Correction: Journal of Translational Medicine (2022) 20:231 10.1186/s12967-022-03383-xFollowing publication of the original article , we have“We recently carried out a clinical validation of full high-risk HPV genotyping HPV Selfy assay [1] according to the international guidelines for DNA HPV test requirements for cervical cancer screening [2] as well as according to the reference protocol for validation of HPV assays for cervical cancer screening on self‑collected samples, i.e. the VALHUDES protocol [3], and we concluded that: (i) HPV Selfy is a high-risk HPV DNA test clinically validated for cervical cancer screening according to Meijer’s guidelines; and (ii) the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimens, implying that HPV Selfy is a test clinically validated for cervical cancer screening on self-collected samples.Our paper demonstrated the non-inferiority of the performance of HPV Selfy obtained on self-collected vaginal samples applying the non-inferiority test recommended by the VALHUDES protocol [4] and, unfortunately, a statistical error has been made.0 equal to 0.98 instead of 0.95 yielding reported T equal to − 0.86 that implies p value > 0.05. The correct calculation using δ0 equal to 0.95 would quantify T equal to 2.11 implying p value < 0.05. This leads to the conclusion that still, relative specificity on self-samples is indeed significantly non-inferior to that observed in paired clinician-collected samples.We noticed that we inverted two numbers applying the recommended formula to assess the non-inferiority of the sensitivity and specificity recorded on self-collected vaginal samples leading to incorrect results, as noticed by Marc Arbyn et al. in their letter to the Editor [5]. In particular, the number of subjects with discordant “self+ /clinician−” and “self−/clinician+” results ; the test reached adequate intra- and inter-laboratory reproducibility.0.97 relative sensitivity and 0.97 relative specificity). Finally, through HPV Selfy genotyping, we were able to describe HPV types prevalence in the study population.Moreover, we demonstrated that the performance of HPV Selfy on self-collected vaginal samples was non-inferior to the performance obtained on clinician-collected cervical specimen Next, we aimed at evaluating HPV Selfy performance on self-collected samples, as indicated by VALHUDES protocol. Hence, we needed to assess whether HPV testing on vaginal self-samples was as accurate as HPV testing on a cervical sample taken by a clinician. To do so, we identified 119 CIN2+ cases (age 25–65 years) and 791 ≤ CIN1 cases, for which we had available paired cervical specimen and self-collected vaginal samples.93/96; relative sensitivity 0.97; 95% CI 0.90–1.04) and specific  to detect CIN2+ in the total study population .HPV Selfy testing in self-collected samples was found similarly sensitive . Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women of 30 years and older.Meijer et al. (2009). VALHUDES: a protocol for validation of human papillomavirus assays and collection devices for HPV testing on self-samples and urine samples.Arbyn et al. (2018). Sample size determination for establishing equivalence/non-inferiority via ratio of two proportions in matched-pair design.Tang et al. (2002). Can HPV Selfy be considered as a clinically validated HPV test for use in cervical cancer screening?Arbyn et al. (2022). Secondary objectives of VALHUDES protocol include the assessment of the absolute accuracy of HR-HPV DNA test applied according to the sampling device and the proportion of adequate samples as determined by amplification of an internal control (a ubiquitous human gene). HPV Selfy assay provides a human beta-globin internal control, used to evaluate sample quality. In the whole study cohort, mean Ct value for the human beta-globin internal control for the HPV Selfy test on self-collected samples, obtained with the direct analysis on self-collected samples, was 26.1 Ct . In the subgroup of women with biopsy-diagnosis of cervical lesions CIN2+, the same analysis resulted in 26.7 Ct . This means that all women were able to self-collect a similar amount of specimen, confirming self-collected samples’ quality adequacy and so the easiness of the self-sampling procedure using the FLOQSwabs® .


The burden of disease caused by cervical cancer ranked second among female tumors in China. The HPV vaccine has been proven to be a cost-effective measure to prevent cervical cancer, but the vaccination rate remained low to date among university students. This study aimed to understand the status quo of HPV vaccine hesitancy among university students across China during the COVID-19 pandemics and systematically analyze determinants of HPV vaccine hesitancy based on the WHO 3Cs model. Cross-sectional data were collected using an online survey of female university students in four cities across China in June 2022. Multinomial logistic regression was adopted to determine factors influencing vaccine hesitancy based on the 3Cs model with three dimensions, namely complacency, convenience, and confidence. Among 1438 female university students surveyed in this study, 89.7% did not hesitate to vaccinate against HPV, only 8.9% hesitated to some extent, and 1.4% refused to vaccinate. The actual vaccination rate for the HPV vaccine was 34.2%. Based on the 3Cs model, this study found that the trust on the efficacy of vaccines, risk perception of being infected by HPV, price, and distance/time were influencing factors of vaccine hesitancy. Knowledge of the HPV vaccine and sociodemographic characteristics, such as education levels, were also statistically relevant. Therefore, it is recommended that relevant scientific knowledge on cervical cancer and the HPV vaccine should be spread on campus, the vaccination appointment procedure should be simplified, and the affordability of vaccination should be increased through strategic purchasing or providing subsidies, so as to reduce HPV vaccine hesitancy. Cervical cancer is one of the most common malignancies among females worldwide, with persistent human papillomavirus (HPV) infection as its necessary cause . In ChinThe HPV vaccine has been proved to be a cost-effective measure to prevent cervical cancer around the world  and is hVaccine hesitancy, which refers to a delay in the acceptance or refusal of vaccines when available , has damA number of previous studies were conducted to understand attitudes towards HPV vaccines among university students in China, which mainly focused on the awareness of cervical cancer and the HPV vaccine, and vaccination willingness ,7,8 17,,19,20. HTo analyze the influencing factors of vaccine hesitancy, the SAGE of the WHO proposed the 3Cs model to comprehensively analyze determinants of vaccine hesitancy from three dimensions, namely complacency, convenience, and confidence . This 3CThis study aimed to understand the status quo of HPV vaccine hesitancy among female university students across China during the COVID-19 pandemics and systematically analyze determinants of HPV vaccine hesitancy based on the WHO 3Cs model. The hypothesis was that 3C factors concerning complacency, confidence, and convenience were associated with HPV vaccine hesitancy.The first HPV vaccine listed in the Chinese market was approved by the National Medical Products Administration (NMPA) in July 2016 as the self-paid bivalent HPV vaccine for women aged 9–45 . SubsequThis study used a multicenter cross-sectional and survey-based research methodology. The independent variables in the study were constructs of the “3Cs” model, while the dependent variable was the intention/hesitancy for taking HPV vaccination. Participants were included in this study if they: (1) were currently enrolled as an undergraduate or graduate student at a sampling university; (2) were 18 years old or older; (3) were female; (4) had access to the Internet via computer or smart phone; and (5) provided informed consent. This study was ethically reviewed and approved by the Institutional Review Board, School of Public Health, Fudan University (IRB#2022-08-0992).The survey instrument, developed by the research group, consisted of items concerning HPV vaccine hesitancy, determinants from the vaccine hesitancy “3Cs” model, knowledge of the HPV vaccine, and socio-demographic characteristics. The question to measure vaccine hesitancy, “if the HPV vaccine was offered to you today, do you have any intention in taking it?”, could be answered with one of the following responses: “acceptance without hesitancy”, “hesitancy”, and “refusal”. Eleven questions concerning “3Cs” factors were developed to measure the participants’ attitudes on complacency, as well as their convenience and confidence . Two queThis study used a stratified sampling method. After comprehensively considering geographical location and socioeconomic development level in China, four cities at different regions were selected as sampling cities, including Shanghai, Guangzhou, Wuhan, and Nanning, with the former as Municipal City and the last three as the capital cities of provinces located in eastern, central, and western regions, respectively. In each city, four to five universities were chosen to cover students with a different education level  and majors .n represents the sample size required for the survey, p = 0.70 into the formula, n would be 322 in each city according to various calculations. Thus, the total sample size of this study was expected to be 1288 female university students for four cities.A priori sample size was calculated with the following formula based on an error α of 0.05 and a permissible error re 68.0%  and 71.8re 68.0% , respectData were collected between 1 June and 30 June 2022 using the Wenjuanxing online survey tool. Students were recruited through advertisements in class announcement. The landing page of the electronic survey instrument provided informed consent and study information. Participants could start to fill out the questionnaire only if they clicked the button which represented that they consented to all information provided and were willing to participate in the study. As for quality control, online questionnaires finished in no less than 180 s with all required items filled were considered as valid. In total, 1498 online questionnaires were collected, and 1438 questionnaires were valid through quality checks.p value < 0.05 was considered statistically significant.The data from online questionnaires were downloaded as a Microsoft Excel file and then imported into IBM SPSS version 26.0  for data analyses. Chi-square tests and multiple logistic regression were used to assess the associations between sociodemographic factors, 3Cs factors, and vaccine hesitancy. The chi-square test was first used to determine factors associated with vaccine hesitancy. Multinomial logistic regression was then adopted to assess the correlations between independent variables and college students’ attitudes toward HPV vaccination . The results of multinominal logistic regression were shown in the form of odds ratios (ORs) and 95% confidence intervals (CIs). A A total of 1438 valid questionnaires were collected in this study. As shown in p < 0.05).Among all female university students, 1290 students (89.7%) were willing to take up the HPV vaccine without hesitancy, 128 (8.9%) were hesitant, and only 20 (1.4%) refused to receive