Health equality is increasingly being considered alongside overall health gain when assessing public health interventions. However, the trade-off between the direct effects of vaccination and herd immunity could lead to unintuitive consequences for the distribution of disease burden within a population. We used a transmission dynamic model of human papillomavirus (HPV) to investigate the effect of ethnic disparities in vaccine and cervical screening uptake on inequality in disease incidence in England.We developed an individual-based model of HPV transmission and disease, parameterising it with the latest data for sexual behaviour  and vaccine and screening uptake by ethnicity  and fitting it to data for HPV prevalence  and HPV-related disease incidence . The outcome of interest was the age-adjusted incidence of HPV-related cancer  in all women in England in view of differences and changes in vaccination and screening uptake by ethnicity in England, over time. We also studied three potential public health interventions aimed at reducing inequality in HPV-related disease incidence: increasing uptake in black and Asian females to match that in whites for vaccination; cervical screening in women who turn 25 in 2018 or later; and cervical screening in all ages.vs 13·4 cases per 100 000 women). Because HPV vaccination uptake is lower in ethnic minorities, we predict an initial widening of this gap, with cervical cancer incidence in Asian women up to 2·5 times higher (95% CI 1·3–4·8) than in white women 20 years after vaccine introduction . In time, we predict that herd immunity benefits will diffuse from the larger white sub-population and the disparity will narrow. Increased cervical screening uptake in vaccinated women from ethnic minorities would lead to rapid improvement in equality with parity in incidence after 20 years of HPV vaccination.In the pre-vaccination era, before 2008, women from ethnic minorities in England reported a disproportionate share of cervical disease. Our model suggests that Asian women were 1·7 times  more likely to be diagnosed with cervical cancer than white women (22·8 Our study suggests that the introduction of HPV vaccination in England will initially widen a pre-existing disparity in the incidence of HPV-related cancer by ethnicity, partly due to herd immunity disproportionately benefiting subgroups with high vaccination rates. Although in time this induced disparity will narrow, increasing cervical screening uptake in girls from ethnic minorities should be encouraged to eliminate the inequality in cervical cancer incidence in the medium term. We recommend that dynamic effects should be considered when estimating the effect of public health programmes on equality.Cancer Research UK. HPV infection is implicated in more than 99% of cervical cancer casesIn 2008, the UK initiated school-based vaccination of 12–13 year-old-girls, with more than 86% of girls receiving a full course of vaccination between 2012 and 2014.Evidence before this studyWe searched PubMed on Sept 16, 2017, with no language or date restrictions for articles seeking to quantify the effects of HPV vaccination on health equality with the following search terms: “HPV”, “model OR model(l)ing”, and “(in)equity OR (in)equality”. In the past 5 years, modelling studies have suggested that HPV vaccination might cause an increase in health inequality but we found none that used behavioural data to assess the interplay between sexual mixing and health-care-seeking behaviour.Added value of this studyWe modelled the interdependency between sexual mixing and vaccination and screening uptake, using data stratified by ethnicity. Data suggest that people are more likely to choose sexual partners from within a group with shared patterns of health-seeking behaviour. We believe this to be the first study to base the evaluation of the dynamic effects of such like-with-like mixing on behavioural data. Our modelling study suggests that the introduction of the HPV vaccine in England will initially widen a pre-existing disparity in the incidence of HPV-related cancer by ethnicity. We predict that over time herd immunity effects will redress this gap at a rate dependent on the degree to which sexual partnerships are formed between groups. We show that increasing cervical screening uptake among vaccinated girls from ethnic minorities could eliminate the inequality in cervical cancer incidence within 30 years.Implications of all the available evidenceThe case study of HPV, cancer, and ethnicity highlights important factors in assessing the effect on health equality of new and existing public health programmes. Our modelling analysis shows that the interplay of infection dynamics with disparities in vaccination and screening uptake could lead to increased health inequality. We recommend that dynamic modelling studies, parameterised with behavioural data, be used to better understand these trade-offs.To understand the complicated nature of infection dynamics and disease progression, and the multiple behavioural and demographic factors such as those associated with HPV and cervical cancer, mathematical models are often used.We developed an individual-based model of HPV infection, cervical disease, and HPV-attributable non-cervical cancer in England, parameterising it with the latest ethnicity-stratified data on sexual behaviour and vaccination and screening uptake. Here we describe the development, parameterisation, and fitting of the model to data on HPV prevalence and incidence of cervical disease and non-cervical cancers; make predictions of the consequences of HPV vaccination on health equality over time; and estimate the effect of three potential public health interventions aiming to reduce inequality in HPV-related disease incidence.We developed a generic individual-based sexually transmitted infection modelWe parameterised the model with demographic data from the Office for National Statistics (ONS)The model population (n=100 000) was split by sex (50:50) and ethnicity .For partnership formation, we modelled three types of non-concurrent heterosexual partnership: casual, steady, and married or cohabiting. Partnership formation followed a female demand model.We modelled 13 high-risk HPV types  and two low-risk type . High-risk types could potentially progress to either squamous cell carcinoma or adenocarcinoma via three stages of intraepithelial neoplasia  and a stage of carcinoma in situ. We established the branching fraction of each type towards squamous cell carcinoma versus adenocarcinoma by the proportion of cancer type attributed to each HPV typeWe implemented the National Health Service (NHS) protocol for England 35 in whiWe implemented the UK vaccination programme, vaccinating a routine cohort (aged 12–13 years) once a year from 2008 and two catch-up cohorts (aged 14–15 years and 16–18 years) between 2009 and 2011 with three doses of bivalent Cervarix . From 2012, the vaccine changed to the quadrivalent Gardasil , reducing to two doses in 2014. Data for HPV vaccination uptake by ethnicity were obtained from eight child health databases stored at the clinical practice research datalink and the primary care trusts . In the We considered the efficacy of two or three doses of either bivalent or quadrivalent vaccine against persistent HPV-16 and HPV-18 infection to be 95% with reduced protection offered by one dose (90% efficacy).For model fitting we used a sequential Monte Carlo approach to estimate transmission probability and rates of disease progression and clearance for each HPV type, assuming that transmission probability and rate of clearance of initial infection to be the same for men as for women. Rates of progression to non-cervical cancers were allowed to differ between men and women. For each scenario, we ran the model for the 100 best-fitting parameter sets.For intervention analyses we modelled alternative scenarios to assess interventions that might reduce inequality. We considered intervening 10 years after vaccine introduction (2018), increasing black and Asian uptake to match white uptake for: vaccination; cervical screening for women who turn 25 in 2018 or later; and cervical screening for all ages.The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.Our model predicts that the age-standardised incidence of cervical cancer will initially remain stable for roughly 10 years before falling sharply, with a time lag