Human papillomaviruses (HPV) infected men causes continued transmission of HPV to women. The prevalence of 15 high-risk HPV strains  and 6 low-risk HPV strains  were evaluated in 935 males with genital warts. Of the 447  HPV DNA positive subjects, 230 (24.6%), 356 (38.1%) and 139 (14.9%) were infected by high-risk, low-risk and both high and low-risk HPV respectively. Of the 356 low-risk HPV infected subjects, 333(93.5%) were infected by single HPV strain; 203 (57.0%), 147 (41.3%), 24 (6.7%) and 5 (1.4%) were infected with HPV genotype 6, 11, CP8304 and 44 respectively; population with age ≤ 20 showed the highest infection rate. High-risk HPV are also highly prevalent in our patients, genotype 16, 58, 51, 39, 52 and 53 are the top five prevalent genotypes with infection rates of 27.4%, 18.7%, 14.3%, 13.9%, 12.6% and 12.6% respectively; only 68.3% subjects were sole infection; subjects with 41 ≤ age ≤ 50 showed the highest infection rate. Both high and low-risk HPV are highly prevalent in men with genital warts, its impact on women HPV control and prevention need further evaluation. Human papillomaviruses (HPV) are small double-stranded DNA viruses that belong to the family of papillomavirus . The ~80Genital warts are evident skin or mucosal growths in the anogenital area display as flat, papular, or pedunculated growths on the genital mucosa . GenitalTreatments on genital warts are mainly divided into medications and surgery, medications include imiquimod, podophyllin, podofilox and trichloroacetic acid, surgery include freezing with liquid nitrogen, electrocautery, surgical excision and laser treatments , 14. AltHPV genotype distribution differs across world regions, even HPV genotypes 16, 18, 31, 52, and 58 are consistently found worldwide, the distribution of a certain HPV genotype still showed huge regional differences . TherefoTotally, HPV DNA data of 935 male patients with genital warts were collected. 488  subjects were negative for HPV DNA. 447  subjects were HPV DNA positive Table . Of the P < 0.05) , 147 (41.3%), 24 (6.7%) and 5 (1.4%) were infected with HPV genotype 6, 11, CP8304 and 44 respectively Figure . The fre) Figure .P < 0.05) , 43 (18.7%), 33 (14.3%), 32 (13.9%), 29 (12.6%), 29 (12.6%), 28 (12.2%), 23 (10.0%), 18 (7.8%), 14 (6.1%), 14 (6.1%), 9 (3.9%), 7 (3.0%), 7 (3.0%) and 6 (2.6%) were infected with HPV genotype 16, 58, 51, 39, 52, 53, 66, 18, 33, 31, 68, 59, 35, 56 and 45 respectively Figure . Genotyp) Figure .To learn any preferendum of HPV co-infection, the co-infection patterns were calculated. As showed in Table Data regarding HPV infection in men is relatively rare and not comprehensive, to compare HPV positivity in this and other studies, we summarized the representative published data. The data include three cross-country studies and one from Brazil –21. As sHPV, as one of the seven notorious carcinogenic viruses, is being studied worldwide . RecentlIn this study, we focused on the HPV prevalence of 15 high-risk HPV strains, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68 and 6 low-risk HPV strains, HPV6, 11, 42, 43, 44 and CP8304 in males with genital warts. Our data showed that, 47.8% subjects were HPV DNA positive, 24.6%, 38.1% and 14.9% of the HPV DNA positive subjects were infected by high-risk, low-risk and high plus low-risk HPV, the prevalence of high-risk HPV is also high in population with genital warts. Secondly, of low-risk HPV infection, HPV 6 and 11 were the dominant genotypes in our population, they account for 93.3% infection; multiple infection among low-risk HPV is relative low, 93.5% were infected by single strain; age ≤ 20 subjects showed that highest infection rate (44.8%). Thirdly, of high-risk HPV infection, HPV 16, 58, 51, 39 and 52 are the top five dominant genotypes, multiple infections with high-risk HPV are common, only 68.3% subjects were sole infection; subjects with 41 ≤ age ≤ 50 showed the highest infection rate (31.5%). Finally, HPV16 is not only the dominant high-risk HPV in our population but also the most frequent co-infection genotype.Although current opinion believe that the 90% genital warts are caused by two low-risk HPV subtypes 6 and 11 , 27. OurThe prevalence of both low-risk HPV and high-risk HPV displayed differences in age groups, this phenomenon was also observed in the HPV infection in women , 30. AltBased on our data, the high-risk HPV are also highly prevalent in the population with genital warts, there are two important information of high-risk HPV infection in our male patients, firstly, since HPV infection is a sexually transmitted disease, efficient HPV control and prevention in women could not ignore that high HPV infections in male; secondly, our data showed that HPV 16, 58, 51, 39 and 52 are the top five dominant genotypes, which differed from the data from a meta-analysis with a large-scale women population, in that report, HPV 16, 18, 31, 52, and 58 are consistently found among the 10 most common types in 5 continents , this dihttp://www.who.int/bulletin/archives/79(4)373.pdf). The Review Board of the Ethics Committee of Medical Research at Shanghai Dermatology Hospital  approved the study protocols. Written informed consents were obtained from all patients according to the guidelines of the Chinese National Ethics Regulation Committee. All patients were informed of their rights to withdraw consent personally or via kin, caretakers, or guardians.This study was conducted in accordance with the World Medical Association Declaration of Helsinki . The kit uses polymerase chain reaction (PCR) with biotinylated PGMY09/11 primer sets that target HPV L1 consensus region, beta-globin was adopted as an internal control for sample amplification. All PCR products were hybridized to the typing strip that included probes for 15 high-risk HPV strains  and 6 low-risk HPV strains . All detection procedures were guided by the manufacturers protocol. Detected HPV strains are defined by genotype not by sub-genotype or quasispecies. The definitions of high-risk HPV and low-risk HPV genotypes were basically based on the Human Papillomavirus Vaccination, Recommendations of the Advisory Committee on Immunization Practices (ACIP) [Biopsies of the outer genital area (scrotum or penis) were performed under local anesthetic. Biopsy samples or surgical specimens were subjected to clinical laboratory for pathological examination. Simultaneously, a part of the biopsy sample of each patient was used for HPV DNA extraction after grinding. DNA extractions were performed using QIAmp Mini Kit , a water blank was processed through all steps of extraction to serve as a contamination control. The extract was stored at –20t-tests, one-way ANOVA, χ2 or Fisher Exact Probability Test according to the characteristics of data distribution. The significance level α was set at 0.05.Statistical analysis was performed using SPSS version 13.0. Continuous variables were presented as means ± standard deviation (SD) and categorical data were presented as numbers (percentage). Differences between groups were examined by using


Human Papilloma Virus (HPV) can play a causative role in the development of sinonasal tract malignancies. In fact, HPV may be the most significant causative agent implicated in sinonasal tumorigenesis and is implicated in as many as 21% of sinonasal carcinomas. To date, there are no definitive, reliable and cost-effective, diagnostic tests approved by the FDA for the unequivocal determination of HPV status in head and neck cancers. We followed an exhaustive algorithm to correctly test HPV infection, including a sequential approach with p16INK4a IHC, viral DNA genotyping and in situ hybridization for E6/E7 mRNA. Here, we report a case of sinonasal carcinoma with discordant results using HPV test assays. The tumor we describe showed an irregular immunoreactivity for p16INK4a, and it tested positive for HPV DNA; nevertheless, it was negative for HR-HPV mRNA. We discuss the possible meaning of this discrepancy. It would be advisable to test HPV transcriptional status of sinonasal carcinoma on a diagnostic routine basis, not only by p16INK4a IHC assay, but also by HPV DNA genotyping and HR-HPV mRNA assessment. Squamous cell carcinoma (SCC) is the most common form of malignant neoplasia in the sinonasal tract, the greatest risk factor in some being smoking tobacco, followed by exposure to solvents, wood dusts, leather dusts and high-risk Human Papilloma Virus (HPV) infection.In 2017 the WHO  updated the classification of Head and Neck Tumors, also revising the chapter on tumors of the nasal cavity, paranasal sinuses and skull base, introducing new entities such as HPV-related sinonasal carcinomas [Sinonasal squamous cell carcinomas are further sub-classified into keratinizing and non-keratinizing squamous cell carcinomas (KSCC versus NKSCC).in trans mechanism [Sinonasal KSCC and NKSCC show significant differences in their clinical, pathological and molecular characteristics, and therefore, their recognition is relevant . Transcrechanism ,9. The Eechanism ,11.Several authors have recently pointed out that the sinonasal tract is the second anatomical site for HR-HPV related tumors to arise, more than a fifth of which show a transcriptionally active HPV ,13,14,15Some reports seem to support the hypothesis of a better outcome for HPV-related sinonasal carcinomas ,16,17Nevertheless, there is an urgent need to collect more data in order to sort out the role of HR-HPV infection on the biological behavior of sinonasal SCC. Several HPV assays are currently available, but none offer optimal sensitivity and specificity levels for HPV detection when used as a single test . In ordeHere, we report a case of sinonasal carcinoma with discordant results at HPV test assays. The tumor we describe showed an irregular immunoreactivity for p16INK4a, and it resulted positive for HPV DNA; nevertheless, it was negative for HR-HPV mRNA. We discuss the possible meaning of this discrepancy.A 63-year-old male patient with a past medical history of a complete and permanent obstruction of the left nasal cavity for the past 1 year, during which he received a contrast-enhanced free computed tomography (CT) scan, which was not diagnostic, and had not received any effective medical treatments, presented to the emergency room for a sudden difficulty in breathing. The patient was sent to the Maxillofacial Surgery Department, where he revealed that he had also performed a previous biopsy of the nasal mass that received a histological diagnosis of sinonasal papilloma with suspected virus-related cytopathic changes. On examination, edema of the left periorbital region was detected, with exophthalmos of the left eye with limitation of visual field in the upper and outer