Cancer of the uterine cervix is the leading cause of cancer-related death among women in Sub-Saharan Africa, but information from the Democratic Republic of the Congo (DRC) is scarce. The study objectives were to: 1/ assess prevalence of (pre)cancerous cervical lesions in adult women in Kinshasa, 2/ identify associated socio-demographic and behavioural factors and 3/ describe human papillomavirus (HPV) types in cervical lesions.A cross-sectional study was conducted in Kinshasa. Between 2006 and 2013, four groups of women were recruited. The first two groups were included at HIV screening centres. Group 1 consisted of HIV-positive and group 2 of HIV-negative women. Group 3 was included in large hospitals and group 4 in primary health centres. Pap smears were studied by monolayer technique (Bethesda classification). Low- or high-grade squamous intraepithelial lesions or carcinoma were classified as LSIL+. HPV types were determined by INNO-LiPA®. Bivariate and multivariable analyses ) were used to assess associations between explanatory variables and LSIL+.n = 131 HIV-positive women), 3.9 % in group 2 (n = 128 HIV-negative women), 3.9 % in group 3 (n = 539) and 4.1 % in group 4 (n = 220). The following variables were included in the GEE model but did not reach statistical significance: history of abortion, ≥3 sexual partners and use of chemical products for vaginal care. In groups 3 and 4 where this information was available, the use of plants for vaginal care was associated with LSIL+ . The most common HPV types among HIV-positive women with ASCUS+ cytology (ASCUS or worse) were HPV68 (12 out of 50 samples tested), HPV35 (12/50), HPV52 (12/50) and HPV16 (10/50). Among women with negative/unknown HIV status, the most common types were HPV52 (10/40), HPV35, (6/40) and HPV18 (5/40).LSIL+ lesions were found in 76 out of 1018 participants. The prevalence was 31.3 % in group 1 (LSIL+ lesions are frequent among women in Kinshasa. The use of plants for vaginal care deserves attention as a possible risk factor for LSIL+. In this setting, HPV16 is not the most frequent genotype in samples of LSIL+ lesions. Cervical cancer constitutes a major health problem worldwide. It is responsible for 530,000 new cases of cancer and causes 270,000 deaths each year , 2. Up tThe link between cervical cancer and HPV infection has been well established , 19–22. In most Sub-Saharan countries, data on the prevalence and mortality of cervical cancer are either sparse or unavailable. Only 17 % of African countries have a national programme and a specific budget for fighting cervical cancer. And where such a cervical cancer programme exists, the effective coverage may be low. In addition, those women at the highest risk of developing cervical cancer may have the most difficult access to care , 34. TheBecause of the high burden of cervical cancer in Sub-Saharan Africa, and presumably also in the DRC, the primary objectives of this study were to: 1/ evaluate the prevalence of (pre)cancerous lesions, 2/ identify associated socio-demographic and behavioural factors and 3/ describe HPV types present among women in Kinshasa.The total number of participants was 1018. One hundred thirty-one HIV-positive women were recruited at HIV screening centres (group 1); 128 HIV-negative women came from the same HIV screening centres (group 2); 539 women were recruited at large hospitals (group 3); and 220 were referred through small health centres (group 4). The HIV status of the women in groups 3 and 4 was unknown.Table Behaviour-related characteristics of the study population are shown in Table In total, 76 of 1018 women were diagnosed with LSIL+ lesions  or invasive cancer, Table P-value of <0.2 were included in a model of multiple logistic regression (one model per study group). The adjusted ORs are given in Table In each of the four groups, bivariate associations were assessed between all explanatory factors  and the presence of LSIL+ lesions. Crude odds ratios (ORs) are given in Table P-value <0.2 on bivariate evaluation were included in a multivariable model. The final GEE model  included history of abortion , more than three sexual partners  and use of chemical products for vaginal care . Adding age to the model did not substantially change the ORs. None of the associations in this model reached statistical significance  to account for clustering within the study groups. Also in the GEE analysis, all factors with a The use of plants for vaginal care could only be evaluated in groups 3 and 4 because this information was not available for the women in the other groups. In a multiple logistic regression model including the use of plants, alcohol consumption and having had three or more sexual partners, the adjusted OR for the association between the use of plants for vaginal care and the presence of LSIL+ lesions was 2.70 , HPV52 (n = 12), HPV68 (n = 12), HPV51 (n = 10), and HPV31 (n = 9). In the groups of women with negative or unknown HIV status, 50 samples were tested with INNO-LiPA of which 40 contained detectable HPV DNA (Table n = 10), HPV35 (n = 6), HPV16 (n = 3), HPV18 (n = 5), HPV51 (n = 5), and HPV54 (n = 5). In addition to the three samples in which HPV16 was detected through the INNO-LiPA test, there were three samples in which both the Abbot Real Time and the GenoID test indicated the presence of HPV16.The following cytology results were classified as ASCUS+ and the corresponding samples were submitted for HPV typing: ASCUS, ASC-H, LSIL, HSIL or carcinoma. In the HIV-positive group, 50 out of the 52 ASCUS+ samples contained HPV DNA. HPV16 was found in 10 out of 50 samples, and HPV18 in 5 Table . Other gNA Table . Here, tIn groups 2, 3 and 4, 55 % of samples which tested positive for HPV DNA contained one single HPV type. Two genotypes were found in 23 % of the samples, three genotypes in 9 %, four in 7 % and more than four in 7 % of HPV-DNA positive samples. In the HIV-positive group (group 1), a single HPV infection occurred only in 20.0 % of the samples. Two genotypes were found in 38 %, three in 9 %, four in 16 % and more than four in 18 % of the samples.The present study was performed to assess the prevalence of LSIL+ lesions and to identify associated factors in different groups of women in Kinshasa. The prevalence of LSIL+ lesions ranged from approximately 4 % in women with unknown or negative HIV status to 31 % in HIV-positive women. We found an association between the practice of intravaginal insertion of plants and the presence of LSIL+. HPV types 16 and 18 which are known to cause cervical cancer in many countries worldwide appear to be less predominant in women in Kinshasa.The prevalence of LSIL+ lesions that we found in the current study in women with unknown or seronegative HIV status (4 %) is consistent with the few studies previously performed in Kinshasa (3 % and 5 %) , 36 and Cervical cancer is known to be more frequent among HIV-positive women. In the DRC, it is estimated that 1.9 % of the adult women are HIV infected, with differences between women living in urban areas (2.4 %) and rural areas (1.0 %) . We founSeveral demographic, economical and behavioural risk factors have been studied in relation to cervical cancer. Most of them may influence the risk of cancer through their effects on the risk of HIV and HPV infection. In the current study, we found a significant association between the intravaginal application of plant products and the presence of LSIL+ lesions. It is a frequent practice in Sub-Saharan Africa to use herbs, leaves and bark of trees to reduce vaginal lubrication and increase friction during sexual intercourse (dry sex) –59. The Most of the studies about the vaginal use of plants have been done in relation to the risk of HIV infection. It has been hypothesised that differences in the vaginal environment may partially explain the different HIV transmission probabilities that are observed across populations . SimilarThe association between HPV infection and cervical cancer is well established, but the specific HPV genotypes that are involved in neoplasia differ across populations. The HPV types that have been most frequently linked to cervical cancer are: 16, 18, 31, 33, 45, 52 and 58 , 65. HPVIn other Sub-Saharan countries, the frequency of HPV16 and 18 varies. Some countries have reported patterns that resemble the situation in Europe and the United States , 66, 67,The DRC is a large country that is facing many complex problems at the same time. As a consequence, cervical cancer is not getting the attention that would be required for adequate disease control. Yet, the burden caused by cervical cancer in the DRC would justify a coordinated control strategy. This study together with a previous report illustrates that women in Kinshasa are willing to participate in prevention and control activities . InterveOur work illustrates that the prevalence of (pre)cancerous lesions in women from different districts in Kinshasa is approximately 4 %. In HIV-positive women, the prevalence is about eight times higher. Traditional practices concerning vaginal hygiene may increase the risk of malignant transformation. More extensive studies, including rural areas, are needed to unravel the contribution of different HPV types in the development of cervical cancer.n = 131) consisted of women who were found to be seropositive for HIV; the second group (n = 128) consisted of HIV-seronegative women. The diagnosis of HIV was based on two rapid tests (Determine®HIV-1/2 (Abbott) and OraQuick®HIV-1/2 (OraSure Technologies)) combined with one of the following serological tests: Vironostika® Uni-Form II Plus O (BioMérieux), Enzygnost® Anti-HIV ½ Plus (Siemens), or Inno-Lia HIVI/II Score . The third group (n = 539) consisted of women who consulted the gynaecology department of the Provincial Reference Hospital of Kinshasa  and the Ngaliema hospital. Both hospitals are large state hospitals in the centre of the city. Data were collected from July to August 2009. At that time, a sensitisation campaign for free cervical screening was broadcast on television, posters advertising cervical cancer screening were shown at the hospitals, and a symposium on cervical cancer took place at the HPGRK. The participants of the fourth group (n = 220) were recruited via primary health care centres in the communities of Kimbanseke, Kisenso, Ndjili and Lemba, located in the poorer suburbs of the city. Data were collected from September 2012 to January 2013 after a campaign in local churches for free cervical cancer screening.We performed a cross-sectional study on the prevalence of precancerous and cancerous lesions of the uterine cervix in four groups of women in Kinshasa. The first two groups consisted of women who participated in a voluntary screening programme for HIV in the ACS/AMO-CONGO centre (Action Communautaire contre le Sida/Avenir Meilleur pour les Orphelins du Sida au Congo) and in the Centre Hospitalier Monkole 3. Women who visited these centres for HIV screening and care between September 2006 and January 2007 were invited to participate in free cervical cancer screening. The first group (All women older than 17 who were willing to participate in the study and for whom a liquid-based cytology (LBC) result was available were included in the analysis.Cytology was the main outcome variable. Cervical smears were collected with Cervex Brush and conserved in ThinPrep solution for LBC and HPV typing. Samples were kept at 4 °C. ThinPrep vials were transferred to the Pathology Laboratory of the University Hospital of Ghent, Belgium, for further analysis. The collected smears were independently read and interpreted by two pathologists. The pathologists were not aware of the HIV and HPV status at the time of reading the microscopy slides. In case of discrepancy, the slides were reread by both pathologists for a final interpretation. The cytology results were reported according to the Bethesda Classification 2001 of cervical pathology. Women with cytology results indicating LSIL , HSIL  and invasive carcinoma were considered to have precancerous or cancerous lesions and classified as LSIL+ (according to ). Women HPV typing was done in participants with ASCUS+ cytology results. Samples from women with ASCUS and ASC-H were included in the HPV evaluation as the European guidelines recommend to determine HPV DNA in these groups. INNO-LiPA HPV Genotyping Extra  was used for HPV typing. It is molecular technique based on the principle of reverse hybridisation designed to recognize fifteen high-risk HPV types , three probable high-risk HPV types , seven low-risk HPV types  and three non-classified HPV types .) The testing strategies varied across the study groups. In groups 1 and 2, INNO-LiPA Genotyping was done for all ASCUS+ cases. In groups 3 and 4, the INNO-LiPA test, due to its high cost, was only done if two other tests gave discordant results  and Full Spectrum PCR HPV Amplification and Detection/Genotyping System ). We present all available INNO-LiPA results because this test detects many different HPV types . Concordversus any consumption), number of pregnancies, number of childbirths, and history of abortion (without discrimination between spontaneous or provoked). Concerning the practice of intravaginal application of plants, information was only available for groups 3 and 4.Socio-economic and behaviour characteristics that were studied in association with the presence or absence of LSIL+ were: age, age of menarche, age of first sexual contact, marital status, formal employment, life-time number of sexual partners, use of products for vaginal care , alcohol consumption  is reported for each of the four groups. Next, we used bivariate and multiple logistic regression to assess the association between socio-demographic and behaviour variables and the presence of LSIL+ lesions within each of the four groups. Finally, we analysed all participants together using generalized estimating equations (GEE) to account for clustering within the groups. The results of all logistic regression and GEE analyses are reported as odds ratios (OR) with 95 % confidence intervals (95 % CI). Data was missing for some participants in some of the explanatory variables; for each of the analyses, the number of included participants is given. We used Stata/IC 10.1 for data analysis.The study protocol on the collection of data and the reporting of data to participants was approved by the Ethics Committee of the School of Health of the University of Kinshasa. After having explained the objectives of the study, all study participants signed a document of informed consent.