of 20 years to achieve an overall 50% reduction in the age-standardised incidence of cervical cancer after 30 years. We estimated a long-term equilibrium incidence of roughly 4·4 cases of cervical cancer per 100 000 women per year  These reIn the pre-vaccination era, our model suggests that cervical cancer incidence was 1·7 times  higher in the Asian than the white population. In absolute terms, this corresponds to an additional 8·5 (7·9–9·1) cases of cervical cancer per 100 000 women-years. After HPV vaccine introduction in 2008, we predict the inequality in cancer incidence between these two groups will become greater . By 2027Findings of our intervention analysis showed that interventions to reduce inequality in cervical disease should focus on aiming for parity in either HPV vaccination or cervical screening uptake. We predict a 2018 campaign raising vaccination uptake in black and Asian girls to levels of their white peers would have little effect on distribution of incident cervical cancer over the following 40 years . HoweverData from our modelling analysis suggest that due to differences in sexual behaviour (including a tendency to choose partners from a similar background as oneself) and uptake of cervical screening, ethnic minorities in the UK are disproportionately affected by HPV-related disease, including cancer. Because uptake of HPV vaccination has been lower in black and Asian girls than in white girls, we predict that there will be increased inequality in HPV-attributable cancer incidence between ethnic minorities over the medium term (in both absolute and relative terms).Initially, the consequences of the HPV vaccination programme are dominated by the direct protection of vaccinated girls themselves. Because vaccination uptake in both routine and catch-up cohorts was higher among white girls, more cancer cases were averted in this sub-population per person than in black and Asian groups. However, over time, indirect effects will also affect cancer incidence. Our modelling analysis suggests that between 20 and 50 years after vaccine introduction, the incidence rate ratio will fall once more until cervical cancer incidence in Asian girls is around 1·9 times that in white girls. Here we see the effect of the herd immunity effect: as the prevalence of HPV circulating in the overall population is reduced, the number of infections in unvaccinated girls of all ethnicities is also reduced and consequently so is HPV-related disease incidence. In the long term, the protective effect of herd immunity will overcome the discrepancy in vaccination uptake.However, our analysis also showed that even if HPV vaccination uptake were increased among black and Asian girls to levels of their white peers, cohort effects and slow progression to cancer would cause the induced inequality to persist for at least 40 years following the intervention. To reduce the resultant inequality more rapidly, it would be necessary to address the difference in cervical screening uptake, thus protecting the unvaccinated. Although it might be easier to encourage vaccinees to attend cervical screening, this would lead to an even greater concentration of unprotected women in the subgroup which is neither vaccinated not screened. It might be preferable to actively seek out unvaccinated women to attend cervical screening.This work builds on a number of other studies that have raised the question of how differences in uptake of vaccination and cervical screening will affect equality in HPV-related disease outcomes. Initial explorations considered hypothetical cases of increasing assortative mixing (assortativity) in partner choice,Our study also has limitations. In line with other HPV modelling studies, we make the simplifying assumption that neither sexual behaviour nor health-protective behaviour varies demographically over time. In fact, the three rounds of Natsal have documented significant time trends, for example showing increasingly younger sexual début.Second, we consider only three ethnic groups, belying more complex behavioural heterogeneity. For example, 88% of people were classified as white, including a substantial (and growing) proportion of non-UK born individuals about whose vaccination and screening behaviour we know relatively little. Furthermore, we did not model migration. The effect of migration on health equality will depend on the relative rates of screening and vaccination uptake compared with the native population and on sexual mixing choices. If migrant populations choose partners from within their own communities or have a relatively low uptake of public health interventions, inequality might be increased further. Furthermore, we only modelled two sexual activity groups because we assumed that the high prevalence of HPV implies its dynamics are not driven by a highly active core group.We made some limiting assumptions about the natural history of HPV. First, we assumed that the infection with one HPV type offers lifetime protection against reinfection with the same type. In forthcoming work, we will reassess this assumption in line with a recent review.Discussion about the cost-effectiveness of including boys in the UK HPV vaccination programme is ongoing. Although achieving parity in HPV vaccination uptake between girls of different ethnicities might not address the increased inequality in cervical cancer incidence, increasing overall vaccine coverage by vaccinating boys could heighten the herd effect sufficiently to hasten its reduction. The Future work should consider the marginal effect on ethnic inequality associated with the introduction of HPV testing as a primary test of HPV infection in view of indications of early studies that psychological burden of infection is associated with social and cultural norms.In conclusion, the case study of HPV, cancer, and ethnicity highlights important factors in assessing the effect on health equality of new and existing public health programmes. Public health interventions should aim to provide not only a population-level health gain but also to maintain, preferably improve, the level of equality, whether on the basis of socioeconomic status, ethnicity, or age. Our modelling analysis shows that the interplay of infection dynamics with disparities in health-protective behaviour can be unintuitive. Direct vaccination benefits will mirror differences in vaccination uptake between groups. Additional herd immunity benefits will be dependent on the degree to which subgroups mix and the extent to which their behaviour differs. We recommend dynamic effects should be considered when estimating the effect of vaccination and screening programmes on health inequality and in planning its minimisation


The association between colorectal cancer and human papillomavirus (HPV) infection isstill unproven. The aim of this study was to investigate the presence of high-riskHPV (HR-HPV) DNA in colorectal tissues from Cuban patients. A total of 63 colorectalformalin-fixed paraffin-embedded tissues were studied . DNA from colorectal sampleswas analysed by quantitative real-time polymerase chain reaction to detect the mostclinically relevant high HR-HPV types .Associations between histologic findings and other risk factors were also analysed.Overall, HPV DNA was detected in 23.8% (15/63) of the samples studied. Viralinfections were detected in 41.7% of adenocarcinoma (10/24) and 27.7% of adenomacases (5/18). HPV DNA was not found in any of the negative cases. An associationbetween histological diagnosis of adenocarcinoma and HPV infection was observed . The only genotypesidentified were HPV 16 and 33. Viral loads were higher in adenocarcinoma, and thesecases were associated with HPV 16. This study provides molecular evidence of HR-HPVinfection in colorectal adenocarcinoma tissues from Cuban patients. Infection with a high-risk human papillomavirus (HR-HPV) type is necessary for thedevelopment of cervical carcinoma .Among tCRC is the fourth most common cancer worldwide. It is estimated to be responsible forapproximately 1,360,602 new cases and 693,881 deaths annually . ApproxiFindings of the present study indicate the presence of HPV DNA in colorectal tumours andsuggest a role for oncogenic HPV types in the establishment of colorectal adenocarcinomas.These results may contribute to an understanding of the aetiology of sporadic CRC.To explore the possible relationship between HPV infection