In squamous cell carcinoma, HPV18+ samples also showed increased median A3B expression when compared to HPV Alpha-9 species or only to HPV16+ samples. Our findings suggest that A3B expression is differentially upregulated in cervical cancer samples infected with HPV18. A3B could be potentially used as a biomarker for HPV infection and as a prognostic tool for clinical outcomes in the context of cervical cancer.The APOBEC3 (A3) proteins are cytidine deaminases that exhibit the ability to insert mutations in DNA and/or RNA sequences. APOBEC3B (A3B) has been evidenced as a DNA mutagen with consistent high expression in several cancer types. Data concerning the A3B influence on HPV infection and cervical cancer are limited and controversial. We investigated the role of A3B expression levels in cervical cancer in affected women positive for infection by different HPV types. Tumor biopsies from cancerous uterine cervix were collected from 216 women registered at Hospital do Câncer II of Instituto Nacional de Câncer, and infecting HPV was typed. A3B expression levels were quantified from RNA samples extracted from cervical biopsies using real-time quantitative PCR. Median A3B expression levels were higher among HPV18 The human papillomaviruses (HPV) are composed of non-enveloped particles containing a double-stranded DNA genome of approximately 8 kb in size . HPV is Cervical cancer is the fourth most incident cancer in women worldwide and in Brazil, responsible for over 8000 deaths in the latter in 2018 according to the last Globocan estimate ,10.The APOBEC family is a group of enzymes with cytosine-to-uracil deaminase activity grouped into five classes, namely AID, APOBEC1, APOBEC2, APOBEC3 and APOBEC4. APOBEC3 is further subclassfied into seven members , characterized by a cytidine deaminase activity on single-stranded DNA ,12. PhysA3B expression is consistently reported at low levels in normal human tissues . HoweverRNA Later , conditioned in liquid nitrogen gallons and transported to Banco Nacional de Tumores e DNA (BNT) at INCA. After 24 h, the RNA Later was removed from the cryotubes and samples were placed in a −80 °C freezer until nucleic acid extraction. This study was approved by the Ethical Committee of INCA (registration # 156/10 and CAAE 53398416.0.0000.5274). An Informed Consent Term was applied to all the patients enrolled in the project.The population studied included 216 women with cervical cancer whose samples were extracted from uterine cervix biopsies. The subjects were registered and followed-up at Hospital do Câncer II of Instituto Nacional de Câncer José de Alencar Gomes da Silva (INCA). Tumor biopsies were collected at surgery rooms before patients initiated any therapy, and were stored in cryotubes with 1 mL of All 216 subjects included in the study were previously investigated with respect to infecting HPV genotypes and HPV16/18 genetic variability ,21. All 2O, supplementing the final volume to 10 µL in order to degrade any DNA traces present in the samples. The mix was then incubated at 37 °C for 30 min and the DNase reaction was interrupted by adding 1 µL RQ1 DNase Stop Solution and proceeding to an incubation at 65 °C for 10 min. Continuing with the RNA treated with DNase, complementary DNA (cDNA) synthesis was performed as follows: 10 µL of input RNA after DNase treatment (1–500 ng/µL), 2 µL of 50 ng random primers (random hexameric primers—Thermo Fisher Scientific), 2 µL of 10 µM dNTPs (for each nucleotide) and ultrapure nuclease-free H2O complementing the final volume to 12 µL. The reaction was incubated at 65 °C for 5 min. After, 4 µL 5X First-Strand Buffer were added for each reaction, as well as 2 µL of 0.1 M DTT. The reaction was incubated at 25 °C for 1 min. For the last step, 2 µL of SuperScript™ II RT were added for each reaction following a cycling with 25 °C for 10 min, 42 °C for 50 min and 70 °C for 15 min.Total RNA extraction from the collected samples was performed using the All Prep DNA RNA kit , following the manufacturer’s protocol. After RNA isolation, samples were quantified using NanoDrop ND-1000 . RNA was kept on ice during all the procedures and stored at −80 °C. All RNA samples were treated with 1 µL RQ1 RNase-Free DNase , 1 µL RQ1 DNase 10X Reaction Buffer, 1–8 µL of input RNA (1–500 ng/µL) and ultrapure nuclease-free HAPOBEC3B mRNA was performed through real-time PCR (qPCR). TaqMan™ probes for GAPDH  and APOBEC3B  were used, and the reactions were carried out in a ViiA 7 Real-Time PCR System apparatus (Thermo Fisher Scientific). Reactions were performed in triplicates containing 5 µL TaqMan™ Gene Expression Master Mix, 0.5 µL TaqMan™ probe for APOBEC3B, 0.25 µL TaqMan™ probe for GAPDH, 2 µL of input cDNA and ultrapure nuclease-free H2O, supplementing the final volume to 10 µL for each reaction. The cycling conditions were as follows: polymerase activation at 50 °C for 2 min, denaturation at 95 °C for 10 min and 40 cycles of denaturation at 95 °C for 15 s, followed by annealing and extension for fluorescence detection at 60 °C for 1 min. The analytical method used for calculating gene expression differences was the 2-ΔCT [The relative quantification of he 2-ΔCT .Statistical Package for the Social Sciences (SPSS) was used for statistical analyses. The Mann–Whitney U test was used to analyze differences in APOBEC3B expression levels between the samples with distinct infecting HPV types and clinical variables . Results were considered significant when p-values < 0.05. A multivariate analysis was also performed in the SPSS software using the Poisson regression model.The software n = 3; 1.4%), HPV45  and HPV59 , and other members of the alpha-9 species: HPV31 and HPV35 , and HPV33, HPV52 and HPV58 . Moreover, we also found four samples positive for HPV73 (1.9%) and one positive for HPV56 (0.5%), belonging to the alpha-11 and alpha-6 species, respectively.In this study, we analyzed a total of 216 women with cervical cancer, all of them previously reported with single infection with different HPV types. The most frequent types observed were HPV16 (Alpha-9), present in 139 samples (64.4%) and HPV18 (Alpha-7), present in 30 samples (13.9%). Besides these, 10 additional HPV types were present in smaller frequencies. These included other members of the alpha-7 HPV species: HPV39 (+ and in 28 (93.3%) of 30 HPV18+ samples. Among the HPV16+ samples, ninety-two (66.2%) belonged to lineage A, twenty-eight (20.1%) to lineage D, six to lineage C (4.3%) and five to lineage B (3.6%). Among the HPV18+ patients, we also observed a higher frequency of lineage A in twenty-six samples (92.9%) and only two samples (7.1%) carried lineage B. HPV18 lineages C and D were not identified in this study. When we performed multiple intratype comparisons between samples carrying different lineages of HPV16 or HPV18 with respect to A3B expression levels, no significant differences were observed .HPV lineages were identified in 131 (94.2%) of 139 HPV16p = 0.753; Kruskal–Wallis). The most frequent histological type observed in our cohort was the squamous cell carcinoma (SCC) (84.8%). Women with HPV16 showed a significantly increased frequency of SCC (89.2%) when compared to those carrying HPV18 (56.7%) given the non-overlapping confidence intervals (respectively 84–94.4% and 38.2–75.2%). Conversely, HPV18-infected women exhibited a higher incidence of adenocarcinoma (ADC) (43.3%) in comparison to women with HPV16 (10.8%) without superposition between the confidence intervals (respectively 24.8–61.8% and 5.6–16%). Most patients presented at intermediary to late stages at diagnosis  and did not differ between HPV types in that regard.Demographic and clinical characteristics of the patients and their relation to HPV are listed in APOBEC3B expression levels in cervical cancer samples, we compared them among distinct HPV type groups. The median APOBEC3B expression level was higher among HPV18+ samples (n = 30) when compared to HPV16+ samples (n = 139) . As expected, HPV18+ samples continued to exhibit significantly increased levels of APOBEC3B expression compared to that group . On the other hand, HPV18+ samples continued to display an increase in the median APOBEC3B expression levels when compared to samples infected with the Alpha-9 (HPV16-related and included) species (n = 148)  species (n = 54)  showed an increased median APOBEC3B expression level when compared to Alpha-9 species (n = 148) . That prompted us to analyze the effect of each variable in the A3B expression levels. A final model was tested in a multivariate analysis in order to simultaneously consider all variables . After multivariate testing .With respect to the clinical variables under study, we did not find any significant association between A3B expression and tumor subtypes or clinical staging using univariate analyses. However, when analyzing age at diagnosis, we found that women above the median age (49 years) displayed higher A3B expression levels than those under the median  showed the uterine cervix to be one of the cancer sites with the greatest A3B expression and APOBEC-related mutational signatures [+ cervical cancer samples exhibited an increased A3B mRNA expression when compared to HPV16+ samples (p = 0.027). These results not only reinforce the association between HPV and A3B expression in cervical cancer, but also suggest that distinct HPV types can modulate A3B expression to different extents.In this study, we describe that eoplasia ,24. Moregnatures ,14. In tOur efforts were concentrated in A3B, and not in other members of the APOBEC3 subfamily, for a number of reasons. In addition to the abovementioned evidence of the importance of A3B in multiple cancers , Burns et al.  showed tHigh-risk persistent HPV infection is well established as the main risk factor for the development of premalignant intraepithelial lesions and cervical cancer progression ,26,27. A+ samples [The data presented herein are consistent with those from previous studies that showed an increase in A3B mRNA levels upon HPV infection ,30. Viei samples . In norm samples .MTA1 mRNA levels and HPV18 E6 could induce its expression. It was also evidenced that transfection of the HPV18 genome induced an increase in DNA deaminase activity, which was proportional to A3B upregulation and required MTA1 participation [A recent report evaluated the potential mechanistic link between metastatin-associated protein 1 (MTA1), whose levels are tightly associated with tumor progression in cervical cancer, and A3B in cervical cancer cells. It was observed that HPV18 infection was significantly associated with cipation .TP53 mutated breast cancer samples [TP53 loss or E6-mediated degradation of p53 prevents the inhibition pathway and possibly leads to A3B upregulation [APOBEC3B [Although a mechanistic model linking HPV to A3B upregulation is notfully established, recent reports suggest promising pathways. Higher A3B mRNA levels were observed among  samples . The p53gulation . MoreoveAPOBEC3B . In thisAPOBEC3B .Another possible link between HPV and A3B overexpression could be through the antiviral responses elicited by the innate immunity. During HPV infection of human keratinocyte cell lines, the viral E5 protein induces the expression of IFN-β . Either + oropharyngeal squamous cell carcinoma [Taken together, different reports indicate A3B upregulation upon HPV infection. However, APOBEC3 deaminase activity can also target the HPV genome, induce hypermutation and limit its infectivity, contributing to virus clearance ,40,42. Iarcinoma . Therefoarcinoma . Interesarcinoma .Our study is limited in the sense that it could not unveil the molecular determinants that govern the differential effect of HPV18 towards A3B upregulation when compared to HPV16 or other high-risk HPV types. We have previously shown that HPV18 infection of human immortalized keratinocytes caused higher levels of A3B induction (1.5-fold) in comparison to HPV16 . In the In conclusion, we found that high APOBEC3B expression was associated with HPV18 infection in women with cervical cancer. Our results suggest that A3B expression is an important biomarker of HPV infection, and potentially a prognostic biomarker of cervical cancer clinical outcomes such as recurrence, response to treatment and overall survival, issues that will require further investigation.


