In this case, 13 studies evaluated HPV detection in tumour tissue, nine studies examined HPV detection in blood samples and five studies evaluated HPV detection in oral samples. Accuracy of HPV detection in tumour tissue was high for all detection methods, with pooled sensitivity ranging from 81.1% (95% CI 71.9–87.8) to 93.1% (95% CI 87.4–96.4) and specificity ranging from 81.1% (95% CI 71.9–87.8) to 94.9% (95% CI 79.1–98.9) depending on detection methods. Overall accuracy of HPV detection in blood samples revealed a sensitivity of 81.4% (95% CI 62.9–91.9) and a specificity of 94.8% (95% CI 91.4–96.9). In oral samples pooled sensitivity and specificity were lower (77.0% (95% CI 68.8–83.6) and 74.0% (95% CI 58.0–85.4)). In conclusion, we found an overall high accuracy for HPV detection in tumour tissue regardless of the HPV detection method used. HPV detection in blood samples may provide a promising new way of HPV detection.The aim of the study was to evaluate the diagnostic accuracy of Human Papillomavirus (HPV) techniques in oropharyngeal cancer. PubMed, EMBASE, the Cochrane Library and clinicaltrials.org were systematically searched for studies reporting methods of HPV detection. Primary outcomes were sensitivity and specificity of HPV detection. In this case, 27 studies were included ( The incidence of oropharyngeal squamous cell carcinomas (OPSCCs) caused by human papillomavirus (HPV) is increasing worldwide ,2. PreviSeveral techniques to evaluate HPV positivity exist. These includes p16 evaluation by immunohistochemistry (IHC), detection of HPV DNA by in situ hybridisation (ISH) or by polymerase chain reaction (PCR), E6/E7 HPV mRNA evaluation by ISH and reverse transcriptase-PCR (RT-PCR), or a combination of the above-mentioned methods. E6/E7 HPV mRNA evaluation is considered the golden standard to assess HPV positivity, as this technique detects oncogenic transcriptional active HPVs, but the test is expensive and technically challenging to perform . On the The definition of HPV+ OPSCC is a critical issue as treatment de-escalation in patients with HPV+ tumours is currently being investigated in clinical trials to avoid unneeded treatment-related side effects, overtreatment, and to minimize the risk of treatment-related acute and long-term morbidity in this patient group. However, this should be performed without misallocating patients with less favourable prognosis to less treatment.In addition, several new techniques for assessing HPV positivity without the need of an invasive biopsy of tumour tissue, e.g., by liquid biopsy using saliva or blood are advancing which would be a readily available way of detecting HPV. Circulating tumur DNA (ctDNA) from virus-induced cancers has previously been shown to be clinically useful as a diagnostic test for oncovirus-driven cancers, such as Hepatitis B virus (HBV)-induced hepatocellular carcinoma  and EpstEvolution in laboratory techniques is rapidly evolving, experience with p16 detection is increasing and new detection standards are continuously being presented. An update on the recent knowledge in HPV detection is a timely needed study. Furthermore, a comparison of the diagnostic accuracy in different specimens and a ranking of these are warranted.The aim of this study was to systematic review the literature on methods of HPV detection and to assess the diagnostic accuracy for HPV detection in patients with OPSCC based on detection methods and in different sample types.This systematic review and meta-analysis was conducted with reference to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement .One author (KKJ) systematically searched the PubMed, EMBASE, Cochrane databases and clinical trials.org for articles in English and Scandinavian language. The search was last updated on the 28 May 2021. We included original studies comprising OPSCC patients investigating diagnostic methods of HPV detection published within the last five years. Studies comprising patients with OPSCC along with other head and neck cancer subsites were included, if they provided information specifying the results of the diagnostic accuracy of HPV detection for the OPSCC patients. Studies were excluded if they included less than 10 OPSCC cases and if the HPV detection method, including the definition of HPV positivity and p16 positivity, was not defined.The search term was phrased broadly to identify relevant references. The following keywords were used to build the search:  AND (HPV or human papillomavirus or human papilloma virus or p16 or papillomavirus or p16 or cdkn2a or cyclin-dependent kinase inhibitor p16 or p16 genes) AND (diagnosis or diagnostic)). The search strategy in PubMed included MeSH terms.We collected information on study type, diagnostic methods, reference methods, sample type, HPV type and the sensitivity and specificity of the diagnostic methods.Statistical analyses were performed using R studio, version 1.2.5. We generated paired forest plots depicting sensitivity and specificity estimates across studies. We conducted a meta-analysis using the bivariate model in R studio by using the mada package function reitsma . The modThe literature search generated 1513 articles, of which 24 were enrolled. Three additional articles were identified through reference lists .n = 975), other reviews, case reports and editorials (n = 218) and lastly, studies regarding HPV diagnostics in other patient groups than OPSCC (n = 196). Thus, 119 articles were assessed by full-text. In this case, 92 studies were not included as they did not concern diagnostic methods (n = 50), did not report diagnostic accuracy (15), did not specify results for OPSCC patients (n = 11), did not define HPV or p16 positivity (n = 9) or included less than 10 patients (n = 7).A total of 1389 articles were excluded based on screening of the title and abstract. Of these, we excluded studies that did not focus on the diagnostic accuracy of HPV detection (n = 229) consisting of head and neck cancer patients with a cancer located at another subsite than the oropharynx (n = 74), healthy controls (n = 75), patients with Warthin’s tumour (n = 20) or branchial cleft cyst (n = 10). The studies including non-OPSCC patients were excluded in the meta-analysis. In this case, 14 studies were European, 10 were US and Canada based and three studies were Asian . Three studies included a non-OPSCC control group  tissue ,27,28,29Of the nine studies evaluating HPV detection in FFPE, six studies evaluated more than one detection method ,26,28,29Specificity ranged considerably, with the lowest specificity reported as 55% (95% CI 38–71) , and thrn = 195) with the majority of patients being HPV+ OPSCC (85.6%). Four of the five included studies reported a specificity of 100% [The five studies evaluating the diagnostic accuracy in FNA comprised few patients  examined the diagnostic accuracy of HPV detection by liquid biopsy using blood samples. One study collected the blood samples both at time of diagnosis and during treatment and evaluated the accuracy of the test regardless of the time of blood collection [Nine studies  . The stuIn the pooled analysis of sensitivity and specificity, the overall sensitivity was 81.4% (95% CI 62.9–91.9), and the overall specificity was 94.8% (95% CI 91.4–96.9) covering all studies regardless of detection method and reference ,41,42,43Five studies evaluated the diagnostic accuracy of HPV detection in oral samples  correspoA considerable variability in sensitivity and specificity estimates were observed among the studies ranging from 60.9% (95% CI 40.8–77.8)  to 86.1%Regardless of oral sample collection method and reference, method the meta-analysis revealed a pooled sensitivity and specificity of 77.0% (95% CI 68.8–83.6) and 74.0% (95% CI 58.0–85.4), respectively. When restricted to the four studies using p16 IHC as a reference, the pooled sensitivity and specificity was 77.6% (95% CI 67.8–78.7) and 72.1% (95% CI 49.1–87.4).This systematic review and meta-analysis evaluated the diagnostic accuracy of HPV detection in patients with OPSCC. As laboratory techniques are evolving rapidly and new detection methods continuously are being introduced, this is an area in need of an update in the current literature. A ranking and comparison of the diagnostic accuracy in different specimens are furthermore needed.As p16-status is an important factor in staging OPSCC , and as We included a total of 27 studies with varying specimens, methods of HPV detection and references for the latter. We first looked at studies evaluating the diagnostic accuracy of HPV detection in tumour tissue. Two studies evaluated the diagnostic accuracy of combining two detection methods, i.e., p16 IHC combined with HPV DNA PCR. Both studies reported high sensitivity of 93% (95% CI 74–98%) and 86% (95% CI 76–92%), respectively. A similar systematic review  also fouOf importance, we excluded nine studies where the definition of p16 positivity was not specified. The exclusion may have had an impact on the results of our review. It is incredibly important to specify the p16 positivity, since it has been shown that to achieve the highest correlation between p16 and HPV results, a staining of >70% of tumour cells to classify the tumour as p16 positive is advised . The enrRecently, the ability to detect HPV in liquid biopsies was introduced as a novel, non-invasive method of HPV detection. The use of a liquid biopsy for cancer detection has shown encouraging results in both colorectal cancer and bladder cancer ,51,52. IWhen looking at the diagnostic accuracy in oral samples obtained from OPSCC patients, our study revealed a lower diagnostic accuracy than the other specimen types with a sensitivity and specificity of 77.6% (95% CI 67.8–78.7) and 72.1% (95% CI 49.1–87.4), respectively. Variability amongst the studies detecting HPV in oral samples varied considerably. A similar meta-analysis  investigIn general, the included studies varied in regards to reference method as well as method of detecting HPV, which comprises a significant limitation when comparing the diagnostic accuracy between studies. This might be one possible explanation for the variation in accuracy across the included studies. The pooled sensitivity and specificity should thus be interpreted with caution as accuracy can vary depending on testing method and reference method. To investigate the accuracy of the specific detection methods in order to circumvent the variability and uncertainty different detection methods might bring to the meta-analyses, we performed a sub-analysis of the diagnostic accuracy stratified on detection method for the studies assessing accuracy in FFPE. We found that sensitivity and specificity in general were high for all detection methods with sensitivity ranging from 81.1 (95% CI 71.9–87.8) to 93.1 (95% CI 87.4–96.4), and specificity ranging from 81.1 (95% CI 71.9–87.8) to 94.9 (95% CI 79.1–98.9). It was not possible to conduct sub-analysis for accuracy in liquid biopsy and FNA due to the lower study numbers. We did not account for the different reference methods in the meta-analysis, as it would have resulted in too few studies to perform a valid meta-analysis. In general, grouping studies regardless of the reference methods when comparing sensitivity and specificity of diagnostic testing in meta-analyses ,55 is a We included studies published within the last five years to avoid excessive variation in the detection methods between studies as p16 positivity previously ranged considerably and a large part of studies used a minimum of 5–69% staining  before AIn conclusion, our systematic review evaluating HPV detection methods in patients with OPSCC showed an overall high sensitivity and specificity of HPV detection in FFPE for both RNA ISH, DNA ISH, DNA PCR and p16 IHC. HPV detection by liquid biopsy and blood samples provides a promising, less invasive method of HPV detection and both sensitivity and specificity were high, thus highlighting HPV detection in blood samples as a promising novel tool of HPV detection. HPV detection in blood samples showed an overall sensitivity of 81.4% (95% CI 62.9–91.9), and an overall specificity of 94.8% (95% CI 91.4–96.9) which is thus comparable to the sensitivity and specificity of HPV detection in FFPE where the sensitivity was ranging from 81.1% (95% CI 71.9–87.8) to 93.1 (95% CI 87.4–96.4) and the specificity was ranging from 81.1 (95% CI 71.9–87.8) to 94.9% (95% CI 79.1–98.9.Lastly, results on the accuracy of HPV detection in FNA and in oral samples were scarce and varied considerably, and evidence on the use of oral samples in HPV detection is currently not substantial enough to highlight it as an acceptable diagnostic tool. In summary, larger studies with homogenous study designs are required to further explore the diagnostic applicability of various HPV detection methods in patients with HPV+ OPSCC.


