Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection.Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test.Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis . Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001).Conclusion: Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL. Human papillomavirus (HPV) is an important cause of cervical cancer and cervical intraepithelial neoplasia (CIN), and is associated with 99% of cervical cancerDespite the high probability of spontaneous regression of ASCUS and LSIL, persistent minor cervical abnormalities are occasionally observed in women with HPV infectionHPV L1, one of the HPV-induced proteins, is a capsid protein produced at the upper layer of the differentiated cervical epithelium at the end of the viral life cycle. HPV is released from cervical keratinocytes after the assembly of viral proteinsThe aim of this study was to find the best predictive model for development of CIN 2+ using the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS, and LSIL with high risk of HPV infection.The medical records of patients who visited the department of obstetrics and gynecology of Seoul St. Mary's Hospital between January 2014 and August 2017 were reviewed. Persistent LSIL or ASCUS was identified by: 1) diagnosis of ASCUS or LSIL on follow up cytology after 1 year, and 2) identification in at least two consecutive cytologic examinations. The inclusion criteria were as follows: 1) persistent ASCUS or LSIL, 2) high risk of HPV infection, and 3) presence of cervical histopathology. The exclusion criteria were as follows: 1) patients who showed high grade squamous intraepithelial lesion (HSIL) or atypical squamous cells with cannot exclude HSIL (ASC-H) at first cytologic examination, 2) low risk of HPV infection, 3) patients who were diagnosed with CIN 2 or higher for the first time, and 4) history of cervical operation or hysterectomy before the study period.TM HPV Genotyping Chip; Pangene CO., Seoul, Korea). The cervical tissue diagnosis was obtained from the records of patients who underwent colposcopy guided biopsy, loop electrosurgical excision procedure (LEEP), or total hysterectomy. Information on age, cervical cytology, HPV genotype, expression status of HPV L1 analyzed by immunocytochemistry, and pathologic diagnosis of the cervix were collected for all patients.A total of 285 cervical cytology samples  were analyzed in this study. Cervical cytology samples were scraped from the exocervix and endocervix with a cervical brush. Cytological diagnosis was made following 2001 Bethesda system. HPV genotype was determined using the HPV DNA chip  of The Catholic University of Korea (KC16RISI0388).® HPV L1 screening set  according to the manufacturer's protocol. The coverslip and mounting medium of Papanicolaou-stained cervical smear slides in xylene were separated, and the residual xylene on the slides was removed with alcohol. To retrieve the antigen, 0.01mol/L citrate buffer (pH 6.0) was used, and the sample was heated in a microwave vacuum histoprocessor  at a temperature of 121°C for 15 minutes. Slides were kept at room temperature for 10 minutes to cool. Subsequently, one droplet of HPV L1 capsid antibody was applied to the cervical smear slides, and slides were incubated for 1 hour at room temperature. One drop of ready -to- use- chromogen solution (3-amino-9-ethylcarbazole) was applied to the slide for 5 minutes. Mayer's hematoxylin counterstain was then applied. At least 1 positive control slide supplied by the manufacturer was included in each batch. Immunocytochemistry was performed using the first specimen.Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed using the Cytoactive2, and analysis of model comparison. All analyses were performed using SAS version 8.0 . P <0.05 was considered statistically significant. Akaike information criterion (AIC) and Schwarz criterion (SC) were used to select of the best model to predict CIN 2+.The expression level of the HPV L1 capsid protein in cervical cytology samples was calculated, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. HPV L1 expression and other variables used to predict CIN2+ were analyzed using Fisher's exact test, χThe sample population for this study included information from 70 patients with ASCUS and 215 patients with LSIL. The general characteristics of the study subjects are shown in Table p<0.05). Further risk of developing CIN2+ was increased five folds in patients with ASCUS compared to that in patients showing LSIL when the HPV L1 expression was absent (Odds ratio (OR) 5.2, 95% confidence interval (CI) 2.2-12.0, p<0.001) (Table p<0.001). In patients with other HPV types, HPV L1 negativity was more frequently observed compared to HPV L1 positivity; however, these differences were not significant (p=0.182). Based on previous results, HPV 16 and 18 infection and lack of HPV L1 protein expression correlated with the development of CIN 2+.Table 1) Table . Table 4p=0.001, OR 6.7, 95% CI 2.8-18.3, p< 0.001, respectively).Table p<0.001).Finally, the diagnostic value of various combinations of cervical cancer screening tools to predict CIN2+ was calculated Table . A lowerASCUS and LSIL are generally considered to be compatible with CIN1. Therefore, a wait and see approach is generally preferred for the management of low grade cervical diseases. However, 4% to 36% of women with persistent ASCUS/LSIL as shown by cervical cytology exhibited high grade histopathologyThe present study found that HPV L1 negative cytology samples correlated with the development of CIN 2+. This finding is consistent with previous reports that the HPV L1 gene expression is decreased in higher grades of CIN. Lack of HPV L1 expression in cervical cytology may be a useful predictive marker for the detection of higher grade CIN. HPV infection is categorized into 2 phases; productive and integrative. The productive phase is characterized by HPV infection of the basal layer through micro-wounds in the epithelium, amplification of HPV viral genes within differentiating epithelial cells and assembly of the HPV L1 capsomere in the upper epithelium et al documented that HPV 16 and 18 infections highly correlated with the development of CIN3p< 0.001) and cancer  between the ages 30 to 64Moreover, HPV infection is associated with an increased risk of development of CIN2+ in patients with ASCUS and LSILObservational data for women with persistent ASCUS and LSIL demonstrated that the risk of progression to CIN 2+ increases 1~2% annuallyDespite the high rate of spontaneous regression of ASCUS/LSIL, patients may experience substantial apprehension about the oncogenic potential of HPV infection, risks associated with low grade cervical abnormality, abnormal colposcopic findings, and additional invasive procedures. Observational management of patients with ASCUS/LSIL can be felt differently among doctors and patients. In general, gynecologists regard observation as way to avoid unnecessary treatment of low grade cervical abnormalities. Patients tend to worry about the possibility that delayed treatment may accelerate the progression to cervical cancers. If cervical biomarkers can offer additional confirmative information on the prognosis of persistent ASCUS and LSIL, the patient's levels of apprehension may be reduced.The present study investigated the capability of immunocytochemistry on cervical cytology samples for the early prediction of high grade cervical disease among women with persistent ASCUS and LSIL. The HPV L1 expression level may provide information on whether observation or treatment is more appropriate for patients with persistent cervical abnormalities. The present results show that lack of HPV L1 expression and presence of HPV type 16 or 18 infection can accurately predict progression to CIN2+ in women showing persistent ASCUS and LSIL. In conclusion, HPV L1 immunocytochemistry and the HPV type test can predict CIN2+ in women with persistent ASCUS or LSIL.


TTNtv). TTN encodes titin, which is the largest protein in the body and is an essential component of the sarcomere. Titin serves as a biological spring, spanning half of the sarcomere and connecting the Z-disk to the M-line, with scaffold and signaling functions. Truncations of titin are believed to lead to either haploinsufficiency and loss-of-function, or to a “poison peptide” effect. However, other titin mechanisms are postulated to influence cardiac function including post-translational modifications, in particular changes in titin phosphorylation that alters the stiffness of the protein, and diversity of alternative splicing that generates different titin isoforms. In this article, we review the role of TTN mutations in development of DCM, how differential expression of titin isoforms relate to DCM pathophysiology, and discuss how post-translational modifications of titin can affect cardiomyocyte function. Current research efforts aim to elucidate the contribution of titin to myofibril assembly, stability, and signal transduction, and how mutant titin leads to cardiac dysfunction and human disease. Future research will need to translate this knowledge toward novel therapeutic approaches that can modulate titin transcriptional and post-translational defects to treat DCM and heart failure.Dilated cardiomyopathy (DCM) is a leading cause of heart failure, sudden cardiac death and heart transplant. DCM is inherited in approximately 50% of cases, in which the most frequent genetic defects are truncation variants of the titin gene (- Titin (TTN) truncation variants are the most frequent cause of dilated cardiomyopathy, one of the main causes of heart failure and heart transplant. Titin is a giant protein, and the mechanisms causing the disease are both complex and still incompletely understood.- This review discusses the role of titin in myocardial function and in disease. In particular, we discuss TTN gene structure, the complexity of genotype-phenotype correlation in human disease, the physiology of TTN and the role of post-translation modification.