I am privileged to guest edit this Frontiers Research Topic addressing HPV infection and cancer disparities with the expert assistance of Dr. Silvia De Sanjose and Dr. Ala-Eddin Al Moustafa. We are, hereby, thankful to authors of manuscripts who had responded and enriched the topic with their valuable contributions. The findings of these manuscripts are interesting and contribute to our understanding of the complexity of HPV infection, socio-economic inequalities, and related cancers in a number of unprivileged populations.Giorgi Rossi and colleagues present an algorithm to model the impact on inequalities of introducing HPV-based screening of cervical cancer . They foTwo papers explore HPV infection and cervical cancer screening in migrant populations. Rodriguez-Sales and colleagues examine the coverage of cervical cancer screening and prevalence of cytological abnormalities in Catalonia by immigration status . They foWood and colleagues report on the Canadian first nations (FN) women who endure a disproportionate burden of ill health in contrast to the mainstream population . The autTwo manuscripts deal with HPV-related cancers in Middle-Eastern countries Saudi Arabia and Syria, both lacking national screening and vaccination programs for cervical cancers , 6. The Hernandez and colleagues assessed the HPV prevalence in 79 invasive penile cancer patients diagnosed in 1998–2005 within 7 U.S. cancer registries (Another interesting manuscript by Vogt and colleagues deals with the concordance of oral and genital HPV infection in South Africa . They stTwo manuscripts deal with HPV in women infected with the HIV , 10. McKThis compilation of manuscripts addresses important health disparities with regard to HPV infection and related cancers. HPV infection and cervical cancer is of international interest, as almost 85% of 530,000 cases diagnosed in 2012 worldwide occurred in low- and middle-income countries. The social and economic impact of HPV infection is substantial, as cervical cancer disproportionately affects more young women. Although health authorities in many countries do not currently address these issues, it is important to emphasize that HPV-related cancers can be diagnosed early with screening and are largely preventable with vaccination.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The APOBEC3 family of cytidine deaminases plays critical roles in host innate immunity. These enzymes function as viral DNA mutators capable of restricting an array of virus infections . Given tHuman papillomavirus (HPV)-associated cancers, including cervical cancer and a subset of head and neck cancers, are enriched in APOBEC3-type mutation signatures . To deteHPV is one of the most common sexually transmitted pathogens. Nevertheless, the vast majority of HPV infections are self-limiting, suggesting that host immunity is efficient at eliminating the virus. We recently found that A3A, but not A3B, inhibits HPV infection in a deaminase dependent manner . It is iElevated A3A expression in HPV-positive cells may limit viral fitness. Thus, HPV must counteract A3A to cope with its restrictive effects. The HIV-1 viral infectivity factor (Vif), one of six accessory proteins, blocks the antiviral activity of APOBEC3G (A3G) by promoting its ubiquitination and proteasome-dependent degradation [http://pave.niaid.nih.gov). We revealed that TC dinucleotides are highly depleted in papillomavirus genomes. The extent of the depletion is dependent on the tropism of the virus, as mucosotropic viruses of the Alphapapillomavirus genus exhibit the greatest degree of TC depletion. Interestingly, mucosal tissues displayed significantly higher expression of various APOBEC3 family members compared to cutaneous skin [APOBEC3s deaminate cytidines with a preferred dinucleotide context. A3G preferentially targets CC dinucleotides, while the remaining family members (APOBEC3A/B/C/D/F/H) target TC dinucleotides . Given tous skin . The higOur studies suggest a model in which the HPV oncoproteins induce A3A and A3B expression during virus persistence. If APOBEC3 activity is sufficiently high for virus restriction, HPV infection is inhibited and viral genomes are eliminated. To avoid the antiviral effects of APOBEC3, HPVs may have evolved to limit the number of TC dinucleotides in their genomes. Surviving viral genomes continue to induce A3A and A3B expression throughout disease progression, which may lead to off-target effects on the host genome that fuel oncogenic mutations Figure .Recent findings raise important questions: What additional factors and mechanisms do lead to deregulation of APOBEC3s? If cancer is considered as an evolving clonal disease, what impact does A3A and/or A3B have during this process? Can APOBEC3 activity serve as a prognostic indicator for risk of cancer progression? Further studies are necessary to understand these dynamic host-pathogen interactions to identify new ways to slow or stop disease progression.


The explanatory variables statistically significant at p≤0.05 were dyskeratosis (p=0.01) and papillomatosis (p=0.04). Forty-nine independent variables  were involved in the second regression analysis. The model correctly classified 85.2% of the lesions and showed a Hosmer-Lemeshow adjustment of p=0.696 and a Nagelkerke’s coefficient of determination of 60.2%. The explanatory variables statistically significant atp≤0.05 were: age younger than 35 years (p=0.001), multiple lesions (p=0.031), hyperorthokeratosis (p=0.019), focal intracellular edema (p=0.002), and the presence of 1 to more than 5 cells with degenerative changes in their nucleus (p=0.048).Conclusions: Toki’s criteria are not adequate to make a diagnosis of lesions by HPV in the mouth, but the logistic regression analysis showed clinical and histopathologic variables which optimize the identification of lesions through the L1 protein. However, a PCR study is advisable when the presence of high-risk HPV is suspected.Objectives: To evaluate the effectiveness of Toki’s criteria in identifying the HPV L1 protein in oral lesions with the use of immunohistochemistry (IHC) and to determine which criteria optimize such identification. Study Design: Retrospective study of 277 cases diagnosed as HPV lesions at 22 years. Tests of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), kappa coefficients, and chi2 values, as well as two logistic regression analyses (p≤0.05), were conducted. Results: Of the lesions studied, 96.4% (267 of 277) were positive for HPV using Toki’s criteria and 28.5% (79 of 277) were positive for L1 by IHC. Toki’s criteria showed sensitivity=93.67%, specificity=2.53%, PPV=6.99%, and NPV=46.55%. Neither concordance nor statistically significant associations were observed between both tests. The logistic regression of Toki’s criteria was useful in the diagnosis of L1, correctly classified 71.8% of the lesions positive for L1, and showed a Hosmer-Lemeshow adjustment of  Key words:HPV, Toki’s criteria. The human papillomavirus (HPV) is a member of the papillomaviridae family , which cIn 1987, Toki defined the use of a scoring system called “HPV score” for detecting HPV in cervical lesions. The system consists of giving a score on six histologic findings: koilocytosis, 4; bi- and multinucleation, 2; dyskeratosis, 1; intraepithelial capillary loops (papillomatosis), 1; basal cell hyperplasia, 1; and acanthosis, 1. The maximum score using this system is 10 points, and a diagnosis of HPV is declared when a score of over 6 points is achieved . HoweverThe HPV contains 7,200 to 8,000bp in a circular double-stranded DNA molecule covered by a capsid of proteins of icosahedral form with 72 capsomeres . Their DThe expression of early genes occurs throughout all epithelial layers . The E1 In infections of high risk, the integration of the viral DNA into the cell genome alters the E2 ORF, which causes the loss of the repressive function of the E2 protein, increasing the expression of the E6 and E7 oncoproteins . This faThe objectives of this study are to assess the efficacy of Toki’s criteria in identifying through IHC the presence of HPV L1 protein in oral mucosal lesions by HPV and to determine the clinical and histopathologic variables that optimize such identification.This is a retrospective and descriptive study of 277 biopsies fixed in 4% formaldehyde buffer, embedded in paraffin, preserved at ambient temperature, and previously diagnosed through histopathologic study as lesions by HPV over a period of 22 years (1990-2012). Lesions with diagnosis of multifocal epithelial hyperplasia (MEH), verruca vulgaris, and condyloma acuminatum, which are considered as lesions by HPV ,11 were The diagnoses were established with the use of clinical and morphologic criteria. Unique or multiple, small, white or pink papular lesions featuring sessile elevations which may show finger-like projections of stratified squamous epithelium with wide variations in the thickness of the keratin layer, usually parakeratinized and koilocytosis microscopically ,11.The clinical characteristics were obtained from the histopathologic applications of patients, and the morphologic data were gathered from the observation of cuts on slides with the use of an Olympus optical microscope. To determine the presence of HPV L1, tissues were stained by IHC.Forty-nine independent variables consisting of 15 clinical and 34 histopathologic characteristics categorized as structural or cellular variables were evaluated.- Clinical variables: Age and gender of the patient, and location, number, contour, base, surface, consistency, color, type, edge, mobility, pain, diameter, and evolution of lesions.- Structural variables: The presence of papillomatosis, pseudoepitheliomatous hyperplasia, acanthosis, and a granular layer ; the presence and intensity of hyperparakeratosis, hyperorthokeratosis, or both keratins (considering only the intensity of the hyperkeratosis presented); epithelial hyperplasia, inflammatory infiltrate, and basal cell hyperplasia; and the presence, intensity, and disposition of both intracellular and extracellular edema were evaluated.