and colorectal carcinogenesis,we investigated the presence of HPV DNA in CRC tissues from Cuban patients and examined therelationship between HR-HPV genotypes and viral loads and adenocarcinomas.Study population - For this purpose, a cross-sectional study to detectHPV infection in colorectal tissues was performed. Study participants were outpatientsat the Cuban National Institute of Gastroenterology between April and August 2014.Patients contributed only one sample for viral detection. The inclusion criteria were noprior surgery, radiation, or cytotoxic therapy for colorectal adenocarcinoma. Diagnosesof samples from colorectal adenomas, adenocarcinomas, and tissues with unremarkablepathological changes  were confirmed by pathologiststhrough standard criteria. In the study, 63 colorectal paraffin-embedded tissues wereincluded: 42 from patients with CRC and 21 from individuals without CRC. Of the 42tumours sampled, 24 were adenocarcinomas and 18 adenomas. Twenty-one specimens diagnosedas chronic inflammatory infiltrates of colorectal mucosa due to benign colitis were usedas the non-malignant control group.Colorectal tissues were obtained from the rectum, rectosigmoid junction, and sigmoidcolon. Complete clinical and pathological data were recorded, including patients’clinical histories and demographics ,family antecedents, grade of tumour differentiation, and tumour location.DNA extraction - HPV detection and genotyping -Formalin-fixed paraffin-embedded (FFPE) colorectal tissues were obtained. Between sevenand 10 slides of approximately 5-µM wide sections of tissue were deparaffinised inxylene and absolute ethanol, and then DNA was obtained using a QIAamp DNA FFPE TissueKit , following the manufacturer’s protocol. The potential forsample-to-sample cross-contamination was limited by performing DNA extractions from onlysix tissue samples per day. DNA quality was evaluated by polymerase chain reaction (PCR)amplification of a fragment from the human β-globin gene using primers PC04/GH20  was performed as previously described  to identHPV 16 and 18 standard curves were constructed from purified genomic DNA from HPVcultures in SiHa and HeLa cell lines, respectively. The curves showed good linearcorrelation  and low error values throughout the range of the six target DNAconcentrations. The assay had a detection limit of 10 copies for HPV DNA for allgenotypes tested. The detection limit for HPV18 and 45 was also of 10 copies of viralDNA. No cross reactions between HPV and other DNA viruses were observed .To identify HR-HPV types and quantify viral loads, single PCR reactions were performedfor each HPV type. To control for DNA quality, β-globin was amplified in each sample.PCR was performed using a LightCycler 1.5 platform . The PCR reaction mixture comprised 5 µL of DNA (up to 50 ng), 4 µL of QuantitativePCR TaqMan Master Mix (Roche Diagnostics), 10 pmol of each primer, and 1-5 pmol of eachprobe in a final volume of 25 µL. The initial denaturation step at 94ºC for 10 min wasfollowed by 45 PCR cycles at 94ºC for 15 s, 50ºC for 20 s, and 60ºC for 40 s each.Statistical analysis - Epidemiological and clinical data were collectedin one-on-one interviews in a private room, using a standardised questionnaire. Datawere processed using IBM SPSS Statistics 20. Proportions were compared by Chi-squaredtesting. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated in aunivariate logistic regression to estimate the relative risk of HPV detection associatedwith different variables, including age at the time of sampling, gender, skin colour,family antecedents, grade of tumour differentiation, and tumour location. Comparisonsbetween viral loads from groups of patients with different histological classificationsor HPV genotypes were made using Kruskall-Wallis and Mann-Whitney tests. All statisticaltests were considered to be significant at a p-value < 0.05.Ethics - This study was approved by the Ethics Committee of theInstitute of Tropical Medicine Pedro Kourí and complies with the principles establishedin the Helsinki Declaration of 1975 and revised in 1983  of the samples tested. HPV infection wasdetected in 41.7% of adenocarcinoma (10/24) and 27.7% of adenoma cases (5/18), but wasnot detected in any of the negative cases (p = 0.002). An association betweenhistological diagnosis of adenocarcinoma and HPV infection was observed . HPV infections were exclusively detected in the rectum and in rectosigmoid junction  of colorectal samples .Among HPV-positive cases, 66.7% had adenocarcinomas (10/15) and 33.3% had adenomas(5/15) . The onl4 copies/µL versus 3 × 106 copies/µL, respectively). HPV 16viral loads were higher than those of HPV 33   .In the present study, the prevalence of HPV infection was higher in the group ofpatients over 60 years of age than in other age groups. However, no statisticalassociation was found (p < 0.05) between HPV infection and age when proportions werecompared by Chi-squared test .The majority of patients with both colorectal lesions, adenomas and adenocarcinomas,were over 60 years of age . Furthermore,among patients with colorectal adenocarcinomas, HPV infection was more frequent in thoseover 60 years  .Family antecedents of familial adenomatous polyposis were associated with the presenceof adenomas .Our study showed that oncogenic HPV types can be detected in colorectal adenocarcinomatissues from Cuban patients. The absence of HPV DNA in non-malignant tissues suggeststhat the presence of HPV is not merely coincidental in colorectal carcinomas, but thatit is a possible cofactor in development of the disease. In our study, an associationbetween a histological diagnosis of adenocarcinoma and HPV infection was observed. Recently, A few studies have also investigated the presence of HPV in premalignant adenomatouspolyps (adenomas). Data on the relationship between HPV infection and other factors,including genetic and molecular changes, and the development of CRC from adenomas arenot conclusive. According to our results, HPV infection was more frequent in females. In the present study, the prevalence of HPV infection was higher in patients over 60years of age than in other age groups. However, no statistical association was found (p< 0.05). Furthermore, in the group of patients over 60 years of age, a higherproportion of HPV-positive cases had a histologic diagnosis of colorectaladenocarcinoma. According to demographic data analysis by In the present study, family antecedents of familial adenomatous polyposis wereassociated with the presence of adenomas. Approximately 95% of CRCs are believed to haveevolved from adenomatous polyps (adenomas). Various genetic and molecular changes occuras these polyps transform from benign to malignant. Familial adenomatous polyposis is aMendelian dominant syndrome with an incidence of 1:11,000 that is caused by analteration in the APC gene. The syndrome is characterised by the presence of adenomatouspolyps in the gastroenteric tract, mostly in the colorectal-junction and duodenum, witha demonstrated adenoma-carcinoma sequence . There iIn our study, HPV infections were exclusively detected in the rectum and rectosigmoidjunction, suggesting that HPV infection may be a result of retrograde viral transmissionfrom the anogenital area. However, this result differs from those of most previouslypublished research, which have shown no relationship between HPV infection and theanatomic location of tumours. In these studies, rates of HPV detection in tissuescollected from the rectum or sigmoid colon were similar to those in tissues obtainedfrom the cecum or ascending colon. Because HPV infection is mainly transmitted by cellsurface contact, the route of viral transmission to the colon remains to be determined.According to Although further studies are needed to reach a definitive conclusion on the involvementof oncogenic HPV types in the establishment of colon adenocarcinomas, our study providesdata that suggests a potential role for HR-HPV infection in the pathogenesis of CRC.Establishing a link between HPV infection and colorectal carcinogenesis, in addition tocontributing to our understanding of the pathogenesis of this disease, could haveimportant implications for patient management and prevention of CRC. If infection withHPV is found to have a causal role in colorectal carcinogenesis, then prophylacticvaccines against HPV could be used to prevent a large proportion of sporadic CRC.


