A cervical cancer HPV non-invasive shunt screening method based on high negative predictive value

Abstract

This invention discloses a non-invasive triage screening method for cervical cancer HPV based on high negative predictive value. The steps include non-invasive sampling, total DNA extraction, HPV detection, and triage interpretation. This invention relies on the non-invasive acquisition of vulvar exfoliated cells and fluorescence PCR detection of hrHPV to construct a multidimensional clinical lesion risk prediction and decision stratification system integrating subject age stratification, HPV molecular typing, and previous HPV treatment history. Based on large-scale paired sample cohort validation, this invention demonstrates high consistency between vulvar and cervical HPV detection. It is the first to propose a triage screening strategy based on high negative predictive value (NPV≥95%), and by introducing age stratification interpretation rules and treatment history correction mechanisms, it achieves accurate screening decision support for women of different ages, physiological states, and medical histories.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a non-invasive triage screening method for cervical cancer HPV based on high negative predictive value. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors in women worldwide, and persistent infection with high-risk HPV (hrHPV) is a major cause of cervical cancer and precancerous lesions (CIN). Early detection of HPV infection status is crucial for timely identification of precancerous lesions and prevention of disease progression. Clinically, there is an urgent need for a low-cost, easy-to-use, highly sensitive, and specific detection method to achieve timely detection of early epithelial mucosal lesions and accurate prediction of the risk of cervical cancer progression.

[0003] Currently, cervical cytology, widely used in clinical practice, has inherent limitations such as low sensitivity and poor repeatability. While molecular testing based on HPV nucleic acid is gradually becoming an important supplement, it still faces many challenges, including high cost, complex operation, and low positive predictive value for CIN. Furthermore, existing methods struggle to effectively differentiate between low-grade and high-grade pathological changes, and also fail to distinguish between carcinogenic and non-carcinogenic viral infections. Additionally, traditional cervical sampling requires exposure of the cervix using a vaginal speculum, which is an invasive procedure, causing significant discomfort or psychological resistance in some participants, leading to poor screening compliance.

[0004] To overcome the aforementioned limitations, self-sampling strategies have received widespread attention in recent years. While vaginal self-sampling can improve screening compliance to some extent, the sampler still needs to be inserted into the vagina, which can cause discomfort or psychological barriers for some women; furthermore, improper operation may damage the mucosa, with the risk of iatrogenic injury being particularly prominent in postmenopausal women whose tissue fragility has increased. In contrast, vulvar sampling is a truly non-invasive method, requiring only gentle rotation of the vulvar skin surface for sampling, completely avoiding instrument insertion and mucosal damage, and theoretically achieving near 100% subject acceptance. However, whether vulvar sampling can reliably reflect cervical HPV infection status still lacks large-scale clinical consensus evidence, and its clinical value needs further validation.

[0005] Besides sampling methods, age-related screening challenges are equally prominent. Postmenopausal and advanced maternal age (≥40 years, especially ≥50 years) women experience a decline in estrogen levels, leading to cervical atrophy and squamocolumnar junction retraction. Traditional cervical sampling often fails to obtain sufficient exfoliated cells, significantly increasing the risk of false negatives. This group is precisely the high-risk population for cervical cancer, and current screening methods are significantly less effective in protecting them. Furthermore, women who have previously undergone cervical conization or physical therapy are at extremely high risk of recurrence due to altered cervical anatomy, and current screening methods lack specific risk correction mechanisms for them.

[0006] In summary, existing cervical cancer screening technologies have significant shortcomings in terms of clinical compliance, applicability to the elderly, and triage accuracy, and urgently need improvement. Summary of the Invention

[0007] Purpose of the invention: The present invention aims to provide a completely non-invasive HPV detection method that is not limited by age or cervical anatomy and can integrate HPV molecular typing and clinical history for precise stratification of cervical cancer risk.

[0008] Technical solution: The present invention provides a non-invasive triage screening method for cervical cancer HPV based on high negative predictive value, comprising the following steps:

[0009] (1) Non-invasive collection of exfoliated cell samples from the vulva of the subjects;

[0010] (2) Extract total DNA from the sample and use human β-globin gene as an internal reference for PCR quality control;

[0011] (3) HPV detection was performed using fluorescent PCR.

[0012] (4) Stratification and triage interpretation are carried out based on the test results and the clinical information of the subjects.

