Screening Methods for Cervical Cancer

Abstract

Cervical cancer seriously affects the health of women worldwide. Persistent infection of high‐risk HPV (Human Papilloma Virus) can lead to cervical cancer. There is a great need for timely and efficient screening methods for cervical cancer. The current screening methods for cervical cancer are mainly based on cervical cytology and HPV testing. Cervical cytology is made of Pap smear and liquid‐based cytology, while HPV testing is based on immunological and nucleic acid level detection methods. This review introduces cervical cancer screening methods based on cytology and human papillomavirus testing in detail. The advantages and limitations of the screening methods are also summarized and compared.

This review mainly introduces and compares cervical cancer screening methods based on cytology, immunology, and nucleic acid levels. Methods based on cytology include Pap smear and liquid‐based cytology. Methods based on immunology include immunohistemistry, immunogold‐agglutination, and serum immunoassay. Methods based on nucleic acid level include PCR, Abbott PCR, Hybrid Capture 2, cobas 4800, REBA HPV‐ID, MassARRAY spectroscopy, HPV sequencing, Aptima HPV assay, etc.

1. Introduction

Cervical cancer is the main health issue in gynecology. ^[1] According to data from 2023, cervical cancer ranked 14th among all cancers and was the fourth most common cancer among women worldwide.2, 3 Most cervical cancer occurs in low and middle‐income countries. ^[4] Human papillomavirus (HPV) is an 8 kb circular double‐stranded DNA virus with heterogeneity and high species specificity. ^[5] HPV plays a major role in the tumorigenesis of cervical cancer. ^[6] Cervical cancer is usually caused by the persistent infection of the oncogenic virus HPV. ^[7] The incidence trend of cervical cancer associated with HPV is more recent than other types of cancer. ^[8] Cervical cancer poses a significant threat to women‘s lives, partly due to the lack of accurate detection indicators to predict the occurrence and development of the disease. ^[9] Also, failure to use screening or its irregular appearance is considered the most important risk factor for developing cervical cancer. ^[10] The World Health Organization called for global initiatives in 2018 to eliminate the public health issue of cervical cancer and to ensure that every woman can receive screening and treatment for precancerous lesions. This requires expanding cervical cancer screening globally.11, 12 Cervical cancer screening can detect cervical cancer and precancerous lesions in a timely manner. Early screening and timely treatment of precancerous lesions can prevent up to 80 % of cervical cancer cases and reduce the mortality rate of cervical cancer.13, 14

The screening methods based on cytology have been very important for cervical cancer detection since the early days. They mainly include Pap smear and liquid‐based cytology. In recent years, cervical cancer screening methods have been continuously improving. The current screening methods for cervical cancer are mainly based on cervical cytology and HPV testing. The operation of this type of method is relatively simple, but the detection sensitivity is relatively low. HPV testing has a higher sensitivity and has developed faster in recent years. Several HPV testing methods have been developed, including nucleic acid hybridization analysis, signal amplification analysis, fluorescence in situ hybridization, nucleic acid‐based analysis etc. ^[15] In this review, the cervical cancer screening methods based on cytology and HPV testing are introduced in detail and compared.

2. Cervical Screening Based on Cytology and HPV Testing

This section gives a detailed review of cervical screening based on cytology and HPV testing for cervical cancer diagnosis, highlighting their strengths and weaknesses.

2.1. Cervical Screening Based on Cytology

For decades, cytology‐based screening has been a traditional screening method, ^[12] with moderate sensitivity to detecting cervical intraepithelial neoplasia 3 (CIN 3) and cervical cancer. ^[16]