Background:No reports exist on genotype-specific human papillomavirus (HPV) acquisition in girls after first sex in sub-Saharan Africa, despite high HPV prevalence and cervical cancer incidence.Methods:We followed 503 HP-unvaccinated girls aged 15-16 years in Mwanza, Tanzania, 3-monthly for 18 months with interviews and self-administered vaginal swabs. Swabs were tested for 13 higHRisk and 24 low-risk HPV genotypes. Incidence, clearance and duration of overall HPV and genotype-specific infections were calculated and associated factors evaluated.Results: A total of 106 participants reported first sex prior to enrolment (N= 29) or during follow-up (N = 77). One was HIV-positive at the final visit. The remaining 105 girls contributed 323 adequate specimens. Incidence of any new HPV genotype was 225/100 person-years (pys), and incidence of vaccine types HPV-6, -11, -16 and -18 were 12, 2, 2 and 7/100 pys, respectively. Reporting sex in the past 3 months and knowing the most recent sexual partner for a longer period before sex were associated with HPV acquisition. Median time from reported sexual debut to first HPVinfection was 5 months, and infection duration was 6 months.Conclusion:This is the first description of HPV acquisition after first sex in sub-Saharan Africa where the incidence of cervical cancer is amongst the highest in the world. HPV incidence was very high after first sex, including some vaccine genotypes, and infection duration was short. This very high HPV incidence may help explain high cervical cancer rates, and supports recommendations that the HPV vaccine should be given to girls before first sex. Key MessagesThis is the first description of HPV acquisition after first sex in sub-Saharan Africa where the incidence of cervical cancer is amongst the highest in the world.HPV incidence was very high after first sex, including of vaccine genotypes.Duration of HPV infection was short in these adolescent girls.,,A number of closely related human papillomavirus (HPV) genotypes are classified by the International Agency for Research on Cancers (IARC) as oncogenic (Group I) or probably oncogenic (Group IIA)Current HPV vaccines are prophylactic, not therapeutic, and should be given before HPV acquisition.The cohort was enrolled as described previously.The London School of Hygiene and Tropical Medicine Ethics Committee and the Medical Research Coordinating Committee, Tanzania, approved the study protocol in 2011. Consent procedures have been previously described.www.hpvcenter.se), and therefore reclassified HPV55 as HPV44, HPV64 as HPV34, and CP6108 as HPV89, and merged HPV-IS39 with HPV82. Therefore, 36 HPV genotypes were detected . For this study, we classified HPV genotypes in IARC groups I (termed carcinogenic) and IIA (termed probably carcinogenic) as HR; HPV -16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68. All remaining genotypes were classified as LR.Swabs were placed dry into cryotubes immediately after collection, stored in cold boxes with ice-packs in the field, submitted daily to the laboratory in Mwanza and stored at -20ºC. They were shipped to the Catalan Institute of Oncology, Barcelona, Spain, where HPV detection and genotyping were performed using the Linear Array HPV genotyping assay . We used the updated HPV types from the International Human Papillomavirus (HPV) Reference Center (IJEonline).Questionnaire data were double-entered into OpenClinica LLC , and analysed using STATA V13.0 . Analyses were restricted to girls whose reported sexual debut was before enrolment or during follow-up . Girls who were HIV-positive were excluded from all analyses. Further detail on statistical methods can be found in theFor each HPV genotype, the number of prevalent infections , new infections  and cleared infections (a new genotype that is no longer detected) was tabulated among all sexually active girls. The genotype-specific prevalence was estimated as the number of visits where the genotype was detected, divided by the number of sexually active visits.Genotype-specific incidence was calculated; person-years (pys) at risk were calculated from enrolment  or date of sexual debut . Kaplan-Meier methods were used to estimate time from sexual debut to first HPV infection among girls who reported sexual debut during follow-up and who were HPV-DNA negative at all visits before reported sexual debut ('HPV naïve').The incidences of all new HPV, new HR-HPV and new LR-HPV infections were calculated among: (i) all sexually active girls; (ii) girls who reported sexual debut during follow-up; and (iii) HPV-naïve girls who reported sexual debut during follow-up. The overall incidence rate and 95% confidence interval (CI) were estimated using random effects Poisson regression to account for clustering of multiple infections within the same girl. Rate ratios (RR) for factors associated with the incidence of new HPV infections among all sexually active girls were estimated using random effects Poisson regression.The genotype-specific clearance rate was calculated among all sexually active girls who had acquired a new genotype; pys at risk were calculated from the date of infection (midway between the last negative and first positive sample for the genotype). Kaplan-Meier methods were used to estimate the median and mean duration of genotype-specific infections and the proportion of infections cleared at 12 months. Cox regression with robust standard errors was used to examine risk factors for clearance.IJEonline). Of those screened, 503 (80.1%) were eligible and enrolled. Overall, 106 (21.1%) participants reported first sex: 29 at enrolment, 77 during follow-up. Among 29 girls whose reported date of first sex was before enrolment, median time from sexual debut to enrolment was 4.2 months (range 0.1-12.4). Among 106 girls reporting sex, 91 (85.8%) attended the final visit (18 months). The median [interquartile range (IQR)] follow-up time was 17.8 (17.4-17.9) months.We located 1177 (75.7%) of 1555 potentially eligible girls on the original school attendance lists. Of these, 801 (68.1%) met the age criteria, of whom 628 (78.4%) consented to be screened  participants accepted HIV testing and 1 (1.1%) was positive. The remaining 105 girls contributed 437 ‘sexually-active visits’  to the analysis; vaginal swabs were provided at 353 of these visits (80.8%), of which 323 (91.5%) were adequate specimens and were genotyped.At enrolment, 71/105 (67.6%) participants were aged 16 years and the others were aged 15. Nearly two-thirds lived in rural areas ;7 (6.7%) were in school; and over half were neither working nor schooling . During the study, 71 (67.6%) reported ever having cleansed inside their vagina, and only 1 girl reported being circumcised.Of the 29 girls who reported ever having had sex at enrolment, 7 (24.1%) had at least one prevalent HPV infection at enrolment. A total of 28 new HR infections and 57 new LR infections were detected during follow-up . The moAmong the 76 girls who reported first sex during follow-up, 35 (46.1%) had at least one HPV infection detected before the reported date of first sex. Among HPV-naïve girls, median time from reported sexual debut to HPV infection was 4.9 months , and toThe overall incidence rate (per 100 pys) of new HPV infections in sexually active girls  was 225In the adjusted analysis  there wDuring follow-up, 33 girls acquired at least one new HPV genotype and contributed 85 new infections to the genotype-specific duration and clearance analysis. In total, 26 of 85 (30.6%) new infections were cleared during follow-up. Median duration of new HPV genotype-specific infections was 6.1 months. This was 6.0 and 6.1 months for new HR and new LR-HPV genotypes, respectively. Overall rate of clearance (per 100 pys) was 