[0013] The hierarchical flow division and interpretation rules in step (4) are as follows:

[0014] If the hrHPV test result is negative and the subject's age is <40 years, then it is interpreted that no invasive cervical examination is required.

[0015] If the hrHPV test result is negative, the subject's age is ≥40 years and the β-globin Ct value is >30, then it is interpreted that resampling and testing or cervical cytology examination is required;

[0016] If the hrHPV test result is negative, the subject's age is ≥40 years and the β-globin Ct value is ≤30, then it is interpreted that a cervical cytology examination or a repeat HPV test after 12 months is required;

[0017] If the hrHPV test result is positive, it indicates that further evaluation or referral is required.

[0018] Furthermore, in step (4), subjects who are hrHPV positive are further stratified:

[0019] If HPV16 and / or HPV18 are detected, it is recommended to refer the patient for colposcopy and cervical biopsy.

[0020] If a non-HPV16 / 18 positive result is detected and the subject has a history of cervical treatment, it is recommended to shorten the follow-up interval to 6–12 months or to directly refer the subject for colposcopy.

[0021] If a positive result is found for non-HPV16 / 18 types and the subject has no history of cervical treatment, it is recommended to have a follow-up HPV test in 12 months.

[0022] Furthermore, the history of cervical treatment includes loop electrosurgical excision procedure (LEEP), cold knife conization, laser therapy, cryotherapy, cervical electrocautery, and any other form of physical destructive treatment of the cervix.

[0023] Furthermore, in step (3), the HPV testing includes both high-risk and low-risk HPV testing. The high-risk HPV types include HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, while the low-risk HPV types include HPV6 and 11. A Ct value < 37 for any high-risk subtype is considered hrHPV positive.

[0024] Furthermore, when the subject's age is ≥40 years, the clinical attention weight for positive / negative results of the vulva is increased in the interpretation of results to compensate for the risk of false negatives in cervical sampling that may be caused by cervical atrophy. Preferably, the increase in the clinical attention weight is 10% (i.e., a weighting coefficient of 1.1). The weighting coefficient is determined based on the fact that in the 40–49 age group, the detection rate of vulvar hrHPV (56.52%) is 10.87% higher than the detection rate of cervical hrHPV (45.65%), therefore the weight for interpreting positive / negative results of the vulva for subjects ≥40 years of age is increased by 10% accordingly.

[0025] Furthermore, in step (1), the sampling site for the exfoliated vulvar cell sample is the surface of the vulvar skin, including the labia majora, labia minora and the vaginal vestibule area.

[0026] Furthermore, the method is applicable to large-scale population screening.

[0027] Furthermore, the population includes women aged ≥40 years, postmenopausal women or women with known cervical atrophy, women with a history of cervical HPV treatment, and / or women who have a fear of traditional speculum examination or refuse to undergo invasive screening.

[0028] This invention provides a method for high-risk HPV detection and cervical lesion risk stratification based on non-invasive sampling of exfoliated vulvar cells. The method encompasses a complete technical chain from biological sample acquisition, molecular extraction quality control, targeted nucleic acid amplification to clinical decision support. Unlike traditional invasive vaginal speculum procedures, this invention achieves highly sensitive capture of HPV sequences by simply collecting exfoliated cells from the lower genital tract, thereby significantly improving the screening willingness and compliance of the test population.

[0029] This invention introduces a high negative predictive value (NPV) mechanism validated by large-scale cohort data, directly eliminating negative vulvar samples from healthy women, thereby significantly reducing unnecessary and invasive cervical biopsies and colposcopy referrals. Addressing the clinical challenge of high false-negative rates in routine screening among postmenopausal and older women due to cervical tissue atrophy and transformation zone invagination, this invention, through monitoring cell abundance internal reference genes, is the first to demonstrate that vulvar sampling has significantly superior molecular pathological sensitivity to cervical sampling in capturing occult viral infections in women aged 40 and above, and based on this, establishes targeted age-stratified interpretation rules.

