The term ‘‘Human Papillomavirus’’ or ‘‘HPV’’ has become synonymous with uterine cervical cancer leading to feminisation of all the preventive measures, especially immunisation. Taking into consideration the rising number of HPV associated cancers among men in many developed countries and the risk of transmission to women, male HPV infection is a serious concern. A systematic review and meta-analysis of literature was performed to determine the global prevalence of HPV among men with oropharyngeal and anogenital cancers. A systematic review and meta-analysis of literature was performed searching electronic databases for published articles in English between January 1984- April 2020 based on standard systematic review guidelines. The meta-analysis component was modified appropriately for the synthesis of prevalence study results. National Institutes of Health checklist for observational, cohort and cross-sectional studies was used to assess the quality of the studies selected after the abstract and content review. The meta-analysis was performed in STATA version 13.0  and the forest plots were constructed using metan package in STATA. Through the electronic search of databases, 3486 original articles were screened for eligibility. Fifty-eight articles were systematically reviewed and 42 articles were qualified for meta-analysis including 4,250 men with oropharyngeal, penile and prostate cancers. The pooled prevalence of HPV DNA in oropharyngeal cancers was 45% (95%CI 24.0%-66.0%). Meanwhile the pooled prevalence rates of 48% (CI 40.0%- 57.0%) and 19% (CI 10.0%-29.0%) were observed in penile and prostate cancers respectively. Even though, articles regarding HPV prevalence in anal cancers were systematically reviewed, none of the studies were qualified for meta-analysis. Higher pooled prevalence of HPV DNA was observed among men with oropharyngeal and penile cancers. Multicentric molecular studies investigating the prevalence of HPV in prostate cancers have to be planned in future. Cancers account for one in six deaths worldwide and the number of cancer cases is much more than the total number of AIDS (Acquired Immunodeficiency Syndrome), tuberculosis and malaria . In 2018 lungs, colorectal, and prostate cancers contributed to 44.4% of all cancers in men . Chronic infection by certain viruses, bacteriae and parasites is the third leading cause of cancers after dietary factors and tobacco . In 2018, 13% of new malignancies were associated with infection by Human Papillomavirus (HPV), Helicobacter pylori, Hepatitis B virus (HBV), and Hepatitis C virus (HCV) .The HPV infection is the most common sexually transmitted viral infection and a causal association was reported in 4.5% of new cancers arising from the cervix, anogenital tract and oropharynx . Persistent high-risk HPV infection leads to almost 12% of female cancers in developing countries and the causal association with cervical cancer was well established (“WHO | Human papillomavirus (HPV),” n.d.). The main determining factors for cervical dysplasia and invasive cervical cancer are early age at sexual debut, multiple sexual partners, young age at first delivery, multiparity and high risk sexual behaviour of male partner .International Agency for Research on Cancer (IARC) has identified HPV-16 as one of the causal agents for oropharyngeal squamous cell cancer (OPSCC) in 2009 . As per IARC monographs on the evaluation of carcinogenic risks to humans, HPV-16 was recognised as the risk factor for oropharyngeal cancers, mainly squamous cell cancers arising from tonsil and base of tongue. IARC does not support a strong and consistent association of oral cavity squamous cell cancers (OCSCC) and laryngeal cancers with HPV (IARC 100B) . In the United States, men are five times more likely to be diagnosed with HPV-associated oropharyngeal cancers than women implying a distinct male gender predominance . The prevalence of male HPV infection is high among men having sex with men (MSM) and men positive for human immunodeficiency virus (HIV) . In the developed countries, the overall incidence of head and neck squamous cell carcinoma (HNSCC) is declining with a concomitant rise of HPV associated oropharyngeal squamous cell carcinoma (OPSCC) . An increase in the annual incidence of HPV associated (OPSCC) among non-smokers and teetotalers, below the age of fifty years was observed in the US. By the year 2045, OPSCC is anticipated to become the third most common cancer among men . Several etiological factors account for penile carcinomas such as poor personal hygiene, phimosis, multiple sexual partners and HPV infections. Despite being rare in developed nations, penile cancer cases in some parts of Africa, South America, and Asia may contribute up to 10% of male genital cancers . Anal cancer is reported frequently among males from less developed nations and females from well-developed countries . The prevalence of HPV DNA in anal cancers vary between 85%-91% and HIV-positive men having sex with men are at greater risk . The link between prostate cancer and HPV has not been established so far. A rise in HPV associated oropharyngeal cancers, as well as anogenital cancers among men from developed countries, indicate high-risk sexual activity and HPV exposure over many decades. This quantitative analysis will be helpful for policymakers and health service providers in generating evidence regarding HPV association among men with anogenital and oropharyngeal cancers facilitating the implementation of appropriate preventive measures. Validated HPV-DNA based screening strategies for early recognition of high-risk HPV infection and HPV vaccination can lead to a significant reduction in the number of HPV-related cancers.The main aim of this meta-analysis is to quantitatively assess the global prevalence of HPV DNA among men with oropharyngeal, penile, prostate and anal cancers. The meta-analysis also investigated whether the HPV-association in oropharyngeal and anogenital cancers differed across various geographical areas and characteristics of study participants. MethodologyA systematic review and meta-analysis of literature was performed searching electronic databases for published articles in English between January 1984-April 2020 based on standard systematic review guidelines prescribed by Cochrane collaboration (htps://www.cochrane.org) and Campbell Collaboration (htps://www.campbellcollaboration.org). The meta-analysis component was modified appropriately for the synthesis of prevalence study results. The present research work comply with the Preferred Reporting Items of Systematic reviews Meta-Analyses (PRISMA) statement .Description of the conditionHPV DNA: Molecular detection of HPV implies prevalent infection, reinfection, auto-inoculation from the oral cavity, anogenital areas or intermittent viral shedding from a latent infection. Persistent infection by high-risk HPV types, is associated with oropharyngeal and anogenital malignancies in men and women .HPV Prevalence among men with cancers: HPV Prevalence is the proportion of men having HPV DNA-associated histologically confirmed oropharyngeal and anogenital cancers at a given time. HPV exhibits epithelial tropism for distinct anatomical sites in both the sexes. The HPV prevalence data among men with certain cancers is vital while undertaking various preventive measures at the population level. Study protocolA comprehensive electronic search of PubMed/MEDLINE and SCOPUS was carried out using search terms such as Human Papillomavirus OR HPV AND men AND oropharyngeal cancer AND Head and neck cancers AND Penile cancer AND anal cancer AND Prostate cancer NOT women NOT cervical cancer NOT vulval cancer NOT vaginal cancer. Hospital-based studies in English, employing fresh or formalin-fixed paraffin-embedded (FFPE) tissue samples from histologically confirmed anogenital and oropharyngeal cancers for molecular detection of HPV were included in the present review. The published articles in English since 1984 regarding HPV association in male cancers were reviewed. Besides, a manual library search for articles in peer-reviewed journals was performed and references of retrieved articles were also appraised to increase the search sensitivity. Inclusion process and criteriaCross-sectional, cohort and case-control studies which screened oropharyngeal and anogenital tissue samples of men with histologically confirmed oropharyngeal and anogenital cancers for HPV DNA were included. Exclusion criteriaStudies not reporting the estimated prevalence of HPV DNA by molecular assays and cancers not confirmed histologically were excluded. All the population-based studies enrolling asymptomatic individuals, estimated prevalence data from controls enrolled in case-control studies and pre-neoplastic lesions were excluded. Studies on HIV-positive men and MSM were not included. Duplicate publications of the same survey and articles published in languages other than English were excluded.Data extractionA validated proforma focusing on the first author, year of publication, region, the period during which samples were archived, sample size, HPV DNA positivity and HPV genotypes was used to extract the data.th April 2020. In the systematic review, the three-stage selection process was carried out for the final inclusion of the studies. One reviewer assessed titles from 3,846 records for the relevance for inclusion in the study. Studies applicable for the review were moved to the second stage and duplicates were excluded . In the second stage, the abstract of the studies (n= 618) was obtained and two reviewers independently analyzed all the abstracts. All the non-relevant studies were rejected (n=465) and the remaining (n=153) studies were moved to the third stage. Full texts of studies (n=153) were retrieved which were again examined by two reviewers independently. Extracted data from full-text articles by the first reviewer was reviewed by a second reviewer independently. All the studies chosen by both the reviewers were involved and the third reviewer adjudged the selection process if there was any difference between the two reviewers. Authors were contacted electronically when there was incomplete or inadequate information. A total of 58 studies were systematically reviewed and 42 studies were qualified for meta-analysis. HPV DNA Prevalence data from males having oropharyngeal and genital cancers from cross-sectional, case-control and cohort studies were included in the quantitative synthesis . The study selection process is depicted in the PRISMA chart . The lasQuality AssessmentNational Institutes of Health checklist  for observational, cohort and cross-sectional studies was used to assess the quality of the studies selected after the abstract and content review,” n.d.). Out of the fourteen questions, question numbers 1, 2, 3, 4, 5 and 11 were applied to our study. The response to the remaining eight questions was marked as not applicable (NA). Each question was categorized as Yes, No, others-CD (can not determine), NA (not applicable), NR (not reported). The studies with six, four /five or less than four “Yes responses” were considered good, fair, and poor quality respectively. Data analysis 2 Statistic to quantify the heterogeneity between the studies.Meta-analysis was performed in STATA version 13.0 . The forest plots were constructed using metan package in STATA. A considerable amount of statistical heterogeneity (variability in the effect sizes) across the studies was anticipated as the included studies were mostly observational. Therefore, a random-effects model for meta-analysis was adapted rather than a fixed-effect model. The pooled prevalence with 95% CI was reported along with IThrough the electronic search of databases, 3,486 original articles were screened for eligibility. Titles of 1084 articles were reviewed and abstracts of 618 articles were retrieved. Full texts of 153 articles were analysed. Fifty-eight articles were systematically reviewed and 42 articles were qualified for meta-analysis including 4,250 men with oropharyngeal, penile and prostate cancers. Oropharyngeal cancersMost of the published literature regarding HPV associated head and neck cancers were narrative reviews, and epidemiologic studies spanning over two to three decades. We observed 91 articles concerning HPV DNA association in oropharyngeal cancer cases between 2001 and 2020. Abstracts of 86 articles were retrieved and full texts of 35 articles were analysed. The characteristics of each study included in the meta-analysis were shown in the In the United States, the association of HPV in OPSCC ranged between 40% and 80%  with a 225% increase in HPV-associated OPSCC since the 1980s and 50% fall in HPV-negative OPSCC . A similar epidemiologic pattern was observed in Australia and Europe also . Meanwhile, almost 