Proficient Human Papillomavirus (HPV) genotyping services are essential to support HPV and cervical cancer elimination strategies, in particular to support HPV vaccine research.To perform a global HPV genotyping proficiency study, with evaluation in relation to previous proficiency studies.The proficiency panel contained 44 coded samples (40 samples containing one or more purified HPV types (HPV6/11/16/18/31/33/35/39/45/51/52/56/58/59/68a/68b) in human DNA, 1 human DNA control and 3 DNA extraction controls). Proficiency required detection of both single and multiple infections of 50 International Units of HPV 16/18, of 500 genome equivalents for other HPV types and no false positivity.One hundred and thirty-two laboratories submitted 211 datasets. Most assays used (182/211 datasets) were commercially available. An all-time high of 75% of the datasets were 100% proficient. One or more false positives were found in 17.5% of datasets. Among laboratories who participated in the 2019 proficiency study, full proficiency increased from 25% in 2019 to 60% in 2021. The high overall proficiency was mostly attributable to a large number of new laboratories, which used similar assays.The worldwide deterioration in comparability and reliability of HPV testing found in 2019 is now reversed and an overall increase in proficiency is found. AFROAfrican Regional OfficeEMROEastern Mediterranean Regional OfficeEUROEuropean Regional OfficeEqualisExternal quality assessment of laboratory medicine in SwedenGEGenome equivalentsHPVHuman papillomavirusInternational HPV Reference CenterIHRCIUInternational unitsLabNetLaboratory NetworkPAHOPan American Health OrganizationPPProficiency panelSEAROSouth-East Asian Regional OfficeWHOWorld Health OrganizationWPROWestern Pacific Regional Office.1In the era of cervical cancer elimination, many laboratories perform Human Papillomavirus (HPV) detection and the number of different assays used has increased exponentially. Accurate and internationally comparable HPV detection is a must for both research and evaluation of HPV vaccination, as well as for screening programs. [3]The International HPV Reference Center (IHRC) coordinated the Global WHO HPV Laboratory Network at the outset and has since 2008 coordinated global HPV genotyping proficiency studies to a) assess the proficiency of HPV typing assays when routinely used in laboratories worldwide, b) evaluate the sensitivity and type-specificity of HPV detection of the different HPV assays when routinely used in laboratories worldwide and, c) identify problems with any assays routinely used. [10]The proficiency panels (PPs) consist of blinded samples with defined amounts of HPV DNA  in a background of human DNA, as well as extraction controls. Laboratories genotype these samples with their standard methods.In 2019 we found a global decrease in proficiency of HPV genotyping 22.1The 2021 PP included 44 coded samples  with 10 ng/ul of human placental DNA (Sigma-Aldrich no 7011), 1 negative control (TE buffer with 10 ng/µl human placenta DNA) and 3 samples of cell lines used as controls for the DNA extraction step in the testing .Table 12The HPV types included were 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68a (HPV 68 prototype) and 68b (ME 180 isolate) at different concentrations /5 ul for HPV 16 and 18, and 500 and 50 HPV genome equivalents (GE)/5ul for the other HPV types). All corresponding proprietors had given approval for the HPV types to be included in the PP.The PP was pre-tested for validation purposes at the Reference Laboratory in Sweden using a modified GP5+/6+ PCR followed by Luminex-based typing and distributed afterwards to laboratories throughout the world. Distribution of panels in November 2021 was performed by Equalis , following the call for participation and requests received. [10]The fee for participation was 1000 Euros for commercial entities and 500 Euros for academic and public health entities. Participants from low and lower-middle income countries could apply for waiving of the fee.All participants were to determine the genotypes in the panel using their standard assays, using their standard sample input volume. There were no particular demands on the assays used and both commercial assays and in-house assays were allowed. In case a participant used several different HPV detection methods, one PP could be used by each assay and one dataset provided from each method.2.2Datasets from laboratories submitted until the 28th of February 2022 were accepted and compiled by Equalis and analysed by the IHRC.Proficiency criteria was established by a WHO consensus meeting 14 years ago, Input with 10 or 15 ul was classified as the same IU/GE content as compared to 5 ul input. The panel also included low concentration samples (5 IU/5 ul for HPV16/18 and 50 GE/5 ul for non- 16/18 types) that were intended for educational purposes, training and for providing information on whether the test exceeded the requirements needed for proficient HPV typing. Detection of these low amounts was not required for proficiency.For assays that did not claim to detect all HPVs included in the PP, evaluation was performed considering only the HPV types that the assay reportedly detected. Consequently, the denominator varies . For assays reporting results only as aggregated types (e.g. “other"), proficiency was considered as long as the assay detected the HPV types targeted by the method. .The results were sent to all participating laboratories that had paid the fee before February 2022.3The 2021 proficiency study was distributed to 144 subscribing laboratories. A total of 132 laboratories returned 211 datasets . Ninety-six laboratories submitted 1 dataset, 13 laboratories submitted 2 datasets, 4 laboratories submitted 3 datasets, 18 laboratories submitted 4 datasets and 1 laboratory submitted 5 datasets.There were 126 participating laboratories that provided data on type of laboratory. There were 83 diagnostic and screening laboratories, 26 laboratories performing HPV surveillance, 8 clinical trial laboratories and 9 diagnostic test manufacturers. The annual number of samples analysed for HPV per laboratory varied from 100 to > 100,000 per year, with 15 laboratories performing 10,000 tests or more.Among datasets that typed for at least one HPV type 158/211 (75%) were 100% proficient for the types claimed to be detected by the test. Of these, 119/158 datasets not only correctly identified the content of all required samples, but also correctly identified the content of the training samples that contained amounts that were lower than required for proficiency (samples that contained only 5IU of HPV16/18 and 50GE of other HPVs). Tests that did not type for all the types in the panel could still be 100% proficient, as the denominator was the number of types claimed to be detected by the test (not the number of types included in the panel).3.1The number of laboratories reporting correct HPV types, with no false positivity, are shown in 3.2n = 59), followed by European Regional Office , Pan American Health Organization , African Regional Office , Eastern Mediterranean Regional Office  and South-East Asian Regional Office .Laboratories submitting datasets belonged to 6 WHO regions, with Western Pacific Regional Office (WPRO) showing the highest number of participating laboratories , Anyplex II HPV 28  and 3 other Hybribio assays (20 laboratories) . Twenty-Twenty-nine datasets were obtained using a variety of in-house assays.Overall, 74.88% of datasets (158/211) were 100% proficient and 82.46% (174/211) of datasets showed no false positivity. There were several assays with all the datasets provided showing 100% proficiency (Hybribio 37 array HPV (Hybribio), HPV-23 Genotyping (Hybribio), Hybribio 14 HR (Hybribio), Real-time PCR MehrViru, Tellgen 27plex 14HR, GenoFlow HPV array (DiagCor) and OncoPredict HPV-DNA (Hiantis)), followed by Hybribio 21 array HPV (Hybribio) with 97.44% of datasets being 100% proficient. None of the datasets obtained with HPV SPF10-LiPA25 (Labo-bio), Abbott m2000 / Alinity M (Abbott), Ampliquality  and VisionArray HPV (ZytoVision) were fully proficient.n = 3) non-proficient .Up to 11 assays  showed no false positivites, including Abbott m2000 / Alinity M (Abbott), VisionArray HPV (ZytoVision), MassArray MALDITOF (Agena), Onclarity (BD) and Hybribio 37 array HPV (Hybribio), in addition to the assays that were 100% proficient as listed above. Only HPV SPF10-LiPA25 (Labo-bio) showed all datasets , but rather due to laboratory conditions of use (e.g. cross-contamination).Comparison of results for laboratories that participated in 2021 as well as in 2008, 2010, 2011, 2013, 2014, 2017 and 2019.In total, 65 laboratories that participated in 2021 had also participated in at least one of the previous proficiency studies. There were 15 laboratories that submitted results in 2021 that had been participating ever since the first PP was issued in 2008, with 10/15 having participated in all eight previous proficiency studies . Some laboratories used the same tests during all years, whereas some laboratories had changed at least one of the tests used.Comparisons of these results were made for each laboratory using the current proficiency criteria for evaluating datasets obtained during all the years. At the outset in 2008 one false positivity had been allowed, but since 2019 no false positivity is required for 100% proficiency. We now retrieved all crude data from previous years and re-calculated proficiency using exactly the same criteria as used since 2019 .The proficiency trend for laboratories participating multiple times is shown in 4We report that