the HPV vaccine. Among those who were willing, 492 students were already vaccinated or already made appointments to become vaccinated, meaning that the total vaccination rate was 34.2%. p < 0.05). It was revealed that higher perceived susceptibility and harm associated with HPV, a higher perceived importance of HPV vaccination, and recommendations from medical staff were significantly related to lower vaccine hesitancy. Trust in the advice of medical staff, and the safety and efficacy of HPV vaccination also became related factors to lower vaccine hesitancy. In the meanwhile, price or distance/time significantly hindered vaccination. However, the impact and relevance of hearing negative vaccine-related information was not significant in our study.Most of the above characteristics were significantly related to vaccine hesitancy . The possibility of HPV vaccine hesitancy was higher for undergraduate students  and junior college students .Based on the WHO vaccine hesitancy 3Cs model, this study comprehensively examined the level of HPV vaccine hesitancy among university students in four cities across China, and systematically investigated its influencing factors.Among the female university students surveyed in this study, 89.7% did not hesitate to receive the vaccine against HPV, only 8.9% hesitated to some extent, and 1.4% refused to vaccinate. Compared with previous studies on HPV vaccine hesitancy or unwillingness to vaccinate among Chinese university students (11.6% to 43.1%) ,21,29,30Our study found that self-confidence, complacency, and convenience have a particular influence on HPV vaccine hesitation among Chinese university students.In the confidence dimension, we found that university students who trusted the advice of medical staff and believed that the HPV vaccine was safe and effective were less likely to hesitate to receive the HPV vaccine. Previous studies have revealed that confidence is the most relevant factor for vaccine hesitancy, and safety and effectiveness were the two most important factors for deciding to receive vaccination ,37. It iIn the complacency dimension, risk perception also had a significant influence on HPV vaccine hesitancy. University students who were more afraid of cervical cancer, conceived that they were more likely to be infected by HPV, and considered that cervical cancer was more harmful often did not hesitate to vaccinate, which was consistent with previous studies . EspeciaIn the convenience dimension, price and time (or distance) were obstacles to HPV vaccination among university students, which were significant in the chi-square test and multinominal logistic regression. As a growing number of cities in China started to offer the two-valent HPV domestic vaccine free for charge for teenagers, the price obstacle for HPV vaccination may be reduced to some extent. In addition, a complicated appointment process was also a disturbing factor affecting HPV vaccination, highlighting the need to simplify the HPV vaccination appointment process according to public demand.Moreover, it is worth mentioning that there is still a gap between the willingness to vaccinate and actual vaccination behavior. The existing literature shows that the willing acceptance rate of vaccination was higher than the actual vaccination rate ,16,21,31To our knowledge, this is the first study to comprehensively examine the vaccine hesitancy and influencing factors based on the 3Cs model among university students across China. By adopting a stratified sampling method to select sample from four cities with different socio-economic development levels, this study has a sufficient sample size and good representativeness. It can provide useful information to help us understand university students’ HPV vaccine hesitancy and develop vaccination strategies under the COVID-19 pandemic circumstances.This study also has some limitations. First, the online survey may have selection bias in the sense that only university students who were interested in the HPV vaccine were investigated. Second, with a cross-sectional design, this study could only reveal the correlation between influencing factors and HPV vaccine hesitancy rather than causal relationships. Third, for investigation convenience, cities located in North China were not included in our sample cities, which may influence the generalization of our results to some extent.The study examined current HPV vaccine hesitancy among female university students in China, revealing that around 89.7% of students were willing to receive the HPV vaccine without hesitancy and that 8.9% had vaccine hesitancy. Based on WHO’s vaccine hesitancy 3Cs model, this study found that trust in relation to the efficacy of vaccines, the risk perception of being infected by HPV, price, and distance/time were associating factors which impacted vaccine hesitancy. HPV knowledge and education levels were also statistically relevant. It is recommended that relevant scientific knowledge of cervical cancer and the HPV vaccine should be spread on campus, that the vaccination appointment procedure should be simplified, and that the affordability of vaccination should be increased through strategic purchasing or providing subsidies, so as to reduce HPV vaccine hesitancy.


The thermal stabilization of blue phases is a subject that has been of scientific and technological interest since their discovery. Meanwhile, carbonaceous nanomaterials such as C60 fullerenes, carbon nanotubes and graphene have generated interdisciplinary interest spanning across solid-state physics, organic chemistry, colloids, all the way to soft matter physics. Herein, the stabilization of liquid crystal blue phases by doping with C60, single-walled carbon nanotubes and graphene oxide is described. All three types of particles are found to extend the combined temperature range of blue phases I and II by a factor of ∼5. Furthermore, mixtures of pairs of different materials, and all three types are shown to stabilize the blue phases. The temperature range of the blue phases is shown to grow at the expense of the cholesteric phase. This leads to a blue phase-cholesteric-smecticA phase triple-point in all cases except that of doping with carbon nanotubes. The mechanisms of this thermal stabilization are discussed in light of theoretical descriptions for other established systems. Carbon nanomaterials of differing dimensionality, namely fullerenes, nanotubes and graphene oxide are shown to stabilize the Blue Phases at the expense of the N* phase until its complete disappearance. A BP–N*–SmA* triple point is observed. Both classes of frustrated phases result from a competition between chirality, thus the formation of a helical superstructure, and thermodynamics. In the case of the Blue Phases (BP) the material would like to preserve an isotropic character, while at the same time forming a helical superstructure. The result is a frustrated phase consisting of double twist cylinders mediated by a cubic structure of disclination lines  the blue phases,on lines .5–7 This opened the gates for display applications of BPs through light modulation via the Kerr effect. Such displays, which were built to prototype level by Samsung,8,9 were much faster in optical response than standard nematic LCDs, with refresh rates up to 250 Hz, excellent viewing characteristics, and most of all the lack of a need for alignment layers, which is a considerable cost factor in production and a source of detrimental dust particles. Nevertheless, polymer stabilization only appeared to work with certain polymer mixtures under very specific conditions. There is thus still the need for a robust methodology of the expansion of the BP temperature regime and a number of different methods were suggested2 with more or less success. The addition of bent-core molecular dopants was shown to widen the blue phase,10 as was the addition of colloidal particles.11–13 Also combinations of different methods had beneficial effects, although not quite being additive.10 A promising approach, not too dissimilar to polymer stabilization, is the additive mixing of a polymer into the BP, which was shown to be increasingly effective the shorter the added polymer was made.14 In fact, extrapolation to a molecular weight equivalent to a dimer, gave a temperature regime of the blue phase, equivalent to what had been observed for some liquid crystal dimers, ∼50 K.15As these defects cost elastic energy, the temperature regime of the blue phase is generally very small, of the order of 1 K. In the past it was thus taken for granted that there were no reasonable applications of these phases. They were regarded of purely academic interest until a method was introduced to widen the BP temperature regime through polymer stabilization.16,17 CoPt nanoparticles,18 aminoazobenzene groups,19,20 or mesogenic units21 in an effort to increase the chemical compatibility with thermotropic liquid crystals at high concentrations of doping. In all cases, the total temperature range of the blue phases was expanded by approximately 2 K or less. Some reports further suggest that as the concentration of the dopant is increased further, the temperature range of the blue phases actually decreases.16,20,21 Furthermore, the concentrations of graphene material that caused the maximal blue phase ranges were rather large, of the order 0.1 wt% or higher.Materials based upon the two-dimensional material graphene have also been investigated as stabilizing additives to the blue phases. Several approaches have used graphene oxide functionalized with either flexible alkyl chains,22,23 These exhibit a discontinuous helical superstructure, which can be commensurate or incommensurate,24 with adjacent smectic blocks being rotated with respect to each other and separated by grain boundaries of regular arrays of screw dislocations  and stored in a refrigerator at 5 °C to mitigate thermal reduction.The liquid crystal used in this study was CE8. Schematic diagrams of the chemical structures of CE8 and each of the nanomaterials used in the investigations are shown in 26 The nanoparticle-doped LCs were then placed between two glass coverslips. In the case of the nanotubes and the graphene oxide, the dispersion of nanomaterial in the LC was heterogeneous, with small aggregates visible in polarized microscopy . Toluene (Sigma Aldrich) was used as the solvent for C60, yielding the distinctive purple-colored solution for which C60 is known.The addition of C60, SWCNTs or GO all increased the width of the blue phases. The isotropic–BP transition and the temperature at which the SmA* phase appeared remained relatively unchanged as larger amounts of nanomaterial were added with the exception of added GO. In this case, the N*–SmA* transition raised slightly in temperature by ∼1 K. The chiral nematic phase decreased in width to accommodate the stabilized blue phases. For the cases of fullerene and GO doping, this reduction in width of the N* phase lead to a BP–N*–SmA* triple point at medium concentrations  to be approximately 60 nm.33 These bundles are larger than the widths of disclination lines in the blue phase and are therefore less effective in reducing the free energy density of the liquid crystal phase. The fullerenes on the other hand can adapt their packing within the disclination lines as to maximise the reduction in free energy. It is however not