Circulating HPV DNA has been previously described in women with advanced stages of cervical cancer and has been suggested to be a prognostic marker of disease recurrences and metastases. Only a few studies have reported the presence of HPV DNA in bloodstream of patients with low grade or precancerous cervical lesions. This study aimed to define if HPV DNA could be detected in plasma samples of 120 women referred for a recent history of cervical dysplasia who presented with lesions ranging from High Squamous Intraepithelial Lesion (H-SIL) to regressed normal cytology. HPV DNA detection was carried out in both plasma and cervical samples using type-specific real-time quantitative PCR assays identifying oncogenic HPV 16, 18, 31, 33, 45, 51 and 52.4 cells and 1,099 copies/ml, respectively.Overall, 34.2% (41/120) of plasma samples were shown to be positive for HPV DNA detection; HPV 45 (46.3%), HPV-51 (29.6%), and HPV 16 (18.5%) were the most frequently identified genotypes. The rate of HPV detection in paired cervical and plasma samples increased with advancing disease stage, ranging from 15.4% in women with regressed lesions to 38.9% in women with HSIL; HPV 16 resulted the most common genotype identified in women found to be HPV DNA positive in both cervical and plasma samples. Moreover, HPV 16 showed the highest median viral load value in both cervical and plasma samples, with 48,313 copies/10Results obtained in this study confirm that HPV DNA can be detected and quantified in plasma samples of women with asymptomatic cervical infection. Further knowledge on HPV dissemination through the blood stream of women with cervical lesions would be very important in better understanding the natural history of HPV infection as well as its potential role in other distant tumors. Cervical Cancer (CC) is one of the most incident cancers in women worldwide, especially in developing countries, with an estimated incidence of 528,000 in 2012 [Infection with high-risk human papillomavirus (hr-HPV) genotypes has been well recognized to be causally associated with almost all CC types. The integration of hr-HPV DNA into the cellular genome and the overexpression of HPV E6 and E7 oncogenes are deemed responsible for different cellular pathways alterations . As thesInfection with hr-HPV has also been associated with the development of oropharyngeal cancer, as well as, more recently, with the occurrence of other extra-genital tumors, such as breast, lung, bladder, and colon cancers –9. How tIn order to better understand the association between bloodstream circulating HPV-DNA and cervical infection in women presenting with a recent history of cervical dysplasia, this study aimed to assess the presence  and agreement between both plasma and cervical samples of seven of the most prevalent hr-HPV types in our geographical area.The study protocol was approved by the Ethics Committee of San Gerardo Hospital, Monza, Italy . All subjects provided written informed consent to participate in the study.A group of women with a recent diagnosis of cervical dysplasia attending the Gynaecology Out-Patients Clinic of San Gerardo Hospital, Monza, Italy were enrolled in the study. Following routine gynaecological examination and repeated Pap smear examination, additional blood and cervical samples were collected from each patient for hr-HPV detection.Immunocompromised patients, women with autoimmune diseases or any diseases involving the immune system, with HIV infection, previously vaccinated against HPV, with a presumed or confirmed pregnancy, with a diagnosis of any malignancies, undergoing or having finished a course of chemotherapy during the six months preceding the study were excluded from the study. Women with a negative Pap test were selected as a control group.Paired cervical liquid cytology and blood samples were collected on the same day from all women visiting the Gynaecology and Obstetrics Department of the San Gerardo Hospital, Monza, Italy.Cytological diagnosis of cervical lesions was performed at the hospital, and findings were assessed according to the 2001 Bethesda System for cervical cytological reporting .Plasma and cervical samples were tested for HPV DNA at the Clinical Microbiology Laboratory of the Department of Medicine and Surgery, University of Milano-Bicocca, Italy.Cervical cytology samples were collected using the Abbott cervi-collect specimen collection kit (Abbott) and transported in ThinPrep® PreservCyt® Solution (HOLOGIC™). 15 ml of cervical samples were centrifuged at 2000 rpm for 15 minutes at 4°C for cervical cells concentration. Cell pellet was suspended in 2.5 ml of PBS and 5 aliquots of 500 μl for each samples were made. One aliquot was used for DNA extraction.Blood samples were obtained by venepuncture from patients using lithium-heparin anti-coagulant and transported to the laboratory at 4°C. In the laboratory, 20 ml of blood were slow pipetted to 20 ml of Lympholyte®-H, sterile liquid (EuroClone) and centrifuged at 400 xg for 40 minutes without brake to obtain blood stratification. Approximately 6 mL of plasma were transferred in a 15 mL centrifuge tube and stored at -80°C. One ml of each sample was used for subsequent DNA extraction.DNA extraction from a 500 μl cell pellet aliquot of cervical sample was carried out using CLART® HPV2 Extraction-Purification and Amplification kit (Genomica) following manufacturer protocol.DNA extraction from 1 mL of plasma sample was performed using NucliSENS® easyMag® (bioMérieux), a second-generation system for automated isolation of nucleic acid from clinical samples. All samples were processed using “specific” protocol, characterized by a higher final elution temperature (about 70°C) and the use of silica beads diluted 1:2, in accordance to the manufacturer’s instructions. The nucleic acids extraction system is fully automated, except for adding the initial sample of plasma (1 ml) and the silica beads (125 μl) 10 minutes after sample lysis, reducing potential risks of sample cross-contamination. The nucleic acids extracts were eluted in 70 μl of NucliSENS® easyMag® elution buffer (bioMérieux).4 cells in cervical samples.HPV DNA amplification was carried out using previously described “in-house” Real-Time quantitative TaqMan PCR assays –22. The All HPV Real-Time quantitative TaqMan PCR assays were previously validated by participation to WHO HPV Laboratory Proficiency Lab-Net 2014.Data were collected on standardized e-forms and analysed using Stata 9.0 . Qualitative and quantitative variables were summarized with absolute (relative) frequencies and medians , respectively. A two-tailed p-value less than 0.05 was considered statistically significant.A group of 120 women  was enrolled. Twenty women were recruited as control group.Normal cervical cytology was detected in 21.7% (26/120) of recruited women with a documented abnormal Pap test in the preceding 6 months (“Regressed”). Abnormal Pap smears at enrolment were detected in 78.3% of the study participants: 25.5% (24/94), 55.3% (52/94), and 19.2% (18/94) showed atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion (HSIL), respectively.4 cells , followed by HPV 51  and HPV 45 .Overall, 44.2% (53/120) of selected women were found to be positive for the detection of one or more of the 7 hr-HPV types investigated. 37.7% (20/53) of HPV DNA positive women showed infection with multiple HPV-types for a total of 82 HPV cervical infections. Data regarding the genotypes identified in cervical samples are reported in HPV DNA was detected in 42.3% (11/26) of women with regressed lesions, 33.3% (8/24) with ASCUS, 42.3% (22/52) with LSIL, and 66.7% (12/18) with HSIL, respectively . No posii.e., HPV 16, 18, 45, 51 and 52); nine of these women  showed the presence of multiple HPV-types for a total of 54 HPV-positivities. Genotypes detected in plasma samples are described in the Overall, 34.2% (41/120) of women were found to be positive in plasma samples for 5 out of the 7 hr-HPV types investigated (HPV DNA was found in 30.8% (8/26) of women with regressed lesions, 33.3% (8/24) with ASCUS, 30.8% (16/52) with LSIL, and 50.0% (9/18) with HSIL, respectively .All samples from women in the control group were found to be HPV DNA negative.Twenty-five  women were found to be HPV-DNA positive both in cervical and plasma samples; among them 44% (11/25) were shown to have the same HPV genotype in both samples . HPV-16 Concomitant detection of the 7 hr-HPV types investigated in both cervical and plasma samples increased with the severity of the disease, ranging from 15.4% (4/26) in women with normal cytology/regressed lesions to 38.9% (7/18) in women with HSIL .Thirteen percent of women (16/120) showed HPV positivity only in plasma samples: the most frequently detected genotypes were HPV 45 , HPV 51 , and HPV 16 . Twenty-eight women  were HPV DNA positive only in cervical samples: the most frequently detected genotypes were HPV 45  HPV 51 , and HPV 16 . The remaining 51 patients resulted negative for the investigated 7 hr-HPV in both sample types.HPV are well-known epitheliotropic viruses requiring differentiating squamous epithelium for their life cycle. The oncogenic potential of hr-HPVs has been associated with the occurrence of cervical, other ano-genital as well as head and neck cancers , 24. MorLimited data are available on the presence of HPV DNA circulating in the bloodstream. Previous reports have described only a limited number of oncogenic HPV types, mostly HPV-16 and -18, in blood samples of women with CC. Moreover different studies investigating the presence of HPV DNA in plasma of women with CC have demonstrated very different PCR detection rates ranging from 6.9% 11/175) HPV 16/18 positivity, as compared to 1.7% (1/60) positivity in controls ; 13.7%  of asymptomatic women with urogenital infections ; ForestaOn this basis, we investigated the presence of HPV DNA in blood samples of 120 asymptomatic women presenting with a recent history of cervical dysplasia. Detection of HPV DNA was performed by the use of previously described highly sensitive hr-HPV type-specific quantitative real-time PCR assays –22. OverConcomitant HPV detection in both cervical and plasma samples was found to increase with the severity of the cervical dysplasia, ranging from 15.4% to 38.9%. Forty percent of women with HPV infection both in cervix and in plasma showed the same genotype, with HPV-16 (20%) being the most commonly identified HPV type. This is in keeping with results of previous studies on women with CC, also reporting HPV-16 as the more prevalent genotype both in blood and cervical samples , 16, altOur study also showed 16 women to be HPV DNA positive only in plasma samples, in the absence of cervical infection. Again, this could result from a cleared cervical infection and/or HPV presence in other non-genital mucosal sites associated with viral persistence and/or replication in the bloodstream , 34, 37.Quantification of HPV viral load in cervical samples could represent a potential marker to discriminate persisting from regressing cervical infections. Data supporting this correlation and HPV viral load prognostic significance have been previously reported , 38.Regarding the role of HPV viral load in blood, few studies have reported a possible correlation between HPV viral load in plasma and tumor clinical stage, suggesting that it could represent a possible marker for advanced cervical disease and/or a genetic marker associated with CC metastasis/recurrences , 17, 32.This study provides further evidence that HPV DNA can be detected and quantified in plasma samples of women with a recent history of low grade cervical dysplasia and/or cleared genital HPV infection. This is contrast with some previous reports correlating circulating HPV DNA with cervical carcinoma or other HPV-related cancers. The identification of hr-HPV DNA in plasma samples of women with apparently cleared cervical infection provides evidence for possible viral persistence and/or replication in anatomical sites other than the genital mucosa. Longitudinal studies are however required to further evaluate the role of HPV DNA detection in the bloodstream of women with asymptomatic infection and/or early stage cervical dysplasia.