- Additional studies will be required to clarify whether missense variants are associated with cardiac disease. While initial studies suggested a role of non-synonymous variants in arrhythmogenic cardiomyopathy, confirmatory investigations have been hampered by the complexity of the protein structure and function. TTN gene that produce a protein of between 27,000 and 33,000 amino acids in length with a molecular weight ranging between 2,900 and 3,800 kDa . TTN is the largest protein in the human body, which is encoded by 364 exons of the ,800 kDa . Structu,800 kDa . The Z-lTTN and produces three major isoforms: N2A, N2B, and N2BA which predominately differ in the lengths of the extensible I-band domains. N2B and N2BA are isoforms expressed within adult cardiomyocytes and differ based on their length and extensibility, with N2BA being a longer, less rigid protein compared to N2B  that presents with ventricular enlargement and systolic dysfunction in the absence of alternative etiologies of cardiomyopathy such as valvular, hypertensive, congenital, or ischemic causes  to correlate truncation location position with phenotype. The data demonstrated that a key predictor of pathogenicity of a TTNtv is whether the affected exon is expressed in cardiac tissue  . CircRNA circRNA . This stControl of RBM20 and subsequent expression of TTN N2BA:N2B ratios is also important in the development of diastolic dysfunction. It is conjectured that decreasing the N2BA:N2B ratio results in stiffening of TTN and a more rigid sarcomere. This in turn may lead to impaired relaxation and a less compliant ventricle consistent with heart failure with preserved ejection fraction (HFpEF). This is seen in a small group of patients who required heart transplantation due to heart failure. The patients with failing hearts but normal systolic function suggestive of diastolic dysfunction had a lower N2BA:N2B ratio compared to patients with failing hearts and reduced systolic function . Furtherhttp://gygi.med.harvard.edu/phosphomouse/index.php (http://www.phosphosite.org/ (http://cpr1.sund.ku.dk/cgi-bin/PTM.pl (In addition to transcriptional modifications, TTN function is also highly regulated by post-translational modifications. These alterations of TTN allow the cardiomyocyte and sarcomere to rapidly adjust to environmental changes within the heart. The most well described modification to TTN involves phosphorylation and de-phosphorylation at unique sites within the protein. Due its size, TTN may have the most phosphorylation sites of any protein. Hundreds of phosphorylation sites on TTN have been predicted based on proteomic analysis. These are listed on online databases: ndex.php , http://ite.org/  or http:n/PTM.pl . While mn/PTM.pl . The effThe PEVK element within the I-band is highly extensible and is characterized by richness in proline, glutamate, valine, and lysine . Phosphoin vitro and in vivo studies where the N2Bus element is phosphorylated demonstrate filament lengthening, reduction in TTN stiffness and reduction in passive tension of the sarcomere and cardiomyocyte (In addition to phosphorylation of the PEVK element, there are several phosphorylation sites within the cardiac specific N2B element. The N2B element is expressed on both cardiac TTN isoforms N2B and N2BA and is composed of three Ig domains as well as a unique sequence, named N2Bus that has many phosphosites . This elomyocyte . While komyocyte .Post-translational modification of TTN via phosphorylation can alter its stiffness and passive tension so that cardiomyocytes can respond to physiologic changes. These alterations may also become pathologic in heart failure where alterations in TTN phosphorylation may lead to worsening of passive and active forces reducing the ability of the heart to function normally. It is likely that in addition to phosphorylation, TTN is modified by many other post-translational pathways. Research is ongoing to identify additional post-translational modifications of TTN and therapeutic targets. In one recent trial, it was suggested that in a mouse model of diastolic dysfunction, metformin improved HFpEF by increasing phosphorylation the N2B element via PKA. It was conjectured that phosphorylation increased TTN compliance and improved diastolic dysfunction as measured by echocardiogram, pressure volume analysis, and exercise tolerance .As the largest protein in the human body, the extent of TTN’s role in cardiac physiology and disease is not yet completely understood. We are still learning how TTN contributes to myofibril assembly, stability, and signal transduction, and how perturbations in these processes can lead to cardiac dysfunction and human disease. We have reviewed how modification of TTN at the genetic, transcriptional, and post-translational levels can affect cardiac function by altering the passive and active forces of the sarcomere and cardiomyocyte. Undoubtedly, future research will continue to elucidate TTN’s large role in cardiac physiology and how transcriptional and post-translational modifications may contribute to DCM as well as normal cardiac physiology. Alteration of TTN represents potential therapeutic targets for genetic and acquired cardiomyopathies.CT wrote the manuscript with contributions of MH and OS. MT, LM, and OS contributed figures and grant support. MH, OS, MT, and LM provided critical insights and editing of the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The number of older adults using mobile devices has doubled over recent years; however, many need assistance in learning how to use their device. To address this gap, we developed patient education materials teaching older Veterans how to download apps and the basics of mobile device and app use. For example, we developed step-by-step guides for three Veteran Affairs mobile apps that target mental health symptoms. Material development involved feedback from providers and older Veterans using a multi-step mixed methods evaluation process. Local technology and geriatric content experts provided initial feedback; all experts agreed the materials would be helpful to teach Veterans about mental health apps. We subsequently interviewed older Veterans (M = 78.5 years) who evaluated the materials. Over 50% of Veterans found the guides clear, articulate, and useful; 83.3% noted they would recommend to others. Lastly, providers who see older Veterans regularly rated the materials; 79% of providers rated the materials as helpful, with an average rating of 4.3 (1 = Strongly Disagree to 5 = Strongly Agree). Providers viewed the materials and apps as useful supplements to psychotherapy and especially useful for individuals who are unable to return to clinic. Overall, both providers and Veterans found the materials easy to understand and valuable for those new using mobile apps or devices. Findings from the evaluation process suggest the design of the materials may be vital to increasing the use of mental health mobile apps among older Veterans.


In the last 20 years, the development of methods to produce virus-like particles (VLPs) and pseudovirions (PsV) along with more physiologically relevant cell- and tissue-based models has facilitated progress in this area. However, many questions regarding HPV infection remain difficult to address experimentally and are, thus, unanswered. Although an obligatory cellular uptake receptor has yet to be identified for any PV species, Rab-GTPases contribute to HPV uptake and transport of viral genomes toward the nucleus. Here, we provide a general overview of the current HPV infection paradigm, the epithelial differentiation-dependent HPV replicative cycle, and review the specifics of how HPVs usurp Rab-related functions during infectious entry. We also suggest other potential interactions based on how HPVs alter cellular activities to complete their replicative-cycle in differentiating epithelium. Understanding how HPVs interface with Rab functions during their complex replicative cycle may provide insight for the development of therapeutic interventions, as current viral counter-measures are solely prophylactic and therapies for HPV-positive individuals remain archaic and limited.Papillomaviruses (PVs) were the first viruses recognized to cause tumors and cancers in mammalian hosts by Shope, nearly a century ago . Over 40 Human papillomaviruses (HPVs) are small, non-enveloped icosahedral viruses of 55 nm containing a circular, ≈8-kb double-stranded DNA genome condensed by cellular histones. All PVs cause benign epithelial hyperproliferative diseases and tumors in mucosal or cutaneous epithelial sites as part of their normal replicative processes. As strictly human pathogens, HPVs have a narrow tropism for human keratinocytes and can only complete their replicative cycles in stratifying and differentiating squamous epithelium . Many HPin vivo  virion attachment to susceptible cells, (2) cellular uptake or virion penetration into the cytoplasm, including release and trafficking of the genome to the replication site, (3) viral genome replication, (4) progeny virion assembly, and (5) release of infectious particles. However, as illustrated in Difficulties in obtaining purified, high-titer infectious HPV stocks from epithelial tissues have impeded efforts to carry out microscopy and genetic assays to investigate early HPV infection events. The most medically relevant  HPVs produce low numbers of infectious virions in human lesions .10–1011 viral genome equivalents per milliliters (vge/ml) that better facilitate attachment and entry studies, particularly those using microscopic localization and virus particle tracking.The vast majority of reports providing insight into HPV entry mechanisms utilized viral particles isolated from monolayer cell cultures ectopically overexpressing HPV L1 and L2 capsid proteins. These viral particles include infectious pseudovirions (PsVs) that carry a “reporter” expression plasmid as a pseudogenome  and quas7–109 vge/ml  present in the extracellular matrix (ECM) and epithelial tissue basement membrane . LamininRab-GTPases are involved in the regulation of Sdc-1 and MMP activities, which may influence the priming of HPV virions on the cell surface. Increased cleavage of the Sdc-1 ectodomain (termed “ectodomain shedding”) is regulated by Rab5, MMP9, and growth factors. Rab5-GDP directly interacts with the cytoplasmic tail of Sdc-1 at the plasma membrane. This interaction prevents Sdc-1 ectodomain shedding until Rab5-GDP exchange to Rab5-GTP via ras-related signaling mediated by growth factor receptors (GFRs), such as the epidermal GFR (EGFR), and heparinase . As willHuman papillomavirus exposure to keratinocytes activates EGFR signaling and leads to Src kinase-mediated phosphorylation of annexin A2 (AnxA2). This leads to the translocation of the AnxA2/S100A10 tetrameric complex (A2t) to the plasma membrane surface where HPV particles colocalize with EGFR and A2t . Given tThe current paradigm suggests that HPV uptake occurs via a receptor complex assembled by tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD151 and CD63, integrins, EGFR and A2t . HPV uptAdditional plasma membrane-associated proteins have been shown to impact infectious HPV entry to some degree. These proteins include α6β4 integrin, the CD9 and CD81 tetraspanins, and EGFR . EGFR sitrans-Golgi network (TGN) and infection  Rab5 trapped virions in EE . Rab5 acHuman papillomaviruses have adapted to use the canonical endocytic process of EE maturation into LE to continue their journey to the nucleus  step 6. During endosomal maturation, the L1 capsid partially dissociates from L2 complexed to vDNA . This isRab7b and Rab9a contribute to trafficking from the LE to the TGN, and siRNA-mediated knockdown or expression of DN versions of both inhibit HPV infection . The HPVThe L2/vDNA complex seems to enter the TGN by at least two routes, one that is Rab7b dependent and one that is Rab9a dependent. Rab7b resides prominently in the TGN and Golgi apparatus, functioning to transport vesicles toward the Golgi . SiRNA kAlternative to HPV transport to the TGN, the activation of macroautophagy, an autophagy variant, impairs HPV infection . Autophacis and cis-medial Golgi until the onset of mitosis - and nuclear domain 10 (ND10)-containing bodies . PMLs anIn the last steps of HPV infectious entry, vDNA localization to ND10/PML bodies is thought to facilitate initial genome transcription, followed by vDNA replication to establish a persistent infection . In earlHuman papillomaviruses’ requirement for tissue-based differentiation and the technical challenges associated with studying membrane function therein have hampered studies of Rab functions during later replication stages. Yet, there is emerging evidence that three of the HPV nonstructural proteins, E5, E6, and E7, each of which have roles in transformation, modulate aspects of endocytic transport pathways (reviewed by In the HPVs’ strict dependence on normal cell functions, viral gene expression disrupts epithelial polarity to enhance cell proliferation in suprabasal cells . As Rab-The multifunctional viral oncoproteins, E6 and E7, are expressed at relatively low levels and predominantly in the lower-to-middle epithelial layers. Therein, they maintain and augment cell cycle and viral genome replicative capacity and promote proliferation as suprabasal cells begin the differentiation program . The undStudies of E6 and E7 cellular localization and how they alter cellular pathways has generally been limited to their ectopic overexpression in monolayer cells. The E6 oncoprotein is localized primarily to the nucleus, but it can also be detected in the cytoplasm . In cervWhile E7 stimulates S-phase entry with E6 in the suprabasal layers, high-risk HPV E6 proteins interact with a number of PDZ-domain-containing cellular proteins involved in cell polarity . High-riConcomitant with altered cell polarity in the middle to upper epithelial layers, increased expression of the HPV E1 and E2 replicative proteins facilitate viral genome amplification to >1000 copies per cell nucleus . This isIn the upper-suprabasal epithelial layers of epithelial tissues and 3D-organotypic tissue models, EGFR and phopho-ERK1/2 signaling gradually diminish , paralleIncreasing differentiation in the upper suprabasal layers drives strong activation of the late viral promoter and heightened expression of the non-structural E4 gene and the capsid genes, L1 and L2 . AssemblProgeny virions do not appear to be fully released from the top epithelial layers of DCCs, which can transmit infection  step 5. Hijacking of common host trafficking pathways is a common theme for many intracellular pathogens . A growiAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although the beneficial effects of vaccines on equity by socioeconomic status and geography are increasingly well-documented, little has been done to extend these analyses to examine the linkage between vaccination and gender equity. In this paper, evidence from the published literature is used to develop a conceptual framework demonstrating the potential impact of vaccination on measures of gender equity. This framework is then applied to human papillomavirus (HPV) vaccination in three countries with different economic and disease burden profiles to establish a proof of concept in a variety of contexts.We conducted a literature review examining evidence on the linkage between health outcomes and dimensions of gender equity. We utilized the Papillomavirus Rapid Interface for Modelling and Economics (PRIME) model to estimate cervical cancer incidence and deaths due to HPV types 16/18 by age in each country. We estimated labor force participation and fertility effects from improvements in health, and converted these into inputs consistent with those used to calculate the United Nations Gender Inequality Index to assess gender equity.In our case study, we found that HPV vaccination among girls could help narrow socioeconomic gender disparities by quantifying the main pathways by which HPV vaccination improves health, which enables improvement in gender equity indicators such as labor force participation and maternal mortality ratios. While these improvements are small when averaged over the entire population, the components measured – labor force participation and maternal mortality ratio – account for 50% of the index scores.This proof of concept model is a starting point to inform future health and economic analyses that might incorporate the impact of gender equity as an additional impact of vaccination in improving the health and well-being of the population. Vaccines are one of the most effective public health innovations in the past century. Every year immunizations save between two and three million lives and avert billions of dollars in costs of illness . Not onlAchieving gender equity and empowering all women and girls is one of the United Nations’ Sustainable Development Goals and a key foundation for a peaceful, prosperous, and sustainable world . Gender The linkage between vaccines and gender equity is particularly relevant in the case of the human papillomavirus (HPV) vaccine. HPV vaccine studies demonstrate an almost 100% protection against the previously high-risk HPV strains that cause cervical cancer (types 16 and 18), and HPV vaccines have been shown to be cost-effective in most settings . While HIn this paper, evidence from the published literature is used to develop a conceptual framework demonstrating the potential impact of vaccination on measures of gender equity. This framework is then applied to the HPV vaccine example in three countries with different economic and disease burden profiles to establish the impact of improved health on gender equity in a variety of development contexts. Our objective is to provide a proof of concept model as a starting point to inform future analyses that seek to estimate the health and economic impact of gender equity.We first conducted a literature review using two databases (PubMed and Web of Science) examining studies on gender equity and health impact published between January 1, 1980 and December 31, 2018. The search strategy used a combination of free text terms across all search fields. Key search terms included those related to gender equity and health impact  AND “impact” AND “health). Record titles and abstracts were screened by a single reviewer to identify studies that met the inclusion criteria. Articles that did not discuss gender equity, did not look at health impact, were not human studies, contained outcomes from a study already reported in another article, or contained a non-English abstract were excluded. Key working papers regarding the topic of gender equity were also included. From each of the included studies, we extracted information on the dimension(s) of gender equity included and the linkage(s) between health outcomes and gender equity.primetool.org. Relevant country- and age-specific inputs for PRIME are provided in the Additional file We modeled the impact that HPV vaccination among girls could have on gender equity in a low-, middle-, and high-income country , where each country represents a different level of disease burden. Compared to the worldwide annual cervical cancer incidence of 14.0 per 100,000 population , TanzaniIn this analysis, we assumed a scenario of 80% coverage of HPV vaccination with a two-dose regimen. The impact of this HPV vaccination scenario on cervical cancer incidence and death projected by PRIME is augmented with the extracted literature review data on the linkages between health outcomes and dimensions of gender equity. Based on evidence from the literature, we developed a conceptual pathway by which HPV vaccination can improve healthy life expectancy in women, which can in turn improve their labor force participation and decrease fertility, and hence narrow a key source of gender disparities in many countries . Labor fTo quantify the magnitude of these relationships, we first converted years of life expectancy to years of healthy life expectancy utilizing life tables and healthy life expectancy at birth and at age 60 from the World Health Organization (WHO) for Tanzania, India, and the United Kingdom . Given cUsing these estimates of healthy life expectancy, we assumed that the decreased cervical cancer incidence and deaths from HPV vaccination yielded decreased years lived with disability (YLDs) and decreased years of life lost (YLLs). The ratio of the decreased YLDs and YLLs to healthy life years gained compared to total YLDs and YLLs to total healthy life expectancy was interpreted as the percentage improvement in health due to HPV vaccination. This percentage improvement in health translated to percentage increases in labor force participation and percentage reductions in fertility rate, utilizing percentages calculated from evidence identified through the literature review. According to a working paper by Bloom, et al., we assumed that a 5% improvement in health from HPV vaccination translated to a 5.9% increase in female labor force participation and a 2.4% reduction in fertility . These pThe impact of HPV vaccination on overall gender disparities was assessed by converting previously estimated labor force participation and fertility effects into inputs consistent with those used to calculate the United Nations (UN) Gender Inequality Index (GII) , 38. TheOnce the relevant inputs were derived, the impact of HPV vaccination on GII scores were calculated by subtracting the current index value from the counterfactual index value under a hypothetical 0% HPV vaccine coverage scenario. Since higher GII scores are associated with greater inequality, the difference between these two values can be interpreted as the reduction in inequality associated with HPV vaccination.Our review of dimensions of gender equity identified five relevant articles , 37, 38 The factors included in these gender equity indices contributed to our second literature review examining linkages between gender equity and health. Our literature search resulted in 205 articles on PubMed and 179 articles on Web of Science, of which 12 met inclusion criteria , 40–49. The results of our healthy life expectancy calculations are shown in Fig. Overall, HPV vaccination is expected to decrease cervical cancer cases and deaths by approximately 80% in Tanzania, India, and the United Kingdom based on PRIME projections. For women of reproductive age (15 to 49 years), the median percentage improvement in healthy life expectancy due to reductions in cervical cancer cases and deaths is 51% in Tanzania, 52% in India, and 49% in the United Kingdom. The impact that HPV vaccination is projected to have in Tanzania, India, and the United Kingdom is shown in Fig. Assuming a 5% improvement in health from HPV vaccination translates to a 5.9% increase in female labor force participation , years oThe years of employment gained from improvements in health due to reduced cervical cancer incidence also contributes to increased economic output. The improvements in economic productivity from years of employment gained by female workers could be approximately $4.7 million in Tanzania, $24 million in India, and $18 million in the United Kingdom (in US$2015).In our sensitivity analysis examining the lower range with half the magnitude of this association, which assumes that a 5% improvement in health from HPV vaccination translate to a 2.95% increase in female labor force participation, years of employment gained among women who averted cervical cancer ranged from 82 to 460 years in Tanzania, 702–4800 years in India, and 19–39 years in the United Kingdom for women aged 15 to 49 years. In terms of economic productivity, this translated to approximately $2.4 million in Tanzania, $12 million in India, and $8.9 million in the United Kingdom. Assuming a 5% improvement in health from HPV vaccination translated to a 1.2% decrease in fertility rate, the associated impact on maternal mortality was 0.72 maternal deaths averted in Tanzania, 2.15 maternal deaths averted in India, and approximately no change in maternal deaths in the United Kingdom.In our sensitivity analysis examining the upper range with double the magnitude of association, which assumed that a 5% improvement in health from HPV vaccination translate to a 11.8% increase in female labor force participation, years of employment gained among women who averted cervical cancer ranged from 330 to 1840 years in Tanzania, 2808–19,200 years in India, and 76–156 years in the United Kingdom for women aged 15 to 49 years. In terms of economic productivity, this translated to approximately $9.4 million in Tanzania, $48 million in India, and $36 million in the United Kingdom. Assuming a 5% improvement in health from HPV vaccination translated to a 4.8% decrease in fertility rate, the associated impact on maternal mortality was 2.9 maternal deaths averted in Tanzania, 8.6 maternal deaths averted in India, and approximately no change in maternal deaths in the United Kingdom.Comparing GII scores calculated based on current HPV vaccination