quadrants. The anterior rhinoscopy revealed a blushed mucosa, no flow through the left nasal cavity, and convex right deviation of the nasal septum. The patient did not refer hyposmia. Due to these findings, the patient underwent a contrast-enhanced magnetic resonance imaging (MRI) scan. The MRI scan confirmed the presence of a lesion occupying the left nasal cavity, associated with a displacing erosion of the surrounding bone walls, extending laterally into the left maxillary sinus, superiorly in the context of the ethmoid cells, posteriorly reaching the sphenoid sinus, supero-laterally protruding inside the left orbitary cavity, infiltrating the lateral and inferior rectus muscles and displacing the optic nerve upwards. The lesion was irregularly enhanced upon contrast medium administration. These findings suggested a diagnosis of a malignant neoplasia. The lesion was biopsied, and the pathology report highlighted the presence of a carcinoma in situ.The patient underwent surgery for resection with left hemimaxillectomy, turbinectomy, exenteratio orbitae and subsequent reconstruction with temporal muscle flap and apposition of orbital filling implants. At gross examination of the specimen, we observed a nodular, whitish lesion of hard-elastic consistency, measuring 4 × 3.5 cm on the two-major axis, macroscopically occupying the maxillary sinus, and infiltrating the orbital floor a. Close H-and-E-stained tissue sections demonstrated a poorly differentiated, infiltrating carcinoma, involving the maxillary bone and the orbital floor, reaching the border of the orbital striated muscles b–d. The To date, the FDA has not yet approved a reliable diagnostic test to assess the HPV status in head and neck cancers. Different techniques are currently used for HPV detection in solid tumors, ranging from specific PCR methods, real-time PCR tests, in situ DNA hybridization (ISH) and immunohistochemical detection of surrogate biomarkers (protein p16INK4a). Since none of these methods offers optimal levels of sensitivity and specificity, there is a need for a multitest approach that can help to unravel discordant results . HPV also plays a causative role in the development of sinonasal tract malignancies, accounting for as many as 21% of sinonasal carcinomas .A crucial role has been recently ascribed to the distinction between HPV-active and HPV-inactive related tumors in those anatomical sites classically associated with HPV infection, such as oropharynx and uterine cervix. Persistent HPV infection is recognized as a causative event in oropharyngeal squamous cell carcinomas , and tumHere, we report a case of head and neck carcinoma with discordant results that emerged from HPV tests. It showed irregular positivity for p16INK4a, positivity for HPV 16 DNA, but negativity for HR-HPV mRNA. Although the data in the literature concerning HPV in the sinonasal tract are not univocal, we believe that even in our unusual case we can hypothesize an initial HPV infection, as evidenced by the previous diagnosis of papilloma, and the positivity for protein p16INK4a of carcinoma in situ. Probably, with the advancement of the neoplasia, the latter became independent of the action of the virus, acquiring a more aggressive phenotype .®) [® could stably complement the current diagnostic algorithms for HPV-related cancer, precisely identifying HPV-driven tumors and efficiently distinguishing between HPV-active and HPV-inactive ones. This case report underlines the importance, in case of suspected HPV infection, to identify the viral transcription status. A key point for this alternative approach is based on the recently proposed modification of RNA in situ hybridization (RNAISH) assay designed to test the transcriptional activity of HPV, detecting E6/E7 mRNA, within tumor cells, of the most common high-risk HPV types via a colorimetric method (RNAscope®) ,33. ThisThe multi test protocol we applied proved to clearly distinguish, on the basis of HPV status, the infiltrating lesion from the papillary, mostly non-infiltrating one. Interestingly, in the latter, the CIS component showed a strong p16INK4a positivity, and lacked it in the infiltrating fraction. We hypothesize an original causative role for high-risk HPV in the onset of the neoplasia, and a progressive lack of dependencies from HPV-related proliferative stimuli during neoplastic progression towards a more aggressive, infiltrating phenotype. There is an urgent need for more data about the clinical outcome and biological behavior of HPV-related carcinomas, in order to unravel the role of viral infection as a prognostic biomarker and predictive of chemo and radiotherapy responses. To date, it is believed that estimates about sinonasal carcinomas HPV status could be inaccurate, since guidelines do not recommend a routine assessment of HPV status in this region and, most importantly, the methods of detection are still extremely variable among different laboratories, with most of the tests used for screening purposes not assessing the transcriptional activity of the virus.®, Advanced Cell Diagnostics, Milano, Italy) to confirm the presence of HPV in tumor cells and its transcriptional activity. Formalin fixed, paraffin-embedded specimens were processed for routine H and E staining. Three different tests were performed for HPV detection: p16 immunohistochemistry (IHC) staining, INNO-LiPA HPV Genotyping v2 Extra test to genotype HPV DNA, and HR-HPV E6/E7 mRNA ISH assay . p16INK4a-IHC was performed on a Ventana Benchmark Ultra  using the CINtec p16 Histology Kit , according to both manufacturers’ recommendations. The previously prepared 4 μm tumor FFPE tissues paraffin sections were deparaffinized and subjected to antigen retrieval using CC1 buffer for 30 min. Subsequently they were consecutively incubated in the prediluted CINtec p16 primary antibody (clone E6H4) for 20 min at room temperature and revealed with Ultra View Universal Alkaline Phosphatase Red Detection Kit .Slides were finally counterstained with Hematoxylin II for 8 min  and Bluing reagent for 4 min and then washed. p16INK4a IHC was scored as positive if there was strong, homogeneous and diffuse nuclear and cytoplasmic staining present in greater than 70% of the malignant cells . For INNO-LiPA HPV Genotyping v2 Extra test, DNA was extracted with QIAmp DNA Mini Kit  following the manufacturer’s instructions and eluted in 100 μL as suggested in the INNO-LiPA assay manufacturer’s instructions. Genomic DNA was analyzed for the presence of specific HPV types by multiplex PCR-based assay (SPF10-modified primers), followed by reverse line blot hybridization, detects and genotypes 28 different HPV genotypes, including 18 high-risk HPVs , 6 low-risk HPVs , and 3 other not-classified HPVs , targeting a 65-bp fragment of the L1 ORF. The assay was carried out following the manufacturer’s instructions. The results were visually interpreted by two independent investigators by comparing them with a template provided with the assay [® 2.5 LS RED assay kit , Italy) on the Leica Biosystems BOND RX platform (LS) and the HPV-HR16 Probe  in accordance with the manufacturer’s instructions. Briefly, 4 μm sections were baked and deparaffinized on the instrument, followed by target retrieval  and protease treatment (15 min at 40 °C). Probes were then hybridized for 2 h at 42 °C followed by RNAscope amplification. Signal detection was performed using fast red (red). Peptidylprolyl isomerase B  (HR-HPV E6/E7 mRNA ISH assay was performed using the automated RNAscopeus gene)  and the ® technique allows to test the HPV transcriptional status via the assessment of E6/E7 mRNAs, the only two HPV viral genes with a recognized oncogenic potential at the cellular level and preserving the tumor morphology on the glass slide.It would be advisable, in our opinion, to test the HPV status of sinonasal carcinoma on a diagnostic routine basis, not only by p16INK4a IHC assay, but taking advantage of those techniques allowing a reliable determination of viral transcriptional status. The RNAscope


OR/RR) with 95% confidence intervals (CI) including subgroup analysis and meta-regression to explore sources of heterogeneity, by Stata 13.0 software. 36 case-control studies, contributing data for 6,980 cases of lung cancer and 7,474 controls from 17 countries and one cohort study with 24,162 exposed and 1,026,986 unexposed from China were included. HPV infection was associated with cancer of lung, pooled OR was 3.64 (95% CI: 2.60–5.08), calculated with the random-effects model. Pooled OR for allogeneic case-control studies, self-matched case-control studies and nested case-control studies were 6.71 (95% CI: 4.07–11.07), 2.59 (95% CI: 1.43–4.69) and 0.92 (95% CI: 0.63–1.36), respectively. Pooled OR for HPV 16 and HPV 18 infection, were 3.14 (95% CI: 2.07–4.76) and 2.25 (95% CI: 1.49–3.40), respectively. We also found that HPV infection may be associated with squamous cell carcinoma, adenocarcinoma and small cell carcinoma. There is evidence that HPV infection, especially HPV 16 and HPV 18 infection, significantly increase the risk of lung cancer. Future research needs to focus attention toward whether an HPV vaccine can effectively reduce the incidence of lung cancer.To estimate the global attributable fraction of human papillomavirus (HPV) in lung cancer, we provided updated information through a system review and meta-analysis. We did a literature search on PubMed, Ovid and Web of Science to identify case-control studies and cohort studies that detected HPV in lung carcinomas. We included studies that tested 30 or more cases and were published before Feb 28, 2017. We collected information about gender, smoking status, HPV detection methods, HPV types, materials and clinical features. If it was not possible to abstract the required information directly from the papers, we contacted the authors. A meta-analysis was performed to calculate the pooled effect sizes ( By 2012, cancer holds the first place of morbidity and mortality in the worldwide, with 14 million new cases and 8 million deaths, which means the age-standardized incidence and mortality were 182 and 102 per 100 000, respectively . ParticuBesides dietary factors and tobacco smoke, infectious diseases represent the third leading cause of cancer in the entire world and the proportion of cancers associated with pathogenic microorganism was estimated to be 16.1% . As wellHPV belongs to the papillomaviridae, a large family of epitheliotropic DNA viruses. HPV gene expression and the viral life cycle are tightly controlled by epithelial cell differentiation. It is assumed that scratching of the epithelial tissue allows the virus to infect undifferentiated cells in the basal layers of stratified squamous epithelium . AccordiIt has been 38 years since Syrjänen who first suggested that HPV could possibly be involved in bronchial squamous cell carcinoma . But in RR = 1.263, 95% CI: 1.015–1.571, P = 0.0367), while no significant association was found among men . However, this cohort-study lacked information on individual lifestyle, smoking, diagnostic criteria for HPV infection, and detailed HPV types.According to the established search strategy and inclusion criteria, after screening and detailed examination, a total of 37 eligible articles were selected Figure . 