Chlamydia trachomatis and Herpes simplex, cause local inflammation, and could contribute to Human Papillomavirus (HPV) and cervical lesion progression. Thus, the aim of this study was to determine any association between the presence of microorganisms of gynecological importance, sexual behavior, clinical and demographical variables to the development and progress of cervical lesions.Some sexually transmitted infectious agents, such as Ureaplasma urealyticum and Ureaplasma parvum. Univariate and multiple logistic regression were performed to measure the association with the cervical lesions, and an odds ratio (OR) with 95% confidence intervals (95%CI) were calculated. The Mann-Whitney U test was also used to compare the microorganism load in the case and control groups. The significance level was 5% in all hypotheses tested.One hundred and thirty-two women between 14 and 78 years and living at Vitória da Conquista, Bahia, Brazil, were included . They answered a questionnaire to provide data for a socioeconomic and sexual activity profile. Samples of cervical swabs were collected and analyzed by PCR to detect genital microorganisms and HPV. Quantitative PCR was used to detect and quantify Trichomonas vaginalis  and Gardnerella vaginalis , adjusted by age and qPCR for U. parvum. U. parvum load showed a statistical difference between the case and control groups .Cervical lesions were associated with: women in a stable sexual relationship , positive PCR for HPV , T. vaginalis, G. vaginalis were associated with cervical lesions in epidemiological studies. U. parvum load was higher in woman with cervical lesions compared with women without lesions. Additional studies are needed to better understand the role of these factors in cervical lesion development.Variables such as stable relationship, HPV, The online version of this article (10.1186/s12879-017-2835-5) contains supplementary material, which is available to authorized users. Cervical cancer is the fourth most common cancer among women in Brazil and seventh worldwide . The higThe development of cervical lesions depends on infection by high risk Human Papillomavirus (HPV), such as HPV-16 and HPV-18, and other factors. These HPV types express the oncoproteins E6 and E7, which interfere with cellular machinery causing cell immortalization and transformation. HPV-related carcinogenesis depends on different factors such as HPV type, virus persistence, sustained viral oncogene expression, viral load and viral genome integration .As in other cancers, cervical cancer is also multifactorial. In addition to identifying HPV as the main etiological agent, additional risk factors such as use of oral contraceptives, smoking, early onset of sexual activity, multiple partners and chronic inflammation due to co-infection with other microorganisms have been strongly associated with cervical lesion progression –5.Cervical inflammation may be associated with high-grade lesions and may be a cofactor for high-grade cervical lesions in women infected with oncogenic HPV. In other cancer types, independent of the causal agent, chronic inflammation exposes the tissue to constant genotoxic damages. In cervical cancer, chronic inflammation added to the initial changes caused by high-risk HPV, may contribute to viral persistence and disease progression , 7.Chlamydia trachomatis and Herpes simplex, cause local inflammation, and could contribute to HPV and cervical lesion progression [Gardnerella vaginalis and Trichomonas vaginalis, as well the species of Mollicutes  are included in this context. These wall-less bacteria have been described for over 30 years as microorganisms related to urogenital disorders. Some species are the main cause of non-gonococcal and non-chlamydial urethritis (NGU-NC), as in the case of bacterial vaginosis (BV). Mollicutes may induce the release of cytokines such as IL-1, IL-6 and TNF-α and chemokines and may cause intense inflammation. This response has been suggested as a possible risk factor for developing precancerous lesions [Mollicutes and HPV may help better understand the role of these bacteria in developing cervical cancer. Therefore, the aim of this study was to analyze the existence of an association between the presence of microorganisms of gynecological importance, sexual behavior, clinical and demographic variables, and the development of cervical lesions in women from Vitória da Conquista city, Bahia, Brazil.Some sexually transmitted infectious agents, such as gression . Similar lesions –10. The From 62 women in the case group, 18 (29.0%) had low-grade squamous intraepithelial lesion (LSIL) described also as Cervical Intraepithelial Neoplasia (CIN I), 16 (25.8%) had high-grade squamous intraepithelial lesion (HSIL)/(CIN II) and 28 (45.2%) had HSIL/CIN III/in situ carcinoma. The detection of the afore mentioned microorganisms by PCR and qPCR methodologies was determined in samples of the case and control groups. The prevalence of the targeted sexually transmitted microorganism was calculated  and 37.6 (SD = 14.4) years, respectively. However, although the general mean age between case and control groups was similar, there was punctual differences in age distributions between the groups. We can observe that among the women without cervical lesion, 40% were less than 31 years old. In women with cervical lesions, a higher frequency was found among those aged 32–42 years (40.3%). This corroborates the fact that the development of cervical lesions are time-dependent and highlights the importance of this variable for the subsequent analyses. Table Mollicutes , U. parvum (qPCR) , T. vaginalis  and G. vaginalis  presented as risk factors for cervical lesions. However, infection by U. parvum serotypes 1  and 6  appeared as protective factors , education level lower than elementary education  and stable sexual relationship . In regards to the microorganisms, positive PCR results to HPV , p > 0.005) were withdrawn from the model one by one, except for the confounding variables or those of biological importance for the model (for example the variables age and presence of U. parvum). The most appropriate model was selected according to the goodness of fit by the Hosmer-Lemeshow test (p = 0.568). The following variables remained in the final model: women in a stable sexual relationship , positive PCR for HPV , T. vaginalis  and G. vaginalis  adjusted by age and qPCR for U. parvum.All the variables that showed association in the univariate analysis were tested together to see if they contributed, together, to the risk of cervical injury. All variables that lost significance in the Wald’s test  between the case and control groups  in the general population, 85.5% (53/62) in the case group and 25.7% (18/71) in the control group. In this study, 85.5% of cases with cervical lesions were positive for HPV. The viral DNA was detected in 24.5% of low-grade lesions (L-SIL/CIN I), 26.4% (CIN II) of intermediate-grade lesions and 49.1% of high-grade lesions . In another case-control study conducted in Recife, Pernambuco, Brazil, women with squamous intraepithelial lesions, high-grade or cervical carcinoma comprised the case group, and women with normal cytology or benign cellular changes comprised the control group. Of the 610 women initially eligible, just 248 (40.7%) were PCR positive for HPV. Among these women, 29.0% (72/248) had a high-grade lesion . Similar2 in the Despite the high risk of developing lesions when infection with high-risk HPV occurs, 18 (25.7%) of 70 women with normal cytology were positive for HPV. This is the case because the samples can be positive for low-risk HPVs that are not responsible for the development of cervical lesions. Besides this, while infection with HPV is a necessary factor in the development of cervical cancer, not all positive cases of virus infection will result in cancer. Furthermore, this virus is commonly detected in the population. In routine cytological examination among women with normal cytology, about 10–30% are positive for any HPV type . In a reThe low rates of cervical cancer precursor lesions, added to high HPV prevalence in the general population and high HPV detection in patients with normal cytology supports the hypothesis that, although a necessary precursor, the presence of HPV alone is not sufficient for developing cancer. First, the development of cervical lesions depends on infection by high risk Human Papillomavirus (HPV), such as HPV-16 and HPV-18. HPV-related carcinogenesis depends on different factors such as HPV type, virus persistence, sustained viral oncogene expression, viral load and viral genome integration. These HPV types express the oncoproteins E6 and E7, which interfere with cellular machinery inhibiting cell death, promoting cell immortalization, and after cell transformation , 18. FurMollicutes, U. parvum and its serotypes, U. urealyticum, T. vaginalis, G. vaginalis, C. trachomatis and N. gonorrhoeae was compared to determine a possible association with cervical lesions. There was a difference for both U. parvum and U. urealyticum between the prevalence observed in PCR and qPCR. These differences can be explained by the higher sensitivity of the qPCR technique compared with PCR, thus allowing a greater detection. Besides, the presence of U. parvum serotype 1  and serotype 6 , in first instance, has been verified as a protective factor for the cervical lesions development. However, when comparing the number of positive samples for U. parvum (32 cases and 35 controls) and the total number of serotypes obtained by adding all positive samples from serotypes 1, 3/14 and 6 (13 cases and 36 controls) we observe that there is no match between the results. This fact, added to the disparity between the results detected by PCR and qPCR, demand for future studies with more sensitive techniques, like qPCR, to verify the real association of such serotypes with the development of cervical lesions.In this study, the detection of U. parvum and cervical lesions in the univariate analysis was detected. However, after logistic regression, the presence of U. parvum lost its association with cervix lesions. There are few and contrasting data in the literature on the relationship between cervical lesion and ureaplasma presence. Ekiel and collaborators conducted a similar study and evaluated 182 women with changes in the cervix: 67 women with atypical squamous cells of undetermined significance (ASCUS), 49 women with LSIL, 22 with HSIL, 39 with normal cytology and 5 with invasive cancer [Mollicutes, but 58 (90.7%) were positive for U. parvum and nine (14.1%) for U. urealyticum. They also found that the ureaplasmas were more frequent among women with HSIL. In another study by Lukic et al., 239 women with different disorders in the cervical region were studied: 66 with ASCUS, 115 with low-grade intraepithelial lesions (LSIL) and 58 with high-grade intraepithelial lesions (HSIL). They did not diagnose U. parvum; however, they found that among women diagnosed with HSIL, 26 (45%) were positive for U. urealyticum [U. parvum than U. urealyticum among women with HSIL. According to Kong et al., U. parvum is more common in clinical isolates of the genital region than U. urealyticum [In addition to the observed prevalence, a positive correlation between alyticum . In thisalyticum .U. parvum, patients with cervical lesions showed higher U. parvum load /μl) than those without cervical lesions  . This can be explained because more microorganisms in the local area could intensify the inflammatory response and/or activate some virulence factor of the pathogen. This could favor infection progression and the development of cervical lesions. Few studies have been conducted focusing on the association of ureaplasma load with disorders of the reproductive system, especially with precursor cervical lesions of cervical cancer [U. parvum and U. urealyticum load in women who had cervicitis, and found a positive relationship between the risk of microbial load and cervicitis [U. urealyticum and the presence of non-gonococcal urethritis and non-chlamydial (NGU-NC) in men but did not observe the same for U. parvum [U. urealyticum load among women with and without cervical lesion.After analyzing the load of l cancer . Lu et arvicitis . Another. parvum . The cau. parvum , 26. HowU. parvum, 3/14 showed greater detection of all serotypes, as described in the literature [U. parvum serotypes 1 and 6 were more frequent among women with lesions, and were inversely associated with the presence of cervical lesions. Furthermore, since the detection of the serotypes was obtained by PCR, it was not possible to observe the microbial load of each of the serotypes in the samples, since the presence of high/low load of microorganisms in a clinical specimen is associated with a stronger inflammatory response and some diseases [Among the four serotypes of terature , 21, butdiseases , 25–27. T. vaginalis was detected in 61.3% of women who had lesions. There was a greater risk related to presence of this agent and the development of cervical lesions. Zhang et al. also reported a positive association between T. vaginalis and the presence of cervical lesion. A total of 16,797 residents in the Jingan district, Shanghai, China, were studied and followed from 1974 to 1985. A risk of 3.31 was found (95% CI = 1.5 to 7.4) [T. vaginalis, and among those with no HSIL detection, and there was no statistically significant association [G. vaginalis was detected in 91.9% of all women who had lesions; a positive association with the presence of injury was also found, showing a higher detection among those with HSIL [C. trachomatis was detected in only 3.2% of women and N. gonorrhoeae was not detected in women with cervical lesions. In this study, these two microorganisms were not associated with cervical lesions possibly due to the low number of cases included in this study. Thus, further studies are needed to better understand the association of these microorganisms with the presence of cervical cancer.It is believed that the inflammatory response caused by the heavy load of microorganisms at the site of infection lead to cell damage suggestive of cervical lesions, as has been observed for other microorganisms. Other sexually transmitted infectious agents were detected.  to 7.4) . Moreoveociation . Verteraociation . In thisith HSIL . In a stith HSIL . C. tracIn this study, women living in the countryside and with an education level below elementary education, showed to be at greater risk for developing cervical lesions. In Recife, a city of the State of Pernambuco, Brazil , women wWomen with irregular sexual activity  were less likely to develop cervical lesions compared to those in a stable relationship . Women in stable relationship, in interview moment, have declared being with only one partner. Thus, one possible explanation to this unexpected fact is that many partners do not use condoms, and if they are involved in extramarital relationships, they are at great risk to contract HPV virus and infect their wives . HoweverT. vaginalis, G. vaginalis were associated with cervical lesions in epidemiological studies. U. parvum load was higher in woman with cervical lesions compared with women without lesion. Despite the evidence presented in this paper on the role of ureaplasmas, T. vaginalis and G. vaginalis in cervical lesions, longitudinal studies with a larger population must be done to precisely define their association with the emergence of cervical injury. In addition, conducting infection tests in in vitro and in vivo study models will further elucidate virulence factors involved in the establishment of cervical lesions. These data are unprecedented for the population under study, and may aid local health authorities in improving prevention measures, and treatment, in order to improve the quality of life of women in the municipality of Vitória da Conquista, Bahia, Brazil. When all aspects involved in pathogenesis are better understood, this knowledge may contribute to improving prevention strategies and treatment.Even if the role of HPV is evident in the development of cervical cancer, further studies are needed to better understand the complexity and multifactorial profile of this condition. In this study, variables such stable relationship, HPV, The study described here is a transversal study of cervical lesions in women (case-control), carried out from 2011 to 2013. One hundred and forty women were initially included . The sample size was determined with statistical power of 95% and 0.05 statistical significance using the reports of cervical lesions and total size of women from Vitória da Conquista . The cases  were from the Family Health Center CAE II of Public Health System (SUS – Brazil) located in Vitória da Conquista city, Bahia, Brazil. Women seeking medical consultations, with a positive diagnosis for cervical lesions  1, 2 and 3 or in situ carcinoma) were selected as the case group. The controls  were from the Primary Care Units of Public Health Clinics (SUS – Brazil) also located in Vitória da Conquista city, Bahia, Brazil. Women with a negative diagnosis were selected for the control group. Screening was conducted among patients aged from 14 to 78 years. Then 140 women (70 cases and 70 controls) initially answered a questionnaire to provide a socioeconomic and sexual activity profile. This study was previously approved by the Ethics Committee on Human Research of the Institute of Biomedical Science – University of São Paulo, Brazil, (protocol number 1089).Cervical samples were taken by cytobrush and swab from the ectocervix and from the endocervix for detecting HPV DNA and bacteriological tests. Swabs were transferred to a tube containing a transport medium .For HPV detection, two microtubes of each sample were sent to the HPV laboratory of the Santa Casa Research Institute of São Paulo . We extracted DNA from all cervical specimens using digestion with 100 μg/mL of proteinase K for 3 h at 55 °C, followed by organic extraction and ethanol precipitation. Extracted DNA in aqueous solution was quantified (ratios 260/280 and 260/230) by NanoDrop 2000 Spectrophotometer . DNA concentrations were normalized to 50 ng/μl to perform β-globin PCR , 500 mM KCl), 0.2 mM of deoxiribonucleotide triphosphate (dNTP), 2.0 mM MgCl2, 1 μM of each primer GP5/GP6 , 0.2 mM of deoxiribonucleotide triphosphate (dNTP), 4.0 mM MgClAs positive control for all PCR, HeLa  cells were used. The products of the targeted amplified DNA and a standard molecular weight of 100 bp were electrophoresed on 1.0% agarose gel stained with 5 μL of GelRed 10,000X . Then the gels were photodocumented.Ureaplasma urealyticum (UU) and Ureaplasma parvum (UP), Trichomonas vaginalis (TV), Gardnerella vaginalis (GV), Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG).For bacteriological tests, genomic DNA samples of vaginal swabs were obtained according to the recommendations of Purelink™ Genomic DNA Mini Kit . DNA was extracted from two microtubes of each sample for detecting 2, 50 pmol of each primer  adding: 1X buffer , 0.2 mM of deoxiribonucleotide triphosphate (dNTP), 2.0 mM MgClUreaplasma urealyticum (UU) and Ureaplasma parvum (UP) stored in our laboratory. These strains were growth in UB medium [Trichomonas vaginalis (TV), Gardnerella vaginalis (GV), Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) were gently seeded by Dr. Jorge Sampaio, (Fleury Institute/São Paulo – Brazil) and directly submitted to DNA extraction. The products of the targeted amplified DNA and a standard molecular weight of 100 bp were electrophoresed on 1.0% agarose gel with ethidium bromide (10 mg/ml). Then the gels were photodocumented.As positive controls, we used strains of B medium . SamplesU. urealyticum and U. parvum DNA load in clinical samples. The assays were performed in duplicate with a Real-Time PCR System 7300 Applied Biosystems platform and TaqMan probes, following the manufacturer’s basic amplification protocol. For constructing DNA standards for absolute quantitation, the ureaplasmas were first cultured in 2 ml at 37 °C and expanded to a 50 ml of UB broth. In a logarithmic growth phase (based in colorimetric changes), the culture was centrifuged at 20,600 g for 30 min at 25 °C. The DNA was extracted using a PureLink™ Genomic DNA Mini Kit. The genomic DNA copy number was calculated relating the amount of DNA (ng) measured by spectrophotometry  with the length of DNA (bp) of the respective strains. Then, 10-fold serial dilutions (107−1 copies/μl) of the ureaplasma DNA standard were prepared and analyzed. The reagents used were: 12.5 μL of Master Mix , 0.75 μL of each primers diluted to 10 pmol  to measure the p-value less than 0.05 were used in the final model, unless the variable was biologically important. The microbial load of case and control groups was compared using the nonparametric Mann-Whitney. Significance level was 5% in all hypotheses tested. All analyses were evaluated by SPSS 20.0 software , R software  and the GraphPad Prism version 5.00 .Categorical variables were shown as frequencies and percentages. In order to evaluate factors associated with cervical lesions, we used the logistic regression model and an odds ratio (OR) and respective 95% confidence intervals (95%CI) were calculated. The selection of variables was done by a stepwise method and OR unadjusted and adjusted were shown in the final model. The goodness of the fit was verified by Hosmer-Lemeshow’s test. Finally, only variables with a Additional file 1: Table S1.Primers used in PCR. (DOC 89 kb)Additional file 2: Table S2.U. urealyticum and U. parvum in cervical samples. (DOC 50 kb)Primers and probes used in qPCR to quantify DNA of


