Plasmodium vivax parasites are the predominant cause of malaria infections in the Brazilian Amazon. Infected individuals are treated with primaquine, which can induce haemolytic anaemia in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals and may lead to severe and fatal complications. This X-linked disorder is distributed globally and is caused by allelic variants with a geographical distribution that closely reflects populations exposed historically to endemic malaria. In Brazil, few studies have reported the frequency of G6PD deficiency (G6PDd) present in malaria-endemic areas. This is particularly important, as G6PDd screening is not currently performed before primaquine treatment. The aim of this study was to determine the prevalence of G6PDd in the region of Alto do Juruá, in the Western Brazilian Amazon, an area characterized by a high prevalence of P. vivax infection.Five-hundred and sixteen male volunteers were screened for G6PDd using the fluorescence spot test (Beutler test) and CareStart™ G6PD Biosensor system. Demographic and clinical-epidemiological data were acquired through an individual interview. To assess the genetic basis of G6PDd, 24 SNPs were genotyped using the Kompetitive Allele Specific PCR assay.Twenty-three (4.5%) individuals were G6PDd. No association was found between G6PDd and the number of malaria cases. An increased risk of reported haemolysis symptoms and blood transfusions was evident among the G6PDd individuals. Twenty-two individuals had the G6PDd A(−) variant and one the G6PD A(+) variant. The Mediterranean variant was not present. Apart from one polymorphism, almost all SNPs were monomorphic or with low frequencies (0–0.04%). No differences were detected among ethnic groups.The data indicates that ~1/23 males from the Alto do Juruá could be G6PD deficient and at risk of haemolytic anaemia if treated with primaquine. G6PD A(−) is the most frequent deficiency allele in this population. These results concur with reported G6PDd in other regions in Brazil. Routine G6PDd screening to personalize primaquine administration should be considered, particularly as complete treatment of patients with vivax malaria using chloroquine and primaquine, is crucial for malaria elimination.The online version of this article (doi:10.1186/s12936-017-1889-6) contains supplementary material, which is available to authorized users. G6PD gene, located on chromosome X, has a high genetic diversity and is highly polymorphic. The majority of mutations do not alter the function of the enzyme, but some can lead to a decrease in the enzymatic activity and cause the G6PD deficiency (G6PDd) phenotype [Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme present in all cells and is involved in the prevention of cellular oxidative damage. This enzyme has a particularly critical function to control the oxidative damage in red blood cells (RBC) and maintain their integrity. In spite of its vital function, the henotype , 2.Plasmodium vivax clinical relapses [G6PDd is very common worldwide and is characterized by a range of enzyme activity levels and associated phenotypes . The majrelapses .Severe G6PDd symptoms, including chronic or intermittent haemolytic anaemia, are in general associated with rare allelic variants. The geographical distribution of G6PDd globally is variable, but it is particularly prevalent in malaria-endemic countries , 6. The G6PD Mediterranean variant (C563T) is predominant in West Asia  [Two genetic variants (G6PD A(−) and G6PD Mediterranean) are among the most frequent and are prevalent in particular populations , 6. The riant C56T is predo India) . In SoutP. vivax is the main malaria parasite in Brazil and PQ, which triggers acute haemolytic anaemia in G6PDd individuals, is the recommended drug to prevent P. vivax clinical relapses. Therefore, to predict the potential incidence of primaquine (PQ)-associated acute haemolytic anaemia and determine the likely need for G6PDd testing, this study aimed to estimate the prevalence of phenotypic G6PDd and identify the main genetic variants in a malaria-endemic area in the Western Brazilian Amazon.In Brazil, a predominance of the African variant (A−) has been reported, and few new mutations identified . To dateA cross-sectional study was carried out from June to August 2016 in the Amazonian region of the Alto do Juruá valley . This region, which has high risk for malaria transmission, includes 5 main cities: Cruzeiro do Sul , Rodrigues Alves , Mâncio Lima , Marechal Thaumaturgo  and Porto Walter , with a total population in 2010 of 131,505 inhabitants  [® portable spectrophotometer for all volunteers. The result of the enzymatic activity was calculated by normalizing the value attained in the Carestart test (U/dl), which is linked to the value of the haemoglobin level measured by the Hemocue (g/dl). All tests were performed immediately after blood collection. At the time of recruitment, for the volunteers who presented signs and/or symptoms of malaria, the diagnosis was made by thick blood smears and read by a local microscopist from the endemic surveillance team as recommended by the Brazilian Ministry of Health. Only 14 individuals were positive. The fact these individuals had malaria at the time of recruitment was not an exclusion factor.Four ml of venous blood were collected from each person using EDTA tubes (BD Vacutainer). The qualitative determination of G6PDd in whole blood was performed by a fluorescence spot test using the method of Beutler  and posiFor molecular characterization of G6PDd, total DNA was obtained from whole blood cells through the use of a commercially available extraction kit , following the manufacturer’s instructions. A total of 24 single nucleotide polymorphisms (SNPs) were genotyped using the Kompetitive Allele Specific PCR (KASP) assay .SNP selection was based on polymorphisms associated with G6PDd. This included SNPs identified in studies performed in Brazil and Latin America, as well as reported by the 1000 Genomes project , 11–13. The statistical analyses were performed using Stata version 14.1 . The prevalence of G6PDd and variants was calculated as proportions with 95% CI (CI 95%). Qualitative data were analysed with the Chi square test or the Fisher Exact-test when indicated. To assess the extent to which ethnic origin, previous malaria exposure and previous clinical complications were associated with G6PDd, odds ratios (OR) with CI 95% were estimated by univariate logistic regression analysis. Adjustment for multiple variables was performed by adding the covariates in a set of multiple logistic-regression models. Test for trend was conducted through the Exact test for trend (modified Wilcoxon test). A two-tailed value of P < 0.05 was considered statistically significant.A total of 516 male volunteers from the Juruá Region in the state of Acre were screened for G6PD activity using the Beutler test. The majority of the individuals were of combined Mestizo (European-Amerindian-African) descent (74.6%), followed by European (14.1%) and Afro-descendants (10.3%) Table . The avep value <0.001; jaundice OR 7.13, CI 3.02–16.85, p value <0.001; black urine OR 4.09, CI 1.75–9.56, p = 0.001), need for hospitalizations  and blood transfusions  reported in the G6PDd group was higher than in the G6PD normal group , Mediterranean, Seatle, Chatam, Santamaria, Betica-Selma, and other SNPs that were polymorphic in South America, according to the 1000 Genome data [G6PD A(−) variant (376 A>G and 202 G>A mutations) . Malaria episodes were reported in both groups at the same frequency, indicating that probably G6PDd does not protect against iewed in ). In coniewed in , 21, andiewed in –17, 22.P. vivax malaria [The prevalence of genetic variants associated with G6PDd were investigated, and 22 out of 23 individuals were detected with the G6PDd A(−) variant, and one individual with the A(+) variant. The prevalence of the G6PDd A(−) variant agrees with data reported in Brazil, where an average of 86% of individuals have the A(−) variant  and Asia (Japan). The frequency of G6PDd among ethnic groups was investigated and an increase of G6PDd was found in Afro-descendants (5.6%) but this difference was not significant when compared to European descendants (4.1%) and those of combined Mestizo ancestry (4.4%) (p = 0.918). The data from this and other studies suggest that the Brazilian population is quite homogeneous regarding G6PDd, regardless of the ethnic groups.P. vivax infected patients to prevent malaria relapse episodes, but in the absence of informative G6PDd screening. The complete treatment of P. vivax malaria advised by the Brazilian Ministry of Health includes a 3 day dose of chloroquine (25 mg/kilo of body weight) and a 7 day (0.5 mg/kg) or 14 day (0.25 mg/kg) scheme of PQ treatment. A short 7 day scheme doubling the PQ dose in order to avoid patients low adhesion to the prolonged treatment is also accepted.This study is one of few that investigated G6PDd in an area endemic for malaria in Brazil. Fewer than one in three studies were performed in malaria-affected areas and focused on Manaus, Acre and Rondônia, with an absence of information available in other malaria-endemic areas. In Brazil, PQ is used in In the Juruá Valley region, the data suggest that one in every 23 male individuals could be G6PDd and, therefore, at risk of haemolysis after PQ treatment. In the Brazilian Amazon several reports have associated G6PDd with a considerably higher risk of haemolysis in patients using PQ, and lethal cases have also been reported (reviewed in ). G6PDd P. vivax, PQ treatment is routinely used to prevent relapses from the dormant hypnozoites in the liver. Consequently, one in each 23 males could be G6PDd and at risk to develop haemolytic anaemia upon PQ administration. In order to decide the importance of routine G6PDd screening, particularly if complete treatment of patients with vivax malaria is needed to accelerate elimination of the disease, it is important to consider the cost and feasibility of screening and the risk of severe haemolytic response at different levels of G6PDd.In this study, the prevalence of G6PDd was evaluated in a malaria-endemic region in the Western Amazon region, and detected a frequency of 4.5%. The G6PDd A(−) variant was responsible for almost all G6PDd . Because this region is endemic for Additional file 1. Flow diagram of the study detailing exclusion criteria.Additional file 2. Characteristics of G6PD deficient individuals and G6PDd screening results.Additional file 3. List of single nucleotide polymorphisms analyzed.