2.1.1. Pap Smear

Pap smear, also known as Pap test, is a cervical smear examination. Pap smear has been used for cancer screening for decades and is a widely used cervical cancer screening method. It involves extracting a small amount of cell samples from the cervix and placing them on a slide to study whether the cells are abnormal under a microscope. ^[17] Cervical cancer examination through Pap smear is carried out through the following process. Clinical doctors perform endoscopic examination and collect cervical cell samples, they apply them onto a glass slide, then send them to the laboratory for analysis by a cell pathologist. The examination of cells can distinguish between normal cells, low‐grade abnormalities, or high‐grade abnormalities. Low‐grade abnormalities, defined as atypical squamous cells of unknown significance (ASC‐US) or low‐grade squamous intraepithelial lesions (LSIL), typically indicate HPV infection but cannot immediately indicate precancerous lesions. High‐grade abnormalities, defined as HSIL, atypical squamous cells implying high‐grade and atypical glandular cells, are highly correlated with high‐grade histological findings and need to be removed to prevent the development of invasive cancer. If high‐grade abnormalities are found, the likelihood of precancerous lesions is high. ^[18] The quality of Pap smear is influenced by collection conditions, operational variability, sampling errors, and cell preservation. ^[19] When using pap smear, all shed epithelial cells and superficial cells can be collected and analyzed. ^[17] During this process, there may be some operational differences in the collection and preservation of cells, and sampling errors may even occur. The subjective difference of observers may also affect the detection results. The sensitivity and specificity of Pap smear sampling for cervical cancer are low, at 55.5 % and 75 %, respectively.17, 20

2.1.2. Liquid‐Based Cytology

Liquid‐based cytology (LBC) has undergone significant changes in the preparation of cervical screening samples compared to Pap smears. When using this technology for cervical cancer detection, plastic equipment (cervical broom or mixed plastic scraper and cervical brush) is used to collect cells from the cervix and transport them to the laboratory in preservatives for processing to produce slide preparations. Cell samples can be processed using liquid‐based cytology systems such as SurePath or Thin Prep. When using SurePath system for processing, cell clumps and mucus are extracted and decomposed using a syringe. The obtained cell suspension is then layered and placed on a density gradient medium. Red blood cells and inflammatory cells are separated from epithelial cells through density gradient centrifugation. Then the cell particles mainly containing epithelial cells is inserted into the robot workstation, where they are resuspended and transferred onto a glass slide. This system can process up to 48 samples at a time. When using ThinPrep system to process cell samples, cell clumps and mucus are decomposed by mechanical stirring, then the liquid preservative solution is filtered through a membrane filter and transferred to a glass slide for staining. ThinPrep 2000 processor can only process one sample at a time, but ThinPrep 3000 and ThinPrep 5000 processors are automated and can process 80 and 160 samples, respectively, at once. ^[21] Then, these slides are screened or automatically imaged by researchers. This method is widely used in the United States, European countries, and many other developed countries. However, it is expensive and heavily reliant on technology. ^[22] LBC screening can reduce sample shortage rates, improve screening capabilities, and provide a platform for the automation of HPV testing. It takes a shorter time with a higher reporting speed and hence reduces women's anxiety. ^[23] The sample quality of LBC method is higher than that of Pap smear, and the probability of an unsatisfactory smear appearance is lower. It is not only a better substitute for traditional smear but also improves the accuracy of diagnosis and detection of abnormal cells in low‐grade squamous intraepithelial lesions (LSIL).24, 25, 26 In addition, residual LBC samples can be used for HPV DNA detection. ^[27]

2.2. Cervical Screening Based on HPV Testing

According to data from 2023, HPV‐18 and HPV‐16 were the causes of 70 % of cervical precancerous lesions and cancers. As shown in Figure 1, there is a close relationship between HPV and the occurrence of cervical cancer. ^[28] Therefore, HPV testing is needed for the diagnosis of cervical cancer. Human papillomaviruses (HPVs) are a family of small epithelial nutritive double‐stranded DNA oncovirus. ^[29] HPVs are divided into low‐risk and high‐risk types. Low‐risk HPVs are also known as non‐carcinogenic HPVs; the most common are HPV types 6 and 11. They usually cause genital warts and respiratory tumors and are associated with low‐grade abnormalities in cervical cells. The most common high‐risk HPV types are 16 and 18, while the less common are 31, 33, 45, 52, and 58. ^[30] High‐risk HPVs such as HPV‐16 and HPV‐18 can lead to chronic infections, cellular abnormalities, and ultimately cancer. ^[31] In addition to cervical cancer, high‐risk HPVs also drive squamous cell carcinoma of the oropharynx, vulva, vagina, anal canal, and penis. ^[32] The detection and genotyping of HPV infection are crucial for the prevention and effective treatment of cervical cancer. ^[33] HPV testing is one of the objective and repeatable of all cervical screening tests and has low requirements for training and quality assurance. ^[34] Detectable markers derived from HPV can be divided into three types: molecular markers based on nucleic acid sequence detection, serological markers based on virus protein antibody detection, and cell markers based on detecting proteins expressed in cells during infection or carcinogenesis. ^[35] Compared to cytological examination, HPV‐based screening has much higher sensitivity and helps women prevent cervical precancerous lesions and cervical cancer. The significant advantage of HPV detection over cytology is that it can be performed on self‐collected specimens, which eliminates the need for personal pelvic examination. ^[36] The detection of the carcinogenic subtypes of HPV makes it possible to predict the risk of future cancer development ^[5]