- Cellular variables: The presence of dyskeratosis, pinocytosis, keratin pearls, and pleomorphism; the presence and number per field of cells with degenerative changes in their nucleus, binucleated cells, and mitosis; and the presence, intensity, disposition, and layer of the koilocytosis were evaluated.The intensity of the histologic characteristics was registered as light, moderate, or severe, and the disposition as isolated, focal, or generalized; the cellular changes and mitosis were counted by examining all fields.- Toki’s criteria: Acanthosis, “1”; dyskeratosis, “1”; basilar hyperplasia, “1”; koilocytosis, “4”; bi- or multinucleated cells, “2”; and papillomatosis, “1” .- Immunohistochemistry: Each of the lesions was cut to 10µm for IHC staining for HPV; monoclonal mouse anti-human papillomavirus (SB24) detects the epitope L1. Deparaffinized sections were immersed in absolute methanol containing 0.03% (v/v) H2O2 at room temperature to block endogenous peroxidase activity. After washing with phosphate-buffered saline , the sections were immersed in 0.01M citrate buffer (pH 6.0) and heated in a pressure cooker for 15 minutes. The sections were incubated in 2% (w/v) bovine serum albumin in 1x PBS for 15 minutes at room temperature to block nonspecific reactions.Appropriately diluted HPV antibody (1:100) was applied to each section for 30 minutes at room temperature. After washing with 1x PBS, the sections were incubated with IgM biotinylated goat anti-rabbit (H+L) antibody  for 10 minutes at room temperature and then washed with 1x PBS. Appropriately diluted streptavidin-peroxidase  was applied to each section for 10 minutes. Then the sections were again washed with 1x PBS, immersed for 3 minutes in 0.05% 3,3’-diaminobenzidine tetrahydrochloride in 0.05M Tris-HCl buffer (pH 7.6) containing 0.01% (v/v) H2O2 (DAB), and counterstained with Mayer´s hematoxylin. Lesions with reaction intensity, light, moderate or severe with a disposition isolated, focal or generalized in the cell nuclei of the spinous layer were considered positive to HPV L1.p≤0.05), odds ratio (OR), and confidence intervals at 95% were obtained, and receiver operating characteristic (ROC) curves were constructed.- Statistical analysis: The values of the lesions according to Toki’s criteria, the clinical and morphologic characteristics, and the positivity for L1 were registered in a data base in the SPSS 20 software . To evaluate the effectiveness of Toki’s criteria in the diagnosis of HPV L1 through IHC, tests of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) with the use of Bayes’ theorem were developed. The association and concordance between the diagnosis of HPV using Toki’s criteria and the identification of the L1 protein by IHC were calculated by the “chi2” and kappa coefficients, considering the valuation of Landis and Koch . Two logDuring the 22-year period considered, 5,037 studies were done in the laboratory, and 277 cases were diagnosed as lesions by HPV in the mouth, presenting a prevalence of 5.49%.The relation between the age range of patients and their gender are show in - Evaluation of Toki’s criteria for the diagnosis of lesions by HPV in the mouth through the identification of L1 by IHC.p=0.155) nor a statistical concordance (kappa coefficient p=0.125) was found between Toki’s criteria and the IHC test for L1.The 96.4% (267 of 277) of lesions had scores greater than or equal to 6 when Toki’s criteria were used, whereas only 28.5% (79 of 277) were found to be positive for L1 by IHC. When compared with the IHC test to identify HPV L1, the Toki’s criteria showed a sensitivity of 93.67%, a specificity of 2.53%, a PPV of 6.99%, and a NPV of 46.55%. Neither a significant association (chi2 p=0.614 and a Nagelkerke’s coefficient of determination of 6.8%. p=0.001). Nevertheless, the cutoff for a sensitivity of 87.3% shows a specificity of 39.8% and a probability of false positives of 60.2% when Toki’s criteria are used in the diagnosis of HPV lesions in the mouth where there is L1 expression.The logistic regression model developed to identify which of the 6 Toki’s criteria are useful in the diagnosis of L1 by IHC correctly classified 71.8% of the lesions positive for HPV L1, and showed a Hosmer-Lemeshow adjustment of - Evaluation of clinical and histopathologic variables for the diagnosis of lesions by HPV in the mouth through the identification of L1 by IHC.p=0.696 and a Nagelkerke’s coefficient of determination of 60.2%. The final model included 20 variables, namely, 6 clinical, 10 structural, and 4 cellular variables, which improved its adjustment: The clinical variables were age of patient, and number, contour, base, surface, and location of the lesions. The structural variables included hyperorthokeratosis, pseudoepitheliomatous hyperplasia, papillomatosis, and granular layer; intensity of the epithelial hyperplasia, hyperkeratosis, and extracellular edema; presence and intensity of the inflammatory infiltrate; and disposition of the intracellular edema. The cellular variables were presence of pinocytosis, presence and number of cells with degenerative changes in their nucleus , and layer of the koilocytosis.The logistic regression model developed correctly classified 85.2% of the lesions positive for HPV L1 infection, and showed a Hosmer-Lemeshow adjustment of p≤0.05. These included age, with patients older of 35±20.2 years having decreased probability of infection by HPV associated with the expression of L1 by IHC; presence of multiple lesions, hyperorthokeratosis, and focal intracellular edema, which increase the risk of infection; and the presence of cells with degenerative changes in their nucleus, which is associated with the infection in general but showed no relevant data in the categories . The ROC curve for the clinical and histopathologic variables shows that they have a very good diagnostic value . The cutoff for a sensitivity of 81% shows a specificity of 85.9% and a probability of false positives of 14.1% when these criteria are used in the diagnosis of HPV lesions in the mouth where there is L1 expression.The rest of the variables presented significance levels of The prevalence of OC has increased in the last decade, and because HPV is the most important biological factor for the disease, the proper diagnosis of HPV is crucial .Toki’s criteria are used for the diagnosis of cervical HPV, and due to the simplicity of the system, its application has been extended to the field of dentistry . HoweverHPV in the mouth sometimes does not behave as theoretically expected, and the HPVs 16 and 18, prevalent in potentially malignant oral lesions, typically show a low viral load and rarely integrate into the cell genome ,16. In fDue to the low number of viral copies that have been found in carcinomas and high-risk lesions, the “hit and run” theory has been developed, which proposes that tumors negative for HPV could develop a precursor with HPV content without requiring the HPV to remain in the malignant state .On the other hand, the IHC tests used for detection of HPV usually use the structural protein L1 as epitope; however, when the lesions increase in severity, the transcription of L1 and L2 tends to disappear ,18, whicThe relatively low levels of detection of L1 by IHC can also be attributed to the antiquity of the samples, which influences the denaturation of L1; a low concentration of antibody used during the technique and the fixation and processing of tissues can have an adverse effect on the epitope .et al., reported that HPV genome does not encode for any DNA methyltransferases and thus it is believed that the viral genome is methylated by DNA methyltransferases of the human host cell (et al., demonstrated the methylation of the HPV16 genome near the 3’ end of the L1 ORF and in the URR, which regulates the expression of E6 and E7 through E2 (et al., found that the sites of methylation within the viral genome are mainly detected at the boundary of the L1/L2 ORFs and within the E1 ORF (In this study, we evaluated the effectiveness of IHC tests in identifying infections by HPV in the oral mucosa. Our results indicate that in people younger than 35±20.2 years, the expression of the L1 protein is more probable. This implies that in children and people younger than 35 years, there is a protective factor that prevents the methylation of the genes E1 and E2 in the URR region, avoiding the action of E6 and E7 and, hence, the integration of the viral DNA with the cellular genome, or may be that in people older than 35 years, there is a risk factor that causes the methylation of the E1 and E2 genes. Regarding this, Fernandez rough E2  and Wilse E1 ORF . TherebyThe clinical presence of multiple lesions appears to represent a less aggressive HPV infection because it is associated with the expression of L1 and is more common in children and those younger than 35 years. An example is the MEH that is a lesion by HPV often presented as a multiple lesion mainly in children which frequently resolves spontaneously .et al., explain that E6 and E7 of the low-risk HPV types can also bind to p53 and pRb respectively. In the case of p53 will not lead to its degradation but in the case of pRb the binding will have a lower affinity, nevertheless may lead to its transformation and it has been reported that E7 of the low-risk virus HPV1 can bind to pRb with the same affinity as E7 of HPV16 (The presence of OC from infections by HPV where integration of the viral DNA to the host DNA does not exist suggests that the expression of E6 and E7 is not necessary for the development of cancer or these proteins can be activated without that integration. Concerning this, Rautava of HPV16 . The PCRof HPV16 , whereasof HPV16 . Althougof HPV16 . Thus, tAlthough Toki’s criteria could be useful in the diagnoses of cervical lesions, they are not appropriate for the diagnosis of oral mucosal lesions by HPV through IHC.We found clinical and histopathologic criteria that optimize the diagnosis of lesions by HPV in the oral mucosa through the identification of the L1 protein by IHC. However, they were few and have to be used prudently.