the global proficiency in HPV genotyping services had increased in 2021. The increased proficiency is seen both in laboratories that had participated in previous studies as well as in laboratories participating for the first time. This improvement suggests that continuing proficiency testing is helpful to sustain accuracy and to avoid a deterioration in proficiency, as found in 2019. [10]There are three major strengths of our proficiency study. Weakness of the study is that the PP was composed for evaluating the HPV genotyping services required to support HPV vaccine research and studies monitoring vaccination impact. However, HPV screening may not need to always separate all different HPV genotypes and the sensitivity requirement may also be different. Similar panels with screening-relevant concentrations of HPV genotypes will be developed to assess and validate the assays (and their use in laboratories) used in cervical screening and triage of high-risk HPV-positive subjects. Another weakness is that we do not have any explanation for the low detectability of HPV68a. Samples were diluted in human DNA as typically found in clinical samples. Further dilution of the samples may have led to a decrease in proficiency. As these samples were readily detectable by a majority of labs, it is unlikely that the low detectability could be caused by a problem with the samples.n = 3; Anyplex II HPV HR Detection, n = 5; BD Onclarity HPV Assay, n = 2; Cobas 4800 HPV Test, n = 3). [12]The proportion of commercial assays has increased over time, from 57% in 2011, 80% in 2019, and now 86% in 2021 (182/211). Surprisingly, only 13/211 datasets had used any one of the 11 assays that in systematic reviews of the literature had been found to have had published validation data fulfilling all validation criteria  from different laboratories, comprising >50% of all fully proficient datasets in the 2021 proficiency study. The widespread use of these assays in laboratories in the WPRO region appears to be a reason why the proficiency was highest in this region. Hybribio was used in 59/132 laboratories, with most of them (44/47) belonging to the WPRO region.The 2021 Global HPV LabNet HPV DNA proficiency study further enables improving and sustaining sensitivity and specificity of different HPV typing assays. Comparing the results in the 2008, 2010, 2011, 2013, 2014, 2017, 2019 and 2021 global HPV DNA PPs, we can both see that it is possible to achieve a global improvement in proficiency of HPV genotyping services and that regular issuing of PPs is needed for sustaining and improving HPV detection. In the efforts to eliminate cervical cancer, the IHRC will continue to issue PP yearly to promote proficiency in HPV testing. Starting in 2022, we will also be issuing HPV screening panels (with screening-relevant concentrations of the HPV genotypes important for screening) to specifically promote proficiency in HPV screening services.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper


P < .0001) or cytology alone . DS showed better risk stratification than cytology‐based strategies and provided high reassurance against pre‐cancers both at baseline and at 1‐year follow‐up, irrespective of the HPV genotype. DS allows for the safe triage of primary screening HPV‐positive women.Triage strategies are needed for primary human papillomavirus (HPV)‐based cervical cancer screening to identify women requiring colposcopy/biopsy. We assessed the performance of p16/Ki‐67 dual‐stained (DS) immunocytochemistry to triage HPV‐positive women and compared it to cytology, with or without HPV16/18 genotyping. A prospective observational screening study enrolled 35 263 women aged 25 to 65 years at 32 U.S. sites. Cervical samples had HPV and cytology testing, with colposcopy/biopsy for women with positive tests. Women without cervical intraepithelial neoplasia Grade 2 or worse (≥CIN2) at baseline (n = 3876) were retested after 1 year. In all, 4927 HPV‐positive women with valid DS results were included in this analysis. DS sensitivity for ≥CIN2 and ≥CIN3 at baseline was 91.2%  and 91.9% (95% CI: 86.1%‐95.4%), respectively, in HPV16/18‐positive women and 83.0% (95% CI: 78.4%‐86.8%) and 86.0% (95% CI: 77.5%‐91.6%) in women with 12 “other” genotypes. Using DS alone to triage HPV‐positive women showed significantly higher sensitivity and specificity than HPV16/18 genotyping with cytology triage of 12 “other” genotypes, and substantially higher sensitivity but lower specificity than using cytology alone. The risk of ≥CIN2 was significantly lower in HPV‐positive, DS‐negative women , compared to triage‐negative women using HPV16/18 genotyping with cytology for 12 “other” genotypes  requires efficient triage of HPV‐positive women to colposcopy and biopsy. In this prospective observational trial in the United States, with 1‐year longitudinal follow‐up, the authors investigated the performance of p16/Ki‐67 dual‐stain cytology for the triage of women identified as HPV‐positive during primary screening. Compared to HPV16/18 genotyping combined with cytological triage of other HPV genotypes, dual‐stain cytology was significantly more sensitive for predicting risk of cervical intraepithelial neoplasia grade 2/3 or worse. The findings indicate that dual‐stain cytology is effective for triage of HPV‐positive women, either alone or when combined with partial HPV genotyping. ACISadenocarcinoma in situAGCatypical glandular cellsASCCPAmerican Society for Colposcopy and Cervical PathologyASC‐Hatypical squamous cells—cannot exclude HSILASC‐USatypical squamous cells of undetermined significanceCIconfidence intervalCINcervical intraepithelial neoplasiaCPRcentral pathology reviewDSdual stainH&Ehematoxylin and eosinHPVhuman papillomavirusHSILhigh‐grade squamous intraepithelial lesionKPNCKaiser Permanente Northern CaliforniaLSILlow‐grade squamous intraepithelial lesionNILMnegative for intraepithelial lesion or malignancyNPVnegative predictive valuePPVpositive predictive valueSDstandard deviation1Molecular testing for human papillomavirus (HPV) is now widely accepted as the preferred approach for cervical cancer screening. A number of countries including Australia, United Kingdom, the Netherlands, Sweden, Denmark and Turkey have phased out Pap cytology (cytology) as the primary cervical cancer screening test and replaced it with primary HPV testing. The biggest challenge to implementing primary HPV screening is managing the large number of women found to have transient HPV infections. In large U.S. cervical cancer screening trials, approximately 14% of women 25 years and older are HPV positive.Testing for the presence of cervical cells showing simultaneous expression of both the cell‐cycle regulator protein p16 and the proliferation‐associated Ki‐67 protein  has been shown in multiple studies to provide good specificity while maintaining high sensitivity when used as a triage test for abnormal cytology or positive HPV screening test results.22.1Women aged 25 to 65 years attending routine cervical cancer screening visits at 32 clinical sites offering cervical cancer screening services, including Planned Parenthood clinics, in 16 states across the United States between September 2017 and November 2018 were invited to join the IMPACT trial, as previously described in detail.The IMPACT trial consisted of two phases, a baseline  and a 1‐year follow‐up phase. Women who met the clinical endpoint  exited the study. Women who did not meet the primary endpoint and/or did not undergo treatment at baseline were invited to participate in the follow‐up phase of the trial. Subjects included in the follow‐up phase underwent an additional round of HPV and cytology testing after 12 months and, analogous to baseline procedures, were referred to colposcopy/biopsy if positive for either of these tests. The flow of the subjects through the baseline and 1‐year follow‐up phases of the IMPACT trial is shown in Figure 2.2Women had one cervical sample collected into a liquid‐based cytology vial  using either spatula/brush or broom‐type collection devices . Specimens were shipped to 1 of 4 central laboratories in the United States participating as clinical laboratory study sites for the trial, and all laboratory testing was performed by these four laboratories.PLUS Cytology kit  on BenchMark ULTRA automated instruments according to the manufacturer's instructions. For the interpretation of p16/Ki‐67 DS slides, at least two cytotechnologists and at least two pathologists from each of the four clinical laboratory sites participated in the review of the slides. Every p16/Ki‐67 DS cytology slide was first interpreted by one cytotechnologist, and the final test result was confirmed by one pathologist.HPV testing using both the cobas 4800 HPV Test and cobas HPV for use on the cobas 6800/8800 Systems  and cytology testing using the ThinPrep Pap Test  were performed on all women enrolled into the IMPACT trial, according to the respective manufacturer's instructions. The use of both cobas 4800 and 6800/8800 HPV tests  on every women allowed for the assessment of the performance of the high‐throughput cobas 6800/8800 HPV test compared to the cobas 4800 HPV test in primary HPV screening, co‐testing with cytology and ASC‐US triage, as described in more detail recently.2.3For the assessment of the performance of DS in triaging HPV‐positive women, only women with positive HPV test results at baseline were included in the analyses. Results for the analysis of DS and comparators as triage tests for women positive for the cobas 6800/8800 HPV test are reported in the main body of the manuscript, whereas results for the cohort of cobas 4800 