completely clear what the mechanism is behind the stabilizing effect of small amounts of nanotubes. On the one hand the elastic deformation energy of the defects and disclination lines are largely reduced, while on the other hand the double-twist cylinder bulk is elastically deformed. This indicates that the maximal thermal stabilization given by the nanotubes should in fact be smaller than that of the fullerenes, which is experimentally observed. Recently, Kemiklioglu and Chien34 reported the effect of the addition of carbon nanotubes to a polymer-stabilized blue phase system. In contrast to our findings, the total width of the blue phases was seen to decrease with increasing nanotube content. This effect could be due to the fact that in the polymer-stabilized system, the disclinations of the blue phases are already filled by the polymer network, and then the addition of further solid material simply leads to elastic deformation of the larger double-twist structure and an increase in the total free energy of the system, which may destabilize the phase.The widths of SWCNTs are of the same order as a fullerene, but their lengths are polydisperse and up to the range of microns, much larger than the size of the BP unit cell (∼300 nm). Furthermore, strong van der Waals and π–π stacking interactions between nanotubes causes them to form bundles, the diameters of which have been measured 35 or via polymer-stabilization in an electric field,36 the cubic blue phases form with multiple domains and grain boundaries . Furthermore, it was found that on cooling and heating, the presence of fullerenes at up to 0.1 wt% had no effect on the width of the TGB phase. A possible explanation for this is that the adjacent phases, the N* and SmA* phases, are much more viscous than the isotropic and blue phases, meaning the fullerenes may be unable to diffuse to, and efficiently fill, the high-energy cores of the screw dislocations that are present in TGB phases.In this work the influence of carbonaceous nanomaterials of different dimensionality, namely 0D fullerenes, 1D nanotubes and 2D graphene oxide sheets, on the stability of liquid crystal blue phases was investigated. Such a study is motivated through the possibility of an easily accessible material for novel display and electro-optic applications with enhanced performance at lower cost than conventional liquid crystals. It was found that small amounts of all of these additives have a positive effect on the thermal stabilization of the blue phases, in some cases leading to a complete vanishing of the cholesteric phase to maximize the blue phase temperature range. Whilst for the 0D case (fullerenes) the effect can be explained in terms of established theoretical models, the use of larger particles like nanotubes and 2D materials for the purposes of stabilizing blue phases do not fit with the idea of filling the lattice of defect lines present in BPI and BPII.There are no conflicts to declare.NA-002-D0NA00276C-s001


Pinus densiflora Sieb. et Zucc. leaf extracts against HPV. The pine needle extracts from each organic solvent were analyzed for antiviral activity. The methylene chloride fraction (PN-MC) showed the highest activity against HPV pseudovirus (PV). The PN-MC extract was more effective before, rather than after treatment, and therefore represents a prophylactic intervention. Mice were pre-treated with PN-MC via genital application or oral administration, followed by a genital or subcutaneous challenge with HPV PV, respectively. The HPV challenge results showed that mice treated via genital application exhibited complete protection against HPV. In conclusion, PN-MC represents a potential topical virucide for HPV infection.Most cervical cancers are associated with high-risk human papillomavirus (HPV) infection. Currently, cervical cancer treatment entails surgical removal of the lesion, but treatment of infection and preventing tissue damage are issues that still remain to be addressed. Herbal medicine and biological studies have focused on developing antiviral drugs from natural sources. In this study, we analyzed the potential antiviral effects of  Human papillomavirus (HPV) is a group of viruses that are extremely common worldwide. There are more than 100 types of HPV, including at least 14 cancer-causing viruses. Although the majority of infections cause no symptoms and are self-limiting, genital HPVs have become a major public health concern because persistent infection with certain types can cause cervical cancer, which kills about 311,000 women worldwide each year [Although commercial vaccines, including Gardasil and Cevarix, represent a remarkable improvement in the fight against cervical disease and other anogenital cancers, vaccinated individuals may remain infected with HPVs that are not targeted by the vaccines. Further, because the vaccines are relatively expensive, they may not be available to all women, especially those in developing countries , 6. CurrRecent studies investigating the prevention and treatment of HPV related to cervical cancer and other genital diseases are attracting increasing attention worldwide . The stuet al., 2019). Approximately 70,000 plant species are used in herbal medicine, including the treatment of HPV infection [®E ointment found effective against HPV-related diseases was formulated with catechins extracted from green tea [Herbal medicine is considered one of the alternative approaches to the treatment of viral diseases . The cells were cultured within Dulbecco’s modified minimal essential medium (DMEM) supplemented with 10% FBS and hygromycin B  at 37°C with 5% CO2 .HPV16 PVs were prepared as described previously via co-transfection of 293TT cells with p16L1/L2 plasmids and pSEAP or pLucf plasmid -23. CellSix-week-old female BALBb/c mice  were randomly divided into four groups, each comprising five mice. The use of animals in these experiments was approved by the Institutional Animal Care and Use Committee of Konkuk University .Pinus densiflora) were purchased at Gyeongdong Market (Korea). Pine needles were extracted with 95% ethanol (10 vol/wt). The extraction was repeated three times, followed by filtration of the extract solution and evaporation (PN-T). The extracts were suspended in distilled water and sequentially sorted into equal amounts of hexane, methylene chloride, ethyl acetate, and n-butanol. Using a separatory funnel, the hexane (PN-H), methylene chloride (PN-MC), ethyl acetate (PN-E), n-butanol (PN-B), and aqueous (PN-W) fractions were obtained.Gangwon-do pine needles  according to the manufacturer’s instructions. The 293TT cells were seeded on a 96-well plate at a density of 1 × 103 cells/well for 24 h. Different extract concentrations were added to the cells for 16 h. The cells were gently washed with PBS once to remove the remaining extract inside the wells. The cells were infected with 1 × 105 relative light units (RLU) of HPV16-SEAP PVs for 1 h. The medium was removed and replaced by a complete culture medium. The cultures were incubated for 48 h at 37°C under 5% CO2, and then the SEAP content in 10 μl of clarified cell supernatant was determined using the Great EscAPe SEAP Chemiluminescence Kit . Each concentration of the extract was assayed for virus inhibition in triplicate.The 293TT cells were grown in 96-well plates at 5 × 102. The supernatant following each pre-treatment was collected at 48 h after infection.Before virus inoculation, non-cytotoxic concentrations (100 μg/mlL and 50 μg/ml) of the extract were added to the cells and incubated for 4, 8, 12, or 16 h. The cells were gently washed with PBS once to remove the remaining extract inside the wells. HPV16-SEAP PV at a multiplicity of infection (MOI) of 20 was introduced for 1 h, and the cells were washed with PBS. The medium was removed and replaced by a complete culture medium. The cultures were incubated for 48 h at 37°C under 5% COCells were pre-adsorbed with HPV16-SEAP PV using different concentrations  of PN-MC extract for 1 h at 37°C. Cells were washed twice with PBS and replaced with culture medium. After 48 h, the antiviral effect by adsorption between virus and extract was evaluated using a SEAP Chemiluminescence Kit.The cells were initially infected with HPV16-SEAP at a MOI of 20. The cells were washed with 200 μl of PBS after 1 h, followed by the addition of different concentrations (800 to 12.5 μg/ml) of extract to the cells, and then incubated for 48 h. After 48 h, the antiviral activity was determined via measurement of SEAP chemiluminescence.The mice were challenged with HPV PV, as described previously . Seven dThe mice received the PN-MC treatment via genital application or oral administration. The genital administration entailed use of the extracts in the vaginal tract and treatment at 3 mg/mouse in 0.5% methylcellulose once a day for 3 days. The oral dose of the extract was 6 mg/mouse in 0.5% methylcellulose  once a day for 5 days. Four hours after the last administration of the PN-MC extract, a challenge test of each mouse was performed.7 infectious forming units (IFU) of HPV16-Lucf PV, each in a 20 μl solution containing 4% carboxymethylcellulose (treatment involved a subcutaneous (SC) challenge with HPV16-Lucf PV in the peritoneal area [The genital treatment involved challenge with 1 × 10eal area , 28.Three days later, all mice were anesthetized via intramuscular injection with 40 mg/kg of Zoletil50 , and 5 mg/kg of Rompun , followed by intravaginal pre-treatment with 20 μl of 4% Nonoxynol-9 (Sigma-Aldrich). The mice were injected intraperitoneally with luciferin (100 μl at 7 mg/ml), and imaged for 10 min with an IVIS 200 bioluminescence imaging system  to visualize the expression of luciferase by measuring the light emission. Equal areas encompassing the site of virus inoculation were analyzed using the Living Image 2.20 software .t-test. P values less than 0.05 were considered statistically significant.All statistical analyses were performed using GraphPad software version 7.0 (GraphPad Software). The significance of differences between treatment groups was analyzed via two-way analysis of variance (ANOVA) or two-tailed Student’s 50) at 25 μg/ml. Therefore, PN-MC was selected as an anti-HPV drug candidate for further study.In order to screen the antiviral activity of the pine needle extract against HPV, pine needles were extracted from each organic solvent. The cytotoxicity of pine needle extract in each organic solvent was measured in the 293TT cell line. None of the extracts showed cytotoxicity to 293TT cells at concentrations less than 1,000 μg/ml . The extA pre-treatment assay was performed to assess the inhibitory activity of PN-MC extract on the entry of HPV into the 293TT cells. The cells were pre-treated with 100 μg/ml or 50 μg/ml of PN-MC for 4, 8, 12, 16, or 24 h to enable its interaction with the cell surface. The anti-HPV effect of 100 μg/ml of PN-MC treatment was observed in the group treated for 12 h or longer. Treatment with 50 μg/ml of PN-MC for 16 h and 24 h in the pre-treatment groups inhibited HPV16 PV by more than 80% . Pre-treVarious concentrations of PN-MC were mixed with HPV PV for 1 h to investigate the inhibitory efficacy of the virus against attachment to host cells. The mixture was then added to 293TT cells. The mixture containing PN-MC and HPV PV showed potent inhibition of HPV attachment at concentrations above 25 