Consistent with a series of reports attributing a causative role of β-HPV at early stages of skin carcinogenesis through ΔNp63 induction and increased keratinocytes stemness, here we provide in vivo evidence that these events are also occurring in the affected skin of KTRs. Due to these β-HPV-driven molecular pathways, the nascent tumor cell is able to acquire a high enough number of carcinogenic insults that its proliferation and survival will eventually become independent of viral gene expression.Many malignancies that occur in high excess in kidney transplant recipients (KTRs) are due to viruses that thrive in the setting of immunosuppression. Keratinocyte carcinoma (KC), the most frequently occurring cancer type in KTR, has been associated with skin infection by human papillomavirus (HPV) from the beta genus. In this report, we extend our previous investigation aimed at identifying the presence of active β-HPV infection in skin tumors from KTRs through detection of viral protein expression. Using a combination of antibodies raised against the E4 and L1 proteins of the β-genotypes, we were able to visualize infection in five tumors  that were all removed from two patients who had been both transplanted twice, had developed multiple KCs, and presented with a long history of immunosuppression (>30 years). These infected tissues displayed intraepidermal hyperplasia and increased expression of the ΔNp63 protein, which extended into the upper epithelial layers. In addition, using a xenograft model system in nude mice displaying a humanized stromal bed in the site of grafting, we successfully engrafted three AKs, two of which were derived from the aforementioned KTRs and displayed β-HPV infection in the original tumor. Of note, one AK-derived xenograft, along with its ensuing lymph node metastasis, was diagnosed as squamous cell carcinoma (SCC). In the latter, both β-HPV infection and ΔNp63 expression were no longer detectable. Although the overall success rate of engrafting was very low, the results of this study show for the first time that β-HPV The prolonged use of immunosuppressive drugs in kidney transplant recipients (KTRs) is a recognized risk factor for infectious diseases and ensuing complications, including infection-related malignancies   from the β-genus, such as HPV5 and HPV8, have long been associated with KC in patients with a specific genetic background and in long-term immunosuppressed individuals , indicating that epithelial cells were actively proliferating in areas of productive viral infection. Thus, based on these findings, we hypothesized that β-HPV-induced basal cell proliferation in the immunosuppressed setting alongside other transforming agents, such as UVB irradiation, might play a causative role at early stages of neoplastic transformation, while it may not be necessarily involved at later stages of the disease , a cellular proliferation marker  analyzing 128 additional skin lesions excised from 29 KTRs; (ii) correlating ΔNp63 expression levels with those of E4 and MCM7 in skin areas of productive HPV infection; and (iii) establishing and characterizing patient-derived xenografts as models to study skin cancer progression of β-HPV-infected skin lesions. In particular, we provide evidence of two KTRs with multiple β-HPVTissue sections were obtained from 171 formalin-fixed and paraffin-embedded blocks (FFPE) corresponding to either the core or the edges of the tumor, collected from 128 skin lesions excised from 29 KTRs attending the dermatology unit in the University Hospital-Novara. Thirty-two tumors were derived from KTRs already described in our previous work  and written informed consent was obtained by all subjects according to the Declaration of Helsinki.3) were xenografted into the stromal bed together with 106 primary human fibroblasts suspended in 100 μl Matrigel  as performed for human skin biopsies.The immunodeficient mice used for xenograft development were male athymic Nude-Foxn1nu . Mice were housed under pathogen-free conditions in our animal facilities in accordance with The Guide for the Care and Use of Laboratory Animals and the experimentation was approved by the CESAPO . Mice were anesthetized with an Ohmeda TEC4 Vaporizer using Isoflurane Vet  and Gelfoam dressing  was implanted into the dorsal sub-cutaneous space together with primary human fibroblasts suspended in 100 μl Matrigel , and wounds were closed with surgical stitches . After 14 days, mice were anesthetized and human skin tumors . β-HPV DNA analysis was carried out using broad spectrum PCR (PM-PCR) in combination with a reverse hybridization system [RHA Kit Skin (beta) HPV ] as previously described , BCC (n = 3), AK (n = 2), or keratoacanthoma (KA) (n = 3), were made available for immunofluorescence analysis. β-HPV+ areas were found in one KA located on the leg, and in two AKs located on the hand and the leg . The cellular proliferation marker MCM7, a subunit of DNA helicase, was more evident in the E4+ area compared with the adjacent epithelium, and extended to suprabasal layers, indicating that cells were primed to enter the cell cycle. In the AK found on the hand, E4 expression was not detected, while nuclei stained with the β-HPV late capsidic protein (L1) were well evident in the upper layers of the epithelium where some differentiation was still occurring, indicating the presence of productive viral infection (data not shown).The first case was a female KTR who underwent a second kidney transplant (KTx) in 2016 due to the failure of her first one after 25 years. She displayed many flat, reddish papular lesions widespread in the total body skin as shown by the representative picture of the forehead Figure  that wereg Table , E4+ celn = 2), AK (n = 2) and seborrheic keratoses . Positivity for β-HPV markers was found in one AK and in the SK removed from the back and the left forearm, respectively. The virus-positive AK displayed highly keratotic epithelium with clear signs of β-HPV-related cytopathic effects  were co-labeled with antibodies against ΔNp63 and MCM7. In addition, six skin lesions  with areas positive for E4 expression from our previous survey were similarly analyzed. As depicted in the representative Figure n = 6) and BCC (n = 6). Of note, in the adjacent β-HPV-infected hyperplastic epithelium as determined by E4 staining  and KA (n = 3), ΔNp63 expression exceeded that of MCM7 as it was still detectable in the more superficial nucleated cells. Quantification of ΔNp63 and MCM7 positive cells confirmed the increase in ΔNp63+ cells in both β-HPV-infected cells and tumors vs. non-pathological epithelium  models. PDXs are developed by implanting tumor samples obtained directly from patients in immunodeficient mice. In this regard, we have previously shown that the creation of a humanized stromal bed, by first implanting Gelfoam dressing along with HDF, facilitates primary human skin SCC xenograft growth in nude mice  turned out to be epidermal cysts filled with cornified material and surrounded by dying epithelial cells. This pitfall prompted us to shorten the length of the growing period in nude mice to 3 months. Using this modified timeline, three out of the 23 skin tumors engrafted gave rise to a tumor mass with well-defined histological features. Three AKs successfully grew in nude mice; two of them displayed a growth curve similar to that of the two BCCs, while one grew faster, especially after week 8 , including those that clinically appeared as benign and/or premalignant. From a total of 42 lesions engrafted following this procedure, we were able to collect 38 xenografts—in four cases no tumor growth was detected. The main characteristics of the KTRs whose skin tumors were grafted into nude mice are shown in Supplementary Table In this study, we extend our previous investigation aimed at identifying the presence of active β-HPV infection in skin tumors from KTRs through the detection of viral protein expression, such as E4 and L1 (Borgogna et al., According to our previous findings, E4 expression was mostly detected at early stages of disease, confirming that virus expansion was taking place in the skin under condition of prolonged immunosuppression, albeit no longer detectable in more advanced stages of disease (Borgogna et al., in vitro and in vivo data linking β-HPV infection to ΔNp63 upregulation (Lanfredini et al., Overall, given the important role of p63 in the proliferation and maintenance of epidermal stem cells, and a number of in vivo evidence that β-HPV infection might be a co-factor of early stages of skin cancer progression. This hypothesis is fully compatible with the “hit-and-run” mechanisms of carcinogenesis, with cutaneous β-HPV playing an important role for tumor initiation and progression, but not required for tumor maintenance. Assuming that viral replication is more active at very early stages of carcinogenesis, or when lesions are not yet even clinically evident and rarely removed, it seems reasonable to propose that at later stages of disease, when lesions require surgical excision, β-HPV infection is no longer detectable or otherwise restricted to some residual areas at the edges of the lesion. These assumptions can explain the low percentage of positivity found in our study. However, the observed reactivation of β-HPV infection occurring in the skin of KTRs alongside the demonstration that these viruses can increase keratinocyte stemness and interfere with the DNA damage response argues in favor of a model whereby the cooperation between β-HPV and UVB, which triggers skin carcinogenesis, is no longer required to maintain the malignant state at later stages of neoplastic development (Hufbauer and Akgül, Emerging evidence clearly indicates that β-HPVs contribute to skin cancer development at early stages and through molecular mechanisms that are different from those reported for high-risk α-types, such as HPV16 and 18 (Howley and Pfister, + and ΔNp63+ intraepidermal hyperplasia can indeed progress to an aggressive SCC, including metastasis. As this SCC developed in 12 weeks in a nude mouse, we cannot rule out that the engrafted AK already harbored small amount of SCC. Nevertheless, cancer cells were originated from the infected epithelium that clinically appeared as field cancerization. Furthermore, the observation that both ΔNp63 and β-HPV are no longer detectable in the SCC suggests that their roles might be more important at premalignant stages where they likely cooperate to create an environment conducive to skin cancer progression.Another important aspect of the current study is the optimization and implementation of a unique xenograft model system in nude mice (Patel et al., Overall, the xenografts obtained following our protocol may prove useful not only in following the fate of each lesion but also in increasing the availability of biological samples for molecular investigations, which is generally limited due to surgical removal of premalignant lesions. In this regard, every effort should be made by clinicians involved in the daily management of KTRs to identify patients with clinically evident virus reactivation in the skin, and as such at increased risk of developing cancer.MG, MD and GP conceived the project. CB and CO designed experiments. CB, CO, SL, FC, RB, and ET performed experiments and collected, analysed data. EZ and PS enrolled patients and collected clinical samples. MG drafted the manuscript with inputs from all authors. CB and MG handled funding, supervision, and coordination. All authors have made final approval for the final version to be submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most of the respondents had the misconception that only females get HPV (95.1%), and that the HPV vaccine eliminates the need for Pap smear tests (68.3%). Most respondents (91.6%) believed that they would not get an HPV infection. Almost half of the respondents (42.9%) held the misconception that HPV infection could not