Figure 1.

HPV infective cycle and cervical carcinogenesis (Permission from MDPI). ^[28]

2.2.1. HPV Testing Based on Immunology

2.2.1.1. Immunohistochemistry

Immunohistochemistry is the study of the localization, qualitative evaluation, and relative quantification of antigens within tissue cells by applying the principle of specific binding between antigens and antibodies. The process consists of chemically reacting the antigen with a chromogenic reagent labeled with the antibody. The immunologic protein markers for cervical cancer includes HPV Osteopontin (OPN), squamous cell carcinoma antigen (SCC−Ag), immunosuppressive acidic protein (IAP), soluble CD44 (sCD44), etc. HPV can be detected by immunohistochemical detection of HPV OPN. OPN is a phosphorylated glycoprotein that serves as a cytokine connecting cells and matrix inorganic substances in the extracellular matrix. ^[37] It is closely related to the occurrence, development, and metastasis of malignant tumors. ^[38] In recent years, some studies have shown that OPN is expressed in cervical cancer.39, 40 BAO et al. used anti‐mouse polyclonal antibody as the first antibody and biotinylated anti‐mouse immunoglobulin G as the second antibody to detect tissue samples by immunohistochemistry. The detection results are shown in Figure 1. The total rate of positive detection was 75.56 %, and the positive expression rate of OPN in HPV‐positive cervical tumor tissues was higher than that in HPV‐negative cervical tumor tissues. ^[41] HPV OPN peptide antibodies have broad‐spectrum reactivity and significant multivalence against HPV infection. ^[41] Detecting the expression of HPV OPN protein in cervical tissues with pathological changes can monitor the proliferation cycle of the virus and indirectly evaluate the infectivity of patients, providing theoretical guidance for the clinical application of HPV OPN protein vaccine and the potential targeted treatment of tumors. ^[42] A study by Origoni et al. showed that the expression of HPV OPN usually indicates that the organism is infected with HPV and that HPV is in the replication stage. ^[43] OPN is highly conserved in HPV types and is the main population specific antigen.44, 45

2.2.1.2. Immunogold‐Agglutination

Detecting HPV‐16 E6 and L1 proteins may be the most suitable tool for screening for pre‐existing cervical lesions and cancer in patients. Bhat Tarakosol et al. directly detected HPV‐16 E6 and HPV‐16 L1 antigens from clinical samples using gold nanoparticles optical assay based on Immunogold‐agglutination technology. They mixed HPV antigens, including E6 and L1, with HPV‐16E6 polyclonal antibodies conjugated with AuNPs or HPV‐16 L1 polyclonal antibodies conjugated with AuNPs at designated times and temperatures for immunogold agglutination testing. The detection results are shown in Figure 2. The immunogold agglutination method has a high sensitivity in detecting HPV‐16E6 infection. The sensitivity of detecting HPV‐16 E6 in CIN‐3+ patients (73.1 %) is higher than that of rapid commercial immunochromatography (53.5 %). The detection limit of HPV‐16 L1 or E6 fusion proteins is 2.5 μg at 15 min. HPV‐16 L1 can be detected at all stages without non‐specific agglutination. The detected HPV‐16 E6 is closely related to the severity of cervical histopathology. This method is easy to operate and very specific. It can present results within an hour, compared with PCR (3–5 hours) and enzyme immunoassay rapid side flow detection (3 hours). ^[46] Ryu et al. reported a method using G protein as the first conjugate and then immobilizing specific antibodies to cardiac marker troponin I (cTnI), which significantly improved the detection sensitivity, up to 10 ng/ml. ^[47]

Figure 2.