HPV positive women are tabulated in the 2.4Clinical endpoints for the study were biopsy‐confirmed ≥CIN2  and ≥CIN3 (secondary endpoint). Formalin‐fixed, paraffin‐embedded biopsy tissue specimens were used for preparation of hematoxylin and eosin (H&E)‐stained slides, as well as for p16 immunohistochemical staining using the CINtec Histology kit  according to the manufacturer's instructions. The pathology review result of the respective clinical laboratory was used for clinical management of the patients. For study purposes, all tissue specimens were subjected to a central pathology review (CPR) as previously described in detail.2.5Co‐primary objectives for the IMPACT trial were (a) to evaluate the performance of DS for identification of ≥CIN2/≥CIN3 when used to triage HPV‐positive women, stratified by HPV16/18 vs 12 “other” HPV genotypes, and (b) to compare the performance of DS to that of cytology when used to triage 12 “other” HPV‐positive women. Acceptable performance of DS for the first objective required 1‐negative predictive value (NPV) for ≥CIN3  to be ≤5% for HPV16/18‐positive women. Acceptable performance for the second objective required the same for 12 “other” HPV‐positive women or, if not met, then 1‐NPV for DS be no worse than cytology for the triage of 12 “other” HPV‐positive women.Statistical analyses were performed on the intended use population of HPV‐positive women included in the IMPACT trial using SAS software, version 9.4.Sensitivity, specificity, PPV, NPV (1‐NPV) and positivity rates were reported as both fractions (n/N) and percentages. Two‐sided 95% confidence intervals (CIs) were calculated using (a) Wilson score method for sensitivity and specificityThere were no missing data for DS results, and unknown CPR reference diagnoses were not imputed. In disposition tables, the number of cases with unsatisfactory DS results is shown, and distributions for CPR results are shown for cases with both satisfactory and unsatisfactory DS results to enable assessment of potential bias.A target sample size of 3500 HPV‐positive women was set in order for 95% CIs for 1‐NPV for co‐primary objectives to span ~3.2%. The obtained sample size of 5250 HPV‐positive women resulted in precision greater than planned.33.1A total of 5250 women with positive cobas 6800/8800 HPV test results at baseline were included in this analysis. For this cohort, the mean age at enrolment was 37.1 years (SD: 10.3), and median age was 34.0 years . The proportion of women aged 25 to 29 years was 29.9% (1568/5250). These and further study population characteristics and descriptive statistics are provided in Table 3.2). Within the cobas 6800/8800 HPV‐positive study population with valid DS results, 536 women with ≥CIN2 were diagnosed at baseline and 632 women were diagnosed with ≥CIN2 cumulatively at baseline and/or year‐1  when used to triage women aged 25 to 65 years with positive primary screening HPV test results, stratified by HPV16/18 vs 12 “other” genotype groups  for ≥CIN3 at baseline were 1.9%, meeting one of the prespecified acceptance criteria for the co‐primary study objective of the trial (1‐NPV: ≤5.0% for ≥CIN3). Overall, similar sensitivity, specificity, PPV and 1‐NPV estimates were observed for cumulative vs baseline disease endpoints, that is, for ≥CIN2 (≥CIN3) detected at baseline and/or after the 1‐year follow‐up (Table P < .0001), cutting the number to less than half  or cytology alone  (Table P < .0001), but significantly lower than observed for cytology alone . Of note, triage with DS alone would have referred significantly fewer women to colposcopy than HPV16/18 genotyping with cytology triage for 12 “other” HPV‐positive women , leading to significantly higher efficiency as shown by the lower number of 4.09 vs 5.35; P < .0001) colposcopies to be performed per ≥CIN2 detected  Table . Similar3.5P = .0181), or cytology alone  . Similar to the results seen in women with 12 “other” genotypes, DS‐negative, HPV16/18‐positive women also had a lower risk of ≥CIN3 than those who were cytology negative, HPV16/18 positive. Similar performance estimates were observed for cross‐sectional data analysis using the baseline colposcopy data and after a 1‐year follow‐up period, and DS triage met the prespecified primary study objectives of the IMPACT trial.The comparative performance of DS vs cytology in the current trial is similar to what was previously reported from the ATHENA study, but differs from what was reported in a 3‐year follow‐up study of HPV‐positive women from Kaiser Permanente Northern California (KPNC).P < .0001). Similar findings were found in the 3‐year KPNC study.Since the risk of ≥CIN2 or ≥CIN3 in DS‐negative, HPV‐positive women was low, irrespective of HPV genotype, we evaluated the performance of DS as a stand‐alone triage test for HPV‐positive women. DS used as the sole triage tool for HPV‐positive women provided significantly better sensitivity and specificity than the current algorithm of HPV16/18 genotyping and cytology triage of 12 “other” genotypes. When DS is used alone to triage HPV‐positive women the cumulative risk (1‐NPV) of ≥CIN3 in triage‐negative women was only 1.4% compared to 2.3% in women who were triage negative using the algorithm of HPV16/18 genotyping with cytology triage of 12 “other” genotypes. Triaging HPV‐positive women with DS alone would have referred a significantly lower number of women to colposcopy vs HPV16/18 genotyping with cytology triage of 12 “other” genotypes  guidelines take a risk‐centered approach to patient management based on a woman's risk of ≥CIN3.Another activity that utilizes considerable screening resources is retesting triage‐negative women at 12 months. Risk cutoffs for returning women to routine screening vary. The ASCCP recommends that only women with ≤0.55% 5‐year risk of ≥CIN3 return to routine screening.Our study has several strengths and limitations. Strengths include that IMPACT was a large prospective study that enrolled women in 32 clinical centers and assessed DS test performance in 4 central laboratories that also performed the cytology and HPV testing. Disease ascertainment was maximized by referring all women who tested positive with either of two HPV tests or who had an abnormal cytology to colposcopy. Women who fulfilled the initial colposcopy referral criteria were followed‐up at 1 year, and those who were either HPV or cytology positive at 1 year were referred for another colposcopy. To eliminate potential study bias, colposcopy was performed blinded to all test results and a nontargeted biopsy was collected when no lesion was identified at colposcopy. A CPR was performed on both H&E and H&E + p16‐stained biopsy specimens. Limitations include the fact that the study follow‐up was limited to 1 year and therefore the assessment of the negative disease prediction of a negative DS for a period longer than 1 year cannot be made.In conclusion, the results of the IMPACT trial demonstrate that DS is safe and effective for the triage of HPV‐positive women identified during primary HPV screening. DS alone or in combination with HPV16/18 genotyping offers an alternative to current triage strategies which are based on cytology, either alone or combined with HPV16/18 genotyping. DS‐based triage provides consistently higher sensitivity than cytology‐based triage, providing better reassurance against ≥CIN2 and ≥CIN3. Using DS alone as the triage reduces the complexity of triage strategies for HPV‐positive women.Drs. Stoler and Wright are consultants to Roche, BD Life Sciences, Inovio, and QSquared Solutions. They are speakers for Roche and BD Life Sciences. The other authors are employees of Roche.ClinicalTrials.gov.All patients enrolled into the trial provided their informed consent before any study procedures. The study protocol and all amendments were approved by an Institutional Review Board (IRB). The trial was conducted in compliance with International Conference on Harmonization (ICH) Good Clinical Practice (GCP) Guidelines, applicable regulations of the U.S. Food and Drug Administration (FDA), and in accordance with the ethical principles originating in the Declaration of Helsinki. This is an observational, noninterventional diagnostic study and in alignment with FDAAA2007, World Health Organization (WHO) and ICMJE was not registered with Supplementary Figure 1 Flow of subjects through baseline and follow‐up phases of the IMPACT trialSupplementary Figure 2. CONSORT diagram. Triage of cobas 4800 HPV‐positive womenSupplementary Figure 3. Risk of ≥CIN3 in cobas 4800 HPV‐positive women dependent on HPV genotype group, cytology, and Dual‐stain resultsSupplementary Table 1. Demographic characteristics and HPV vaccination status by Dual‐stain results of the cobas 6800/8800 HPV‐positive study populationSupplementary Table 2. HPV genotype and Dual‐stain results by baseline cytology and cumulative 1‐year histology results among cobas 6800/8800 HPV‐positive womenSupplementary Table 3. HPV genotype and Dual‐stain results by cytology and baseline histology results among cobas 4800 HPV‐positive womenSupplementary Table 4. HPV genotype and Dual‐stain results by baseline cytology and cumulative 1‐year histology results among cobas 4800 HPV‐positive womenSupplementary Table 5. Triage of cobas 4800 HPV‐positive women with Dual‐stain and cytology by HPV genotype group: baseline and cumulative 1‐year data for ≥CIN2 and ≥ CIN3Supplementary Table 6. Triage performance of Dual‐stain and cytology, alone or in combination with HPV16/18 genotyping for detecting ≥CIN2 and ≥ CIN3 in cobas 4800 HPV‐positive women: baseline and cumulative 1‐year dataClick here for additional data file.