Chemical Science. These articles cover diverse nanomaterials, including organic, inorganic, organic–inorganic hybrid and superstructured nanomaterials. The details based on the composition are as follows:Owing to its fast development, chiral nanoscience has become one of the core research topics in chemistry, physics, medicine and materials science. Recently, many efforts have been focused on constructing new types of chiral nanomaterials with unique optical activity. This themed issue highlights the state-of-the art progress in this field, based on a series of invited and selected articles published in et al.’s work on constructing pillar[5]arene-based chiral nanotubes via pre-regulation of the building blocks’ chirality (DOI: 10.1039/D1SC00074H), the surface-induced enantiomorphic crystallization of achiral fullerene derivatives in thin films done by Keisuke Tajima et al. (DOI: 10.1039/D0SC01163K), and chiral polymer hosts for circularly polarized electroluminescence devices realized by Changsoon Kim, Youngmin You and coworkers (DOI: 10.1039/D1SC02095A).On the topic of organic nanomaterials, one can refer to Tomoki Ogoshi 10.1039/C9SC04239C). Minghua Liu, Shimei Jiang and coworkers presented multicolor tunable circularly polarized luminescence in a single AIE system (DOI: 10.1039/C9SC05643B). Qinghua Lu, Hailiang Zhang, Quan Li and coworkers showed solvent polarity driven helicity inversion and circularly polarized luminescence in chiral AIE fluorophores (DOI: 10.1039/D0SC04179C). A corresponding review about the chiral assembly of organic luminogens with AIE properties was given by Hai-Tao Feng, Ben Zhong Tang and coworkers (DOI: 10.1039/D1SC02305E).Specific to organic nanomaterials with chiral aggregate-induced emission (AIE) properties, Yanhua Cheng, Ben Zhong Tang and coworkers reported the polymorph selectivity of an AIE luminogen under nano-confinement to visualize polymer microstructures . Yoshitaka Aramaki, Takashi Ooi, Masakazu Nambo, Cathleen M. Crudden and coworkers introduced the synthesis and enantioseparation of chiral Au13 nanoclusters protected by bis-N-heterocyclic carbene ligands (DOI: 10.1039/D1SC03076K). Georg H. Mehl showed the development of two helices from one chiral center in self-organized disc-shaped chiral nanoparticles (DOI: 10.1039/D0SC05100D). A review on template-assisted self-assembly of achiral plasmonic nanoparticles into chiral structures was written by Luis M. Liz-Marzán (DOI: 10.1039/D1SC03327A).On the topic of inorganic nanomaterials, Masahiro Ehara, Takuya Nakashima and coworkers reported enantioseparation and chiral induction in Ag10.1039/C9SC02670C). Xiaogang Qu fabricated a series of stereoselective nanozymes (Fe3O4@poly(AA)) using a ferromagnetic nanoparticle yolk as the catalytic core and amino acid-appended chiral polymer shell as the chiral selector (DOI: 10.1039/D0SC03082A). Yongsheng Zhao, Chuanlang Zhan, Jiannian Yao and coworkers reported lanthanide MOFs for inducing the molecular chirality of achiral stilbazolium with strong circularly polarized luminescence and efficient energy transfer for color tuning (DOI: 10.1039/D0SC02856H).On the topic of organic–inorganic hybrid nanomaterials, Shu Kobayashi prepared heterogeneous Rh and Rh/Ag bimetallic nanoparticle catalysts immobilized on chiral polymers with high-to-excellent yields and enantioselectivities (DOI: 10.1039/C9SC02336D). De-Liang Long, Leroy Cronin and coworkers fabricated peptide sequence mediated molybdenum blue nanowheel superstructures (DOI: 10.1039/D0SC06098D). Kazuhiko Nakatani, Chikara Dohno, Ben L. Feringa and coworkers designed a photoswitchable DNA glue with high regulatory function and supramolecular chirality transfer (DOI: 10.1039/D1SC02194J). A related review of hierarchical self-assembly into chiral nanostructures was presented by Minghua Liu (DOI: 10.1039/D1SC03561D).On the topic of superstructures, Zeyuan Dong constructed a helical supramolecular polymer nanotube by manipulating strong noncovalent interactions (DOI: The above articles and reviews provide a complete picture of the construction of various chiral nanomaterials. We hope that the readers will quickly grasp the entire concept of this field with the help of this themed issue.


After 3 years of follow-up, the sensitivity for CIN2+ (n = 133) and CIN3+ (n = 74) were both similar between DH3 and HC2 . The respective specificity for CIN2+ or CIN3+ did not differ between the two tests. A noninferiority test showed that both sensitivity and specificity of DH3 for CIN2+ and CIN3+ were noninferior to those of HC2 at baseline and after 3-year follow-up, respectively . When used in primary screening strategy, the DH3 assay would yield an immediate sensitivity of 92% for CIN2+. DH3 HPV performs equally to HC2 for the detection of high-grade lesions in cervical cancer screening and has a potential advantage in primary screening strategy due to HPV16/18 genotyping.The authors compared the clinical performance of DH3 human papillomavirus (HPV) assay, which detects 14 high-risk HPVs with 16/18 genotyping based on hybrid capture technique, and Hybrid Capture 2 (HC2) test for women undergoing cervical cancer screening. A total of 7, 263 residual cytology specimens from an adjudicated cohort with 3-year follow-up were tested by the DH3 assay and the HC2 test. Assay results were compared with each other and to histology review. The overall agreement between the DH3 assay and the HC2 test was 99.2% (κ = 0.938). At baseline, DH3 had the equal sensitivity to that of HC2 for cervical intraepithelial neoplasia (CIN) grade 2 or higher  in cervical cancer screening have already been demonstrated. Hybrid Capture 2 (HC2) is the best validated HPV assay and has been considered the gold standard for hrHPV testing. However, HC2 cannot discriminate HPV16 and 18 from the other hrHPV types, which greatly limited the application of HC2 in cervical cancer screening. The DH3 human papillomavirus (HPV) is a recently developed assay based on hybrid capture technique like to HC2, which can specifically identify HPV 16/18 on the basis of detecting the 13 hrHPV types targeted by HC2 as well as HPV66. This comparative study of the two assays for detection of hrHPV infection in residual cytology samples from cervical cancer screening setting reveals that DH3 HPV provides a perfect alternative to HC2 in detecting hrHPV infection and identifying cervical precancer, while allowing concurrent HPV 16/18 genotyping. Cervical cancer is the fourth most common cancer in women worldwide, with over 500,000 new cases annually, most of which occur in developing countries . Almost Hybrid Capture 2  hrHPV DNA test, the first hrHPV test approved by the U.S. Food and Drug Administration (FDA), was designed to detect these 13 hrHPV types. HC2 assay is based on hybrid capture technique without PCR, which can reduce the risk of false-positive caused by cross-contamination. Detection of full-length human papillomavirus (HPV) DNA is another advantage of HC2, which can reduce to miss HPV that has been disrupted due to viral integration or point mutations . In factThe DH3  hrHPV DNA test is a recently developed assay based on hybrid capture technique like to HC2 , 12. It In this study we compared the concordance of DH3 HPV to the HC2 test using residual cytology samples from cervical cancer screening setting. In addition, we present here the first longitudinal data of 3 years follow-up regarding the clinical performance for the detection of high-grade cervical intraepithelial neoplasia (CIN) by the DH3 assay in comparison to the HC2 test.The mean age of the 7,263 women was 47.2 years , with 63.6% aged 45 years or older. At baseline, of the 7263 samples, 691 (9.51%) were DH3 HPV-positive, including 152 (2.09%) DH3 HPV16/18-positive, whereas 663 (9.12%) were HC2 HPV-positive. In total, 43 samples were DH3 HPV-positive but HC2 HPV-negative, 15 samples were DH3 HPV-negative but HC2 HPV-positive. The overall agreement between the DH3 HPV assay and the HC2 test was 99.20%, the positive agreement was 97.74%, and the negative agreement was 99.35% . The kapCervical disease status of this cohort and the corresponding HPV results at baseline are shown in P = 0.416 and P = 0.427, respectively). A noninferiority test revealed that both the clinical sensitivity  and specificity  of DH3 HPV for the detection of CIN2+ were noninferior to those of HC2 at baseline. Likewise, we found that both the clinical sensitivity  and specificity  of DH3 HPV for the detection of CIN3+ were noninferior to those of HC2 at baseline.The performance of DH3 HPV and HC2 test for identifying CIN2+ or CIN3+ at baseline and over 3-year follow-up are presented in P = 0.776 and P = 1.0, respectively). The specificity for detection of CIN2+ and CIN3+ were also similar between the two HPV assays . A noninferiority test showed that both the clinical sensitivity and specificity of DH3 HPV for detection of CIN2+  and CIN3+  were also noninferior to those of HC2 over 3-year follow-up, respectively.After 3 years of follow-up, the sensitivity for detection of CIN2+ and CIN3+ were both similar between DH3 and HC2 . There were only 58 (0.8%) discrepant specimens between the two assays, 43 of which were DH3 HPV-positive but HC2 HPV-negative. This is not unexpected, because the additional HPV 66 is covered by DH3 assay. A retrospective study showed that the prevalence of HPV-66 was approximately 0.94% among women attending cervical cancer screening in the same province of China .As expected, the analytical concordance was quite good between the DH3 assay and the HC2 test. We found that the overall agreement was 99.20% between the two assays, the positive agreement was 97.74%, and the negative agreement was 99.35%. The kappa coefficient for the overall agreement was 0.938, indicating almost perfect interrater agreement. These results showed that specifically identifying HPV16/18 on the hybrid capture platform