The rate of positive hrHPV results in primary screening has been reported to range from 4.6% to 15.3% [ 4 , 11 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. It depends on geographic variations and the type of tests used. Among those who tested positive, the immediate detection rate of CIN2 + ranged from 4.3% to 18.9% [ 4 , 17 , 18 , 19 , 21 , 22 , 24 ]. The findings of the current study are consistent with these published data. Effective triage tests are essential for optimizing colposcopy referral and enhancing the detection of cervical precancer. Three triage strategies are recommended: partial genotyping for HPV16/18, reflex cytology, or dual staining with a history of hrHPV or CIN2 + detection [ 2 ]. Extended HPV genotyping has been investigated further for refined risk stratification. The Becton Dickinson (BD) Onclarity and Improving Primary Screening and Colposcopy Triage (IMPACT) cervical cancer screening trials used receiver operating characteristic (ROC) analysis to identify an optimal cutoff for balancing sensitivity and specificity in CIN3 + detection. This threshold lay between women who tested positive for HPV18/31 but negative for HPV16, and those who tested positive for HPV33/52/58 with NILM, excluding HPV16/18/31 [ 11 ]. Data from the New Technologies for Cervical Cancer Screening 2 (NTCC2) trial highlighted three risk groups for CIN3 + based on hrHPV status [ 12 ]. The highest‐risk group requiring immediate colposcopy included women positive for HPV16/18, those with high‐grade cytology (e.g., atypical squamous cells cannot exclude HSIL [ASC‐H], HSIL, adenocarcinoma in situ [AIS]), or those who were dual staining‐positive but negative for HPV35/39/51/56/59/66/68. A 1‐year follow‐up for hrHPV testing was advised for individuals with HPV16/18 but negative dual staining, those with HPV31/33/45/52/58 and NILM or negative dual staining, and those with HPV35/39/51/56/59/66/68 plus low‐grade cytology (NILM, atypical squamous cells of undetermined significance [ASC‐US], or atypical glandular cells [AGC]) or positive dual staining. A 3‐year follow‐up was recommended for women infected with HPV35/39/51/56/59/66/68 who had NILM or negative dual staining. Geographic variations in HPV genotype distribution have been noted in screening results. The ATHENA trial reported that the most prevalent hrHPV genotypes were HPV16 (1.6%), HPV52 (1.0%), HPV51 (0.8%), HPV18 (0.7%), HPV39 (0.7%), and HPV58 (0.7%) [ 19 ]. In Greece, screening of 30 445 women identified HPV16 (2.5%), HPV31 (2.3%), HPV68 (1.9%), HPV52 (1.4%), and HPV66 (1.4%) as the most common hrHPV genotypes [ 20 ]. Among Chinese women, the five most prevalent genotypes were HPV52 (3.45%), HPV16 (3.43%), HPV58 (2.94%), HPV68 (2.07%), and HPV51 (1.94%) [ 4 ]. The present findings are similar, as HPV52, HPV16, and HPV58 were the most commonly detected genotypes, consistent with trends seen in Chinese populations. Regional differences have also been observed in CIN2 + histopathology. Among 2703 Chinese women who tested positive for hrHPV, 117 (4.3%) were diagnosed with CIN2 + . The most prevalent hrHPV genotypes in these CIN2 + cases were HPV16 (57.3%), HPV58 (16.2%), HPV52 (15.3%), HPV31 (11.1%), and HPV18 (9.4%) [ 4 ]. Another study examining 440 women with histologically confirmed CIN2 + found HPV16 (63.4%), HPV58 (15.0%), HPV52 (10.3%), HPV33 (8.8%), and HPV18 (7.9%) to be the most frequent [ 24 ]. The current study likewise identified HPV16 as the most commonly detected hrHPV genotype among women with CIN2 + . Various hrHPV genotypes differ in their potential to induce carcinogenesis. Huh et al. reported that the cumulative 10‐year risk of CIN3+ was 15%‒20% for HPV16, 3% for HPV18, and 1% for other hrHPV types [ 25 ]. In the Population Based Screening Study Amsterdam (POBASCAM), single infections of HPV16 (OR 4.10, 95% CI 2.98‒5.64), HPV33 (OR 2.68, 95% CI 1.39‒5.15), and HPV58 (OR 2.15, 95% CI 1.04‒4.43) significantly increased the risk of CIN2 + . The positive predictive values (PPVs) for CIN2 + of HPV16, HPV33, HPV18, and HPV31 were 20.8%, 14.4%, 7.4%, and 7.1%, respectively [ 18 , 26 ]. The ATHENA trial reported PPVs for CIN2 + of 21.9% for HPV16, 19.7% for HPV31, 12.2% for HPV33, 10.1% for HPV18, 9.6% for HPV35, and 9.1% for HPV52 [ 19 ]. At KPNC, the PPVs for HPV16, HPV31, HPV18, and HPV33 were 16.7%, 10.2%, 9.4%, and 