levels and under the counterfactual scenario of 0% HPV vaccination, improvements in labor force participation and maternal survival were both associated with a decrease in the level of gender inequality  in the three countries of interest. Compared to the status quo GII scores in Tanzania, India, and the United Kingdom , the absolute changes in GII scores were small (0.0000021 (0.0000019–0.000013), 0.0000017 (0.0000017–0.0000032), and 0.0000002 (0.0000002–0.0000004), respectively) because cervical cancer is a rare outcome and GII index scores are estimated on a 0–1 scale. While the index comprises multiple dimensions, labor force participation and maternal mortality ratio account for approximately 50% of the index score.We developed a conceptual framework that links vaccination and gender equity utilizing existing literature. In our case study, we found that HPV vaccination could help narrow socioeconomic gender disparities by quantifying the main pathways by which HPV vaccination improves health, which enables improvement in gender equity indicators such as labor force participation and maternal mortality ratios. Specifically, we found that the years of employment gained among women with averted cervical cancer ranged from 165 to 920 years in Tanzania, 1404–9600 years in India, and 38–78 years in the United Kingdom for women aged 15 to 49 years. The averted maternal deaths among women with averted cervical cancer was 1.4 in Tanzania and 2.3 in India. These improvements likewise translate to improvements in gender equity. While these improvements are small when averaged over the entire population, the components measured – labor force participation and maternal mortality ratio – account for 50% of the index score.While the magnitude of improvement we found is small due to the rarity of cervical cancer, we have likely underestimated the benefits. This is because our model did not include many other benefits of HPV vaccination such as indirect (herd) effects, social stigma associated with cervical and other HPV-related cancers, reductions in time-consuming and anxiety-inducing cervical screening and treatment rounds, as well as effects on non-cervical HPV-related disease such as vulvar, vaginal, anal and oropharyngeal cancers , 51. We In this analysis, we selected HPV vaccination as a case study for gender equity given that its health benefits are predominantly in women . If we eEquity is a key criterion for health technology assessments in many countries , 56 and Given the importance of gender equity to development, this suggests that decision-making bodies such as national immunization technical advisory groups (NITAGs) and donors may wish to consider vaccine impact on gender equity as one of the decision-making criteria around new vaccine introductions. Future research should be conducted to examine these gender equity benefits beyond this case study of three countries from the lens of HPV vaccination.This analysis presents a number of limitations. First, our conceptual framework and quantitative associations rely on the available published literature, which remains limited on this topic. Specifically, we rely heavily on Bloom, et al. , which rWhile our analysis focused on reductions in gender inequity from overall improvements to healthy life expectancy, Finlay and Lee (2018) have examined the causal linkages between improvements in reproductive health, specifically, and the economic empowerment dimensions of gender equity . This anGender equity is one of the United Nations’ Sustainable Development Goals and a key dimension of inclusive economic growth. It has been associated with improvements in health outcomes for women and children, in addition to economic growth. This proof of concept model is a starting point to inform future health and economic analyses that might incorporate the impact of gender equity as an additional impact of vaccination in improving the health and well-being of the population. Reduced HPV incidence may lead to improved gender equity through improvements in female contributions to the labor market and economic productivity, improvements in female education, lower fertility, and reduced youth dependency.Additional file 1: Appendix A. Papillomavirus Rapid Interface for Modelling and Economics (PRIME) tool country-level inputs for India, Tanzania, and the United Kingdom. Appendix B. Cervical cancer incidence and mortality and all-cause mortality by age for India, Tanzania, and the United Kingdom. Appendix C. List of linkages between gender equity and health.


New strategies are needed to combat the high incidence of cervical cancer in resource-limited settings such as sub-Saharan Africa. Screening for high-risk human papillomavirus (hrHPV) DNA is sensitive for pre-cancer, but its lack of specificity results in substantial overtreatment in low resource settings where additional testing  is rarely available. Testing for hrHPV E6/E7 mRNA may enhance specificity, but little is known about its performance characteristics in resource-limited settings.In a series of community health fairs in rural Uganda, women aged 25 to 49 years provided self-collected vaginal samples, which were tested for hrHPV  E6/E7 mRNA with the Aptima® assay. Positive specimens underwent testing for HPV-16 and 18/45. After excluding pregnant women, all women testing positive for any hrHPV subsequently were offered cervical biopsy to determine pathology.A total of 1892 women provided a vaginal sample for hrHPV testing during 24 health fairs. The median age was 34 years, HIV prevalence was 10, and 95% had not been previously screened. Prevalence of any hrHPV E6/E7 mRNA was 21% : 19 to 23%); the prevalence of HPV-16 was 2.6%, HPV-18/45 1.9%, and HPV 16 and 18/45 were jointly found in 0.1% of the study population. Younger age, pregnancy and HIV-positivity were independently associated with any hrHPV infection. Of the 255 evaluable cervical biopsies, the positive predictive value of detecting any hrHPV E6/E7 mRNA for presence of cervical intraepithelial neoplasia grade 2 or higher (“CIN 2+”) was 8.2% (95% CI: 5.1 to 12%). The positive predictive value associated with detection of HPV-16 mRNA (15%) or HPV-18/45 mRNA (15%) was only slightly higher.Among community-based women in Uganda, the prevalence of any hrHPV E6/E7 mRNA in vaginal samples was high, but the prevalence of the most oncogenic HPV types  was substantially lower. Positive predictive value of hrHPV mRNA-positivity for CIN 2+ was also low, including when restricting to HPV 16/18/45-positivity. The findings emphasize the need to identify more specific screening approaches for cervical cancer. Sub-Saharan Africa has the highest rates of cervical cancer worldwide , and as One potential strategy to improve specificity of cervical cancer screening is testing for the mRNA of the oncogenic proteins E6 and E7 from hrHPV types. In data from mainly European and American populations, such hrHPV mRNA tests have demonstrated sensitivity and specificity above 90% for the detection of CIN grade 2 or higher (“CIN 2+”) . DetectiIn order to better understand the utility of hrHPV mRNA testing for population-based screening programs in sub-Saharan Africa, we set forth in Uganda — one of the countries most affected by cervical cancer in the region — to determine a community-based estimate of the prevalence and correlates of hrHPV E6/E7 mRNA detection and the positive predictive value of detection of this biomarker for pre-cancer or cancer.We carried out a cross-sectional study of the prevalence and correlates of hrHPV E6/E7 mRNA in self-collected vaginal specimens among women attending community-based cervical cancer screening fairs in rural western Uganda. Women with detectable hrHPV mRNA subsequently underwent a cervical biopsy to determine presence of CIN or cervical cancer, thus allowing estimation of the positive predictive value of E6/E7 mRNA detection.The target population was women residing in either the Kiboga or Kyankwanzi districts in western Uganda. Both districts are characterized as rural with the largest population center holding 8342 residents. In a randomly chosen 16 communities in these districts, we engaged Village Health Team members (VHTs) to inform and mobilize women to attend cervical cancer screening fairs in central locations in their communities. VHTs are lay citizens that receive basic training by the Ministry of Health and are utilized by the Ministry of Health to promote health programs within their communities. The VHTs made announcements on local radio and at churches, burial ceremonies, markets, and community centers. The mobilization message informed residents about the importance of screening for cervical cancer and the upcoming screening fairs in their communities. On the day of the fair, women attended an interactive educational session about cervical cancer, HPV infection and screening, and all women 25–49 years residing in the two districts were asked to participate in a research study about screening; women with a history of cervical cancer or prior hysterectomy were excluded. The study was approved by institutional review boards in both Uganda and the United States., and all women provided written informed consent to participate.We used an interviewer-administered questionnaire to ask participants about their demographic, socioeconomic, and clinical characteristics, including extensive contact information for result communication and subsequent treatment if necessary.o C prior to testing.Participants were provided with instructions about self-collection of a vaginal specimen, including a graphic illustration. Each woman was given the testing kit  containing a cytobrush and a capped vial containing PreservCyt® medium. They were instructed to insert the cytobrush into their vagina until it met resistance, rotate three times, and then place the brush into the vial and recap. From the field, the specimens were transported at room temperature to Kampala within 1 week and then frozen at -20Vaginal samples were tested using the Aptima® HPV assay (Hologic Inc.) at the University of Washington-University of Nairobi STI Laboratory, Mombasa Kenya, based on manufacturer’s instructions. Briefly, the Aptima® assay is a nucleic acid amplification test for the qualitative detection of E6/E7 mRNA from the 14 hrHPV types . Assay results are interpreted on the basis of the signal-to-cut-off ratio for the analyte, and specimens with ratios ≥1.0 were considered positive. All hrHPV-positive samples were further genotyped for HPV-16, HPV-18 and HPV-45 using the Aptima® HPV 16 18/45 genotype assay, also performed according to manufacturer’s instructions.Women who tested positive for any hrHPV mRNA were asked to return to a mobile treatment unit in their community. Women who presented during their menses were asked to return when their cycle had stopped. After excluding women who were self-reporting or suspicious for pregnancy and confirmed with a positive pregnancy test , we performed Visual Assessment for Treatment (VAT) using 3–5% acetic acid. Women with lesions felt to be inappropriate for cryotherapy or suspicious for cancer were referred for alternative management at Mulago Hospital in Kampala; all others underwent a 2 or 3 mm cervical biopsy  before receiving cryotherapy. In other words, in order for us to strive for complete ascertainment of CIN 2+ in all hrHPV RNA-positive women, VAT was only used to determine which women should have a therapy different than cryotherapy, and for these women a biopsy was also sought prior to therapy.Biopsies (including those obtained from the referred women) were processed at the Department of Pathology of the Makerere College of Health Sciences; they were determined