36 of tn = 287) and Oceania studies represented 1.5% of all cases in the pooled dataset (n = 104). The size of studies in the pooled dataset varied from 30 to 1,634 cases. The full list of the included case-control publications was shown in Table 36 case-control studies were conducted further meta-analysis. The majority of cases in the pooled dataset were from Europe  and Asia . North American studies represented 4.1% of all cases in the pooled dataset , P < 0.001, estimate of between-study variance Tau2 = 0.47 . Significantly higher HPV infection rates were found in lung cancer patients, compared to controls, in both male  and female . And a significant difference in pooled OR between male and female was not found in HPV infection and lung cancer (P > 0.05). However, significantly higher HPV infection rates were found in lung cancer patients, compared to controls, in non-smokers , but not in smoker subgroup (P > 0.05). Stratified by HPV detection method, significantly higher HPV infection rates were found in polymerase chain reaction (PCR), in situ hybridization (ISH), southern blot/dot blot (SB/DB), sequencing and bead-based multiplex serology method (BMSM), but not in multiplex liquid bead microarray antibody assay (LBMA). Stratified by HPV type, significantly higher HPV infection rates were found in lung cancer, compared to controls, in both HPV 16 , HPV 18  and HPV 11 , but not in HPV 6 and HPV 31 (P > 0.05). There was a significant difference in pooled OR between tissue and blood (P < 0.05). Using tissues as detection materials, we found that HPV infection was a risk factor of lung cancer. While using blood, a significant association was not found between HPV infection and lung cancer. We also pooled HPV DNA positive rates in lung cancer patients and controls by sample type  and tissue . Stratified by histological type, significantly higher HPV infection rates were found in lung cancer, compared to controls, in both adenocarcinoma , squamous cell carcinoma  and small cell carcinoma , but not in adenosquamous carcinoma and large cell carcinoma (P > 0.05). Regardless of the clinical stage and differentiated grade, HPV infection was a risk factor of lung cancer. Subgroup analysis results of continent, gender, smoking, detection method, HPV type, material, histological type, clinical stage and differentiated grade for the relationships between HPV infection and lung cancer were shown in Table In different regions: HPV infection in lung cancer patients were significantly higher compared to controls only among the Asian subjects .OR 95% CI: 1.82 (1.69–1.97), P < 0.001) and random effects model (pooled OR 95% CI: 3.64 (2.60–5.08), P < 0.001), we found the results of the two models were different, indicating that the small sample studies had an effect on combined effect. Therefore, after we excluded the studies with sample size of case group or control group less than 100, the pooled effect size of fixed effects model (pooled OR 95% CI: 1.49 (1.37–1.63), P < 0.001) was similar with random effects model (pooled OR 95% CI: 1.87 (1.19–2.95), P = 0.007). We used iterative methods to estimate the number of missing studies, and 15 studies were trimmed and filled with recalculating of pooled OR and 95% CI , P < 0.001) and that of random effects model (pooled OR 95% CI: 1.71 (1.22–2.39), P = 0.002) did not occur significantly reversed which meant the results of the meta-analysis were robust. It was less likely that the results would change with new studies reported in the future.Sensitivity analysis using fixed effects model revealed that there was no significant difference between the studies, and that the effect of a single study removed on the combined results was not significantly changed. When the pooled effect size was compared between fixed effects model (pooled Pcontinuity corrected = 0.796). However, the result of Harbord's modified linear regression test (P = 0.001) and Peters’ regression test (P = 0.011) were significant. Furthermore, the limited number of studies (n = 36) indicated a potential publication bias. In Figure P > 0.10. Hence, it was plausible that publication bias was a part of the cause of the observed asymmetry in this funnel plot. However, seven of 15 studies were in the region of P < 0.05, then confounding factors that cause systematic differences in the results of large and small studies was a likely cause of the funnel asymmetry. Nfs0.05 = 2326, which was much larger than the number of eligible studies. Even if there was a publication bias, the results were still relatively stable.Although some asymmetry occurred in the contour-enhanced funnel plots Figure , Beggs’ dies n = 6 indicatHPV is a non-coated double-stranded epitheliotropic DNA virus . There aThe molecular mechanism of HPV infection leading to lung cancer has in recent years been an active research field, and a number of reviews have described the pathogenesis in detail –56. HPV OR and 95% CI: 3.64 (2.60–5.08). Zhai et al. [OR is 5.67 . Moreover, an international pooled analysis about HPV-associated lung cancers shows that pooled OR is 3.86 (95% CI: 2.87–5.19) [OR of this study is smaller, which is possibly due to the inclusion of nested case-control studies leading to weak correlations. The pooled OR of allogeneic case-control studies is significantly higher than that of nested case-control studies, and HPV infection do not increase the risk of primary lung cancer in nested case-control studies. The lack of association between HPV antibody positivity and increased risk of lung cancer can be explained as a high level of serum HPV antibodies reflecting a strong host immune response that prevents the recurrence or reactivation of HPV infection and thereby hinders the development of HPV-associated lung cancer [Growing evidence supports the link between HPV infection and lung cancer, but the relationship is still debatable. To date, seven independent systematic reviews and meta-analyses , 61–66 hg cancer . This hyP = 0.02) [OR of Asian is higher than European and American. It is closely related to the higher positive rate of HPV in lung cancer tissues in Asia  have a higher risk of lung cancer than smokers. A meta-analysis of four case-control studies shows pooled OR in never-smokers is 4.78 (95% CI: 2.25–10.15) [ORs of tissue detection methods  are generally higher than those of serological detection methods (LBMA and BMSM), possibly due to the low amount of HPV in the blood circulation and the low sensitivity and specificity of serological detection methods. The sensitivity and false positive rate of PCR are higher than other methods [OR of PCR is higher than other methods, resulting from the selection of primers and the small sample sizes of other detection methods. In this study, HPV 16, HPV 18 and HPV 11 infection significantly increase the risk of lung cancer, while HPV 6 and HPV 31 infection are not significantly associated with lung cancer. A meta-analysis suggests that the average infection rates of HPV 16 and HPV 18 in lung cancer patients are 19.80% and 18.59%, compared with non-cancer controls, the combined ORs are 5.84 (95% CI: 3.14–10.86) and 4.29 (95% CI: 2.34–7.86) [OR of tissue is significantly higher than that of blood. The point estimated OR of lung squamous cell carcinoma (5.66) is slightly higher than that of lung adenocarcinoma (5.39). Because of the affinity to squamous cell, HPV infection rate of squamous cell carcinoma is higher than that of adenocarcinoma. The result is consistent with the results of Syrjänen [At present, the conclusion of HPV infection and lung cancer is still inconsistent. The possible influencing factors are: research population, test samples, sample processing methods and detection methods . Therefo = 0.02) . Accordisia (28.1-35.7%[615–10.15) , which i5–10.15)  and Euro5–10.15)  is signi5–10.15) , 63. Whe methods , 68. How34–7.86) . There i34–7.86) . It is g34–7.86) . However34–7.86) . TherefoSyrjänen  and Zhaicinoma 5. is slighIn this article, we ensure the high recall ratio through multi-databases and multi- approaches, and improve the precision ratio with the strict inclusion and exclusion criteria. Eligible studies are selected by two people who paid attention to control the quality as much as possible to reduce the search bias. Subgroup and sensitivity analyses show that the result of meta-analysis is stable and the conclusion is reliable. However, in the process of meta-analysis, there are still some limitations: (1) Potential bias cannot be completely ruled out, because HPV infection depends largely on the sensitivity, specificity and HPV subtype of detection methods. Subgroup analysis can explain partial bias, but there are still unknown bias. (2) There is a slight publication bias. According to the funnel plot, there is still a lack of small samples and no statistically significance unpublished articles. (3) There may be multiple publication bias, because it is difficult to distinguish whether a study published repeatedly.In summary, our meta-analysis indicates that HPV infection, especially HPV 16 and 18, increases lung cancer risk, particularly in squamous cell carcinoma and small cell carcinoma. The development of international standard laboratory contributes to the favorable combination of multi-center experimental results and increases the reliability of causal inference. Case-control studies are difficult to determine the time sequence, so well-designed cohort study or randomized controlled trial urgent need to clarify the relationship between HPV and lung cancer. Although HPV vaccines can theoretically prevent the development of lung cancer, future research needs to focus attention toward whether an HPV vaccine can effectively reduce the incidence of lung cancer.We identified eligible studies either in English or Chinese published up to Feb 28, 2017 by searching the MEDLINE (PubMed), Embase (OVID) and Web of Science. Search terms were “human papillomavirus”, “HPV”, “lung carcinoma”, “lung neoplasm” and “lung lesions”. The search was limited to studies that had been conducted on human subjects. Meeting abstracts were excluded because of limited data they offered. Reference lists of the retrieved