90% of oropharyngeal squamous cell cancers are attributable to HPV in Sweden . The higher estimate of HPV DNA prevalence in OPSCC varying between 38%-56% was reported from North America, Japan, Australia, Northern Europe, Western Europe and Eastern Europe. Meanwhile, 17% prevalence was observed in Southern Europe and 13% in the rest of the world . The studies from Asian countries also reported HPV DNA prevalence in the range of 30%-50% in OPSCC  which is more prevalent among middle-aged, non-smoking white men with multiple sexual partners . National cancer registry data from South Korea, located in East Asia having a distinct cultural and ethnic background also reported a rise in HPV associated oropharyngeal cancers in the last two decades .Penile cancersBetween 1989 and 2017 a total of 347 articles were published including reviews and meta-analyses. For the systematic review, 76 original articles were included. Twenty-three articles comprising 2,531 penile cancer cases were qualified for meta-analysis. The prevalence of HPV DNA ranged between 25%-90% in penile cancers . StudiesA higher prevalence rate of penile cancer case was reported from low-income countries in Asia, Africa and South America contributing up to 10% of cancers in males . Even though penile cancer is less common in developed countries, a significant rise of incidence in the last fifty years with a constant survival rate was reported from Germany . In the US, HPV prevalence in penile cancers was observed to be increasing since 1998. There is a great disparity in the geographical distribution of penile cancer with the lowest incidence being reported in Israel . The highest number of cases were reported from certain regions in Brazil with age-standardised incidence rate of 2.9-6.15/100,000 . Penile cancers are mainly squamous cell carcinomas, out of which 49%, 17%, 8%, 6% and 4% are keratinising, mixed warty-basaloid, verrucous, warty and basaloid types respectively. Meanwhile, non-HPV related cancers include other subtypes such as verrucous and papillary squamous cell carcinomas. The highest pooled HPV prevalence of 84.0% (95% CI 71.0 %-93.0%) was reported in the basaloid squamous cell carcinoma, followed by warty-basaloid squamous cell tumors having a pooled HPV DNA prevalence of 75.7% (70.1%-81.0%) . Prostate cancersAbout 270 articles were published between 1990 and 2019 regarding HPV DNA association in prostate cancers. Full texts of seventy-six articles were retrieved and nine studies carried out among 876 prostate cancer cases were included for quantitative synthesis. Prostate cancer is the most common cancer and the second leading cause of cancer deaths among men in the United States . The first study by Dodd et al reported a higher HPV association in 14 benign prostatic hyperplasia cases and four prostate cancers suggesting prostate gland, a reservoir of HPV in 1990 . High-risk HPV infection was detected in prostate cancer cases from North America, Asia-Pacific region, Europe, South America, New Zealand and Australia . The high-risk types commonly detected in prostate cancer were HPV-16,-18,-31,-33 and -58 . The studies from Canada and Japan supported a causal association of HPV in prostatic adenocarcinoma . Recent studies from Greece, North India, Australia and Mexico also reinforced these findings . Malaysia reported HPV DNA association in more than 50% of prostate cancer cases diagnosed at advanced stages . A higher incidence rate was also reported among Asians living in the United Studies than their counterparts in the native countries . The studies from the US, Mexico, Brazil, Australia, India and Iran were included for meta-analysis. A recent study from Mexico with no clear definition of the study population and an Indian study in which there was no mention of the exact time during which the samples were procured were classified as fair . Two earlier studies from the US reporting low prevalence rates used southern Hybridisation for HPV DNA detection which was much less sensitive than Polymerase Chain Reaction (PCR) . The HPV prevalence estimate varied between 2.3%-41.1% in tissue samples as in In Asia, a significant number of men with prostate cancer are diagnosed at advanced stages of cancer . The incidence varies vastly between countries with the lowest rate of 9.1/100,000 in Iran to the highest rate of 22.9/100,000 in Philippines . Based on population-based cancer registries, prostate cancer was the fifth most common type of cancer among Indian men with 33,000 incident and 112,000 prevalent cases respectively in the year 2016 . Globally, recent molecular studies have reported significant HPV DNA association in tissue samples of prostate cancer cases. The overall pooled prevalence of HPV DNA in prostate cancers  was 19% (CI 10.0%-29.0%) as in Anal cancersThere were 376 articles regarding HPV DNA prevalence in men with anal cancers published between 1984 and 2020 January. This included 61 narrative reviews, ten systematic reviews and five meta-analyses. Out of the 376 articles, 297 studies had included HIV-positive men having sex with men. Anal cancer is rare globally with a worldwide incidence of 1 per 100,000 cases and every year 27,000 new cases are reported . Almost 90% of anal squamous cell cancers are attributed to HPV infection . However, a recent study reported a rise in advanced anal squamous cell cancers with a higher mortality rate in the United States since 2001 . Only one cross-sectional study enrolled immunocompetent men as well as HIV-positive men with anal cancers . There were no publications of HPV DNA prevalence among immunocompetent men with anal cancers globally. There are cross-sectional studies on HPV prevalence in the community including HIV positive men and negative men from different parts of the world. The meta-analyses have been published regarding HPV DNA positivity among HIV-positive men or MSM with anal cancers. None of the studies was qualified for quantitative synthesis.Almost all the published studies regarding HPV associated male anal cancers were performed among MSM or men living with HIV from Europe and America . A high incidence rate of anal cancers was reported among French men on antiretroviral therapy since 1996 . Multicentric study enrolling cohorts from different parts of North America reported higher anal cancer rates among HIV positive men having sex with men when compared to HIV positive women . A recent international study reported HPV DNA prevalence of 95.4% in invasive anal squamous cell cancers with a predominance in warty basaloid types in tissue samples from 24 countries . There was no statistically significant gender disparity in anal cancer prevalence rates even up to 10 years of diagnosis. The present study observed vast variation in the HPV DNA prevalence estimates among men with oropharyngeal or anogenital cancers which was mainly attributed to the variation in the geographical areas. Earlier studies carried out in the 1990s used less sensitive molecular assays such as southern hybridization or in situ hybridization. All the studies combined for quantitative synthesis concerning HPV DNA association in penile cancer cases employed FFPE for HPV DNA testing. Meanwhile, the studies included in the quantitative synthesis of oropharyngeal and prostate cancers used fresh frozen tissues or FFPE for molecular analysis. The meta-analysis by Mehanna et al observed no significant differences in the prevalence estimates when fresh frozen samples or FFPE tissue samples were used . A Systematic Review of the worldwide prevalence of HPV DNA reported that one-third oropharyngeal and one-fourth oral squamous cell carcinomas have an association with this oncogenic virus . Almost 90% of the HPV associated oropharyngeal tumours are positive for HPV-16 . The prevalence of HPV DNA in oropharyngeal cancers was stated to be rising significantly, especially in North America and Europe . The pooled prevalence estimates varied from 25% in oropharyngeal cancer cases in Europe to more than 50% in the US and Canada . Our estimate was in agreement with a recent meta-analysis of studies published between 2004 and 2014 which reported a pooled prevalence of 42.6%. Even though HPV molecular tests in tissue biopsy samples are being used for staging of oropharyngeal cancers, Centres for Disease Control and Prevention (CDC) do not recommend routine HPV testing of men for screening purpose of throat, anal and penile cancers. Stratified analysis of oncogenic and non-oncogenic HPV prevalence was not performed in the meta-analysis as majority of the studies reported only oncogenic types. As shown in Norwegian cancer registry-based study spanning more than sixty years reported a consistent and moderate increase in the incidence of penile cancers. One systematic review reported HPV association of 47%-48% in penile cancers. The present meta-analysis was in accordance with two recent meta-analyses reporting a pooled HPV DNA prevalence of 51% in penile cancers. For the past twenty years, a rise in prostate cancers has been reported in many developing countries mainly due to rapid urbanisation, changing lifestyles and migration from rural to urban areas. Molecular Studies in the last two decades have suggested a causal association between prostate cancer and HPV. We observed comparatively less number of meta-analyses evaluating the etiologic role of HPV in prostate cancers. One meta-analysis observed a possible pathogenic association between HPV infection and the development of prostate cancer. The pooled analysis of prevalence data by Lin et al based on both serological tests and molecular tests observed no significant association between HPV infection and prostate cancer. Meanwhile, recent meta-analyses from Asia reported a statistically significant association of HPV DNA with prostate cancers . Serologic assays are not recommended for detection of HPV infection as antibody response to this oncogenic virus is minimal and standardised WHO HPV assays based on VLP (Virus Like Particles) are not commercially available. Human Papillomavirus infection in men (HIM) study initiated at different centres in Brazil, the United States and Mexico at the beginning of the twenty-first century witnessed an overall HPV DNA prevalence of 50.5% in the oropharynx and anogenital areas . Unlike oropharyngeal cancers, there was no increased occurrence of HPV-attributable anogenital cancers among middle-aged men who are non-drinkers and non-smokers. Almost all HPV-associated male oropharyngeal cancer tissue samples are positive for either HPV-16 or HPV-18 . The US FDA has approved quadrivalent HPV vaccine for prevention of anal cancers among men as well as women aged 9-26 years in 2010 . However, there are no data regarding HPV association in anal cancers from many developing countries including India as per Human Papillomavirus related Diseases report-India published in 17th June 2019 (https:// hpvcentre.net/ statistics/ reports/ IND. pdf). In developing countries anal cancers are often under-reported and advanced cancers are often misdiagnosed as rectal cancers. 2 ≥ 50%, enormous heterogeneity was perceived between studies. The considerable discrepancy was noted in the time period during which cases were recruited. Another major disparity was in the various molecular assays employed for HPV DNA detection as well as genotyping. Most of the HPV type-specific molecular tests detect the two most common oncogenic types, HPV-16 and HPV-18. The detection of co-infection with multiple HPV types in tissue samples also varies depending on the molecular tests used. Quality of evidence: This is a comprehensive systematic review of HPV DNA prevalence among men with oropharyngeal, penile, anal and prostate cancers. The present meta-analysis incorporated high-quality peer-reviewed studies published in indexed journals. As the IHowever, there were not many dissimilarities in the type of samples tested, molecular assays, and quality of the studies. In conclusion, there exists significant association of HPV with oropharyngeal and genital cancers occurring among men. The present systematic review and meta-analysis calls for policymakers to roll-out gender neutral HPV immunisation globally and to develop HPV DNA based validated screening strategies for oropharyngeal and anogenital cancers in men. SS and VK and conceptualised the study and developed the research protocol; SS, NR, PVB, PB and G A identified articles for full-text review, extracted data from studies, and matched inclusion criteria. N R did the statistical analyses. SS drafted the study. All authors critically revised the paper, approved the final study, and VK agreed to be accountable for all aspects of the work in ensuring that questions related to the integrity of any part of the work are appropriately investigated and resolved.