In the literature, data on the prevalence of human papillomavirus (HPV) infection in men vary significantly and the exact distribution of specific genotypes is still unclear. As infections usually occur without symptoms, men might only attend their hospital clinic when they have a specific concern, being in most cases genital warts (condylomas), which are often caused by low-risk HPV genotypes. The aim of this study was to assess HPV genotype distribution and prevalence among men attending hospital for HPV-associated conditions and to evaluate infection-associated factors.Samples from men with clinical manifestations of HPV-related infections seen during 2007–2012 at the Clinical Microbiology and Infectious Control Department at Basurto University Hospital were genotyped using Linear Array HPV Genotyping Test kit . Data on probable risk factors were collected and investigated for possible association.Of 184 anogenital samples, 138 (75 %) were tested as positive for HPV; 57 (41.3 %) single HPV infections and 81 (58.7 %) multiple infections. Only 45.6 % of HPV-positive samples presented low-risk genotypes 6 or 11, whereas 71/138 (51.4 %) had at least one oncogenic (high-risk) genotype. Oncogenic genotypes and multiple HPV infections were both associated with a higher number of lifetime sexual partners and their incidence appeared to increase with patient age.Although it is accepted that HPV 6 and 11 genotypes are main causes of condylomas, our findings show a high incidence of multiple infections and high-risk genotypes in men with benign HPV manifestations. The fact that the condyloma is a skin lesion facilitates the entry of virus into cells and thus cancer progression; therefore, monitoring for HPV is important, especially in those patients with high-risk genotypes (regardless of whether they cause condyloma). At present, human papillomavirus (HPV) infections are the most commonly diagnosed sexually transmitted disease . Most inAs infections are sexually transmitted, both men and women can be carriers of HPV. Infections in men usually occur without symptoms, but, unlike women, there are no recommended or approved screening programs for asymptomatic infections in men –6. FurthNinety percent of condyloma acuminata are caused by two low oncogenic risk genotypes, HPV 6 and 11 , 8. HoweIn the literature, data on HPV infection prevalence in men vary significantly (1.3 % to 72.9 %) and the exact distribution of specific HPV genotypes remains unclear , 11. ConThe aim of this study was to assess HPV genotype distribution among men with benign clinical manifestations of HPV-related infections and to evaluate factors associated with HPV infection.This study was performed with the approval of Clinical Research Ethics Committee at Basurto University Hospital  and all procedures were in accordance with the guidelines and ethical standards for experimental investigation with human subjects required by the Helsinki Declaration of 1975, as revised in 2000.All patients gave written and informed consent prior their inclusion in the study and parents provided written informed consent on behalf of children (<18 years old) enrolled in our study.All men attending the Consultation of Sexually Transmitted Diseases (CSTD) clinic at Basurto University Hospital from 2007 to 2012 due to a lesion in the genital area that urologists suspected as being potentially caused by HPV were eligible for recruitment to the study. No selected groups were used.Lesions were tape-stripped to remove possible environmental contaminations  and sampData on medical diagnosis, age at diagnosis, location of lesion, sexual behaviour  and number of lifetime sexual partners were part of the medical history collected from each individual during the medical consultation at the time of sample collection.L1 gene.DNA was isolated and molecular genotyping was carried out using a Linear Array HPV Genotyping Test kit , according to manufacturer’s instructions. HPV genotyping was performed by amplifying a fragment of the This genotyping kit is capable of identifying up to 37 genotypes. HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82 were considered to be high-risk types, whereas HPV 26, 53 and HPV 66 were defined as probable high-risk types [Men were considered to have a high-risk HPV infection if they were positive for one or more high-risk HPV genotypes – whether or not they were also positive for one or more low-risk HPV genotypes. On the other hand, men were considered to have a low-risk HPV infection if they were positive only to low-risk HPV genotypes.Genotyping was repeated twice  to confirm the result when a multiple genotype HPV infection was detected or a negative result was obtained. Negative and positive controls were provided with the kit and used in every test.p-value below 0.05 was considered to be statistically significant.The association between the different variables was determined using the chi-square test. Fisher’s exact test was also performed when the expected numbers were small as it is more accurate than the chi-square test in this situation. SPSS 11.0.1 software  was used for the data analysis and a Principal Component Analysis (PCA) was performed to compare the genotypes of the HPV infections based on the age, number of lifetime sexual partners and sexual behaviour of the men, In order to do this, men were classified into three age groups  and stratified into one of five number of sexual partners groups . Regarding sexual behaviour, a man was categorised as MSM (Men that had Sex with Men) if the participant reported having had sex  with another man in his lifetime. All of these PCA analyses were performed using the Unscrambler® software (CAMO).A total of 184 β-globin positive anogenital samples were obtained for the study (1 sample per patient) and all specimens were classified by urologists as cauliflower-like growths. No flat lesions or stem-like protrusions were detected.One hundred and thirty eight specimens (75.0 %) tested positive for HPV Table , includiLow-risk genotypes HPV 6 and/or HPV 11 were detected in 63/138 (45.6 %) of the positive samples. Sixty two of these (44.9 %) showed the presence of only low-risk HPV genotypes and single infections were detected in 45 of these cases (68.1 %). The main HPV genotypes detected in these low-risk infection specimens were: HPV 6 (32/62), 11 (11/62) and 62 (7/62). In contrast, 71 samples (51.4 % of HPV-positive samples) showed the presence of at least one oncogenic (high-risk) HPV genotype, and multiple HPV infection was detected in 60 (84.5 %) of these cases. High-risk HPV predominant types in these samples were: HPV 16 (30/71), 52 (22/71), and 33 (8/71). In this group, the predominant low-risk genotypes associated with high-risk multiple infections were HPVs 6 and 84. The combination of HPV 6 or 11 plus at least one high-risk genotype was detected in 24 specimens.vs. multiple infection), a significant difference was found (p < 0.0001). High-risk genotypes were more frequently found in multiple infections, while low-risk types were more commonly detected in single infections  and the anal area (32 multiple infections vs. 16 single infections) and the proportion of multiple infections seemed to be higher in anal location. However, these results were not statistically significant and could not be used to establish an association between the type of infection and condyloma location.Overall, multiple infections showed predominance in both the penile area . Similarly, the proportion of patients with at least one high-risk genotype also appeared to increase with age ; however, none of these differences were considered statistically significant (vs. multiple) or risk  at the time of diagnosis . For example, only 10/33 (30.3 %) with <2 lifetime sexual partners had a high-risk HPV genotype , while this was 43/83 (52 %) in those with 2–5 partners  but for HPV 16 differences were statistically significant. HPV 16 presence was more prevalent as the number of lifetime sexual partners increased .Fifty four men 39.1 %) confirmed that they had sexual relationships with other men while 84 (60.9 %) confirmed themselves as heterosexual. In general, men in the MSM group had a higher number of sexual partners than heterosexuals and as such the risk of becoming infected with HPV 16 or another high-risk HPV genotype as well as having a multiple infection was higher  were more frequent in heterosexuals than men in the MSM group . Men in the MSM group tended to have a higher risk of being infected with HPV 16  and 51.4 % of total samples were infected with at least one high-risk HPV genotype. Furthermore, high-risk genotypes were more often detected in multiple infections, while single infections showed a predominance of low-risk genotypes (p < 0.01).Benign lesions are often caused by low-risk HPV genotypes with genotypes 6 and 11 causing 90 % of genital warts . HoweverData on the prevalence of HPV infections in men with genital warts is unclear; however, a high prevalence of some high-risk HPV genotypes, such as HPV 16, in asymptomatic men has been reported , 20. In It is important to note that these data do not imply that most lesions are caused by high-risk genotypes. It is very likely that low-risk genotypes (HPV 6 and/or 11) are the genotypes responsible for causing the lesion and that high-risk genotypes are present due to patients’ sexual activity. In this study, we showed that presence of HPV 16 was significantly higher in men who had had more lifetime sexual partners.et al. used metagenomic sequencing methods for testing apparently negative HPV condyloma acuminata and found out that most of them were HPV positive [In our study, HPV DNA was not detected in 46 samples. Negativity with the Linear Array HPV Genotyping Test kit used may be due to specimen inadequacy, genotyping method insensitivity, or the inability of the kit to detect the existence of HPV genotypes other than the 37 genotypes that the kit is capable of identifying. Johansson positive . This mapositive . We atteet al. found a strong association between the acquisition of a new HPV infection and the number of sexual partners [p < 0.05). Men in the MSM group had had more lifetime sexual partners and as such the risk of having HPV 16 or at least one oncogenic HPV genotype as well as a multiple HPV infection were also higher in the MSM group.It is confirmed that prevalence of HPV infection increases with the number of sexual partners , 23–27. partners . In thisAccording to the results of this study, there was no association between the prevalence of any HPV genotype and age, confirming previous findings , 27, 28;These results confirm that HPV infection and reinfection are strongly associated with sexual activity, and support earlier data suggesting that this, and not differences in natural immunity, is the most important factor regarding infection/reinfection risk .There is a wide variation in clearance rates of the various oncogenic HPV genotypes; however, it is well known that the clearance rates for high-risk HPV types are lower than for non-oncogenic types, but clearance of all genotypes increases with age  – which There are no available approved routine screening tests for genital warts, anogenital cancer, or HPV status in general , 6, 10. One method to estimate HPV prevalence involves collecting specimens from patients who attend hospital clinics with suspected HPV-related conditions, as we have done in this study. However, as infections usually occur without symptoms and most individuals only go to the hospital when a clinical manifestation, such as a genital wart or cancer, is apparent, this method is likely to be inaccurate to estimate HPV general prevalence in men.More knowledge on the HPV distribution in men is essential for three main reasons: i) the information collected is valuable from the point of view of HPV association with anogenital cancers and genital warts (with follow up studies), ii) the fact that men act as transmitters of HPV infection to both women and men and, iii) to establish a database about the diversity and pathogenicity of different HPV genotypes, which may help to design and optimize treatment and vaccination protocols in order to reduce the disease in men, and consequently in women.Although it is accepted that HPV 6 and 11 genotypes are main causes of benign condylomas, our findings show a high incidence of multiple infections and high oncogenic risk genotypes in men with benign HPV manifestations.


There are errors in the Funding section. The correct funding information is as follows: Financial support for this particular investigation derived from the Timor-Leste Family Study dataset was provided by the School of Population Health, The University of Queensland. This work has been produced with the assistance of funding provided by the Department of Veterans’ Affairs. However, the views expressed in this version of the Work do not necessarily represent the views of the Minister for Veterans’ Affairs or the Department of Veterans’ Affairs. The Commonwealth does not give any warranty nor accept any liability in relation to the contents of this Work. The Department of Veterans’ Affairs had no role in study design, analysis, decision to publish, or preparation of the manuscript for this particular investigation.