HPV antigen immunogold‐agglutination test. Tube 1 and 2 are the reactions between AuNPs coated with normal goat antibodies and L1 and E6 fusion proteins, respectively. Tube 3 is a reaction between goat anti‐HPV‐16E6 coated AuNPs and E6 fusion protein. Tube 4 is a reaction between goat anti‐HPV‐16 L1 coated AuNPs and L1 fusion protein. (Permission from Elsevier). ^[46]

2.2.1.3. Serum Immunoassay

Identifying biomarkers for high‐risk HPV infection can enable early detection and treatment of cervical diseases. Nucleic acid programmable protein array (NAPPA)48, 49 can quickly analyze serum antibody response in cases of HPV infections50, 51 and cervical cancer.52, 53, 54 Ew aisha et al. used HPV protein microarrays NAPPA48, 49 to detect the specific IgG Abs of two low‐risk (HPV6 and 11) and ten high‐risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58) ^[55] to characterize the diversity of immune responses in cervical cancer and pre‐invasive cervical diseases. They expressed the full‐length cDNA encoding 12 types of HPV proteomes as the C‐terminal GST fusion protein and captured it on the surface of the slide. ^[56] Immunoreactivity to E4 protein from HPV16, 31, 35 and 45 and positive control EBV EBNA‐1 protein is shown in Figure 3. The results showed that 20–46 % of CIN and ICC patients had a wide range of antibodies to HPV early proteins in their serum, which were associated with the severity of cervical disease. ^[19] Research on serum collected before the diagnosis of cervical cancer has shown that the presence of E6 and E7 specific antibodies is associated with an increased relative risk of cervical cancer, which can be detected 5 years before diagnosis.46, 57

Figure 3.

Immunoreactivity to E4 protein from HPV16, 31, 35 and 45 and positive control EBV EBNA‐1 protein is shown. (Permission from Ivyspring International Publisher). ^[19]

When limited to cases where HPV16 (as opposed to any other oncogenic HPV) DNA is known to be detected, the sensitivity (proportion of cases where at least one HPV ag specific Ab is detected) is equivalent. CIN II/III is 59.6 % vs 59.8 %. Among all women (regardless of HPV DNA status). And the specificity is 78.6 %. HPV serology has the potential for disease diagnosis and prognosis. However, its clinical application is limited by HPV heterogeneity, detection variability, and viral immune escape. ^[19] The serological response of genital HPV infection mainly targets the conformational epitopes on the main capsid protein L1 of the virus. ^[46] Only 50–70 % of infected women have serum conversion,58, 59 and the proportion of serologically false negative women depends on the antibody testing method.46, 60, 61, 62, 63, 64

2.2.2. HPV Testing Based on the Nucleic Acid Level

HrHPV genotypes include HPV‐16, 18, 31, 33, and 35. Among these genotypes, HPV‐16 and 18 are the two most common types.65, 66 With the advancement of methodological technology, various methods which can detect HrHPV infection in the early stage and prompt patients for timely treatment, have also been introduced. ^[67] The Cobas HPV test approved by the US Food and Drug Administration (FDA), Hybrid Capture 2 (HC2), and Cervista can perform qualitative testing of virus DNA sequences. ^[68] HrHPV testing has been recommended for routine screening of cervical cancer. ^[69] Compared to tests that detect HPV RNA, tests based on HPV DNA detection have lower specificity for diseases confirmed by histopathology. ^[70]

2.2.2.1. Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR) is a selective technique for detecting HPV, it can exponentially replicate and increase the number of target HPV sequences present in biological specimens after repeated amplification cycles. ^[35] It involves the use of Taq DNA polymerase derived from the thermophilic bacterium Thermos aquaticus, as it can maintain activity at relatively high temperatures, thereby improving primer specificity. PCR is made of three core steps. The first step is denaturation, and it involves heating the PCR tube components at high temperatures (94–96 °C). This weakens the DNA and separates the two complementary strands. The second step is annealing, and it involves cooling the PCR tube component at 55 °C, to allow DNA primers to bind to complementary sites on the template strand. The third step, expansion, consists in heating the PCR tube component at 72 °C, which allows DNA polymerase to replicate the template strand by adding nucleotides to the end of the primer thus producing two double‐stranded DNA molecules. The number of target DNA is usually multiplied, by repeating multiple cycles. ^[71] PCR‐based detection has a wide range of specificity and sensitivity, with a sensitivity range of 75 % to 100 % and a specificity range of 85 % to 97 %. ^[72] They are determined by factors such as the size of the PCR product, the amplified HPV DNA profile, the ability to detect multiple types, the selected primer set, reaction conditions, and the performance of the polymerase in the reaction. ^[73] Most primer groups aim to target L1 genes or E6 and E7 oncogenes. ^[74] PCR primers targeting the E6 or E7 regions are described as preferred, as the L1/E1 region is often lost during the integration of viral DNA into the host genome DNA, and targeting the L1 or E1 region may miss out on advanced diseases. ^[75]