Variations in human papillomavirus (HPV) E6 and E7 have been shown to be closely related to the persistence of the virus and the occurrence and development of cervical cancer. Long control region (LCR) of HPV has been shown multiple functions on regulating viral transcription. In recent years, there have been reports on E6/E7/LCR of HPV-16 and HPV-58, but there are few studies on HPV-52, especially for LCR. In this study, we focused on gene polymorphism of the HPV-52 E6/E7/LCR sequences, assessed the effects of variations on the immune recognition of viral E6 and E7 antigens, predicted the effect of LCR variations on transcription factor binding sites and provided more basic date for further study of E6/E7/LCR in Chengdu, China.LCR/E6/E7 of the HPV-52 were amplified and sequenced to do polymorphic and phylogenetic analysis. Sequences were aligned with the reference sequence by MEGA 7.0 to identify SNP. A neighbor-joining phylogenetic tree was constructed by MEGA 7.0, followed by the secondary structure prediction of the related proteins using PSIPRED 4.0. The selection pressure of E6 and E7 coding regions were estimated by Bayes empirical Bayes analysis of PAML 4.9. The HLA class-I and II binding peptides were predicted by the Immune Epitope Database server. The B cell epitopes were predicted by ABCpred server. Transcription factor binding sites in LCR were predicted by JASPAR database.50 SNP sites  were found. From the most variable to the least variable, the nucleotide variations were LCR > E7 > E6. Two deletions were found between the nucleotide sites 7387–7391 (TTATG) and 7698–7700 (CTT) in all samples. A deletion was found between the nucleotide sites 7287–7288 (TG) in 97.56% (40/41) of the samples. The combinations of all the SNP sites and deletions resulted in 12 unique sequences. As shown in the neighbor-joining phylogenetic tree, except for one belonging to sub-lineage C2, others sequences clustered into sub-lineage B2. No positive selection was observed in E6 and E7. 8 non-synonymous amino acid substitutions  were potential affecting multiple putative epitopes for both CD4+ and CD8+ T-cells and B-cells. A7168G was the most variable site (100%) and the binding sites for transcription factor VAX1 in LCR. In addition, the prediction results showed that LCR had the high probability binding sites for transcription factors SOX9, FOS, RAX, HOXA5, VAX1 and SRY.This study provides basic data for understanding the relation among E6/E7/LCR mutations, lineages and carcinogenesis. Furthermore, it provides an insight into the intrinsic geographical relatedness and biological differences of the HPV-52 variants, and contributes to further research on the HPV-52 therapeutic vaccine development.The online version contains supplementary material available at 10.1186/s12985-021-01565-5. According to statistics, about 1.4 million women worldwide suffer from cervical cancer, which is the second most common cancer among women in the world and the main cause of cancer death in some developing countries . CervicaHuman papillomavirus (HPV) is a capsid-enclosed small circular double-stranded type of DNA virus of approximately 8 kb in size . It can The genome of HPV is comprised of an early region , a late region (L1 and L2), a small non-coding region (NCR) and a long control region (LCR) , 17. AmoThere have been reports about the E6, E7 and LCR of HPV-16 and HPV-58 in recent years. However, studies of the HPV-52 are few, especially for the LCR. In addition, statistics shown that the infection rate of HPV-52 among Sichuan women has increased year by year. Therefore, it is necessary to carry out in-depth research on the nucleotide polymorphisms with the HPV-52 E6, E7 and LCR. This study identified the single nucleotide polymorphism (SNP) in the HPV-52 E6, E7 and LCR in Sichuan, southwest China, assessed the possible association of polymorphisms in the HPV-52 E6, E7 and LCR with the virus infection, propagation and replication, predicted the high affinity antigen epitope of HPV-52 E6 and E7, and analyzed the binding sites of the transcription factors in the LCR. Our results could provide basic data for further studies on the HPV-52 epidemiology, prevention, and therapeutic vaccine development.All participants were informed of the study aims, and a written informed consent was received from each patient before sample collection, and the patients' privacy have been fully protected. This study was approved by education and research committee and Ethics Committee of Sichuan University, China , and was carried out in line with the Helsinki Declaration.3432 cervical scrape cell samples were collected from outpatients who underwent routine cervical screenings at The Affiliate Reproductive Hospital of Sichuan Genitalia Hygiene Research Center, Chengdu SongZiNiao Sterility Hospital and Chengdu Medical College Affiliated Infertility Hospital from December 2018 to December 2019. The sample was stored at − 20 °C in cell preservation solution and all methods were performed in accordance with the relevant guidelines and regulations.HPV DNA was extracted and genotyped using Advanced Fragment Analysis (AFA) based on capillary electrophoresis system with the commercial Human Papillomavirus Genotyping Kit for 25 types  according to the manufacturer’s guidelines. This kit is able to classify 25 different HPV types . The existence of other types of HPV may have a certain impact on HPV-52, only samples that tested positive for single infection of HPV-52 were picked out for amplification, sequencing and following study to ensure the single of variables.The primer pairs were designed by Primer premier 5.0 according to the HPV-52 reference sequence (GenBank: X74481.1) and the primer sequences were: HPV-52 E6 F: 5′-ACCCACAACCACTTTTTTTTAT-3′, HPV-52 E6 R: 5′-CACCATCTGTATCCTCCTCATC-3′; HPV-52 E7 F: 5′-TTGTCAAACGCCATTATGTCCT-3′, HPV-52 E7 R: 5′-TTGCCTCTACTTCAAACCAGCC-3′; HPV-52 LCR F: 5′-GCCCAAACTAAAACGCCCT-3′, HPV-52 LCR R: 5′-CACCGATTCTTCCAGCACC-3′. All the primers were synthesized by TSINGKE, China. The E6, E7 and LCR fragments were amplified in 50 μl PCR reaction volumes containing 18 μl of extracted DNA, 25 μl Taq 2X PCR Master Mix with Dye , 1 μl forward primer, 1 μl reverse primer and 5 μl Nuclease-free water.The PCR amplification was performed under the following conditions: an initial 30 s denaturation step at 95 °C, followed by 30 amplification cycles, with each cycle including a 30 s denaturation step at 95 °C, a 30 s annealing step at 52–55 °C , and a 35–65 s  elongation step at 68 °C, and then 5 min final extension at 68 °C, ended up and held at 4 °C. The PCR products were examined under UV light after electrophoretic separation on a 2% agarose gel. The positive fragments were subjected to the bi-directional DNA sequencing  for the further analysis.http://bioinf.cs.ucl.ac.uk/psipred/) using the default parameters.To identify the SNP in E6, E7 and LCR, the HPV-52 E6/E7/LCR sequences were aligned to the HPV-52 reference sequence (GenBank: X74481.1) by MEGA 7.0 after sequencing. The secondary structures of the HPV-52 E6 and E7 proteins were predicted by PSIPRED 4.0 , HQ537739 (A2), HQ537740 (B1), HQ537743 (B2), HQ537744 (C1), HQ537746 (C2), HQ537748 (D) .To estimate the selection pressure acting on the HPV-52 E6 and E7 protein coding regions, the non-synonymous and synonymous nucleotide divergence were calculated by CODEML in PAML 4.9. Using the Bayes empirical Bayes (BEB) analysis, the sites with the posterior probability > 95% were identified as the positively selected sites .http://www.iedb.org/) was used to predict the major HLA-I and HLA-II binding peptides [http://www.allelefrequencies.net/hla6006a.asp) and 27 HLA-II allele [The Immune Epitope Database (IEDB) server  was used to predict B cell antigen epitopes of HPV-52 E6/E7 reference and variant sequences according to the default parameters[The ABCpred server  was used [To analyze the transcription factor binding sites in the LCR of HPV-52, the JASPAR database  were HPV positive including single infection  and coinfections . Up to six infections were found in coinfections, double infection accounted for 19.06%, triple infection accounted for 6.54%, quadruple infection accounted for 2.42%, fifth infection accounted for 1.42%, and sixth infection accounted for 0.14%  was 447 bp and that of E7 was 300 bp, which were consistent with the reference sequence. LCR was only 879 bp, with a 10 bp deletion compared with the reference sequence in 97.56% (40/41) samples. A total of 50 SNP sites and 3 deletions were identified across the E6, E7 and LCR. The combinations of all these SNP sites made for 12 unique E6/E7/LCR sequences  of the samples, respectively. G108C and A379G, leading to the amino acids substitution of E3Q and K93R, respectively. A378C and A379G together led to the amino acid substitution of K93R. G350T, G356A and A530G were synonymous mutation.For E7, 10 SNP sites were observed compared with the reference sequence and the combinations of all these SNP made for 2 unique E7 sequences. A801G and C751T were the most variable sites and were observed in 100% and 97.56% (40/41) of the samples, respectively. C662T, AG706/707GA, T727G, C733T, G742A and T848G, leading to the amino acids substitution of T37I, S52D, Y59D, H61Y, D64N and L99R, respectively. T573A, C751T and A801G were synonymous mutation. No deletion or insertion mutation sites were found in either E6 or E7 sequences. The secondary structures also had not changed in either E6 or E7 protein.For LCR, 34 SNP sites and 3 deletions were observed compared with the reference sequence and the combinations of all these mutations made for 9 unique LCR sequences. G7622A, T7624G, T9659C, G7712C and G7861A were observed in all samples. G7168C, C7207A, G7371, A7657C, A7865G and T13C were observed in 97.56% (40/41) of the samples. Two deletions were found between the nucleotide sites 7387 to 7391 (TTATG) and 7698 to 7700 (CTT) in all samples. A deletion was found between the nucleotide sites 7287 to 7288 (TG) in 97.56% (40/41) of the samples. Also, T7933C and A7938G were the common variable sites and were observed in 36.59% (15/41) and 60.98% (25/41) of the samples, respectively.The neighbor-joining phylogenetic trees were constructed by MEGA 7.0, using the 12 unique HPV-52 E6/E7/LCR variant sequences and 7 sub-lineages reference sequences. The phylogenetic tree  analysis of PAML 4.9 were indicated that: in the HPV-52 E6 and E7 protein coding sequences, no positive selection sites were observed.86–94KTLEERVKK), HLA-A*33:03 (127–135NIMGRWTGR) and HLA-A*30:01 (91–99RVRKPLSEI/91–99RVKKPLSEI) showed the best binding affinity (PR = 0.01). 134 and 114 E7 HLA I predicted epitopes were selected form the E7 reference and variant sequence, respectively , HLA-B*44:03 (47–55AEQATSNYY/47–55AEQATDNYY) and HLA-B*15:01 (45–54GQAEQATSNY) showed the best binding affinity (PR = 0.01).To evaluate the impacts of the HPV-52 E6 and E7 sequence polymorphisms on the immune recognition of antigens, the binding peptides for both HLA I and HLA II were predicted by the IEDB servers. Based on the principle of epitope selection described in the methods section, 273 and 275 E6 HLA I predicted epitopes were selected form the E6 reference and variant sequences, respectively  were considered as the major eradicators of both HPV-infected cells and cervical cancer . HPV oncMutations on LCR may influence the binding sites and the function of it. The HPV LCR which contains the binding sites for both viral and cellular factors, has shown regulatory functions on replication of HPV, transcriptional activity of the E6/E7 and the other interaction through the virus life cycle , 38. In In addition, SOX10, a transcription factor of the sex determining region Y (SRY)-related high motility group (HMG)-box gene family, playing an important role in cancer progression, including tumorigenesis, changes in the tumor microenvironment, and metastasis , 51. StuIn conclusion, this study investigated the gene polymorphisms and phylogeny of high-risk HPV-52 E6/E7/LCR, the possible influence of non-synonymous substitutions in E6/E7 on the T-cell and B-cell response and the impacts of the LCR variations on the bindings of the cellular transcription factors from Southwest China. Knowledge of genetic variation in HPV may be useful as an epidemiologic correlate of cervical cancer risk, or may even provide critical information for developing diagnostic probes. Although our study showed some limitations on sample capacity and source, it provided more basic data for the further immunotherapeutic approaches and vaccine development strategies. It also helps performing further study to demonstrate the biological function of HPV-52 E6/E7/LCR variants and the effect of multiple infection of high-risk HPV on tumor progression. The TFBS we found are still need deeper exploration for the potential of them to be marker in diagnosis and therapy.Additional file 1: Table S1. HLA-I alleles and lengths selected for prediction. Table S2. HLA-II alleles and lengths selected for prediction.Additional file 2: Table S3. Prediction results for HPV52 E6 reference sequence HLA-I epitope peptides. Table S4. Prediction results for HPV52 E6 variant sequence HLA-I epitope peptides.Additional file 3: Table S5. Prediction results for HPV-52 E7 reference sequence HLA-I epitope peptides. Table S6. Prediction results for HPV-52 E7 variant sequence HLA-I epitope peptides.Additional file 4: Table S7. Prediction results for HPV-52 E6 reference sequence HLA-II epitope peptides. Table S8. Prediction results for HPV-52 E6 variant sequence HLA-II epitope peptides.Additional file 5: Table S9. Prediction results for HPV-52 E7 reference sequence HLA-II epitope peptides. Table S10. Prediction results for HPV-52 E7 variant sequence HLA-II epitope peptides.Additional file 6: Table S11. Predicted linear B cell epitopes of HPV-52 E6. Table S12. Predicted linear B cell epitopes of HPV-52 E7.