The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions.Human papillomavirus (HPV) inactivates the Sixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP.p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%).HPV frequency was associated with cervical cancer cases  for this article can be found here:  Cervical cancer represents an important public health problem, as it is the fourth most common type of carcinoma in women worldwide. This disease was responsible for 265,000 deaths in 2012, of which 87% occurred in developing countries  r. (2005)  reportedp = 0.005), as corroborated by the literature. In spite of that proved correlation, this infection alone is probably not sufficient to develop the disease. On the other hand, the samples in which HPV was not detected also presented cervical lesions. This molecular event was explained by Han et al. (2006) [We detected HPV with a higher frequency among the case samples (66.0%) than the control (26.6%) in which 77.8% of the cancer group samples presented HPV. We found a very statistically significant association between HPV infection and cervical cancer (. (2006)  and Sotl. (2006) . These s. (2006) -28 about. (2006) , which mEBV infection was found in a series of cases and control groups in which the rate of detection frequency was not considered statistically significant p = 0.732). This finding indicates that EBV cannot be responsible for cervical cancer progression alone. Nevertheless, cancer was revealed to be associated with HPV and EBV coinfection (p = 0.027), showing its possible role as a cofactor in cervical cancer progression. Some preliminary studies found similar results  than in the other lesions and controls (6.7% for RB1 and 13.3% for CDH1 in controls). These data for CDH1 are consistent with findings in the literature , and these groups had the lowest frequencies for the unmethylated status in both genes . Our findings also agree with those of Narayan et al. (2003) , who anaRB1/CDH1 methylation status and HPV/EBV infection. Our findings showed an important increase in the methylation status of both genes, RB1 and CDH1, with a pathological change seen with HPV/EBV infection. It is important to note that the methylated status of RB1 was considered to be associated with HPV infection (p = 0.042), revealing an important role for HPV in cervical cancer epigenetics.We also analyzed the potential association between The elucidation of the molecular relationships between viral and host proteins and their epigenetic modifications could improve the process of cervical cancer screening. Indeed, the detection of possible biomarkers and cofactors for the possibility of cervical cancer could allow for molecular differentiation between initial and precursor lesions.RB1 methylation and cervical cancer. With EBV, the methylation of RB1 was a tumor-associated event, where only the cancer group presented RB1 methylation in the presence of this virus. HPV infection and cancer progression were significantly associated. The global frequency of methylation was higher when HPV was present, showing its important role in this epigenetic mechanism in cervical carcinogenesis. Nevertheless, EBV seems to have a cofactor role in this process, which needs to be further investigated.In conclusion, our results showed that the methylation status of both genes increased with disease progression, revealing a significant correlation between


FAM19A4 and mir124-2 loci, to detect CIN3+ using both HPV-positive lavage- and brush self-samples.DNA methylation analysis of cancer-related genes is a promising tool for HPV-positive women to identify those with cervical (pre)cancer (CIN3+) in need of treatment. However, clinical performance of methylation markers can be influenced by the sample type utilized. We describe a multiplex quantitative methylation-specific PCR that targets We determined methylation thresholds for clinical classification using HPV-positive training sets comprising lavage self-samples of 182 women (including 40 with CIN3+) and brush self-samples of 224 women (including 61 with CIN3+). Subsequently, independent HPV-positive validation sets of 389 lavage self-samples (including 78 with CIN3+), and 254 brush self-samples (including 72 with CIN3+) were tested using the preset thresholds. Furthermore, the clinical performance of combined methylation analysis and HPV16/18 genotyping was determined.FAM19A4 and mir124-2 thresholds for both self-sample types to yield highest CIN3+ sensitivity at 70% specificity. Validation set analysis resulted in a CIN3+ sensitivity of 70.5% (95%CI: 60.4–80.6) at a specificity of 67.8% (95%CI: 62.7–73.0) for lavage self-samples, and a CIN3+ sensitivity of 69.4% (95%CI: 58.8–80.1) at a 76.4% (95%CI: 70.2–82.6) specificity for brush self-samples. In combination with HPV16/18 genotyping, CIN3+ sensitivity and specificity were 88.5% (95%CI: 81.4–95.6) and 46.0% (95%CI: 40.4–51.5) for lavage self-samples, and 84.7% (95%CI: 76.4–93.0) and 54.9% (95%CI: 47.7–62.2) for brush self-samples.Training set analysis revealed similar FAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive lavage- and brush self-samples to identify women with CIN3+. In combination with HPV16/18 genotyping, significantly higher CIN3+ sensitivities are obtained, at decreased specificity. •FAM19A4/mir124-2 triage of HPV-positive self-samples is a feasible and promising tool•FAM19A4/mir124-2 analysis identifies HPV-positive women at risk of cervical cancer.•FAM19A4/mir124-2 analysis performs equally well in lavage and brush self-samples.•FAM19A4/mir124-2 triage is warrantedFurther validation of HPV self-sampling with  Therefore, additional testing  is essential to identify HPV-positive women with CIN lesions in need of treatment. This approach will reduce over-referral, unnecessary colposcopies, and overtreatment of women without clinically meaningful HPV infections. Currently, various triage strategies for hrHPV-positive women have been considered including repeat cytology testing FAM19A4 and mir124-2, increases with cervical disease severity FAM19A4 and mir124-2 are particularly high in women with cervical cancer and advanced high-grade lesions, the latter characterized by a longer duration (≥ 5 years) of a preceding high-risk HPV infection MAL and mir124-2 loci has reported to be clinically non-inferior to cytology triage on a subsequent physician-taken scrape in a recent randomized controlled trial Although HPV16/18 genotyping as molecular test is directly applicable to self-samples and detects women with increased cervical cancer risk, a non-negligible fraction of (pre)cancers associated with non-HPV16/18 high-risk HPV types will be missed when using genotyping alone With an increasing interest in self-collection for HPV-based cervical screening programs FAM19A4 and mir124-2 loci. The performance of the qMSP assay in terms of analytical sensitivity, analytical specificity, and daily-use reproducibility is described. In addition, the clinical performance of the assay, either or not combined with HPV16/18 genotyping, for detection of cervical (pre)cancer was determined on both self-sample types.Here, we conducted a post-hoc analysis on 1049 HPV-positive self-samples from previous PROHTECT studies that had been collected by either lavage- and or brush-based self-collection devices 22.12.1.1Since CIN2 lesions often represent a misclassified CIN1 or CIN3, women with CIN2 lesions (n = 36) were excluded from the training set. From the remaining 215 women, 182 had sufficient quantities of the self-sample with qualitatively adequate DNA left for qMSP analysis. Of them, 40 were histologically diagnosed with CIN3+  with a median age of 38 years (range 33–58). The remaining 142 women had no evidence of CIN2 + . Of these, 42 had histologically-confirmed absence of CIN, 36 had CIN1 and 64 women had both a negative HPV test and normal cytology at follow-up The median age of women with ≤ CIN1 was 38 years (range 33–64).For the training set, 375 HPV-positive (GP5 +/6 + PCR) cervico-vaginal lavage self-samples collected with the second generation Delphi-screener  from non-responder women participating in the PROHTECT-3B trial were available. The flowchart of this training set is shown in For the validation set, 515 HPV-positive (GP5 +/6 + − PCR) lavage self-samples, collected with the first generation Delphi-screener  were available from non-responder women participating in the methylation triage arm of the PROHTECT-3A trial (NTR2606). The first and second generation Delphi-screener perform equal in DNA yield and HPV-detection 2.1.2n = 25, as explained above), and of whom sufficient quantities were left of the self-sample with qualitatively adequate DNA for qMSP analysis, were included in the training set. The remaining series of 224 women included 61 women who were histologically diagnosed with a CIN3+ lesion  with a median age of 38 years (range 33–59) The other 163 women had no evidence of CIN2 +. Of these, 57 had histologically-confirmed absence of CIN, 34 had CIN1, and 72 women had both a negative HPV test and normal cytology at follow-up The median age of women with ≤ CIN1 was 38 years (range 33–63).For the training set, 409 HPV-positive (GP5 +/6 + PCR) vaginal brush self-samples collected with the Evalyn brush  from non-responder women participating in the PROHTECT-3B trial n = 254). Of these, 72 women were histologically diagnosed with a CIN3+ lesion  and had a median age of 36 years (range 31–61). 