This study proposed a new theoretical framework that combines the protection motivation theory and the elaboration likelihood model to examine how health information processing patterns influence parents' vaccination decision-making on behalf of their daughters. Based on survey data from 359 parents of girls aged 9–15, we tested the theoretical model by using structural equation model. The results showed that the central route, represented by information quality, affected the parents' perceptions of HPV severity and susceptibility; the peripheral route, represented by source credibility, influenced their perceptions of HPV severity, HPV susceptibility, vaccine response efficacy, and secondary risks. Also, Chinese parents' perceptions of HPV vaccines, not perceptions of HPV, affected their intention to vaccinate their daughters. The study suggests in addition to improving the quality of health information, the peripheral route, such as the release of vaccination photos, public immunization evaluations, and case narratives, should also be used to change parents' perceptions. Besides, reducing the traditional stigmatization of female sexuality and improving parents' understanding of the new generation's sexual attitudes will increase parents' intention to have their daughters vaccinated against HPV. China Family Development Report, adolescents' first sexual experience takes place on average at the age of 15.9 years, and in recent years this average has trended upward  to vaccination behavior and position it within the framework of the elaboration likelihood model (ELM) to derive hypotheses about the effects of different information processing routes using data from Chinese parents of girls aged 9–15.Theoretically, the study explains the influence of parents' health information processing patterns on the health decisions they make for their children; it finds a shift in the explanatory model of media effects from media exposure to media information processing. Empirically, the quality and sources of information are also likely to influence child-protective behavior; thus, health organizations should focus on providing high-quality information and countering online disinformation through credible media.PMT has been frequently used in recent years to explain people's attitudes and behaviors toward vaccination . The thePMT has been widely used in health behavior studies. Studies have examined the relationship between various elements of PMT and treatment behaviors in the areas of pregnancy exercise , malariaPMT emphasizes the role of cognitive mediating processes in health behavior change. Threat appraisal and coping appraisal are initiated when people confront information sources . Threat HPV vaccination behaviors are influenced by perceived HPV severity, perceived HPV susceptibility, and vaccine response efficacy from PMT . These aHowever, PMT does not consider the current risk culture, where people adopt protective behaviors while worrying about a range of secondary risks associated with the protective behaviors. Pawlowski  argued tIn China, the news is often criticized for acting as a “magnifying glass” for vaccine risks . The newH1a: Perceived HPV severity is positively associated with parents' intention to vaccinate their daughters against HPV.H1b: Perceived HPV susceptibility is positively associated with parents' intention to vaccinate their daughters against HPV.H1c: Perceived response efficacy of HPV vaccines is positively associated with parents' intention to vaccinate their daughters against HPV.H1d: Perceived secondary risk is negatively associated with parents' intention to vaccinate their daughters against HPV.The media ecosystem is considered an information source in the PMT, influencing individuals' cognitive mediation process. Individuals generally do not have a systematic knowledge of viruses or vaccines, and the media has become the main channel for individuals to acquire health knowledge , 32 and Although media exposure profoundly influences people's cognitive mediation processes and subsequent health-protective behaviors as an information source, how individuals process the health information they encounter and the effects of information processing patterns remain unknown . Petty aWhen individuals consider health information in depth, the information is often processed through the central route in a way that produces a stronger persuasive effect. In this case, individuals' assessment of health information quality occurs through the central processing route, which significantly influences their willingness to adopt the information . AccuracHowever, individuals do not always process health information thoughtfully. They may be exposed to it passively or unconsciously , such asWhen parents process HPV-related health information through the central route, they focus on information quality. However, when parents do not expend efforts to obtain high-quality information or fail to assess information quality, source credibility becomes the most commonly used peripheral cue to influence their perceptions. The underestimation of HPV threats and the perceived vaccine risks mainly come from misleading information or the inherent impression from previous vaccine risk events . When paH2a: Information quality is positively associated with parents' perception of HPV severity.H2b: Information quality is positively associated with parents' perception of HPV susceptibility.H2c: Information quality is positively associated with parents' perception of the response efficacy of HPV vaccines.H2d: Information quality is negatively associated with parents' perception of the secondary risk of HPV vaccination.H3a: Source credibility is positively associated with parents' perception of HPV severity.H3b: Source credibility is positively associated with parents' perception of HPV susceptibility.H3c: Source credibility is positively associated with parents' perception of the response efficacy of HPV vaccines.H3d: Source credibility was negatively associated with parents' perception of the secondary risk of HPV vaccination.This study combines the ELM and the PMT to explain how parents' processing of HPV-related health information affects their protection motivation and thus influences their decision to immunize their daughters against HPV. In the integrated model, parents in the pseudo-environment constructed by the media (information source) activate either the central route  or the peripheral route (source credibility assessment) for processing HPV-related information. Both processing routes may influence their threat perceptions of HPV, response efficacy perceptions of the vaccine, and risk perceptions (cognitive mediating processes), ultimately influencing their decisions whether to have their daughters vaccinated against HPV (coping mode). The study framework is shown in We selected three primary and four junior high schools in Hubei Province by stratified sampling and then 24 classes from Grades Four through Nine (four classes per grade) by generating random numbers. Questionnaires were distributed to the girls' parents, who filled them out on a volunteer basis; they were given a gift worth about 10 RMB as a small honorarium. We obtained informed consent forms from all the participants. This study was approved by the Research Ethics Committee of the corresponding's author's university.The questionnaire was divided into two parts with a total of 43 items. The first part measured demographic variables, and the second part measured the research variables across seven dimensions: information quality as the central processing route, source credibility as the peripheral processing route, HPV severity, HPV susceptibility, secondary risk, vaccine response efficacy, and intention to vaccinate their daughters. The questionnaire was adapted from previous studies, and items were assessed on a 5-point Likert scale, with 1 = “strongly disagree” and 5 = “strongly agree.”The online HPV and vaccine information is exactly what I need.”), understandability , adequacy , and usefulness . In the exploratory factor analysis, only one factor was extracted from the items of information quality. Therefore, these four dimensions did not appear as variables, respectively.The information quality and source credibility scales were mainly drawn from Lim and Kim 's questiThe information about HPV vaccination released by relevant government agencies  is very credible” and “The information about HPV vaccination released by the official media  is very credible.”Source credibility (Cronbach α = 0.830) assessed parents' trust in the source of health information. The items included “HPV is scary to girls” and “HPV infection can have a serious impact on a girl's health.”The HPV severity and HPV susceptibility scales were based on Patty et al. 's study.HPV is highly contagious for girls” and “There is a high possibility of HPV infection after having sex for girls.”HPV susceptibility was measured with two items (Cronbach α = 0.715) that assessed parents' perception of the probability of their daughters being infected with HPV. The items included “Side effects of HPV vaccines can cause serious illness to girls” and “Side effects of HPV vaccines can affect girls' daily activities.” Secondary susceptibility was measured with four items such as “Girls who receive HPV vaccination are prone to side effects” and “Juveniles who receive HPV vaccination are prone to side effects.”Secondary risk was measured with eight items (Cronbach α = 0.933), which focused on parents' perceptions of side effects of HPV vaccines and included two dimensions. Secondary severity was measured with four items such as “HPV vaccines can increase girls' resistance to HPV” and “HPV vaccines can effectively prevent girls' genital warts, cervical cancer, and other diseases in future.”Vaccine response efficacy was measured with three items (Cronbach α = 0.886) that examined parents' confidence in HPV vaccines' benefits, including “I am considering getting my daughter vaccinated against HPV” and “I will get my daughter vaccinated against HPV according to the provided vaccination schedule.”Intention to vaccinate was measured with three items (Cronbach α = 0.956) that assessed parents' consent regarding the HPV vaccination of their daughters. The scale was adopted from Yang , and incIn addition, demographic variables were included in the study as control variables, including parents' gender, age, place of residence, education, occupation, income, and number of children, and the ages of their daughters.Response rate was 38.6 % and a total of 435 questionnaires were collected. After data screening, 76 invalid questionnaires were excluded because the answers to different questions were too similar, and 359 questionnaires were finally included in the data analysis. The sample consisted of 72 fathers (20.1%) and 282 mothers (78.6%), and 5 respondents did not provide their gender (1.4%). The mean age of the parents was 40.95 years old (SD = 4.05); 58.8% of the parents had only one daughter, 15.9% had two daughters, and 22.6% had one son and one daughter. The mean age of the daughters was 12.96 years old (SD = 1.40). 