High risk human papillomavirus (HR-HPV) infection in males is a health issue with implications for HPV-related lesions in their partners. The identification of risk factors for male infection may improve our understanding of HR-HPV transmission and prevention. The aim of this study was to evaluate the relationships between lifestyle, genital warts and HR-HPV infection. The study was focused on men with an increased risk of HR-HPV infection: male sexual partners of women diagnosed with high-grade squamous intraepithelial cervical lesions.Men were enrolled and prospectively recruited within the first six months after diagnosis of cervical lesions in their female partners . Epidemiological and sexual behaviour data were obtained. The presence of genital warts was established by visual inspection. Detection and genotyping of HR-HPV infection in genital samples were performed with a Linear Array HPV Genotyping Test. All HR-HPV positive men were offered a follow-up exam at 12 months. SPSS version 19 was used for statistical analysis.p = 0.015), smoking habits  and sexual debut before 16 years old  were associated with an increased risk for HR-HPV infection . This association was also observed for genital warts and smoking status in a multivariate analysis. The same genotype was found after one year in 71.4% (20/28) of subjects.The prevalence of HR-HPV infection in men was 45.1% (79/175). Genital warts were observed in 10.3% (18/175) of the subjects. Detection of genital warts (OR 3.5, The presence of genital warts and smoking habits seem to be associated with a higher risk of HR-HPV infection in males. Earlier sexual debut may increase this risk. Extensive knowledge of the natural history of HR-HPV infection in males is an absolute requirement for the design and implementation of prevention strategies for the general population as well as for specific populations such as couples after treatment for high-grade cervical lesions. Human papillomavirus (HPV) infection is one of the most common sexually transmitted infections. It is known that up to 70% of both men and women of the world population will be infected during their lifetime. Approximately 60 HPV genotypes are involved in infection of the genital tract, and at least 12 genotypes are considered to be high risk (HR-HPV) or oncogenic [being which is one of the leading causes of cancer-related mortality in women [The main consequence of HR-HPV infection is cervical cancer, in women . This rein women . Moreovein women . Althougin women . About oin women . Male inin women ,9, and tSince the risk of HPV infection and cervical cancer in females is significantly influenced by male behaviour ,11, a grSome of the most consistently reported risk factors for the acquisition of HPV infection in men are higher number of current and past sex partners (SP) , lack ofThe strength of the reported associations vary depending on the features of the populations under examination and the HPV genotype involved, i.e., oncogenic, non-oncogenic or both.Since it is known that HPV vaccination is a potential preventive approach for men, additional insight from epidemiological studies of HPV infection in men is needed.The aim of this study was to establish relationships between lifestyle factors, genital warts (GW) and HR-HPV infection in a high-risk population: male sexual partners of women diagnosed with high-grade squamous cervical lesions.A cross-sectional study was conducted by the Urology Department of the University Hospital of Vigo. Between January 2014 and December 2016, 202 asymptomatic men older than 18 years were invited to participate in this study because their current partners  presented HGCL  in the previous six months. These couples were selected from the Cervical Cancer Screening Consulting, Department of Obstetrics and Gynaecology, Vigo, Spain. Only 175 of 202 (86.6%) couples met the inclusion criteria for the study. Women were diagnosed by cytology, colposcopy and histological examination. HPV genotypes associated with lesions in women were found in 171 cases. After physical examination, a penile scraping was obtained and submitted to PCR assay to identify HPV carriers. The institutional review board approved the study . Information concerning the research project was provided to all participants. Written informed consent was obtained from all couples. A genital examination was done to identify signs of flat, macular and acuminate lesions (GW). Men were invited to fill in a self-administered questionnaire on lifestyle, including sexual behaviour (SB). Because most men are not knowledgeable about the implications of a positive HPV test, the staff spent the necessary time educating men about HPV. We explained that a positive test for the virus does not necessarily put them at risk for disease. Participants did not receive any incentive for study involvement.All HR-HPV positive men were invited to a clinical follow-up at 12 months. Condom use was recommended to all couples. Women were advised to take the HPV vaccine in accordance with Spanish recommendations to prevent new infections by different HR-HPV genotypes . All womThree, dry cytobrushes for each male were used to scrape the genitalia three times to collect exfoliated cells from different penile areas, including the dorsal and ventral penile area, external and internal surface of prepuce, coronal sulcus, glans and distal urethra. The three samples were combined and collected into one single vial containing molecular biology grade TE buffer pH 8.0 . Samples were maintained at 2–8°C and processed within 24–72 hours after collection.The following variables were studied: age, nationality, HR-HPV infection and specific HPV genotype, age at first sexual intercourse (FSI), number of sexual partners (SP) in the year preceding the study, number of sexual partners up to the date of the study, smoking in the last year, circumcision, regular condom use in the last year, and presentation of genital warts. An extended questionnaire  was valiDNA was isolated using the QIAamp MinElute Media Kit . The extracted nucleic acids were stored at -20°C. An aliquot of the original sample was also stored at -20°C. Amplification and detection were carried out using the Linear Array HPV Genotyping Test  according to the manufacturer's instructions. We described the distribution of 21 HR-HPV genotypes classified as HR  or probable/possible HR   by the Iodds ratio (OR) and corresponding 95% confidence interval (95% CI) were calculated. A p value <0.05 was considered statistically significant. Multivariate logistic regression analysis was used to evaluate factors that were independently associated with HR-HPV detection and with 12 months persistence of HR-HPV infection in males (step-wise logistic regression models). Only variables with p ≤0.25 in the bivariate analysis were included in the multivariate analysis. A manual stratified analysis was conducted to explore potential interactions and additive effects among variables. SPSS version 19.0  was used for statistical analysis.Age and standard deviation were calculated. Qualitative variables were compared using the Chi-square test. The A total of 175 couples were included. The average age of men was 38.6 ± 8.7 (22–70) years. The average age of women was 36.1 ± 8.0 (22–65) years. The prevalence of HR-HPV infection was 45.1% (79/175) in men and 97.7% (167/171) in their female partners. Their distribution is shown in vs. 34.1% (28/82), OR 2.3; 95%CI: 1.2–4.3) and the presence of genital warts (77.2% (13/18) vs. 42% (66/157), OR 3.5; 95%CI: 1.2–10.5) were associated with an increased risk for HR-HPV infection in men in the univariate analysis. Being under 16 years old at first sexual intercourse was also associated with an increased risk for HR-HPV infection in men (Smoker status (54.8% (51/93) n in men . Male cin in men .p = 0.686), smoking status (p = 0.615), genital warts (p = 0.311), male circumcision (p = 0.503), condom use (p = 1), lifetime sexual partners (p = 1) and monogamous relationship with an HPV-infected woman in the last year (p = 0.295) were not associated with an increased risk for HR-HPV infection at the one-year follow-up.A total of 55 HR-HPV positive patients fulfilled criteria for a one-year follow-up. All HPV positive men received a letter of reminder for the follow-up date. Among them, 28 (50.9%) agreed to complete the follow-up and attended the scheduled appointment. Mean age, HPV16 infection and severity of female histological alterations were similar between those who agreed to complete the follow-up and those who did not. The same HR-HPV genotype was found in 71.4% (20/28). HPV16 was found in 55% (11/20) of patients with the same genotype infection at the 12-month follow-up. Their distribution is shown in The objective of this study was to assess the risk factors associated with HR-HPV infection in a high-risk male population. Male SPs of women with HGCL have been the focus of previous studies ,19–21. IIt is still unclear whether a reinfection pattern between couples could occur . It was In addition to HPV16, which is the most common genotype , other gThe clearance rates for HPV infection have not been extensively investigated; however, findings suggest that HPV infection clearance is quicker in men than in women . NeverthAs we reported previously , in our In the present study, the prevalence of genital warts was 10.3%, which was lower than that shown in previous studies (17%) . GenitalIn this study, certain sexual behaviours were associated with HR-HPV infection in men, similarly to previous reports ,35. For et al. reported that 64.4% of positive subjects had ≥5 lifetime sexual partners in their population [Burchell pulation , which ipulation  and Mexipulation . One expet al. found an association between HPV infection and number of sexual partners during the previous three months. These results might also be explained by the inclusion of low-risk genotypes in their study or by the shorter time after infection that reduced the possibility of clearing the viral infection. These two factors may have led to a higher overall prevalence.Although 81.8% of HPV infected men in our population were monogamous in the previous year, this fact was not associated with higher or lower HR-HPV prevalence. In contrast, Nielson et al. have provided epidemiologic evidence that male circumcision was associated with a reduced risk of genital infection in males [As in other studies ,40 we diin males . Even thin males . Removalin males . Our resin males .We did not find the expected protective effect of condom use on HPV prevalence. The effectiveness of condom use is still under discussion. Given that the virus is transmitted by skin to skin contact, condom usage has been assumed to be less effective against HPV infection compared to other sexual diseases transmitted by semen or vaginal secretions. Some authors have also reported high HPV prevalence even among those cases who report regular condom use . By contOne limitation of our study was the small sample size. Moreover, in our study, female lifestyle habits  and histories of other sexually transmitted diseases were unknown and therefore could not be assessed. Differences between this study and previously published studies in sample collection, DNA extraction and HPV genotyping methods may also have influenced the results between studies, making it difficult to directly compare results.Males are not only vectors but may also play a major role in the prevention of HPV-related diseases . Men canAlthough similar studies have been conducted in different countries, it is important to evaluate the different features of HPV infection in men from different populations to confirm these results.Future trials on HPV vaccines in males should be carried out to assess the potential impact not only on the presence of penile HPV infection but also on penile subclinical lesions as a surrogate measure for the efficacy of vaccination.The presence of genital warts and smoking status seem to be associated with a higher risk of HR-HPV infection in male, and earlier sexual debut could increase this risk. Extensive knowledge of the natural history of HR-HPV infection in males could be an important goal for the design and implementation of potential prevention strategies for HPV-related disease in men, specifically for high-risk populations such as males in relationships with females after treatment for high-grade cervical lesions. Our results could contribute to improve the existing HPV prevention strategies, including targeted strategies.S1 Appendix(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