[0030] Furthermore, this invention integrates specific HPV molecular typing (pathogenicity differences between HPV16 / 18 and other non-16 / 18 subtypes) and the subject's history of previous cervical physical destructive treatments to construct a dynamic error correction mechanism, ensuring the rigor, evidence-based nature, and individualized accuracy of molecular pathology diagnostic reports.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0032] 1. Extremely high negative predictive value: Based on validation of 719 clinical samples, the negative predictive value of vulvar hrHPV testing reaches 95.72%. A negative result can safely avoid more than 95% of invasive cervical procedures (colposcopy, biopsy), significantly reducing over-treatment and patient suffering.

[0033] 2. Solving the screening challenge for older women: For women aged ≥40 years, a sample quality assessment and age stratification interpretation rule based on β-globin internal reference is proposed for the first time, overcoming the false negatives of traditional sampling caused by cervical atrophy and improving the screening accuracy for this population;

[0034] 3. Precise Risk Stratification: Integrating HPV genotyping (HPV16 / 18 vs. non-16 / 18) and previous treatment history information clarifies the clinical management pathway for vulvar-cervical inconsistencies. For those with a history of treatment, a negative vulvar test cannot simply rule out cervical infection (V- / C+ group accounts for 93.75%). For those who are non-16 / 18 positive without a history of treatment, follow-up is recommended rather than immediate referral (V+ / C- group accounts for 91.67%) to avoid unnecessary medical intervention.

[0035] 4. Completely non-invasive and with high compliance: Vulvar sampling does not require a speculum and has no risk of mucosal damage, making it suitable for large-scale population screening, especially for postmenopausal women, those who are afraid of invasive examinations, and those in resource-scarce areas.

[0036] 5. Simple operation and controllable cost: Fluorescent PCR technology is mature and the reagent cost is lower than that of sequencing or hybridization capture methods, making it easy to promote and apply in primary healthcare institutions;

[0037] In summary, this invention, based on large-scale cohort verification of high consistency in vulvar-cervical HPV testing, proposes for the first time a triage screening strategy based on high negative predictive value (NPV≥95%), and introduces an age-stratified interpretation algorithm and a treatment history correction mechanism to provide accurate and efficient screening decision support for women of different ages, physiological states, and medical histories. Attached Figure Description

[0038] Figure 1 This is a flowchart of the research process (showing the inclusion, detection, and consistency analysis of 719 pairs of samples).

[0039] Figure 2 The distribution of HPV infection in vulvar and cervical samples in different age groups was shown in Figure A (positive rate of high-risk HPV (hrHPV) in each age group); Figure B (positive rate of low-risk HPV (lrHPV) and positive rate of mixed infection of hrHPV and lrHPV in each age group); Figure C (comparison of β-globin Ct values ​​in cervical samples from women <40 years old and ≥40 years old (mean ± standard error; <40 years old: 21.81 ± 0.056; ≥40 years old: 22.14 ± 0.0567; t test, P < 0.01)). Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0041] Example 1

[0042] This embodiment provides a non-invasive triage screening method for cervical cancer HPV based on high negative predictive value, the steps of which include: (1) non-invasive sampling; (2) extraction of total DNA and quality control; (3) HPV detection; and (4) interpretation and triage.

[0043] The method is as follows.

[0044] Step (1), Sample Collection: Using a disposable sterile sampling brush (brush head made of nylon or polyester fiber), rotate the brush 5 to 10 times on the surface of the subject's vulvar skin (including the labia majora, labia minora, and vaginal vestibule area) with moderate force to ensure that the brush head fully contacts the vulvar epithelial surface to collect exfoliated cells. The sampling process does not require the use of a speculum or insertion into the vagina; it is painless and non-invasive throughout. After sampling, place the brush head in a centrifuge tube containing cell preservation solution, seal it, label the subject's information, and store it temporarily at 4°C or send it directly for testing.

[0045] Step (2), Total DNA Extraction and Preservation: Take out the sample to be processed, vortex to mix, and transfer 1 ml of liquid to a 1.5 ml centrifuge tube. Centrifuge at 13,000 rpm for 5 minutes and discard the supernatant. (Note: If the sample contains too much blood, add 1 ml of physiological saline to the precipitate, mix well, centrifuge at 13,000 rpm for 5 minutes, and discard the supernatant). Add 50 μl of DNA extraction solution to the precipitate, mix thoroughly, and incubate at 100℃ for 10 minutes; centrifuge at 13,000 rpm for 10 minutes, and retain the supernatant (DNA sample) for later use. Add 50 μl of DNA extraction solution directly to the control tube of the kit, mix thoroughly, incubate at 100℃ for 10 minutes, centrifuge at 13,000 rpm for 10 minutes, and retain the supernatant for later use. It is recommended that DNA samples be used for PCR detection immediately; otherwise, store at 4℃. Samples not to be tested on the same day should be stored at -20℃ for no more than 30 days.