Not much is known about the role of scientific knowledge in vaccination decision making. This study is based on previous findings that the concern about the human papillomavirus (HPV) agent mutating back to a virulent HPV was common among Swiss student teachers and turned out to be one factor of vaccine hesitancy. The study investigate the impact of a standard public health brochure describing the effectiveness, safety, and importance of HPV vaccination on young student teachers, and the additional effect of supplementing the standard brochure with biological arguments against the mutation concerns. It uses a pre-posttest design and assigns participants randomly to two groups, one reviewing a standard public health brochure, the other the same brochure enhanced with additional biological information. Participants in both groups showed a significant positive change in their beliefs about vaccination safety, effectiveness, and importance in preventing cervical cancer. Post hoc analysis showed significant safety beliefs gain for the subgroup of participants who received the biology-enhanced text and held moderate, rather than high or low, pretest safety beliefs—the so-called fencesitters. We conclude that these fencesitters may particularly profit from even minimal  interventions, an effect that should receive more attention in future research. Few studies have explored the effect of scientific knowledge on health reasoning and decision-making. As a consequence, we know little about the specific health situations in which scientific knowledge is beneficial, the desired content and depth of this knowledge, and the optimal ways to impart it.The study described in this paper is a further step in a series of studies about vaccination hesitancy that has been conducted with Swiss schools, students, and pre-service teachers ,2,3,4. WThe current study was prompted by the findings of two qualitative Swiss studies on HPV vaccination. The first of these studies  found thTo the best of our knowledge, so far, no studies have pointed out, not to mention investigated, biological concerns as reasons for vaccination hesitancy. Nevertheless, the two abovementioned studies suggest that biological information—in this case about the character of the vaccination particles as genetically engineered non-virulent markers that cannot mutate into viruses—could make a difference when it comes to the decision making of two key players for HPV vaccination in school. These two key players are the students’ teachers and mothers.As far as we are aware, such a hypothesis has never been tested. Generally, the role of scientific knowledge in health decision making is unclear . It is aGiven all these uncertainties, we opted for minimal intervention. This study explores if just integrating a paragraph about biological information on HPV vaccine particles may influence reported safety judgments in young student teachers. We gave the student teachers no introduction, no context, and no personal communication about the issue. The information was simply distributed, and the effect of this minimal intervention was measured. Actually, this is quite realistic, as it often is the only way how youths and their parents are informed about HPV and the HPV vaccination.HPV is the most frequently occurring sexually transmitted infection . While mFirst available in 2006, the HPV vaccination is a safe and effective preventative measure against cancers caused by HPV . The vacEven though the HPV vaccine can prevent cervical cancer, many parents are reluctant for their adolescents to be vaccinated. In the US, according to the Centers for Disease Control and Prevention, only 43% of adolescents are up to date on their HPV vaccination, as defined by having received both of the two recommended doses . The estAlthough we know that concerns about safety are the primary cause of refusing vaccination, very few pre-COVID-19 studies focus on understanding specific beliefs about safety and the knowledge that underlies them. Reviews of anti-vaccination websites over the span of a decade suggest that they endorse views that vaccines cause idiopathic diseases, include poisonous additives and ingredients, and erode the body’s natural immunity ,18. ThusIncreased attention to vaccine hesitancy during the COVID-19 pandemic highlighted that many misconceptions about safety have to do with vaccine biology , as well as mistrust of governments, science authority, and healthcare establishment [Gust and colleagues warn health professionals against the dichotomous categorization of parents into ‘vaccine supporters’ and ‘vaccine deniers’ . They caIn the aforementioned qualitative study, Zeyer and Sidler  intervieGust and colleagues  and ZeyeOne place where individuals encounter information about complex biological mechanisms pertaining to cells and viruses is the school science classroom. However, science education systems in most countries do not include in-depth coverage of microbiology and immunology that would form the conceptual basis for understanding vaccination ,23. StudAnother common misconception found among adolescents  and younDecisions about how to decrease misconceptions about HPV vaccination and increase vaccination rates must take into account not only what information to provide, but also in what contexts and situations to provide information. Should this be carried out in the science classroom, the health classroom, or through out-of-school public health channels? At the moment, the wall between health communication and science education is difficult to scale on both sides . On the At present, information about HPV and HPV vaccination is most likely to be communicated to young people and their parents outside of school through public health brochures and websites. Little research has been conducted on the impact of HPV brochures on safety beliefs and vaccination attitudes. The present study aims to answer two questions. First, it quantitatively examines whether a standard public health brochure affects student teachers’ perception of the effectiveness, safety, and importance of HPV vaccination. Second, it investigates whether supplementing a traditional HPV information brochure with text about relevant biological concepts is more effective than the brochure alone in communicating the safety of the vaccine. In particular, the study investigates the impact on beliefs of a biological explanation about the mechanism by which HPV vaccination acts on the immune system. It focuses on beliefs about vaccination importance, safety, effectiveness, and, specifically, the possibility of the vaccine causing HPV infection. While the study is exploratory, we hypothesize that the biology-enhanced text will have a greater positive effect on safety beliefs than the traditional brochure. The study aims to generate questions that will set a research agenda for further examining the relationship between health and science education and between formal education and public health communication.Participants in this study were enrolled students from the Lucerne University of Teacher Education in Switzerland. They were all enrolled in a course titled ‘Health and Illness in School’ in the autumn of 2016 taught by the second author. The aim of the course was to prepare students, future Swiss school teachers, for promoting health and addressing health concerns among Swiss school children. The course enrollment included 350 students. Three hundred and eleven students chose to participate in the study. Of these, 232 completed pretest and posttest questionnaires and were included in the analysis (130 in the standard brochure and 102 in the extended brochure condition). Twenty-nine percent of the participants were male and seventy percent were female. Ninety-five percent were between twenty and twenty-eight years old. The majority (70%) were training to be primary school teachers.The study focused on future teachers both because most of them were within the age range recommended for HPV vaccination and because they would soon be in a position to influence the health decisions of others. When it comes to addressing HPV vaccination in the classroom, this may be a concern because student teachers’ understanding of antibodies and immunity is not free from misconceptions . https://www.cdc.gov/hpv/parents/questions-answers.html (accessed on 12 January 2022). Based on the literature review and our prior findings of concerns related to misconceptions around vaccination-related risks, the primary focus of our questionnaire was on beliefs about vaccination safety. To establish content validity of the questionnaire, two health professionals—a physician and a nurse—were asked to map each of the ten statements in the questionnaire to one of three relevant concepts of the HBM  or to mark the statement as the overall summative statement. The health professionals mapped the statements with 100% agreement. In addition, when asked to add possible additional safety concerns that someone may have about HPV vaccination, both judged the coverage of statements 5 to 9 as comprehensive. The pretest questionnaire also included demographic questions about the participants’ age, gender, and educational focus.The study protocol was exempt from IRB review by the Office of Human Subjects Research of the US National Institutes of Health. Participants provided informed consent in writing. A pretest–posttest design was used in this study. At the outset, participants completed a questionnaire where they were asked to rate ten statements see  on a 5-pBox 11. Without HPV vaccination, a woman may get cervical cancer.2. HPV vaccination is important.3. HPV vaccination is safe.4. HPV vaccination is effective against cervical cancer.5. Serious side effects can occur with HPV vaccination.6. In some cases, HPV vaccine may cause HPV infection.7. In some cases, HPV vaccination may cause cervical cancer.8. In some cases, HPV vaccination may cause fertility problems.9. In some cases, HPV vaccination may weaken the recipient’s immune system.10. The benefits of HPV vaccination outweigh the risks of HPV vaccines.https://www.cdc.gov/std/hpv/stdfact-hpv-vaccine-young-women.htm (accessed on 12 January 2022). The CDC brochure was selected as an authoritative document that could provide a basis for further cross-cultural comparisons. The brochure was shortened and edited to focus on the information pertaining to the questions in What is HPV?; What is HPV vaccination?; How effective are the HPV Vaccines?; Are HPV vaccines safe? The information in these sections was presented in a clear factual manner, providing references to authoritative sources and statistics, but biological mechanisms were not described .After the pretest, participants were given a brochure about HPV vaccination to read. They read either the