articles, reviews and editorials were also screened to find all additional eligible studies. This meta-analysis was performed in accordance with PRISMA guidelines.OR or RR with 95% CI; (4) there were no restrictions based on patients’ nationality, ethnicity or gender; (5) when an overlap of patients was found in several studies, only the study with the largest sample size and detailed information or the study that met the above criteria was included.The studies selected had to meet the following criteria: (1) case–control or cohort studies compared HPV infection among lung neoplasms patients and non-cancer controls; (2) histological diagnosis of cases and controls were established; (3) sufficient information was provided to calculate An initial screening of the title and abstract was performed in the first step, followed by a further screening based on a full text review. Information was independently extracted from all eligible publications by two investigators (Wei-min Xiong and Fei He), and discrepancies were resolved through discussion or via a third researcher. Literatures quality evaluation were evaluated using the Newcastle-Ottawascale (NOS) scale . For stuORs or RRs with corresponding 95% CIs were calculated. If there are no events in either the case or control arms of the trial, the trial should be discarded from the meta-analysis [P value and I2 value[I2 is less than or equal to 50%, that suggested that it was homogeneity and the fixed effects model  of metad method ) should d method .OR/RRs and 95% CIs was conducted to test the meta-analysis results of stability. Using Contour-enhanced funnel plots (confunnel with filled studies from metatrim) [0.05 = (Σz/1.64)2 - K, where z is the z value of each independent study and K is the number of studies. When Nfs > 5K + 10, it is judged that there is no publication bias [Moreover, we did pre-specified exploratory meta-regression and subgroup analyses to investigate the effect of selected study and participant characteristics on the results, including study design, research area, gender, smoking status, detection methods, HPV subtypes, material, histological type, clinical stage and tissue differentiation. Sensitivity analysis by sequential omission of individual studies and trim and fill method  with recetatrim) , Beggs’ etatrim) , Harbordetatrim)  and Peteetatrim)  to detecetatrim) . Nfs0.05ion bias .All statistical tests were performed with the Stata 13.0 . All above analyses were two-sided test and the significance level was 0.05.


E6™ Cervical Test (OncoE6 Test) is a rapid, easy-to-use lateral flow method detecting HPV16/18 E6 oncoproteins that has proven to detect high-grade cervical lesions with high specificity. If compatible with self-collection samples, this technology might allow for decentralized screening of hard-to-reach populations.The paradigm shift from cytological screening to Human Papillomavirus (HPV)-based screening for cervical cancer allows the introduction of new technologies in sample collection and diagnostics. The OncoFor technical validation, cervicovaginal lavages were collected from 20 patients with confirmed HPV16+ or HPV18+ invasive cervical cancer. Cervical smears were collected by polyester-tipped swabs and cytobrushes. All samples were applied to the OncoE6 Test and cytobrush samples additionally genotyped.Lavage, swab, and cytobrush revealed concordant outcome in 18/20 samples. HPV types corresponded with the HPV genotyping by GP5+/6+ PCR analyses. Due to a rare mutation found in the E6 antibody binding site one sample was not detected, another sample had very low cellularity.Overall, vaginal lavages are technically adequate for the OncoE6 Test. Combining self-sampling with oncoprotein rapid testing to detect women with highest risk for severe dysplasia or cancer may allow for secondary cancer prevention in settings where other screening modalities were unsuccessful to date. Despite successes in reduction of cervical cancer related mortality by the introduction of screening programs in developed regions, the worldwide cervical cancer incidence remains high, especially in developing countries. The World Health Organization (WHO) estimates more than 550.000 new cases annually . CervicaThe association of cervical cancer with hr-HPV  has motivated the development of molecular screening tests detecting presence of HPV, and WHO recommends the introduction of such screening tools . GeneralE6™ Cervical Test (OncoE6 Test), a technology directly detecting elevated levels of the E6 oncoprotein of HPV types 16 and 18 [A necessary pathogenic event during cervical cancer carcinogenesis is the upregulation of HPV encoded oncoprotein expression. The maintained expression of HPV oncoproteins E6 and E7 is a prerequisite for invasive cervical cancer to develop, while levels of the E6 and E7 oncoproteins and the corresponding mRNAs are at a very low level in non-transforming HPV infections and low grade dysplasia . These f6 and 18 . The Onc6 and 18 .careHPV and VIA) at 98.9%; the sensitivity of the OncoE6 Test for CIN3+ was 53.5% for all lesions detected and 70.3% when stratified for lesions positive for the HPV types 16/18/45. In this study, the OncoE6 Test had by far the highest positive predictive value (PPV) for CIN3 of 40.8%, while the PPV of HPV testing and VIA was < 10%; the negative predictive value was 99.4% [The OncoE6 lateral flow assay was validated in numerous clinical studies, and initially only physician collected specimens were used in those studies. For example, in a large (~ 7500 subjects enrolled) screening study conducted in several rural areas in China, the OncoE6 Test had the highest specificity for detection of CIN3 and cervical cancer among all tests involved .From each patient three different samples were collected in the following order: a vaginal lavage sample (1) taken with the self-sampling device Delphi Screener , a dry sample (2) collected with a sterile polyester tipped swab  and a cytobrush sample (3) that was applied to PreservCyt storage media . Before the swab and cytobrush sampling, a speculum was inserted and these two samples were collected from the cervix or, if visible, directly by touching the tumour. All three samples were collected by a physician. The samples were stored at − 20 °C until processing.E6™ Cervical Test  following the manufacturer’s instructions. The lavage sample (1) was centrifuged and the whole cell pellet re-suspended in lysis buffer. The entire lavage sample was used for the OncoE6 Test to assure highest yield of cells and hence the best possible results. The swab sample (2), was transferred into a tube for lysis without any further preparation, according to the standard protocol provided for the OncoE6 Test. Of the cytobrush sample (3) in PreservCyt storage media, 5 out of 20 ml of cell suspension were centrifuged and the cell pellet was re-suspended in lysis buffer and subsequently applied to the OncoE6 Test. The protocol provided by Arbor Vita Corporation for PreservCyt samples recommends as little as 2 ml cell suspension. We used 5 ml to assure the expected amount of cells would be sufficient.All three samples were applied to the OncoThe OncoE6 Test results for all three sample types were compared to the HPV genotyping outcome, and subsequently the lavage and cytobrush sample results were compared to the swab-based test result.HPV genotyping was performed using the cytobrush sample (3) kept in storage media; 1 ml of this cell suspension was used. DNA was extracted using the QIAmp DNA mini kit  and following the manufacturer’s instructions. The extracted DNA was submitted to genotyping using the BSGP5+/6+ PCR approach followed by a Luminex-based readout . The outFor all cytobrush samples DNA concentrations were measured using the NanoDrop 2000  device. It was not possible to measure DNA concentration from the lavage and swab sample, as those were fully used for the OncoE6 test.One sample (#5) was applied to DNA sequencing of the E6/E7 gene region in the HPV genome . Primers encompassed the E6 region of HPV 16 (NCBI accession #: NC_001526) and had the following nucleotide sequences:Forward: TTGAACCGAAACCGGTTAGTReverse: AGATCAGTTGTCTCTGGTTGCComplete sample sets  were obtained from 20 patients with histologically confirmed invasive cervical cancers; subjects had been pre-selected for infections with HPV types 16 and/or HPV 18; the patients presented at the Clinic for Gynaecology or Radiology, Charité University Hospital in Berlin for treatment. The median patient age was 45 years, ranging from 26 years to 64 years. The patients came to the clinic for surgery and/or radiochemotherapy, at which occasion the samples were collected. None of the patients had previously received treatment for cervical cancer .The genotyping results obtained at the time of sample collection confirmed that 15 out of the 20 cervical cancer patients were positive for HPV 16 and the remaining 5 patients for HPV 18. Three patients each were also positive for additional high-risk HPV types albeit at lower virus load or with a borderline positive result. None was positive for HPV 16 and HPV 18 simultaneously.The outcome of the OncoE6 Test for lavage, swab and cytobrush samples was highly consistent (see Table Sample set # 5 (see Table The second discordant sample set (# 15), tested negative for HPV 16 with the Delphi Screener lavage sample and the cytobrush sample, but tested positive for HPV 16 from the swab sample (the latter in concordance with the control genotyping). Possible reasons for these false negative results could be insufficient cellularity in the respective samples. To judge cellularity of the false negative sample we compared DNA content of the cytobrush samples by NanoDrop measurements as an indirect measure of possible cellularity problems. A low DNA concentration was seen in the cytobrush collected sample with only 6 ng/μl while positive samples had a mean content of 51.9 ng/μl (range 2.8 to 208.9). Sample # 9 and #18 had low DNA concentrations in the cytobrush samples as well with 2.8 and 6 ng/μl, respectively, while the OncoE6 results for these samples were positive.Based on these test results the sensitivity to detect HPV 16 or 18 positive cervical cancers by the OncoE6 Test with the Delphi Screener lavage and the cytobrush sample in PreservCyt media was determined at 90% and for the swab sample at 95%. After exclusion of sample set #5 due to the E6 epitope mutation described above, sensitivity can be adjusted to 95%. For the swab sample, sensitivity was 100% Table . SpecifiThe shift from cytology-based cervical cancer screening to molecular-based screening opens the path for self-sampling in conjunction with nucleic acid based HPV tests and potentially also with HPV