[0046] Step (3), HPV Nucleic Acid Detection: This embodiment uses fluorescent PCR technology to detect HPV. The kit used is the 14 hrHPV nucleic acid typing detection kit (fluorescent PCR method) produced by Jiangsu Mojin Biotechnology Co., Ltd. This kit can simultaneously detect the following 14 high-risk subtypes: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, and can report positive results for HPV16 and HPV18 separately. In addition, the low-risk HPV (6 / 11) nucleic acid detection kit from Wuhan Baitai Gene Engineering Co., Ltd. is used as an auxiliary detection.

[0047] PCR reaction system (total volume 25 μl): DNA template 2 μl, PCR reaction solution 22.4 μl, Taq enzyme 0.5 μl, UNG enzyme 0.1 μl. Each reaction plate included a positive control (containing HPV plasmid), a negative control (sterile water), and an internal control. PCR amplification and detection parameters are shown in Table 1.

[0048] Table 1 PCR amplification parameters

[0049]

[0050] Internal control quality control: The human β-globin gene is used as an internal control for sample cell abundance. β-globin primer and probe sequences are provided by the kit. The β-globin Ct value needs to be measured for each sample. Judgment criteria: If the β-globin Ct value is ≤30, it indicates sufficient sample cell quantity and reliable test results; if the Ct value is >30, it suggests insufficient sample cell quantity or low DNA extraction efficiency, and a message "Sample unqualified, resampling recommended" will be output.

[0051] Step (4), Result Interpretation: Determine the presence and genotype of hrHPV in the sample based on the PCR amplification curve and Ct value. A Ct value < 37 for any high-risk subtype is considered positive.

[0052] Example 2

[0053] This embodiment provides a triage and interpretation method based on age stratification and its application in large-scale population screening.

[0054] Based on validation data from 719 clinical samples (aged 14-71 years), the inventors found that in the <40-year-old age group, the NPV of vulvar hrHPV detection was 95.44%, with a concordance κ=0.827 compared to cervical detection; in the ≥40-year-old age group, the NPV of vulvar hrHPV detection was 97.78%, with a concordance κ=0.781, but the β-globin Ct value of cervical samples was significantly higher than that of the <40-year-old group (22.14±0.056 vs. 21.81±0.056, P<0.01), indicating that the cell count in cervical samples decreases with age, posing a risk of false negatives. The validation data are shown in Table 2 (subject demographics) and Table 3 (consistency analysis).

[0055]

[0056]

[0057] Based on the above findings, this embodiment provides the following age stratification interpretation method:

[0058] Step D1: Obtain the subject's age information (in years).

[0059] Step D2: If the hrHPV test result is negative and the subject's age is <40 years, the message "No invasive cervical examination (colposcopy or cervical biopsy) is output directly. This interpretation is based on the fact that the NPV rate in this population is 95.44%, and a negative vulvar result can safely rule out the vast majority of cervical hrHPV infections.

[0060] Step D3: If the hrHPV test result is negative, but the subject's age is ≥40 years, further evaluate the Ct value of β-globin in the vulvar sample or the cervical sample collected at the same time;

[0061] If the β-globin Ct value is ≤30, the output will be "Vulvar hrHPV negative, but age ≥40 years, it is recommended to combine cervical cytology examination or repeat HPV testing after 12 months";

[0062] If the β-globin Ct value is >30, the output will be "Insufficient sample cell count, cervical atrophy may lead to false negatives, it is recommended to resample or directly perform cervical cytology examination".

[0063] Step D4: If the hrHPV test result is positive, regardless of age, a prompt message "further evaluation or referral is required" will be output, and the process will proceed to the subtype analysis and treatment history correction process in Example 3.

[0064] Specific case: A subject, aged 45, had a negative hrHPV test result on a vulvar sample, with a β-globin Ct value of 25.3. The system output: "Vulvar hrHPV negative, but you are ≥40 years old. It is recommended to combine this with a cervical liquid-based thin-layer cytology test (TCT) or a follow-up HPV test in 12 months."