27 women were diagnosed with CIN2 and had a median age of 36 years (range 31–56). The remaining 155 women had no evidence of CIN2 +. Of these women, 24 had histologically-confirmed absence of CIN, 24 had CIN1, and 107 women had both a negative HPV test and normal cytology at follow-up. The median age of this group was 36 years (range 30–62).For the validation set, 541 HPV-positive (GP5 +/6 + PCR) vaginal brush self-samples from non-responder women participating in the PROHTECT-2 trial (NTR1851), were available. These samples were collected with the VibaBrush . The flowchart of the validation set is shown in All PROHTECT trials had ethical approval by the National Health Council and all participants gave informed consent.2.2Women with an HPV-positive self-sample were referred for a colposcopy-directed biopsy in case of a positive triage test at baseline. In case of a negative triage test, HPV-positive women were invited for a repeat co-test (HPV and cytology) after 6–12 months. If at least one of these tests was abnormal  or hrHPV-positive), women were referred for colposcopy-directed biopsy as well. Biopsies taken at colposcopy were histologically assessed in participating hospitals and classified as normal (CIN0), CIN1, CIN2, CIN3 or invasive cancer, according to international criteria 2.2.1DNA from lavage and brush self-samples was isolated using the Nucleo-Mag 96 Tissue kit  and Microlab Star robotic system  according to manufacturers' protocol 2.2.2FAM19A4 and mir124-2 loci, as well as ACTB as a sample quality control. Analyses were performed on an ABI 7500 real-time PCR-system . For each target, Quantification Cycle (Cq) values were measured at a fixed fluorescence threshold. The result of a sample is expressed in ΔΔCq ratio being a measure for hypermethylation using the 2-ΔΔCq method ACTB, FAM19A4, and mir124-2) was used as amplification target to assess the analytical sensitivity. Serial 10-fold plasmid dilutions  were analysed in duplicate in two independent qMSP runs. The analytical sensitivity was determined as the lowest dilution giving 4 out of 4 positive results (Cq < 40). To assess the analytical specificity, bisulphite-converted unmethylated DNA from primary keratinocytes and unmodified DNA  were analysed in quadruplicate. The reproducibility of the assay based on ΔΔCq values was evaluated by duplicate measurement in independent qMSP runs of bisulphite-converted DNA of 30 HPV-positive cervical samples.Isolated DNA was subjected to bisulphite treatment using the EZ DNA Methylation Kit  as described previously 2.2.3FAM19A4/mir124-2 methylation analysis combined with HPV16/18 genotyping. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined for outcome CIN3+ and CIN2+ with 95% Wald confidence intervals. The colposcopy referral rate was calculated as the proportion of HPV-positive women who had a positive triage test result. Analyses were performed in the R package (version 2.15). Additional calculations were performed in IBM SPSS Statistics 20 and Excel.First, both training sets  were used to define clinical decision points, i.e., thresholds for positivity of the triage assay. At a predefined specificity value of 70% for CIN3+, an optimization procedure was performed to calculate the maximum corresponding CIN3+ sensitivity over all possible threshold values for the combined marker panel. The defined methylation thresholds converted the test result into a categorical variable leading to an optimal CIN3+ sensitivity at 70% CIN3+ specificity level. The thresholds were subsequently evaluated in the two independent validation sets . The primary outcome was CIN3+ detection and the secondary outcome was CIN2 + detection. In the validation sets, all samples were additionally evaluated for the clinical performance of 33.1FAM19A4 and mir124-2, a multiplex qMSP assay was used. The analytical sensitivity of the assay was 2.5 copies of methylated target per PCR reaction for both FAM19A4 and mir124-2, and the reference gene ACTB. The assay revealed no positive signals with bisulphite-converted unmethylated DNA nor non-bisulphite treated DNA of cervical samples, showing a high analytical specificity. The assay demonstrated a high reproducibility on HPV-positive cervical samples with Pearson correlation coefficients between the ΔΔCq values of 0.988 for FAM19A4 . According to the thresholds, samples with ΔΔCq ratios above the respective threshold for at least one of the targets (FAM19A4 and/or mir124-2) were rated as test-positive, while samples with ΔΔCq ratio of both targets below their threshold were rated as test-negative. The use of these thresholds resulted in CIN3+ sensitivities of 75.0% (95%CI: 61.6–88.4) and 72.1% (95%CI: 60.9–83.4) in the training sets of HPV-positive lavage self-samples and brush self-samples, respectively.Using the training sets of HPV-positive lavage and brush self-samples separately, methylation thresholds for n = 13; brush, n = 12) were scored methylation positive on their self-sample by the FAM19A4/mir124-2 methylation marker assay at the predefined 70% specificity threshold.Next, the clinical performance of the multiplex qMSP assay was validated on independent sets of HPV-positive lavage and brush self-samples. Sensitivities, specificities, PPV, NPV and referral rate for colposcopy for endpoints CIN3+ and CIN2+ are presented in FAM19A4/mir124-2 methylation analysis with HPV16/18 genotyping, CIN3 + sensitivities increased and specificities decreased for both self-sample types. In lavage self-samples, a CIN3 + sensitivity of 88.5% (95%CI: 81.4–95.6) at a specificity of 46.0% (95%CI: 40.4–51.5) was observed. In brush self-samples, a CIN3 + sensitivity of 84.7% (95%CI: 76.4–93.0) at a specificity of 54.9% (95%CI: 47.7–62.2) was observed cancer in HPV-positive women. In comparison to the MAL/mir124-2 triage test that was prospectively evaluated in HPV-positive lavage self-samples in a screening setting FAM19A4/mir124-2 assay features likewise clinical performance in HPV-positive brush-collected self-samples as well as lavage-collected self-samples, it can be considered as a promising, universal triage test for HPV-positive (cervico-)vaginal self-samples collected by different self-collection devices. In combination with HPV16/18 genotyping, significantly higher sensitivities were obtained, yet at the cost of decreased specificities. Our study findings support further validation of cervical screening studies with HPV testing combined with triage by FAM19A4/mir124-2 methylation analysis on self-collected (cervico-)vaginal specimens. To our knowledge this study is the first to evaluate in large sample series whether DNA methylation analysis is equally applicable to both HPV-positive lavage- and brush-collected self-samples for CIN3+ detection. Several studies have described DNA methylation of promotor regions of genes as candidate biomarkers JAM3/EPB4/TERT/C13ORF18 marker panel showed good feasibility on lavage self-samples MAL/mir124-2 marker panel performed non-inferior to cytology triage via a physician-taken cervical scrape for the detection of CIN2+ JAM3/EPB4/TERT/C13ORF18 methylation has been published FAM19A4/mir124-2 methylation analysis in both HPV-positive lavage and brush self-samples as assessed on large sample series herein, suggests that this assay is an attractive, directly applicable molecular triage tool for self-samples, independent of the collection device used. Further prospective studies are warranted to clinically validate HPV testing combined with FAM19A4/mir124-2 methylation-based triage on self-collected (cervico-)vaginal samples in cervical screening studies.In this study, we evaluated The qMSP assay used in this study allows fast and reliable read-out of multiple methylation markers and a reference gene in one assay. As such, the methylation test safes clinical material, time and costs and improves quality control. Using a dichotomized outcome, the assay provides a clinical decision point to refer an HPV-positive woman for colposcopy or not. The advantages of our study are the evaluation of large, independent series of different self-sample types, and the use of a standardized assay. A limitation of our study can be seen in the difference in follow-up time between the cohorts used in this study . However, in the PROHTECT-2 cohort, the far majority of women  women developed CIN3+ during the first 18 months of follow-up and only 3/72 women during the 2nd 18 months of follow-up. As the number of women diagnosed with CIN3+ during the 2nd 18 months in PROHTECT-2 is limited, we feel that this has no meaningful influence on the clinical performance figures presented in this work for both cohorts.FAM19A4/mir124-2 assay described herein. Cytology is a widely accepted triage test for HPV-positive women, but cytology triage following HPV self-sampling requires an additional cervical scrape taken by a physician n = 13 and n = 12 for lavage and brush self-samples, respectively). Nonetheless, part of CIN2 and few CIN3 lesions are likely to remain undetected when a methylation marker-based triage strategy is used. Based on our previous work, these lesions are likely early-onset or incident lesions with a low progression-risk to invasive cancer FAM19A4/mir124-2 methylation marker analysis and HPV16/18 genotyping may be considered for triage of women with an HPV-positive self-sample FAM19A4/mir124-2 methylation analysis. These findings are in line with Verhoef et al. MAL/mir124-2 and HPV16/18 genotyping on HPV-positive lavage self-samples leads to significantly higher sensitivities for CIN3+, yet at the cost of a lower specificity.To the best of our knowledge, no other methodology is currently available that can be reliably applied directly to self-collected (cervico-)vaginal specimens, and has similar clinical sensitivity and clinical specificity figures for the triage of HPV-positive women following self-sampling as the 5FAM19A4/mir124-2 methylation analysis performs equally well in HPV-positive lavage- and brush self-samples to identify women with CIN3+. In combination with HPV16/18 genotyping, significantly higher CIN3+ sensitivities are obtained, at decreased specificity. Further validation of molecular cervical screening with self-sampling in population-based study is warranted.FAM19A4/mir124-2 methylation marker analysis, HPV16/18 genotyping and the combination of both triage tests for outcome CIN2+ in the validation sets stratified by self-sample type.Clinical performance of The following is the supplementary data related to this article.CJLMM, PJFS, RDMS and DAMH have minority stake in Self-Screen B.V., a spin-off company of VU University Medical Center Amsterdam. ATH is an employee of Self-screen B.V. CJLMM has participated in the sponsored speaker's bureau of Merck, GSK, Qiagen, Menarini, Segeene, and Roche, and served occasionally on the scientific advisory board of GSK, Qiagen, Merck, and Roche. CJLMM has occasionally been consultant for Qiagen and Genticel and is a minority shareholder of Diassay B.V. Formerly CJLMM was a minority shareholder of Delphi Biosciences. PJFS has been on the speaker's bureau of Roche, Abbott, Gen-Probe, Qiagen and Seegene. He is consultant for Crucell Holland B.V. DAMH has been on the speaker's bureau of Hologic/Gen-Probe and serves occasionally on the scientific advisory boards of AMGEN and Pfizer. JB has played an advisory role for Merck and Roche and has been on the speakers' bureau of Qiagen. RLMB received grants from Roche and SP-MSD and has participated in the speakers' bureau of Roche. All other authors declare that they have no conflicts of interest.