oncoprotein tests . The DelTwo of the 20 samples revealed discordant outcome resulting in a sensitivity of 90% for the self-sampling device, however, this may not be applicable with regard to cervical cancer screening by the OncoE6 Test in a population wide setting, as substantiated by the outcome of our further analysis.One sample set (# 5) was negative in OncoE6 testing for all three sampling methods, despite a strong positive signal for HPV 16 in the L1-based genotyping assay. Sequencing of the E6-specific epitope coding gene segment revealed a non-silent mutation within the E6 gene sequence, suggesting that the resulting epitope has reduced binding strength to the anti E6 mAb used in the OncoE6 Test. Review of the literature for description of E6 specific mutations revealed several studies from various countries . The mutation we found in our sample set # 5, however, was not described in any of the studies –25. We tA major objective of our study was to assess whether or not the Delphi Screener lavage sample self-sampling device is in principle technically compatible with the OncoE6 Test; we therefore focused on samples from HPV 16/18 positive histologically confirmed cervical cancer patients. Excluding the one inadequate sample from our analysis, sensitivity for the self-sampling device among cervical cancer patients selected for this technical validation is at 95% (18/19 samples correctly detected) for the detection of HPV 16 or 18 induced cervical cancers using the Delphi Screener lavage. It can be expected that such very rare mutations do not reduce sensitivity of the OncoE6 test in population-based screening significantly.The second sample set (# 15) resulting in discrepant outcome showed negative results for the lavage sample as well as the cytobrush sample with the OncoE6 Test, while the result for the swab sample was positive for E6 oncoprotein. It was noticeable that HPV genotyping performed from the cytobrush sample in PreservCyt media showed very low signal strength with the semi-quantitative BSGP5+/6+ PCR with Luminex readout, suggesting the sample was very low on HPV positive cells. To investigate further, we measured the DNA concentration in the cytobrush samples and this sample had a DNA concentration of only 6 ng/μl. Potentially, only few cancer cells were collected, or the tumour was not directly accessible for sample collection, resulting in negative test results for the cytobrush and the lavage sample. It is possible, therefore, that the number of E6 expressing cells was too low, thus resulting in a negative OncoE6 Test. In contrast, sample #9 and #18 showed a low DNA concentration, suggesting low cellularity of the sample, but in this case it did not seem to have an impact on the genotyping results, which presented positive with high signal strength. As hypothesized previously, self-collected samples bear the risk of not containing enough biological material . Also thWeighing the advantages of wider population benefits due to extended screening possibilities against the drawbacks of a potentially limited sensitivity, we conclude that the Delphi Screener-based lavage sampling represents a very promising approach in conjunction with E6 oncoprotein testing. The undisputed advantages in ease of use and acceptance by the use of self-sampling in conjunction with the high positive predictive value and high specificity of disease detection, as demonstrated for the OncoE6 Test in population-based sampling studies , represeOur study outcome warrants further investigation in larger population-based studies. The focus of our investigation was foremost to prove the technical feasibility of this approach, and we therefore concentrated on patients with invasive disease positive for HPV 16 or 18. This does not allow any conclusion on the sensitivity and specificity to be found among women with CIN2 or CIN3 lesions. Future studies should include a more typical screening population and thus allow determination of the clinical specificity of self-collection in conjunction with the OncoE6 Test and assess its usability as a screening test for early detection of lesions. The lower specificity of self-sampling devices has already been mentioned by Arbyn et al.  and shouUsing self-sampling devices and E6 oncoprotein testing for cervical cancer screening would allow highly effective selection of women with a high-risk for cervical lesions. Reaching out by self-sampling to women who normally may not benefit from the screening activities or even avoid participation due to cultural barriers could enhance success of preventive strategies . This isThis technical validation shows promising results for E6 oncoprotein testing from lavage samples obtained by a self-collection device, from swab samples and from cytobrush samples, yet further studies are be needed to investigate the feasibility in the general population. The feasibility of self-sampling in conjunction with the highly specific oncoprotein-based test may constitute an important element of cervical cancer secondary prevention in regions and resource constrained settings that could not be served in the past.


Human papillomavirus (HPV), has been recognized as an vital preliminary event in the oncogenesis of cervical cancer. But the prognostic value is not well defined, because of past studies showing conflicting results. So we conducted this meta-analysis to evaluate whether HPV DNA status was associated with prognosis in cervical cancer.P = 0.001; disease free survival [DFS]: pooled HR = 0.362, 95% CI = 0.252−0.519, P < 0.001). Furthermore, in subgroup analysis, the results revealed that the association between HPV DNA positive cervical cancers and better OS  in Mongoloid patients. Similarly, it existed in good OS  and DFS  in Caucasian patients.A total of 17 previously published eligible studies including 2,838 cases were identified and included in this meta-analysis. Positive HPV DNA was associated with good prognosis in patients with cervical cancer  = 0.610, 95% confidence interval (CI) = 0.457−0.814, HPV DNA status in cervical cancer may be a useful prognostic biomarker before carcinomas are treated. However, larger sample sizes and more comprehensive studies are required in the future studies to verify our findings. Cervical cancer is one of the most common malignancies in women worldwide. Based on the results of epidemiologic studies supported by basic experimental findings, the infection of human papillomavirus (HPV), especially high-risk HPV , has been recognized as an vital preliminary event in the oncogenesis of cervical cancer . It has Over the past decades, a large number of studies tried to evaluate the prognostic value of HPV status in cervical cancer, however, the exact mechanism about the prognostic factor of HPV was not totally understood. In HPV-positive cervical cancer, HPV sequences may be integrated in cells and thus enhance the expression of E6/E7 viral oncogenes to active oncogenes and alter cell growth . While iThe prognostic significance of HPV status in cervical cancer before treatment have been got conflicted results and remained unclear. The size of the sample in each study was limited. Therefore, we performed this comprehensive metaanalysis to evaluate the prognostic value of HPV status in cervical cancer, so as to illustrate which kinds of patients may require careful follow-up for recurrence and need include additional treatment.A total of 1,524 articles were retrieved by a literature search of the PubMed, Embase, and Web of Science databases, using different combinations of key terms. As shown in the search flow diagram  became the major race of literatures including Europe (n = 10) and North America (n = 1), followed by Mongoloid (n = 6) including Japan (n = 3), China (n = 2) and Korea (n = 1), respectively.To assess the relevance between HPV DNA and cervical cancer prognosis, 2,838 patients met the inclusion standard were finally selected for the meta-analysis. The sample-size was with a wide range from 32 to 515. Among the 17 cohorts for HPV DNA status, 11 focused on OS, 3 focused on DFS and rest 3 focused on both OS and DFS; and after categorized by races, Caucasian   , as well as in Caucasian .The meta-analysis of HPV DNA status was based on two outcome endpoints: OS and DFS. For OS, 14 studies in total were included in the meta-analysis, inside which, given the fact that the P = 0.792; P = 0.171)  Figure .HPV has been identified as the key causative agent for the development of cervical cancer. Most studies had determined whether HPV DNA status before treatment was a prognostic factor in cervical carcinoma patients. However, as large centers were hardly able to recruit patients with large sample size, the conclusions from every center remained non-comprehensive.P < 0.000) and better OS . In subgroup analysis, the results revealed that the association between HPV DNA positive cervical cancer patients and better OS  in Mongoloid patients. Similarly, it existed in good OS  and DFS  in Caucasian patients.To our knowledge, current meta-analysis is the first complete overview showing the prognosis significant of HPV DNA status in cervical cancer. We evaluated the survival data of 2,838 cervical cancer patients about HPV DNA status and included in total 17 different studies systematically. As a whole, the results clearly determine that positive HPV DNA is a better prognostic factor in cervical cancer, with better DFS  evaluation of HPV DNA status for predicting prognosis in cervical cancer, (b) provide hazard ratios (HRs) with 95% confidence intervals (CIs) or enable calculation of these statistics from the data presented, (c) classify HPV status as ‘positive’ and ‘negative’.in vitro or in vivo, but not based on patients; (c) articles were without the HRs, 95% CI, or not dealing with OS, DFS, or the K-M survival curves; (d) the follow-up duration was shorter than 3 years.Exclusion criteria were: (a) literatures were published as letters, editorials, abstracts, reviews, case reports and expert opinions; (b) experiments were performed Information was carefully and independently extracted from all eligible publications by two authors using a standardized form. Disagreement was resolved through independently extracting data from the original article by the third author, and consensus was reached by discussions. The meta-analysis of this study was based on two outcome endpoints: OS and DFS. As stated by the inclusion and exclusion criteria above, the following items were extracted from each study, including the first author’s name, publication year, country of origin, number of patients analyzed, tumor stage, clinicopathologic factors, source of tissue, HPV detection method, HPV genotype detected, OS and DFS. The main features of these eligible studies were summarized in Table 2 statistic [P value > 0.10 and I2 < 40% suggested a lack of heterogeneity among studies. According to the absence or presence of heterogeneity, random effects model or fixed effects model was used to merge HR, respectively.The data collected from each qualified paper was used to evaluate the association between HPV DNA status and cervical cancer prognosis through meta-analysis. Pooled HRs and 95% CIs for their outcome endpoints (OS and DFS) were calculated. Subgroup analysis was performed when there were at least three studies in each subgroup. Statistical heterogeneity was assessed using Cochran’s Q test and Higgins’s Itatistic , P valueP values for comparisons were two-tailed and statistical signiﬁcance was deﬁned as p < 0.05 for all tests, except those ones with heterogeneity.Funnel plots and the Egger’s test were employed to evaluate the possible publication bias . If a pu