2.2.2.2. Abbott PCR and Hybrid Capture 2 (HC2)

Abbott PCR Assay is a real‐time PCR detection that can detect DNA from 14 HrHPV genotypes and distinguish between HPV16, HPV18, and “non 16/18” genotypes. This method uses a mixture of primers and probes to detect human globulin genes in cervical cells and HPV DNA in cervical swab samples. HPV and internal control probes are single‐stranded DNA oligonucleotides covalently linked to both ends of the probe, with fluorescence and quenching parts.76, 77 HPV 16, 18, non 16/18, and internal control signals can be detected and distinguished simultaneously in a single reaction. HC2 is widely recognized as the gold standard for HrHPV detection ^[78] and is a sandwich capture molecular hybridization detection method that provides semi‐quantitative results using chemiluminescence detection. HPV DNA was denatured and hybridized with a mixture of single‐stranded whole genome length RNA probes specific to 13 HR HPV genotypes. Alkaline phosphatase coupled antibodies were added to the RNA DNA hybrid, and chemiluminescence substrates were added to detect the immobilized hybrid. However, HC2 cannot distinguish HPV16 and 18 from other HrHPV types. The coincidence rate between HC2 and Abbott PCR results in Ko et al.′s study is shown in Table 1. The sensitivity and specificity of high‐risk genotype testing are shown in Table 2. Compared to Abbott PCR, HC2 has more false negative results. ^[79]

Table 1.

Concordance rate between HC2 and Abbott PCR. ^[79]

		HC2		Coincidence	Kappa	95 %CI(%)
		Positive	Negative	rate (%)	coefficient
Abbott	Positive	106	15	97.3	0.879	0.832–0.926
	Negative	10	810

Table 2.

Sensitivity and specificity for HR HPV genotypes (n=941). ^[79]

Assay	HPV genotype	Sensitivity (%)	95 % CI(%)	Specificity (%)	95 % CI(%)
HC2	All HR	90.0	81.4–95.0	95.9	94.3–97.1
Abbott	All HR	98.0	92.–99.6	96.8	95.3–97.8
	16	95.8	76.9–99.8	99.2	98.4–99.7
	18[	88.9	50.7–99.4	99.8	99.1–100

2.2.2.3. Cobas 4800 HPV‐DNA Assay and REBA HPV‐ID HPV‐DNA Assay

Cobas 4800 is based on real‐time PCR and uses a β‐ globin primer set that serves as a genomic DNA reference to ensure sample size and quality. It can specifically detect the combined presence of HPV16, HPV18, and 12 other HR HPVs. Its detection sensitivity for HPV16 and HPV18 is high. Cobas 4800 is easy to apply, has good repeatability, large sample size, and all steps can be completed on a fully automated machine. It is being used for cervical cancer screening in many countries. Clinical evaluation shows that cobas 4800 has significant sensitivity in detecting CIN2+ lesions.80, 81 Cobas 4800 analysis has been approved by the US Food and Drug Administration as an independent analysis for cancer screening. ^[82] REBA HPV‐ID is based on reverse line hybridization. The membrane strip hybridization control line is used to verify the presence of color reactions and reagents, and the β‐globin positive control line is used to verify the quality of the collected samples. Automated REBA HPV‐ID® can be used for HPV genotyping system implementation. This method can detect 32 types of HPV, including 18 HR types, 1 possible HR type (HPV 34), and other types. There are significant differences between these two detection methods in detecting HPV‐positive cases. Though Cobas®4800 can detect single or mixed HPV16 infection better than REBA HPV‐ID®”, the two methods provide good HPV infection detection.83, 84, 85 They help screen for persistent HR‐HPV infection in women, and predict an increased risk of progression to cervical cancer. ^[86]