To examine cultural barriers and participant solutions regarding acceptance and uptake of the human papillomavirus (HPV) vaccine from the perspective of Black African, White-Caribbean, Arab, Indian, Bangladeshi and Pakistani young people.In total, 40 young people from minority ethnic communities in Scotland took part in a qualitative study, involving seven focus groups and four paired interviews, to explore their views and experiences of the HPV vaccine. Using critical discursive psychology, the analysis focused on young people’s accounts of barriers and enablers to information, access and uptake of the HPV vaccination programme.Participants suggested innovative strategies to tackle intergenerational concerns, information design and accessibility, and public health communications across diverse contexts. A cross-cultural community engagement model was developed, embracing diversity and contradiction across different ethnic groups. This included four inter-related strategies: providing targeted and flexible information for young people, vaccine provision across the life-course, intergenerational information and specific cross-cultural communications.This is the first HPV cross-cultural model inductively derived from accounts of young people from different ethnic communities. We recommend public health practitioners and policymakers consider using the processes and strategies within this model to increase dialogue around public engagement, awareness and receptivity towards HPV vaccination. Human papillomavirus (HPV) is the most prevalent sexually transmitted infection among young people . Three vSince 2008, the UK immunisation programme has offered girls aged 9–14 years protection against two oncogenic types – HPV16 and HPV18 – which are responsible for 70% of cervical cancer cases. More recently, a quadrivalent vaccine has been introduced, which also provides protection against HPV6 and HPV11, which cause anogenital warts . In ScotHPV vaccination programmes are delivered within varying contexts, such as schools and primary care . InterveEvidence also reveals that knowledge and acceptability of the vaccine are lower in some ethnic minority groups . MeaningDuring a catch-up campaign in England, over six hundred 16-year-old girls from a further education college were recruited into a prospective study. Ethnicity was independently associated with vaccine uptake. Those from Black or Other ethnic backgrounds were less likely to have been vaccinated than those from White backgrounds . Using aCommunity engagement is one important facet of any health promotion approach that attempts to address health inequalities : 278. ThMisunderstandings and lack of knowledge regarding HPV vaccination have been associated with poor health literacy . PromotiAdopting a bottom-up approach can inform the design and delivery of effective communication strategies, providing downstream benefit to improved public health . The devNational HPV vaccination programmes tend to adopt universal delivery strategies rather than targeted cultural and demographic strategies. Thus, relational, institutional, cultural and contextual factors that shape sexual health practices are often ignored. Qualitative methodologies, such as discursive psychology, could address this gap, where issues of intersectionality and participation are key. This study examined the accounts of young people from Black, Asian and Minority Ethnic (BAME) communities in order to examine understandings and explanations for HPV-related health behaviours within differing cultural contexts. Drawing on inductively produced participant solutions, we outline a cross-cultural community engagement model for HPV which encompasses diversity across a range of BAME populations.The study involved seven focus groups and four paired interviews with a purposive sample of 40 young people from BAME communities, recruited from three Scottish geographical regions between June and November 2015. Underpinned by a critical public health paradigm and utilising critical discursive psychology , we explAnalysis centred on the ways in which the young people used interaction, language and different discourses to construct their opinions, identities and social worlds, as well as cultural barriers and culturally sensitive solutions regarding access and inclusion within the HPV vaccination programme.n = 21), Sikh (n = 12), and Christian (n = 3). Three were married and 12 stated they had been in a sexual relationship; 18 described themselves as students, 9 were employed, 8 unemployed and 5 still at school; and 23 of the women had received the vaccine.Over 80 community organisations and networks were contacted to invite their members (aged 16–26 years) to participate in the study. In total, 40 participants  took part in the study. This number was large enough to include a diverse range of participants with differing characteristics, yet small enough to allow for an in-depth analysis of the data. Of the 40 participants, 24 were aged 16–19 years and 16 were aged 20–26 years. Participants lived in urban, suburban and semi-rural areas. In all, 28 described themselves as being of South Asian descent, 6 of Arab descent and 6 of Black African descent. Religious beliefs and practices were important to the majority of participants: Muslim  established links with ethnic community groups, societies and networks, who promoted the study and assisted with recruitment. Prospective participants were given a study participant information sheet that provided assurances about confidentiality and anonymity, and written informed consent was obtained.Participants were invited to a gender-specific focus group or paired interview where they could feel comfortable and engage in shared activities, dialogue and debate . The resThe topic guide see  includedFocus groups and paired interviews were audio-recorded, transcribed verbatim and anonymised, and participants were given pseudonyms. The data sets did not differ substantially, so they were analysed as one corpus data set. To enhance credibility and rigour, two of the nine members of the research team coded each transcript for recurring topics and themes, discourses and counter-discourses , signifiParticipants initially revealed minimal understanding of HPV or the purpose of the vaccine, despite acknowledging that information was provided around the time of vaccination. Accounts suggested that sex education had failed to include information about HPV and there were few, if any, opportunities to discuss or learn about HPV and/or related cancers at school or elsewhere. Most were unaware how HPV affected men and were surprised to learn that other countries offer this vaccination to boys/young men. These discussions prompted participants to provide ideas about how HPV education could be delivered in a more impactful way.Similarly, after HPV educational information was provided, participants were surprised regarding the magnitude of the virus. Many were confused by the different types of the virus, its links with cancer and how that develops, and they voiced concern about its prevalence and obscurity. In contrast, some expressed little or no anxieties about HPV, citing religious grounds, suggesting a distancing from the virus by those with specific moral positions. Several Muslim women, aged less than 20, questioned the implications of the virus for them if they were celibate and intending a monogamous life after marriage. Young Sikh men expressed surprise about men not being offered the vaccine and highlighted barriers to discussing HPV transmission routes openly within their community. Similarly, young Muslim men discussed how HPV challenges cultural or religious norms which arouse parental fears of promiscuity, which in turn led to parental disapproval of the vaccine and potential stigmatisation of families who accept vaccination. Accounts of participants of Indian/Pakistan descent and/or Muslim/Sikh religion were contested by men and women of African descent who felt they could talk relatively about sexual practices from a young age. These discussions prompted participants to talk about solutions to overcoming intergenerational concerns and culturally sensitive strategies for different ‘ages and stages’, genders and settings.Having identified cultural barriers, participants discussed how practitioners and policymakers could raise the profile of HPV across diverse communities. These factors and processes are illustrated within the community engagement model  and follRelationships between sexual activity, transmission and cancer were largely unclear to participants and discussions centred on whether practitioners should emphasise cancer or sexually transmitted infections. A staged approach was offered as a solution – delivering information at age-appropriate and differing developmental stages during school and extending this across the lifespan – for example, in contexts such as further education institutions and primary care. These settings were deemed to be ‘neutral’ contexts where cultural sensitivities or conflicts could be avoided and where cultural norms (i.e. marriage) could be enablers. These would offer additional opportunities for young people from minority ethnic communities to consider receiving the vaccine, if they had previously refused or missed the opportunity as adolescents:Amberash:Maybe later on … sex education isn’t for, like, 12, 13 year olds. I think it’s more older, that’s when they can introduce it and make the connection.Chidimma:I don’t think I would have processed the information at that time, even at 16. Priya:I think when you’re that age [9–14 years] … I’d want to know the general information but I wouldn’t want to go into such detail because I wouldn’t be that interested. Miriam:In the Muslim community, round about the 20s is when most people begin to get married … I think that’s a good age to actually raise awareness of it. In relation to health literacy, participants commented on a contradiction within public health provision – the scale of infection and yet the lack of accessible information. They offered notions of what might be concise and clear information since HPV appeared to challenge commonly accepted HIV-related safer sex messages . ParticiFaiza:Quite a lot of people at my school didn’t get it because we didn’t actually know how important it was … we didn’t really know … if it was, like, vital.Basima:It’s because we never really went into depth about why we need it. Ayotunde:I think they are trying to make it look, like, worse than it is … because some of them … are cured by immune system. They don’t need to tell us, it’s a hundred [different genotypes].Chinaka:They don’t need to arouse the fear in people. A flexible age-appropriate and culturally sensitive approach, offering targeted vaccine information, was proposed by participants. Medical and public health practitioners could provide a range of information opportunities across the life-course to raise awareness, regardless of current boundaries of a school-based, gender-specific programme.Providing the vaccine within a variety of contexts was deemed necessary in order to offer more choice and privacy to young people when considering HPV information and deciding whether to receive the vaccination. Participants favoured continuing the current school’s vaccination programme with the inclusion of boys, but widening the programme to offer the vaccine in colleges and primary care. Thus, they advocated a flexible, staged approach, extending the vaccination programme to people older than are currently included. The use of trusted sources  seemed important, acknowledging young adolescents’ engagement in sexual activity in general, and more specifically one that reflects their age of sexual debut around the time of marriage in order to be culturally congruent:Sukhbir:If they get it when they’re 12, they’re just going to be like, oh yes, I got a jag [injection] today, but I don’t know what it was for. But, if they get it later on it will like affect their lifestyle in a way.Achint:Just reiterate it’s not just girls can get it as well, guys can get it, [and] … if they’re not going to give vaccine to boys at least tell them about what HPV is, not have exclusive talks with girls. Mandeep:For me, it would be the later years, even as late as university … If someone offered me the HPV vaccination right now, it might be no. Why do I need another injection for something which I personally deem as probably very low risk to me. Miriam:Because