8.9%, respectively [ 26 ]. Another study reported that HPV16, HPV33, HPV35, HPV58, HPV31, and HPV68 had PPVs for CIN2 + of 27.4%, 27.0%, 26.5%, 24.4%, 23.6%, and 22.4%, respectively [ 24 ]. In the present study, the PPVs for CIN2 + of HPV16, HPV35, HPV18, HPV52, HPV51, HPV58, and HPV33 were 20.4%, 12.0%, 11.9%, 8.9%, 7.6%, 7.2%, and 6.5%, respectively. These findings underscore the importance of incorporating extended hrHPV genotyping into screening protocols. Based on the current study, women with HPV16, HPV35, and HPV18 had an immediate CIN2 + risk exceeding 11%. Previous studies have highlighted the high oncogenic potential of certain hrHPV genotypes following HPV16 and HPV18. Considering these findings, the threshold for colposcopy referral may warrant expansion to include additional genotypes. Elevated risks have been reported for HPV31 [ 19 , 21 ], HPV33 [ 3 , 18 , 24 ], HPV45 [ 21 ], and HPV35—particularly among African [ 2 ] and Thai women. Additionally, other genotypes such as HPV52, HPV51, HPV58, HPV45, HPV33, and HPV59 were associated with an immediate CIN2 + risk ranging from greater than 5%–9%. These findings highlight the importance of implementing effective triage strategies for women with these genotypes. The present study also demonstrated that the immediate risk of CIN2 + in women with HPV31, HPV39, HPV56, HPV66, and HPV68 was relatively low. Notably, HPV66 is no longer classified as a high‐risk type according to the latest International Agency for Research on Cancer evaluation [ 1 ]. These results support extending re‐screening intervals to 3–5 years for HPV66. In addition, HPV56 and HPV68 have been reclassified by IARC as lower‐risk hrHPV types, suggesting a 1‐year re‐screening interval for these infections. This approach aligns with the 2025 American Society for Colposcopy and Cervical Pathology guidelines, which recommend annual follow‐up HPV testing for women positive for HPV56/59/66 in the absence of high‐grade cytologic abnormalities, no history of CIN2+ within the past year, and no persistent hrHPV infection [ 2 ]. In cervical cancer screening settings, single hrHPV infections are detected more frequently than multiple hrHPV genotype infections. The reported prevalence of multiple hrHPV infections ranges from 14.0% to 39.8%, which aligns with the current findings [ 18 , 19 , 21 , 24 ]. The impact of multiple hrHPV infections on carcinogenesis and their association with increased CIN2 + risk remains controversial. One study reported that multiple hrHPV infections were significantly associated with a higher risk of persistent infection (30.5%) compared to single hrHPV infections (16.6%, p  <  0.001) [ 21 ]. In contrast, another study found no significant difference in the risk of CIN2 + between single and multiple hrHPV infections, regardless of the number of genotypes involved [ 24 ]. These findings are consistent with results from the present study. A key strength of this study is that it not only evaluated hrHPV genotyping but also retrieved subsequent histopathological findings, all interpreted by specialized gynecologic pathologists at a single institution. Nevertheless, the study's retrospective design led to some missing data due to incomplete medical records. Information on participants' HPV vaccination history was unavailable. In addition, follow‐up data from later screening rounds were not analyzed. Future studies should focus on population‐based or nationwide analyses across diverse regions, particularly in low‐resource settings where the burden of cervical cancer remains high. Long‐term follow‐up is essential to assess outcomes from subsequent screening rounds, including HPV clearance rates, re‐infection rates, and the significance of genotype‐specific persistence or type‐switching infections. These data will be critical for accurately estimating the absolute risk of CIN2+ by HPV genotype and for informing the development of effective HPV‐based screening algorithms. Viral and host factors associated with later pathological diagnoses should also be explored. Furthermore, the cost‐effectiveness of extended hrHPV genotyping and its potential triage strategies warrants investigation. In conclusion, incorporating genotype‐specific hrHPV testing into healthcare systems can improve risk stratification, reduce unnecessary colposcopy referrals, and enhance patient care.