standard or extended version of the brochure, which were both developed using a US CDC brochure ?Viruses, like HPV, are single-cell sub-microscopic organisms that can infect humans. As organisms, viruses are simpler than most living cells. They lack many of the parts (organelles) that cells usually have. Viruses can be thought of as containers with DNA that contains instructions for building new viruses. Like all living things, viruses can reproduce, making more of themselves. But viruses are so simple that they so can only reproduce by entering a host cell. This is where our trouble begins. Viruses infect (enter) host cells, take over their reproductive machinery to make more viruses, and damage them in the process. That’s how viral infections make us ill.2.2 How does HPV vaccination protect against HPV?Vaccination makes a person resistant to an infection by teaching the immune system to recognize it and trigger a response. The immune system is the body’s system for destroying intruders, such as viruses. This system works partly by producing proteins called “antibodies” that connect to the intruders’ proteins called “antigens.” These antigen-antibody complexes signal other mechanisms of the immune system to destroy the intruder. Different viruses have different antigens that require different specialized antibodies. Because of this, the immune system is often more effective in responding to previously encountered antigens. Vaccines contain antigens, giving the immune system the opportunity to learn to recognize viruses and other infectious agents. Vaccines can introduce antigens as part of weakened viruses (never fully strong viruses), dead viruses, or through genetically engineering of harmless viral antigens. The HPV vaccine contains only antigen molecules of HPV. These molecules can stimulate the immune system to produce HPV antibodies, but cannot enter host cells, and thus can’t use their reproductive machinery and damage them. Therefore, HPV vaccination can never cause HPV infection. It can, however, prevent HPV infections which cause cervical cancer.After completing the pretest and reading their texts, students handed these materials to the proctor. The posttest, taken at the end of the class, approximately one hour after reading the brochures, was identical to the pretest, with the exception of the demographic questions.All study materials were originally created in English and later translated into German by the second author, who is fluent in both languages. An independent bilingual speaker verified the translation’s accuracy by back-translating materials into English and comparing translations with the original.Susceptibility to HPV Infection (Susceptibility). This single-item variable, ranging in value from 1–5, reflected the rating of the statement ‘Without HPV vaccination, a woman may get cervical cancer’.Importance of HPV Vaccination (Importance). This two-item variable, ranging in value from 2–10, reflected the sum of ratings of the following two statements: ‘HPV vaccination is important’, and ‘The benefits of HPV vaccination outweigh the risks of HPV vaccines’. As measured by Crohnbach’s alpha, the internal consistency of the importance variable was α = 0.636 in the pretest and α = 0.786 in the posttest.Safety of HPV Vaccination (Safety). This 6-item variable, ranging in value from 6–30 reflected the sum of the ratings of the ‘HPV vaccination is safe’ statement and inverse ratings of the following five statements: ‘Serious safe effects can occur with HPV vaccination’, ‘In some cases, HPV vaccine may cause HPV infection’, ‘In some cases, HPV vaccination may cause cervical cancer’, ‘In some cases, HPV vaccination may cause fertility problems’, and ‘In some cases, HPV vaccination may weaken the recipient’s immune system’. As measured by Cronbach’s alpha, the internal consistency of the safety variable was α = 0.717 in the pretest and α = 0.864 in the posttest.Effectiveness of HPV Vaccination (Effectiveness). This single-item variable, ranging in value from 1–5, reflected the rating of the statement ‘HPV vaccination is effective against cervical cancer’.The analysis was conducted for the following dependent variables, which were developed to reflect theoretical HBM components on the basis of students’ responses:The data were analyzed through repeated-measures MANOVA and repeated-measures ANOVA, with time elapsed between the pretest and posttest as the within-subject factor and type of text as the between-subject factor.Statistical tests are two-tailed, unless otherwise specified.p < 0.001), but no significant effect of time by text type interaction. Similarly, univariate analysis of each factor produced a significant time effect and no significant interaction effect  = 2783.26, fect see .In addition, repeated-measures ANOVA was performed on participants’ rating of the ‘In some cases, HPV vaccination may cause HPV infection’ statement, as this statement was the most directly targeted by the biological text of the extended version. Similar to the overall safety factor effects, there was a significant effect of time, but not of time by text interaction .After obtaining the whole-sample results, we further hypothesized that participants with different initial beliefs about HPV vaccination safety might be affected differently by the biology in the extended text. Specifically, we hypothesized that participants who viewed vaccination as very safe at the outset may be close to the ceiling level and not demonstrate much of a change in their safety perceptions scores, while participants with a very high level of initial skepticism may have their beliefs grounded in a complexity of factors not easily affected by a single biological passage. Participants with moderate or uncertain initial views, on the other hand, may be most likely to change their views in response to a biological explanation.To test this hypothesis, we divided the original sample into three groups based on participants’ pretest perceived safety scores. A participant answering ‘neither agree, nor disagree’ to all six safety questions would have attained a score of 18. That score (18), achieved by 57 of the 268 participants who provided complete data for the safety factor questions, also represented a very distinctive mode in our data set. The second most frequent score (of 16) was obtained by 23 participants. We, thus, defined the ‘initial moderate safety belief’ group as those whose pretest scores fell within +/−2 points of 18, ranging from 16 to 20. Participants with pretest safety scores of 15 or below were defined as the ‘initial low safety belief’ group, and participants with pretest safety scores of 21 or above were defined as the ‘initial high safety belief’ group. Notice that these are average scores. For example, participants with a score of 18 could agree or disagree with some of the six safety questions, as long as they had an average score of 3, i.e., a total score of 18, for all six questions.p = 0.025) for the Initial Moderate Safety Belief Group and not significant for the other two groups.The perceived safety of HPV vaccination was analyzed for each group using repeated-measures ANOVA with time as a within-subject factor and type of text as a between-subject factor. The results are presented in In order to calculate the size of this effect for the Initial Moderate Safety Belief Group, Cohen’s d for pretest–posttest-controlled studies  was applIn this study, we chose to estimate the size of the intervention effect by calculating Cohen’s d  for pretWhile Lenhard and Lenhard standardize both pretest and posttest standard deviations, another common approach standardizes pretest standard deviations only . This tyNevertheless, we prefer using Lenhard and Lenhard’s method, because scales are used in two different ways in our study. On the one hand, they provide a continuous measure in comparing pretest and posttest scores. On the other, they are used as an indicator of an underlying categorical variable. By selecting a subsample as described, the variance will necessarily be reduced for the pretest scores for the selected group and the calculation of effect size will be inflated. Overall, Cohen’s d calculated according to Lenhard and Lenhard will surely be on the safe side, and it can be expected that the substantial value of this variable will be rather larger than the value of 0.49 that we are using in our argumentation.This study examined the impact of a standard public health brochure on young adults’ perception of the effectiveness, safety, and importance of HPV vaccination. Furthermore, it also investigated the added effect of supplementing the standard brochure with a biological explanation of vaccine-induced immunity.Participants in both conditions showed a significant positive change in their beliefs about vaccination safety, effectiveness, and importance in preventing cervical cancer. These results agree with Zeyer and Knierim’s  quantitafencesitters, i.e., participants whose pretest safety beliefs were moderate, rather than high or low. Although the overall impact is limited, a single read of a standard brochure may positively impact some individuals’ beliefs about HPV vaccination. Furthermore, the biological explanation had an additional positive impact on the subgroup of those concerned about vaccine safety whose initial negative attitudes were not too strong. Further research should explore the potential of in-depth science-based explanations, as well as the best strategies and contexts for delivering them.Contrary to our hypothesis, adding biology-enhanced text to the standard brochure did not have an overall positive effect on participants’ beliefs about HPV vaccination safety. However, post hoc analysis showed a significant gain in safety beliefs for the biology-enhanced text in the subgroup comprised of fencesitters, i.e., those whose initial beliefs about HPV vaccination safety were neutral. This finding is strengthened through the study’s use of a restricted data range and by a considerably high effect size of the intervention. While beyond the scope of the present study, further research may illuminate how individuals with different initial perceptions of vaccination and different levels of certainty respond to biological explanations. It also suggests that focusing educational and explanatory efforts on fencesitters may have positive practical implications and should be explored in other contexts, such as promoting COVID-19 vaccination.When it comes to addressing the role of conceptual science knowledge, the study reveals several possibilities that merit further investigation. It presents an interesting finding, though highly exploratory in nature, about the impact of a biological explanation on participants’ beliefs about the safety of HPV. While there was no overall effect of enhancing the brochure with biological explanations, such an in-depth explanation obviously may have played a role for With regard to seeking out a larger and more definitive impact of biological knowledge, it appears that a couple of printed paragraphs of biological explanation presented in isolation from the rest of relevant biology did not affect participants’ beliefs over and above the factual text. More research, with a qualitative focus, is needed to determine why this was the case. One limitation of the current design was its inability to assess text comprehension. It is possible that the readers understood the text but found it unconvincing or that it did not address all of their concerns about vaccination safety. Vaccine hesitancy is a complex phenomenon that includes many cognitive and non-cognitive components. Most effective public health programs for addressing vaccine hesitancy intervene on multiple levels , involve multiple components and materials, and unfold over a period of time . It is aWe often vainly search for spectacular interactions between science education and health behavior, while missing the more delicate, but no less important, benefits on a deeper level. This may be the significance of this study, even if, at a first glance, one might be disappointed with the findings. In fact, important biomedical information on HPV vaccination does not result in dramatic changes in beliefs among students. One might even be tempted to discard the intervention tested in this study.However, this might be an important