μg/ml . The resPost-treatment assay was performed to evaluate whether PN-MC inhibited viral proliferation. Various concentrations of PN-MC were used for the treatment. In contrast to pre-treatment, the results of post-treatment showed no HPV inhibitory effect. Only a PN-MC concentration of 800 μg/ml showed 48.3% inhibition of HPV PVs . As a reA murine model was used to evaluate the antiviral activity of PN-MC against HPV infection. Mice were pre-treated with PN-MC via genital or oral administration and then challenged with HPV PV to the genitals or subcutaneously, respectively. Mice treated via genitals showed complete protection against HPV. p < 0.001). Luminescence levels of genitally treated mice were 7-fold less than those of orally treated mice. At the levels of protection, genital treatment resulted in more than 97% protection against HPV PV. By contrast, in the case of SC infection after oral administration with PN-MC, the level of protection from HPV PV infection was 46%. Taken together, topical administration of PN-MC to the genitals inhibits HPV PV infection.Here, we investigated the anti-HPV effect of pine needle extracts in vitro and in vivo. First, an in vitro antiviral assay was performed to determine the anti-HPV effects of various pine needle extracts. Based on the screening results, we selected PN-MC as a candidate agent with anti-HPV activity. Pre-treatment and cell adsorption of PN-MC effectively inhibited viral invasion. The results in the inhibition test of viral attachment to host cells indicate that PN-MC blocks viral attachment to cells by inhibiting the interaction with HPV and its receptors. However, post-treatment of extracts did not show a viral reduction. This phenomenon is related to the binding affinity of the extract to the HPV receptor, which appears to inhibit the entry rather than the viral replication. These results suggest that pine needle extract is an effective agent for preventing HPV infection, and therefore we used the PN-MC extract for pre-treatment in an animal model. During the in vivo test, mice were effectively protected against HPV when PN-MC was topically administered via the vaginal tract.Previously, we isolated the compounds from the pine needle. Twelve compounds were isolated and registered in patent . However, the yield of the compounds was so low that anti-HPV analysis could not be performed. Among the various organic extracts, large amounts of substances with anti-HPV function could be obtained in the methylene chloride layer.Sexually transmitted diseases (STDs) can be transmitted via genital, orogenital, or anogenital contacts, and are a public health concern worldwide. Approximately one million people around the world are newly infected with STIs each day. Although several preventive strategies such as vaccination and a range of therapeutic drugs have drastically reduced the risk of contracting STIs, these infections continue to spread .Although currently available HPV vaccines have significantly reduced the incidence of HPV infection, it is impossible to prevent the spread of more than 150 types of HPV. The HPV vaccine is an exceptional primary prevention tool, but the question of adequate secondary prevention strategies remains open. Topical virucides have been proposed and pursued as a secondary prevention strategy . Other sA number of important factors such as safety, efficacy, cost, acceptability, efficiency, drug delivery patterns, and tolerance primarily influence the development of virucides. A variety of plant extracts showed proven antiviral activity. Optimizing plant-based antiviral agents requires a better understanding of the structure and antiviral mechanism of various extracts. Our results also provide insight into viral treatment using plant extract. It can be concluded that pine needle extracts exhibit antiviral effect against HPV. In addition, due to the abundance of pine needles available as a natural resource, the production cost is relatively low compared to other chemically synthesized drugs. The low cost of microbicides from PN-MC provides a better approach to popular prevention.In conclusion, treatment with the PN-MC extract derived from the pine needles reduced HPV in vitro and in a mouse model. Therefore, the findings indicate that the PN-MC extract can be used as a preventive agent against HPV and developed as an active ingredient in topical virucides against HPV infections. Further studies are needed to determine the precise antiviral mechanism of PN-MC, develop appropriate PN-MC formulations, and administer topical treatments to inhibit HPV.


Two validated questionnaires were used to collect data. Results: Participants demonstrated moderate knowledge about HPV infection and vaccination, with a mean knowledge score of 53.26 (SD ± 20.65) and 38.92 (SD ± 17.58), respectively. Cronbach’s alpha value was 0.77 and 0.80. Female participants were better informed than males. Approximately 52.3% of respondents had been vaccinated and 65.5% were willing to get vaccinated in the future. Vaccination rate was significantly associated with gender , knowledge about the HPV vaccine  and age . Reasons for vaccine refusal were insufficient information (36.8%) and fear of side effects (19%). Correlates of positive vaccination intention were knowledge about HPV . Conclusions: The findings suggest that the Greek government’s continuing HPV promotion efforts and education on the risks of HPV infection among young people are likely to increase vaccination acceptance among this group.Introduction: Human papilloma virus (HPV) is one of the most common sexually transmitted infections and is widely known as the main causative agent for cervical cancer. The aim of this study was to investigate the perceptions, knowledge and attitudes of young Greek adults concerning prevention of HPV infection and HPV immunization. Material and Methods: This constitutes a cross-sectional online survey. A convenience sample of young Greek adults ( Human papilloma virus (HPV) is one of the most common sexually transmitted infections, with an estimated 80% of people being infected with HPV in their lifetime . HPV is There are today three licensed prophylactic vaccines against HPV, exhibiting an excellent safety and immunogenicity profile. The quadrivalent vaccine  was first licensed in 2006, the bivalent vaccine (targeting HPV types 16 and 18) in 2007, and the 9-valent vaccine  in 2014 [In Greece, since the introduction date in 2008, all vaccines have been fully financed by the national authorities and are offered to females 11–18 years old and to young men and women 18–26 years old who belong to special risk groups, according to the Greek National Immunization Programme (NIP) ,8,9. ThiIn order to achieve high vaccination coverage, it is necessary to obtain an overview of the knowledge about and attitude toward HPV vaccination in the general population. Previous studies have revealed that despite the high prevalence of HPV, there are misconceptions or else a lack of awareness about HPV even among otherwise well-informed people while, by contrast, there is evidence that vaccination uptake is increased when the targeted population is well-informed about the risks and benefits ,12,13. TThe aim of this study was to investigate the perceptions, knowledge, and attitudes of young Greek adults concerning the prevention of HPV infection and HPV immunization.This study is based on a cross-sectional online survey.A convenience sample was used to implement the present study. The study sample consisted of young Greek adults aged 17 to more than 35 years, living in Athens and urban areas. Participants who did not consent to participate in the study and those outside the study age range (under 17 years and over 40 years) were excluded. A total of 883 out of 926 people participated in the study. The inclusion criteria for participants were to be students attending medical schools and other higher education institutions and health professionals and be able to speak, read, and write in Greek. Exclusion criteria were inadequate language skills and age under 17 years.The data collection was carried out through the completion of two anonymous questionnaires, after receiving informed consent from each participant. The questionnaires were converted into an online file via an electronic questionnaire creation platform and then posted in an electronic communication environment. Research data were collected from December 2020 to March 2021. Participation was voluntary and anonymity was assured. At the beginning of the research study, participants were asked to study the consent text, and only if they agreed would they complete the other sections. There was another consent question at the end of the questionnaire and answers were recorded only if the participants consented.Before collecting data, we obtained approval by the Research Committee of the Hellenic Open University.The research tools which evaluated the participants’ perceptions, knowledge, and attitudes concerning the prevention of HPV infection and HPV immunization were as follows:A structured questionnaire indicated by Dafermou et al.  which inA questionnaire indicated by Agorastos et al.  which in2 test or Fisher’s exact test was used to compare ratios where necessary. Student’s t-test was used to compare quantitative variables between two groups. Parametric dispersion analysis (ANOVA) was used to compare quantitative variables between more than two groups. To identify a type-I error due to multiple comparisons, Bonferroni correction was used, according to which the significance level was set at 0.05/k (k = number of comparisons). Linear regression analysis was used to find independent factors related to the knowledge scores, from which dependence coefficients (b) and their standard errors (standard errors = SE) were derived. In order to find independent factors related to vaccination and vaccination intention, a logistic regression analysis was performed, and the odds ratio (OR) was derived with 99% confidence intervals (99% DE). The internal reliability of the knowledge questionnaire was tested using Cronbach’s alpha coefficient. The significance levels were bilateral, and the statistical significance was set at 0.01.The Statistical Package for Social Sciences version 22.0 was used to perform the statistical analysis. Mean values and standard deviations (SD) were used to describe the quantitative variables. Absolute (N) and relative (%) frequencies were used to describe the qualitative variables. Pearson’s χn = 763) were women. A total of 62.5% (n = 552) were students in higher education and 23.4% were full-time employees. Inhabitants of urban areas accounted for 73.5% of the study population and the remaining 24.6% lived in the capital city of Athens. In total, 863 participants (97.7%) were ethnic Greek. A total of 48.8% of respondents were unmarried, while 32.5% were in a relationship. In addition, 56.4% (n = 498) had a sibling and 46.5% had at least one parent with a higher-education qualification. Finally, 22.9% of the participants had a monthly family income of over EUR 2000.In total, 926 people participated in the survey, of whom 883 consented to participate; the response rate being 95.5% . The meap = 0.006). About 16.5% of respondents (n = 146) reported that they had been infected by HPV in the past, while male participants had a significantly