93.3% of the parents were currently living in an urban area.The results of the descriptive analysis are shown in t-test, parents differed significantly only in the assessment of source credibility, as shown by the fact that fathers  trusted online sources significantly more than mothers . The rest of the variables did not show any difference between fathers and mothers.According to the results of the independent sample The model fit was assessed using the ratio of the chi-square value and the degree of freedom value, root mean square error of approximation (RMSEA), Tucker-Lewis index (TLI), and comparative fit index (CFI). The results showed a chi-square value of 2694.727, a degree of freedom of 819, and a ratio of 3.29. The TLI value of 0.838, CFI value of 0.848, and RMSEA value of 0.08 indicated an acceptable model fit.The reliability of the measurement model was assessed using the composite reliability (CR), with a CR value above 0.7 indicating good intrinsic model quality . The resDiscriminant validity was evaluated by comparing the square root of the AVE values with the absolute values of the correlation coefficients between different variables. The square roots of the AVE values in this study were all greater than the absolute values of the correlation coefficients between the corresponding latent variables and other variables ; the dis and HPV susceptibility , confirming hypotheses H2a and H2b. Source credibility was positively correlated with perceived HPV severity , HPV susceptibility , and vaccine response efficacy , confirming hypotheses H3a, H3b, and H3c. Source credibility was negatively correlated with the perceived secondary risk of HPV vaccines , confirming hypothesis H3d. Perceived HPV vaccine response efficacy was positively correlated with parents' intention to vaccinate their children against HPV , confirming hypothesis H1c. Perceived secondary risk was negatively correlated with parents' vaccination intention , confirming hypothesis H1d. The relationships between source credibility and vaccine response efficacy  and between source credibility and secondary risk  were insignificant; hypotheses H2c and H2d were therefore not confirmed. The relationships between HPV severity , HPV susceptibility , and vaccination intention were not significant either, and therefore hypotheses H1a and H1b were not confirmed. Thus, 8 out of 12 paths in the hypothesized model were valid.The relationships between the different latent variables were assessed by examining the structural model. The hypothesized model and standardized model coefficients (standard errors) results are shown in Structural equation model testing was separately used to examine the mediating effects of vaccine response efficacy and secondary risk between source credibility and parents' intention to vaccinate their daughters; model 4 in PROCESS 3.2 was used, with a confidence interval of 0.05. If the lower and upper limits of the confidence interval do not contain 0, the mediating effect of the variables is confirmed.As This study examines how the information processing patterns of individuals affect their cognition and produce persuasive effects. The results show that the central route, represented by information quality, influences parents' assessment of HPV severity and susceptibility. The peripheral route, represented by source credibility, affects parents' perceptions of HPV severity, susceptibility, vaccine response efficacy, and secondary risk simultaneously. Perceptions of vaccine response efficacy and secondary risk are associated with parents' intention to have their daughters vaccinated against HPV. No significant relationships were found between HPV severity and susceptibility and vaccination intention. In addition, this paper shifts the persuasion effect of health information from media exposure to information processing. Previous studies have focused on the influence of media exposure on perceptions of vaccination and subsequent behaviors –56. ThisWhen parents processed information about HPV and vaccines using the central route, information quality positively predicted their perceptions of HPV severity and susceptibility. This finding suggests that parents' thoughtful processing of vaccine-related information influences their assessment of HPV severity and susceptibility, and that higher information quality is associated with stronger perceptions of virus severity and susceptibility. This is consistent with previous research , which hHowever, information quality did not predict parents' perceptions of vaccine protection and risks, i.e., the central route failed to influence parents' perceptions of protective behaviors. This contradicts a previous study of the factors that influence the level of employee engagement in information security protective behaviors . That stFirst, vaccine knowledge is more complex and multidimensional than perceptions of virus severity and susceptibility. High-quality information may not give “laypeople” a comprehensive knowledge of vaccines, and it is difficult to obtain systematic explanations of vaccines from the news. Respondents said, “There are too many medical terms in the news about vaccines, which are quite obscure”; “I don't know much about vaccines, mRNA, or protein.”Second, most parents were born in the 1970s and remembered the vaccine incidents widely reported by the media. It is difficult to dispose of stereotypes even with high-quality news explanations. “When I was a child, there were reports that the hepatitis A vaccines seemed to have killed a few elementary school students. Vaccines involve injecting the virus into bodies after all, so I still have to be careful.”Third, vaccines cannot be disentangled from other areas of society, such as the political and commercial spheres. Vaccine information is a complex mediator in the knowledge production of biosciences, market organizations, and other social groups . Social The level of trust in the information source can simultaneously influence parents' perceptions of HPV severity, susceptibility, vaccine response efficacy, and secondary risk, suggesting that the peripheral route significantly influences parents' perceptions of the virus and vaccines. This result is similar to the findings of previous research  into thePeople's Daily says it works, so it should work, right?” “Dr. Wenhong Zhang also took his daughter to get HPV vaccines, so it should be safe.” Therefore, compared to the central route, rich peripheral cues help parents form correct perceptions of HPV and vaccines.Further, when the media that parents trust release health information, their attitudes toward viruses and vaccines may be influenced even though they have not processed the information in depth. “Overall, this study suggests that perceptions of HPV are not significantly associated with parents' intention to vaccinate their daughters. Instead, perceptions of the vaccines predict parents' intention. Specifically, although parents recognize HPV as dangerous and easily transmitted, it does not mean that they will have their daughters vaccinated. Parents are only likely to immunize their daughters when they perceive HPV vaccines to be an effective, low-risk defense against the virus. In Chinese folk tradition, HPV is associated with female infidelity, and HPV infection has been stigmatized as a “sign of dishonor.” Traditional gender attitudes are deeply rooted, and it would be a family scandal if a daughter were infected with HPV.The interviews reveal that while parents believed HPV is a serious, easily transmitted disease, they also believe the chances of their own daughters being infected to be extremely low. Respondents said, “My daughter is clean, so it is impossible for her to get HPV”; “HPV is sexually transmitted, but my daughter is very well behaved.” PMT provides a multiplicative model for the Chinese context. If any of the four components is missing, the protection motivation will be weakened and the intention to vaccinate will be reduced. One of the interviewed parents said, “My daughter is unlikely to be infected with HPV. The vaccine is a virus after all, it is safer not to vaccinate. Human genes can be changed by the vaccine”; another remarked, “The HPV vaccines are given to those who have frequent sex.” This shows that parents will only increase their intention to vaccinate their daughters if they believe that their daughters are at risk for HPV infection and that the vaccine is effective and safe.This study shows that perceived vaccine response efficacy and perceived secondary risks have significant mediating effects on the relationship between source credibility perceptions and vaccination intention. This suggests that after processing vaccine information through the peripheral route, parents consider having their daughters vaccinated when they believe the vaccines are effective and safe. The mediating effects demonstrate the existence of a “media-cognition/motivation-behavioral intention” influence model. Parents immersed in the information sources reconsider and change their perceptions of the vaccines after processing the information through central and peripheral routes, and may then adopt vaccination behavior.This study has some shortcomings. First, we used the self-reporting method to obtain the subjects' behavioral data, which is subjective . The scaThis study shows that the central and peripheral routes affect different aspects of protection motivation. The central route affects parents' perceptions of HPV severity and susceptibility and the peripheral route influences parents' perceptions of HPV severity, HPV susceptibility, vaccine response efficacy, and secondary risks. Therefore, in addition to improving the quality of health information, the peripheral route should also be used to change parents' perceptions. Studies have shown that pictures and photos, price, and narrative approaches are also considered rich peripheral cues . As partIn addition, perceptions of HPV do not affect parents' intention to vaccinate their daughters. Even if Chinese parents fully learn about the severity and infectiousness of HPV, they still believe that their daughters will not be infected due their conservative attitudes. Nonetheless, the age of young women's first sexual experience in China is now 15 years old, and sexual attitudes tend to be open and tolerant generally . ReducinThe datasets generated and analysed during the current study are not publicly available due to the consideration of participants' privacy, but are available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by the Research Ethics Committee of Zhongnan University of Economics and Law. The patients/participants provided their written informed consent to participate in this study.Research methodology: QW, FZ, and WZ. Formal analysis: FZ and QW. Survey and data collation: CT and WZ. Writing–original draft preparation: FZ. Writing–review and editing: QW. Supervision: WZ. Funding acquisition: QW and WZ. All authors have reviewed and agreed to the published version of the manuscript.This study was supported by the Ministry of Education of China, Humanities and Social Science Foundation Research Fund (No. 21YJC760081).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cervical cancer is estimated to cause 341,831 deaths each year, with 9 of 10 deaths occurring in developing countries. Over the past decade, there has been a significant increase in cervical cancer incidence among women in the Philippines. Persistent infection with high-risk human papillomavirus (HPV) is the well-established necessary cause of cervical cancer. Based on limited studies conducted in the Philippines, the prevalence of infection with any HPV genotype was 93.8% for cervical squamous cell carcinoma and 90.9% for cervical adenocarcinomas. HPV types 16 and 18 were the most common HPV genotypes among Filipino patients with cervical cancer. On the other hand, the incidence of HPV infection among Filipino women with normal cervices was 9.2%. The World Health Organization has launched a global agenda of eliminating HPV infection by 2030. One of its key milestones is to vaccinate 90% of girls with the HPV vaccine by 15 years. However, the HPV vaccination rate among Filipino women remains to be unsatisfactory. HPV vaccination has only been included in the Philippine Department of Health's community-based National Immunization Program in 2015. Despite these efforts, the Philippines currently ranks last on HPV program coverage among low-middle income countries, with coverage of only 23% of the target female population for the first dose and 5% for the final dose. The principal reason for the non-acceptance of HPV vaccines was the perceived high cost of vaccination. The low utilization of available cervical cancer screening tests such as Pap smear and visual inspection with acetic acid hampered the Philippines' control and prevention of HPV infection and cervical cancer. Among those diagnosed with cervical cancer in the Philippines, only an estimated 50% to 60% receive some form of treatment. To this end, we