[0065] The above methods are suitable for large-scale population screening applications, and are particularly suitable for: women aged ≥40 years; postmenopausal women or women with known cervical atrophy; women with a history of cervical HPV treatment; and women who have a fear of traditional speculum examination or refuse to undergo invasive screening.

[0066] The screening process is as follows:

[0067] 1. Subjects signed informed consent forms;

[0068] 2. Vulvar exfoliated cells were collected using a vulvar sampling brush by a trained nurse or the subject themselves (self-sampling);

[0069] 3. Samples are sent to the laboratory for DNA extraction and fluorescent PCR detection.

[0070] 4. The detection system automatically outputs β-globin Ct value, hrHPV positive / negative status, and genotyping results;

[0071] 5. The system automatically executes the above interpretation rules and the correction process of Example 3 based on age and treatment history (obtained from electronic health records or self-reported by the subjects) to generate the final screening report;

[0072] 6. The report includes clear recommendations for next steps (such as "non-invasive examination", "follow-up examination in 12 months", "referral for colposcopy", etc.).

[0073] Example 3

[0074] This embodiment corrects the interpretation of HPV typing and past treatment history for positive hrHPV test results in the age stratification interpretation method of Embodiment 2.

[0075] 1. Specific handling of HPV16 / 18: If HPV16 or HPV18 subtypes (single or mixed infection) are detected in the vulvar sample, regardless of the subject's age and other factors, a direct prompt message of "recommendation for colposcopy and cervical biopsy" is issued. This is because HPV16 / 18 has the highest carcinogenic risk, and in this study, the vulvar-cervical concordance for HPV16 / 18 was extremely high (HPV16 κ=0.839, HPV18 κ=0.892), and a positive or negative vulvar result strongly suggests a positive cervical result.

[0076] 2. Management of hrHPV positive (non-HPV16 / 18): If a vulvar sample tests positive for hrHPV subtypes other than HPV16 / 18 (such as HPV31, 33, 52, 58, etc.), the subject's previous cervical treatment history will be further obtained. This previous cervical treatment history includes, but is not limited to: LEEP (Loop Electrosurgical Excision Procedure), cold knife conization, laser therapy, cryotherapy, cervical electrocautery, or any form of physical destructive treatment of the cervix.

[0077] (1) History of treatment: Output "You have previously received cervical HPV treatment, and your vulva test is currently positive for non-16 / 18 type hrHPV. It is recommended to shorten the follow-up interval to 6-12 months, or directly refer you for colposcopy." Basis for this interpretation: In this study, among the 16 samples with negative vulva / positive cervix (V- / C+), 15 cases (93.75%) had a history of HPV treatment, indicating that treatment history is an important risk factor for vulvar-cervical inconsistency.

[0078] (2) No history of treatment: Output "You tested positive for non-16 / 18 type hrHPV, but have no history of cervical treatment. It is recommended to retest for HPV in 12 months." The basis for this interpretation is: Among the 48 samples with positive external genitalia / negative cervical genitalia (V+ / C-), 91.67% were non-16 / 18 type, and most had no history of treatment, suggesting that such infections are mostly transient or local colonization, with a low risk of progression.

[0079] Specific Case: A subject, aged 32, tested positive for HPV52 (not 16 / 18) in a vulvar sample. The system inquired about her treatment history: she had undergone LEEP surgery two years prior. The system output: "You have previously received cervical treatment and are currently HPV52 positive. It is recommended that you have a follow-up HPV test in 6 months or be directly referred for colposcopy."

[0080] Example 4

[0081] This embodiment provides verification data from 719 pairs of samples on which the present invention is based, to demonstrate the technical effects of the present invention.

[0082] Study subjects: 719 female patients aged 14-71 years who visited the Department of Dermatology, Nanjing University School of Medicine Affiliated Gulou Hospital from June 2021 to May 2024.

[0083] Detection method: In the validation study, vulvar samples were tested according to the method in Example 1, and cervical samples were collected simultaneously for comparative testing.

[0084] result:

[0085] 1. The positive rate of vulvar hrHPV was 47.98% (345 / 719), and the positive rate of cervical hrHPV was 43.53% (313 / 719). The overall concordance rate was 91.10%, and κ=0.821.