in the Asian community, people need it later, when they’re in their 20s, mid-20s. Say the vaccine and testing is available from your GP if they want to get it. Just so they know they haven’t missed their opportunity. Regardless of the religious persuasion, young women were particularly concerned to discover that men are at risk of developing HPV-related cancers and transmitting the virus to others. Repeated questioning about routes of transmission and pleas for equality – both sexes to be vaccinated – suggested some underlying anxiety about perceived risk of acquiring HPV:Chinaka:And then the older guys are still sexually active. What about them? It should just be mandatory like tax.Ebele:Any guy should be able to get it, whether they’re 60 or they’re 13. Public health policy was frequently problematised, especially regarding how HPV information and the vaccine are delivered inside or outside a school programme where mass vaccinations are administered within a defined immunisation schedule. Having considered the delivery of the immunisation programme in secular contexts such as schools and health centres, participants discussed additional strategies to address tensions within their own cultural contexts.Social norms within families and communities and places of worship were emphasised. Young men from South Asian communities positioned themselves as powerless to resolve intergenerational tensions regarding ‘acceptable’ or open dialogue around HPV. In contrast, the women suggested various opportunities to promote intergenerational information. Across all cultural groups, participants advocated ongoing education for the older generation:Amitt:The older generation, culturally don’t like to talk about how it is. They like to talk about how it should be … Depends on where you put this information. If you put it in universities … you wouldn’t be as wary of cultural effects, but if you’re trying to target a mosque or something like that, be very cautious of the way you approach it. Aminah:It’s like a youth group, but for elderly women, so look for groups like that, or other women’s classes … I think they would be more comfortable … and keener to listen … because they might feel a bit embarrassed, and then they can ask questions. To avoid alienating parents, Muslim men suggested greater transparency and a more personalised approach, for example, by offering parents different ways to contact the school about the vaccine:Khaliq:School newsletters is a good way, put up a little section explaining it, and offer them a contact line or an email, … to enquire about or if you’re worried about HPV just phone this [number], or come to school, or talk to us. In comparison, respondents in a group of Black men argued for a broader approach, educating everyone without fear of repercussions:Amberash:I think parents as well need to be educated about this, so if kids are educated in school, parents have first-hand information already. What I want to see is some leaflets distributed in health centres as well talking about this, which is similar to HIV. As a whole, participants required targeted education to address intergenerational concerns and HPV promotion strategies delivered across a range of settings. Notably, they recommended different modes of communication across South Asian and Black African cultures .Finally, participants called for a broad, diverse and comprehensive approach to tackle the dearth of public awareness about HPV within their communities. This included community outreach for public health practitioners to contact and work at a local and national level; exploiting opportunities such as local community and public events; delivering information in a sensitive manner engendering discussion, trust and dialogue; striving to desexualise and destigmatise the message; and taking it to gender-specific arenas such as established women’s groups in religious or cultural centres . CommuniAmberash:The community, government, realised they find it difficult to deal with us, so we decided to create our own organisation. Now they do approach us and find ways and answers from us … Just train somebody who has more knowledge, who has ideas of minorities, approach more people from minorities, have seminars so people can put their view forward. Participants across all communities suggested a public campaign about HPV. This suited some men and women from South Asian communities who preferred messages to be delivered in ‘neutral’ territory:Mandeep:There are definitely Muslims with alcohol problems, or problems related with STIs … but in a mosque environment, it’s very hard to get them to admit it. That would be like one of the last places they would go to look for information. In an educational environment, in school or universities, student union, all of those kind of places are I think much better suited. Layla:I wouldn’t hand it out in a mosque, no.Faiza:I think they might find that a bit … offensive, yeah. I think they should stick to high schools. Mandeep’s account acknowledges ‘Muslims with alcohol problems, or problems related with STIs’, as a hidden group within cultural norms and expectations. This points to tensions around those individuals who are at risk of HPV through covert sexual practices versus those individuals who are protected from risk through cultural expectations around marriage as sexual debut. Inherent within the account is the precarious notion of deeming marriage as protective for sexual health within such communities.Men from the Gurdwara suggested that peer support from within their community was preferable to relying on outsiders to interpret and explain the vaccine. They warned aligning it with HIV would stigmatise the virus within their community. Muslim young men also raised the issue of stigma if the vaccine was associated with men who have sex with men:Ramindar:I don’t know if it’s as bad as HIV though, that might kind of confuse things. HIV was regarded as a big taboo, it still kind of is. If someone had HIV or someone mentioned it, it was something which caused a divide, and it might be, by aligning it with that you kind of … Mandeep:If you’re aiming it at a specific, like a Muslim culture, it would be more receptive if it was sold almost like the common cold where anyone can get it through physical contact, but you’re more likely to get it if you have sex.Kumar:[the] consequence of rolling it out to gay men first … it becomes something seen as, oh this is important for gay men. So it almost gets, not stigma, but … from a cultural perspective, we might tend to say, that’s only important for gay men, so it’s not really important. In contrast to this fear of offending elders or causing disrespect, some women asked for presentations to be delivered to women’s groups within the religious community and mosque/Gurdwara complexes:Deepti:I think we should start something in the Gurdwara itself for the younger kids that come here.Gurpreet:Or just for women, like younger women …They don’t want to talk about the same subject with men.Divya:If they’re not comfortable speaking about it maybe we can have like wee closed groups like this. An HPV community engagement model, derived from our analysis, conceptualises a process of engagement and empowerment recognising context and complexity as a way forward for policy and practice. Participants provided strategies for tailored public health interventions – how they could be delivered, by whom, when and where. The model embraces cultural sensitivities and diversity with the purpose of raising acceptability of the HPV vaccine and promoting self-determinism. Differing participant views shed light on the question of whether HPV messages should be gendered, culturally specific, general or neutral. As indicated, the context of delivery will determine specificity and tone of the information – each mode requires negotiation with a discrete community and is important for informed decision-making for the vaccine.Participants called for accessible, interactive, experiential and memorable information. This is crucial from a policy perspective because in Scotland, from 1 June 2016, the age of first cervical smear will be offered at 25 years of age – 14 years after HPV vaccination – leading to a potential ‘disconnect’ between HPV, cancer and screening. Some educational interventions show promising results in raising awareness of cancer and healthy lifestyles . One intn = 100) where 39% believed the appropriate age for receiving the vaccine was between 25 and 30 years (White Northern hemisphere norms and assumptions about age of sexual debut were challenged by our participants but left some ‘hidden’ individuals vulnerable within cultural expectations. This indicates a need for transparency regarding immunological reasoning and rationale behind current age of vaccination as well as inviting communities to attend at culturally appropriate stages, through to early adulthood. This issue was echoed by a survey of female family practitioners in Pakistan (30 years .Furthermore, our findings strongly suggest that intergenerational issues need attention. Knowledge transfer may be influenced by how older people interpret HPV information or act as gatekeepers for the community. Participants raised the possibility of consulting directly with elders about HPV during future initiatives. Targeting the vaccine towards men who have sex with men could sexualise the vaccine and lead to further misunderstandings, as highlighted by our Muslim participants. Working in partnership with communities will be essential if policymakers are to devise appropriate non-stigmatising risk communication messages – for example, how much information ought to be disclosed taking into account HPV risk factors, morbidity and mortality rates . A trialParticipant accounts revealed a tension between the need for social integration and localised or ‘neutral’ information for specific communities. The cross-cultural model provides a tool for practitioners and policymakers to engage with community fora, grassroots organisations and advocacy organisations. It stresses the importance of working with people who are in proximity both physically and culturally to discrete communities. A community-engaged research process was described by The apparent need for health information to be made available in a variety of formats and settings was mirrored in a US study of mothers and adolescents from Somali, Eritrean and Hispanic communities . All thrA key strength of our proposed model is that it comprises insights from in-depth discussions with several minority ethnic communities and therefore contributes to a neglected area in research, policy and practice. Participants signalled four areas for practitioners and policymakers to focus on: a targeted information strategy, an extended and flexible gender-neutral vaccination programme, proactive culturally sensitive education across all age groups and partnership working to adopt guidance from each community.We acknowledge that this is a tentative model which will require further testing and refinement with more diverse cultural groups and within different cultural settings, as well as parents, carers and other family members. This study involved a relatively small sample of young people from three geographical regions in Scotland, whose demographic and ethnic backgrounds were varied. Our sample cannot be considered representative of the wider population of young people from all ethnic groups in Scotland or elsewhere. Although our findings are not generalisable, age, gender and generational status were explored and the model and its processes may be transferrable to other contexts. These and other key influences, such as socio-economic status, are likely to shape perceptions and intentions. An understanding of these intersectional influences within different ethnic groups is essential to inform strategies which promote vaccination uptake. Finally, the pragmatics of these suggestions need to be further explored along with the effectiveness in reaching targeted audiences.This paper illustrates a novel HPV community engagement model, inductively constructed from the accounts of young people from Black, Asian and other minority ethnic communities. Implementation of the model could enable dialogue with diverse communities, combining a bottom-up with a top-down approach where priorities can be set by policymakers with the delivery guided by the needs of each specific community. Tackling diversity in this way could increase the profile and future uptake of HPV vaccines.