did not affect the overall efficacy of hrHPV detection. DH3 HPV provides a perfect alternative to HC2 in detecting hrHPV infection, while allowing concurrent HPV 16/18 genotyping.In addition to the assessment of analytical concordance, the most important consideration when evaluating a new assay for detection of hrHPV in cervical screening is the clinical performance. A clinically useful hrHPV assay should have balanced the sensitivity and specificity for CIN2+ to ensure reliable detection of women with high-grade lesions and to minimize HPV-positive results in those without disease. It has been recommended that the candidate assay should have a clinical sensitivity and specificity for CIN2+ of not less than 90% and 98% of those of HC2, respectively .In this study, of 7,263 women, 75 CIN2+ and 45 CIN3+ were identified at baseline. Both the clinical sensitivity and specificity of DH3 HPV for detection of CIN2+ and CIN3+ were noninferior to those of HC2, respectively. Furthermore, the sensitivity of DH3 HPV in identifying CIN2+ and CIN3+ was equal to that of HC2, 98.67% and 97.78%, respectively. The favorable clinical performance of DH3 was also confirmed in 3-year follow-up study, which comprised 5840 women with 133 CIN2+ and 74 CIN3+. These results indicate that the clinical value of DH3 HPV in identifying high-grade CIN is very similar to that of HC2.There is growing evidence that HPV-based screening is more cost-effective than cytology or co-testing . BecauseConsidering the ability of concurrent HPV16/18 genotyping of DH3 assay, the potential value of DH3 HPV in primary cervical cancer screening was investigated in the present study. For this cohort, primary screening strategy with triaging HC2-positive women using reflex cytology would yield an immediate sensitivity for CIN2+ of only 74.67%. These results suggest that about 1/4 of high-grade lesions would be missed if HC2 primary screening was performed in areas with medium quality of cytology. If DH3 HPV primary screening was carried out with triaging DH3-positive women using the synchronous HPV16/18 genotyping and/or reflex cytology, the immediate sensitivity would increase to 92.0%. It should be noted that only slightly more women would be referred in DH3 HPV primary screening but 23.2% of additional CIN2+ cases would be identified immediately. These results support that DH3 HPV has a potential advantage in primary cervical screening. Consistently, our cross-sectional study reported previously in a routine screening population suggested that DH3 HPV performed similarly to Cobas 4800 in primary screening strategy for women aged 25–65 years .There are several limitations of this adjudicated cohort study. Firstly, the specimens were stored for up to 3 years before they were retested with the DH3 HPV and the HC2 assay. However, the values obtained for HPV prevalence in this study are in agreement with our previous study showing a DH3 HPV positive of 9.9% in a cross-sectional screening . SecondlIn addition to the perfect analytical agreement, evaluation of DH3 HPV in cervical cancer screening setting with baseline and 3-year longitudinal data showed that the clinical performance of DH3 HPV is not inferior to that of HC2. A considerable benefit of DH3 HPV test is the concurrent HPV16/18 genotyping which could serve as a valuable additional tool in patient risk stratification and management. In fact, triaging DH3-positive women using its inherent HPV16/18 genotyping and/or LBC would yield an immediate sensitivity for CIN2+ of 92%, which was significantly higher than triaging HC2-positive women using LBC. In summary, our results demonstrate that the DH3 HPV performs equally as well as HC2 for the detection of CIN2+ lesions in cervical cancer screening setting. Moreover, DH3 HPV has a potential advantage in primary screening strategy due to HPV16/18 genotyping and can be considered as a primary cervical screening option. Further evidence for the applicability of the DH3 HPV test in primary screening needs to be investigated.For this study, all specimens were from the residual cell preservation solution  of two screening projects with 3-year follow-up in Zhejiang province, China. After cytological examinations were routinely performed, the 7263 residual specimens of liquid-based cytology (LBC) in PreservCyt solution at baseline were stored in one walk-in refrigerator with 4°C for up to 3 years prior to HPV testing with both a DH3 test and an HC2 assay.in situ), and carcinoma. Due to the ethical considerations, almost all of the women with negative co-testing results were not referred to colposcopy and were all regarded as LSIL or less.According to the respective referral indications of the two projects, colposcopy was performed by the same team from our hospital. Patients with visible acetic-white iodine-negative lesions were subjected to biopsy. Patients with abnormal cytology results and no visible acetic-white iodine-negative lesions were subjected to endocervical curettage. Patients who had a normal cytology and showed no visible lesion during colposcopy were not subjected to biopsy and were considered “no lesion”. The histological diagnoses of cervical lesions were divided into normal, low-grade squamous intraepithelial lesion (LSIL/CIN1), high-grade squamous intraepithelial lesion (HSIL)/CIN2, HSIL/CIN3 . DH3 HPV testing was performed as described previously . The resP values less than 0·05 were considered to indicate statistical significance.The level of agreement between the two HPV tests was assessed by Cohen’s kappa statistics. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) were calculated by using the conventional contingency tables, and 95% confidence intervals (95% CI) were computed using Wilson score method. The chi-square test was used for intercomparison of proportions. Noninferiority of the clinical performance of DH3 HPV test versus HC2 assay was evaluated by noninferiority test for proportion (one-sided U test). The margins used for noninferiority were sensitivity for the detection of CIN2+ lesions of at least 90% and specificity for the detection of lesions less severe than CIN2 of at least 98% relative to the results of HC2, as previously described . Statist


Pubmed and Embase. Studies investigating the impact of HPV vaccines on oral or oropharyngeal HPV infection were enrolled. This review reports the relative prevention percentage (RPP), including a risk of bias assessment as well as a quality assessment study. Nine studies were included : five cross-sectional studies; one randomized community trial study (RCT); one longitudinal cohort study; and two case-control studies. A significant mean RPP of 83.9% (66.6–97.8%) was calculated from the cross-sectional studies, 82.4% in the included RCT and 83% in the longitudinal cohort study. Further, two case-control studies that measured antibody response in participants immunized with HPV vaccines were included. Respectively, 100% and 93.2% of participants developed HPV-16 Immunoglobulin G (IgG) antibodies in oral fluids post-vaccination. Analysis of the studies identified a significant decrease in vaccine-type oral or oropharyngeal HPV infections in study participants immunized with HPV vaccines across study designs and heterogenous populations. Further, a significant percentage of participants developed IgG antibodies in oral fluid post-vaccination.Human papillomavirus (HPV) imposes an increased risk of developing cervical, anal and oropharyngeal cancer. In the Western world, HPV infection is currently the major cause of oropharyngeal cancer. The effectiveness of HPV vaccines for oral or oropharyngeal HPV infection is yet to be determined. This study conducted a systematic literature search in  Human papillomavirus (HPV) imposes a risk of developing cervical, anal and oropharyngeal cancer. HPV infection is currently the major cause of oropharyngeal cancer in the Western world and the incidence of oral and oropharyngeal HPV infection is increasing ,3,4,5,6.The effect of prophylactic HPV vaccines is well documented for prevention of certain HPV types in cervical and anal cancer , but a pThe prevalence of oral and oropharyngeal HPV infections varies greatly amongst countries ,9,10, anThis review calculated/extracted a relative prevention percentage (RPP) in oral and oropharyngeal HPV infection amongst vaccinated and unvaccinated individuals as a surrogate goal for vaccine effectiveness, including studies that examine antibody response to vaccination as an alternative surrogate goal for immunization, with the aim of providing an analysis of established evidence. The aim of the study was to provide a systematic analysis and comparison of existing studies in order to provide evidence on the effect of HPV vaccines on oral and oropharyngeal HPV infection.This systematic review was conducted with reference to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement  12]..12].One author (KN) systematically searched PubMed and EMBASE for articles in the English and Scandinavian languages. The search was last updated on ultimo January 2021. Studies published in the period from January 2016 to March 2021 were included. The following keywords (MeSH terms included in PubMed) were used: HPV OR Human papillomavirus OR Papillomaviridae AND Vaccine OR HPV-vaccine OR Quadrivalent vaccine OR Gardasil-9 OR Cervarix AND Oral OR Mouth OR Oropharyngeal. Inclusion criteria were studies that investigated the association between HPV vaccines and oral and/or oropharyngeal HPV infection through one or both of the following designs: (1) using a population of individuals who have received the HPV vaccine and comparing incidence of oral or oropharyngeal HPV infection to a matched population or (2) assessment of antibody response following administration of HPV vaccines. Studies with other objectives were excluded.For the randomized control studies, the Cochrane risk of bias tool RoB-2  was