summarize the burden of HPV infection and cervical cancer on Filipinos and the risk factors associated with the disease. We present the current screening, diagnostics, treatment, and prevention of HPV-related diseases in the Philippines. Lastly, we also propose solutions on how each building block in health systems can be improved to eliminate HPV infection and reduce the burden of cervical cancer in the Philippines. Human papillomavirus (HPV) infection is the most common sexually transmitted infection worldwide. Aside from causing anogenital warts, persistent infection with high-risk HPV genotypes is the well-established necessary cause of cervical cancer . It is eCurrently, cervical cancer is the fourth leading cancer in women worldwide and the second most common cancer among women of reproductive age . CervicaThe World Health Organization (WHO) has launched a global agenda of eliminating HPV by 2030. One of its key milestones is to vaccinate 90% of girls with HPV vaccines by 15 years of age . DespiteIn the Philippines, 37.8 million women are at risk for cervical cancer. The country has an annual burden of 7,897 cervical cancer cases and 4,052 deaths . Despitevia cervical cytology, 42.1% among those with high-grade squamous intraepithelial lesions (HSIL), and 58.6% among women with cervical cancer  1 to CIN 3 remains unclear, it is hypothesized that increased expression of E6 and E7 by high-risk HPV genotypes influences cellular progression to carcinogenic phenotypes. This contrasts with the low E6 and E7 expression levels in CIN 1 lesions . AccumulCervical cancer is the second leading cancer among women in the Philippines despite being the fourth leading cancer among women globally, next to breast, colorectum, and lung cancers . This maHuman immunodeficiency virus (HIV) infection is a significant risk factor for HPV infection, persistence, and associated cancers. This is related to suppression of the immune system, which usually clears HPV in most infected women, and increases the risk of exposure among sexually active adults . A meta-Chlamydia trachomatis is also an essential co-factor for cervical carcinogenesis. It can cause chronic inflammation, disrupt epithelial integrity, and induce cervical metaplasia. These effects may lead to HPV viral load enhancing, genome integration, and genomic instability, synergizing cervical cancer transformation (C. trachomatis seropositive women (odds ratio (OR) 1.80; 95% confidence interval (CI) 1.22–2.66), which increased with higher C. trachomatis titers in women under 55 years of age  between cervical cancer and control group (chronic cervicitis patients). However, this study showed that 22.73% of HPV-positive patients were co-infected with Ureaplasma spp. and 9.09% with Mycoplasma spp.   . Propose2–12.47) , 43.Early age at first sexual intercourse is associated with an increased risk of HPV infection due to increased exposure and riskier sexual behavior such as having unprotected sex and having multiple sexual partners . A meta-Age at first parity is a significant risk factor in cervical cancer . One posHigh parity is also considered a significant risk factor for cervical cancer, as shown in a multi-center study from eight case-control studies showing a direct association of high parity with HPV infection. The odds ratio for seven full-term pregnancies or more was at 3.82 (95% CI 2.66–5.48) as compared to nulliparous women, and 2.25 (95% CI 1.57–3.22) as compared to women with one or two pregnancies . This waThe use of oral contraceptive pills (OCP) is associated with an increased risk of invasive cervical cancer. This risk increases with increasing duration of use and declines after cessation of OCP use. In less developed countries, the use of OCP for 10 years from around age 20–30 was estimated to increase the cumulative incidence of invasive cervical cancer from 7.3 cases per 1,000 women to 8.3 cases per 1,000 women by age 50 . A multiSmoking is a well-established risk factor for cervical cancer, as supported by several studies , 50–53. Although the molecular mechanisms of HPV-associated cancer development are not well established, there have been several immune response genes from host innate immunity and adaptive immune response associated with regression, persistence, or progression of HPV. Since most individuals can eliminate the virus in 12–24 months without intervention, the role of host genetic differences will be deemed necessary in determining the risk of developing cancer. Polymorphisms and variations in these genes may confer susceptibility or prediction to the development of cervical cancer. However, these findings need further evaluation . To our TP53 on HPV-related cancers. TP53 codes for p53, a nuclear transcription factor which activates target genes that facilitate cell cycle arrest, allowing cells to either repair damaged DNA or undergo apoptosis in the presence of irreparable DNA damage , BRCA1-associated ring domain protein 1 (BARD1) gene, primary microRNA-218 (pri-miR-218), and laminin-5 β3 (LAMB3) were also associated with increased risk of cervical cancer , which is essential in defense against viruses and intracellular pathogens and induction of immune-mediated inflammatory responses, has also been associated with increased cervical cancer risk , tumor necrosis factor-alpha (TNF-α), and interleukin 12 (IL-12), have been implicated in cervical carcinogenesis , 63–67. cer risk , 72. Stuesponses , 74.PIK3CA mutation is associated with poor treatment response and low survival rate, while MDM2 is associated with the development of cervical cancer and poor prognosis  was associated with lower cervical cancer rates than TT genotype  are overlooked health factors. However, the most fundamental causes of health disparity can be attributed to socioeconomic inequalities . SocioecPoor accessibility of health care is reflected in the high mortality of patients with cervical cancer. The Philippines has the fourth highest age-standardized mortality rate in Southeast Asia with 7.89 per 100,000 women per year, next to Myanmar and Indonesia (14.4), Timor-Leste 8.76), and Cambodia (8.33) . The ove, and CamRelated to health care access is health-seeking behavior and health literacy, as they are also intimately linked to socioeconomic factors. Health literacy is described as the ability to access, understand, appraise, and apply health information in terms of health care, disease prevention, and health promotion . AccordiFinally, the social environment, which is greatly affected by socioeconomic status, largely affects the behavioral risk factors for HPV infection as previously discussed above. The risk of sexually transmitted infections may increase due to lack of access to condoms and health education, and early pregnancy and marriage are related to education . AccordiOverall, there is a lack of local studies that portray how sociodemographic disparities directly contribute to the high burden of cervical cancer in the Philippines. However, their proxy variables, e.g., low income and educational attainment, high poverty incidence, and poor health literacy, may reflect how women of lower socioeconomic status in the country are rendered more vulnerable to cervical cancer.via the Bethesda system  is an acceptable alternative approach to pap smear in low-resource settings. It is a simple test with locally available supplies and can be performed by trained health workers. The result is immediate, appearing as acetowhitening, which allows VIA to be combined with treatment procedures for early cervical lesions such as cryotherapy , 97. NumDespite the introduction of pap smear in the Philippines during the 1990s and subsequent promotion of VIA as an alternative screening approach to a pap smear in 2005, screening utilization remains low. In a household health survey done in 2003, the estimated coverage of cervical cancer screening in women aged 18–69 was 7.7%, with 9.3% coverage in urban areas and 5% coverage in rural areas . More reAmong the factors identified that can be attributed to the failure of cervical cancer screening were (1) lack of knowledge about symptomatology of cervical cancer, (2) perception that cancer is fatal and lack of awareness that cervical cancer is treatable, (3) unavailable screening and treatment facilities and expertise, and (4) inconsistent patient adherence to follow-up consults and treatment . There avia Proclamation No. 368, which declares May as Cervical Cancer Awareness Month , which is responsible for implementing universal health coverage, started coverage of basic outpatient services including pap smear  guidelines emphasize a risk-based strategy aligned with current knowledge on HPV natural history and cervical carcinogenesis . More frIn their clinical practice guidelines, the Society of Gynecologic Oncologists of the Philippines (SGOP) advises post-treatment monitoring after 6 months then annually for 3 years. They further support the use of vaccination against HPV types 16 and 18 as efficacious against persistent HPV infection and CIN 2/3 , 129. DuEvidence supports post-treatment or therapeutic vaccination to reduce the risk of clinical disease relapse after treatment . A studyLactobacillus spp., in inhibiting cellular cervical pathogenesis by producing bacteriocins, lactic acid, and hydrogen peroxide. Ongoing studies may soon further elucidate the underlying physiology of microbe- and host-microbe interactions with HPV infection  Cancer Report, with an update published in 2021 . This program is funded by the Cancer Assistance Fund, established under Republic Act 11215 or the National Integrated Cancer Control Act passed in 2019  in 2019 , cervicaWith the information collated and analyzed in this review, we propose the following national research agenda related to cervical cancer and HPV infection in the Philippines. First, implementation research should be done to develop a locally applicable system on how to implement a national program on cervical cancer screening and HPV vaccination. These include highlighting opportunities and addressing challenges in quality of health service delivery, availability of medical supplies including vaccines and screening materials, presence of trained health personnel to administer the services, establishment of a national registry that can provide timely data to implementers and policymakers, and cost-effectiveness of different interventions . Second,RL, OT, and SdP-S edited and proofread the manuscript. All authors read, approved the final manuscript, and drafted the manuscript.RL, LC, GP, OT, IT, and SdP-S are past and current trainees of the MD-Ph.D. in Molecular Medicine Program, supported by Department of Science and Technology—Philippine Council for Health Research and Development (DOST-PCHRD), and administered through University of the Philippines Manila. Funding support for this publication was provided by MD-Ph.D. in Molecular Medicine Program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cervical cancer has become a worldwide concern owing to its high incidence and mortality rates. To date, high-altitude areas of Tibet have not benefited from any large-scale cervical cancer screening programs. Therefore, we initiated a screening program to investigate the prevalence of human papilloma virus (HPV) and HPV genotype distribution to reveal cervical cancer and its precursor which lead to morbidity among women in the city of Nagqu in northern Tib3et.A total of 25,173 women were recruited to undergo HPV genotype tests between June and December 2019. Women infected with HPV 16 and/or 18 underwent colposcopy and histological examination. Women with other high-risk HPV type (hr-HPV) underwent cytological tests to determine whether to conduct further colposcopy and histological examination for diagnosis. HPV prevalence was calculated in the total population and further stratified according to various parameters, such as age group, area location , and single or mixed infection status. The HPV genotype distribution was also investigated accordingly. Cervical lesions revealed by further colposcopic findings were also analyzed; high-grade