lower HPV incidence rate compared to women (13.3% vs. 17%) (p < 0.001). The highest HPV infection rate according to age was found in the 26–35 age group (31.1%) (p < 0.001).Most participants (81.1%) stated that they take precautions during sexual intercourse . Males in = 431) had visited a gynecologist at least once in the past year. The frequency of visits to the gynecologist was found to be highest in the age group 26–35 years old (72.9%) and the lowest in the age group under 25 years old (22.0%) (p < 0.001). The majority (98.8%) knew about the Pap test, having been informed mainly by their family and the doctor . It was found that, as participants’ age increased, the information rate via the gynecologist increased, while information via the Internet decreased (p < 0.001). Nearly 66.2% of the female population regularly had a Pap test, of whom 79.8% did it once a year, while 24% had been infected with HPV at some point in their lives. The Pap test rate differed significantly between the three age groups, with the lowest rate being in the age group of 25 years and under (50.4%) and the highest among women over 35 years (97.1%) (p < 0.001). Moreover, the rate of HPV infection among women who had cervical screening differed significantly between the age groups, with the lowest being in the group under 25 years of age (16.4%) and the highest in the 26–35 age group . The main reasons for not being screened were not being sexually active (62.5%), followed by the absence of symptoms (20.3%).Approximately 57.2% of female participants (p = 0.001), employed (p = 0.001), married (p = 0.006), living in Athens (p = 0.001) and having previously been infected with HPV (p < 0.001). Moreover, there were significant differences in overall mean knowledge scores among the three age groups and the frequency of gynecologist visits. Participants who were 26–35 years old were more likely to report that they had heard of HPV, compared to those under 25 years (p = 0.001). Female participants who had cervical screening, visited the doctor regularly, and knew about the Pap test and HPV-DNA test had a higher score, indicating more knowledge regarding HPV (p < 0.001). In the multivariable model, the following factors were positively associated with HPV awareness: female sex , residence in the region of Athens  and positive HPV infection . Parents’ educational level and safe sexual behavior were not associated with HPV awareness.The majority of the study population was aware of HPV (90%) . The meap < 0.001), married (p = 0.009), and women who had cervical screening and knew about the HPV-DNA test (p < 0.001). Participants 26–35 years old were better informed about the HPV vaccine than those over 35 years (p = 0.002). In the multivariable model, the factor of female sex was positively associated with vaccine knowledge . Occupation, residence, parents’ educational level, sexual behavior and HPV prevalence were not associated with HPV vaccine awareness.Similarly, most respondents (88.6%) were aware of HPV vaccination. The mean HPV vaccine knowledge score was 38.92 out of 100 (SD ± 17.58). Internal consistency regarding the HPV-vaccine-knowledge item was 0.80. The most common information resources reported concerning the vaccine were the gynecologist (50.6%), followed by the Internet (43.6%) and the family (39.9%). Bivariate correlations of positive HPV vaccine awareness included the following: female sex (n = 462) had already been vaccinated. Approximately 50.3% did not know which vaccine type they had received, while 60.9% had received all three HPV injections. Of those who had refused, a total of 36.8% reported lack of information as the main reason, followed by the fear of side effects (19%) and not being sexually active (15%). Of the participants, almost 52.3% (p < 0.001) (p < 0.001). Vaccinated participants knew significantly more about HPV and the vaccination (p < 0.001). In the logistic regression model, age, sex and the HPV-vaccine-knowledge score were positively associated with HPV acceptance. The results showed that the factors associated with higher vaccination rate in the entire sample were vaccinated participants being up to 25 years , being female  and knowing better about the vaccine . Screening factors for women, residence, marital status, parents’ educational background, sexual behavior and HPV infection were not associated with vaccination uptake, as had been hypothesized.Bivariate correlates of HPV vaccination included the following: female sex, students, age up to 25 years, and HPV and HPV vaccination knowledge (< 0.001) . Vaccinan = 284) indicated that they would like to receive the vaccine if they were to decide in the future. According to bivariate correlation, the following factors significantly contributed to the participant’s vaccination intention: age, occupation, safe sexual behavior and greater knowledge about HPV (p < 0.002). Students as well as those who take precautions during sexual intercourse had a significantly higher intention rate (p = 0.005). Moreover, participants who intended to get vaccinated knew better about HPV, compared to those who had no such intention (p = 0.001). Multiple logistic regression was performed to assess predictors of intention. The results indicated that the more participants knew about HPV, the more likely they were to have the intention to be vaccinated . Screening for women, sex, the vaccine knowledge score and previous HPV infection did not appear to influence participants’ intention to be vaccinated.Among the non-vaccinated respondents, 67.5%  stated that young people should be informed about the association between HPV with sexual behavior and cervical cancer before being vaccinated. Approximately 57.2% of participants believed that vaccination is beneficial for people who have already been infected by a specific HPV type, by offering protection from other HPV types.This study aimed to investigate the perceptions, knowledge, and attitudes of young Greek adults concerning the prevention of HPV infection and HPV immunization. The study results were encouraging, as they showed a favorable attitude toward vaccination and a moderate knowledge score for HPV and the vaccine. More than half of the study population had been vaccinated and about two out of three of the non-vaccinated were willing to receive the vaccine in the future.The research findings revealed the moderate knowledge level of participants about HPV. Although the majority was aware of HPV, fewer than half knew of the possible transmission modes and only a quarter of the overall sample about the HPV–cancer association. To our knowledge, few similar studies on young adults have been conducted in Greece. One study on Greek students 15–18 years old observed limited knowledge about HPV , while aVaccination-related knowledge was at a lower level than HPV knowledge. More than three-quarters of respondents stated that they were aware of the HPV vaccine, but only one-third answered all the vaccine questions correctly. Similarly, a previous Greek study by Vaidakis et al.  found thDespite the relatively high level of awareness of HPV, only half of participants had been vaccinated. This rate is, nevertheless, well above the estimated national vaccination coverage of 9%. These results correspond to those of previous Greek research, which demonstrated above-average vaccination coverage ,38,39. FThe study findings shed light on the need for the provision of more information on HPV, given that the main reason reported for vaccination reluctance was insufficient information, followed by fear of side effects. This can partly be attributed to misinformation spread by the Internet and the media, which leads to misconception about the safety and efficacy of the HPV vaccine. In fact, many international studies have determined that inadequate information about the vaccine and safety issues are common among both parents and adolescents ,48,49.Vaccination intention among non-vaccinated participants proved to be high, with more than half of participants stating that they were willing to be vaccinated in the future. Intention rate was higher among young participants, given that this age group (20–24 years old) are at the highest risk for contracting HPV, while older participants were less positive about vaccination, probably due to the perception of their low risk of HPV infection and being in a monogamous relationship. Moreover, people who followed safe sexual behavior were found to have a higher intention rate, the latter surely reflecting their awareness of and sensitivity to preventive measures. Xiao  demonstrRegarding the source of information and trust, respondents stated that the gynecologist along with the Internet and family provided most of their information and were the most trusted. However, as mentioned, given that the Internet does not always provide accurate information, this finding suggests that governments must consider taking steps so that the Internet becomes part of a communication strategy that will promote the dissemination on the Web of reliable, scientifically evaluated information to young people and to the population at large. Meanwhile, healthcare providers can play a pivotal role in promoting vaccination, by cooperating with schools in providing health education to adolescents.With respect to screening, two out of three female participants regularly had a Pap test, the majority of whom were over 35 years old. This is likely explained by the inclusion in the questionnaire of older women (over 35), who appear to have greater awareness of health issues and to routinely apply preventive behaviors. This finding is in-line with other Greek studies ,53. PartOne limitation of the current study is its Web-based nature. Online surveys may have advantages related to the high speed and low cost of data collection. However, they may be biased by limited and selective participation, as certain populations with no access are unable to participate. Furthermore, using a convenience sample means that the sample is not representative of the general population so that there is the risk of bias. In terms of external validity, a larger sample size including more male participants as well as inhabitants of capital cities would render more valid results. One strength of the study is that the results may be used in the implementation of future research as well as in awareness campaigns concerning HPV prevention.The aim of the present study was to investigate young Greek adults’ awareness of and attitudes toward HPV vaccination. In conclusion, the participants’ general attitude proved positive, as about half had been vaccinated and more than half were willing to receive the vaccine in the future. Vaccination uptake was significantly associated with knowledge of HPV and its vaccine, as well as other factors. The knowledge level was moderate and related to sex, residence, and previous HPV infection. The main vaccination barrier was inadequate information. Consequently, more educational programs are needed to raise awareness regarding HPV and the vaccination. These programs should be targeted both at health professionals and the general public. Future research studies need to collect and use data from a representative sample of the Greek population to provide a more in-depth evaluation of the views and predictors influencing HPV vaccination uptake in Greece.