usedThe literature search generated 1530 studies of which we included five cross-sectional studies ,10,15,16Four cross-sectional studies ,10,15,16The enrolled studies used different methods for collecting data and biological material from participants. One study  collecten = 312) found no correlation between HPV prevalence and vaccination status. The authors of this study claimed that results regarding oral and oropharyngeal infection proved inconclusive due to only four participants testing positive for any type of oral and oropharyngeal HPV infection.One cross-sectional study   examined the effectiveness of the AS04-adjuvanted HPV-16/18 vaccine in reducing oral HPV infection in young females. Three arms of eleven communities  were enrolled and participants were blinded regarding vaccine allocation. The trial showed a significant reduction in oral HPV-16/18 and found an RPP of 82.4% in HPV-16/18 six years post-vaccination.The community randomized trial   investigated antibody response in oral and oropharyngeal mucosal fluids and sera pre- and post-immunization with HPV vaccines. Both studies showed that a significant percentage of the population developed a vaccine-specific antibody response post-immunization. Sera and saliva were collected using mouthwash and Merocel sponges at both day one and month seven, and were tested for anti-HPV 16/18 immunoglobulin G (IgG) levels by an L1 virus-like particle-based enzyme-linked immunosorbent assay. In one [n = 150) seroconverted and the majority of participants developed anti-HPV16/18 antibodies in the oral cavity following vaccination: 96% for anti-HPV 16 and 72% for anti-HPV 18. One study also showed that in vitro antibodies were able to neutralize HPV pseudovirions [The two case-control studies ,20  seroconverted and developed IgG antibodies in oral fluids against vaccine-targeted HPV, in this case HPV 6, 16 and 18, post-vaccine. In vitro testing showed that antibodies were capable of neutralizing HPV pseudovirions. In the other case-control study [n = 150) seroconverted and 93.2% developed anti HPV-16 antibodies while 72.1% developed anti HPV-18 antibodies within seven months. After seven months, antibodies in oral fluids were significantly correlated to serum levels.In the aforementioned case-control studies, neutralizing HPV-targeted serum and saliva antibodies were collected from participants before and after immunization. In one study , 100% ofol study , similarOverall, the comparative studies showed a high HPV vaccine effectiveness on oral and oropharyngeal HPV infection regardless of study design. The cross-sectional studies had a mean relative reduction percentage of 83.9%, the RCT  had a reIn Denmark, the HPV vaccine is offered to girls and boys between the ages of 12 and 18 years and is a part of the tax-funded vaccination program. Until ultimo 2021, men who had sex with men between the ages of 18 and 26 years could elect for a tax-funded vaccine as well. [It is worth considering that a persistent oral or oropharyngeal HPV infection is rare in the general population, even amongst risk population e.g., partners of cervical HPV positive women . HoweverNo standardized procedures have been decided on for the collection of oral and oropharyngeal samples regarding determination of HPV infections. This has caused inconsistencies in the methods used to obtain oral and oropharyngeal samples. In the included studies, the following methods were used: Floq Swab; ; oral riThis systematic review has several strengths. Studies with different designs were enrolled, i.e., RCT, cross-sectional, case-control and cohort studies, in order to undertake a broad comparative analysis with the same predetermined outcome-specific assessment. In the same way, studies with different methods of assessing incidence and prevalence of oral and oropharyngeal HPV infection participated in the analysis. The relevant data was extracted and the RPP of each study was calculated in order to perform a diverse comparative analysis. A similarity in results across different methods supports the legitimacy of the outcome. Furthermore, the studies consisted of heterogenic populations, making it possible to determine the general effectiveness of HPV vaccines on oral and oropharyngeal HPV infections.One limitation of this study is that the averages calculated in the RPP of the cross-sectional studies are not weighted averages. This is due to some of the studies not having full transparency regarding which participants had oral and oropharyngeal HPV infection as opposed to any HPV infection, thereby making it difficult to calculate a weighted average. Another consideration is the inconsistent sample techniques as mentioned above.A further consideration is the inability to distinguish between samples from the oral cavity and from the oropharynx. This results in uncertainty and makes it difficult isolating vaccine effectiveness on oropharyngeal HPV infection. In future studies, one way of isolating oropharyngeal material would be to biopsy tissue from the tongue basis and tonsil and analyze it for HPV infection, which only one of the studies did . RegardiThe RPP of HPV vaccines  on oral and oropharyngeal infection proved significant, though it is still relevant to discuss why the RPP is not closer to 100%. One reason for this could be human error or inadequate technique in sample collection. Another explanation could be recall bias in the studies where vaccine status was self-reported. A third explanation could be participants having contracted oral or oropharyngeal HPV infection prior to the vaccination. In many communities, particularly religious communities, there is a taboo surrounding premarital sexual relations, which in theory could inhibit participants from truthfully reporting sexual status prior to vaccination trials. A final explanation could be some participants not developing adequate antibody response post-immunization. To investigate this hypothesis, further studies are needed.A significant decrease in vaccine-type oral and oropharyngeal HPV infection in study participants immunized with HPV vaccines was calculated or extracted, across study design and heterogenous populations. Similarly, a significant percentage of participants in the case-control studies developed IgG antibodies in the oral cavity post-immunizations with HPV vaccines, which presents an alternative surrogate goal for vaccine effectiveness. Furthermore, the similarity in RPP across study designs supports the legitimacy of the results.


HPV-positive HNSCCs are characterized by a different biology and demonstrate better therapy response and survival compared to alcohol/tobacco-related HNSCCs. Although we have a better understanding of the biology of both groups of HNSCC, the biological factors, especially environmental factors associated with the increased radiotherapy response, are still unclear. In this manuscript, we review the effects of an important microenvironmental factor, namely, low oxygen levels, also known as hypoxia, on the radiotherapy response and the tumor biology of HPV-positive and HPV-negative HNSCCs. In addition, we provide an overview of the current strategies to detect and target hypoxia, with a description of important clinical trials.Head and neck squamous cancers are a heterogeneous group of cancers that arise from the upper aerodigestive tract. Etiologically, these tumors are linked to alcohol/tobacco abuse and infections with high-risk human papillomavirus (HPV). HPV-positive HNSCCs are characterized by a different biology and also demonstrate better therapy response and survival compared to alcohol/tobacco-related HNSCCs. Despite this advantageous therapy response and the clear biological differences, all locally advanced HNSCCs are treated with the same chemo-radiotherapy schedules. Although we have a better understanding of the biology of both groups of HNSCC, the biological factors associated with the increased radiotherapy response are still unclear. Hypoxia, i.e., low oxygen levels because of an imbalance between oxygen demand and supply, is an important biological factor associated with radiotherapy response and has been linked with HPV infections. In this review, we discuss the effects of hypoxia on radiotherapy response, on the tumor biology, and the tumor microenvironment of HPV-positive and HPV-negative HNSCCs by pointing out the differences between these two tumor types. In addition, we provide an overview of the current strategies to detect and target hypoxia. HNSCC arises from epithelial cells and occurs in the upper aero-digestive tract. It is the sixth most common cancer worldwide and the incidence is still rising . HNSCC iOn the biological level, infection of the squamous epithelium with a high-risk human papillomavirus, mainly HPV-16 and HPV-18, can cause malignant transformation through the action of the two main oncoproteins E6 and E7. The E6 oncoprotein disrupts the p53 pathway, which leads to cell cycle dysregulation and uncontrolled cell cycle progression. The E7 oncoprotein induces inhibition of the retinoblastoma protein, leading to activation of the transcription factor E2F. This causes the initiation of cell cycle progression and expression of p16, a surrogate marker for HPV infections. In contrast, HPV-negative tumors have typical p53 mutations ,5.On the clinical level, patients with HPV-positive tumors have a significantly better overall prognosis, partly due to a higher radiosensitivity ,7. UntilAs previously mentioned, the better overall prognosis of HPV-positive HNSCC can, at least partly, be explained by the increased intrinsic radiosensitivity of these tumors ,11,12,13Another key factor that is linked with the radiation response is hypoxia. Hypoxia is a common feature in solid tumors and is caused by a discrepancy between oxygen supply and demand, since the abnormal and inefficient tumor vasculature cannot fulfil the highly demanding