lead to serious illness. Findings revealed poor knowledge about both HPV and the HPV vaccine, low perceived susceptibility to HPV infection and misinformation about HPV infection among HPV-vaccinated girls. Therefore, it is essential to increase the knowledge and awareness of health risks regarding HPV infection among teenaged girls who have received the HPV vaccine.The National HPV Immunization Programme, which offers free human papillomavirus (HPV) vaccines to teenaged female students, was launched in Malaysia in 2010. HPV vaccination paired with adequate knowledge about HPV infection provides the best protection against cervical cancer. To identify the level of knowledge and the health beliefs towards HPV and the HPV vaccine among HPV-vaccinated female students in Malaysia. A nationwide cross-sectional survey among 14 years old female students who had received three doses of the HPV vaccine was conducted in 32 randomly selected schools from 13 states and 3 federal territories in Malaysia between February 2013 and April 2013. Among 2482 respondents, knowledge about HPV infection and the HPV vaccine was extremely poor. The mean total knowledge score was only 3.56 (SD ± 1.76), out of a possible score of 10. The majority of respondents were unaware that  Cervical cancer is one of the leading causes of death in women around the world . AccordiVaccines are now available to prevent HPV types 16 and 18 which most commonly cause cervical cancer and its precursors . HPV vacAlong with knowledge, health beliefs about HPV are also important. The Health Belief Model (HBM) is the most widely used theoretical framework to study personal beliefs that predict disease knowledge and human health behaviours, including risky sexual behaviour, intention to receive HPV vaccine, and practice of preventive measures against HPV , 13. A lA nationwide survey was conducted by simple random sampling to select two secondary schools from each of the 13 states and 3 federal territories in Malaysia, giving a total of 32 schools. All Form Two Malaysian female students (aged 14 years) in the sampled schools who had completed the three doses of the HPV vaccine as part of National HPV Immunization Programme in the previous year were invited to participate in the survey. The study was carried out between February 2013 and April 2013.A standardized questionnaire was given to each student. The questionnaire assessed demographic characteristics, and knowledge and health beliefs related to HPV infection and vaccination. In the knowledge section, respondents were asked a series of questions regarding HPV infection, its relationship to cervical cancer and genital warts, HPV vaccine and the effectiveness of the HPV vaccine , and were asked to choose only one reply from three given choices of ‘true’, ‘false’ and ‘don’t know’. A correct response was given a score of one, and an incorrect or ‘don’t know’ was scored as zero. The possible total knowledge score ranged from 0 to 10, with higher scores representing higher levels of knowledge.The second section of the questionnaire assessed respondents’ health beliefs towards HPV vaccination using the HBM construct as the theoretical framework , which iThe third section of the questionnaire assessed respondents’ attitudes towards HPV vaccination. Seven statements were presented, and respondents could choose only one from three response categories of ‘agree’, ‘don’t know’ and ‘disagree’.The questionnaire was in three languages: Bahasa Melayu , Chinese and English, and was pretested before commencing the study. At each selected school, a survey administrator was appointed and briefed on how to correctly administer the survey questionnaires.The questionnaire was content validated by three experts from the Department of Social and Preventive Medicine of the University of Malaya to ensure the relevance (or the intent) and clarity of the questions. After minor amendments, the questionnaire was pilot tested on 20 randomly sampled students who were not included in the actual study. Pilot test participants were included from the three main ethnic groups, the Malays, Chinese and Indians. The questionnaire was revised again according to the students’ comprehension of the questions. Then, the questionnaire was construct validated on 150 randomly sampled students and factor loading was calculated to indicate the level of each specific knowledge item. The internal consistency  of the score for knowledge items was found to be 0.653. After two weeks, test-retest reliability was assessed on the same 80 participants, and found to be 0.2 to 0.5, with most kappa values greater than 0.1. Based on the outcomes of the construct validity and test-retest, the questionnaire was revised again before the actual study was done.The study was approved by the Medical Ethics Committee at the University of Malaya Medical Centre, Kuala Lumpur, Malaysia and the Ministry of Education, Malaysia (reference: number 968.3). Written permission was obtained from the State Education Department in each of the states and federal territories. Written permission was further obtained from the principals of the selected schools. Written informed consent was also obtained from the students, and their participation in the study was voluntary.p < 0.05. Descriptive statistics include the calculation of frequencies for demographic variables, knowledge and health belief items, and mean scores for the knowledge items. Independent t-test and one way analysis of variance (ANOVA) were used for comparison of means and to test the associations between the total knowledge score and demographic variables.All statistical analyses were performed with Statistical Package for the Social Sciences version 20.0 . The significance level was set at A summary of the respondent characteristics is provided in The respondents had received information about HPV infection or vaccination from a variety of sources, including a doctor (53.8%), a teacher (53.4%), television or radio (29.6%), parents (28%), newspapers (21.3%), friends (13.4%), and the internet (11.5%) .only females get HPV infection and more than two-thirds (72.4%) did not know that genital warts are caused by HPV. Nearly half of the respondents disagreed with the statement that vaccinating boys against HPV can help to protect female students against HPV infection (42.2%). Half of the respondents did not know the following that: the HPV vaccine does not eliminate the need for Pap smear tests (51.2%), HPV is a sexually transmitted infection (47.6%), HPV cannot be cured (45.2%) and HPV infections are common and many people have been infected (43.8%). A majority correctly responded that vaccines are available to prevent HPV infection (79.6%), HPV can cause cervical cancer (71.1%) and most people who become infected with HPV do not even know it (66.4%).p = 0.0001). It was also found that the mean total knowledge score was significantly higher among respondents with mothers in professional and managerial capacities  and likewise fathers  than among others. The total knowledge score was also significantly higher among respondents from households with a monthly income above MYR 3000 than from households with an income < MYR 3000 .Across the sample, the mean knowledge score (10 items) was only 3.56  out of a possible score of 10. As shown in the HPV vaccine is good for one’s health just like all other vaccines, and 80.1% believed that taking the HPV vaccine is a good idea as it is recommended by the government. The majority of respondents (84.5%) believed that the HPV vaccination is beneficial. More than half (67%) were worried about getting infected with HPV and had the belief that infection with HPV can lead to serious illness (57.1%). Only 8.4% of respondents felt that they were susceptible to getting infected with HPV. Most respondents (84.2%) reported that they thought that HPV vaccine shots will prevent HPV infection.My religion prohibits me from receiving the HPV vaccine because it is sex related (1.7%), HPV vaccine shots may encourage people to have multiple sexual partners (3.5%), My parents might not allow me to get the HPV vaccine (4.1%), and HPV vaccines are not safe for me (7.2%).The study demonstrates a profound knowledge deficit concerning HPV among HPV-vaccinated female students in Malaysia. Teenagers and women who have given informed consent to receive the HPV vaccine would be expected to have improved their knowledge about HPV , 17. As HPV is a common sexually transmitted infection, this may lead to a low perceived susceptibility to HPV infection. Correcting such a fallacious perception is crucial as it may lead to a greater inclination to engage in protective behaviour connected to their low perceived susceptibility to HPV infection. Therefore, health promotion programmes should emphasize that HPV vaccination reduces, but does not eliminate susceptibility to HPV infections [only females get HPV and did not know that vaccinating boys can help to protect girls. Educational campaigns at the school level will help people to realize that both genders are equally susceptible to HPV infection [Our study revealed that that the students had low perceived susceptibility to HPV. As most of the respondents were unaware that fections . Furthernfection .the HPV vaccine will prevent all HPV infection, and did not know that HPV vaccine does not remove the need for Pap smears. This indicates poor awareness that the vaccine does not protect against all HPV types that are associated with cervical cancer [Many respondents held the misbelief that l cancer , 27 and l cancer . Howeverl cancer . EducatiOnly half of the respondents reported hearing about HPV from teacher, may perhaps imply that targeted public health information provided by school health teams along with the vaccine did not reach all the girls that have received the HPV vaccine. Findings are in concordance with our inquiries prior to the study where some schools reported that HPV information was not provided to the students. Our findings suggest that the health information about HPV should be made compulsory along with the provision of the HPV vaccine. Over half reported receiving HPV information from doctors may imply provision of information by HPV recommending physician during their encounter with HPV recommending physicians . Less thThe key limitation of this study is that all measures were based on the participants’ self-reports, which may cause reporting bias. Despite this limitation, the uniqueness of this study is that it includes a large sample from all parts of Malaysia, allowing for a reliable assessment of HPV knowledge and beliefs among the key population of HPV-vaccinated female students.Despite the provision of a free National HPV Vaccination Programme since 2010, poor knowledge and misconceptions regarding HPV infection and vaccination still exist, along with low perceived susceptibility to HPV among vaccinated teenaged female students in Malaysia. More effective education programmes are much needed to enhance knowledge and heath beliefs, and to ensure sustainable reductions of HPV infection and associated diseases.S1 File(DOCX)Click here for additional data file.S2 File(SAV)Click here for additional data file.