AP‐1, NF‐κB, and STAT3 signaling in oral cancer. Differential expression/activation of specific members of these transcription factors has been documented in HPV‐positive oral lesions that respond better to therapy. We performed a comprehensive analysis of differentially expressed, transcriptionally active members of these pivotal signaling mediators to develop specific signatures of HPV‐positive and HPV‐negative oral lesions by immunohistochemical method that is applicable in low‐resource settings. We examined a total of 31 prospective and 30 formalin‐fixed, paraffin‐embedded tissues from treatment‐naïve, histopathologically and clinically confirmed cases diagnosed as oral or oropharyngeal squamous cell carcinoma (OSCC/OPSCC). Following determination of their HPV status by GP5 + /GP6 +  PCR, the sequential sections of the tissues were evaluated for expression of JunB, JunD, c‐Fos, p50, p65, STAT3, and pSTAT3(Y705), along with two key regulatory proteins pEGFR and p16 by IHC. Independent analysis of JunB and p65 showed direct correlation with HPV positivity, whereas STAT3 and pSTAT3 were inversely correlated. A combined analysis of transcription factors revealed a more restrictive combination, characterized by the presence of AP‐1 and NF‐κB lacking involvement of STAT3 that strongly correlated with HPV‐positive tumors. Presence of STAT3/pSTAT3 with NF‐κB irrespective of the presence or absence of AP‐1 members was present in HPV‐negative lesions. Expression of pSTAT3 strongly correlated with all the AP‐1/NF‐κB members (except JunD), its upstream activator YpEGFR1092, and HPV infection‐related negative regulator p16. Overall, we show a simple combination of AP‐1, NF‐κB, and STAT3 members’ expression that may serve as molecular signature of HPV‐positive lesions or more broadly the tumors that show better prognosis.Prior studies established constitutively active  Despite being most preventable malignancies associated with tobacco use, squamous cell carcinoma of oral cavity retains the 11th position among the most common cancers. Oral cancer alone accounted for an estimated 300,000 new cases and 145,000 deaths in 2012 with an estimated burden of 702,000 prevalent cases over a period of 5 years κB, and STAT3 that derive or regulate viral gene expression by interacting with the enhancers present in the URR region in vicinity of viral promoters κB κB family that participated in DNA binding κB p50 was found overexpressed and activated in oral cancer; though participation of p65 was detected particularly in HPV‐positive oral cancer κB, and STAT3. Therefore, changes in expression pattern of these key transcription factors may be responsible for biologic variability in lesional behavior and varied treatment response of HPV‐positive and HPV‐negative oral cancer patients Interestingly, tumors with transcriptionally inactive HPV infection  behaved clinically and pathologically similar to HPV DNA—negative tumors, as they had similar gene expression profiles and showed similar 5‐year survival ratesκB, and STAT3 that independently and/or in conjunction with each other control several downstream genes involved in the overall manifestation of carcinogenesis Considering HPV transmission is solely through sexual contact The aforementioned observations prompted us to examine oral lesions for collective expression of a set of transcriptionally relevant factors in context of the HPV‐positive status to define molecular signatures of HPV‐positive lesions. These signatures can prove useful to discriminate lesions which could show better prognosis irrespective of the HPV status.A total of 116 prospectively collected fresh biopsies and 30 formalin‐fixed paraffin‐embedded preserved tissues (retrospective) from treatment‐naïve, histopathologically and clinically confirmed cases diagnosed as oral or oropharyngeal squamous cell carcinoma (OSCC/OPSCC) were obtained from three tertiary cancer care hospitals  and a dental hospital . In addition, paraffin‐embedded tissue sections were also available for 36 oral cancer cases Figure β‐actin) and ABC staining kits were from Santa Cruz Biotechnology . Antibodies and their clonal description are listed in Table S2. Primers/oligonucleotides were custom‐synthesized from Microsynth  or Eurogentec  listed in Table S3. Immobilon‐P PVDF membranes was from Millipore Corporation , xylene from Fisher Scientific , absolute ethanol and H2O2 were procured from Merck . Tween‐20, 3, 30‐Diaminobenzidine (DAB), Meyer's hematoxylin and all other reagents used in the study were of analytical and molecular biology grade and were procured from Sigma Aldrich  unless specified otherwise.All antibodies (except μm thickness) was performed according to xylene‐free method with slight modification μL reaction mixture containing 100 ng DNA, 10 mmol/L Tris‐HCl (pH 8.4), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 125 mmol/L of each dNTPs , 5pmol of oligonucleotide primers, and 0.5U Taq DNA polymerase (B. Genei (Merck), Indi) in ABI2720 Thermalcycler [Applied Biosystems Inc. (Thermo Fisher)]. The temperature profile used for amplification constituted an initial denaturation at 95°C for 4 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min, which was extended for 5 min at the final cycle. Details of primers used for HPV detection and genotyping for HPV16 and HPV18 are presented in Table S3.Genomic DNA was isolated from freshly collected OSCC biopsies by the standard proteinase K digestion followed by phenol‐chloroform extraction and PCR amplification performed following the procedure described earlier Total proteins from biopsies were prepared by the method described previously Immunoblotting was performed as per routine laboratory method described earlier Immunohistochemistry was performed as per routine laboratory method described earlier Two independent pathologists performed the histopathological evaluation of hematoxylin and eosin‐stained tissue sections as per routine procedure. Scoring of IHC staining was performed by two independent investigators (GV and AT). In cancer cases, all the cells were analyzed for c‐Fos, JunB, JunD, c‐Jun, p50, p65, p16, STAT3, pSTAT3, and EGFR immunostaining. Overall inter‐observer difference varied between 5–10%. Discrepant scores were resolved by third evaluation (ACB). Every IHC‐stained tissue was scored as reported earlier P ≤ 0.05 was considered significant.To determine association between the epidemiological and clinicopathological characteristics, Mann–Whitney test and Chi‐square test were employed, using SPSS Statistics Software version 2015 . Chi‐square test and Fisher's exact test  were employed to analyze association between the expression of proteins among different histopathological grades of tissue biopsies using SPSS Statistics Software version 2015 (IBM Chicago). For all analyses, P = 0.038). Histologically, majority of the cases were either well‐differentiated or moderately differentiated and were in their advanced stages. HPV positivity did not display any specific distribution differences with respect to the clinical stage or histopathological grade. Analysis of type‐specific HPV infection by PCR revealed HPV16 positivity of 29% (9/31) and HPV18 positivity of 16.1% (5/31). HPV16 was detected in tumors of different subsites of oral region, whereas HPV18 was confined to buccal mucosa, alveolar/gingiva, and retro‐orbital space, or the oral cavity. HPV infection was absent in normal control tissues.A total of 135 tissues out of 146 qualified for amplification of internal control for subsequent HPV analysis by GP5 + /GP6 +  PCR. Overall, 22.9% (31/135) samples were positive for HPV infection. The distribution of HPV positives was 7/30 in formalin group and 24/104 in fresh biopsies group. The clinicoepidemiological and demographic distribution of samples along with their status of HPV infection is presented in Table Expression of selected AP‐1 members, c‐Jun, JunB, JunD, and c‐Fos was evaluated. Immunoblot analysis of tissues from histopathological grade‐matched tumors from HPV‐positive and HPV‐negative cases demonstrated an increased expression of all the AP‐1 family members tested compared to control tissues. However, JunB, JunD, and c‐Fos showed a further increase in their expression in HPV‐positive oral cancers, whereas c‐Jun did not show a differential expression among HPV‐positive and HPV‐negative tissues . In situ examination of tumor tissues for expression of JunB, JunD, and c‐Fos by immunohistochemical analysis also confirmed presence of the AP‐1 members but their expression varied in HPV‐positive and HPV‐negative tissues on case to case basis . Statistical evaluation of different grades for AP‐1 members’ expression showed a strong correlation of JunB expression with HPV‐positive tumor tissues, whereas JunD and c‐Fos expression did not show any significant association with HPV positivity Table .