proliferating tumor cells. Most tumors exhibit median oxygen levels <2%, while normal oxygen levels, i.e., normoxia, is defined as 20% oxygen . HypoxiaThe revelation of the importance of hypoxia in the radiation response, together with the knowledge of the higher radiosensitivity of HPV-positive HNSCC, resulted in studies investigating the link between the amount of hypoxia and HPV status. This hypothesis was further strengthened by a sub-analysis of a large, randomized trial testing the use of the hypoxia modifier nimorazole with radiotherapy in HNSCC  . This stOverall, these findings suggest that the cellular and molecular mechanisms of hypoxia in HPV-positive and HPV-negative tumors are more important for the radiotherapy response than solely the oxygen fixation theory.Hypoxia does not merely modulate the radiation response, but additionally has various biological, molecular, and genomic effects on the tumor and micro-environment. The main goal of these hypoxia-induced effects is the survival and even proliferation of cancer cells in unfavorable hypoxic circumstances, leading to tumor progression, invasive growth, and cancer cell spreading . In addiBroadly, the hypoxia-induced adaptational processes have two main objectives . The firAs a second objective, the oxygen and energy consumption of cancer cells must be lowered. Since mRNA translation is one of the most consuming processes, the overall protein synthesis decreases during hypoxic stress as a way of energy conservation ,66,67,68Another attempt to lower the oxygen consumption involves metabolic transition to anaerobic glycolysis ,63,72,73The DNA damage response is a complex overlapping network of genes that sense and repair DNA damage. The role of DDR genes, especially sensors such as ATM and ATR, and DNA repair pathways such as non-homologous end-joining (NHEJ) and homologous recombination (HR), in radiotherapy response has been established for several tumor types, including HNSCC. Moreover, as mentioned before, we and others have shown that HPV-negative and HPV-positive HNSCCs show differences in the DDR after radiotherapy. Hypoxia affects all DNA damage response pathways, in particular the mismatch repair (MMR) and HR pathways, by downregulating key genes in these pathways such as MSH1, MLH2, and RAD51 ,75,76. TRecent NGS studies ,80 have As mentioned earlier, the DDR differs between HPV-positive and HPV-negative HNSCC, with lower rates of HR and more error-prone NHEJ in HPV-positive tumors, resulting in genomic instability and radiosensitivity ,57,58,59Tumors elicit complex immune responses, and hypoxia seems to play a significant role in this by promoting an immunosuppressive microenvironment through the recruitment and modulation of immune cells ,83,84,85Severe stress and/or unrepairable damage can induce various cell death mechanisms, such as apoptosis, autophagy, necrosis, necroptosis, and ferroptosis. Hypoxia has direct effects on these mechanisms and modulates the interactions between the different pathways . ApoptosNot only HPV infections, but also hypoxia, seem to influence p53 function in cancer cells. However, studies report contradictory findings about the effects of hypoxia on apoptosis depending on the oxygen level . Severe In order to overcome the negative influence of hypoxia, various strategies to modify or target hypoxia in combination with RT have been extensively investigated in preclinical and clinical settings since the 1950s. Since most solid tumors are characterized by hypoxia, these trials investigating hypoxic modification and detection involved a variety of tumor sites, such as bladder, cervix, and lung. However, the head and neck area is one of the most investigated tumor sites regarding hypoxia . An overThe first hypoxic modification method that has been explored is hyperbaric oxygen (HBO) breathing, which leads to a physical increase of the blood oxygen levels ,103,104.18F-MISO PET, treatment with TPZ did decrease the ratio of recurrences in the hypoxic subpopulation [Other hypoxic modification methods are based on hypoxia-activated prodrugs (HAP), which are selectively activated in hypoxic circumstances by enzymatic reduction ,109,110.pulation . Anotherpulation ,111,112.pulation . Howeverpulation ,101,113.pulation . The byspulation . These fpulation .Up till now, the oxygen mimetic nimorazole is the only hypoxic radiosensitizer that has been implemented in clinical practice, since a large phase III trial (DAHANCA 5) showed a significant outcome benefit by combining radiotherapy with nimorazole in HNSCC ,37. HoweAs summarized above, only one clinical trial  could deTumors display different degrees and distributions of hypoxia, leading to variable responses to hypoxia-targeting strategies. Therefore, information about the extent of hypoxia is crucial in order to select patients who will likely benefit from hypoxic modification. In addition, pretreatment information about the hypoxic status has proven prognostic value ,118. SinThe first and the oldest detection method includes the direct measurement of tumor oxygen concentration using polarographic needle electrodes, for example the Eppendorf electrode. The key advantage of this method lies in the fact that it measures oxygen levels directly and that it has a proven correlation with prognosis in multiple cancer types, including HNSCC ,125,126.Later techniques are based on detecting and monitoring hypoxia markers in biopsies or resection specimens. These markers can be endogenous, like hypoxia-inducible factor 1α (HIF-1α), carbonic anhydrase IX (CAIX), glucose transporter 1 (GLUT-1), and vascular endothelial growth factor (VEGF) . OverexpA third biopsy-based detection strategy assesses gene expression signatures, which consist of a selection of genes specifically upregulated under hypoxia. This upregulation is a result of the cellular adaptation and transcriptional response to hypoxia, mainly regulated by the HIF pathway ,139,140.Despite their benefits, both the electrode- and tissue-based measurements are less user friendly, since they involve invasive techniques and only inform on oxygen levels at a specific time point, unless repeated samples are taken. This makes them less suitable for monitoring tumor oxygenation and/or treatment response, underlining the need for non-invasive approaches like hypoxia imaging .Positron emission tomography (PET) with hypoxia-selective tracers is more useful in clinical practice, allows hypoxia monitoring during treatment, and offers a reliable and reproducible visualization of tumor oxygenation, as demonstrated in various tumor types ,146,147.18fluoromisonidazole (18F-MISO), which consists of labeled 2-nitroimidazoles, which selectively bind to macromolecules in hypoxic cells, is the one most commonly used and investigated [18F-MISO PET. In this study, patients with hypoxic tumors had a higher risk of locoregional recurrences, demonstrating the prognostic value of 18F-MISO PET. As mentioned before, the addition of tirapazamine to chemoradiation significantly improved the outcome for these patients [Various hypoxia PET tracers have been developed, but stigated ,148. In stigated , the hyppatients .18F-fluoroazomycin arabinoside (FAZA), with promising prognostic potential for hypoxia detection in HNSCC as well [18F-FETNIM, 18F-EF3, 18F-EF5, and 18F-HX4 [A more novel hypoxia-selective PET tracer is  as well . Other n 18F-HX4 ,154,155. 18F-HX4 .The current techniques to detect hypoxia show promising results regarding their prognostic and predictive value; however, each has its inherent weaknesses. Further validation of these techniques is warranted in different tumor types and treatment regimens. Additionally, the ideal timing for hypoxia detection still remains a subject for debate. The focus of future research should lie on patient selection with reliable hypoxia detection methods in combination with testing of hypoxia-targeting agents in the hypoxic subgroup.HPV-positive and HPV-negative HNSCCs differ at the biological and clinical level, with a better overall prognosis for the HPV-positive group. This improved prognosis is, at least partly, attributable to the increased radiosensitivity and thus better radiation response of HPV-positive tumors. Besides HPV infections, hypoxia is also an important modulator of the radiation response. The most well-known explanation for this interplay lies in the ‘oxygen fixation theory’, which arose many decades ago. Nevertheless, more recently, it has become clear that hypoxia induces a variety of cellular processes that also, and probably to a more important extent, seem to influence the therapy response. Moreover, these cellular changes affect the biological behavior and phenotype of hypoxic cancer cells. In addition, recent NGS studies show that hypoxia causes genetic instability, which can be linked to a poorer prognosis. However, it remains unclear whether and how these cellular and genetic alterations influence the therapy response and the increased radiosensitivity of certain tumor types like HPV-positive HNSCC. Further studies investigating the fate of hypoxic cells and the hypoxia-induced cellular adaptions and genetic alterations, with and without radiotherapy, can shed light on the importance and interplay of these various known biological processes.Because of its negative impact on tumor responsiveness and overall prognosis, hypoxia remains an important therapeutic target to be exploited. Unfortunately, up till now, the clinical successes of hypoxia-targeting strategies are poor. Some recent HAPs show improved targeting abilities in several preclinical trials; however, the responses are still heterogeneous, underlining the need for proper hypoxia stratification. The existing detection methods are promising, but need further validation in prospective randomized trials. Ideally, these should be combined with the testing of novel hypoxia-targeting strategies.