[0086] 2. Using cervical examination as a reference standard, the sensitivity of vulvar hrHPV detection was 94.89%, the specificity was 88.18%, the positive predictive value was 86.09%, and the negative predictive value (NPV) was 95.72% (see Table 3).

[0087] 3. HPV18 subtype showed the highest concordance (κ=0.892, NPV=99.41%), followed by HPV16 (κ=0.839), with low-risk HPV at 0.746.

[0088] 4. All 5 pathologically confirmed CIN cases (4 CIN I cases and 1 CIN II-III case) had completely consistent HPV test results on the vulva and cervix.

[0089] 5. Age stratification: NPV = 95.44% and κ = 0.827 for the <40-year-old group; NPV = 97.78% and κ = 0.781 for the ≥40-year-old group. The β-globin Ct value of cervical samples in the ≥40-year-old group was significantly higher (22.14 vs. 21.81, P < 0.01).

[0090] 6. Analysis of inconsistent samples: 16 cases in the V- / C+ group (vulva negative / cervix positive), of which 15 cases (93.75%) had a history of HPV treatment; 48 cases in the V+ / C- group (vulva negative / cervix negative), of which 44 cases (91.67%) were non-HPV16 / 18 types.

[0091] The above data fully demonstrate that the present invention has an extremely high negative predictive value (≥95.72%), which can safely exclude the vast majority of cervical hrHPV infections; the correction rules for women aged ≥40 years and those with a history of treatment significantly improve the accuracy of screening.

Claims

1. A non-invasive triage screening method for cervical cancer HPV based on high negative predictive value, characterized in that, Includes the following steps: (1) Non-invasive collection of exfoliated cell samples from the vulva of the subjects; (2) Extract total DNA from the sample and perform PCR quality control, with the human β-globin gene as an internal reference; (3) HPV detection was performed using fluorescent PCR; (4) Stratified triage interpretation based on test results and clinical information of subjects: If the hrHPV test result is negative and the subject's age is <40 years, then it is interpreted that no invasive cervical examination is required. If the hrHPV test result is negative, the subject's age is ≥40 years and the β-globin Ct value is >30, then it is interpreted that resampling and testing or cervical cytology examination is required; If the hrHPV test result is negative, the subject's age is ≥40 years and the β-globin Ct value is ≤30, then it is interpreted that a cervical cytology examination or a repeat HPV test after 12 months is required; If the hrHPV test result is positive, it indicates that further evaluation or referral is required.

2. The method according to claim 1, characterized in that, In step (4), subjects who are hrHPV positive are further stratified: If HPV16 and / or 18 are detected, it is recommended to refer the patient for colposcopy and cervical biopsy. If a positive result is found for a type other than 16 / 18 and the patient has a history of cervical treatment, the interpretation is to shorten the follow-up interval to 6-12 months, or to directly refer the patient for colposcopy. If the test result is not positive for HPV type 16 / 18 and there is no history of cervical treatment, then it is interpreted as a follow-up HPV test after 12 months.

3. The method according to claim 2, characterized in that, The cervical treatments include LEEP (cervical conization), cold knife conization, laser therapy, cryotherapy, cervical electrocautery, or any form of physical destructive treatment of the cervix.

4. The method according to claim 1, characterized in that, In step (3), the HPV detection is for high-risk HPV and low-risk HPV. The high-risk HPV includes HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68. The low-risk HPV includes HPV types 6 and 11.

5. The method according to claim 4, characterized in that, A Ct value <37 for any high-risk subtype is considered hrHPV positive.

6. The method according to claim 1, characterized in that, When the subject is ≥40 years old, the clinical weighting of positive and negative results should be increased in the interpretation of results.

7. The method according to claim 6, characterized in that, The clinical attention weighting is increased by 10% to compensate for the risk of false negatives in cervical sampling that may result from cervical atrophy.

8. The method according to claim 1, characterized in that, In step (1), the sampling site for the exfoliated cells of the vulva is the surface of the vulvar skin, including the labia majora, labia minora and the vestibule area of ​​the vagina.

9. The method according to any one of claims 1-8, characterized in that, The method is applicable to large-scale population screening.

10. The method according to claim 9, characterized in that, The target population includes women aged 40 years or older, postmenopausal women or women with known cervical atrophy, women with a history of cervical HPV treatment, and women who have a fear of traditional speculum examination or refuse to undergo invasive screening.

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