INK4A is a surrogate signature compensating for the specificity and/or sensitivity deficiencies of the human papillomavirus (HPV) DNA and Papanicolaou smear (Pap) co‐test for detecting high‐grade cervical squamous intraepithelial lesions or worse (HSIL+). However, traditional p16INK4A immunostaining is labour intensive and skill demanding, and subjective biases cannot be avoided. Herein, we created a high‐throughput, quantitative diagnostic device, p16INK4A flow cytometry (FCM) and assessed its performances in cervical cancer screening and prevention.P16INK4A FCM was built upon a novel antibody clone and a series of positive and negative (p16INK4A‐knockout) standards. Since 2018, 24 100‐women  have been enrolled nationwide for two‐tier validation work. In cross‐sectional studies, age‐ and viral genotype‐dependent expression of p16INK4A was investigated, and optimal diagnostic parameter cut‐offs (using colposcopy and biopsy as a gold standard) were obtained. In cohort studies, the 2‐year prognostic values of p16INK4A were investigated with other risk factors by multivariate regression analyses in three cervicopathological conditions: HPV‐positive Pap‐normal, Pap‐abnormal biopsy‐negative and biopsy‐confirmed LSIL.P16INK4A FCM detected a minimal ratio of 0.01% positive cells. The p16INK4A‐positive ratio was 13.9 ± 1.8% among HPV‐negative NILM women and peaked at the ages of 40–49 years; after HPV infection, the ratio increased to 15.1 ± 1.6%, varying with the carcinogenesis of the viral genotype. Further increments were found in women with neoplastic lesions . Extremely low expression of p16INK4A was observed in women with HSILs. As the HPV‐combined double‐cut‐off‐ratio criterion was adopted, a Youden's index of 0.78 was obtained, which was significantly higher than that (0.72) of the HPV and Pap co‐test. The p16INK4A‐abnormal situation was an independent HSIL+ risk factor for 2‐year outcomes in all three cervicopathological conditions investigated (hazard ratios: 4.3–7.2).P16INK4A quantification offers a better choice for conveniently and precisely monitoring the occurrence of HSIL+ and directing risk‐stratification‐based interventions.FCM‐based p16 INK4A FCM was established for triaging cervical high‐grade neoplasia/cancer.• The quantification detection system—p16INK4A FCM co‐test is significantly higher than that of HPV and Pap co‐test.• 24 100‐women cross‐sectional studies revealed that HSIL+‐triaging efficacy of HPV and p16INK4A‐abnormal women would run a HSIL+‐risk > 4‐fold higher than those with a p16INK4A‐normal level.• 2‐year cohort studies indicated that HPV‐positive/Pap‐abnormal p16 Based on the p16INK4A quantification system developed, which was modified from a traditional FCM platform, most random measurement errors, such as those caused by the optoelectronic fluctuations of FCM signal‐processing or biophysical heterogeneity of the exfoliated cells, were satisfactorily controlled by a parallelly prepared reference sample (blank‐labelled); additionally, the systemic errors caused during sample‐harvesting/labelling processes and/or FCM read in/readout procedures were rectified by a set of external standards . With these refinements, the utility of FCM in detecting abnormal cells of extremely low abundance (1/10 000–10/10 000) that were labelled with a difficult‐to‐measure dose of fluorescence signals was enabled. Herein, we report the diagnostic performance of this novel system. The physiological and pathological expression patterns of p16INK4A in normal/abnormal cervical cells were quantitatively investigated. The age‐, HPV genotype‐ and lesion‐dependent expression of p16INK4A was precisely appraised, and the use of the p16INK4A‐positive ratio for assessing the risks of high‐grade lesions was validated. The obtained FCM data provided important evidence for the applicability of p16INK4A‐based quantitative pathology in the risk stratification‐oriented management of women threatened with neoplastic lesions.In recent years, we have sought to construct a reliable detection system to quantitatively profile the p16INK4A FCM, namely, (1) why was the ‘p16INK4A‐positive ratio’ selected as a more sensitive measurement scale for p16INK4A FCM; (2) what is the normal expression pattern of p16INK4A at the cervical epithelium; (3) what changes of cervical p16INK4A expression pattern could be detected by FCM after HPV infection; (4) how is the diagnostic performance of p16INK4A FCM in identifying histological HSIL+ lesions; (5) could the ‘p16INK4A‐positive ratio’ be applied to predict the 2‐year pre‐cancer/cancer risks of women with abnormal HPV/Pap tests, especially under complex pathological conditions.In general, the current study aimed to address the critical questions that might be encountered during clinical application of p1622.1INK4A FCM. At each medical centre, eligible HPV‐positive and/or Pap‐abnormal women who attended during the same period were systematically enrolled as a candidate pool, and the information and cervical samples of these women were properly kept until phase 2 or 3. During phase 2, after the age compositions of the enrolled HPV‐negative cervical healthy women were determined, age‐matched HPV‐positive women who had normal Pap results were selected from the candidate pool and called back to participate in our observational study; their informed consent was obtained before the first round of follow‐up. In addition, the candidate pool was continuously expanded by collecting eligible HPV‐positive and/or Pap‐abnormal women during the same phase. Phase 2 enrolment ceased on 20 September 2019, and the study quickly entered phase 3 as the HPV genotyping process had been completed for each of the phase 2 women . In phase 3, HPV‐positive/‐negative Pap‐abnormal women were selected from the pool based on the following rules: (1) the enrolled HPV‐negative Pap‐abnormal women should comprise a group with the same/similar age composition as that of the group of HPV‐negative Pap‐normal women; (2) the enrolled HPV‐positive Pap‐abnormal women should comprise a group with the same/similar age composition and HPV genotype composition as those of the group of HPV‐positive Pap‐normal women; (3) the women were randomly selected and called to determine their willingness to participate if they met the age and HPV genotype requirements; informed consent was obtained from the enrolled women before the first round of follow‐up; and (4) if there were still vacancies regarding a specific age and/or HPV genotype condition that could not be fulfilled by the pool, the candidates were selected and enrolled directly from outpatient departments of the participating centres before 20 March 2020. Upon the enrolment deadline, the remnant and redundant candidates in the pool were released, and their information and cervical samples were deleted or discarded under surveillance. The study protocol  between 20 September 2018 and 20 March 2020, at 10 local medical centres of mainland China, namely, (1) Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, (2) Pudong Hospital, Fudan University, Shanghai, (3) Songjiang Hospital , Songjiang, Shanghai, (4) Qilu Hospital, Shandong University, Jinan, Shandong, (5) Zouping People's Hospital, Zouping, Shandong, (6) The Central Hospital of Zibo Mining Group Co. Ltd., Zibo, Shandong, (7) Chifeng College Affiliated Hospital, Chifeng, Inner Mongolia, (8) The Second People's Hospital, Three Gorges University, Yichang, Hubei, (9) Yongcheng People's Hospital, Yongcheng, Henan and (10) Taiyuan Maternal and Child Health Care Hospital, Taiyuan, Shanxi. Women were excluded if they were pregnant, a virgin (or <16 years old), had undergone hysterectomy or had been treated for cervical intraepithelial neoplasia (CIN) or any malignant diseases during the past 5 years. Women with severe acute or chronic mental/infectious/internal/surgical diseases as well as those with autoimmune/posttransplant diseases who relied on daily immunosuppressive drugs or those with endocrinological syndromes/abnormal uterine bleeding who had to use hormone medicines long term were also excluded. The enrolment process contained three phases Figure . For phal Figure  was appr2.2INK4A‐specific monoclonal antibodies used for FCM purposes were prepared from a set of novel hybridomas . There were no clones for FCM purposes commercially available to us before this work (even at the time of publication). To build anti‐human p16INK4A antibody hybridomas, the CDKN2A cDNA sequence (NM 000077.4) was synthesised and inserted into the prokaryotic expression vector pET‐28a, which was then transferred into Escherichia coli strain BL21 to produce the full‐length p16INK4A antigen. The E. coli‐expressed product was purified, concentrated and applied to immunise BALB/c mice. The obtained novel hybridoma clones were sequentially tested for their immune reactivity and specificity against human p16INK4A protein by ELISA and Western blotting , an immunochemistry‐purposed mouse anti‐human p16INK4A monoclonal antibody  or an anti‐human β‐actin antibody . The immunoreactions were then detected with a (secondary) HRP‐conjugated rabbit anti‐mouse IgG polyclonal antibody  and visualised with ECL Western Blotting Detection reagents .Whole‐cell protein extracts of HeLa and HeLa16 cells were separated in 10% SDS‐PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes . The protein bands were first immunoreacted with our novel p162.4INK4A FCM, each of the cell suspensions  was divided into two equal parts; one part  was sequentially immunoreacted with one of the three novel p16INK4A‐specific (primary) monoclonal antibodies  and a (secondary) FITC‐conjugated rabbit anti‐mouse IgG monoclonal antibody  in pH 7.4 phosphate‐buffered saline (PBS) at room temperature for 0.5–2 h; and the other  was immunoreacted with the secondary FITC‐conjugated rabbit anti‐mouse IgG antibody in PBS at room temperature for 0.5 h. A NovoCyte 2060 flow cytometer  was used for testing, and