No study has combined tumour and clinical covariates for survival to construct an individual risk-profile for overall survival (OS), time to progression (TTP), and survival after progression (SAP) in patients with HPV+ and HPV– oropharyngeal squamous cell carcinoma (OPSCC). Based on the largest-to-date, unselected, population-based cohort of patients diagnosed with OPSCC, we performed a comprehensive analysis of long-term OS, TTP, and SAP and constructed novel nomograms to evaluate patients' prognoses.P < 0.01). 376 patients failed to maintain disease control with a median TTP of 13 months in the HPV+/p16+ subgroup vs. 8.5 months in the HPV–/p16– subgroup (P < 0.05). HPV combined with p16 status remained one of the most informative covariates in the final Cox regression model for OS, TTP, and SAP.At a median follow-up of 4.0 years (range: 0.8–15.8 yrs.), 690 deaths were recorded. The 5-year OS, TTP, and SAP for the HPV+/p16+ subgroup were 77%, 82%, and 33, vs. 30%, 66%, and 6% for the HPV–/p16– group  between 2000–2014 in Eastern Denmark. Survival rates were estimated by the Kaplan-Meier method. A multivariate Cox regression model was used to construct predictive, internally validated nomograms.We included all patients diagnosed with OPSCC (The HPV+/p16+ subgroup had improved OS, TTP, and SAP compared with other combinations of HPV and p16 after adjusting for covariates. Nomograms were constructed for 1-, 5- and 10-year survival probability. Models may aid patients and clinicians in their clinical decision making as well as in counselling, research, and trial design. In the Western world, the main risk factor for developing oropharyngeal squamous cell carcinoma (OPSCC) is infection with high-risk human papillomavirus (HPV); while, a smaller proportion is related to the high consumption of alcohol and smoking tobacco. Patients with HPV-associated OPSCCs have improved survival, which may be related to a different mutational profile , 2, histProgression is a strong predictor of survival in patients with OPSCCs. The subgroup of patients who are HPV and p16-positive have a lower risk of recurrence and improved SAP compared with patients without HPV/p16 –12. ThesTo avoid selection-bias, we aimed to include all patients diagnosed with OPSCC in the Eastern part of Denmark, which has an approximate population size of 2.4 million inhabitants. The cohort is well described and includes detailed information on both tumour  as well as clinical characteristics  with virtually no patients lost to follow-up. The primary aim was to identify tumour- and patient-factors associated with OS, TTP, and SAP. Due to our unique, complete population-based cohort spanning 15-years, we were able to construct and validate reliable, predictive nomograms.A total of 1,542 patients diagnosed with OPSCC between 2000–2014 were included, and their characteristics are summarized in Table p < 0.05). Other factors significantly associated with a poorer OS in the univariate analysis were: pack years of smoking, treatment, tumour location, and T, N, and M classification  were included, with a total of 690 deaths. In the total cohort, 1,336 patients were treated with curative regimes; of these patients, 543 died during follow-up. The subgroup of HPV+/p16+ had a markedly better prognosis compared with the other combinations of HPV/p16 Table  . The subgroup of HPV+/p16+ had a significantly longer TTP compared with the other combinations of HPV/p16 . The median TTP for the total cohort was 9.7 months. The median TTP was 13 months for the HPV+/p16+ subgroup and 8.5 months (P < 0.05) for the HPV−/p16− subgroup. Median time to loco-regional progression was 7.6 months for the HPV+/p16 subgroup and 7.4 months for the HPV−/p16− group. The corresponding figures for distant progression were 18 and 11 months, respectively, (P < 0.05). The univariate analysis of factors influencing TTP included: pack years of smoking, HPV/16 status, and T, N, and M classification of tumours  in the HPV+/p16+ subgroup vs. 182 (36%) in the HPV−/p16− subgroup . The HPV+/p16+ status also independently influenced SAP positively with a hazard ratio of 2.2 (95% CI: 1.6–2.9) compared with the HPV–/p16– subgroup ; index score 5 to 7, intermediate (< 80% and > 20% probability of surviving 5 years); and index score 8 or greater, high risk (≤ 20% probability of surviving 5 years)  and specified- and non-specified tonsillar squamous cell carcinomas (STSCC and NSTSCC). The latter was according to the certainty of origin. The pathologist validated the diagnosis of OPSCC. The p16 staining was considered positive if there was a strong and diffuse nuclear and cytoplasmic reaction in more than 75% of the tumour cells . The HPVGreen System (GSOpen). Progression was only considered if it was biopsy-verified. Data on progression stem from the DPDR. The TTP was defined as the time from diagnosis of OPSCC to time of progression at any site. In the analysis of TTP, patients were censored either at the last date of follow-up or at time of death. Patients alive at the last date of follow-up were censored in survival analyses for OS and SAP. The date of diagnosis was used to reflect the date of treatment, since the vast majority of Danish patients initiate treatment within 1 month [http://www.statistikbanken.dk/statbank5a/default.asp?w=1344). Missing data were left out of the analysis, and variables with missing values are illustrated in Table p value < 0.05 was considered significant. Data were analysed with SPSS version 23  and R statistics version 3.03.The date of the last follow-up was the 1st of November 2015. The OS was defined as the time from diagnosis of OPSCC to death due to any cause. Data on vital status (death) stems from the real-time, national, patient-administrative  1 month , 41. The 1 month . The mod 1 month . Additio 1 month . The anaWhen performing multivariate analysis of survival, it is of interest to examine how well the models can explain the variation in survival observed in the cohort. The Somers' D for the final model for OS was 0.60, which corresponds to an AUC of 0.80. This was validated with bootstrapping to prevent possible over fitting using the Somers' D rank correlation for the predicted log hazard and observed survival time. The optimism in the model was 0.0064, and the expected bias-corrected AUC was 0.79, suggesting that the model was not over fitted. Finally, the models were evaluated for calibration accuracy in predicting the probability of surviving 5 years. This was performed with bootstrapping to estimate the optimism from the models for how the predicted 5-year survival estimates compared with the observed survival estimates . The cor


The primary objective was to determine human papilloma virus (HPV) clearance rate after cervical biopsy among women with persistent high-risk HPV infection compared with spontaneous HPV clearance rate in the absence of biopsy.We collected data from a dedicated screening program of women aged 30–70 years old. Inclusion criteria for the baseline non-interventional cohort were a positive HPV test  and normal cytology. In the baseline cohort women were followed with approximately yearly HPV-tests and cytology until HPV regressed (one negative HPV test) or interventions in the form of diagnostic biopsies or therapy. Women who had a diagnostic biopsy were included in the biopsy cohort and followed until HPV regression or therapy. Observed HPV regression rates and time to HPV regression were compared between baseline and biopsy cohorts. For the comparison, we used Fisher’s exact test for the HPV regression rates and interval-censored, accelerated failure time model for time to HPV regression.Among the 1079 women included in the baseline cohort, 499 (46.3%) had HPV clearance and 475 were referred for colposcopy with biopsy. The biopsy cohort comprised all women who were not treated and had at least one HC2 test after biopsy . Of those, 138 (68.7%) experienced HPV regression. In the biopsy cohort, time to clearance of HPV infection was approximately halved  compared with the baseline cohort. This result was robust in a wide range of sensitivity analyses.A higher proportion of women cleared their HPV infection, and time to HPV clearance was shorter in the biopsy cohort than in the baseline cohort. It is reassuring for clinicians to know that conservative management of patients with HPV persistency is successful when colposcopy with biopsies excludes high-grade disease. It is well established that infection of the uterine cervix with high-risk human papillomavirus (HPV) types needs to persist for many years or even decades before invasive cervical cancer develops. There are different approaches to estimate the risk of malignant progression associated with HPV infections and the overall risk of cervical cancer. The life-time risk for cervical cancer reaches 6.5% in some areas without cervical cancer screening . HoweverStudies on the clinical course of HPV infections of the uterine cervix require colposcopy with histological assessment (biopsy) at recruitment because cytology or colposcopic impression alone may under- or overestimate the grade of associated CIN lesions . AlthougLong-term observational studies of women with incident or prevalent HPV infections suggest a typical clearance pattern with a high regression rate in the first year of observation and lower regression rates in subsequent follow-up years . It was Wolfsburg pilot project for better prevention of cervical cancer with Primary HPV Screening (WOLPHSCREEN) has been described in detail elsewhere [The lsewhere . In brielsewhere . AbnormaWomen with positive HC2 tests and normal cytology are followed in yearly intervals. After each year, HC2 testing is repeated and those women with a persistent positive HC2 test may be referred for colposcopy. The colposcopy protocol mandates biopsies for all women with a persistent, positive HC2 test result after 1 year, including those with normal Pap smear test results; therefore, more than 90% of referred women undergo histological assessment.The ethics committee of Lower Saxony in Hannover was consulted but approval and/or trial registration was not required because statutory health insurances Deutsche BKK and Audi BKK use the pilot project as their routine screening program. WOLPHSCREEN is managed by Klinikum Wolfsburg, the central database is located in the department of gynecology. The contract between health insurances, gynecologists in private practice, other partners, and Klinikum Wolfsburg encourages the use of WOLPHSCREEN data for research and gives all rights for analyses and publications to the head of the department of obstetrics and gynecology as leader of the scientific team. Participants need to give written consent to be included in WOLPHSCREEN. The written consent includes an agreement that pseudonymized data and left-over material may be used for research.We followed women aged 30 to 70 years with a positive HC2 test and normal Pap smear at recruitment and at least one further HC2 prior to any intervention or colposcopy to determine the natural course of HPV infection. Women stay in the baseline cohort until regression of HPV infection (the first negative HC2 result), cervical biopsy or therapy, or their last HC2 test, whichever occurs first. Women from the baseline group with a positive HC2 test for at least 1 year can be subsequently referred for colposcopy/biopsy and then included in the ‘biopsy cohort’. In the biopsy cohort, women were observed until HPV regression, further biopsy or therapy, or their last HC2 test (the same criteria as in the baseline cohort). The ‘biopsy cohort’, therefore, comprises a subsample of women from the baseline group at a different time point.First, the distribution of sociodemographic and clinical characteristics was compared for both cohorts by means of tabulation and chi-squared test. In this analysis, we did not exclude women who contributed more than one baseline period . Furthermore, we ignored that women included in the biopsy cohort were a subsample of women in the baseline cohort. In the next step, we used an accelerated failure time (AFT) regression model to compare regression of HPV infection in the biopsy cohort with the baseline cohort. AFT is an alternative approach to Cox regression . In the The effects of changing the inclusion criteria for baseline and/or biopsy cohort were studied in four sensitivity analyses (1) further biopsies were no longer considered a censoring event in the biopsy cohort and were included in the analysis; (2) women with ASC-US or more at the time of inclusion in the biopsy cohort were excluded after biopsy; (3) women developing CIN3 at any point during follow-up were excluded in both cohorts; and (4) only the first follow-up period per woman was included in the analysis.From 29,140 follow-up periods available for 22,029 women, 1079 follow-up periods matched all inclusion criteria for the baseline cohort  showed spontaneous HPV regression without any intervention, 10 (0.9%) underwent immediate excisional therapy, and 95 (8.8%) had persistent HPV infection at the end of follow up, but did not have immediate colposcopy for a variety of reasons . The remaining 475 women were referred for biopsy. Among them 201 (42.3%) had follow-up periods with additional HC2 test results and were included in the ‘biopsy cohort’. Of these, 138 (68.7%) experienced HPV clearance and 53 (26.4%) required further biopsies  148 women were referred for excisional treatment without further HPV tests between biopsy and treatment; b) 38 women had a second biopsy before subsequent HPV testing (in these women HPV infection cleared in 27 cases); these 38 cases were included in the sensitivity analysis ‘multiple biopsies allowed’; c) 43 cases whose last HPV test before transition to the biopsy cohort was positive and their first HPV test after the biopsy was negative; these cases represent spontaneous regression, however, we adopted this conservative approach (reducing the possible effects of biopsies) because HPV testing was not done at the time of biopsy and, therefore, it was not clear if clearance occurred in the baseline cohort or after the transition to the biopsy cohort; d) the remaining 45 women were lost to follow-up, mostly because the time for their next scheduled visit was not reached yet at the time of our analysis.The proportion of women who cleared HPV infection was consistently higher in the biopsy cohort than in the baseline cohort Fig. . In the Overall, the results confirm our a priori hypothesis that taking biopsies increases the clearance rate of high-risk HPV infections. The biopsy cohort comprised women with persistent HPV infection from the start of observation for at least 1 year; despite this persistent infection, we