2.2.2.4. MassARRAY Spectroscopy

MassARRAY spectroscopy is used to detect specifically 15 high‐risk carcinogenic types of HPV, HPV‐16, ‐18, ‐31, ‐33, ‐35, ‐39, ‐45, ‐51, ‐52, ‐56, ‐58, ‐59, ‐66, ‐68, and ‐73. When HPV detection is performed using MassARRAY spectroscopy, HPV DNA is extracted for PCR amplification, followed by primer extension reaction. Analyze primer extension products using MALDI‐TOF MS and classify genotypes based on the quality of each allele. ^[87] Schematic diagram of MALDI‐TOF MS workflow is shown in Figure 4. ^[88]

Figure 4.

Schematic diagram of MALDI‐TOF MS workflow. (Permission from Frontiers Media S.A.). ^[88]

MassARRAY spectroscopy is a highly sensitive, accurate, fully automated, and high‐throughput method that can process up to 2000 samples in approximately 11 hours. This method can be used to screen oncogenic HPV types ^[89] and quantify viral load ^[90] in samples containing trace amounts of viruses. However, its practicality and clinical application have not been fully evaluated. This method is based on real‐time amplification analysis of multiple nucleic acid sequences, using molecular beacon probes to perform isothermal detection, amplification, and detection of E6/E7 mRNA of oncogenic HPV types 16, 18, 31, 33, and 45. It is a unique and specific test because it is the only test in its class that can detect E6/E7 mRNA in each of the five most common carcinogenic types of HPV‐16, ‐18, ‐31, ‐33, and ‐45 with high specificity for identifying these five types.91, 92 However, this test is more specific for detecting high‐level CIN2 compared to other HPV tests.70, 93

2.2.2.5. HPV Sequencing (HPV‐Seq)

HPV circulating tumor DNA (ctDNA) is proposed as a biomarker for detecting and monitoring HPV related cancers. ^[94] Scott V. Bratman's research team has developed HPV‐seq, a quantitative detection method for ctDNA, with better performance than dPCR, and can reveal qualitative information about ctDNA. This method is an optimized method for the hybridization, capture and sequencing of HPV virus genome. Scott V. Bratman's research team applied HPV‐seq to locally advanced cervical cancer patients, demonstrating its performance in detecting ctDNA with less than one copy and achieving sensitive detection of HPV‐specific ctDNA. HPV‐seq can detect 0.01 % and 0.003 % of SiHa DNA, corresponding to 0.6 and 0.2 copies, respectively, indicating the presence of strong LLOD at less than one copy. The bait length of the HPV‐16 gene increases the likelihood of detecting HPV and improves analytical sensitivity. HPV‐seq can perform hypersensitive detection and qualitative analysis of ctDNA in cervical cancer. It can genotype samples that have not passed dPCR detection and have relatively low ctDNA levels, as well as report the size of ctDNA fragments. HPV seq can have an impact on the treatment and monitoring of HPV related cancers, providing new ideas for the potential clinical application of HPV ctDNA analysis. ^[95]

2.2.2.6. Aptima HPV Assay

Aptima HPV detection (AHPV) is an amplified nucleic acid test that can qualitatively detect HPV E6/E7 mRNA in 14 high‐risk HPV types in cervical specimens. ^[96] The practicality of AHPV detection comes from the possibility that sustained infection with high‐risk HPV may lead to virus integration into the host cell genome, inducing the overexpression overexpression of E6/E7 mRNA. ^[97] AHPV detection mainly includes three steps: cell lysis, using tar‐specific oligomers connected to magnetic particles to capture targeted E6/E7 HPV mRNA; Direct amplification of target RNA using transcription mediators and hybridization protection analysis in which chemiluminescence labelled probes are used to detect amplified products. Adding internal controls to all samples during the target capture step can reduce the risk of false negative results. The selective capture, amplification, and detection of HPV RNA by AHPV can improve the specificity of HPV molecular detection. ^[96] Table 3 provides the comparison of cervical screening methods based on HPV testing.