a total of 10 000 events were recorded for each run of the sample. The p16INK4A‐positive ratio was defined as the ratio of cells in the test portion of the sample with FITC signals stronger than a reference cut‐off. The reference cut‐off was defined as an FITC signal intensity equal to the lower limit of signal intensity of the top 10, 1 or 0.1% autofluorescent/falsely labelled cells. The FITC signal intensity data of each run were exported into Excel tables  to calculate p16INK4A‐positive ratios at a given reference cut‐off. The cellular mixtures with HeLa/(HeLa+HeLa16) ratios of 0 , 1, 5, 25, 50 and 100%  were used as a ladder that was tested in parallel to calibrate the p16INK4A FCM result in each run  and HPV DNA testing (5 mL), that is, approximately 15 mL in total.Cervical cytological samples were collected in ThinPrep fixative  for the liquid‐based Pap test.2.7HPV‐positive and/or Pap‐abnormal women were followed up regularly at a 3‐month interval for 2 years. Women with HPV infections or cytologically diagnosed with ASC‐US or worse lesions  were referred to colposcopy before initiating a regular follow‐up schedule. If their biopsy results indicated an intraepithelial squamous lesion lower than HSIL , the women were then involved in one of the three follow‐up cohorts, namely, HPV‐positive Pap‐normal, Pap‐abnormal biopsy‐negative and biopsy‐confirmed LSIL, based on their initial HPV, cytological and biopsy diagnoses. During follow‐up, the women were offered regular HPV DNA and Pap tests. If their regular Pap results revealed a persistent LSIL lesion over 1 year or a lesion worse than their initial cytological/biopsy diagnoses, the women were referred to colposcopy again. For women whose HPV DNA test revealed evidence of an infection with a novel viral genotype(s) or a relapse of former viral infection following an intermittent negative viral DNA test, colposcopy and biopsy (if necessary) were also referred. The (primary) endpoint event was biopsy‐confirmed HSIL or invasive cervical cancer within 2 years of follow‐up.2.8χ2 test/Fisher's exact test and ANOVA/Student's t‐test were used to compare enumeration  and measurement  data, respectively. The linear relationship between the actual and FCM‐detected nominal p16INK4A‐positive ratios as well as that between the viral genotype‐specific p16INK4A increments and multiple‐infection‐induced variations of genotype‐specific p16INK4A‐positive ratios were analysed using Pearson's product‐moment correlation coefficient. The changes in p16INK4A increments between two paired sets of groups of women, for example, HPV‐negative versus HPV‐positive women (age‐group pairs), single infections versus multiple infections  and Pap‐normal versus Pap‐abnormal women (age‐group pairs), were analysed using the Wilcoxon signed‐rank test. The cumulative risk of high‐grade lesions in p16INK4A‐normal versus ‐abnormal women was compared using Kaplan–Meier curves and the log‐rank test. The independencies of the hazards (presented as hazard ratios [HRs]) of clinical‐pathological risk factors  contributing to the occurrence of high‐grade lesions during follow‐up were analysed using a univariate or multivariate Cox regression model. The receiver operating characteristic curve was used to determine the optimal cut‐offs of the p16INK4A‐positive ratio in diagnosing p16INK4A‐abnormal cases among p16INK4A expression higher‐than‐normal  and lower‐than‐normal populations. SPSS 18.0 software  was used for analyses, and p < .05 was considered statistically significant.Two‐sided 33.1INK4A monoclonal antibody obtained by immunising the animal with E. coli‐expressed full‐length p16INK4A protein, was selected as a candidate clone to detect p16INK4A‐positive cells in the modified FCM platform. The p16INK4A protein‐binding ability (specificity and sensitivity) of this antibody was examined by Western blotting. For whole‐cell protein extracts of HeLa at a loading amount equal to 104 cells/lane (loading volume: 20 μL), clone 1H1087 showed no nonspecific bands on the PVDF membrane, while the other two clones, 1A72 and 1B517, which were prepared in parallel, detected bands for irrelevant proteins in the lysates of HeLa or HeLa16 cells  detected by p16INK4A FCM exhibited a perfect linear relationship with the actual numbers , strong‐positive (reference‐cut‐off = 1%) and extremely strong‐positive (reference‐cut‐off = 0.1%) cells and their actual numbers (percentages) reached 0.9961, 0.9944 and 0.9973, respectively, and were highly close to the performance (r = 0.9917) of the ΔMFIs  using FCM reached 0.1% at the reference cut‐off of 10% , while this ratio dropped in women aged 60 years  in the cervix. We applied an age‐ and HPV genotype‐matching strategy to build study cohorts  varied with the age of the women  than that among their HPV‐negative counterparts . According to the age‐ and HPV infection‐dependent distributional patterns of the aforementioned 17562  and 3596 cases , age‐ and viral genotype‐matched women with abnormal Pap tests were assessed and comprised each study cohort based on TBS categories  than in those with HSILs (5.6%) and were lowest in those with LSILs  than in those with LSILs (3.9%), the detected increments reached the highest (7.5%) in those with HSILs, justifying the utility of p16INK4A as a signature for triaging women with high‐grade lesions in this subsituation  and applied a double‐cut‐off‐ratio criterion , 21.7% (p16INK4A FCM vs. LSIL), 57.0% (p16INK4A FCM vs. ASC‐H) and 60.9% (p16INK4A FCM vs. HSIL)  and 11.9% (lower cut‐off) for HPV‐positive cases and 18.6% (upper cut‐off) and 11.2% (lower cut‐off) for HPV‐negative cases, by which a maximal Youden's index of 0.78 was obtained, superior to that of the HPV DNA and Pap co‐test by 5.6%  Table . Alterna6% Table . In totan Tables  and S7.3.5INK4A‐positive ratios who had negative colposcopy‐guided biopsy results . Additionally, there were women with biopsy‐confirmed LSILs  who were followed up to 2 years as required by the American Society of Colposcopy and Cervical Pathology (ASCCP) guidelines. Therefore, we conducted a prospective study of these women to investigate whether they would run a higher risk of HSIL+ . As expected, for all three conditions, namely, the HPV‐positive Pap‐normal (NILM) condition, Pap‐abnormal biopsy‐negative condition and biopsy‐confirmed LSIL condition, women with an abnormal p16INK4A status exhibited a significantly higher incidence rate of histological  HSILs  and were statistically significant in multivariate analyses  cohorts, their cumulative risks (PPVs at year 2: 13.2–15.6%) of histological HSILs exceeded the ASCCP‐recommended threshold for colposcopy referral , while in the biopsy‐confirmed LSIL cohort HPV‐positive subsituation, this risk was further doubled for p16INK4A‐abnormal women , which were close to the ASCCP‐recommended ‘1‐year return’ threshold  in 2 years  while remaining below the colposcopy referral threshold at year 2  with abnormal p16s Tables . In the  LSILs 46 in total LSILs 46 in total LSILs 46 in total4INK4A as a qualitative pathological signature has been fully documented. Previously, pathological and clinical guidelines and/or instructions were issued for labelling p16INK4A on cytological (Pap) or histological (biopsy) slides with proper immunostaining techniques as well as for evaluating the severity of pre‐cancerous/cancerous lesions  with qualitative criteria of p16INK4A.INK4A pathology to women with premorbid, ambiguous or transitional pathological statuses of cervical lesions , which enabled both immediate and short‐term (2‐year) risk assessments of high‐grade lesions in more specified and difficult clinical settings. Our data showed that p16INK4A FCM  can be used to quantitatively and precisely determine the cervical p16INK4A expression level, by which both age‐dependent physiological and infection‐induced aberrant expression of p16INK4A can be measured; therefore, the related intracellular molecular changes upon HPV infection  can be surveilled. These findings justify the utility of p16INK4A‐based quantitative pathology for detecting/predicting cervical carcinogenesis and directing early interventions  among high‐risk women.For cervical cancer, the implication of p16INK4A as an immunostaining signature for cervical pre‐cancer/cancer lesions has been 20 years.INK4A was originally proposed to eliminate discrepancies between pathologists as they interpreted Pap or biopsy findings into TBS‐categorised terms. As the initial inventors of the p16INK4A immunostaining technique, Klaes and his colleagues observed intensive and diffuse immunohistochemical staining of p16INK4A in 100% (60 out of 60) of cases of HSIL and 97% (58 out of 60) of cases of invasive cancer.INK4A to be labelled on thin‐layer, liquid‐based Paps, that the qualitative correlation between immunocytochemical staining of p16INK4A and histological HSIL+ lesions was formally established.INK4A immunocytochemistry is still a labour‐intensive and skill‐demanding technique that requires pathologists to carefully discern truly stained cells from false‐positive cells under a nonspecific immunostaining background. Moreover, per Bibbo's approach, the semiquantitative count of true‐positive cells is a prerequisite for predicting HSIL+ lesions. Sahebali et al. subsequently created a holo‐quantitative scale for p16INK4A‐based detection of HSILs by counting the exact number of positive cells in a Pap, which obtained a 21.2% PPV at the 95% detection sensitivity.INK4A‐positive cervical cells should be proportional to the risk of high‐grade lesions. This principle can be summarised as a ‘pyramid theory’ with an emphasis that the cervical pre‐cancer/cancer should originate from a vast number of morphologically normal but molecularly altered cells . In this study, we applied this principle to triage potential lesions; nevertheless, our technique avoided the burden of manually counting p16INK4A‐positive cells under a microscope. The