The purpose of this study was to test the feasibility of undertaking a full population investigation into the prevalence, incidence, and persistence of oral Human Papilloma Virus (HPV) in Scotland via dental settings. Male and female patients aged 16–69 years were recruited by Research Nurses in 3 primary care and dental outreach teaching centres and 2 General Dental Practices (GDPs), and by Dental Care Teams in 2 further GDPs. Participants completed a questionnaire (via an online tablet computer or paper) with socioeconomic, lifestyle, and sexual history items; and were followed up at 6-months for further questionnaire through appointment or post/online. Saline oral gargle/rinse samples, collected at baseline and follow-up, were subject to molecular HPV genotyping centrally. 1213 dental patients were approached and 402 individuals consented (participation rate 33.1%). 390 completed the baseline questionnaire and 380 provided a baseline oral specimen. Follow-up rate was 61.6% at 6 months. While recruitment was no different in Research Nurse vs Dental Care Team models the Nurse model ensured more rapid recruitment. There were relatively few missing responses in the questionnaire and high levels of disclosure of risk behaviours . Data linkage of participant data to routine health records including HPV vaccination data was successful with 99.1% matching. Oral rinse/gargle sample collection and subsequent HPV testing was feasible. Preliminary analyses found over 95% of samples to be valid for molecular HPV detection prevalence of oral HPV infection of 5.5% . It is feasible to recruit and follow-up dental patients largely representative / reflective of the wider population, suggesting it would be possible to undertake a study to investigate the prevalence, incidence, and determinants of oral HPV infection in dental settings. Oropharyngeal (throat) cancer (OPC) is among the cancers with the fastest increasing incidence in the UK, with a near 3-fold rise in the past 15 years and incidence rates around 5 per 100,000 , 3. ThesHPV infection is increasingly implicated in the rising incidence of OPC , with HPThere have been only two European studies, both small. In the first, a 9.3% prevalence was found in a Swedish sexual health clinic-based study (n = 483), of adults aged 15–23 years, with reported high cervical HPV prevalence (70%) among the female participants (n = 408) .Oral HPV prevalence or incidence have rarely been investigated in relation to HPV vaccination. One proof-of-principle study demonstrated the prevention potential of the HPV vaccination against oral HPV infection . A recenThe National HPV Immunisation Programme in Scotland began in 12–13 year old females in 2008 as an intervention to reduce cervical cancer. Uptake of the HPV vaccine has been impressive, with sustained high levels of coverage (>90%) and a catch-up campaign, running over 3 years, which offered vaccination to all females aged 13–18 years old with an uptake of 66% . It is fThe primary aim of the HOPSCOTCH project was to test the feasibility of using dental settings, to undertake a population-wide (16–69 years) epidemiological study of the incidence, prevalence, and determinants of oral HPV infection in the Scottish population, which could ultimately allow assessment of the impact of the HPV vaccination programme in Scotland on oral HPV infection in young women and potential herd immunity in men aged 16–25 years old. The specific objectives of the study were to assess the feasibility and best methods of recruiting dental patients and capturing complete questionnaire data ; and to examine the practical / logistical aspects of taking and transferring oral gargle/rinse samples from the dental facility to the Scottish HPV Reference Laboratory in a state suitable for assay. NHS dental facilities  in Scotland are potentially ideal settings for this study as patients are generally healthy, attend regularly for routine dental appointments , and theBaseline recruitment commenced in November 2013 and continued to March 2015. Potential outreach teaching centres and General Dental Practitioners (GDPs) registered with the Scottish Dental Practice Based Research Network (SDPBRN) as Rapid Evaluation Practices (REPs) were sent a Study Information Sheet and invitation letter and subsequently contacted by telephone to confirm suitability for participation. All potential sites were assessed for suitability regarding their geographical location, patient numbers/profile, wi-fi/internet accessibility and availability of private space. Baseline patient recruitment and data collection took place in seven sites (three outreach teaching centres and four GDPs). We tested recruitment and data collection conducted by Research Nurses in five sites  versus recruitment and data collection conducted entirely by the dental practice clinical team (in two further practices). Sites were based in NHS Greater Glasgow & Clyde (GG&C) and NHS Tayside Health Boards . RelevanA paper-based participation/screening log was used to record the details of all patients screened for eligibility. Screened patients were either: i) sent the study invitation materials  two weeks prior to routine dental appointment; or ii) given the materials at the time of appointment, provided this was at least 30 minutes ahead of the approach for potential recruitment to the study. The method of approach varied by site, related to local patient appointment processes. Participating patients were assigned a study identification number and contact information was stored for the 6-month follow-up. For non-participants, reasons for non-participation, age, sex and home postcode (to assign the area-based socioeconomic deprivation index score—Scottish Index Multiple Deprivation (SIMD) )–were loInformed consent was sought by Research Nurses at five sites, and by a trained Dental Nurse from the Dental Care Team at two sites. Consent was sought for study participation (including 6-month follow-up), specimen storage in the Scottish HPV Archive (Edinburgh), participation in data linkage, and willingness to be approached to participate in future studies.We developed a bespoke, web-based questionnaire with the University of Glasgow Robertson Centre for Biostatistics (RCB). Responses were stored directly onto the RCB secure servers. Participants self-completed the questionnaire via an online tablet computer in a private area  of all participants (both recruitment models) was followed up via a postal/online questionnaire, with self-administration of the oral gargle/rinse and posting of the sample. Those who failed to attend their follow-up appointment were also offered this option.The study examined the feasibility of successfully linking patient participant data to routine health datasets. A unique identifier relating to each participant who consented to record linkage to national datasets, including the Scottish Immunisation & Recall System (SIRS) and Child Health Schools Programme-System (CHSP-S) for HPV vaccination data, was assigned to both his/her consent form and questionnaire. Data linkage was performed by the Electronic Data Research and Innovation Service (eDRIS), Information Services Division, NHS National Services Scotland.Participants provided a bio specimen comprising a 10 ml oral rinse sample in saline mouth rinse at two time points, 6 months apart . Particihttp://www.shine.mvm.ed.ac.uk/archive.shtml) for use in anonymised research in the future.Cell pellets were obtained from 10 ml rinses via centrifugation at 2900 ± 150 X g for 15 ± 2 mins., supernatants were discarded and the cellular pellets frozen in tissue stabilisation buffer  pending NA extraction. NA was extracted using a system initially optimised for downstream HPV detection of cervical and urine samples  using thHPV amplification and genotyping were performed using a luminex based HPV genotyping assay, currently used for national HPV immunisation surveillance , 28. ThiStatistical analysis of participant data was carried out using R version 3.1.1. Confidence intervals for the participation proportions, follow-up rates and HPV positivity percentages were calculated using Wilson’s method using the Hmisc package in R. 95% confidence intervals are reported in all cases. Chi square tests were used to test for associations between the outcomes and demographic variables. Simulation tests based upon 5000 replications were used to calculate the p values. Post stratification weights were estimated using raking to adjust the HPV prevalence to address any imbalance between sample and population demographics. This was carried out using the survey package in R.NHS West of Scotland Research Ethics Committee (REC) specifically approved this study [Reference no. 13/WS/0166:19/09/13], and NHS National Services Scotland Privacy Advisory Committee (PAC) [PAC37/14:4/02/15] approved the data linkage.A total of 1212 individuals, aged 16–69, were approached to take part in the study from seven centres in two health board areas—NHS GG&C and NHS Tayside. Of these, 59% were female compared to the Scottish population of which 51% in this age-group are female . The ageIn total, 402 individuals agreed to participate in the study giving an overall participation rate of 33.1% with no imbalance in participation rates between men and women. Among the 1072 with known age, participation was significantly associated with age (p = 0.010) and was lower among those aged 16–25. There was no apparent association (p = 0.827) with participation and SIMD .There were significant differences among the participation rates according to research site—(p<0.0001), with relatively low participation in two centres . Participation was not significantly (p = 0.072) associated with the use of a Research Nurse as opposed to a Dental Care