90.4 for any HPV genotype. After adjustment for age, there were no significant associations with any examined factors .In this study, we demonstrate an extremely high incidence of vaginal HPV infection after first sex in adolescent Tanzanian girls. Acquisition was rapid in the initial months after first reported sex, and over half of the girls were positive for any HPV DNA in these first 6 months. These findings support current recommendations that adolescent girls should ideally be vaccinated before first sex.Few studies have examined HPV incidence in young women after sexual debut. First acquisition of HPV (which predominantly occurs in the months after first penetrative sex) is a unique opportunity to document HPV genotypes to which young women are exposed and which may then become latent (and therefore un-detectable) until reactivation later in life. Current molecular testing cannot differentiate reactivation from first acquisition or re-infection and therefore all studies of HPV incidence in sexually active women can only record presumed incidence of HPV infections, since some apparent new infections may actually be re-activations. HPV84, -83, -61, -66 and CP-108 were the most common genotypes seen in our study. This is in contrast to global prevalence data in cytologically normal women that have reported HPV16, -18, -52, -31 and -58 as the most prevalent genotypes.,,,,The overall HPV incidence in our study (187/1000 person-months) was far higher than that reported in already sexually active women. A cohort study of sexually active women in Brazil, median age 33 years, reported an incidence of 13.4/1000 person-months,,Comparing the incidence of individual genotypes in our Tanzanian study with a study in women in the USA aged 16-23 years, 5% of whom reported never having had sex,Not working was associated with increased HPV incidence compared with being employed or in vocational training. Girls not working may be at increased risk of engaging in sex in exchange for gifts or money or of forced sex, which are risk factors for HIV and other STIs,Limitations of our study include the use of self-administered swabs rather than clinician-collected cervical swabs. We used self-administered swabs since speculum examination was undesirable in girls who had not passed sexual debut. Over 90% were β-globin positive, indicating adequate sampling.,Unobserved intervals  of over 180 days were removed from the analysis. However, sensitivity analyses, where girls were assumed to be uninfected with a new genotype during those intervals, gave similar results . Samples negative or missing for a given genotype, but which had been taken between two samples positive for that genotype, were classified as positive since studies describing long-term persistence have demonstrated sporadic detection of the same genotype early in the course of a persistent infection.,,,,,Median time from first reported sex to acquisition of any HPV was 5 months. This is longer than 2.4 months reported in college students in the USA tested 3-monthly.We report a rapid acquisition of HPV infection, extremely high incidence and rapid clearance in young women after their first reported sex. This study was carried out in a region with one of the highest incidences of cervical cancer in the world, and our findings may help to explain these high rates of cervical cancer and the high HPV prevalence observed in East AfricaThis work was supported by the Wellcome Trust [grant number ITCRBE30] and the WHO Collaborating Centre for HIV Surveillance in Zagreb, Croatia, via a grant from the Croatian Ministry of Foreign and European Affairs. K.B. and R.J.H. receive support from Medical Research Council (MRC) and Department for International Development (DFID) [grant number G0700837].Supplementary DataClick here for additional data file.


We also immunostained 61 OPSCC tissue samples with anti-TLR4. Results showed lower TLR1 and TLR6 mRNA expression and higher TLR9 protein expression in HPV+ when compared to HPV–OPSCC cells. TLR4 expression did not vary by HPV status in OPSCC cells, but TLR4 expression was significantly lower in HPV+OPSCC tissues. After stimulation with PGN, only one cell line (HPV+) did not secrete IL-6 or IL-8. Furthermore, HPV+OPSCC lines showed no IL-6 or IL-8 production on treatment with LPS/LPS-UP. The data suggest changes in TLR4 signaling in HPV+OPSCC, since we have shown lower tissue expression of TLR4 and no pro-inflammatory response after stimulation with LPS and LPS-UP. Also, it suggests that OPSCC may respond to HPV infection by increased expression of TLR9. This study demonstrates differences in expression and function of TLRs in OPSCC, which are dependent on HPV status, and may indicate subversion of the innate immune response by HPV infection.Toll-like receptors (TLRs) have been widely investigated due to their importance in the inflammatory response and possible links to tumor promotion/regression and prognosis. In cancers with an infective etiology, such as human papillomavirus (HPV)-associated Oropharyngeal Squamous Cell Carcinoma (OPSCC), TLR responses may be activated and play a role in tumorigenesis. Our aim was to assess the expression of all TLRs in OPSCC cell lines (both HPV The innate immune response detects pathogenic microorganisms through a number of mechanisms, including recognition of pathogen-associated molecular patterns (PAMP) by pattern-recognition receptors (PRRs), which include the Toll-like receptors (TLR) . Most epOver 150 years ago, Virchow demonstrated a connection between inflammation and cancer by observing leucocytes within tumor tissue. Today, there is a wide consensus on the importance of the inflammatory response in tumor propagation and as a risk factor for carcinogenesis . The linAlthough the TLR system has the capacity to respond to a range of microbial factors, individual TLRs demonstrate relatively high ligand specificity. TLR1, TLR2 and TLR6 primarily recognize peptidoglycan (PGN), lipoteichoic acid and zymogen; TLR3, double stranded RNA; TLR4, lipopolysaccharides (LPS) of Gram-negative bacteria; TLR5, bacterial flagellum; TLR7 and TLR8, single-stranded RNA; TLR9, bacterial and viral unmethylated CpG DNA [Changes in the expression of TLRs or their signaling pathways may lead to progression or regression of the tumor, depending on cancer type . Many st+ OPSCC have a better prognosis than those with HPV-negative disease [Patients with HPV disease , 12, but disease . In addi disease , 15. In  disease , 16.+ and HPV– OPSCC, and determine the function of selected receptors after stimulation with LPS and PGN, in order to determine the extent of alterations in TLR expression and function in OPSCC.Our overall aim was to describe the patterns of expression of all TLRs in HPV-) when compared to lines SCC2 and SCC90 (HPV+), p < 0.0001. Significant differences of TLR1 gene expression were also noted between HPV– cell lines (SCC72 and SCC89), p < 0.05 , 1 Figure . Signifi1 Figure .p < 0.001  did not demonstrate significant change in IL-6 expression  demonstrated an increase in IL-6 mRNA expression  demonstrated higher levels of IL-6 mRNA in response to PGN  did not express IL-6 or IL-8 at either the mRNA or protein level under either basal or stimulated conditions, p < 0.001  was the only OPSCC cell line tested which did not show any pro-inflammatory response to PGN stimulation; cell line SCC2 did exhibit increased IL-8 mRNA and protein expression after stimulation with 1 µg/mL PGN , 1 Figure . No sign1 Figure .p < 0.01), however there was no consistent difference between HPV+ and HPV– groups  increased production of IL-6 or IL-8  with either LPS or LPS-UP, led to both increased IL-8 mRNA expression , after stimulation with 1 µg/mL of LPS or LPS-UP, higher gene expression of IL-6 was detected compared to unstimulated control  compared to HPV– cell lines (SCC89 and SCC90), as measured by flow cytometry   , also a d SCC90) . TLR7 diHPV-associated OPSCC shows clinical, pathological and biologically distinct features, due to viral activity within tumour tissue , 18. HPV– when compared to HPV+ cell lines. Despite this finding, there were no significant differences in membranous TLR1 and TLR6 expression on comparing HPV+ and HPV– OPSCC cells. De Carlo reported a similar decrease in TLR1 gene expression in HPV+ cervical cancers [In this study, where we evaluated TLR 1-10 mRNA expression by qPCR, we observed that only TLR1 and TLR6 mRNA are more expressed in HPV cancers . In an a cancers . The con cancers .