inadequate for histological evaluation if they were too small for processing. Adequate biopsies were interpreted with hematoxylin and eosin staining. Specimens were read as normal, cervicitis, CIN 1, CIN 2/3 or invasive cancer.After determining the descriptive parameters for the population, we evaluated the participants’ various sociodemographic and clinical characteristics for their association with hrHPV mRNA-positivity . We used prevalence ratios as the measure of association and used log-binomial regression to estimate the prevalence ratios adjusted for other factors. We used a directed acyclic graph (DAG) to depict background knowledge and inform variable selection in the multivariable regression models , 18. TheA total of 2142 women attended and registered for cervical cancer screening at one of 24 health fairs within 16 communities between March and November 2016. Two hundred and forty women were ineligible for the research study due to age (96%), symptoms or exam suggestive of cervical cancer (2.5%), or prior hysterectomy (1.3%). Of the 1902 eligible women, 1892 (99%) provided a self-collected vaginal specimen while 1890 provided both a specimen and a completed questionnaire : 19 to 23%) (Table P < 0.001). Women who were pregnant were 1.37 times as likely to be hrHPV-positive than women who were not pregnant . HIV-infected participants were over two times more likely to be hrHPV-positive than those who were HIV-uninfected . When evaluating HPV 16, 18, or 45 mRNA-positivity, we found, in adjusted analyses, that only younger age and HIV infection status were significantly related to hrHPV mRNA detection.In evaluating independent correlates of the presence of E6/E7 HPV mRNA, we constructed a directed acyclic graph to depict the system and inform what factors to adjust for Fig. . We firsA total of 301 of 393 (77%) hrHPV mRNA-positive participants returned for treatment, of whom 38 did not receive a cervical biopsy or cryotherapy because of pregnancy (71%), vaginal prolapse (5.7%), inability to safely administer cryotherapy due to cervical position (11%), referral to Mulago Hospital for alternative management but subsequent loss to follow-up (11%), or deferral because of concurrent systemic illness (2.7%). There was no significant difference in age, marital status, educational level, parity and HIV status between those women who returned and those who did not (data not shown). Of the 263 participants who underwent a biopsy, 255 biopsy samples  were adequate for interpretation. Among these, 8.2% (95% CI: 5.1 to 12%) displayed CIN 2+ , making the positive predictive value of detecting any hrHPV E6/E7 mRNA for the presence of CIN 2+ 8.4% , but there was a much lower prevalence (4.6%) of HPV-16 or 18/45, the types traditionally recognized as most oncogenic. The positive predictive value of hrHPV E6/E7-positiivity for CIN 2+ was also low (8.2%) without much improvement when limited to HPV-16 or HPV-18/45 E6/E7 mRNA-positivity. Younger age, pregnancy and HIV-positivity were significantly associated with harbouring any hrHPV mRNA.The prevalence of hrHPV mRNA in our study was substantially lower than what was found by other hrHPV mRNA studies within Africa. In a study by Adamson et al. among 325 HIV-infected women in South Africa, the prevalence of hrHPV mRNA was 37% . Ting etThe positive predictive value of the presence of any hrHPV mRNA for CIN 2+ that we observed was low but very similar to what was observed by Ting et al. in Kenya (10%)  and withWe found that younger age, pregnancy and HIV-positivity were significantly associated with detecting hrHPV mRNA. The association with HIV is consistent with multiple reports in which HIV infection results in increased incidence of HPV infection , 34, decOur approach sought to estimate the prevalence, correlates and predictive value of hrHPV mRNA at the community level, but there were limitations. First, our participants freely responded to a community mobilization campaign, which may have selectively enriched the study population for those who perceived themselves to be at risk of HPV infection or may be more likely to engage in prevention behaviours. It is therefore difficult to assess whether the participants were strictly representative of the general Ugandan rural population. Second, we could not with our study design estimate the negative predictive value of non-detection of hrHPV mRNA. Doing so would have required biopsy of all or a representative sample of the mRNA-negative women, which was beyond the scope of our work. Third, because of the nature of our recruitment and screening (large numbers of participants screened simultaneously), we were not able to comprehensively measure some of the constructs  potentially related to hrHPV-positivity. Thus, our lack of finding a role for some of the constructs may have been because of misclassification. Finally, the cross-sectional nature of the study precluded a better understanding of the meaning of our correlations  or of the positive predictive value of mRNA-positivity for CIN 2+ over a longer duration.In this community-based sample of women from Uganda, the prevalence of any hrHPV E6/E7 mRNA was high, but the prevalence of the most oncogenic HPV types  was substantially lower. Positive predictive value of hrHPV mRNA-positivity for CIN2+ was also low, including when limited to just HPV 16/18/45-positivity. The findings emphasize the need to identify more specific screening approaches for the prevention and early detection of cervical cancer.


Head and neck cancer is the sixth most common malignancy worldwide, predominantly developing from squamous cell epithelia (HNSCC). The main HNSCC risk factors are tobacco, excessive alcohol use, and the presence of human papillomavirus (HPV). HPV positive (+) cancers are etiologically different from other HNSCC and often show better prognosis. The current knowledge regarding HNSCC miRNA profiles is still incomplete especially in the context of HPV+ cancer. Thus, we analyzed 61 freshly collected primary oral (OSCC) and oropharyngeal (OPSCC) SCC samples. HPV DNA and RNA was found in 21% cases. The Illumina whole-genome small-RNA profiling by next-generation sequencing was done on 22 samples and revealed 7 specific miRNAs to HPV+ OSCC, 77 to HPV+ OPSCC, and additional 3 shared with both; 51 miRNAs were specific to HPV− OPSCC, 62 to HPV− OSCC, and 31 shared with both. The results for 9 miRNAs  were assessed by reverse transcription-quantitative polymerase chain reaction on the whole study population. The data was additionally confirmed by reanalyzing publicly available miRNA sequencing Cancer Genome Atlas consortium (TCGA) HNSCC data. Cell signaling pathway analysis revealed differences between HPV+ and HPV− HNSCC. Our findings compared with literature data revealed extensive heterogeneity of miRNA deregulation with only several miRNAs consistently affected, and miR-9 being the most likely HPV related miRNA. Most HNC develop from squamous cell epithelia, which accounts for 95% of head and neck carcinoma (HNSCC)3. HNSCC often gets diagnosed in a late phase, when it is difficult to treat, with 5-year survival of only 40–50%5. HNSCC are further characterized according to their primary site of origin, with most common sites being oral cavity, oropharynx, pharynx, larynx, and sinonasal tract6. Globally, HNSCC accounts for approximately 550,000 cases annually7, while in Croatia, 896 new cases were estimated in 20158.Head and neck cancer (HNC) is the sixth most common malignancy worldwide, predominantly arising within the mucosal linings of the upper aerodigestive tract10. In western countries, tobacco and alcohol induced HNSCC is declining, while HPV-driven HNSCC, especially oropharyngeal, is increasing in younger individuals10. HPV type 16 has been found in the majority of HPV associated HNSCC, and it is capable of transforming infected cells into cancerous by expressing oncoproteins E6 and E7, which bind, among others, to two important tumor suppressor proteins, p53 and pRB, respectively10.The main risk factors for HNSCC development are smoking and excessive alcohol use. Furthermore, the role of human papillomavirus (HPV) has emerged in recent years, particularly in oropharyngeal tumors10. Even though these two groups are etiologically different, the treatment remains the same7. However, there are indications that the treatment could be optimized for each groups of patients. Therefore, it is crucial to find more sensitive and specific biomarkers, which could enable development of better diagnostic, prognostic and therapeutic approaches for HNSCC. The HPV positive oropharyngeal cancer in particular was found to be so different from other HNSCC subtypes that the new TNM classifications11 and the specific staging guidelines12 were made specifically for this subset of tumors. In 2015, The Cancer Genome Atlas (TCGA) consortium published a comprehensive molecular catalogue on HNSCC13. Frequent mutations of novel druggable oncogenes were not demonstrated, but the difference between the HPV associated and non-viral groups was confirmed. The TCGA study revealed that HNSCC lacked predominant gain-of-function mutations in oncogenes, whereas an essential role of epigenetics in oncogenesis has become apparent. The study of Masuda et al.14 emphasizes that HNSCC seems to be an epigenetic disease, rather than genetic. Studies on the epigenetic changes in HNSCC such as miRNA profiling are promising to find specific biomarkers for both groups of tumor patients15.Based on the HPV presence, HNSCC is broadly divided in two groups: HPV positive (+) with better prognosis and HPV negative (−) tumors with worse prognosis16. Previous reviews21 have already noticed a discrepancy of miRNA findings across studies focusing on HNC, and there is no consensus on specific miRs that are associated to HPV+ HNSCC. Therefore, more thorough studies on a homogeneous and well described study population, with distinguishing tumor site of origin and etiology are necessary to get a clearer picture.Small non-coding RNAs, such as miRNA (miR) are highly conserved and about 22 nucleotides long, with important role in a variety of processes, including development, cell proliferation, and differentiationIn this study, we collected and analyzed fresh tissue of primary oral and oropharyngeal HNSCC patients, determined the HPV status and performed whole-genome miRNA profiling to identify differences in miRNA expression levels. We also performed a literature review on similar medium and high throughput miRNA studies on HNSCC populations to evaluate our findings in this context. Furthermore, we have reanalyzed the publicly available TCGA miRNA sequencing data in the same context to further validate our findings.HNSCC patients with primary oral (O) and oropharyngeal (OP) tumors treated at the Clinic of Maxillofacial Surgery of the Clinical Hospital Dubrava between 2015 and 2017 were enrolled in the study. The study was approved by the Bioethical Board of the Ruđer Bošković Institute (BEP-3748/2-2014) and the Ethical Board of the Clinical Hospital Dubrava (EP- KBD-10.06.2014). The experiments were performed in accordance with relevant guidelines and regulations. Informed consent to participate in the study was obtained for 65 cases. Four cases were excluded because 2 tumors were not primary and 2 were not squamous cell carcinoma. Thus, the total of 61 primary cancer samples have been included in this study. The majority (75%) of tumors were of oral origin , while a quarter were of oropharyngeal origin .All patients underwent surgery as primary treatment and 2 pieces of tumor tissue were stored in 700 μL of G2 lysis buffer (Qiagen) or RNA later (Ambion) solutions for the isolation