conceptual mistake. Rather, vaccination hesitancy is a dynamic and challenging period of indecision around whether or not to accept vaccination . The speYet, and this seems to be suggested by our results, for the ‘moderates,’ who lie in-between the accepters and rejecters, information about the biomedical safety of HPV vaccination may be just the right nudge towards a health literate decision. It may be in this unspectacular way that scientific literacy supports health decisions in everyday life and that the science classroom can become an important setting for health education. Today, individuals are expected to be active participants in their healthcare, collaborating with health professionals in choosing the course of action in care, treatment, and prevention. Participatory medicine encourages patients to ask questions and to engage in shared decision-making . Our stuThis study looked at public health texts, which were translated from English to German. They were enhanced with additional information that had been identified as useful in a local Swiss study. Targeted health communication is and will remain an important mission of public health. Long, interactive discussions with patients and consumers are not feasible in every setting where health information delivery takes place. Consequently, health information brochures and handouts are and will always be important tools in delivering health information. At the same time, this study makes a case for fostering alliances between public health and school science education and for bringing health topics into science education. While the impact of conceptual knowledge on daily life needs to be further explored, it is certainly reasonable to assume that it is more likely to be impactful when delivered in a connected, comprehensive, expertly facilitated characteristic of the science classroom ,30,43. ALastly, in the COVID-19 era, when vaccination is hotly debated and often doubted by the public of many countries, it is important to remember that when it comes to scientific literacy, conceptual knowledge is only one component. Individuals’ scientific knowledge and their attitudes towards science and scientists play a very important role in building trust and acceptance of vaccines, especially in a context in which health becomes politicized . Therefo


Head and neck cancer (HNC) is the sixth most common cancer worldwide, with a general prognosis of 50% disease-specific survival (DSS). The subgroup of oropharyngeal (OP) cancers are of interest because HPV infection is one of several causative agents and carries favorable prognosis. Influxes of inflammatory cells into tumors may vary with prognosis. T lymphocytes are important regarding specific immune defense. Within the immune system T regulatory cells (Foxp3 positive) co-governs this process. We have therefore primarily studied levels of Foxp3 (+) cells in malignant tumors from 170 patients related to prognosis of the patients. Higher levels of T lymphocyte Foxp3 (+) cells predicted better 5-year DSS. This case was unique relative to age, gender, TNM stage, and HPV infection; but more so among tumor HPV (+) than HPV (−) patients. The results encourage further study into the use of immune-based therapy in HNC patients.Head and neck cancer (HNC) is the sixth most common cancer worldwide. Oropharyngeal (OP) cancers are of special interest because of possible underlying HPV infection which is tied to prognosis. Influxes of inflammatory cells into tumors may vary with prognoses. We wanted to study whether the number of tumor-infiltrating lymphocytes (TIL) and tumor-associated macrophages (TAM) in tumors correlated to HPV status and predicted 5-year disease-specific survival (DSS). Formalin-fixed paraffin-embedded (FFPE) biopsies cut sections from 170 patients treated for OP cancer were stained by immunohistochemistry and evaluated for the number of CD68 (+) TAMs, CD3 (+), and Foxp3 (+) (T regulatory) TILs. From FFPE slides HPV by PCR and p16 by immunohistochemistry were established. From FFPE Hematoxylin-Eosin slides, levels of tumor nuclear polymorphism, tumor invasion, desmoplasia, and inflammation were determined as previously published. Levels of TIL CD3 (+) and TIL Foxp3 (+) were increased among the HPV (+) compared to the HPV (−) patients. High levels of TIL Foxp3 (+) and CD68 (+) macrophages predicted better 5-year DSS. TIL Foxp3 (+) levels predicted independent of age, gender, TNM stage, and HPV infection as well as level of stromal desmoplasia, tumor invasion, and nuclear polymorphism, but more pronounced among tumor HPV (+) than HPV (−) patients. Head and neck cancer (HNC) is the sixth most common cancer group worldwide , and hasBoth epidemiological and experimental evidence suggest links between inflammation, specific immunity, and cancer, regarding both defense against , and estLevels of inflammatory cells within tumors predict prognosis in various malignant tumors .The numbCD3 is a general marker for T lymphocytes . The T cThe mononuclear phagocyte (MNP) system includes monocytes, macrophages, and some dendritic cells . MNP in When measured from basic HE slides, the degree of stromal desmoplasia, the level of nuclear polymorphism, and invasion and lymphocyte infiltration relate to prognosis in OPSCC patients . It shouThe extent of disease, commonly described by the TNM stage of the disease , yields We have aimed to study the five-year disease-specific survival (DSS) of newly diagnosed OPSCC patients dependent on the activation level of TILs measured by level of CD3 and Foxp3-positive TILs, as well as the level of tumor-associated macrophages (TAM) measured by CD68-positive cells. Furthermore, we have studied the TIL Foxp3 infiltration level five-year DSS adjusted by the degree of stromal desmoplasia, tumor lymphocyte infiltration, nuclear polymorphism, and degree of tumor invasion.Since 1 January 1992 all patients diagnosed with head and neck cancer (HNC) at the Department of Otolaryngology/Head & Neck Surgery, Haukeland University Hospital (HUH), Bergen, Norway have been registered in a hospital-based HNC registry. HUH treats all cases of HNC in the Western Health Care Region of Norway. This region includes approximately one million inhabitants. All HN squamous cell carcinoma (HNSCC) patients were subjected to standardized diagnostic work-up, which consists of clinical examination, CT/MRI scans of the primary tumor site, neck, thorax, and liver, and ultra-sonographic examination of the neck including, if indicated, fine needle aspiration cytology. Diagnostic endoscopic examination under general anesthesia is performed if possible. From this population, patients with OPSCC diagnosed in the period from 1992 to 1 July 2008 were identified. The treatment of the patient cohort from which the included OPSCC patients were drawn, have been published in detail . In shorTumor tissue samples from oropharyngeal squamous cell carcinomas were fixed in buffered formalin and paraffin embedded. Sections were cut at 4 μm. The following antibodies were used in this study: Anti-CD68 , anti-CD3 , and anti-Foxp3 . Antigen retrieval and staining were performed fully automated in a Ventana Benchmark Ultra according to the manufacturer’s instructions.Part of the material was evaluated concomitantly in a two-headed microscope by the primary investigator (BL) and an experienced histopathologist (LH), the remaining by the primary investigator alone. Scoring was carried out by counting the number of positive cells per HPF at 630× magnification in a Zeiss Axio microscope, calculating the mean number of positive cells in five neighboring fields, randomly chosen. If the counts in two fields exceeded 200 cells, calculation of the mean number was based on those fields. The tumor stroma and tumor epithelium were evaluated separately and mean values calculated in each tissue compartment .All sections were evaluated blindly, without knowledge of HPV status or histo-morphologic parameters as described below. An interim analysis was performed showing that all survival prognosis was tied to number of counted cells found in the tumor epithelium. Analyses from tumor neighboring fields were therefore discontinued, and the presented results are from tumor epithelial counting only.The histology grading has been scored as reported in detail previously . The scoAll tumor samples were reviewed by an expert in pathology (OKV), and representative tissue samples were selected. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) specimens.We have previously published this method in detail . All othp < 0.05 were considered statistically significant. All p values represent two-sided tests. Prognostic variables with disease-specific survival (DSS) were determined using a Kaplan–Meier estimator (log rank option) and/or Cox proportional hazards regression models. The five-year survivals are given as relative risk (RR) and confidence intervals (CI).The statistical program package PASW was employed  employing analyses as indicated. Results yielding n = 170) represents a subgroup of the entire group of patients diagnosed in Western Norway with OPSCC (n = 280) in the period from 1992 until 1 July 2008. The present cohort (p < 0.001) and regarding Foxp3 17.7 ± 2.22 versus 9.7 ± 1.32 (p < 0.001). Regarding CD68-positive cells a trend was observed towards more cells among HPV (+) than HPV (−) tumors.Magnitude of CD3-, CD68-, and Foxp3-positive cells divided by tumor HPV status are shown in Regarding the level of lymphocyte infiltration, the degree of desmoplasia, and nuclear polymorphism and invasion, significant positive correlations were shown to CD3 and CD68 levels. Regarding Foxp3 levels, the associations were more definitely shown among the HPV (−) patients than among the HPV (+) patients .p < 0.001) (p < 0.001) (p = 0.005), but not among HPV (−) patients. The smoking history predicted five-year DSS among the HPV (−) patients. TNM stage predicted five-year DSS among the HPV (−) patients only. Five-year DSS was predicted by tumor site (tonsils/base of tongue versus elsewhere OP) among HPV (+) patients only  and 92 had tumors that were HPV (+). In addition, two patients had unknown HPV status. HPV infection status strongly predicted better five-year disease-specific survival (DSS) (< 0.001)  left. p1< 0.001)  right. Aonly All .p < 0.05) (p < 0.001) (p = 0.025) (We found that patients with a high tumor number of CD3-positive TILs had better five-year DSS than those with low such numbers ( < 0.05) . For Fox< 0.001) . Concern= 0.025) .p = 0.004) were still determined (p = 0.012). If the patients were stratified by whether they had HPV-infected tumors, five-year DSS prediction regarding Foxp3-positive TILs (termined . Foxp3 Tp = 0.007). If the patients were stratified by tumor median CD3 levels, the five-year DSS survival prediction by Foxp3 TIL levels were still valid (p = 0.007) (Results not shown).If the patients were stratified by CD68 density, the Foxp3 survival prediction was maintained  . The levSubsequently, we studied the same Cox regression as above cited, but with the addition of the various histological parameters. The Foxp3 survival prediction remained . The samA Cox stepwise regression five-year DSS analysis was performed including age at diagnosis, T stage of tumor, Foxp3-positive TIL level, and whether HPV and/or p16 positive . The resAround the year 2000, the treatment strategy of OPSCC patients changed from mainly RT to surgery followed by RT. A Cox multivariate analysis was performed including HPV status, T stage, TIL Foxp3 positivity level, age of the patient, and whether treated in the first or last inclusion period of the study . The resLevels of TIL CD3 (+)s and TIL Foxp3 (+)s were increased among the HPV (+) compared to the HPV (−) patients. High levels of TIL Foxp3, CD3 positive, and TAM  predicted better five-year DSS. This was the case with TIL Foxp3 levels unique relative to age, gender, TNM stage, HPV infection as well as rate of stromal desmoplasia, nuclear polymorphism, and tumor invasion.Level of lymphocyte infiltration as measured by HE histology has been recognized as a positive marker for survival in OPSCC patients . In bothFoxp3 is a transcription factor belonging to the family of forkhead box (FOX) proteins. Foxp3 is associated with T lymphocyte development; in particular T regulatory lymphocytes (Tregs) . It has Tregs expressing