κB members, p50 and p65 was evaluated. Immunoblot analysis of tissues from histopathological grade‐matched tumors from HPV‐positive and HPV‐negative cases demonstrated an increased expression of p50 compared to control tissues. However, p65 showed a detectable expression in HPV‐positive oral cancers . In situ examination of tumor tissues for p50 and p65 expression by immunohistochemical analysis confirmed variable presence of both the members of NF‐κB family in HPV‐positive and HPV‐negative tissues . Statistical evaluation of the p50 and p65 expression with HPV positivity showed a strong correlation only between p65 and HPV infection in the tumor tissues but the frequency of overexpressed p50 was equally distributed among HPV‐positive and HPV‐negative tumors and did not show any association with HPV positivity . In situ examination of tumor tissues confirmed varied presence of the STAT3 and pSTAT3 in oral tissues. Most of the HPV‐positive tissues demonstrated nil or weak expression , whereas a majority of HPV‐negative lesions expressed moderate/strong STAT3 (57.7%) or pSTAT3 (75.5%) , and STAT3  were scored 0 for nil or weak expression and 1 for moderate or strong expression. Individual molecular marker scores were combined and their distribution was evaluated in HPV‐positive and negative oral cancer lesions by Fisher's test (Table κB (p50 and p65), and STAT3  were scored 0 for nil or weak expression and 1 for moderate or strong expression. Any marker in category AP‐1/NF‐κB/STAT3 if scored as 1 in immunohistochemical analysis, indicated involvement of the respective category of the transcription factor. Based on the representation of each class (AP‐1/NF‐κB/STAT3) of transcription factors, individual tumor tissues were segregated in different categories and their distribution was evaluated by paired t‐test. HPV‐positive oral lesions demonstrated a specific presence of AP‐1 in combination with NF‐κB but with an absence of STAT3 , NF‐T3 Table . The preP < 0.001; OR‐28.16) (Table P = 0.033) and it negatively correlated with HPV infection status (OR – 0.27)  that activates STAT3  that actκB, and STAT3 family and to develop their profile as diagnostic/prognostic signatures of HPV‐positive oral cancer lesions that may show better outcome by applying routine IHC methodology. The investigation revealed a simple combination of AP‐1 (any JunB/JunD/c‐Fos) and NF‐κB (p50/p65) lacking STAT3 (STAT3/pSTAT3Y705), a signature that was associated with HPV‐positive tumors. On the contrary, presence of STAT3/pSTAT3 with NF‐κB lacking p65 irrespective of the presence or absence of AP‐1 members was predominantly associated with HPV‐negative lesions. Although IHC is a subjective and qualitative method, it is a routine and reliable technique in most of the pathology departments and requires minimal infrastructure that can be established in low‐resource settings with relative ease.In this study, we attempted to establish a correlation between key members of AP‐1, NF‐In the study, HPV‐positive cases were of younger age group and HPV positivity was found to be typically over‐represented in males as reported by others HPV infection among different oral subsites varied considerably. This is again explainable with tissue‐specific variations in expression/activity of transcription factors. Tumors from oral cavity collectively showed lesser HPV positivity than tumors of oropharyngeal region. A higher frequency of detecting HPV in oropharyngeal region is well documented Our analysis of full spectrum of AP‐1 member in oral carcinogenesis showed involvement of AP‐1 members c‐Jun, JunB, JunD, and c‐Fos during oral carcinogenesis κB family, p50, and p65, whereas presence of p65 was differentially over‐represented in HPV‐positive oral cancers. NF‐κB interacts with viral promoter via a potential NF‐κB binding site κB increase in oral cancer depending on disease severity irrespective of the HPV infection κB complex. However, nuclear p65 phosphorylated at Ser 276 is found to contribute to malignant phenotype and mediate chemoresistance κB along with AP‐1 members showed a strong cooperativity κB constituted by p50/p65 is essentially required for c‐Fos expression Aside from AP‐1, HPV‐positive oral cancers also expressed key members of NF‐κB κB and EGFR/AP1 networks of signaling pathways and contribute to the malignant phenotype and the intrinsic or acquired therapeutic resistance of HNSCC Examination of activated STAT3 (pSTAT3Y705) showed a negative association of this markers with HPV positivity in oral lesions. The results obtained are in sharp contrast to the carcinogenic role of STAT3 in HPV infection of cervical carcinogenesis κB but with an absence of STAT3. It is hypothesized that the cocktail of the transcription factors from AP‐1, NF‐кB, and STAT3 family collectively determines expression of the components of each transcription factor that further controls distinct sets of genes involved in oral cancer progression. The specific composition of these transcription factors are not only required for active HPV transcription but will differentially control the spectrum of genes expressed in the cell. High‐throughput screens of HPV‐positive and negative head and neck cancers showed differences in the gene expression patterns, which correlated to STAT3/NF‐кB/AP‐1 signaling pathway Correlative assessment of differentially expressed members of AP‐1, NF‐кB, and STAT3 signaling in HPV‐positive and negative tumors revealed specific differences in member profiles. HPV‐positive oral cancers demonstrated a more restricted spread. HPV‐positive oral lesions demonstrated a specific presence of AP‐1 in combination with NF‐P = 0.008). High pEGFR expression was accompanied with pSTAT3 levels in HPV‐negative tumors. Interestingly, HPV infection had no impact on treatment response to EGFR blockers in head and neck cancer and these inhibitors are recommended independently of HPV status Along with active STAT3, pEGFR1092, and p16, being positive and negative regulators of STAT3 signaling, were examined. Our data showed an negative association of pEGFR with HPV infection in oral cancer. EGFR is known to regulate HPV16 transcription via AP‐1. However, in case of oral cancer, we observed its negative correlation particularly with JunB  (1) Representative immunoblots of total cellular proteins (50 μg/lane) from histopathological grade‐matched HPV‐positive and HPV‐negative biopsies from oral cancer patients, tested for the expression of c‐Jun, JunB, JunD, and c‐Fos. The blots were stripped and reprobed for β‐actin and evaluated as input control. (2) Aggregated mean (±S.D.) abundance ratios of the band intensity of indicated proteins in HPV‐positive and HPV‐negative oral cancer tissue normalized to β‐actin in three independent experiments. *P ≤ 0.05 versus expression of the respective proteins in control normal tissues. #P ≤ 0.05 versus expression of the respective proteins in control HPV‐negative oral cancer biopsies. (B) Representative photomicrographs of immunohistochemical analysis of c‐Fos, c‐Jun, JunB, and JunD in histopathological grade‐matched HPV‐positive and HPV‐negative biopsies. Freshly fixed, paraffin‐embedded sections (5 μm) of oral tissues were processed for IHC and probed for c‐Fos, c‐Jun, JunB, and JunD with respective antibodies and detected by HRP‐DAB method. Brown precipitate indicates immunopositive cells, blue stain represent nuclei, and co‐localization of brown and blue stain represents nuclear localization of respective AP‐1 family members.Figure S2. Level of key components of NF‐кB family members, p50 and p65, in HPV‐positive and HPV‐negative oral cancer: (A) (1) Representative immunoblots of total cellular proteins (50 μg/lane) from histopathological grade‐matched HPV‐positive and HPV‐negative biopsies from oral cancer patients, tested for the expression of p50 and p65. The blots were stripped and reprobed for β‐actin and evaluated as input control. (2) Aggregated mean (±SD) abundance ratios of the band intensity of indicated proteins in HPV‐positive and HPV‐negative oral cancer tissue normalized to β‐actin in three independent experiments. *P ≤ 0.05 versus expression of the respective proteins in control normal tissues. #P ≤ 0.05 versus expression of the respective proteins in control HPV‐negative oral cancer biopsies. (B) Representative photomicrographs of immunohistochemical analysis of p50 and p65 in histopathological grade‐matched HPV‐positive and HPV‐negative biopsies. Freshly fixed, paraffin‐embedded sections (5 μm) of oral tissues were processed for IHC and probed for p50 and p65 with respective antibodies and detected by HRP‐DAB method. Brown precipitate indicates immunopositive cells, blue stain represent nuclei, and co‐localization of brown and blue stain represents nuclear localization of respective NF‐кB family members.Figure S3. Level of key components of STAT3 and pSTAT3 in HPV‐positive and HPV‐negative oral cancer: (A). (1) Representative immunoblots of total cellular proteins (50 μg/lane) from histopathological grade‐matched HPV‐positive and HPV‐negative biopsies from oral cancer patients, tested for the expression of STAT3 and pSTAT2 (Y705). The blots were stripped and reprobed for β‐actin and evaluated as input control. (2) Aggregated mean (±SD) abundance ratios of the band intensity of indicated proteins in HPV‐positive and HPV‐negative oral cancer tissue normalized to β‐actin in three independent experiments. *P ≤ 0.05 versus expression of the respective proteins in control normal tissues. #P ≤ 0.05 versus expression of the respective proteins in control HPV‐negative oral cancer biopsies. (B) Representative photomicrographs of immunohistochemical analysis of STAT3 and pSTAT2 (Y705) in histopathological grade‐matched HPV‐positive and HPV‐negative biopsies. Freshly fixed, paraffin‐embedded sections (5 μm) of oral tissues were processed for IHC and probed for STAT3 and pSTAT2 (Y705) with respective antibodies and detected by HRP‐DAB method. Brown precipitate indicates immunopositive cells, blue stain represent nuclei, and co‐localization of brown and blue stain represents nuclear localization of respective STAT3.Figure S4. Distribution of samples according to the individual score of expression: Each member of AP‐1 , NF‐кB (p50 and p65), and STAT3  were scored 0 for Nil/Weak expression and 1 for Moderate/Strong expression). Individual molecular marker scores were added and their distribution was evaluated in HPV‐positive and HPV‐negative oral cancer lesions by paired t‐test.Figure S5. Expression of important regulators involved HPV‐mediated carcinogenesis in HPV‐positive and HPV‐negative oral cancer: (A) (1) Representative immunoblots of total cellular proteins (50 μg/lane) from histopathological grade‐matched HPV‐positive and HPV‐negative biopsies from oral cancer patients, tested for the expression of p16 and EGFR. The blots were stripped and reprobed for β‐actin and evaluated as input control. (2) Aggregated mean (±SD) abundance ratios of the band intensity of indicated proteins in HPV‐positive and HPV‐negative oral cancer tissue normalized to β‐actin in three independent experiments. *P ≤ 0.05 versus expression of the respective proteins in control normal tissues. #P ≤ 0.05 versus expression of the respective proteins in control HPV‐negative oral cancer biopsies. (B) Representative photomicrographs of immunohistochemical analysis of p16 and EGFR in histopathological grade‐matched HPV‐positive and HPV‐negative biopsies. Freshly fixed, paraffin‐embedded sections (5 μm) of oral tissues were processed for IHC and probed for p16 and EGFR with respective antibodies and detected by HRP‐DAB method. Brown precipitate indicates immunopositive cells, blue stain represent nuclei, and co‐localization of brown and blue stain represents nuclear localization of respective p16 and EGFR.Click here for additional data file.Table S1. Overall and investigation‐wise distribution of clinical specimen and respective clinicopathological characteristics of oral cancer patients.Table S2. List of antibodies used in this study.Table S3. List of primers used, their amplicon size, and the annealing temperatures.Click here for additional data file.Data S1. PPTx1.Click here for additional data file.Data S2. PPTx1.Click here for additional data file.Data S3. Material and Method.Click here for additional data file.