Background. Human papillomavirus (HPV) vaccines are recommended for girls prior to sexual debut because they are most effective if administered before girls acquire HPV. Little research has been done on HPV prevalence in girls who report not having passed sexual debut in high HPV-prevalence countries.Methods. Using attendance registers of randomly selected primary schools in the Mwanza region of Tanzania, we enrolled girls aged 15–16 years who reported not having passed sexual debut. A face-to-face interview on sexual behavior and intravaginal practices, and a nurse-assisted self-administered vaginal swab were performed. Swabs were tested for 13 high-risk and 24 low-risk HPV genotypes.Results. HPV was detected in 40/474  girls. Ten different high-risk and 21 different low-risk genotypes were detected. High-risk genotypes were detected in 5.3% . In multivariable analysis, only intravaginal cleansing (practiced by 20.9%) was associated with HPV detection .Conclusion. This cohort of adolescent Tanzanian girls had a high HPV prevalence prior to self-reported sexual debut, and this was associated with intravaginal cleansing. This most likely reflects underreporting of sexual activity, and it is possible that intravaginal cleansing is a marker for unreported sexual debut or nonpenetrative sexual behaviors. Cervical cancer is the most common form of cancer in women in sub–Saharan Africa and the highest age-standardized incidence of cervical cancer in the world is found in East Africa at over 30.0 per 100 000 person-years . AlthougThe 2 available HPV vaccines, Gardasil (MSD) and Cervarix , cover the 2 most common cancer-causing HPV genotypes HPV-16 and -18. Additionally, Gardasil covers HPV-6 and -11, which, though low-risk for cervical cancer, are common causes of genital warts. HPV vaccination is most effective if administered prior to acquiring infection with these vaccine-related genotypes , 9. HoweTo address the gaps in knowledge, we present baseline cross-sectional results from a cohort study in Tanzania that enrolled adolescent girls who reported that they had not passed sexual debut, and who were tested for vaginal HPV DNA. This study is the first to describe HPV prevalence in girls in sub–Saharan Africa who report no previous sexual intercourse, and the associations between reported sexual behaviors, vaginal practices, and HPV infection.The cohort was enrolled between January and August 2012 from previously prepared attendance lists of randomly selected government primary schools in 3 districts of the Mwanza Region in Tanzania. These lists, covering all primary schools in the districts, had been drawn up in 2010 to prepare for a trial of delivery methods for HPV vaccine . The lisEligibility criteria included being aged 15 or 16 years at enrolment, enrolled in class 6 in 2010 in 1 of the selected schools, not pregnant (self-reported), planning to stay in the study area or able to travel to appointments, self-reporting never having had vaginal sex, and being willing to self-administer vaginal swabs. A subsample of 26 of the 82 schools were randomly selected and, in order to prevent stigmatization of girls who reported sex, at each of these 26 schools the first girl who reported ever having had sex was enrolled. An additional eligibility requirement for these girls was having passed sexual debut within the past 12 months.The London School of Hygiene and Tropical Medicine Ethics Committee and the Medical Research Coordinating Committee, Tanzania, approved the study protocol. Because all potential participants were considered minors in Tanzania (under 18 years old), written informed consent was required from a parent/guardian with subsequent participant informed assent. Consent for enrolment was taken before any assessment of previous sexual debut. The consent procedure involved separate face-to-face explanations of the study to parents and daughters by a study nurse, provision of written information, and time to ask questions. Individuals unable to read were consented in the presence of an independent witness who provided an additional signature. Individuals unable to write provided a thumbprint. Parents/guardians were compensated for their time with Tsh5,000 (approximately $3), and participants were provided with a toothbrush and toothpaste.After enrolment, girls had a face-to-face interview with a female study nurse. Interviews were carried out in private at the participant's home, school, or local health center, depending on participant preference. Interviews were carried out in Swahili using a structured paper questionnaire, which had been translated and back-translated from English. Questions covered demographic and socioeconomic details, menstrual and vaginal hygiene practices, nonpenetrative sexual behaviors, and details of previous penetrative-sex frequency and partners. Interviews included colloquial terms for sexual behaviors that had been collected during focus group discussions with similar-aged girls. One self-administered vaginal Dacron swab was obtained after instructions from a study nurse, who remained in the room and provided verbal and positional hand guidance if necessary. Girls are being followed every 3 months for 18 months. We report results from the enrolment visit.Immediately after collection, swabs were stored dry in cryotubes and placed into cold boxes with ice packs. They were submitted daily to the reference laboratory in Mwanza and stored at −20°C until they were shipped to the Catalan Institute of Oncology, Barcelona, Spain. HPV detection and genotyping were performed using the Linear Array HPV genotyping assay  which detects 37 HPV genotypes . For this study, 13 HPV genotypes, HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68, were classified high-risk [Briefly, DNA was extracted from the specimens by silica-gel-based methods . Extracted material was amplified with the PGMY PCR system, and the generated amplicons were detected and typed by reverse-line blot reaction . Polymerase chain reaction in this assay is based on a multiplex system, including human β-globin amplification primers. This provides an internal quality control for sample material. Specimens consistently negative for β-globin amplification were excluded because it was assumed that vaginal sampling was unsuccessful. All protocols were performed according to manufacturer's instructions: each step was performed in separate rooms and negative controls were used.Questionnaire data were double-entered into OpenClinica LLC , and analyzed using STATA V12.0 . Baseline cohort characteristics were examined, and the prevalence of any HPV infection and prevalence of individual HPV genotypes were calculated. Household wealth was estimated using a score based on the number of possessions owned by the head of the household in which the participant currently resided. Median age of menarche in the cohort was calculated using Kaplan–Meier survival methods to account for those who had not reached menarche.P < .1 after adjustment for age were included in a multivariable model. Vaginal cleansing was investigated as a binary term for ever having cleansed, and as frequency of cleansing categorized as never, ≤2, and ≥3 times per day. All P values are from likelihood ratio tests.Factors associated with prevalent HPV infection (treated as a binary outcome for any HPV genotype vs none) were identified using logistic regression models. Age was considered an a priori potential confounder, and factors reaching Of 1555 potentially eligible girls on the original school attendance lists, 1177 (76.5%) were located. From these, 801 (68.1%) were confirmed to be the appropriate age, and 628 (78.4%) consented to be screened for enrollment  and the remainder (52.5%) were 16 years old . In total, 24  girls had a high-risk HPV genotype. Multiple genotype infections were detected in 20  girls, comprising 50.0% of those with HPV DNA. The 2 most commonly detected genotypes were HPV-42 and HPV-58, with a prevalence of 1.9% (N = 9) and 1.1% (N = 5), respectively , and high-risk HPV was detected in 22.7% . Though the numbers involved were small, their demographic profile was similar to that of girls who reported no previous sex (data not shown).A total of 22 eligible girls who reported having passed sexual debut within the last year were enrolled from 26 randomly selected schools. HPV DNA was detected in 7  of these 22 girls, significantly higher than in those who reported never having had sex (P = .10). Only the reporting of ever having practiced intravaginal cleansing was associated with HPV DNA detection in univariable analysis, and this persisted after adjustment for age  (Table P = .002). However, among girls who reported cleansing, there was no evidence of an association of HPV detection with method (fingers vs cloth), or substance used   based on the testing of self-administered vaginal swabs. This is higher than in 2 previous studies among women who reported no previous sex: 1 study of 130 Swedish women aged 10–25 years reported a prevalence of 1.5%, and 1 longitudinal study of American women aged 18–20 years reported that 1.7% of specimens were found to have HPV DNA . A furthPrevious studies have confirmed vaginal sex as the predominant method for acquisition of cervical HPV infection in women, as demonstrated by rapid HPV acquisition after reported sexual debut, and a significant increase in the risk of HPV infection with increased number of partners and with high-risk partners , 15. LacAlternative explanations for the presence of vaginal HPV DNA in girls and women who report no previous sex include mother-to-child transmission (MTCT) of HPV, nonpenetrative sex practices, or transmission via fomites . MTCT isIn the age-adjusted analysis in our study, disclosure of ever having practiced intravaginal cleansing was associated with more than doubled odds of prevalent HPV. Furthermore, a positive dose-response relationship was seen with increasing frequency of vaginal cleansing. Commonly, women use intravaginal practices (IVP) to manage menstruation, as part of their sexual practice , and to improve genital hygiene. IVP includes both cleansing inside the vagina with water, soap, or other products, and the insertion of products into the vagina  . IVP is Trichomonas vaginalis in Zambian girls aged 13–16 who reported no previous sexual contact [HPV has been detected in fingernail and fingertip specimens of young sexually active women in the United States, as well as on toilet seats in several European airports, and on surfaces in a sexual health clinic in the United Kingdom , 33. HPV contact . Intrava contact .An important and likely explanation for the observed association between intravaginal cleansing and HPV is that intravaginal cleansing may be a marker of unreported sexual debut or nonpenetrative sex behaviors. Girls may be more likely to start vaginal cleansing after sexual debut because they believe that this may prevent pregnancy or STIs, relieve STI-related symptoms, or because they have been taught that this practice is appropriate for sexually active women . IncreasMultiple HPV-genotype infections were as common as single genotype infections in this study, consistent with observations in adolescent girls in previous studies in the United States , 39. MorMajor strengths of this study include the interview method, in which the questionnaire included local age-specific colloquial terms for sexual practices, and was administered by trained nurses experienced in adolescent sexual behavior research. In addition, vaginal swabs were self-administered but collection was directly observed by nurses. Self-administered vaginal swabs in a previous local study (median age 17) demonstrated collection of epithelial cells in 233/244 (95%) of swabs , and 99%One of the limitations of this study is that the enrollment list came from primary schools. Although primary school attendance is a legal requirement in Tanzania, and has been reported as 97% , it may The proposed HPV vaccination program in Tanzania will target girls in primary school class 4, where the median age is 10 years, and a catch-up campaign in older girls has not thus far been proposed. This study provides useful evidence for policymakers in relation to the likely effectiveness of such a campaign. Overall, 80.3% of 15–16 year old girls reported no previous sex, 99.2% of whom did not have infection with either of the 2 high-risk HPV genotypes included in current HPV vaccines (HPV-16/18). The WHO recommend including older girls in catch-up campaigns if a significant proportion of girls are naive to HPV vaccine types [The data we present strongly link prevalent HPV infection with reported intravaginal cleansing. Intravaginal cleansing may be a marker for undisclosed sexual activity in our population, but could alternatively be a novel mechanism for the nonsexual transmission of HPV. If this is the case, the identification of this potentially modifiable risk factor would be highly relevant for a country with one of the highest rates of cervical cancer in the world. Further research to confirm and understand the link between intravaginal cleansing and HPV is required and is currently underway in Mwanza.