+ and HPV– cell lines, led us to assess the functionality of TLR2 after stimulation with PGN (a TLR2 agonist). PGN from S. aureus is an activator of TLR2 [– lines SCC72 and SCC89 expressed IL-8 and IL-6, respectively, after stimulation with PGN. The HPV+ cell line SCC2 also expressed IL-8 after stimulation. This suggests functional TLR2 in HPV– and HPV+ cells. However, intracellular receptors such as NOD (nucleotide-binding oligomerization domain) 1 and NOD2 can recognized peptides derived from the degradation of PGN. If NOD1 or NOD2 are stimulated, it could induce secretion of interleukins [The lack of any difference in TLR2 expression at either gene or protein level, between HPV of TLR2  and incr of TLR2 . HPV– lirleukins , 27, thu+ tumors compared to HPV– tumors ex-vivo. This finding is consistent with previous work assessing HPV- associated HNC [in vitro findings suggested no clear relationship between TLR4 gene expression in HPV+ versus HPV– cell lines; all cell lines expressed TLR4 when assessed by flow cytometry. Despite conflicting with ex-vivo findings, our in vitro work is also consistent with data published by Jouhi et al [TLR4, a cell membrane receptor, has also been implicated in tumor promotion . Immunohated HNC . Our in hi et al .+ cell lines, although we noted variable increased secretion of IL-6 and IL-8 in HPV– cell lines. The lack of response in HPV+ lines may suggest changes in the TLR4 signaling pathway as a consequence of viral infection. In cervical cancer, the reported changes in expression of TLR4 are a source of controversy and effects seen may be related to the HPV type and different material and methods used; the cervical carcinoma lines SiHa (HPV16+) show higher expression of TLR4 than HeLa (HPV18+), moreover SiHa, but not HeLa display resistance to apoptosis following treatment with LPS, via TLR4 [Unlike the upregulated release of inflammatory mediators observed in response to PGN, stimulation with LPS and LPS-UP resulted in no change in IL-6 or IL-8 expression in HPVvia TLR4 . In cervvia TLR4 , 28, whivia TLR4 . Howevervia TLR4 –33, respvia TLR4 , 12, 34.+ OPSCC.Many carcinomas have been found to express high levels of IL-6 and/or IL-8, suggesting an important role of these cytokines in the tumor microenvironment , 36. IL-Polymorphisms of TLRs have been associated with many diseases, and may be a risk factor linked to cervical cancer . In addi+ lines and lower expression in HPV– lines.TLR9 has been the focus of numerous studies into cervical carcinoma, due to this receptor acting as a method of HPV recognition . A decre+ tumors. In our work, TLR9 was more highly expressed in HPV+ OPSCC when compared with HPV– and p16 expression was also higher in HPV+ cell lines and tumor specimens (data not shown). Moreover, HPV+ OPSCC show better prognosis which could be influenced by the slowdown of the cell cycle caused by p16ink4a, as demonstrated by these authors.Interestingly, Parroche et al., 2016, demonstrated downregulation of TLR9 in HNC (HPV negative) and showed that TLR9 induced a slowdown in the S-phase in HNC mediated by p16ink4a , a prote+ cervical disease [In cervical cancer, studies have also shown increased expression of TLR9 in those patients who eliminated the virus , however disease .There are many possible explanations for the poor correlation we have observed between mRNA and protein expression in TLRs 1, 2, 4, 6 and 9 including variations in mRNA half-life , 49, and+ OPSCC, in vivo [+ [TLRs 3, 5, 7 and 8 appear to exert anti-tumor effects by converting immune tolerance into anti-tumor immunity [ in vivo . TLR5 al vivo [+ . TLR7 in vivo [+ , but lit+ lineages compared to HPV-, and that TLR1, 2, 4, 6 and 9 proteins were expressed in all cell lines, irrespective of HPV-status. TLR9 was upregulated in HPV+ OPSCC cell lines, which may represent a cellular response to viral infection. After stimulation with PGN, both HPV– and one HPV+ cell lines expressed IL-6 and/or IL-8. We have furthermore demonstrated lower expression of TLR4 in HPV+ OPSCC compared to HPV– OPSCC tumor tissue, as well as a lack of IL-6 and IL-8 expression in HPV+ cell lines after stimulation with both LPS and LPS-UP, inferring functional changes in the TLR4 signaling pathway of HPV+ disease. This work demonstrates a comprehensive survey of TLR expression and function in both HPV+ and HPV– OPSCC.In summary, we report that of the TLRs, only TLR1 and TLR6 demonstrated reduced mRNA expression in HPVEthical approval was obtained from the School of Dentistry/University of São Paulo Ethical Committee.°C.Experiments were carried out using two HPV16-associated oropharyngeal carcinoma cell lines (SCC2 and SCC90) and two HPV negative oropharyngeal carcinoma cell lines (SCC72 and SCC89) . The cel°C for 10 min, 37°C for 2 h, 85°C for 5 min and then kept at 4°C using the DNA Engine Dyad thermal cycler.RNA was isolated using RNeasy Mini Kit  and treated with DNase I . The quantity and quality of RNA were analyzed by NanoDrop Spectrophotometry . Reverse transcription was performed in 300 ng/ml of total RNA using High Capacity cDNA Reverse Transcription  in a final volume of 10 mL. Samples were incubated at 25°C for 2 min, 95°C for 10 min, 95°C for 15s, 60°C for 1 min, using dissociation curve analysis to confirm no bimodal curve or abnormal amplification . In the case of TLR2, TLR4, IL-6 and IL-8 expression, differences were assessed between control and stimulated cells (PGN or LPS or LPS-UP) from the same cell line. Ct values exceeding 35 cycles were not considered amplified. Negative control consisted of the master mix, primers and sterile water.5 cells/ml in cold FACs buffer on ice. Cells were divided into 4 aliquots and incubated with either PE-conjugated anti-human CD281 , PE-eFluor 610-conjugated anti human CD282 , Alexa Fluor700-conjugated anti human CD284  or anti-human CD286 biotinylated  for 20 min on ice, in the dark, followed by FACS buffer washes. Samples previously incubated with anti-TLR6 biotinylated antibody were incubated for another 20 min with Straptavidin FITC . Cells were then washed, centrifuged and fixed with 1% paraformaldehyde. For intracellular staining, cells were fixed in 1% paraformaldehyde, washed with FACS buffer, centrifuged and incubated with 0.1% saponin for 15 min at room temperature and then washed, centrifuged, re-suspended in cold FACS buffer. Cell suspensions were then divided into 2 aliquots and incubated in the dark and on ice with either APC-conjugated anti human CD289  or APC-conjugated IgG2a K Isotype control . Unstained cells were used as a further control. Experiments were undertaken in triplicate and data is presented as median fluorescence intensity (MFI). Flow cytometry was performed using the LSRII Flow Cytometer System and data analyzed using Flowing Software 2.5.Adherent cells were washed with FACs buffer  and detached non-enzymatically, centrifuged and re-suspended at 1 × 10°C with primary monoclonal antibody anti-TLR2  , or incubated for 1h with primary anti-b-actin . Membranes were then incubated in horseradish peroxidase-conjugated anti-rabbit IgG (1:3000) for 1 h and developed with SuperSignal West Pico chemiluminescent substrate . THP1 lysate was used as a positive control.Cells were lysed using RIPA buffer  containing protease and phosphatase inhibitors . Samples were centrifuged and the supernatant assayed for total protein using BCA Protein Quantitation as per manufacturer’s protocol . 