Foxp3 are hypothesized to play an important part in immunological homeostasis and peripheral self-tolerance . Tregs aFoxp3 TIL expression levels may also be associated with prognosis in cancer patients. High levels of Foxp3-positive lymphocytes have been linked to worse prognosis, e.g., colorectal cancer, melanomas, and lung carcinomas . For othIn HNC, two different causes of cancer are determined with HPV or smoking/alcohol-related OPSCC  etiologyThe underlying causes as to why HPV-generated cancer exists remain unknown. One obvious suggestion is that the immune system in tonsillar and base of the tongue (BOT) tissue merit a privileged status for the epithelial cells . This isThe mechanisms underlying the carcinogenesis of the HPV virus are not fully known . p16 indWe have previously shown that a high degree of lymphocyte infiltration in OPSCC indicate better prognosis . This isOne could hypothesize that the surgical removal of most of the tumor mass may tip the balance in the host towards the immune system than compared to relying only on RT-based tumor removal. We have therefore studied DSS in regard to whether the patients had been treated with surgical removal of the main tumor mass or not. We could not find any difference, and thus found no support in favor of such a hypothesis. In this study, the counting of the immunohistochemically stained slides were mainly carried out by one blinded person. More information could be gathered with morphometry, and this remains a future goal. The study reports from a cohort with limited number of patients. With more patients, survival results regarding HPV (+) patients in particular, could have been strengthened. The patients were from one hospital, and a multi-center study would be preferable. The patients were, however, drawn from most of the eligible patients from one geographical region and therefore are not strongly biased. Each patient has been closely followed up and therefore the results about the cause of death are accurate. The staining from all slides has been performed as a single procedure diminishing drift in the method over time. This study is one of several that encourages further studies about immune treatment of OPSCC. Immunotherapy is a therapy with potentially profound efficiency, but not all patients experience this, and this underlines the importance of choosing the correct patients . StudyinIn conclusion, we have shown that high levels of CD3, CD68, and Foxp3 cells within the tumors of OPSCC patients better predict prognosis. The TIL Foxp3 prediction is best among HPV (+) tumors. The TIL Foxp3 survival prediction was to some extent independent of TNM stages, age, and gender as well as levels of tumor nuclear polymorphism, tumor invasion, desmoplasia, general lymphocyte infiltration, and TAM levels. The present results support the notion to carry out more studies about the Foxp3 system in OPSCC patients.


It is believed that HPV infection can result in the death of placental trophoblasts and cause miscarriages or preterm birth. In clinical cases of placental villi positive for HPV DNA reported by other authors, contamination is suspected in the act of crossing the cervical canal. We analyzed placental samples of women who resorted to elective abortion obtained by hysterosuction of ovular material, bypassing any contact with the cervical canal and vagina.We studied the chorionic villi of the placenta of 64 women who resorted to voluntary termination of pregnancy, in the first trimester. To avoid contamination of the villi by the cervical canal, we analyzed placental samples obtained by hysterosuction of ovular material, bypassing any contact with the cervical canal and vagina. All samples of chorionic villi were manually selected from the aborted material and subjected to research for HPV DNA.HPV DNA was detected in 10 out of 60 women (16.6%). The HPV DNA identified in the placenta belonged to genotypes 6, 16, 35, 53, and 90.The study shows that papillomavirus DNA can infect the placenta and that placenta HPV infection can occur as early as the first trimester of pregnancy.The online version contains supplementary material available at 10.1186/s12884-021-03937-9. Human papillomavirus infection (HPV) is a sexually transmitted disease associated with cervical carcinogenesis whose prevalence of genotypes shows significant differences worldwide , 2. In iRecently, it was discovered that the HPV virus is capable of infecting trophoblast cells as well as squamous cells . This diEvidence of HPV transmission through the placenta is not without controversy; in fact, HPV DNA was detected with a wide prevalence range of 4 to 75% in the placenta of women who miscarried and in 20–24% of women who underwent voluntary termination of pregnancy –12. EppeWe investigated the presence of HPV in the placenta of women in the first trimester of pregnancy, who asked for termination of pregnancy (TOP), in order to verify the hypothesis that the placenta may be infected with HPV and that this manifests as early as the first trimester of pregnancy.From May 2019 to June 2020 we studied the chorionic villi of the placenta of 64 women who asked for TOP. The gestational age of women was between the 7th and 12th week. The decision on whether or not to participate in the study was at the patient’s discretion and the patient was not disadvantaged in any other way for refusing to participate in the study. Subjects were excluded if they had evidence of immunosuppression  or had been vaccinated against HPV. The women were invited to participate regardless of whether they had a history of HPV genital infection. The viral status  of the mother was not reported deliberately, first, because the aim of the research is to demonstrate the presence of HPV DNA in the placenta, second, because some data are incomplete and the risk is to reduce the number of women of the study group, already small. The prospective observational study was approved by the local institutional review committee (n. 154/2019/PO) and written informed consent was obtained from all study participants. Participants responded to a questionnaire with demographic and clinical data in order to characterize the study group .Since other studies on vertical HPV transmission were often hampered by the possible contamination of the placenta with vaginal cells from an infected birth canal, other authors used the transabdominal pathway, whereas we analyzed placental samples obtained by hysterosuction of ovular material, bypassing any contact with the cervical canal and vagina.Women were administred prostaglandins (geneprost 1 mg) by vaginal pessaries to dilate the cervix to such an extent that a cannula (Karman’s cannula) could be introduced in the uterus, which was connected to an aspirator to aspirate ovular material. In order to avoid contamination of the ovular material with HPV coming from the maternal vagina, we resorted to a particular method. We inserted a 10 mm Karman cannula into the already dilated cervix. This cannula acts as a shirt, through which a 6 mm Karman cannula reaches the uterine cavity and aspirates the ovular material, without contaminating it. Furthermore, the cervical canal lined with cylindrical cells, is hardly contaminated by papillomavirus as it has a particular tropism for the squamous epithelial cells that line the vagina and the uterine portio; this is even more so in pregnant women whose cervix is characterized by a physiological eversion of the endocervical mucosa on the uterine portio. Moreover, the cervix represents not only an anatomical barrier but also an immunological barrier against ascending infection through mucus production, inflammatory cytokines and antimicrobial peptides. All samples of chorionic villi were manually selected from the aborted material and subjected to research for HPV DNA.We have chosen to work on fresh, non-paraffinized placental tissue: in order to make the sample as homogeneous as possible. The placental tissue was processed in a sterile hood and, using a petri dish as a support, reduced to very small pieces using a scalpel from which 3 small sample sections from 3 different points were taken and placed into an Eppendorf type test tube. Following this, the treatment continued with cellular lysis by adding the lysis buffer (ATL buffer) and proteinase K with overnight incubation at 55 °C.The day after the extraction continued using the commercial HIGH Pure PCR Template (ROCHE) kit which is based on the ability of DNA to bind to inert media or filters contained in columns; subsequently the nucleic acids bound to the filter were eluted through elution buffer and the eluate was stored at − 80 °C until the moment of use.For the amplification of HPV DNA the Ampliquality kit HPV-TYPE EXPRESS v 3.0  was used. The kit is based on a rapid system for the identification of human Papillomavirus by single step PCR: in the preparation of the PCR session, in addition to the samples, a positive amplification and typing control (HPV 61 DNA) as well as a negative control (sterile distilled water) were also used; in addition, the kit can evaluate the suitability of the DNA extracted by the amplification of the TST gene (thiosulfate sulfurtransferase rhodanese).The PCR mix was divided into a sufficient number of test tubes for the analysis of samples and controls, as follows:HPV-TYPE EXPRESS MIX 20ul.Extracted DNA 5ul.The test tubes were then transferred to a thermocycler with the previously programmed amplification profile.a-Denaturing: the marked amplified product is denatured by chemical or thermal denaturation (incubation at 95 °C);b-Hybridization: the strip is incubated, under specific temperature conditions, agitation and phj with the solution containing the marked and denatured amplified product;c-Washes: to remove the excess marked amplified product and then to define the “astringency” of the hybridization;d-Detection: Displays the hybrid that was formed based on the type of marked probe (NBT/BCIP coloring solution).The subsequent genotyping of the amplified product was carried out by an allele-specific hybridization technique; it is a technique that includes a solid phase support in which specific probes (Reverse Line Blot) have been used and includes:The kit used for genotyping was Ampliquality HPV-TYPE EXPRESS, able to determine the presence of 40 types of Papillomavirus, in particular: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, 66, 67, 6, 11, 40, 42, 43, 61, 69, 70, 44, 54, 55, 62, 64, 71, 72, 81, 83, 84, 87, 89, 90.The statistical analysis of the data was carried out with the software package SPSS 15.0 . Descriptive statistics were expressed by frequency, arithmetic mean and percentages.n = 2/10) had two different types of HPV DNA. HPV 16 was the most frequent genotype in this study. Genotype 6 was the second most frequent . The HPV DNA identified in the placenta belonged to genotypes 6, 16, 35, 53, and 90. The villi of five placentas (50%) had high carcinogenic HPV  and two  in the cervical epithelium can detect the presence of microorganisms and elicit an innate immune response characterized by the production of cytokines and AMP .Another possibility could be intrauterine transmission at the time of fertilization by latent HPV-carrying sperm; growing evidence indicates that HPV is found in the sperm of infertile males , 19.The other way is hematogenic as HPV has been detected in the mononucleate cells of peripheral blood, suggesting a hematogenic diffusion path , 21; howThe role of papillomavirus in the genesis of cervical cancer is now certain and one important factor is genotyping  since geThe human papillomavirus (HPV), was detected in the cervix in 15–25% of pregnant women , 28 and There is clearly a need for further research regarding the relationship between HPV infection and the abnormal course of early pregnancy leading to miscarriage or fetal defects.While on the one hand our research has a limit represented by the small size of the sample studied, on the other it has a strong point represented by the particular method used to avoid contamination of the placenta with HPV coming from the maternal vagina.Our study shows that papillomavirus DNA can infect the placenta and that placenta HPV infection can occur as early as the first trimester of pregnancy. The overall clinical implication of these results remains to be clarified.Additional file 1.