of DNA and RNA, respectively. Samples were stored at +4 °C and delivered to the laboratory at the end of the day where they were stored at −20 °C for up to 2 weeks before DNA and RNA isolation.22.To supplement the samples, the analysis included 3 additional HPV+ (DNA and RNA) tonsil carcinoma samples and 3 additional HPV− normal tonsillar tissue, collected at the Motol University hospital  and previously analyzed by TaqMan miRNA Cards A and BFor nucleic acid extraction, smaller (~20 mg) pieces of tumor tissue were used. The DNA was isolated with the EZ1 Biorobot using EZ1 DNA tissue kit (Qiagen) following the manufacturer’s protocol. The total RNA was isolated using miRNeasy mini kit (Qiagen) also following the manufacturer’s protocol. Both DNA and RNA quality and quantity were analyzed on nanospectrofotometer (Implen). Furthermore, RNA quality was assessed by Bioanalyser RNA6000 Nano kit (Agilent) to determine the RIN number.23. Briefly, 50 ng of sample DNA was amplified by PGMY, GP and/or SPF10 primers and with primers specific for HPV16. To determine sample adequacy human beta-globin specific PCR was performed. Samples with any discrepancy (n = 14) in HPV testing were further analyzed using INNO-LiPA HPV Genotyping Extra (Fujirebio) according to the manufacturer’s protocol.The presence of HPV DNA was assessed using three types of consensus and one type specific PCR primer pair as described previously24 and the amplicons  were visualized on 3%-agarose gel electrophoresis. CaSki cell line cDNA was used as positive control, while the negative control contained all PCR reagents without cDNA. The suitability of cDNA for amplification was confirmed by beta-actin PCR25. In this study, samples positive for both HPV DNA, and E6 mRNA were considered HPV positive.HPV16 E6 mRNA analysis was performed on HPV DNA positive samples. Briefly, one μg of RNA was reverse transcribed using QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s protocol. The presence of full length or most abundant splice variant of the HPV16 E6 open reading frame (E6*I) was detected by PCRet al.26. Briefly, low risk group is defined as HPV positive tumors from non-smoking patients or from smoking patients with lower nodal stage. High risk group consists of HPV negative smokers or tumors with high T classification in nonsmokers, while the intermediate risk group consisted of smoking patients with HPV positive N2b+ tumors or non-smoking HPV negative tumors with T classification less than 4.To try separating relevant HPV infections from those where other factors might confound HPV activity, patients were also classified into risk groups according to Ang et al.26 Twenty-two NGS libraries were constructed with TrueSeq Small RNA Library prep kit (Illumina) according to the manufacturer’s protocol. For multiplexing and library pooling, index pools A (1–12) and B (13–22) were used. Bioanalyser (Agilent) was used for quality control of indicated steps as recommended by the manufacturer. Library sequencing was done on NextSeq 500 sequencer (Illumina) using NextSeq 500 Mid output kit (Illumina).A subset of samples (19 cancer samples and 3 controls) was selected for high-throughput miRNA analysis by next generation sequencing (NGS). Samples with poor RIN (<7) scores were excluded. Thus, the following samples were selected for NGS library preparation: 6 HPV+ (DNA and RNA) and 4 HPV− oropharyngeal cancer samples ; 3 HPV+ (DNA and RNA) and 6 HPV− oral cancer samples ; and 3 healthy tonsil tissue samples (controls). Out of 6 selected OP+ samples, two were classified as intermediate risk group according to Ang Raw sequences were trimmed of adapter sequences using FastQ toolkit Basespace App (Illumina) by selecting TrueSeq Small RNA adapter sequences from the relevant app menu. Sequencing data was analyzed using Small RNA Basespace App v1.0.1 (Illumina) to determine significantly different miRNA expression between groups. The automated pipeline uses Bowtie to align reads against reference databases to determine counts, which are then assessed for differential expression using DESeq2. Within the pipeline, miRNA sequences with mean normalized counts across all samples ≤10 are filtered out before statistical analysis. Further analyses were performed by importing Small RNA Basespace App count data into R and independently analyzed by the DESeq2 package.−ddCt method27. Briefly, dCt values were obtained by normalizing to the referent control sample, i.e. obtained by subtracting mean replicate Ct values of the combined referent sample from the mean replicate Ct value of each sample for each miR tested. Subsequently, dCt values for each miR were normalized to referent miRs in each sample to obtain ddCt value. The fold change was calculated by 2−ddCt formula. The statistical difference was tested by t-test on dCt values of each miR compared to dCt values of the referent miR within each subgroup of samples.The NGS results were validated by real-time quantitative Reverse Transcription PCR (qRT-PCR) on the same samples tested by NGS. For technical validation of the NGS experiment, we have selected 9 miRNAs that were found to be differentially expressed by NGS . Assays were designed to cover both over and under expressed miRNA. Priority was given to miRs found in HPV positive samples but without being found as significant in HPV negative samples. In addition, miRNAs -21-3p, -31-5p, -100-5p were chosen, since they were often reported in many different cancer types and could represent positive control targets. The TaqMan Advanced miRNA synthesis kit (Applied Biosystems) was used to convert isolated RNA to cDNA following the manufacturers protocol. Following conversion, 5 μl of diluted cDNA was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using TaqMan Advanced miRNA single tube assays (Applied Biosystems). The three normal tonsillar samples were pooled in equal concentration before cDNA synthesis to be used as normal reference. Assays for miR-16-5p and -191-5p were evaluated as internal reference control (manufacturer’s recommendation) as well as miR-181a-5p that showed very low intra-sample variation in the NGS experiment. Calculations were performed using each of the 3 referent miRs individually (data not shown) and as average of all 3 values. As the results were similar, the final analysis was performed with the average value of all 3 reference miRs. The fold changes were calculated using the standard 2For further validation of potentially relevant miRs, clinical samples not tested by NGS  were tested with qRT-PCR individual assays in the same way as for technical validation. For clinically relevant validation, priority was given to miRNAs with at least 100 normalized mean count in OP+ subset since low count (expression) might lead to inconsistent results on routine samples. Even though NGS analysis indicated a very limited number of miRNAs exclusively associated with HPV, the following miRNAs were selected for analysis: miR-9-5p, -21-3p, -29a-3p, -100-5p, -106b-5p, -143-3p, and -145-5p. The miRNAs miR-9-5p, -106b-5p and -29a-3p were selected as they were deregulated in our OP+ subset and not found significant in HPV negative samples. As for technical validation, miRs -21-3p and -100-5p were chosen because of their relevance in different cancer types. Specifically miRs -143-3p and -145-5p were selected as they were most commonly found by other studies to be downregulated in HNSCC cases even though they were not found to be significant in our HPV positive samples. In both cases, the purpose was to assess the utility of selected miRs as potential biomarkers. Internal reference controls and combined sample pool of healthy tonsil samples was used as referent sample for fold change calculations; as done for the technical validation. Since the initial NGS set was selected with overrepresentation of HPV positive and oropharyngeal samples, the independent set was underrepresented in those samples and consisted of 35 O−, 6 OP−, 3 O+ and only 2 OP+ samples. To increase robustness, analysis was done on the independent set (n = 46) or the total set (n = 61) of clinical cancer samples.13. We were able to match miRNA sequencing data and relevant information for 72 cancer samples (Supplementary Dataset SD1). There were 40 samples from oral cancer (12 HPV RNA positive) and 32 oropharyngeal cancer (21 HPV RNA positive) patients. We were also able to find miRNA sequencing results for matched normal solid tissue from two oropharyngeal cancer and two oral cancer patients. However, we chose to include only oropharyngeal tissue normal controls to make the control groups comparable to our sequencing experiment, where we have also used oropharyngeal normal samples as control. Briefly, raw counts of all miRNA sequences were tabulated (including isomiR sequences) and imported to R alongside annotation data (Supplementary Dataset SD1) for the analysis with DESeq2 package using identical R pipeline as for our samples. As before, miRNA sequences with 10 or less normalized reads across all samples were removed. Samples were also additionally classified according to Ang et al.26 risk factors from available clinical data, which included smoking and pack/year data for the majority of cases.To assess the validity of the results in a completely unrelated set of patients, we accessed publicly available miRNA sequencing data from TCGA data portal for oral and oropharyngeal cancer samples. Detailed clinical data and HPV status for cases with available miRNA sequencing data was obtained from the TCGA data portal as well as the TCGA consortium HNSCC focused publication29. Normalized counts of our and TCGA data were imported to R as “reads per million miRNA mapped”. Our sequencing data was used either as a training set for TCGA data classification, or as test set after training the classifier on TCGA data. Another set of classifier models were created where TCGA dataset was split in half with the first half used for training and the second for testing. Classification was performed for several variables: sample group , HPV RNA presence  and risk group  according to Ang et al.26.In attempt to create a miRNA classifier from the NGS data, we used multinomial sparse group lasso method as implemented within msgl R package30 or msgl classifier training and testing.Data management and basic analysis was done in Microsoft Excel, while statistical testing was done in Medcalc (v 11.4.2). R studio (v 1.1.383) was used to interface with R (v 3.4.2.) and perform miRNA differential expression using DEseq2 (1.18.1)21. Thus, to help us determine the relevance of particular miRNAs we decided to reexamine complete data from medium- and high-throughput miRNA HNSCC studies. To this end, we searched the NCBI PubMed database for such studies using combinations of terms HNSCC, HNC, head and neck cancer, OPSCC, oropharyngeal, miRNA, microRNA, small RNA, microarray, NGS, and next generation sequencing. Studies were accessed and wherever possible raw or supplemental data including all significantly deregulated miRNAs were extracted and tabulated. Where data was not available in the manuscript or Supplement material, we sent queries to the corresponding authors but rarely got answers. Where the information regarding -5′ or -3′ strand was not given, we considered the miRNA to belong to the more abundant form according to miRbase. If a miRNA was marked with the asterisk (*), we considered it to correspond to less abundant form according to miRbase. In addition, included articles as well as published reviews referring to the topic were searched for references to primary publications. However, reviews themselves were not included unless summarizing primarily low-throughput