20 μg of total protein was loaded onto 4–12% polyacrylamide precast gels . After transfer to nitrocellulose membranes using an iBlot Dry Blotting System  for 7 min, the membranes were washed with Tris buffer and blocked with 5% dried milk in tris buffered saline containing 0,05% tween-20, for 1 h and incubated overnight at 4Escherichia coli 0111:B4 , lipopolysaccharide ultra-pure (LPS-UP: a specific TLR4 agonist) from E. coli , and peptidoglycan from Staphylococcus aureus (PGN: a TLR2 agonist) . For ELISA, cells were stimulated with 0.01–1 µg/mL of LPS; 0.01-1 µg/mL of LPS-UP and 0.1–10 µg/mL of PGN [°C for 24 h. Experiments were undertaken using triplicate biological repeats.SCC24, SCC90, SCC72, SCC89 were stimulated with various concentrations of lipopolysaccharide (LPS: a non-specific TLR4 agonist) from L of PGN , 56. ForLPS, LPS-UP and PGN were added to keratinocyte monolayers  in T25 flasks. Culture supernatants were collected in order to measure cytokine levels of IL-6 and IL-8, using BD OptEIA Human IL-6 and BD OptEIA Human IL-8 ELISA kits .+ oropharyngeal carcinomas  at 95°C for 30 mins, and quenching in 3% hydrogen peroxide for 15 min. Blocking of nonspecific binding was undertaken using 5% BSA at room temperature for 30 mins. The tissue was then incubated overnight at 4°C with Monoclonal Anti-TLR4 , followed by the Envision Dual Link System horseradish peroxidase method. Staining was then revealed by the addition of di-aminobenzidine (DAB) substrate-chromogen and the TMA was counterstained, dehydrated and mounted.We analyzed TLR4 expression in 61 FFPE oropharyngeal tumor samples in a tissue microarray (TMA) consisting of 31 HPVrcinomas  and 30 Hrcinomas  . Tumor area was then calculated using ImageJ software, by selecting the region of interest using the “measure” tool. Each spot image was submitted to color deconvolution  to separU-test. Graphs are expressed as pg/mL and mean ± SD. The Mann-Whitney U test was also used to compare TLR4 staining between HPV+ and HPV– tumors. All statistical tests were undertaken with the significance level set at p < 0.05.Statistical analysis was undertaken using GraphPad Prism software version 6.0 . For qPCR data, results are expressed as mean ± SEM, and statistical significance assessed using one-way ANOVA post hoc Tukey analysis to compare more than 2 groups and t-Student to compare two independent groups. For flow cytometry data, after failure of normality testing, the non-parametric Kruskal-Wallis test with post hoc Dunn-Bonferroni correction was used. Graphs depict mean MFI ± SD. Analysis of ELISA data was undertaken through comparison of stimulated samples  against unstimulated control, using the Mann-Whitney


It is well known that persistent infection with high-risk HPV (hr-HPV), mostly HPV-16 and 18, is the main cause of cervical cancer development. Manganese superoxide dismutase (MnSOD or SOD2) are highly expressed in different neoplasia. The present study investigated SOD2 protein expression and the presence of hr-HPV types in 297 cervical samples including non-neoplastic tissue, cervical intraepithelial neoplasia grade 3 (CIN3), squamous cell carcinoma (SCC) and adenocarcinoma (ADC). Strong SOD2 expression was significantly higher in ADC (82%) than CIN3 (52%) or SCC (64%). There was no association between SOD2 expression and HPV 16 and/or 18 detection for every lesion analyzed. Binary Logist Regression revealed that strong SOD2 expression  and HPV 16 and/or HPV 18  were independently more associated with CIN3 than non-neoplastic cervix. Strong SOD2 expression  and HPV 16 and/or HPV 18  were independently more associated with ADC than SCC. Similar findings for SOD2 expression were observed by the Cochran Mantel-Haenszel test, controlling for HPV-16 and/or HPV 18. In conclusion, the expression of SOD2 was increased in CIN3 and SCC, and more increased in cervical ADC than in SCC. Strong SOD2 expression was statistically independent of the presence of HPV 16 and/or 18. These findings suggest that the mitochondrial antioxidant system and HPV infection could follow independent pathways in the carcinogenesis of cervical epithelium and in the differentiation to SCC or ADC of the cervix. Cervical cancer is the second most common neoplasia in women, with estimated 500,000 cases occurring each year, globally resulting in >250,000 deaths –3. SquamVirtually 100% of cervical cancers are associated with human papillomavirus (HPV) infection . Twelve SOD1 gene), manganese superoxide dismutase (encoded by the SOD2 gene) and extracellular superoxide dismutase (encoded by the SOD3 gene). Moreover, SOD2 is found exclusively in the mitochondrial matrix and is an evolutionary conserved enzyme in a variety of organisms [In parallel, several cervical carcinoma-derived cell lines have been shown to be resistant to TNF anti-proliferative effect, suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. Analyzing the global transcription profile of normal and HPV-immortalized keratinocytes after TNFα treatment, using a microarray approach, manganese superoxide dismutase (MnSOD or SOD2) was identified as one of the differentially expressed genes in association with inflammatory response . Superoxrganisms .The pathway by which SOD2 expression might contribute to cancer development is not clear, but some researchers have suggested that, as a pro-oxidant protein, it promotes the accumulation of hydrogen peroxide, which can further lead to activation of various oncogenic pathways , 17. SucThere is some evidence that oxidative stress and altered redox homeostasis might play a role as co-factors in cervical carcinogenesis , 20. TerIn summary, HPV infection has been considered a necessary event for cervical cancer occurrence, although not sufficient to trigger its development. Other factors not directly dependent of these viruses should act for the cells to achieve the malignant phenotype; oxidative stress could be one of them and the SOD2 protein expression a marker of this condition. The goal of the present study was to analyze if SOD2 protein expression level is associated with HPV infection, focusing on the 16 and 18 types, in ADC, SCC and cervical intraepithelial neoplasia 3 (CIN3) samples.HPV prevalence and type distribution according to the histopathological diagnosis are presented in Table p < 0.00001) and SCC (p = 0.02) when compared with CIN3. In addition, these two viral types were significantly more associated with ADC than SCC (p = 0.02).Figure The expression pattern of SOD2 protein was determined in the different histopathological samples using the non-neoplastic tissues as the control. As a general rule, SOD2 positive cells display a granular cytoplasmic staining pattern. Overall, a strong cytoplasmic SOD2 staining was observed in CIN3, SCC and ADC throughout the lesion, while no staining was observed in the stromal cells. However, in some cases, SOD2 expression was also observed in inflammatory cells infiltrating both epithelium and stroma. Representative examples of SOD2 staining in CIN3, SCC and ADC are presented in Figure p < 0.00001) when compared with non-neoplastic tissues and was significantly higher in ADC than in CIN3 (p < 0.0001) and SCC (p = 0.02). No statistical difference was found between SCC and CIN3 (p=0.15).Most samples of non-neoplastic epithelia and normal