In an essay, Paul Bloem and Ikechukwu Ogbuanu discuss the public health implications of HPV vaccination. A large proportion of the disease burden related to infection with human papillomavirus (HPV), the most common sexually transmitted infection, can be prevented through vaccination.There is inequity in access to HPV vaccines, and populations with the largest burden of HPV-related diseases have the least access to the vaccines.Affordability and sustainable financing of HPV vaccination are barriers to introduction in low- and middle-income countries because of the relatively high cost of the vaccine and its delivery to a nontraditional target population.Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) and can cause HPV-related cancers, most of which are cervical cancers, and genital warts. Although the majority of HPV infections are asymptomatic with spontaneous resolution, disease may result from persistent infection with high-risk HPV genotypes. While infection with a high-risk oncogenic HPV type is the underlying cause of virtually all cases of cervical cancer, most infections with high-risk HPV types do not lead to cancer. This is because infection persists only in a small percentage of people; only a small proportion of these persistent infections progress to precancer, and, of these, even fewer lead to invasive cancer .Two HPV types (16 and 18) cause 70% of cervical cancers and contribute to other, less prevalent, noncervical cancers. HPV infections are responsible for nearly all of the 528,000 estimated cervical cancer cases in women each year, accounting for nearly 266,000 deaths yearly [Two additional HPV types 6 and 11) are responsible for up to 90% of anogenital warts 1 are res. Rates aHPV infections are also associated with other cancers, including vaginal, vulvar, penile, anal, and oropharyngeal cancers. Of the estimated 27,000 cases of anal cancers globally, 88%  were caused by HPV infections, predominantly by HPV types 16 and 18 .For the first time in the history of STI prevention and control programmes, an intervention is available to prevent one of the most common STIs, HPV infection. Vaccines to prevent infection by high-risk oncogenic HPV types 16 and 18) and HPV types that cause anogenital warts (6 and 11) have been in use since 2006. HPV vaccines are most effective when administered before the onset of sexual activity. By preventing HPV infection, they could greatly reduce the morbidity and mortality from cervical cancers 6 and 18 .There are currently 3 HPV vaccines available. The bi- and quadrivalent vaccines have been on the market since 2006; both vaccines protect against the most common oncogenic genotypes of HPV types 16 and 18). In 2014, a 9-valent vaccine was licensed, which protects against 5 additional oncogenic HPV types, in addition to types 16 and 18. The quadri- and 9-valent vaccines also protect against HPV types 6 and 11, which cause genital warts; both are licensed for use in males [6 and 18.Originally requiring 3 doses for full protection, the HPV vaccination schedule was revised in 2014. Adolescents 14 years or younger now require just 2 doses for full protection, with an interval of at least 6 months and up to 12 or 15 months between doses .Given 10 years of availability and the documented effectiveness of HPV vaccines, there remain wide global disparities in access to this intervention. Globally, by the end of 2016, nearly 70 countries  have introduced the vaccine into their national immunisation schedule, either nationally or in a part of the country ,10. UnfoSimilarly, the countries with the highest morbidity owing to cervical cancer and anogenital warts are also the ones least likely to have introduced the vaccine. By June 2016, 71% of high-income countries, 35% of upper-middle-income countries, 8% of lower-middle-income countries, and 6% of low-income countries had introduced the HPV vaccine [In a recent analysis by WHO, the proportion of girls living in countries with access to publicly funded HPV vaccination differed markedly by region. Only in the Americas and the European region do the majority of girls currently live in countries with access to free HPV vaccination (86% in the Americas and 62% in the European region). This proportion drops to 10% or less in all other regions .Current WHO recommendations prioritise vaccine access for girls for the purpose of cervical cancer prevention. This recommendation was based on modelling data showing that, for the prevention of cervical cancer, reaching high coverage in girls is more cost-effective than attaining lower coverage in both boys and girls . HoweverThe magnitude of the burden of cervical cancer coupled with the importance of anogenital warts and their impact on quality of  life, as well as the availability of effective and safe vaccines for prevention, have recently led to considerable policy attention in support of the introduction of HPV vaccines. In the context of the Decade of Vaccines, the Global Vaccine Action Plan promotes the introduction of new vaccines, including HPV vaccine . In 2012In low-income countries, all introductions to date have been supported by donors . Many of the first wave of countries implemented a Gavi-supported demonstration programme. The demonstration programmes aimed to assess the feasibility and cost implications of different vaccine delivery strategies for adolescent girls, a group that presents unique challenges and opportunities to most immunisation programmes in low-income countries that traditionally focus on infant vaccination . To dateIn addition to vaccine cost, a major barrier to HPV vaccine uptake is the lack of experience delivering a 2-dose vaccine to 9- to 14-year-old girls through routine immunisation programmes. Country experiences have shown that high coverage can be achieved in multiple settings. Several countries in different income categories have reported vaccination coverage levels above 80%  or 90% . On the other hand, there are also examples of countries in which full-dose coverage remains below 50% more than 5 years following introduction  [HPV vaccines have been shown to be highly effective in real-world settings. Impact studies have shown significant reductions in the circulation of oncogenic and other genotypes included in the vaccine and reductions in the incidence of genital warts and high-grade precancerous lesions . In addiA key challenge for LMICs is the sustainable financing of HPV vaccine introduction. This is driven by 2 factors—vaccine price and delivery cost. Gavi-eligible countries are relatively (and temporarily) shielded from the high vaccine cost, as they finance the vaccine at a fraction of the Gavi price of $4.50 per dose  , acceptance in some other countries remains a challenge  . While oAlthough the majority of LMICs introduce the HPV vaccine for cervical cancer control, many do not have a well-performing cervical cancer screening programme for women. Similarly, many LMICs do not have a sustainable preventive health delivery platform for adolescents. The introduction of HPV vaccination could be an opportunity to address both of these deficiencies. For instance, HPV vaccine introduction provides a rare opportunity to build or strengthen school health and adolescent health platforms. These platforms could offer additional preventive and curative health interventions for adolescents. Such interventions could include the provision of tetanus boosters, meningococcal vaccines, or the new dengue vaccine, as relevant, but also deworming tablets, iron and folic acid supplementation, or health education messages, including on sexual and reproductive health, STIs, and HIV .In the 10 years since its introduction, HPV vaccination has seen many positive developments. A reduced number of doses and more flexible schedules have reduced costs and facilitated programme implementation. Recent clinical trials are assessing the efficacy and duration of protection offered by 1 dose of HPV vaccine, based on recent promising data on efficacy , 25. TheAt the currently projected pace of HPV vaccine introduction and uptake, a significant impact of the HPV vaccine on cancer prevention will not be seen until 15 to 20 years postintroduction. Scenarios for faster uptake and impact have been proposed in literature, including combinations of vaccination and HPV DNA testing . Until sSeveral factors are combining to accelerate uptake and coverage of HPV vaccines among LMICs. Most notable are the increased policy attention to STI and cervical cancer prevention and control, the ongoing support from the Gavi Alliance to low-income countries, and the continued downward pressure on HPV vaccine prices. The potential impact of increased access to this vaccine on STI and cervical cancer prevention is enormous. We are confident that, over the next decade, the HPV vaccine will fulfil its promise and reach its full public health potential through broader availability.