studies. Thus, care has been taken to include each primary study only once within the final Table (Supplementary Dataset SD2). The exception was in case when a particular study presented the differential expression results of several comparisons in which case significantly deregulated miRNAs from each comparison are listed as a separate column. In addition, studies examining miRNA deregulation in cervical cancer but included in the HNSCC focused reviews were also included to allow cross tissue examination of HPV infection related miRNAs. The prepared table (Supplementary Dataset SD2) served to both demonstrate heterogeneity of previous studies (Supplementary literature), to inform decisions for the current study, and to put the results in context.So far, overlap of published results on miRNA deregulation in HNSCC, when only the validated or the most relevant miRs from each manuscript are considered, is relatively lowet al.26; only 3 HPV positive samples were classified as intermediate risk.A total of 61 primary oral and oropharyngeal cancer samples were included in the study. Cases were grouped according to the HPV presence and patient characteristics are summarized in Table To analyze miRNA differences in expression profiles, a total of 22 NGS sequencing libraries were made from: 6 HPV+ and 4 HPV− oropharyngeal cancer samples (OP+ and OP−); 3 HPV+ and 6 HPV− oral cancer samples (O+ and O−); and 3 normal tonsil tissue samples. Complete sample annotation table is provided as Supplementary Table Final analysis revealed differential regulation of 501 different unique miRNA sequences  which were significantly deregulated in 1154 comparisons across all sample group comparisons (Supplementary Dataset SD3). However, out of 501 unique sequences only 101 fully corresponded to mature miRNA sequences, while the rest were isomiRs with either sequence mismatches or with slightly different size.Based on the final results and on all miRNA sequences, samples did not form distinguishing clusters on heatmap or PCA plots Fig.  except f31 . The overall concordance between NGS and RT-qPCR data was almost complete with only 5 of 36 comparisons giving discordant statistical testing results . In addition, the whole set of clinical samples were also assessed Fig. .Figure 4To increase robustness, only miRs with at least 100 normalized counts were considered. The following miRs were chosen miR-9-5p, -29a-3p, -106b-5p, 143-3p and -145-5p. Furthermore, generally cancer-related relevant miR-21 and miR-100 were also chosen for this analysis. The analysis reinforced relevance of miR-9 (p = 0.001), miR-21 (p < 0.0001), miR-29a (p = 0.038), miR-100 (p = 0.0029), miR-143 (p = 0.050) and miR-145 (p = 0.006) , while several isomiRs of the same miR were significant in O+ subset. On the other hand, neither mature miR-9 nor its isomiRs were found as significant in OP− or O− subsets. Another very consistent result was the significant upregulation of miR-21-3p in all comparisons; again, indicating the global relevance of this miR. On the other hand, mature miR-29a, -100, -143 and -145 were not found to be significantly deregulated in either set, but their isomiRs were. In the case of OP subset, the direction of change was the same as in our samples and in some cases isomiRs were abundant. Average normalized count of miR-29a isomiR was 3600 , of miR-100 isomiR was 10000 , of miR-106b isomiR was 1980  and of miR-143 isomiR was 56000 . It is also important to note that the deregulation of miR-29a-3p and its isomiR, which was significant in OP+, were however not found to be significantly deregulated in OP− or O− subsets. However, unlike our findings, the same miRNA was not significantly deregulated in O+ subset of TCGA data. The only miRNA that we identified in our samples that couldn’t be confirmed in the reanalysis of TCGA data was miR-27a-5p, which was not found to be significantly deregulated in the TCGA data in any of the comparisons on the mature or any of the isomiR forms.et al.26 risk levels (complete or without intermediate risk samples). Twelve different classification models were created 33 pathways appear to be significantly associated with miRNA targeted genes. This gene list was based on gene-miRNA validated interactions (Supplementary Dataset SD6). Similar analysis of miRNAs specific for HPV− subsets provided a list of 88 associated cell signaling pathways. Table To evaluate potential role of miRNA significantly differently expressed exclusively in HPV+ subsets of HNSCC, all such miRs (including isomiRs) with at least 100 normalized counts on average were entered in Diana tools miRPath (v3.0)34. However, it has to be noted that median ages of HPV positive and HPV negative groups overall were similar, although in general HPV related HNSCC are usually more associated with lower age10.The literature regarding miRNA expression in HNSCC is significant. However, there is no clear consensus on significance of individual miRNA sequences found as deregulated. Specifically, while there are strong overlaps in literature for some miRs  it is difficult to pinpoint a miRNA sequence or set of sequences that would distinguish HPV positive and HPV negative HNSCC to help differentiate those two etiologically different subtypes of HNSCC. In most cases, potential miRNAs are found equally often by studies focusing on HPV positive but also by studies focusing on HPV negative HNSCC reducing the chance that deregulation of such a miRNA is HPV related. To add to the current knowledge, we assessed the miR profiles in a well described set of clinical HNSCC samples. The study group consisted of primary oral and oropharyngeal tumors with known HPV status on both DNA and RNA level. The overall HPV prevalence (DNA and RNA positive) was 23% but that increased in tonsil subset (57%). HPV prevalence and overall structure of patients fits well with current literature on HNSCC37, thus the isomiRs cannot be simply disregarded.It is still unclear why miRNA profiles exhibit such large differences among studies. One of the likely reasons is the overall heterogeneity of HNSCC as a group leading different authors to include differently structured sample groups in their studies. Furthermore, unstandardized methods of HPV detection or inference of its presence  also led to different groupings if at all considered. Added to that were methodological differences in miRNA detection, quantitation and normalization in each study. However, another less evident reason for discrepancies could be the isomiRs. In our main NGS sequencing experiment significant differential regulation of 1145 miRNA sequences was detected across comparisons but the majority were actually isomiR sequences (Supplementry Dataset S3). It is currently unknown to which extent do these sometimes very abundant forms affect results of other studies where they cannot be distinguished from mature miRNA when utilizing methods other than NGS. On the other hand, several studies have thus far pointed out the independent importance of isomiRs in gene regulationet al.26, three distinct survival profiles were observed. The greatest risk was for HPV negative OPSCC patients, however, HPV positive patients were in the lowest risk group only if they didn’t smoke or had tumors with lower nodal stage. It is possible that inclusion of samples classified as intermediate risk group might confound miRNA results and associations both herein and in other previous studies. Indeed, 2 of our 9 sequenced HPV positive samples could be classified as intermediate risk group, where in addition to HPV, other factors like smoking might further influence the miRNA profiles. However, classifying samples only according to Ang et al.26 risk factors still did not resolve the issue of suboptimal separation either in our samples or those in TCGA.Another important factor possibly confounding both previous and current findings is the fact that HPV itself does not need be a causal factor in tumor development even if found in the tumor as other factors like smoking might have a stronger impact. As emphasized in the study by Ang 38. While it is possible that the detected form of unspliced mRNA is due to DNA carryover, this is unlikely as RNAse-free DNAse step during RNA isolation was performed to minimize such possibility. It is also interesting to note that this sample was classified as an intermediate risk group sample  and all clinical samples by qRT-PCR. The concordance of two methods on the same samples was very good . Furthermore, both miRNAs were thoroughly reviewed very recently in the context of different cancer types39. However, its roles are very dependent on the context. Other studies have shown miR-9 to be upregulated in recurrent HNSCC42, but also as a potentially good salivary43 and even methylation biomarker44. It was one of the few overlapping miRs identified between HNSCC and cervical cancer22. More importantly, previous functional studies have already shown that miR-9 seems to be the miRNA most activated by HPV E6 protein in cervical cancer45.Briefly, miR-9 appears to be upregulated by HPV-E6 and when upregulated, it blocks keratinocyte differentiation and induces proliferation and migration40, but it is most often downregulated across cancers, and thus might be considered tumor suppressive. It was shown to influence proliferation, apoptosis, angiogenesis and metastasis depending on cancer type. Interestingly, miR-29a was also shown to induce drug resistance40, which might also explain some of the survival differences between HPV positive and HPV negative cancers if it is more often (but not exclusively) affected in HPV positive cancers.MiR-29a was also shown to behave differently depending on the contextet al.26 risk levels). We created different models where classifier was trained either on our data or TCGA data and then used to classify the other set. Since the maximal success rate was 64%, no classifier model was robust enough. Interestingly, models trained on one half of TCGA data and tested on the other half of TCGA data were also not successful even though their cross validation error rates were very low. It appears that large sample heterogeneity, even in the independent sample sets such as TCGA, might in part explain the discrepancies in previous literature. It probably is interesting to note that classifiers based on miRNA expression might need further specialized statistical modeling to decrease the impact of “normal tissue contamination” on the classifier29; however, this was beyond the scope of the current study.Following examination of individual miRNAs, we also attempted to produce classifiers based on combinations of several miRNA sequences , different methods  and different grouping of samples during analysis . Despite this, some miRNAs show consistency, in particular miRs -21, -100 and -145 are overall relatively consistently detected in all HNSCC studies. However, of HPV specific miRs, only miR-9 seems to be consistently found in HPV positive and rarely in HPV negative subsets of HNSCC, including results from clinical samples in this study and specifically the analysis of TCGA data. Thus, miR-9 is the most likely miRNA specific for the HNSCC with the HPV etiology. While overall miRNA profiles also show lack of consistency and do not easily allow classification based on specific patterns, it appears that miRNAs identified in HPV positive and HPV negative cancers possibly affect cancer relevant pathways differently hence, reinforcing their etiological differences.Supplementary InformationDataset 1Dataset 2Dataset 3Dataset 4Dataset 5Dataset 6