Table 3.

Comparison of cervical screening methods based on HPV testing.

HPV detection methods	Sensitivity	Specificity	LOD	Advantages	Disadvantages/challenges	Ref
Immunohis‐tochemistry	Positive detection rate, 75.56 %	/	/	Broad‐spectrum reactivity and significant multivalence against HPV infection	/	[41]
Immunogold Agglutination	73.1 %	>90 %	10 ng/ml	High sensitivity; Simple operation and fast results	Low sensitivity; Mixture of immunogold specific for each HPV types may be a challenge	[46–47]
Serum Immuno	59.8 %	78.6 %	/	With the potential for disease diagnosis and prognosis	Limited by HPV heterogeneity, detection variability, and viral immune escape	[19]
PCR	75 %~100 %	85.0 %~97.0 %	/	Exponentially replicate and increase the number of target HPV sequences	The sensitivity and specificity are determined by factors such as the size of the PCR product, the amplified HPV DNA profile, etc	[35,72–73]
Abbott PCR	98.0 %	96.8 %	/	Detect DNA from 14 HrHPV genotypes and distinguish between HPV16, HPV18, and “non 16/18” genotypes	Need to evaluate the clinical performance in CIN2+ specimens with cytological abnormalities	[79]
HC2	90.0 %	95.9 %	0.2 pg/ml	Gold standard for HrHPV detection	HPV16 and 18 cannot be distinguished from other HrHPVs; Compared to Abbott PCR, there are more false negative results	[78–79]
cobas 4800	100.0 %	24.0 %	150 copies/ml	Is easy to apply; Has good repeatability and a large sample size; All steps can be completed on a fully automated machine	Can only specifically detect HPV16, 18, and 12 other HR‐HPVs	[86]
REBA HPV‐ID	75.0 %	100.0 %	100 copies/PCR reaction (50 μL)	Can detect 32 types of HPV; The hybridization step could be performed using an automated machine; Be used for epidemiological study of HPV type distribution	Detection of single or mixed HPV16 infection is not as good as cobas 4800	[83,85–86]
MassARRAY spectroscopy	Detection rate, 94.8 %	/	/	Highly sensitive, accurate, fully automated; Quantify the viral load in samples containing trace amounts of viruses; Detect the E6/E7 mRNA of each of the five most common carcinogenic types HPV‐16, −18, −31, −33, and −45, with high specificity for identifying these five types	The practicality and clinical application have not been fully evaluated	[87,89–91]
HPV Sequencing	100.0 %	67.0 %	<0.6 copies	Perform hypersensitive detection and qualitative analysis of ctDNA in cervical cancer; Genotype samples that have relatively low ctDNA levels and report the size of ctDNA fragments	The clinical practicality of quantitative and qualitative ctDNA analysis needs to be confirmed	[95]
Aptima HPV Assay	95.2 %	42.2 %	/	Qualitatively detect HPV E6/E7 mRNA in 14 high‐risk HPV types in cervical specimens; Selective capture, amplification, and detection of HPV RNA can improve the specificity of HPV molecular detection	Relatively few HPV positive samples available for cytological diagnosis	[96]

2.2.2.7. Other HPV Testing Methods

In situ hybridization (ISH) provides a convenient and reliable method for detecting HPV infection in formalin‐fixed and paraffin‐embedded tissues. HR HPV was detected in relatively small amounts of CIN and SCC through RNA in situ hybridization, but there was no clear association with atopy or other clinical and histopathological features. Long term follow‐up analysis of more cases is needed to confirm that in situ hybridization may be helpful for prognostic evaluation. ^[98] DH3 HPV detection is based on hybrid capture technology, ^[99] which can specifically identify HPV16 and 18 based on detecting 13 HrHPV types targeted by HC2 and HPV66. It can replace HC2 in detecting HrHPV infection and identifying cervical precancerous lesions. It can also perform HPV 16 and 18 genotyping simultaneously. ^[78] The performance of DH3 HPV in identifying high‐level CIN in cervical cancer screening is similar to that of Cobas HPV. ^[99] PanHPV detection is a next‐generation sequencing (NGS) method that includes two primer libraries covering different regions of eight high‐risk HPV genomes. PanHPV detection is a highly sensitive and specific detection that recognizes cHPV DNA in plasma. ^[101] ChapterDx HPV‐STI NGS detection is easy to automate and cost‐effective. This method can detect and quantify 29 HPVs in single tube and single‐step PCR reactions and can be applied to tens of thousands of samples in one sequencing. ^[102] The detection of HPV ctDNA through droplet digital polymerase chain reaction (ddPCR) is highly sensitive and specific. It can be used for early detection screening trials and disease response monitoring. ^[33]