Based on the proposed mechanism of oxidative polymerization of methylene blue and the data of MALDI, EPR, and IR spectroscopy methods, the structure of the polymethylene blue chain is proposed. It has been shown that polymethylene blue has a metallic luster, and its electrical conductivity is probably the result of conjugation over extended chain sections and the formation of charge transfer complexes. It was found that polymethylene blue is resistant to heating up to a temperature of 440 K and then enters into exothermic transformations without significant weight loss. When the temperature rises above 480 K, polymethylene blue is subject to endothermic degradation and retains 75% of its mass up to 1000 K.The kinetic regularities of the initial stage of chemical oxidative polymerization of methylene blue under the action of ammonium peroxodisulfate in an aqueous medium have been established by the method of potentiometry. It was shown that the methylene blue polymerization mechanism includes the stages of chain initiation and growth. It was found that the rate of the initial stage of the reaction obeys the kinetic equation of the first order with the activation energy 49 kJ · mol Polymet glasses .Although the area of possible application of polymethylene blue is rapidly expanding, the chain structure of this polymer remains a matter of debate ,15,16,17−1 in a nitrogen flow (70 mL · min−1) on an STA 449 F3 Yupiter instrument . XRD was recorded using a Malvern Panalytical Aeris XRD benchtop X-ray diffractometer .Methylene blue and ammonium peroxodisulfate were purchased from Sigma–Aldrich. Double-distilled water was used as a reaction medium. Vibrational spectra of the samples were recorded in potassium bromide pellets using a Nicolet 380 spectrometer . MALDI spectra were recorded on an Ultraflex II mass spectrometer  with an accelerating voltage of 25 kV with Nd: YAG laser (355 nm) desorption from a DHB matrix. EPR spectra of solid samples of methylene blue and polymethylene blue were recorded on a commercial spectrometer Bruker EMX  . Photographs of methylene blue samples and the product of its oxidative polymerization were obtained using a stereoscopic microscope Lomo MSP-2-2SD equipped with an MC-5 electronic camera ; images were processed using the MCview  software package. TGA and DSC were recorded at a heating rate of 10 degrees · minAn amount of 0.320 g of methylene blue was dissolved in 50 mL of bidistillate  was used to accelerate the dissolution). A solution of 0.912 g of ammonium peroxodisulfate in 50 mL of bidistillate was also prepared. The solutions were mixed, and the reaction was carried out at a temperature of 298 K for 18 h. The resulting precipitate was filtered off, washed on a filter with 25 portions of water, 100 mL each, and then dried at a temperature of 323 K.An amount of 0.160 g of methylene blue was dissolved in 50 mL of bidistillate. A solution of 0.456 g of ammonium peroxodisulfate was also prepared and dissolved in 50 mL of bidistillate. The electrode of an F20-Standard pH meter manufactured by Mettler Toledo was placed in an aqueous solution of a monomer, and a solution of an oxidizing agent was quickly added, recording the pH values at specified time intervals. Kinetic studies were carried out under thermostating conditions at temperatures of 298 K, 308 K, and 318 K.The time dependences of proton concentrations obtained under the conditions of oxidative polymerization of methylene blue at different temperatures are shown in −3, 3.52 · 10−3, and 6.31 · 10−3 mol · L−1 at temperatures of 298 K, 308 K, and 318 K, respectively.After a short period of rapid increase in the concentration of protons, the rate of their release decreases significantly . In thisIt is noteworthy that the kinetic data for the initial stage of the oxidative polymerization of methylene blue are in satisfactory agreement with the first-order equation of the reaction rate (1).The concentration of the monomer reacted during the initial stage should be proportional to the change in the concentration of protons; therefore, the fulfillment of Equation (2) should be expected.After substitution of (2) into (1), separation of variables and subsequent integration from Kinetic Equation (3) satisfactorily agrees with the experimental data for the initial stage of methylene blue polymerization .−3, 3.8 · 10−3, and 7.2 · 10−3 s−1 at temperatures of 298, 308, and 318 K, respectively. The temperature dependence of the rate constant of the oxidative polymerization of methylene blue is linear in the coordinates “lnk vs. T−1”  and the peroxodisulfate anion. The calculated activation parameters are consistent with the rapid approach of these ions so that the ion pair actually functions as a monomer. A similar case was described earlier for the oxidation of dopamine hydrochloride by the action of ammonium peroxodisulfate [It is known that methylene blue is capable of forming molecular associates in aqueous solutions, among which the dimeric form predominates . The firisulfate .We assume that the subsequent formation of polymer chains is associated with the slow diffusion of methylene blue to the radical cations of methylene blue dimers, as shown in Thus, considering the mechanism of methylene blue polymerization, one can expect the formation of dicationic units of the polymer chain, as well as monocationic fragments as a result of nucleophilic demethylation .The structure of the polymethylene blue chain is a subject of constant discussion in the literature and varies greatly in the works of various scientific teams ,15,16,17For polymethylene blue under conditions of laser desorption from the DHB matrix, chain destruction can be expected in accordance with the mechanism presented in The pattern of the isotope distribution of the 284.17 signal  indicateThe fraction of quaternized nitrogen in the main chain of polymethylene blue is probably small, and conjugation is realized in sections of the chain of considerable length. At the same time, even a small number of dicationic units can lead to a dominant signal in MALDI , becauseThe above assumptions are in indirect agreement with reports on the high electrical conductivity of polymethylene blue , as well16 spin · g−1.The mechanism of the onset of electrical conductivity may possibly also include the inter-chain contribution associated with the formation of complexes with charge transfer. EPR spectroscopic data are consistent with this assumption. First, methylene blue, used as a monomer, has an EPR signal with a line width of ∆H = 15.9 G and a g-factor of ~2.0045 A. A polyAccording to XRD data, polymethylene blue resulting from the chemical oxidative polymerization of methylene blue contains a significant amount of amorphous phase .−1 for most of the stretching and bending vibration bands in the range 1000–1700 cm−1 after polymerization of methylene blue (−1 in the region of high wavenumbers, a change in the position of the bands of out-of-plane bending vibrations of C-H bonds, and a decrease in their number are consistent with an increase in the resonance energy and the participation of C-H bonds in the oxidative polymerization of methylene blue.IR spectroscopic data indicate an increase in wavenumbers by 10–50 cmene blue . This ciIf methylene blue begins to decompose slowly at temperatures above 373–383 K , then poAlthough polymethylene blue is unstable when heated above 440 K, low-volatility compounds predominate among its decomposition products. Thus, when heated to 1000 K, the sample retains about 75 wt% of the initial mass.−1. The first kinetic order is presumably a consequence of the rapid convergence of the molecular dimer of methylene blue, stabilized by charge transfer, and the peroxodisulfate anion, followed by a slow one-electron transfer within the ion pair. The implementation of one-electron transfer within the ion pair is also indicated by the low value of the preexponential factor 7.9 · 105 s−1 and the negative value of the activation entropy −140 J · mol−1 · K−1. As a result, at the initiation stage, a radical cation of the covalent methylene blue dimer is formed, which is capable of subsequent addition of monomer molecules. Taking into account electronic and spatial factors, as well as kinetic data, a mechanism for the polymerization of methylene blue is proposed, which implies the formation of a chain linked through position 8 of the ring and quaternized nitrogen atoms, and the possibility of nucleophilic demethylation of the initially formed polymer is considered. The proposed structure of the polymethylene blue chain assumes the presence of extended conjugated sections of the chains and is consistent with its metallic luster and the data of MALDI, EPR, and IR spectroscopy methods. It was found that polymethylene blue can maintain stability up to 440 K; however, with a further increase in temperature up to 1000 K in an inert atmosphere, only 25 wt% of products are formed, subject to entrainment.It is shown that the rate of the initial stage of chemical oxidative polymerization of methylene blue obeys a first-order kinetic equation with an activation energy of 49 kJ · mol


The Alphapapillomavirus 9 (α-9 HPV) is a member of the Alphapapillomavirus genus and Papillomaviridae family. These viruses are almost all carcinogenic HPV, which is closely related to 75% of invasive cervical cancer worldwide, and has a high prevalence in Sichuan. The carcinogenic function is mainly realized by its E6 oncoprotein.E6 gene was sequenced and analyzed. The positive selection sites of HPV E6 genes were estimated by PAML 4.8 server. The secondary and tertiary structure of E6 protein were predicted by PSIPred and Swiss-model. The T-cell antigen epitopes of E6 protein were predicted by IEDB.Cell samples were collected by cervical scraped for HPV detecting and typing.HPV-16, HPV-31, HPV-33, HPV-52, HPV-58 5 α-9 genus HPV subtype positive samples were selected, their E6, 96 cases HPV-31 E6, 216 cases HPV-33 E6, 288 cases HPV-52 E6 and 405 cases HPV-58 E6 were successfully amplified, 17, 6, 6, 13, and 4 non-synonymous nucleotide mutations were respectively detected in HPV-16, 31, 33, 52, and 58 E6, 7 positive selection sites of α-9 HPV E6 were selected out . The structure and antigen epitopes of E6 protein with amino acid substitution differ from those of wild-type E6 protein, especially for the mutation located in the E6 positive selection site.α-9 HPV has a high prevalence in Sichuan, China. From 2012 to 2017, 18,067 cell cervical samples were collected, and 3135 were detected with α-9 HPV infection. Among which, 250 cases HPV-16 E6 nucleotide non-synonymous mutation in the positive selection site influence the protein structure and decrease the antigen epitopes affinity of the E6 protein overall, making it more difficult for the HPV-infected cells to be detected by the immune system, and enhancing the HPV adaptability to the environment. Mutations influence the validity of HPV clinical diagnostic probes, the polymorphism analysis of α-9 HPV E6 enrich the data of HR-risk HPV in Sichuan China, and the detection probes designed with the polymorphism data in mind can improve the efficiency of clinical detection; Mutations influence epitopes affinity, the association of E6 polymorphism and epitope affinity can improve the design of therapeutic vaccine with good immunity and high generality antigen epitope; The above study all provide a good theoretical basis for the prevention and treatment of HPV-related diseases.HPV The online version contains supplementary material available at 10.1186/s12985-021-01728-4. E6 and E7. The carcinogenicity of HPV E6 protein is more evident than that of E7 protein, in terms of the cell cycle changes and the efficiency of HPV infected cells to permanent biochemical transformations [Cervical cancer is the second most common cancer among women aged 15–44, 99.7% of the cervical cancers were found to be associated with high-risk (HR) Human Papilloma Virus (HPV) persistent infection [rmations , 3. WithAt present, there is no specific drug for HPV treatment and mainly relies on the body's immune system to detect and eliminate the virus. Human leukocyte antigen (HLA) has the function of recognizing itself target by recognizing and stimulating CD8+ cytotoxic T lymphocytes (CTL), CD4+ helper T lymphocytes (Th) as well as binding antigen polypeptide to