observed a faster regression rate of HPV infections in the biopsy cohort than among the baseline cohort. Our data indicate a direct impact of taking biopsies on the subsequent clinical course of HPV infection. In studies examining the clinical course of prevalent HPV infections, the majority of those reporting high regression rates during the first year of observation referred all or at least a substantial proportion of participants for colposcopy with biopsy at the time of recruitment or shortly thereafter , 11–13. The strengths of this study are the large sample size and its population-based character. Despite the large size, all investigations were conducted by a single team using a standardized protocol. The main limitation is that the data do not come from a clinical trial but from real-life cervical cancer screening. Therefore, the probability of censoring (due to treatment or biopsy) is not independent of the results of the HPV tests. In many cases, it is not clear if this under- or overestimates the effect of biopsy, but in cases where the direction of effect is clear, it results in an underestimation and, therefore, we consider our final estimate of biopsy effects as conservative. Furthermore, women screened in the WOLPHSCREEN program are followed up consistently and systematically according to published protocols . Given tHPV persistency over one or more years identifies women at increased risk for long-term viral persistence and the development of CIN3+ , 14. By The findings were also consistent in the sensitivity analyses. The inclusion of follow-up periods with multiple biopsies, most likely indicating HPV persistence, introduces potential bias because these cases were included only in the biopsy cohort and not in the baseline cohort. Consequently, and as expected, in the sensitivity analyses allowing multiple biopsies, the estimates for the effect of biopsy were smaller (indicative of a bias in a conservative direction), but still significant. The advantage of HPV screening is the detection of CIN3 lesions and cancers (CIN3+) that are missed by cytology. These cases of underlying CIN3+ in women with HPV persistence are assumed to have a very low regression rate. As the risk of CIN3+ was higher in the baseline cohort, we did a sensitivity analysis that excluded all cases of CIN3+ in all cohorts to avoid a possible bias related to the different nature of these cases. However, we still observed an increased regression rate of formerly persisting HPV infections in the biopsy cohort compared with the prevalent HPV infections in the baseline cohort. The finding of faster clearance of persistent HPV infection after biopsy among older women is unexpected and intriguing. Previously, we have shown that HC2-testing with a sensitive relative light unit threshold (RLU 1.0) seems reliable even in older women because of cross-hybridization with some potentially oncogenic HPV subtypes . NeverthOur findings are relevant for clinicians and patients because they suggest that observational management of women with HPV persistency is an effective approach. When high-grade disease is excluded by colposcopy with biopsies, the remaining risk of CIN3 during long-term observation is very low , 16, andWe conclude that all studies of the natural history of HPV face a dilemma. On the one hand, the exact identification of HPV-associated disease depends on high-quality diagnoses during follow-up, which in the case of cervical disease are based on colposcopy; however, the accuracy of colposcopy is determined by an appropriate number of punch biopsies , 18. On


While the majority of Human papillomavirus (HPV) infections are transient and cleared within a couple of years following exposure, 10–20% of infections persist latently, leading to disease progression and, ultimately, various forms of invasive cancer. Despite the clinical efficiency of recently developed multivalent prophylactic HPV vaccines, these preventive measures are not effective against pre-existing infection. Additionally, considering that the burden associated with HPV is greatest in regions with limited access to preventative vaccination, the development of effective therapies targeting persistent infection remains imperative. This review discusses not only the mechanisms underlying persistent HPV infection, but also the promise of immunomodulatory therapeutic vaccines and small-molecular inhibitors, which aim to augment the host immune response against the viral infection as well as obstruct critical viral–host interactions. E1, E2, E4, E5, E6, and E7), two late genes (L1 and L2), along with a non-coding region (E6 and E7 are of particular significance due to their roles in inactivation of host tumor-suppressor genes and oncogenic progression [Human papillomavirus (HPV) is a small, double-stranded DNA virus with a genome consisting of approximately 8000 base pairs. The HPV genome encodes six early genes  is one such genetic marker, of which certain alleles seem to have a more prominent association with an inability to clear HPV infection and the subsequent development of cervical cancer ,14,15. GGiven the prevalence of infection with more than one type of HPV among patients, co-infection with multiple HPV types was investigated as a potential predictor of subsequent persistent infection. Results suggest that previous infection with HPV increases the chances of acquiring another HPV infection ,18. HoweApproximately 95% of cervical cancer biopsies contain HPV viral genomes ,22,23. WHPVs are classified into two major subcategories depending on the site of primary infection. Alpha HPVs generally infect genital epithelia and are further designated as high-risk and low-risk depending on their ability to induce cancer . High-riIn addition to being associated with cervical and anogenital cancers, persistent HPV infection has also been linked to head and neck cancers ,27,28. SIn cases of persistent HPV infection, an environment of genomic instability increases the likelihood of viral genome integration into the host genome . In apprE2 open reading frame (ORF) [E6/E7 promoter, is able to displace other transcriptional factors and thus prevent the formation of a transcription initiation complex [E2 expression is associated with the oncogenic progression of HPV as it consequently deregulates and increases the expression of viral oncogenic proteins, E6 and E7, which are known to disrupt tumor-suppressor genes p53 and pRB, respectively [A proposed consequence of viral genome integration that can result in oncogenic progression is the disruption of the me (ORF) . Normallme (ORF) . It was  complex ,37. The ectively ,39. It hectively . Integraectively ,34. UltiAfter escaping the initial immune response, viruses must maintain their genomes within the host nucleus in order to achieve persistent infection. While integration into the host genome is an option favored by many chronic viruses, papillomaviruses like HPV maintain their genomes as extrachromosomal episomes that tether to host DNA . By contIn order to ensure the viral genome is not lost during cell division, there is a tethering mechanism in place that attaches the viral genome to host mitotic chromosomes through protein intermediates . TetheriInterestingly, one study demonstrated that E2 protein encoded by HPV 11, 16 and 18 has the property to directly interact with the mitotic spindles, thereby maintaining HPV ori-containing DNA as “mini-chromosomes” in dividing cells. The likely region of HPV E2 involved in this mitotic spindle interaction is thought to be a 14-amino acid sequence that is highly divergent from its analogous sequence in BPV 1 E2 . It is lBecause of the critical role E2 plays in maintaining persistent infection, its interactions with host proteins are of particular interest. Though nearly all papillomaviruses express E2 and maintain their genomes through episomal tethering to mitotic chromatin, the host proteins targeted by the viral E2 protein are largely dependent upon the specific papillomavirus type .An early proteomic study identified Bromodomain-containing protein 4 (BRD4), also known as mitotic chromosome-associated protein (MCAP), as a critical binding partner for E2 . BRD4 isIn addition to episome maintenance, it has been demonstrated that BRD4 is vital for E2’s ability to activate transcription in all papillomaviruses ,59,60. AThe diversity within the papillomavirus clade supports the ability of its viruses to interact with various host cellular partners. Researchers have shown that α and β HPV E2 proteins likely interact with cellular targets in addition to BRD4 and furthermore, bind to distinct regions of the host chromosome ,64. In aIn addition to TopBP1, ChIR1 has been shown to be a key cellular partner of E2. Parish et al. demonstrated through intracellular co-localization experiments that HPV 11 and BPV 1 E2s co-localize with ChIR1 on the chromosome during prophase ,68. WhilThe development of prophylactic vaccines, such as Gardasil which targets HPV 6, 11, 16 and 18, marked a turning point in the HPV field as it provided an efficient preventative public health measure against HPV . HoweverNational two-dose vaccine programs remain both cost-effective and successful in preventing persistent HPV infection . In receCurrently, clinical studies are also being conducted on potential therapeutic vaccines, which, unlike prophylactic vaccines, fight HPV infection via immunotherapy . Most caE6 and E7 genes within the DNA vaccine is shuffled to render the recombinant proteins incapable of degrading p53 and pRb [Numerous DNA-based vaccines have been developed to target persistent HPV infection and are currently in various stages of clinical study. These vaccines function by introducing a significant amount of viral DNA intradermally or intramuscularly to myocytes, which then express the antigen encoded by the DNA. The expressed, secreted antigen is then recognized and engulfed by dendritic cells and is subsequently expressed on MHC complexes, which are presented to CD8+ T-cells ,78 FiguA. GX-188 and pRb . GX-188E and pRb . HoweverElectroporation, alongside DNA vaccination, has been shown to augment vaccine efficiency by modifying the cell membrane to increase DNA uptake and promoting inflammation and recruitment of APC’s at the site of vaccination . In vitrTo counter this problem of low vaccine efficacy, new models of therapeutic vaccines have been proposed. For instance, a recent study utilizing live attenuated influenza A virus as the vaccine vector for the expression of HPV E6–E7 fusion transgenes demonstrated its ability to elicit a broader immune response relative to vaccination with the recombinant protein alone . In thisClinical studies have demonstrated the potential of utilizing Modified Vaccinia Ankara (MVA) as an alternative attenuated viral vector for designing therapeutic vaccines as well. Recombinant MVA engineered to express BPV E2 (MVA E2) has demonstrated its potential to serve as an alternative intralesional treatment for HPV-induced lesions ,83. FollDendritic whole-cell based (DC) vaccines have also been proposed as a therapeutic strategy for patients with early stages of cervical cancer D. These Despite the advances made, there are significant challenges in the development of therapeutic vaccines . Many fo®, a zinc-citrate compound, was effective in eliminating several types of cervical high-risk HPV infection [An interesting pilot study has found that an intra-vaginal infusion of CIZARnfection . Althougnfection . FurtherAs may be inferred from previous discussion, protein–protein interactions provide more specific therapeutic windows to clearing persistent HPV replication. By interrupting processes critical to the HPV life cycle such as DNA replication, episome maintenance, or viral transcription, latent infection may be terminated because the virus is unable to maintain its genome in dividing cells. Small molecular inhibitors and nucleic acid-based treatments that target these interactions have shown to be promising non-conventional therapeutics and are currently undergoing clinical trial . HoweverThe interaction between E2 and its host binding partners is a strong prospective target for therapeutic development because of its role in episomal maintenance, as well as viral transcription and replication. For example, blocking the BPV E2–BRD4 interaction prevents mitotic chromosomal localization of E2 and viral genome, thereby preventing episomal maintenance . In lateNevertheless, it is crucial to consider that therapeutics targeting E2 and host interactions may prove only to be relevant in the stages of HPV infection in which integration has yet to occur in order to eliminate episomal HPV. Although proposed E1–E2 inhibitors have the same theoretical effect in terms of inhibiting persistent infection by preventing viral replication, the nuanced variations in E1–E2 structural binding between papillomavirus genera make it a difficult therapeutic target . Due to While the advancement of Gardasil and other multivalent prophylactic vaccines provide preventative public health measures against HPV infection, the burden associated with HPV remains high in regions where access to regular screening and vaccination are unavailable. Therefore, the development of antiviral agents along with therapeutic vaccines to treat HPV post-infection remains imperative.Interrupting virus-host interactions critical to HPV persistence demonstrates great potential to terminate persistent HPV infection, which may otherwise proceed to malignant cancer. Notably, these protein–protein interactions are relatively divergent between PV types and miniscule changes in relevant target protein amino acid sequences can drastically affect drug efficiency. Therefore, the likelihood of developing a pan-HPV antiviral currently remains low. Targeting the better-conserved cellular proteins with which HPV proteins interact provides an alternative option for developing a pan-HPV therapeutic; however, the deleterious effects caused by interrupting these proteins during normal cellular processes may outweigh the potential benefits derived from interfering with viral function. Specific drug delivery measures and research into the comprehensive cellular pathways associated with HPV pathogenesis may minimize these unfavorable outcomes. Practically speaking, multiple classes of antiviral drugs may be necessary in order to efficiently treat persistent HPV infection based on HPV types, stages of infection, and sites of infection. Despite these reservations, insights into the molecular mechanisms of HPV pathogenesis revealed in the last decade hold promise for the development of highly specific and effective antiviral agents and immunomodulatory vaccines for treating HPV persistent infection.