and malignant lesion morbidities were calculated in total and in each county. Most data were collected and analyzed using descriptive and consistency check statistical methods, and a risk factor investigation for HPV infection was performed using logistic regression models.The total HPV infection rate among women in Nagqu was 13.42%. Of the 25,173 women in the study, 999 (3.97%) were HPV 16/18 positive, 2,379 (9.45%) were other hr-HPV-positive, and 21,795 (86.58%) were HPV-negative. The five most common HPV genotypes, accounting for more than 60% of all HPV infections in Nagqu people, were HPV 16, 58, 31, 18, and 52. Tibetan women younger than 20 years and older than 60 years were the two age groups with the highest rates of HPV infection, 26.7% and 19.8%, respectively. Among the HPV-positive women, 2,656 (78.33%) were infected with a single strain and 732 (21.67%) were infected with multiple strains (more than two genotypes). HPV prevalence increased in high-altitude areas  and decreased in relatively low-altitude areas . Multiple analyses showed that age, parity, age at first delivery, and altitude of residence were independent factors facilitating HPV infection in Tibetan women. High-grade and malignant cervical lesions revealed by histological findings were different among living locations, with the highest rates in Xainza, Baingoin, and Nyainrong, these being 2.019%, 1.820%, and 1.116%, respectively, among women in these areas.Our survey provides an overall perspective on HPV genotype infection and cervical lesions in women in northern Tibet. The data not only provide useful information for the treatment of cervical lesions but also has great value in terms of the primary and secondary prevention measures that can be taken for women living in these regions.www.chictr.org.cn, indentifier ChiCTR2000035061. Cervical cancer is the third most common cancer worldwide and the fourth most common cancer among Chinese women. Reportedly, there are 34,000 and 275,000 cervical cancer-related deaths in China and globally, respectively . Human pSeveral cervical screening programs have been developed and applied to populations in various countries to identify precursors of cervical cancer. Multiple strategies are available for cervical cancer screening. The cheapest and simplest method of visual inspection with acetic acid has a high false-positive rate, requires a large number of trained clinicians, and is time-consuming, all of which make it difficult to access in Tibet despite its relative simplicity and low cost. The pap smear or cytology test has been used for several decades , 4 ; howIn most parts of China, for cervical cancer screening, we adopt the principle of “Opportunistic screening.” That is, when a woman comes to the hospital for various reasons, doctors suggest additional cervical cancer screening. Usually, combined cytology and HPV tests are used in most Chinese medical affiliations, and patients can afford the expense by themselves , 14. HowThis is the first large-scale cervical cancer screening program conducted in the city of Nagqu, northern Tibet. This study aimed to investigate the prevalence and distribution of HPV genotypes and cervical lesions in Nagqu women. This is the first regional epidemiological study to elucidate the incidence of cervical cancer and its precursor which leads to morbidity in Nagqu.A total of 25,173 married women aged 18–65 years in Nagqu City, Tibet Autonomous Region of China, who were available at home and who consented to undergo screening between June and December 2019 were recruited in this study. HPV genotype testing was the first step for all these participants. Women with positive results were divided into two groups. The first group included those with HPV 16 and/or HPV 18 infections, and the second group included high-risk HPV (hr-HPV) infections with genotypes other than 16 and 18. Women with HPV 16 and/or 18 infection in the first group underwent colposcopy and histological examination at the People’s Hospital of Nagqu. Women in the second group with other types of hr-HPV infection underwent cytology testing initially. If cytology results showed ASCUS  or more in these women, they were requested to undergo further colposcopy and histological examination for diagnosis. The flow diagram . HPV testing was performed using an HPV genotyping sequencing system. This assay could detect 16 hr-HPV genotypes , as described here in detail , 18. BriWomen who required colposcopy examinations were referred to a single expert gynecologist at the People’s Hospital of Nagqu, Tibet. All examinations were conducted according to routine procedures described previously . HematoxSPSS 21.0  was used for statistical analysis. All participants were divided by age group and county to calculate the prevalence of the overall HPV genotype, HPV 16/18, and other hr-HPV genotypes. Additionally, a stratifying strategy was used to analyze the cytology and histology results. To compare differences in HPV infection prevalence and cervical lesion rates among various locations and age groups, the chi-square test was used accordingly. Also, a logistic regression model was used to investigate factors influencing HPV infection. The p value was considered significant when p < 0.05.The total HPV infection rate among women in Nagqu was 13.42%. Of the 25,173 women in the study, 999 (3.97%) were HPV 16/18 positive, 2,379 (9.45%) were other hr-HPV HPV positive, and 21,795 (86.58%) were HPV negative  were singly infected, 575 (17.02%) were doubly infected, 130 (3.85%) were triple infected, 24 (0.71%) were quadruply infected, and only three patients (0.01%) were quintuply infected  had a lower positive rate (12.6%), and the difference was statistically significant.Based on the age groups, as shown in 2, and its 11 counties have different altitudes and different modes of production and life, we calculated the HPV infection rate in women according to each of the 11 areas. Generally, women living in eastern areas with lower altitudes had lower HPV infection rates than those living in western areas with higher altitudes (p = 0.001). Correlation analysis also indicated a significant correlation between altitude and HPV infection rates, p = 0.001, gamma = -0.251 , and 739 women (78.8%) were diagnosed with HPV 16/18. Meanwhile, among other hr-HPV-positive women, only 53.5%  were diagnosed with LSIL. Additionally, in cytology tests of HPV 16/18 and other hr-HPV-positive patients, the diagnosis of atypical squamous cells of unknown significance (ASCUS), atypical squamous cells cannot exclude HSIL (ASCH), high-grade squamous intraepithelial lesion (HSIL), and cancer was very rare. The most dominant difference was the proportion of Negative for Intra-epithelial Lesions or malignancies (NILM). In the 16/18-positive patients, only 3.1% (29 women) were diagnosed with NILM, whereas in other hr-HPV-positive patients, nearly 40% were diagnosed with NILM. The results are summarized in p = 0.001).A total of 2,315 women underwent histological examinations, as shown in p = 0.001.As there were different HPV infection rates in different counties, we next investigated the distribution of HSIL and cervical cancer lesions in each of the 1 different counties. The analysis indicated that, consistent with the HPV infection profiles, patients with HSIL and cancer lesions seemed to have a similar distribution  in high-altitude areas of Ecuador, a Latin American country with a largely high altitude, although it was a hospital-based rather than a community-based investigation . As mentTherefore, we assumed that possible reasons may be attributed to the following two elements: hypoxia from high altitude and cold weather because of extreme climate, both of which facilitate HPV transmission.Although a few studies have suggested a higher risk of HPV prevalence and cervical cancer in women living in high-altitude areas, there is solid evidence that many types of cancer are more common in high-altitude-living people, such as gastric cancer and breast cancer. The mechanism, which has been comprehensively reviewed , is compResearchers have also revealed an association between cold weather and a high risk of many cancers, such as breast, prostate, and colon cancer. The reasons may be related to evolutionary adaption, high concentrations of certain air pollutants, and low levels of serum vitamin D, although any single factor cannot explain this epidemiological phenomenon .Another possible reason we suspected, although with no scientific data, may be attributed to the social and living styles of Tibetan women, especially in pastoral areas. From our examinations and the subsequent interviews, women in pastoral areas in northern Tibet had very poor sanitation, and many of them did not clean their anogenital areas with tissue or other materials after defecation. In addition, in many places, polyandry and premature sex are common in society. Compared with Han women, Tibetan women have a lower probability of using prenatal care or receiving maternal and child healthcare , and TibIn our study, we found that the main form of HPV infection was a single infection, while multiple HPV infections were less common, and the results were consistent with those of previous studies. The five most common HPV genotypes in Nagqu people were HPV 16, 58, 31, 18, and 52, accounting for approximately 60% of all infections. This result differed slightly from those of previous studies on the top five HPV genotypes in China . FortunaAs indicated in other studies, our survey also demonstrated that the age-stratified HPV genotype prevalence has a U-shaped pattern. That is, the infection rate was highest in both the youngest and oldest groups and lowest in the median group . This waAlthough the incidence of HSIL and cervical cancer was nearly the same between the HPV 16/18 infection group and the other hr-HPV infection group, the LSIL frequencies were significantly different . In the As mentioned above, the HPV infection rate correlated with altitude; a similar phenomenon was also observed in the histological results , which wThe strength of this study is highlighted by the fact that it showed a higher prevalence of HPV infection in women residing at higher altitudes in Tibet. Further analysis indicated that HPV infection and cervical lesion incidence might have a certain correlation with location altitude and it might also vary in different age groups. This information not only provides useful information for the treatment of cervical lesions but also has great value in primary and secondary cervical cancer prevention measures for women residing in high-altitude areas.The weakness of our survey included missing some important information of the women participating in the study such as the number of sex partner, smoking or alcohol drinking habits, and economic status, so that statistical analyses could not be performed to investigate more risk factors facilitating HPV infection and cervical precancer pathogenesis. Further studies are needed not only for more rigorous and comprehensive investigations but also for long-term surveillance of HPV-positive Tibetan women.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Nagqu people’s Hospital. The patients/participants provided their written informed consent to participate in this study.QW, YH, FL, CL, ZS, and DG contributed equally to this work and shared first authorship. DZ, LB, ZH, ZS, NJ, XH, JL, KY, SB, and MT helped in writing this article together. ZX and XX are the authors of this article. All authors have made significant contributions to this article. All authors agree to be responsible for the content of this article and have submitted the manuscript.National Natural Science Foundation of Liaoning, China, 2019-BS-073 and Public health scientific research project of Futian District, Shenzhen hospital of Guangzhou University of Chinese Medicine, NO. FTWS2019104. Interregion collaborative innovation science and technology program, QYXTZX-NQ2022-03.Science Foundation of Liaoning Provincial Department of Education LZ2019044.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.