regulate the immune response, control and eliminate HPV infection . The antE6 oncogene is strong, its non-synonymous mutation changed E6 protein amino acid composition, which may relate to the differences in immune response and pathogenicity [HPV is a high infectious and mutable virus, with different epidemic trends and mutation types in different regions and populations . The polgenicity . Some mugenicity . For exagenicity –23. In tgenicity .HPV has strong regional and population differences, the prevalence of α-9 HPV and the harmfulness of E6 oncoprotein are extremely high, and E6 polymorphism is closely related to the difference of immunogenicity, adaptability, and pathogenicity. Therefore, it’s urgent to study the genetic diversity, positive selection sites, antigen epitope, the protein structure of α-9 HPV E6 for providing data to realize the effective prevention and control of the disease in this region.6H5CO2Na, 1 L H2O).The study was ethically approved by the Education and Research Committee and Ethics Committee of Sichuan University, Sichuan, China. Eighteen thousand sixty-seven specimens were randomly collected from January 2012 to December 2017 in Chengdu Women and Children's Center Hospital, Chengdu Jinjiang District Women and Children's Hospital, Angel Women's and Children's Hospital, Affiliated Hospital of Sichuan Reproductive Health Research Center, Sichuan Reproductive Health Research Center Affiliated Hospital, Shuangnan Hospital, Chengdu Song zi niao Sterility Hospital, Infertility Hospital Affiliated to Chengdu Medical College and Chengdu Jinsha hospital. Before sample collection, written informed consent was obtained from all patients or their guardians, and patient privacy is strictly protected. The cell specimens were collected randomly by cervical scraped and placed in − 20 °C antiseptic buffer  according to the manufacturer’s guidelines.E6 were designed by PRIMER version 5.0 and NCBI  Primer Blast based on the reference sequences, the primers and reference sequences used for the molecular characterization analysis of α-9 HPV E6 were shown in Additional file 2O, 1 µl primers, 0.4 µl TransTaq DNA polymerase, 2.5 µl dNTPs, and 3 µl buffer. The reaction conditions were shown in Additional file E6 were purified and sequenced by TSINGKE at least twice .The primers of α-9 HPV E6 sequence were determined according to the reference sequence in GenBank  was used to calculate the ratio (ω = dN/dS) between non-synonymous mutation rate (dN) and synonymous mutation rate (dS) to determine the α-9 HPV E6 gene-positive selection sites.Phylogenetic Analysis by Maximum Likelihood 4.8  and Swiss-model were used to analyze the secondary and tertiary structure of E6 protein.Mega6.0 software was used to translate the http://www.iedb.org/). According to the method recommended by IEDB, lower the percentile rank (PR) of antigen epitopes is better the affinity, peptides with PR < 1.0 for HLA-I and peptides with PR < 5.0 for HLA-II were deemed to meaningful as well as selected for further analysis.According to the Chinese major histocompatibility complex database (dbMHC) average frequency of HLA alleles, 13 HLA-I and 6 HLA-II alleles were selected , 25 (0.41%), 137 (2.25%), 438 (7.19%), 571 , 413 (6.78%), 3270 (53.68%), 1021 (16.76%), 50 (0.82%) respectively  HPV-16 E6 samples were variants, and 17 non-synonymous mutations were detected. 96 HPV-31 E6 were successfully amplified, 68 (70.80%) variants and 6 non-synonymous mutations were detected. 216 HPV-33 E6 were successfully amplified, 76 (35.19%) variants and 6 non-synonymous mutations were detected. 288 HPV-52 E6 were successfully amplified, 250 (86.80%) variants and 13 non-synonymous mutations were detected. 405 HPV-58 E6 were successfully amplified, 356 (87.90%) variants and 4 non-synonymous mutations were detected. Details of α-9 HPV E6 nucleotide polymorphisms were shown in Tables 250 HPV-16 ed, 162 6.80% HPV-Calculated by Codeml software using Naive NEB and Bayes Empirical Bayes models, seven positive selection sites of α-9 HPV E6 were detected,there were D32E of HPV-16 E6, K35N, K93N, R145I of HPV-33 E6, K93R of HPV-52 E6, K93N, R145K of HPV-58 E6. In contrast, no reliable HPV-31 E6 positive selection site was selected out , one C-terminal (residues 147–158) and two Zinc fingers -CxxC) three domains. The two Zinc finger binding domains form a deep pocket, which can mediate the most important tumor suppressor protein p53 ubiquitination degradation by binding to the "LXXLL" sequence of E6AP protein , 25. 145x-CxxC) tPositive selection sites make the gene frequency of the corresponding amino acid increasingly stable and enhance the species' adaptability to the environment . AccordiHPV E6 protein plays a key role in cervical cancer development. During HPV infection, the immune system will treat E6 protein as an antigen presentation to eliminate HPV infection and reduce the occurrence risk of HPV-related diseases with the help of body immunity . Some spE6 mutation on protein conformational and immunogenicity. We discussed the relationship between protein structure, positive selection site, antigen epitope and pathogenicity of α-9 HPV E6 protein in Sichuan was discussed for the first time. Amino acid substitution in positive selection sites may affect the virus infection efficiency, immunogenicity, and pathogenicity by altering their T-cell epitopes affinity to improve the survival ability of α-9 HPV as well as an adaptation to evolution. These results help explore the relationship between HPV E6 polymorphism and HPV affection capacity and its action mechanism to improve the therapeutic vaccine of α-9 HPV in Sichuan regions of China.Studies have found that mutations affect the efficiency of HPV vaccine , the proα-9 HPV is extremely prevalent in Sichuan, China. The positive selection site K93N of HPV-33 E6, K93R of HPV-52 E6 and K93N of HPV-58 E6 all occurred in the 93rd amino acid of E6 protein. N86H, R145I (positive selection site) of HPV-33 E6 and D86E, R145K (positive selection site) of HPV-58 E6 occurred in the same location of E6. α-9 HPV E6 positive selection sites that adaptive to the environment D32E, K35N, K93N, R145I, K93R, R145K have been widely spread, they all located in the E6 protein active region and altered their protein structure, as well as overall reduce the immunogenicity of the E6 protein, so that HPV infected cells are more difficult to be detected by the immune system and enhance the adaptability of α-9 HPV to the environment.E6 mutations in positive selection sites may affect the virus infection efficiency, immunogenicity by altering their protein structure, epitopes affinity to improve the survival ability of HPV.Additional file 1: Table S1. Primers and PCR condition used for the molecular characterization of α-9 HPV E6. Table S2. Average frequency of HLA-I and HLA-II alleles (> 5%) across the Chinese population. Table S3. The HLA-I predicted epitopes of HPV-16 E6. Table S4. The HLA-II predicted epitopes of HPV-16 E6. Table S5. The HLA-I predicted epitopes of HPV-31 E6. Table S6. The HLA-II predicted epitopes of HPV-31 E6. Table S7. The HLA-I predicted epitopes of HPV-33 E6. Table S8. The HLA-II predicted epitopes of HPV-33 E6. Table S9. The HLA-I predicted epitopes of HPV-52 E6. Table S10. The HLA-II predicted epitopes of HPV-52 E6. Table S11. The HLA-I predicted epitopes of HPV-58 E6. Table S12. The HLA-II predicted epitopes of HPV-58 E6.


HPV Vaccination NOW: This is Our Moment social media campaign is an initiative of the South Carolina Cancer Alliance (SCCA) and Hollings Cancer Center at the Medical University of South Carolina (MUSC). This statewide social media campaign aimed to increase parental awareness of and build vaccine confidence around HPV vaccination in S.C. The ten-week campaign was strategically implemented between June and August 2019 to encourage HPV vaccination at back-to-school medical appointments. A process evaluation showed that the campaign resulted in over 370,000 total impressions, reached over 33,000 individuals, and culminated with over 1122 followers. There were over 2700 engagements on Facebook and Twitter. A qualitative content analysis indicated that pro-vaccine and anti-vaccine comments were dominated by personal stories. Comments promoting misinformation about the HPV vaccine were often countered through peer-to-peer dialogue. Findings suggest that creating opportunities for the target audience to engage with campaign messages effectively corrected misinformation.The human papillomavirus (HPV) vaccine provides protection from six HPV-related cancers. Approximately half of South Carolina adolescents have not completed the vaccination series, representing a missed opportunity to prevent cancer. The  In the U.S., HPV causes approximately 35,900 of the 45,300 new cases of HPV-associated cancer among men and women each year [The United States (U.S.) could eliminate cervical cancer by 2059 through increasing human papillomavirus (HPV) vaccination and improving participation in cervical cancer screening and treatment . Severalach year . Althougach year , effortsDespite having been available since 2006 in the U.S., uptake of the HPV vaccine remains lower than other adolescent vaccines. In the U.S., HPV vaccination rates are below those of other adolescent vaccines available, and rates remain lower than in other developed nations . AustralAlmost half of South Carolina adolescents have not completed the vaccination series, representing a missed opportunity to prevent cancer. In 2016, South Carolina had the lowest rate of HPV vaccine completion among adolescent girls in the U.S. (30.8%) and the second lowest rate of up-to-date vaccination among adolescent boys (27.4%). Although South Carolina has made significant improvements in HPV vaccination initiation (71.8%) and completion (53.0%), completion rates remain slightly below the U.S. average (54.2%) . Data shSocial media remains a promising, although underutilized, tool in HPV vaccination promotion with limited evidence evaluating extant interventions ,17. ExpeIn addition to serving as a primary source of health information, scholars argue social media may provide an effective platform to correct misinformation and address myths about vaccination . Some stHPV Vaccination NOW social media campaign addressed HPV vaccination misinformation online and fostered message co-creation with the target audience. Formative audience research and a pilot campaign guided the development and implementation of the social media campaign. The campaign aimed to raise the voices and stories of parents in South Carolina. Messaging focused on compassion and understanding, recognizing that all parents want to do what is best for their children and families. This approach was designed to empower parents and appealed to their moral responsibility and personal choice to protect children from cancer. The research study was designed to answer two research questions: (1) What myths and misinformation about HPV vaccination emerged through the HPV Vaccination NOW social media campaign? and (2) How did the campaign correct misinformation to help mitigate its impact?The purpose of this study was to investigate how the https://muschealth.org/hpv (accessed on 1 January 2021). The campaign’s call-to-action included prompts to “Like”/“Follow” social media platforms. Researchers employed a mixed methods approach to evaluation, including (1) a campaign process evaluation, including message split-testing and social media metrics and (2) a qualitative content analysis of user-generated content.The ten-week campaign was strategically implemented between 5 June and 10 August, 2019 to encourage HPV vaccination at back-to-school medical appointments. Two Social media platforms, Facebook and Twitter, were selected to target parents in South Carolina by providing information and addressing misinformation. A new Medical University of South Carolina (MUSC) website was created to address misinformation and offered information for making an appointment to get the HPV vaccine at MUSC, through a primary care provider, or at a Department of Health public health clinic , and the National Cancer Institute-designated Hollings Cancer Center at MUSC based in Charleston, South Carolina. The HPV Vaccination NOW campaign tagline was “This is Our Moment,” and the slogan was “South Carolina is gaining momentum. We