Data were analyzed using IBM SPSS Statistics, version 29 (IBM Corp, Armonk, NY, USA). Categorical data were reported as numbers and percentages. Continuous data were reported as mean (SD) or median (IQR). Between‐group comparisons were performed using the χ 2 test or Fisher′s exact test, as appropriate. A p value less than 0.05 was considered statistically significant.

Cervical microtrauma and infection with high‐risk human papillomavirus (hrHPV) are major causes of cervical neoplasia. Recently, 13 HPV genotypes were recognized as oncogenic and were categorized into four risk groups [ 1 ]. HPV16 is classified as the “highest” risk genotype. HPV18 and HPV45 are considered “high” risk. HPV31, HPV33, HPV35, HPV52, and HPV58 are categorized as “medium” risk, and HPV39, HPV51, HPV56, HPV59, and HPV68 are labeled “lower” risk. Geographic variations exist in hrHPV genotype distribution, and these differences influence the risk of cervical intraepithelial neoplasia grade 2 or worse (CIN2 + ) [ 2 ]. For instance, HPV35 is more prevalent among African women and has been linked to a higher risk of cervical cancer. At Kaiser Permanente Northern California (KPNC), women infected with HPV16 and HPV33 had the highest 7‐year cumulative risk of progression to CIN3 + , reported at 21.5% and 18.4%, respectively [ 3 ]. In the Shanxi Province Cervical Cancer Screening Study (SPOCCS), the most commonly detected hrHPV genotypes were HPV52, HPV16, HPV58, HPV68, and HPV51. However, among women diagnosed with CIN2 + , the most prevalent hrHPV genotypes were HPV16, HPV58, HPV52, HPV33, and HPV31 [ 4 ]. Primary hrHPV testing is globally recognized for its high sensitivity in cervical screening. It significantly increases the detection of cervical neoplasia and is recommended in multiple clinical guidelines [ 5 , 6 , 7 ]. Optimizing the detection of CIN2 + while reducing unnecessary colposcopy referrals requires robust triage strategies. Currently recommended triage methods for hrHPV‐positive women include partial genotyping for HPV16/18, reflex cytology, or p16/Ki67 dual staining [ 5 , 7 , 8 , 9 , 10 ]. Other triage approaches, including host or viral methylation assays, have also been investigated. Some studies suggest the potential value of extended HPV genotyping; however, no specific guidelines exist for managing women who test positive for individual hrHPV genotypes [ 2 , 3 , 11 , 12 ]. Traditionally, primary screening in Thailand relied on conventional and liquid‐based cytology. When abnormal cytology results occur, the prevalence of CIN2 + pathology tends to be higher than in Western countries [ 13 ]. Primary hrHPV testing was implemented in Thailand in 2020 [ 14 ]. The primary objective of this study was to investigate histopathological outcomes among women who tested positive for hrHPV. The secondary objective was to assess the significance of each hrHPV genotype.

The authors declare no conflicts of interest.

This retrospective study reviewed medical records from a single institution. The Siriraj Institutional Review Board approved the study protocol (COA no. 033/2024). The study was conducted in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.