Worldwide, 96,000 cases of oropharyngeal cancer (OPC) occurred in 2012. Human papillomavirus (HPV) is a risk factor for OPC. Data on oropharyngeal HPV infection are limited. There is no consensus on the best sampling method for detecting the infection. We describe the prevalence of oropharyngeal HPV infection among HIV-infected men and compare the performance of oral rinses and swabs in detecting oropharyngeal HPV infection. Paired oral rinses and swabs for 181 men were tested for HPV DNA using the Roche Linear Array. Performance was determined by the number of infections detected and the percentage of samples with adequate DNA extraction. Agreement between sampling methods was assessed by the kappa statistic. Prevalence of oropharyngeal HPV infection with rinse samples was 1.8% (three infections) and 0.6% (one infection) with swabs (p = 0.06). Adequate cellular DNA extraction was more likely with rinse (93.4%) than swab samples . There was moderate agreement between the methods (kappa = 0.49). The prevalence of oropharyngeal HPV DNA infection among this predominantly heterosexual sample of men living with HIV was low and consistent with the infrequent oral sex practices. Oral rinse performed better than oral swab in detecting oropharyngeal HPV DNA infection and might contribute to screening for OPCs. Women are more likely to generate antibodies following genital HPV infection than men, with these antibodies offering some protection against oral HPV infection.4 In addition, women have higher genital HPV viral loads, making transmission higher with male–female oral sex than female–male.4 HIV-infected men have a higher risk for oropharyngeal HPV infection and OPCs than HIV-negative men.6Human papillomavirus (HPV) plays an aetiological role in OPCs, with 25–50% of OPCs related to HPV.7 A study in Pretoria, South Africa documented a prevalence of 6% among 125 men, of whom 4% were HIV infected.8 Another study of 34 men in Soweto, South Africa, of whom 9% were HIV infected, reported a prevalence of 18%.9 It is difficult to directly compare prevalence estimates, as sexual orientation, HIV sero-status, sample sizes and HPV sampling methods vary between studies. Using baseline data from a cohort of men living with HIV infection (MLWH) in Johannesburg, South Africa, we document the prevalence of oropharyngeal HPV and compare the performance of oral rinses and oral swabs in detecting these infections.Data on prevalence of oropharyngeal HPV infection from low- and middle-income countries are limited. The prevalence among healthy men in Brazil was reported as 2%.10 Briefly, men were enrolled if 18 years or older, HIV-positive and reported sex in the past three months.10 We collected socio-demographic and behavioural data using a questionnaire. Participants completed sensitive questions on sexual behaviour using computer-assisted self-interview (CASI). The primary aim of the cohort study was to evaluate the natural history of HPV infection and disease in HIV-infected men in South Africa to help inform the selection of HPV prevention interventions in this population. The sample size of 181 was deemed adequate to compare the performance of oral rinse and oral swabs with sensitivity of 70%, specificity of 90% and precision of 10%. Calculations were based on oral HPV prevalence of 20%. We assumed that the prevalence of oropharyngeal HPV infection in men who were all HIV-positive would be higher than the 18% in men in nearby Soweto, of whom only 9% were HIV-positive.9 The study was approved by Wits Human Research Ethics Committee . Written, informed consent was obtained from participants after explanation of study objectives and procedures.The cohort study enrolled 304 men, of whom 181 were randomly selected for this sub-study using random numbers generated in Microsoft Excel. Full details of study procedures are described elsewhere.A comprehensive oral examination was conducted by medical officers, which included inspection of the gums, tonsillar pillars, tonsils, retropharyngeal wall and palate. Oral rinses were first collected by asking participants to gargle 15 mL of normal saline for 15 s before spitting the fluid into a container and centrifuging it at 3000 r/min for 10 min to obtain sediment. A cotton swab was then rubbed against the palatine tonsil and oropharynx. At the same visit, to assess anogenital-oropharyngeal HPV genotypic concordance, a genital swab was collected by rubbing a cotton swab around the glans penis, coronal sulcus and ventral surface of the penis. Intra-anal swabs were collected by blindly inserting a Dacron swab 3 cm into the anal canal and removing it while rotating and applying pressure on the walls of the canal. The swabs and sediment were stored at −70°C before HPV DNA testing. Venous blood was collected for CD4 cell count and HIV-1 plasma viral load (PVL) testing.The MagNA Pure LC DNA Isolation Kit I  was used to extract HPV DNA from the swabs and sediment. HPV genotype distributions were assessed by the Roche Linear Array assay . The human β-globin gene served as an internal control for cellular adequacy, extraction efficiency and amplification. Therefore, if the internal control is positive, then HPV will be detected if present. All HPV test strips were interpreted separately by two people to minimise reading errors.a + d)/N, where a = number positive by both assays, d = number negative by both assays and N = total sample and the kappa statistic. Since the contingency table was unbalanced, we calculated percentage positive and negative agreements, defined as a/[0.5 × (N+ a−d)] and d/[0.5 × (N − a + d)], respectively.11 Stata™ Version 13 was used for all analyses.Fisher’s exact test was used to compare the percentages of samples with adequate cellular DNA extraction and HPV DNA positive as well as characteristics of participants with and without oropharyngeal HPV infection. Agreement between the two sampling methods was assessed by the percentage of crude agreement, defined as p = . More than one-fourth smoked (28%) and half drank alcohol (53%). Eighty per cent were taking antiretroviral therapy (ART), of whom 55% had undetectable PVL. The median number of life-time sexual partners was 30, with 27% reporting more than 100 partners. A quarter had had more than one sexual partner in the preceding three months (27%). Smaller proportions reported ever having oral sex: 15% oral-genital and 4% oral-anal contact. Only 7% reported ever having sex with men (MSM).Oral rinse samples were more likely to have adequate cellular DNA extraction than the oral swab samples . Oral rinse samples identified three HPV infections, compared to only one with oral swabs (p = 0.06). Overall, 82% of the samples gave concordant results, kappa = 0.09, indicating minimal agreement. When analysis was restricted to the 151 samples with adequate DNA extraction, there was moderate agreement between the sampling methods . Corresponding percentages of positive and negative agreement were 50% and 99%, respectively .Overall, prevalence of HPV DNA was 1.8% . The three participants with oropharyngeal infections had high-risk sexual behaviour. They all reported a sexual debut aged under 18 years, more than one sexual partner in the past three months and had not used a condom with their most recent partner. They were all clinically stable: on ART for more than 18 months, with CD4 cell count above 500 cells/µL and PVL less than 50 copies/mL. No macroscopic oropharyngeal lesions were found on examination of these three participants .We also examined anal and genital samples of the three men who had oropharyngeal HPV infection . Partici7 but is several fold lower than the two previous estimates from South Africa.9 These variations likely reflect differences in sexual behaviours between the study populations since all three studies used oral rinse samples. For example, 80% of men in the Soweto study reported oral sex compared to only 15% in our study.9 Men in our study reported higher numbers of lifetime sexual partners than the average (10) documented for Southern Africa.12 The reason is unclear, but CASI compared to interviewer-administered questionnaire (used in the Pretoria study) and self-administered questionnaire (used in the Soweto study) could have minimised social desirability bias. This is plausible because being an MSM is still stigmatised within South African communities despite the decriminalisation of homosexuality in 1994.13Our study shows that oropharyngeal HPV prevalence in this population is low and that the detection of infection is influenced by sampling method. The prevalence of infection was similar to the 2% reported among men in Brazil,2 This is in keeping with our finding that the anal or genital HPV infection status per se was not associated with oropharyngeal infection. However, HPV auto-inoculation through non-sexual means, such as contaminated fomites or fingers is possible.14 It is noteworthy that no HPV type 16 infection, which is mainly implicated in OPC, was isolated. The implications of this finding are unclear but could relate to the generally low prevalence of oropharyngeal HPV infection among these men who all had no OPC. Future studies which test the HPV status of histologically-confirmed OPC cases in South Africa are required to better understand the role of HPV type 16 in the causation of these tumours.We found concordant HPV DNA genotypes from anogenital and oral samples in two participants who reported oral sex. This supports the contention that oral sex is responsible for anogenital to oropharyngeal transmission of HPV infection, and that the observed rise in OPC associated with HPV may be attributed to changes in sexual practices.15 This is likely due to the rinse washing out exfoliated cells from around the oral cavity, while oral swabs may be unable to access areas like the tonsillar crypts.15 Overall, there was minimal–moderate agreement between the two sampling methods. The difference between the high crude agreement and moderate agreement from kappa is explained by the percentage positive and negative agreements which show that the main area of disagreement between the sampling methods was in positive results.11Similar to previous studies, we found that oral rinsing is better than oral swabbing for ensuring adequate cellular DNA extraction and detecting oropharyngeal HPV infection.The main limitations of this study are that it is cross-sectional and the prevalence of oropharyngeal HPV infection is low, making it difficult to detect associations. Additionally, it consisted of mainly heterosexual HIV-positive men, and findings may not be extrapolated to other populations. Concordance of genotypes from anogenital and oral samples does not necessarily imply that these are the identical viruses and it is possible that these were two separate infections with the same HPV type since viral gene sequencing was not done. Despite these limitations, it contributes literature on the prevalence of oropharyngeal HPV infection among men in South Africa and informs decisions about oropharyngeal sampling.The prevalence of oropharyngeal infection among this predominantly heterosexual sample of MLWH was low and consistent with the infrequent oral sex practices. Oral rinse performed better than oral swab in detecting oropharyngeal HPV DNA infection and might contribute to screening for OPCs.Prevalence of oropharyngeal infection among this predominantly heterosexual sample HIV-positive is low.Oral sex is a risk factor for oropharyngeal HPV infection.Oral rinse performed better in detecting oropharyngeal HPV DNA infection and might contribute to screening for OPCs.The dataset generated during and/or analysed during the current study is not publicly available as it contains longitudinal data which are yet to be published but are available from the corresponding author on reasonable request.