Misiones Province in northeastern Argentina is considered to be a region with a high prevalence of HPV infection and a high mortality rate due to cervical cancer. The reasons for this epidemiological trend are not completely understood. To gain insight into this problem, we explored the relationship between mitochondrial DNA (mtDNA) ancestry, HPV infection, and development of cervical lesions/cancer in women from the city of Posadas in Misiones Province.Two hundred and sixty-one women, including 92 cases of patients diagnosed with cervical lesions and 169 controls, were analyzed. mtDNA ancestry was assessed through HVS1 sequencing, while the detection and typing of HPV infection was conducted through nested multiplex PCR analysis. Multivariate logistic regression was conducted with the resulting data to estimate the odds ratios (ORs) adjusted by socio-demographic variables.The study participants showed 68.6% Amerindian, 26.1% European and 5.3% African mtDNA ancestry, respectively. Multiple regression analysis showed that women with African mtDNAs were three times more likely to develop a cervical lesion than those with Native American or European mtDNAs [OR of 3.8 (1.2–11.5) for ancestry and OR of 3.5 (1.0–12.0) for L haplogroups], although the associated p values were not significant when tested under more complex multivariate models. HPV infection and the development of cervical lesions/cancer were significant for all tested models, with the highest OR values for HPV16 [OR of 24.2 (9.3–62.7)] and HPV-58 [OR of 19.0 (2.4–147.7)].HPV infection remains a central risk factor for cervical cancer in the Posadas population. The potential role of African mtDNA ancestry opens a new avenue for future medical association studies in multiethnic populations, and will require further confirmation in large-scale studies. Papillomaviridae ; (2) mtDNA ancestry, including three categories ; (3) mtDNA haplogroups, which included eight categories , with haplogroups occurring at a frequency less than 5% being “clustered” into larger monophyletic clades such as UK, JT, HV and L  called “phylogroups”; and (4) HPV types, including 10 categories , in which those HPVs occurring at frequencies less than 5% were clustered into larger groups . For the analysis, HPV types were tested by ordering them according to their increase in the OR according to Muñoz et al., 2003 ;  mtDNA haTwo hundred and sixty one samples, including 92 cases of patients diagnosed with cervical lesions  and 169 control subjects were analyzed. The average age of the H-SIL+ cases was 32.1 years (age range 20–54 years), that of the L-SIL cases was 31.5 years (20–50 years), and that of controls was 36.0 years (10–83 years).A total of 130 samples were identified as positive for HPV (49.8%) and 11 different viral types  were identified in them. HPV infection was higher in women with cervical lesions (70%) compared to those without lesions (37.9%), a pattern consistent with its role in cervical cancer development. Briefly, HPV16 infection had the highest frequency among study groups , followed by HPV6/11 . Multiple infections (double and triple) were present in 11.1% of women while 8% of the viral infections remained untyped. Details about HPV types and their frequencies are shown in The results of the association analysis are shown in The mtDNA ancestry of the study population was 68.6% Amerindian, 26.1% European and 5.3% African in origin, respectively. There were statistically significant differences in mtDNA ancestry among the study groups, with women having European mtDNAs being more frequently found in the cervical lesion groups (L-SIL and H-SIL+) than those women with Amerindian mtDNAs (p<0.05). In addition, we explored the effect of socio-demographic variables on the mtDNA distribution within this population . NotablyWe identified 17 different mtDNA haplogroups in the study population, all of which corresponded to the major maternal lineages that have contributed to Argentinean history. These included A2, B2, C1, D1 (Amerindian), H, HV, I, J, K, T, U, V, X2 (European) and L0, L1, L2, L3 (African) . mtDNA hThe association between mtDNA ancestry and cervical lesion development was estimated using logistic regression models adjusted by significant socio-demographic variables . The results are shown in We ran an additional test that included HPV infection in the regression models . The results are shown in Multi-ethnic populations offer an opportunity to test the effects of ancestry on disease within the same population . Based oMoreover, there is growing evidence that mtDNA variation is deeply structured in worldwide populations and also more susceptible to false-positive findings in association studies than autosomal SNPs , 45. In In Argentina, there are no previous reports on the relationship between mtDNA ancestry and cervical cancer, and only three publications have addressed this issue in the published literature , 46, 47.An additional study limitation was the unsuccessful mtDNA typing in 18.4% of the samples, a value above the usual threshold of 5–10%. The result can be attributed to the use of DNA from formalin-fixed, paraffin-embedded tissue in the group of H-SIL+ samples. This source of DNA has been reported to have a lower DNA quality/quantity compared to cervical scrapes, due to several potential problems including DNA cross-linking, DNA fragmentation and the presence of PCR inhibitors , 49. SomNevertheless, epidemiological data derived from US cancer registries indicated that African Americans, referred to as ‘black’ in them, have a higher incidence of cervical cancer and a lesser survival rate from all combined malignancies relative to individuals of the ‘white’ population . These dOur results may help to provide new working hypotheses about these risk factors. Argentina is an area of considerable interest in this respect, as blacks have historically contributed to its population but today are not phenotypically “visible” in its inhabitants . As a reth-19th centuries), when African slaves from Brazil escaped by crossing the Uruguay River and hiding themselves in the forest of Misiones in search of freedom. In addition, the War of the Triple Alliance (1865–1870) involved soldiers of Afro-Brazilian origin, with some remaining in Posadas after the end of the war. From 1900 onward, African descendants from Brazil migrated to Misiones as a product of the dynamic socio-political and economical situation of the two bordering countries [As a final note, the African genetic component in this study population was higher than the nationwide average of 2% , 27. Thiountries .In spite of these historical records, the 2010 census indicated that only 0.3% of individuals from the Misiones population consider themselves to be African descendants . HoweverDifferent from the mtDNA results, HPV infection was strongly associated with the development of cervical lesions for all the tested models, a finding that highlights the biological meaning of such an association.We have characterized the genetic ancestry of a population from Posadas in Misiones Province, northeastern Argentina, and identified a potential association between African mtDNA ancestry and cervical cancer, a result that will require future replication in an independent larger sample. HPV infection remains a central risk factor for cervical cancer in Posadas population, a finding that is of utmost importance in the vaccine era.S1 TableLegend: SNPs positions are denoted using the CRS sequence as reference.(DOCX)Click here for additional data file.S2 TableaO.R. adjusted by sample center location and nationality (Model II). Significant associations are in boldface.Legend: (DOCX)Click here for additional data file.S3 TableaO.R. adjusted by sample center location and nationality (Model III). Significant associations are in boldface.Legend: (DOCX)Click here for additional data file.S4 TableaO.R. adjusted by socio-demographic variables (Model IV). Significant associations are in boldface.bIncluding: HPV18, HPV59, HPV45 (specie A7).Legend: (DOCX)Click here for additional data file.S5 Tableboldface.Legend: O.R. adjusted by socio-demographic variables (Model V). Significant associations are shown in (DOCX)Click here for additional data file.