Team. Similarly, it was not associated (p = 0.432) with the type of dental setting . ReasonsOf those who had agreed to participate (n = 402): (a) all participants had initialled the box on the consent form indicating they had understood the Participant Information Sheet and agreed to participate in the baseline and 6-month follow-up; (b) 16 (3.9%) did not give consent for the data linkage element of the study; (c) 23 (5.7%) participants did not wish to be contacted about future studies. The Research Nurse model was more effective in terms of achieving the target recruitment numbers on time in three of the four sites , while the Dental Care Team model did not reach the target despite recruiting in almost twice the number of recruiting weeks . The average cost per recruitment was substantially lower in the Dental Care Team model (£26.23)–under half those associated with the Research Nurse (£66.46).A total of 390 participants (from 402 who consented) completed the questionnaire ; 263 (67.4%) participants completed all 30 questions which should have been answered by everyone, 354 (90.8%) omitted less than 2 questions. The most non-responses (12.1%) were for the income question . Only 7 The self-report vaccine data were comparable to the health administrative datasets. Data linkage of our study data to the Community Health Index (CHI) number  was successful on 99.7% (n = 384) of the 385 participants who’s data were submitted to ISD—all of whom had consented for data linkage. For the purposes of preliminary feasibility, linkage to the SIRS database was tested. Of the 375 individuals who completed the questionnaire and linked to SIRS database, 15 had received the HPV vaccination . There were no HPV vaccination records for men in the SIRS data. Among the 221 questionnaires from female participants, 18 reported having had the HPV vaccine , 202 reported no HPV vaccine, with 1 missing value. Three women stated that they had the HPV vaccine  but there is no corresponding record in SIRS; these three women were aged between 35–50 years and it is unlikely that they received the vaccine. Among the 21 women aged 16–25 there was an exact match between the questionnaire and SIRS for 17. Of the remainder, one reported that she had received three doses but has only one record in the SIRS data, and two stated that they had three doses but there is no record in SIRS.Follow-up is primarily assessed among the 380 participants who had a valid baseline oral gargle/rinse specimen, as the principal aim of the follow-up is in the determination of new/incident acquisitions of oral HPV among participants who were negative at baseline and clearance of HPV among participants who were positive for HPV at baseline. Participants who had a follow-up oral specimen submitted to SHPVRL were considered to have satisfied the conditions for a successful follow-up. Overall follow-up rate was 61.6%, with similar rates among men and women. Follow-up was significantly better among older patients (p<0.0001). There was no evidence (p = 0.186) to suggest that follow-up was associated with deprivation of the participant . The eviGenerally, operational processes between source collection points and the central testing laboratory were good with few significant issues. Of the samples received at SHPVRL, only 11 could not be processed for HPV testing: 7/11 were discarded due to a packaging error where the transit receptacle was used to contain the sample instead of the primary (inner) tube and 4/11 were labelled inadequately. These issues related to 2 tranches of samples rather than 11 separate errors.The automated NA extraction procedure, initially designed for cervical cytology samples, was amenable to oral wash pellets and <5% showed clogging/blocking on the automated platform. This level was manageable from a laboratory-process point of view. Quantity of extracted NA varied according to sample and associated cellularity. Quantitative assessment of showed that the mean NA yield was 64ng/ul (range 27ng/ul to 130ng/ul). Yield was not affected according to whether the sample was transported as a postal kit or via the practice and, as described, was more dependent on the individual sample. Purity of the NA as measured by 260/280 absorbance ratios showed between sample variation; however, adequate quality for the key downstream application [HPV PCR testing] was evidenced by amplification of the cellular in-house control (beta globin) in 98% of samples.The present study was not powered to examine HPV prevalence of HPV types including in subgroups or in relation to risk associations.There were 380 participants with an unequivocal HPV test result at baseline . At baseOral HPV positivity did not vary with gender but there is a suggestion of an association with age (p = 0.081) and with deprivation, (p = 0.037). Oral HPV positivity was greatest in the 26–39 age-group where the rate was 10.4%  compared to a rate of 2.6%  in the 16–25 age group.Out of the 380 who gave a valid oral swab at baseline, 234 had a valid swab at follow-up, 61.6% . Among those with both a baseline and follow up HPV result, 4 out of 223 HPV negative at baseline acquired HPV, 1.8% . There were 11 HPV positive individuals at baseline and 8 were clear of HPV at follow-up, giving a clearance percentage of 72.7% .In summary, our multidisciplinary research group has demonstrated feasibility of recruiting participants using dental care facilities, a centralised laboratory (SHPVRL), and data-linkage resources, to undertake epidemiological studies of oral HPV infection. The experience and data gained now make us well placed to design and implement a population study which would measure the impact of the HPV vaccine on oral HPV among women and assess the implications for extending the vaccine to boys.We achieved an overall participation rate of 33.1%, which was somewhat lower than the 67% participation rate in the National Health and Nutrition Survey (NHANES) used in the US oral prevalence study , but is The consent rate to the data linkage component (96%) was substantially higher than anticipated or observed elsewhere—a systematic review and meta-analysis of the international literature estimated 67% consent rate to use linkage to medical records . MoreoveTo our knowledge this was the first observational epidemiological studies in Scotland to recruit patients through dental settings with support of Research Nurses. While both the Research Nurses and Dental Teams faced the same challenges in terms of converting patient approaches to study participants, the Research Nurses were largely able to work more intensively, recruit more participants, and meet their targets on time. Dental Care Teams managed to recruit participants at the same patient participation rate, but were not able to recruit sufficient numbers in the allotted time-frame. The higher costs for the Research Nurse model were due to them being employed and their salary costs being paid directly for this study. They were then deployed to work within the different dental settings. The Dental Care Teams were fitting this study into their clinical daily practice routine and were compensated accordingly on an individual participant recruitment basis which was based on payment of similar tasks on their contract. Completion of the questionnaire on the tablet computer was disappointingly much lower than anticipated. The reasons were that one centre offered the paper version as an option rather than as a back-up, and there were technical reasons, mainly related to internet connectivity issues in two centres, which could be addressed prior to a larger study.Postal follow-up, in which patients provided their own oral gargle/rinse specimen and posted it  was less effective (36.4%) than a follow-up appointment, However this response was much higher than that observed in an earlier Scottish survey to facilitate HPV vaccine monitoring where response rate to a self-taken urine or vaginal swab was (13.2%) . It is wParticipants were able to complete a questionnaire covering a wide-range of factors including demographic, socioeconomic, lifestyle, and sexual behaviour/health. While a third of participants gave feedback that they were uncomfortable with the sexual activity questions, completion rates were high, and participants were able to disclose risk behaviours. Capture of this information may have been facilitated by self-completion in a private area or perhaps  by the online questionnaire.In terms of sexual history, we were able to access a significantly at-risk sample. For example, 39% of male participants reported recreational drug use, 34% of males reported >10 partners, 36% of males reported insertive anal sex with a female partner and 24% of women reported receptive anal sex. The proportions reporting >10 partners is broadly similar to those reported by the NATSAL study . In addiOur preliminary estimate of oral HPV prevalence  is remarkably in-keeping with the overall prevalence in the US population  , and theOur study demonstrates that it is feasible to investigate the prevalence, incidence, and determinants of oral HPV infection in dental settings. We now know what resources and methods are needed to expand this approach to assess the impact of the school-based HPV vaccination programme in Scotland on oral HPV in girls/women who have received the vaccine and to examine if there has been any herd immunity in boys/men. Crucially we have sufficient data to power a full-population approach. We have provided the first preliminary estimate of oral HPV prevalence in the Scottish population. In our preliminary analysis we found an overall prevalence of oral HPV infection of 5.5% and a higher prevalence among 26–39 year olds of 10.4%.We demonstrated that the Research Nurse recruitment model was more effective than Dental Care Teams in terms of baseline and follow-up recruitment and meeting recruitment targets on time, but that the former model was more expensive.To undertake a fully powered study to assess the impact of the HPV vaccination programme in Scotland on oral HPV infection in men and women, we would need to enhance recruitment of 16–25 year olds. This could be established in dental settings serving populations with a high proportion of young people, including community and Further/Higher education settings.We have also shown that the boundaries of the nature of research undertaken in dental settings can be stretched beyond current traditional dental care and research remits. This points to the importance of further research into barriers and facilitators to implement other risk factor assessment / prevention, screening, and care activities not necessarily associated with current practice .S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.