A single nucleotide polymorphism (SNP) 35 kb upstream of the HLA-C gene is associated with HLA-C expression, and the high expressing genotype (CC) has been associated with HIV-I control. HLA-C is unique among the classical MHC class I molecules for its role in the control of viral infections and recognition of abnormal or missing self. This immunosurveillance is central to the pathogenesis of non-melanoma skin cancer (NMSC), and of squamous cell carcinoma (SCC) in particular. While sun exposure is a major risk factor for these cancers, cutaneous infections with genus β-HPV have been implicated in the development of SCC. We hypothesized that the high expression HLA-C genotype is associated with β-HPV infections. Therefore, we investigated the association between β-HPV serology and the −35 kb SNP (rs9264942) in a population-based case-control study of 510 SCC cases and 608 controls. Among controls, the high expression −35 kb SNP genotype (CC) reduced the likelihood of positive serology for multiple (≥2) β-HPV infections , and β-HPV species 2 infection . However, no association with β-HPV status was observed among SCC cases. Our findings suggest that underlying immunogenotype plays an important role in differential control of β-HPV in SCC cases and controls. Non-melanoma skin cancers (NMSC) represent the most common malignancies in the world with increasing incidence rates The connection between immunity and NMSC is well described in individuals undergoing immunosuppressive therapies TMC8 is associated with seropositivity to multiple β-HPV types among healthy individuals Cutaneous HPV include alpha, beta, gamma, mu, and nu genera, which are further grouped into species, type, and subtype, according to the extent of sequence identity HLA-C is unique among the classical MHC class I molecules for its role in the control of viral infections and recognition of abnormal or missing self, by presenting antigens to cytotoxic T lymphocytes (CTL) and providing an inhibitory signal to NK cells Study protocols and materials for recruitment of cases and controls were approved by the Committee for the Protection of Human Subjects at Dartmouth College. Informed consent was obtained from all study subjects.Newly diagnosed cases of histologically confirmed SCC in New Hampshire were identified using an incident survey as previously described g for 20 min at 4°C. Plasma, red blood cells and the buffy coat were stored separately at −80°C until analysis. Each specimen was labeled with a type code  and a unique identifier so that the staff was masked to the disease status associated with all samples. Frozen plasma samples were previously analyzed for antibodies to the major capsid protein L1 of genus β human papillomavirus types 5, 8, 9, 15, 17, 20, 23, 24, 36, 38, 49, 75, 76, 92, 96, and 107 S-transferase capture enzyme-linked immunosorbent assay (ELISA), in combination with fluorescent bead technology From all participants a venous blood sample of 20–30 mL was collected in heparinized tubes. Blood was separated by centrifugation at 2,500 DNA was extracted from buffy coat using Qiagen genomic DNA extraction kits. We collected data on (T>C) coding SNP located in the 5′ region of the HLA-C gene, 35 kb away from the transcription initiation site (rs9264942), which is a marker of level of HLA-C messenger RNA transcript and cell surface expression. Genotyping of the −35 kb SNP polymorphism was performed using the Taqman allelic discrimination technique .We examined the association of the −35 kb SNP genotype with seropositivity to β-HPV among SCC cases and controls. This included an analysis of multiple β-HPV (≥2) and β species: β1 , β2 , and β3 . In addition we tested the association of the −35 kb SNP genotype with case status, including an analysis stratified by β-HPV species. For all models odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression, adjusted for age at diagnosis, sex, and skin type. The measure of skin type was defined as the reaction to 1 hour of sun exposure the first time in the summer. For greater statistical power the four level response variable was collapsed to two levels; those who responded that they had a severe sunburn with blistering, or painful sunburn for a few days followed by peeling were assigned in the Burn category, and those that responded that they tanned without any sunburn, or had a mild burn followed by tanning, were assigned Tan category Genotype data for the −35 SNP were available for 1118 white participants, 608 controls, and 510 SCC cases. Allele frequencies were in Hardy-Weinberg equilibrium. The prevalence of the C allele was 39% in controls and 38% in the SCC group, consistent with the published prevalence in white populations Next, we examined the association of the −35 kb SNP genotype with β-HPV positive serology stratified by β-HPV species , among controls and SCC cases . In contFinally, we investigated the association of the −35 kb SNP genotype with SCC case risk. No significant differences in the prevalence of the C allele were observed between SCC cases and controls . StratifWe investigated the association of an HLA-C expression genotype with presence of β-HPV seropositivity in SCC cases and controls. Among controls the high expression (CC) genotype was associated with reduced prevalence of multiple β-HPV and species 2 β-HPV serologic response, compared to controls with negative β-HPV serology. However, no significant associations with β-HPV status were observed among SCC cases. Our findings indicate that the −35 kb SNP is implicated in β-HPV control in healthy individuals, but not in SCC patients. This suggests a deregulation of HLA-C mediated immunity in individuals with SCC, that potentially relates to the development of skin tumors.Previous studies of the HLA-C −35 kb expression SNP have been focused on HIV infection control. While β-HPV and HIV infections are different in their mode of action and target cell type, the initial immune response in early viral control and maintenance of chronic infection are important for both. In the context of HIV, the protective effect of the high expressing (CC) genotype has been attributed to better antigen presentation to CTL, especially in early HIV control, and effective education and licensing of the maturing NK cells Given its dual functionality in interactions with innate and adaptive immunity, HLA-C plays a key role in immunosurveillance of both virally infected cells and recognition and elimination of tumor transformed cells. Tumor immunosurveillance is highlighted in the immunosupressed OTR and EV patients with deregulated innate and cell mediated immune responses While the mechanism of SCC development and its relationship to β-HPV positivity is yet to be determined, our results show a lack of association between HLA-C expression genotype and β-HPV among SCC cases, suggesting a potential deregulation of HLA-C mediated immunosurveillance. This is further corroborated by our β-HPV stratified case-control analysis, where SCC was positively associated with genotype only among those with multiple or β-HPV species 2 positive serology, an association driven by differences in genotype distribution among controls and not the cases. Thus, the underlying immunogenotype, which is implicated in control of β-HPV, may be a potential susceptibility factor for SCC, and individuals with this underlying deregulated immunity may be predisposed to the development of tumors.Interestingly, the HLA-C locus has been reported as a major determinant of susceptibility to psoriasis, an inflammatory skin disorder with a strong genetic component, and the low expressing T allele of the −35 kb SNP is associated with an increased risk of the disease Previous investigations of β-HPV control have shown a link between common genetic variation and positive β-HPV serology In this study we only measured a single genetic marker, the −35 kb SNP. The MHC-I region and the polymorphic HLA-C loci contain extensive LD. Moreover, −35 kb SNP has been shown to be in partial LD with the HLA complex P5 variant, which, in turn, is in strong LD with the HLA-B*5701 allele that is strongly associated with viral control of HIV


Voice dysfunction or dysphonia may be associated with several clinical conditions. Among these, laryngeal human papillomavirus (HPV)-induced lesions should be considered as a possible causative factor. We report a case of dysphonia in a patient presenting with an HPV laryngeal lesion. We also discuss the clinical features of the disease, its histopathological findings, and treatment and rigorous follow-up.We report a case of laryngeal papilloma in a 29-year-old, Afro-descendant, male patient with dysphonia. He was a non-smoker and was not a drug user. Videolaryngostroboscopy revealed signs suggestive of pharyngolaryngeal reflux. The right vocal fold presented with a papillomatous aspect in the posterior third, which underwent excision. Histopathological examination showed a nodular lesion of the right vocal fold, conclusive of squamous papilloma with absence of malignancy.Patients presenting with persistent voice dysfunction or dysphonia should be investigated for possible laryngeal HPV infection. Diagnostic confirmation by HPV genotyping is important for follow-up of potential recurrence. Voice dysfunction or dysphonia has widespread effects and has been frequently studied. Functional and neoplastic dysphonia is among the many causative factors; however, it may also be associated with neurological and systemic diseases, as well as other diseases such as oropharyngeal papilloma, which is a benign tumor caused by human papillomavirus (HPV). More than 200 HPV types have been described and 40 are capable of infecting the anogenital and oral epithelia. HPV may be clinically characterized by appearing in proliferative processes such as oral papilloma, commonly associated with low-risk genotypes 6 and 11. It has also been considered as the major etiological agent in development of neoplasia of the anogenital tract, such as cervical, anal, and penile cancer, as well as oral tumors, where there is a higher prevalence of high-risk genotypes 16 and 18[The World Health Organization (WHO) considers HPV infection to be an epidemic of high relevance for public health.The most frequently observed oral manifestations associated with HPV infection there include: condyloma acuminatum, focal epithelial hyperplasia, verruca vulgaris, and oral squamous papilloma. These lesions are all benign and are normally associated with infection with low-risk HPVs, among which subtypes 6 and 11 are more prevalent, 4.HPV infection may also be associated with oropharyngeal carcinoma development, among which, squamous cell carcinoma is the eighth most common type of cancer in men. Additionally, persistent virus infection, together with the use of alcohol and tobacco are predisposing factors for development of oropharyngeal cancer, 5.One of the main complaints with these types of lesions is speech alteration (dysphonia), as reported in the present case, and deglutition (dysphagia). XimenesHere, we describe a case of dysphonia in a patient diagnosed with vocal chord papilloma. This article addresses an uncommon issue of dysphonia related to HPV-induced lesions in an adult patient, highlighting the need for further investigation of dysphonia.A 29-year-old Afro-descendant, single male, undergraduate biological sciences student, who lives in Itaboraí-Rio de Janeiro (RJ) and works as a waiter, was referred by the stomatologist to the Department of Otorhinolaryngology of a private hospital, reporting dysphonia and hoarseness for 2 months.He is homosexual and reported nonexclusive sexual partnerships in the previous year, and casual condom use during oral and anal intercourse. Regarding HPV infection, his partner already had virus-induced lesions in the anal region, although he had been treated. The patient denied smoking, social drinking, and illicit drug use. He had a verruca vulgaris in his right hand that disappeared without surgery or medication when he was a child. He reported being born by cesarean section.Lesions were not observed during the intra and extra-oral physical examination, and neither was cervical lymphadenopathy noted. Oral hygiene was satisfactory and there was no biofilm retention, caries, or gingivitis. Body examination revealed a wart in the foot.Videolaryngostroboscopy presented signs suggestive of pharyngolaryngeal reflux, and retrocricoid and interarytenoidal edema. Remaining unchanged anatomical pharyngolaryngeal sites were noteworthy Figure A,B ThereAfter routine preoperative examinations, the patient was referred for surgical excision of the nodular lesion of the right vocal fold. Histopathological examination showed a nodular lesion of the right vocal fold, with several small, firm and elastic brown tissue fragments, with some papillomatous areas measuring 1.7 × 0.8 × 0.2 cm, conclusive of squamous papilloma with absence of malignancy  at-risk population. He admitted to having a sexual partner who had already presented with condylomatous anal injury. This may have contributed to the emergence of HPV laryngeal infection, because our patient has maintained relationships with the previously infected individual, and the practice of fellatio is positively associated with HPV infection. A literLaryngeal papillomatosis is a disease that is characterized by a verruca, whose main etiological agent is HPV. The injury is typically diagnosed as benign squamous papilloma and can lead to discomfort in speaking, affecting mainly the vocal folds, epiglottis and vestibular folds, with possible recurrence–11.Histopathological diagnosis indicated a nodular lesion on the vocal folds, characterizing papillomatosis that was confirmed as squamous papilloma with absence of malignancy. This corroborates the majority of studies in which the main viral types found in this injury are HPV 6 and 11, which are considered low-risk oncogenic types.Histopathological diagnosis indicated squamous papilloma. In addition, PCR based molecular biology techniques by PCR confirmed HPV 6 infection in this particular case.There is evidence to suggest the need to improve prevention strategies in order to reduce the prevalence of STDs and related complications.In conclusion, patients presenting with persistent voice dysfunction or dysphonia should be investigated for possible laryngeal HPV infection. Diagnostic conformation by HPV genotyping is important for follow-up of potential recurrence.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent form is available for review by the Editor-in-Chief of this journal.