C.trachomatis are the most prevalent, viral and bacterial STI worldwide. Both commonly have an asymptomatic development and can evolve into a persistent infection which, added to coinfections, may be important cofactors for the oncogenic transformation.HPV and C.trachomatis infection in women with normal and abnormal cervical cytology.Evaluate the prevalence of oral and genital HPV and C.trachomatis infections were detected using PCR with specific primers.The cross-sectional study included 200 swabs, 100 oral and 100 cervical from 50 women with normal and 50 with abnormal cervical cytology. HPV and n = 27/100) of women with normal and abnormal cytology. Out of 100 genital samples we detected HPV DNA in 18% (n = 18/100) and 14% (n = 14/100) out of 100 oral samples. HPV genotypes detected were genotype 6 of low-risk and 16, 31, 52, 58 and 16–31 coinfection of high-risk. C.trachomatis DNA was detected in 49% (n = 49/100) of patients. Out of 100 genital samples we detected C.trachomatis in 35% (n = 35/100) and 31% (n = 31) out of 100 oral samples. There is statistically significant (p < 0.05) between cytology and HPV and C.trachomatis infection but there is no statistically significant between cytology and the other characteristics.HPV DNA was detected in 27% (C.trachomatis detection and specific treatment in asymptomatic women because this infection may increase the risk of HPV persistence and coinfection induces a pro-inflammatory environment that may promote the carcinogenesis.Since the histology of oral mucosa resembles that of the uterine cervix, we can anticipate the presence of HPV and other STI which are detected in different lesions of genital areas and the oral mucosa. Therefore, is important  Chlamydia trachomatis (C.trachomatis) are the most frequent, viral and bacterial respectively, sexually transmitted infections (STIs) worldwide and HPV is the necessary but no sufficient cause for cervical cancer [C.trachomatis could act as a cofactor that may lead to epithelial disruption and facilitate HPV entry which facilitates HPV infection and contributes to the viral persistence, increasing the risk of developing cervical neoplasia [C.trachomatis infection [C.trachomatis can cause different diseases such as cervicitis, endometritis, pelvic inflammatory disease and ectopic pregnancy [Human Papillomavirus (HPV) and l cancer . Coinfeceoplasia , 3. Whilnfection . C.trachregnancy .C.trachomatis may be a cofactor for HPV associated oropharyngeal cancer [C.trachomatis in the oral cavity varies widely among published studies. This variability can be explained by the varied biological samples, the lack of global standardization techniques and the diversity of population study groups [Despite the well-established role of HPV in cervical cancer, evidence suggest that HPV may also be an independent risk factor for oral cancer  and it il cancer , 7. A pol cancer . The frey groups , 10.Many risk factors such as age, tobacco, alcohol, hormonal contraceptive use and parity are associated with development cervical precancer and cancer, while cervical microenvironment, may also influence the natural history of HPV infection , 11, 12.C.trachomatis infection in women with normal and abnormal cervical cytology.The present cross-sectional study aimed to evaluate the prevalence of oral and genital HPV and The cross-sectional study included oral and cervical swabs from 50 women with normal cervical cytology and 50 women with abnormal cervical cytology (Bethesda criteria), who had been referred to Maternity and Neonatology University Hospital- Argentina. Immunocompromised women were excluded. Patients included in the study, signed an informed consent form.A short questionnaire was used to collect data about age, number of sexual partners, oral and anal sex, number of pregnancies, history of STIs, oral lesion, contraceptive method, tobacco use and HPV vaccine.The total number of cigarettes smoked throughout the patient’s life was calculated considering smoker to the person who smoked more than 100,000 units .Cervical swabs were collected by a gynecologist using a brush and oral swabs were collected by a dentist, who did a bilateral scraping of oral cavity. Specimens were put in a sterile tube containing 1-ml phosphate buffer solution with antibiotic and antifungal agents to be processed. Both samples were collected by professional doctors on medical visit to Maternity and Neonatology University Hospital.This study was approved by the Committee of Ethics, National Hospital of Clinics- Argentina (RePIS2548) according to the ethical principles stated in the declaration of Helsinki.C.trachomatis was detected using CTP1 and CTP2 primers to cryptic plasmid (201 bp) [DNA was extracted using the commercial AccuPrep Genomic DNA Extraction Kit-Bioneer, following the manufacturer’s instructions. HPV L1 genomic region (450 bp) was amplified with degenerate primers MY09 and MY11 following Manos’s protocol . HPV-DNA(201 bp) . The PCR2) and Fisher Exact with a significance level 5% (95% CI), Epi info 3.5.4, CDC software (2012).Results were analyzed using Chi-square . The women studied were divided according to normal and abnormal cytopathological findings. Normal papapanicolaou test include infections and inflammation and abnormal results includes atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) according to Bethesda system [p > 0.05), Table C.trachomatis). Concerning history of STI, 2 patients with normal cytology had a history of Trichomonas vaginalis and Herpes Simplex Virus (HSV) infection. While in patients with abnormal cytology, 2 of them had a history of C.trachomatis, 1 of them HSV and 1 had history of Treponema pallidum.There is no statistically significance between normal and abnormal groups and risk factors such as age, number of sexual partners, oral sex, anal sex, pregnancies, history of STIs, oral lesion, contraceptive method and tobacco use . HPV genotypes detected were genotype 6 of low-risk and genotypes 16, 31, 52, 58 and 16–31 coinfection of high-risk. A total of 10 samples HPV DNA positive were genotype not identified. The more frequent genotype was the type 16 in normal and abnormal cytology as well as oral and genital mucosa. The only HPV type detected in oral mucosa was 16  with normal and abnormal cytology (Table 00 with nC.trachomatis DNA was detected in 49% of patients (49/100) (Table C.trachomatis in 35% (35/100) of these and out 100 oral samples we detect C.trachomatis DNA in 31% (31/100); 17 patients with both mucosa infected- 8 NG and 9 AG .ients 49/00  in comparison with NG . There is statistically significance (p < 0.05) between cytology and HPV and C.trachomatis infection .HPV was detected more frequent in NG 36%, 18/50) in comparison with AG , and in the different way 8/50 in cC.trachomatis  patients with the two infections, HPV and is Table . Out of C.trachomatis infection [C.trachomatis infection in unclear [When cervical HPV infection is investigated, different sites such as the oral cavity is not routinely investigated, except in the presence of visible lesions, as well neither nfection . Althoug unclear , 19. Bot unclear .n = 18) that detected oral HPV DNA in 4581 cancer-free subjects [The present results showed a total prevalence of 27% for HPV in the 100 patients studied. In reference to oral mucosa, we detect 14% of prevalence. Data about oral HPV, in asymptomatic patient is still on debate. Kreimer reported that asymptomatic oral HPV16 infection was found in 1.3% of a systematically reviewed and abstracted data from published studies  in which a prevalence of 36.09% was detected in women with normal cytology who used public health services  and loweIn women with abnormal cytology we detected 18% HPV DNA positive, surprisingly prevalence lower than women with normal cytology but in agreement with Ji, who detected 18.4% HPV in women with abnormal cytology, who visited the department of gynecology of the hospital from China . To diffAlthough HPV18 genotype is the second more frequent high risk type detected worldwide, in this study, we not detected HPV18 genotype in genital as well as oral mucosa. This is in agreeing with previous studies in oral mucosa in Argentina , 23, 29.C.trachomatis and cytology status, we detect a prevalence of 38% in women with normal cytology and 60% in abnormal cytology, being more frequent in genital area. These results are in concordance with a study from India, which detected more prevalence of C.trachomatis in patients with abnormal cytology (31.5%) in comparison with normal cytology (5.8%) in women with complaints of vaginal discharge attending STI clinic in a tertiary care hospital in New Delhi [C.trachomatis prevalence in symptomatic  and asymptomatic women with a request for a vaginal exudate study [C.trachomatis in women with normal and abnormal cytology, 7.1 and 7.2%, respectively, in China [C.trachomatis in 9.02% in women who used public health services with abnormal cytology [Concerning ew Delhi . On the cytology .5% in cote study , all rescytology .C.trachomatis, are in concordance with previous result of our group and results of other authors, other prevalence are very different. The detection rate of HPV and C. trachomatis in the oral cavity specially, is known to vary widely in different places of the world. These differences could be associated with the different types of samples collected in each study, the type of detection technique used, the different habits of the populations studied, the oral pathologies present in the patients studied, and the prevalence and circulation microorganism of the area under study [So, although some of our results, both HPV and er study  As well er study .C.trachomatis infection, although HPV was more frequent in normal cytology. Therefore, C.trachomatis detection and specific treatment in asymptomatic women, is important to prevent future sequelae. However, some studies reported that C.trachomatis infection may increase the risk of HPV persistence and coinfection induces a pro-inflammatory environment that may promote the carcinogenesis [C.trachomatis coinfection was found in 14%, predominantly in genital mucosa. This result is in concordance to Costa Lira, who detected 12.5% of coinfection in cervical swabs of the women studied [The current study showed an association between cytology status and HPV and ogenesis . In the  studied . The pre studied .There are a lot of risk factors for STIs, specially for cervical lesions , howeverC.trachomatis, identified by many studies as possible cofactors for oncogenesis. There is evidence that suggest screening for C.trachomatis is cost effective when its prevalence is above 3% [This study highlights the importance of HPV diagnosis and the sexually transmitted pathogens such as above 3% . These r