data we obtained were quite objective due to the nature of machine‐reading and automatic analysis of p16INK4A FCM, which were also improved by the addition of external standards to calibrate this detection system (see section Methods). We, therefore, obtained a better diagnostic performance with p16INK4A with a much higher Youden's index (0.78) compared with earlier works .INK4A‐positive cells rather than microscopically assessing their morphology.The history of p16INK4A in the cervix varied with the woman's age. The overall expression of p16INK4A was significantly lower in women aged 20–29 and 50–59 years than in women aged 30–39 and 40–49 years, suggesting that normal levels of female hormones  might be essential for maintaining the physiological expression of p16INK4A, while for women at the menopausal period, diminished proliferation activity of the cervical epithelium was associated with decreased p16INK4A expression . The possible reasons for their neglect might include that (1) they overlooked the importance of age‐related p16INK4A expression in pursuing a proper cut‐off ratio for cervical neoplasia and that (2) the traditional techniques did not allow probing a tiny change in protein expression within a normal range or below the detection threshold . Owing to the precise quantification ability of p16INK4A FCM, we characterised the age‐related variations in p16INK4A expression in normal cervixes . Therefore, directly mixing p16INK4A‐positive ratios of younger women (20‐29 years old) with those of elderly women (≥30 years old) would result in a significantly lowered cut‐off ratio to be set for neoplastic lesions, leading to raised false‐positive cases in age groups of 30–39 and 40–49 years. Such data would undermine the diagnostic accuracy of p16INK4A. We therefore adopted age‐adjusted p16INK4A criteria and achieved an improved PPV for HSILs  First, in previous reports, the 5‐year cumulative incidence of HSIL+ presented a first peak at 11% in women aged 40–44 years and a second at 10% in women aged 60–64 years.INK4A increment in women aged 40–49 years and a minor peak in women aged 60–69 years  Second, we found that women in the 20–29 years age group, who were reported to have the lowest risk of HSIL+ lesions, had a minimal increase in p16INK4A after HPV infection. Additionally, current clinical experiences have indicated that HPV infections scarcely cause severe neoplastic changes in the cervixes of women aged 20–24 years.INK4A dysregulation. (3) Third, after reviewing the literature, we noted that the peak serum oestrogen and progesterone concentrations emerge at ages 40–49 years,INK4A positive ratios/increments in the observed age group in our study , specificity (88.5 vs. 86.4%), PPV (13.6 vs. 11.3%) and NPV , where even a nuance of positive ratio/increment between single and multiple infections could be discriminated . Sahebali et al.INK4A overexpression events than did other HR genotypes, while LR HPVs caused the lowest p16INK4A overexpression. Therefore, both previous reports and our own data pointed to a common fact that p16INK4A, as a downstream element of RB, can be used to measure the harm an HPV infection could do to the host cells. The hazards of each HPV genotype, therefore, can be ranked per the p16INK4A increments they have induced. However, we have other profound findings. We observed that an extra infection of less dangerous genotypes can interfere with the carcinogenetic capacity of a more dangerous genotype , and in two of such cases, p16INK4A immunochemistry was even negative. Similar p16INK4A‐negative HSIL+ cases have also been reported in other studies.INK4A FCM in detecting HPV‐positive HSILs has been ensured after adopting a lower cut‐off ratio . The existence of these cases lowered the overall expression level of p16INK4A in HSIL cases and hence influenced the diagnostic accuracy of p16INK4A FCM, especially when a single‐cut‐off‐ratio strategy was applied. Previously, a study conducted by Nuovo et al.INK4A overexpression per se; the other is CDKN2A promoter hypermethylation, which could be reflected by the extremely low expression of p16INK4A. The latter produces a distinctive population of neoplastic lesions associated with a poor prognosis.INK4A expression by FCM‐based detection  was adopted, the sensitivity and Youden's index of p16INK4A FCM were improved by 4.3% and 0.04, respectively , should be focused on creating a fitter nonlinear algorithm for p16INK4A to predict more carcinogenesis events.Our study described the practical performance of p16ed Table . The datn Figure . This fie Figure . Our epie Figure . SimilarINK4A FCM in Pap‐/biopsy‐normal (or LSIL) populations. The roles of p16INK4A in predicting women with cervicopathologically normal or low‐grade abnormal status are of interest to gynaecological clinicians worldwide. However, the outcomes of p16INK4A‐abnormal populations with different initial situations of HPV DNA, Pap and/or biopsy were not clear. Before 2008, Carozzi et al.INK4A in triaging biopsy‐negative/LSIL women threatened with increased risks of high‐grade lesions during 3‐year follow‐up. The design of the NTCC study was basically consistent with ours with a few exceptions: (1) in the NTCC study, only HPV‐positive women were referred to colposcopy, whereas in our work, both those with HPV infections and those with abnormal Pap results (even if HPV‐negative) underwent colposcopy; and (2) in the NTCC study, CIN2+  cases detected by initial colposcopy and biopsy were mixed with those diagnosed during follow‐up for calculating p16INK4A predictive values; however, in our study, cumulative risks of HSILs were calculated independent of initial colposcopy/biopsy examination results and were studied separately for three particular conditions , providing more detailed information for p16INK4A‐based prognosis. Carozzi et al.INK4A immunocytochemistry could improve both immediate and short‐term assessments for CIN2/3+ risks , and the calculated relative cumulative 3‐year risk for histological CIN2/3+ (HSIL+) lesions was 3.74 in p16INK4A‐positive women compared with negative controls. Later, in 2012, Wentzensen et al.INK4A/Ki‐67 dual‐staining technique.INK4A diagnostic specificity could be enhanced. They finally obtained a 5‐year risk assessment performance by dual‐staining for HSIL+ lesions with sensitivity 83%, specificity 59%, PPV 21% and NPV 96%, which were very close to the parameters reported by Carozzi et al.,INK4A prognosis (approximately 1%). In our study, the 2‐year HSIL‐risk predictive performances of p16INK4A differed with study cohorts. The sensitivity (100%) and NPV (100%) of p16INK4A FCM reached the highest levels in the HPV‐negative biopsy‐confirmed LSIL women, while the highest specificity (98.2%) was found in the HPV‐positive NILM women. Furthermore, in the biopsy‐confirmed LSIL cases with positive HPV DNA tests, the PPV  of p16INK4A‐abnormal cases reached its highest level (29.3%), implying this particular population of women with LSILs had a much greater risk of HSIL+ than had been suggested by the ASCCP guidelines ,INK4A to be interpreted on an individualised basis where both viral infection history and Pap/biopsy status need to be co‐weighed. This has mirrored the spirit of the 2019 version of the ASCCP guidelines.Our study investigated the prognostic value of p16INK4A detection system, allowing the quantitative depiction of the expression level and pathological increment of p16INK4A in cervical epithelial cells; (2) a large population‐based p16INK4A quantification database, revealing the age‐ and viral genotype‐dependent expression of p16INK4A in the cervix, necessitating an age‐ and HPV DNA test‐adjusted p16INK4A diagnostic criterion to be made; (3) an improved performance in detecting/predicting high‐grade lesions in the cervix, allowing precise colposcopy referral and early intervention among high‐risk women; and (4) insights into the relationships between p16INK4A quantification and the ASCCP guidelines, promoting evidence‐based decisions for treating women with ambiguous HPV and/or Pap results.The main strengths of this study include the following: (1) the establishment of a novel p16INK4A, we applied an age‐ and genotype‐matching strategy to enrol the study populations. Although HPV‐negative Pap‐normal cases were collected consecutively, the matched HPV‐positive Pap‐normal/abnormal cases were semi‐objectively enrolled, which might disrupt their natural time‐line continuity. (2) We did not determine the association between infection titre and p16INK4A increment. Although the viral infection titres could be detected via quantitative PCR (qPCR), there are currently no United States Food and Drug Administration‐approved HPV DNA qPCR detection kit available worldwide. (3) No HPV‐negative Pap‐normal women were referred for colposcopy, and a few women with HPV‐negative HSIL might be misdiagnosed. However, considering that the number of such cases could be extremely low and the reported incidence was <0.04%,INK4A FCM, should not be affected.This study has limitations. (1) Due to the need to evaluate age‐ and HPV genotype‐specific expression of p16INK4A qualitative pathology into a quantifiable and interlaboratory cross‐verifiable IVD, which was implemented through an FCM platform. This technological innovation built a bridge for pathologists to reach a more objective and consistent opinion on the nature of Pap abnormalities and inform clinicians of the molecular stage of HPV infection(s). The same technique can be applied to other known immunochemical signatures with clear clinical or pathological significance, such as Ki‐67, hTERT, EGFR, HER2 and ALK, which could provide more precise and quantitative information for pre‐therapy evaluation and post‐therapy surveillance.In summary, our work has developed the traditional immunochemistry‐based p16The authors declare no conflicts of interest.Supporting InformationClick here for additional data file.