In Sweden, where screening ends at the age of 60, about 30% of the cervical cancer cases occur in women older than 60. The aim of the present study was to investigate the prevalence of HPV and cervical dysplasia in women of 60 years and above.From September 2013 until June 2015, 1051 women aged 60–89 years (mean 68 years) were sampled for an HPV test when attending an outpatient gynecology clinic. Women with positive results had a second HPV test and liquid based cytology (LBC), after 3.5 months on average. Those with a positive second HPV test were examined by colposcopy, and biopsy and a sample for LBC was obtained.The prevalence of HPV was 4.1%,  at the first test, and at the second test 2.6% remained positive . The majority of women positive in both HPV tests, had dysplasia in histology, 81.5% (22/27) . HPV-related dysplasia was found in 2.1%,  of the 1051 women. Four of the 22 women with positive HPV tests also had abnormal cytology, one ASCUS and three CIN 1. No cancer or glandular dysplasia was detected.A significant proportion of elderly women were found to have a persistent cervical HPV infection. Among them there was a high prevalence of CIN diagnosed by histology. The HPV test showed high sensitivity and specificity in detecting CIN in elderly women, while cytology showed extremely low sensitivity. There is a lack of knowledge concerning the prevalence of infection with oncogenic types of human papilloma virus (HPV), and cervical dysplasia in women of 60 years and older. Older women often ask for continued screening for cervical cancer (CC) after the organized screening is terminated. In Sweden, a combination of organized and opportunistic Pap smear screening has reduced the incidence of squamous cell cancer in cohorts most regularly screened, by around 70% . The prePersistent infection with oncogenic types of human papilloma virus (HPV) is the major cause of cervical cancer . As HPV In post-menopausal women, due to hormonal changes, the transformation zone where precursor lesions develop, is situated in the cervical canal and is therefore not accessible for proper examination and sampling . As a coThis retrospective study is based on 1051 women aged 60–89 years (mean age 68 years), attending an outpatient gynecology clinic and having an HPV test as part of a gynecological examination . The stuAll LBC specimens were screened by cyto-technicians and those considered as abnormal were reviewed by a surgical pathologist. For cervical cytology The Thin Prep Pap Test was used. The cervical smear was collected with a plastic spatula and a cyto-brush. LBC specimens were placed in PreserveCyt solution and processed in the Thin Prep 5000-processor, HologicCytyc Corporation, Boxborough, Mass.) . SpecialThe HPV test was performed using a multiplex real-time PCR assay (hpViR) as earlier described, which detects the following high-risk HPV types 16,18,31,33,35,39,45,51,52,56,58 and 59 . A cervical sample was collected using a cytobrush. The sample was applied to a filter paper matrix, an indicating FTA elute card . Data was accessed anonymously. This retrospective study on clinical data did not require patient consent according to the Ethical Review Board. The HPV positive women have given written informed consent for historical data as approved by the Regional Ethical Review Board, Uppsala, Sweden (Dnr 2016/441).For statistical analysis the Statistical Package for Social Sciences (SPSS) version 22 for Windows and Excel were used. A p-value less than 0.05 was considered statistically significant. The age groups 60–64 years (n = 277), 65–69 years (n = 394), 70–74 years (n = 235), 75–79 years (n = 95), 80–84 years (n = 41) 85–89 years (n = 9) and 60–69 years (n = 671), 70–79 years (n = 330), 80–89 years (n = 50) were compared. For statistical significance testing between age groups the chi-squared test in SPSS was used. Confidence intervals (CI) of proportions (Fleiss) were calculated using Excel .All 1051 women offered testing, accepted an HPV test, and all samples were considered adequate and analyzed. The study design and HPV and dysplasia occurrence is shown in Of the 27 women that were positive for HPV in the second test, four had either atypical squamous cells of undetermined significance (ASC-US) or CIN 1 in cytology. One of them had benign histology and turned HPV and cytology negative after one year. Of the 22 women with dysplasia in histology, 19 (86.4%) had a normal cytology while 3 (13.6%) had dysplasia  also in the cytology. Women who were HPV negative in the second HPV test, 37.2% (16/43), all had a normal cytology and were scheduled for a follow up HPV test after one year.In the third HPV test of 15 women after one year , 2 (13.3%) women were HPV positive with the same HPV type as in the first test. Cytology and histology showed CIN I in one woman and was normal in the other. There was no atypia of glandular cells, adenocarcinoma in situ or adenocarcinoma found. In screening history (from 1986 and onward) of the 43 women that had a positive first HPV-test 16 (37.2%) had a history of dysplasia . Ten of these women (23.3%) had dysplasia before 60 years of age and 6 women when older than 60.All HPV types tested for were found . MultiplAt colposcopy of 26 of the 27 women who were also HPV positive in the second HPV test, the transformation zone (TZ) was not visible in 17 (65.4%), and partly visible in 9 (34.6%) of the cases. No one had a fully visible TZ. Vaginal lesions were found with colposcopy in three cases and biopsy for histological evaluation showed vaginal intraepithelial dysplasia VAIN I in all three cases. All three women with VAIN also had CIN I.There were no differences in prevalence of HPV or dysplasia between age groups (5 years and 10 years), and there were no HPV positive women above the age of 80 (n = 50). The most prevalent HPV types were HPV 16 (27.9%) 12/43, followed by HPV 33/52/58 (20.9%) and HPV 31 (14.0%) . The disTo our knowledge this is the first study on the prevalence of HPV and the association between HPV and dysplasia in elderly women. We found that the HPV prevalence was rather low in older women, but if they also tested positive in a second HPV test, the risk of cervical dysplasia was high. Since dysplasia was not detected by cytology in the vast majority of cases, LBC does not seem to be an appropriate method for screening women older than 60. In Sweden women are invited to the screening program until the age of 60, meaning that the current study focused on women who are no longer screened.In the current study 4.1% of the women tested were HPV positive. As a comparison Ferenczy et al found that only 1% were HPV positive, out of 306 tested postmenopausal women aged 50–70 years . The lowHPV clearance between test one and test two was as high as 37.2% after on average 3.5 months. This is surprisingly high since a study on women aged 30–50 years showed 29% clearance after on average 4.4 months . In another study on women aged 30–65 years the clearance was 41% with 2.7 months between test one and test two . It has We found poor correlation between LBC and histology, with only three with a positive cytology out of the 22 with dysplasia in histology, i.e. 13%. Cytology is thus not an appropriate method for the detection of cervical dysplasia in this age group. We also found that almost half of the women had a nonvisible TZ and none had a fully visible TZ. Colposcopy thus gave poor guidance for taking biopsy, as the transformation zone was not fully visible. We suggest this constitutes a potential sampling error for both cytology and histology due to an inability to visualize and sample from the TZ, which is the origin for precancerous lesions ,30,31.Biopsies lacking the TZ are not reliable and thus diagnostic conization should be offered to women with negative biopsies. In our experience there are additional difficulties in diagnostics in older women, due to vaginal atrophy and sometimes also vulvar disease such as lichen sclerosis.Despite accounting for known risk factors, Charlton et al. were unable to predict individual patients' probability for progression of low grade squamous epithelial dysplasia (LSIL), to high grade dysplasia (HSIL) and adenocarcinoma in situ (AIS), on the basis of available data in women 16–66 years (mean 30.5 years) . In younThere is a need for future studies to form a basis for a screening algorithm adapted for postmenopausal women. In our experience Swedish women exit the screening program sometimes with the misleading information that they are not at risk for cervical cancer. Recently the Swedish Board of Health and Welfare has recommended a screening program based on HPV testing, which is extended until the age of 64 . Our cliWe hope that our results will serve as motivation to conduct studies focusing on older women also, in order to effectively reduce the prevalence of cervical cancer in this age group. Moreover it is clear that persistent cervical HPV infection is common in this age group and that the vast majority have cervical dysplasia. It is also clear that cytologically-based screening is not meaningful in this age group due to the low sensitivity of this method. The next step would be to introduce repeat HPV testing as a screening method and to find algorithms for sampling strategies such as self-sampling, test intervals and treatment options.S1 DatasetRaw data PLOS ONE.(XLSX)Click here for additional data file.