Importantly, the cross-neutralizing antibody response was maintained at least 82 weeks in mice or 42 weeks in rabbits, and complete protection against HPV58 was observed at week 85 in mice. Our data demonstrate that HPV16L1-58L2 cVLP is an excellent pan-HPV vaccine candidate.The neutralizing antibodies elicited by human papillomavirus (HPV) major capsid protein L1 virus-like particle (VLP)-based vaccines are largely type-specific. An HPV vaccine inducing cross-neutralizing antibodies broadly will be cost-effective and of great value. To this end, we constructed HPV16L1-58L2 chimeric VLP (cVLP) by displaying HPV58 L2 aa.16-37 on the DE surface region of HPV16 L1. We found that vaccination with the HPV16L1-58L2 cVLP formulated with alum plus monophosphoryl lipid A (Alum-MPL) adjuvant elicited robust neutralizing antibodies in both mice and rabbits against all tested HPV types including HPV16/31/33/35/52/58 (genus  Persγ, μ, v) , 6. Low-omatosis , 8. Cutaus warts , 10. Infciformis .Three available HPV L1 VLP-based prophylactic vaccines, bivalent Cervarix (contains HPV16/18 VLPs), quadrivalent Gardasil (contains HPV16/18/6/11 VLPs), and nonavalent Gardasil-9 (contains HPV16/18/31/33/45/52/58/6/11 VLPs), have shown to provide protection against vaccine types –16. TherAn alternative approach to fill in the gap between cross-protection and cost is focused on developing a vaccine using the minor capsid protein L2. Vaccination with the N-terminus of L2 induces cross-neutralizing antibodies, but the antibody titers against the homologous HPVs are lower than that induced by L1 VLPs –27. ApprHPV16 is the most common oncogenic type worldwide and accounts for approximately 60% of invasive cervical cancer (ICC) , 42. HowBecause HPV58 is highly prevalent in China, we have examined the potential of HPV58 L2 aa.15-37  and HPV6  RG1 peptides are also able to do so. HPV58 and HPV6 RG1 peptides were synthesized and conjugated with KLH . Two rabbits per group were immunized four times with KLH-58RG1 or KLH-6RG1 and sera were collected two weeks after the fourth immunization to determine the neutralizing antibody titers against 13 types of HPV pseudoviruses (PsVs).α9, α7, α10 and β1 genera, HPV58 RG1 peptide was chosen to develop chimeric VLPs.As shown in Table To potentially enhance and broaden cross-neutralizing responses induced by the HPV58 RG1 peptide, we engineered an HPV16L1-58L2 chimeric protein by displaying HPV58 L2 aa.16-37 on the DE loop of HPV16 L1 and expressed it in baculovirus expression system. The HPV16L1-58L2 protein was highly expressed similar to HPV16L1 protein as analyzed by SDS-PAGE with coomassie blue staining Figure . The HPVP>0.05, Figure α9) , HPV18/39/45/59/68 (α7) , low-risk mucosal HPV6/11 (α10) , as well as cutaneous HPV2/27/57 (α4)  and HPV5 (β1) . However, sera from mice immunized with HPV16 L1 VLPs had very weak cross-neutralizing activity against HPV31 and non-detectable against other HPV types tested (not shown). Taken together, HPV16L1-58L2 cVLPs formulates with human-applicable Alum-MPL adjuvant were able to induce cross-neutralizing antibody responses to common high/low-risk mucosal and cutaneous HPV types in mice.To test the ability of HPV16L1-58L2 cVLPs to induce neutralizing antibodies, sera from mice (n=5) immunized with Alum-MPL adjuvanted HPV16L1-58L2 cVLPs were analyzed by standard PsV-based neutralization assay (PBNA). Immunization with HPV16L1-58L2 cVLPs induced potent neutralizing antibody against HPV16 with mean titer of 166,400, which was comparable with that by HPV16 L1 VLPs  by PBNA. As shown in Figure To further explore the long-term duration of neutralizing antibodies induced by Alum-MPL adjuvanted HPV16L1-58L2 cVLP vaccine, rabbits (n=4) and mice (n=5) were immunized at weeks 0, 4, 7 and 10. Rabbit sera were collected at weeks 12 and 42, and mouse sera were collected at weeks12 and 82 to determine the cross-neutralizing antibody levels. Although the titers were 6-51 folds lower than those at week 12, antisera from HPV16L1-58L2 cVLP immunized rabbits at week 42 were still able to neutralize all 15 tested HPV PsVs Figure , includiAs shown in Figure HPV L1 VLPs are highly immunogenic but induce mainly type-specific neutralizing antibodies. The recently approved Gardasil-9 contains HPV L1 VLPs from 7 types of high-risk HPVs and 2 types of low-risk HPVs. Thus, an HPV vaccine that can be produced cost-effectively and covers a broader spectrum of HPV types than Gardasil-9 is desired. In this study, we used HPV16 L1 VLP as the backbone to display HPV58 L2 aa.16-37 into HPV16 L1 DE loop. We found that the chimeric VLPs induced broadly neutralizing antibodies against over 17 HPV types. Thus, we believe that it is a promising candidate for a new generation of HPV vaccine.Pseudomonas aeroginosa) each displaying one type of HPV L2 aa.17-31 (including HPV58), provided only partial in vivo protection against HPV58, and the neutralizing activity of the antisera was not known. To our knowledge, the immunogenicity of HPV58 RG1 peptide to elicit cross-neutralizing antibody responses is not well studied. Here we found that immunization of KLH-58RG1 peptide could induce cross-neutralizing antibody responses against all 13 tested HPV types with relatively high titers whereas KLH-6RG1 antisera neutralized only 10 HPVs with very low titers. These data indicated that the immunogenicity of any HPV type RG1 peptide is difficult to be empirically predicted.Although RG1 sequences from different HPVs are relatively conserved  or HPV6 L2 aa.16-39 were synthesized by GL Biochem LTD (Shanghai). Each peptide was conjugated to keyhole limpethemocyanin (KLH) via the N-(3-Dimethylaminopropyl)-N’-ethylcarbonate linker.HPV16L1 delta C31 gene (accession number: GU556964) expressed HPV16 L1 VLP at high level in baculovirus expression system [HPV58 L2 aa.16-37 gene sequence was genetically engineered into the DE loop of the above HPV16 L1 between nt.408 and nt.412 (aa.136 and aa.138) with three point mutations in the flanking sequence  by PCR strategy. The HPV16L1-58L2 construct was subcloned into pFastBac1 vector and expressed in the Bac-to-Bac baculovirus expression system according to the manufacturer's instructions (Invitrogen).Our previous work showed that the optimized n system . In thisHPV16L1-58L2 chimeric proteins were purified by CsCl ultracentrifugation as described previously  with minDLS measurements were performed on a Malvern Zetasizer Nano ZS . Samples were filtered through a 0.45 μm filter followed by equilibration to 25°C. The hydrodynamic size was recorded as Z-average hydrodynamic diameter. All data reported here are the averages of three measurements of the same sample.For TEM analysis, purified proteins were adsorbed on a carbon-coated grid for 1 minute, rinsed with distilled water, negatively stained with 1% uranyl acetate for 3 minutes and examined with a TEM-1400 electron microscope operating at 80 kV with a magnification of 80,000×.New Zealand white (NZW) rabbits and BALB/c mice were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and kept in the animal facility of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.NZW rabbits (n=2) were immunized subcutaneously on days 0, 14, 28 and 42 with 1 mg of synthetic HPV58 L2 aa.15-37 or HPV6 L2 aa.16-39 coupled to KLH  in complete Freund's adjuvant (CFA) with the primary dose and 500 μg of antigens in incomplete Freund's adjuvant (IFA) with boost doses. Sera were collected two weeks after the fourth immunization and stored at −20°C.Female BALB/c mice (n=5) or NZW rabbits (n=4) were vaccinated subcutaneously at weeks 0, 4, 7 and 10 with 10 or 50 μg of HPV16L1-58L2 cVLPs formulated in Alum-MPL, respectively. HPV16 L1 VLPs adjuvanted with Alum-MPL was used as a control. Sera were collected at weeks 12, 42 or 82 and stored at −20°C.PsVs of HPV2/5/6/11/16/18/27/31/33/35/39/45/ 52/57/58/59/68 with encapsidated reporter plasmid pfwB which encoding green fluorescence protein (GFP) or pLucf which encoding both luciferase and GFP were produced in 293TT cells as previously described [P values <0.05 were considered to be statistically significant.Standard PBNA was performed as described previously . Every s5 IU of PsVs (encapsidated reporter plasmid pLucf) and 1% carboxymethyl cellulose  was intravaginally instilled using a positive-displacement pipette. Forty-eight hours post-PsV challenge, mice were vaginally instilled with 0.4 mg of 5′-F-Luciferin . Three minutes later, luciferase signals were acquired for 10 min with an IVIS 200 bioluminescent imaging system .Murine vaginal HPV PsV challenge was performed as described previously , 36. Mic