have made significant improvements in HPV vaccination rates. This is our moment. #HPVvaxNOW #OurMoment.” See The HPV Vaccination NOW aimed to raise the voices and stories of parents in the state as primary, trusted messengers. Messages focused on compassion and the understanding that all parents want to do what is best for their children and families. The campaign also focused on empowerment and appealing to parents’ moral responsibility and personal choice to protect their children against cancer. The campaign capitalized on progress and momentum by focusing on South Carolina’s significant improvement in vaccination rates. In line with national campaigns, HPV Vaccination NOW emphasized cancer prevention, normalizing HPV vaccination as a routine part of the vaccination series, and the safety, effectiveness, and long-lasting protection of the HPV vaccination. A social media editorial calendar was created with 3–5 posts per day on Facebook and Twitter with a different theme each week “Process evaluation measured the implementation of the campaign, including reach and frequency of message exposure or dose . ResearcHPV Vaccination NOW social media platforms. Content analysis is a well-established method to investigate vaccine misinformation on social media [A qualitative content analysis was performed to analyze public posts on the al media ,35. All al media . To ensual media . Making al media . The resHPV Vaccination NOW social media campaign included parents of pre-teens and teenagers, specifically women and men between the ages of 30 and 50 across the state of South Carolina. Since social media allows anonymity, identifying the exact demographic breakdown of the target audience was not possible. The campaign’s followers with public demographic information suggested that the campaign reached the intended target audience. The majority of followers on Facebook were between the ages of 25 and 44 years. The gender breakdown was 88% women and 12% men. On Twitter, the gender breakdown was 61% women and 39% men. Individuals interacted with the campaign from 30 out of 46 counties in South Carolina, or at least 65% of the state. There was robust engagement across each region of South Carolina, including the Upstate, Midlands, Pee Dee, and the Lowcountry regions.The primary target audience of the HPV Vaccination NOW campaign resulted in significant exposure and strong saturation of the campaign messages  addressing misinformation and (2) stimulating peer-to-peer dialogue.The content analysis revealed myths and misinformation about HPV vaccination that emerged through the “Got my son and daughter vaccinated against HPV. I know too many women diagnosed with cervical cancer or pre-cancerous conditions not to want to protect my own kids from this virus. Vaccination rates are much higher in other developed countries while Americans continue to deny the benefits of life saving vaccines to their children over false information and anti-science viewpoints.”Pro-vaccine and anti-vaccine comments were dominated by personal stories. Pro-vaccine comments often mentioned the importance of vaccinating sons and daughters based on personal experiences with cancer. According to one parent:“Worst thing I ever did for my daughter. Made her gain 60 pounds and caused what looks like permanent body aches and has so much trouble sleeping since then. They are terrible, I would never recommend them. Never.”Anti-vaccine comments frequently described personal experiences with vaccinations. One parent said that HPV vaccination was the:HPV Vaccination NOW campaign did not delete comments such as this one. Researchers deleted and/or blocked spam, hate speech, inappropriate/offensive language, personal attacks, and trolls . All other comments were allowed and researchers replied to all posts, while also providing an opportunity for the target audience to engage with the campaign messages.The The most common posts featuring misinformation about the HPV vaccination described concerns about safety and side effects, effectiveness, and sexual activity. Misinformation related to safety often focused on the HPV vaccination as new and untested. According to one parent, “Because it isn’t safe at all…They haven’t had enough clinical trials to make sure that it’s safe. Everything is for profit with side effects being overlooked.” Many comments opposed the HPV vaccination because it prevented transmission of a sexually transmitted infection (STI). According to one post, “How about teach kids about safe sex not vaccines to prevent std?” Researchers replied to all comments with empathy and provided credible information from third-party sources, such as the Centers for Disease Control and Prevention (CDC). Responses started with an acknowledgment that researchers welcomed engagement with the campaign and reinforced our core value of compassion. For example, many posts started with, “We appreciate your comment. We believe that all parents in South Carolina want to do what is best for their children and families.” If a parent shared a negative vaccine experience, the response began, “We appreciate hearing from you and understand your son/daughter had a negative experience.” Comments promoting misinformation about the HPV vaccine were often countered through peer-to-peer dialogue. Campaign followers on Facebook and Twitter often provided direct rebuttals to misinformation. When one parent shared a negative vaccine experience, “my son almost fainted, couldn’t raise his arm for two days and slept for about ten hours straight. Won’t be getting it again,” another parent posted, “sure beats brain and throat cancer though.” In another exchange, when one parent described an inaccurate side effect, “it causes early infertility too!” another parent countered with “cervical cancer leads to infertility.” In another discussion, one parent linked the HPV vaccine to sexual activity, “my children won’t be getting it. Unless you have sex, you can’t get the disease. And my children are raised to wait until marriage. Shame on y’all promoting this junk.” Another parent responded, “HILARIOUS…so…because they wait until marriage…they can’t get HPV??? UUUMMM WRONG ANSWER! You don’t have to be a child!! This is not promoting children having sex. This vaccine is to prevent HPV related cancers. Vaccines = Prevent.”Many campaign followers engaged in peer-to-peer dialogue by sharing their reasons for vaccinating their children. In response to parents who posted about adverse vaccine events, many parents posted positive experiences. According to one parent, “Had both my daughters vaccinated and there were no issues!” Other parents described the HPV vaccine as an opportunity to protect their children and keep them safe. According to one parent, “Made sure my daughter got this vaccine. It’s another step I was able to take to ensure her safety.” Parents reinforced campaign messages by sharing their own stories. According to one parent, “I had my youngest son and daughter vaccinated. It is important.”HPV Vaccination NOW: This is Our Moment campaign demonstrated significant exposure and strong saturation of the campaign messages. In only ten weeks, the campaign resulted in over 370,000 total impressions, reached over 33,000 individuals, and culminated with over 1122 followers. There were over 2700 engagements on social media. In addition, Facebook and Twitter offered an excellent return on investment with a cost-per-click (CPC) rate below the national average. Reach decreased in weeks 7 and 8, while beginning to increase again in weeks 9 and 10. It is likely that these numbers reflect real-time campaign adjustments designed to avoid message fatigue and audience burn-out. Furthermore, these results were achieved with a total advertising budget of approximately USD 2000. The HPV Vaccination NOW social media campaign provides a cost-effective model for comprehensive cancer control coalitions and other non-profit organizations to make a minimal investment for a substantial return on investment. This approach could be scaled to maximize impact on HPV vaccination and build resilience against anti-vaccination myths and misinformation.The results from the process evaluation indicate that the HPV Vaccination NOW social media campaign offers best practices for effectively responding to HPV vaccination misinformation in order to limit the impact of false information online. The content analysis revealed strategies that were most effective to engage the target audience. The top social media posts uniformly engaged trends and current conversations, including Follow Friday #FF, Woman Crush Wednesday #WCW and Thankful Thursday #TT. This finding highlights the importance of providing content that offers value to the target audience in order to increase engagement. Split-testing revealed a preference for messages paired with a photograph, instead of a graphic. In line with current research, a photograph may increase the emotional value of the advertisement and resonate more strongly with parents [The  parents .HPV Vaccination NOW social media campaign and how the campaign corrected misinformation to help mitigate its impact. The most common posts featuring misinformation about the HPV vaccination described concerns about safety and side effects, effectiveness, and sexual activity. In particular, safety concerns were often based on the vaccine as “new and untested” with some posts expressing a lack of trust for pharmaceutical companies and the government because the vaccine was created “for profit with side effects being overlooked.” These sentiments reflected recent research that found antivaccine messages on Twitter focused on safety concerns and conspiracy theories [The content analysis also identified myths and misinformation about HPV vaccination that emerged through the theories . As a reFindings revealed that pro-vaccine and anti-vaccine comments were dominated by personal stories. These narratives often described health impacts of the vaccine or HPV-related cancer. In line with recent research calling for campaigns to include stories of HPV vaccine preventable diseases , resultsHPV Vaccination NOW social media campaign created opportunities for the target audience to engage with campaign messages. The campaign created a welcoming space for parents to discuss the HPV vaccine by focusing on compassion and understanding, recognizing that all parents want to do what is best for their children and families. Researchers ensured a safe space for discussion by responding to and engaging with all posts. However, researchers avoided responding to individual comments, choosing to provide responses at the post-level provided an opportunity to correct misinformation among readers without amplifying anti-vaccine comments. This finding extends recent research on the importance of developing a community management strategy to facilitate dialogue about HPV vaccination [HPV Vaccination NOW campaign fostered message co-creation with the target audience. Peer-to-peer dialogue transformed the campaign into a space for parents to interact with one another, which effectively corrected misinformation. By empowering parents to share accurate information and supporting parents as spokespeople in support of HPV vaccination, the campaign leveraged peer-to-peer dialogue to mitigate the spread of misinformation.The cination . By encoHPV Vaccination NOW social media campaign through a series of invited expert panels, the U.S. Department of Health and Human Services’ (HHS) Office on Women’s Health (OWH) launched the HPV VAX NOW campaign to increase HPV vaccination rates among young adults ages 18–26 in Mississippi, South Carolina, and Texas in January 2021. Despite the strengths and success of the campaign, the results are limited by the real-world conditions in which this study was conducted. A qualitative content analysis highlights robust qualitative data in the form of quotes embedded and contextualized in the results, which contributes to the credibility and transparency of qualitative research [HPV vaccination NOW social media campaign, as well as how campaign strategies mitigated the spread of misinformation.This study contributed to the call by scholars to better understand public health strategies to address misinformation and disseminate accurate information on social media . After lresearch . Howeverresearch ,38. Desp