3. Summary and Outlook

Cervical cancer is a tumor disease that has serious adverse effects on women. Human papillomavirus infection is a necessary condition for cervical precancerous lesions and invasive cervical cancer. By conducting cervical cancer screening, precancerous lesions and cervical cancer can be detected. In recent years, cervical cancer screening methods have continuously improved, which has significantly reduced the incidence rate and mortality of cervical cancer. ^[36] This review provides a detailed introduction to commonly used and potential cervical cancer screening methods based on cytology and HPV testing. The disadvantages of current cervical cancer screening methods include cumbersome operations, low throughput, and low sensitivity.

In the present and future, it is expected to develop more sensitive, specific, and high‐throughput cervical cancer screening methods, as well as interdisciplinary screening methods that combine biology with materials science, biochips, nucleic acid aptamers, chemistry, etc. Wang et al. (2023) prepared a simple label‐free electrochemical immunosensor (ECIS) and achieved ultrasensitive detection of HPV16 L1 protein. They studied graphene oxide and developed quasi spherical structures on graphene oxide using UV visible spectroscopy, transmission electron microscopy, and energy dispersive X‐ray spectroscopy Ag@Au Core‐shell nanoparticles (Ag@AuNPs‐GO). As a current response amplifier, Staphylococcal protein A is used to immobilize the Fc portion of a monoclonal antibody specific to HPV16 L1 protein. The electrochemical and interfacial kinetic properties were verified using cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy detection limit of 0.002 ng mL⁻¹ and a wide linear relationship in the range of 0.005‐400 ng mL⁻¹at a regression coefficient (R²) of 0.9948. This ECIS has acceptable accuracy, good reproducibility, stability, and selectivity. ^[103] Also, high sensitivity detection of HPV can be achieved in the future by preparing a novel graphene oxide polymer sensor and binding nucleic acid aptamers on the surface of graphene oxide. The specific method includes the following steps: Detection of HPV based on physically adsorbed DNA sequences on graphene oxide surfaces. The end of the nucleic acid adapter is labeled with FAM fluorescence, and the graphene oxide surface physically adsorbs the sequence and quenches the fluorescence on DNA. After binding with HPV, the fluorescence recovers, allowing for specific detection of HPV. To detect HPV based on surface fixed DNA sequences on graphene oxide chips: Fix graphene oxide on a glass slide, the modified amino group at the other end of DNA forms an amide bond with the carboxyl group on the surface of graphene oxide. When HPV is added, specific detection of HPV is achieved by changing the fluorescence intensity on DNA. The detection of HPV in actual samples based on graphene oxide sensors consist in detecting different concentrations of HPV using graphene oxide polymer sensors, then calculate RSD, and obtain the actual effect and stability of the sensor through RSD.

Conflict of Interests

The authors declare no conflict of interest.

Biographical Information

Jingyan Li, obtained a Bachelor's degree in Biological Science from Huzhong Agricultural University and is about to obtain a Master's degree in Biology from Jiangsu University. Her research focuses on Biochemistry and Molecular Biology, and she has published an SCI paper titled Sensors Based on the Carbon Nanotube Field Effect Transformers for Chemical and Biological Analysis. She is applying for a patent for a paper‐based method for preparing graphene DNA sensors and detecting mercury ions; Detection of AFB1 by Au NaCl gel and DSAI labeled nucleic acid aptamer fluorescence sensor. Phone: 15207191050, Email: lijingyan202103@163.com.

Biographical Information

Sidonie Darina Adobo, born in Lomé( Togo), is passionnate of biology. she obtained a professionnal bachelor degree in medical and biological analyses in University of Lomé (Togo) in 2019. Currently a master‘s students in Jiangsu university majoring in biology. She is interseted in biosensors and working on aptamers based biosensors for metal ions detection.