glandular cells presented weak or negative staining for SOD2. Strong SOD2 expression (>50% positive cells) was observed in 3%, 52%, 64% and 82% of non-neoplastic samples, CIN3, SCC and ADC, respectively  and HPV-16 and/or HPV 18  were independently associated with CIN3 when compared with non-neoplastic diagnosis. Strong SOD2 expression  and HPV-16/18 positivity  were also independently associated with ADC when compared with SCC. Similar findings were observed for ADC when CIN3 was considered as reference. Strong SOD2 expression was not associated with SCC when compared with CIN3 . The Cochran Mantel-Haenszel test showed similar findings for strong SOD2 expression in all interdiagnosis comparisons, controlling for HPV-16 and/or HPV 18 , preventing programmed cell death or necrosis onset, thus favoring the malignant phenotype [The role of SOD2 in cancer initiation and progression is not well understood. henotype . Reductihenotype –25. On thenotype , 15, 26.2O2 [A recent review on SOD2 and carcinogenesis summarized that ROS largely originating in the mitochondria play essential roles in the metabolic and (epi) genetic reprogramming of cancer cell evolution towards more aggressive phenotypes. Therefore, there is likely a dichotomy where SOD2 can be considered a protective antioxidant reducing superoxide, as well as a pro-oxidant factor during cancer progression, with these effects depending on the accumulation and detoxification of H2O2 . Neverth2O2 -15, 27. 2O2 , 29, gli2O2 , bladder2O2 , lung [32O2 , breast 2O2 , penile 2O2 , gastric2O2 , esophag2O2  and oral2O2 .In relation to cervical cancer and its precursor lesions, Termini et al.  examinedOur study showed that the detection rate of HPV 16 and/or 18 increased with the squamous lesion severity, and this finding was expected  that shoAlthough the HPV types 16 and 18 are more typically prevalent in more severe cervical neoplastic lesions, such as strong SOD2 expression, no association was observed between strong SOD2 expression and HPV-16 and/or 18 in non-neoplastic tissue, CIN3, SCC or ADC samples. This information is new and suggests that SOD2 and HPV-16 and/or 18 infections could act in the carcinogenic process through different pathways, and not as co-factors. Additionally, this study showed that, controlling for the presence of HPV-16 and/or 18, strong SOD2 expression was significantly associated with CIN3 in relation to non-neoplastic cervical tissue, but there was no association between CIN3 and SCC. These findings are also new and reinforce the hypothesis that SOD2 could have a relevant and independent role in CIN3 development, but the data available do not support this assumption for the CIN3 progression to SCC.Cervical ADC, in relation to SCC, occurs more frequently in younger and Caucasian women, and is generally associated with worse prognosis , 41. TheThis study showed that SOD2 expression is especially higher in ADC in relation to other cervical samples, including SCC. Eighty-two percent of ADC and 64% of SCC cases revealed strong SOD2 expression, and no ADC cases showed negative or moderate expression. Termini et al.  describe2O2 produced in the mitochondria seems indispensable for hypoxic adaptation and energetic and/or metabolic homeostasis, and, therefore, it seems relevant to elucidate how much this process depends on and is controlled by SOD2. In this way, the better understanding of redox hubs in the mitochondria will likely lead to new and improved therapeutics of a number of diseases, including cancer [The pathway by which some types of HPV or variants could be associated with ADC or SCC is not clear. Also, no information was found in the literature about the SOD2 expression in non-neoplastic glandular epithelium of the cervix. This study indicates that the oxidative stress is higher in the ADC and the antioxidant system is strongly activated. At this point, it seems not possible to indicate how much the oxidative stress is an agent promoting the carcinogenesis or how much it is consequence of the cancer progression. The Hg cancer , 47.In conclusion, our results showed that the expression of SOD2 was increased in CIN3 and SCC, and more increased in cervical ADC than in SCC, and this pattern of SOD2 expression was statistically independent of the presence of HPV 16 and/or 18, the most prevalent types in the cervical cancer. These findings suggest that the mitochondrial antioxidant system and HPV infection could follow independent pathways in the carcinogenesis of cervical epithelium and in the differentiation to SCC or ADC of the cervix.All tissue samples were originally collected for diagnostic purposes and were thoroughly anonymized before the use in this study. This study was approved by the State University of Campinas ethics committee (Protocol No. 04500146000-10). The institutional ethics committee waived the need for signed consent because this was a risk-free retrospective study and it was no longer possible to contact many of the enrolled women.Case selection was based on pathology reports obtained from 2005 from women consecutively attended to at the Women’s Hospital Prof. Dr. José Aristodemo Pinotti, State University of Campinas, Brazil. Haematoxylin-eosin (H&E) paraffin-embedded sections were reviewed and the best representative samples were identified. Specimens were obtained by punch biopsies, large loop excisional cervical procedures, cones or hysterectomies. All women with non-neoplastic lesion and CIN3 had previous abnormal Pap test and they had undergone conization or loop excision of transformation zone of the cervix due to suspicion of squamous high-grade lesion.Fixed and paraffin embedded tissues were cut into 3-μm sections and stained with H&E for histological analysis and selection of the appropriate tissue area for further investigations. This study included 297 cervical samples: 73 ADC, 61 SCC, 103 CIN3 and 60 non-neoplastic cervical tissue.The sample size estimation was based on the data from the Termini et al. study of SOD2 expression in different stages of cervical neoplasia . Those aCervical specimens with a confirmed histological diagnosis were analyzed for the presence of HPV DNA. Paraffin sections were systematically obtained from each block using a sandwich method . Total DNA was extracted using a proteinase K lysis procedure, as previously described . The micThe Innogenetics  INNO-LiPA SPF-10 HPV Genotyping Extra assay was used according to the manufacturer’s instructions. This version of the assay allows the simultaneous and separate detection of 15 high-risk HPV types  and 10 low-risk HPV types .After deparaffinization in xylene and rehydration in alcohol, antigen retrieval was performed by incubation in boiling 10mM citrate buffer, pH 6.0, for 20 minutes. Samples were incubated with an anti-SOD2 mouse polyclonal antibody , at a 1:1500 dilution in 10% horse serum phosphate buffered saline solution for 18 hours at 4°C. Immunohistochemistry for SOD2 was performed according to Polymer Detection System , as described elsewhere [The immunohistochemical assays were evaluated considering the percentage of stained epithelial cells. SOD2 expression was categorized based on cytoplasm positive reactions, as previously reported ; samplesAll statistical analyses were performed with the Statistical Package for Social Sciences (SPSS) 15.0 software. Samples diagnosed as non-neoplastic were used as the reference category for comparisons with CIN3, SCC and ADC. Furthermore, samples diagnosed as CIN3 and SCC were used as reference for ADC analysis. For the statistical analyses, the SOD2 expression was scored as less or equal to 50% of stained cells (negative to moderate expression) or more than 50% of stained cells (strong expression). The analysis of SOD2 expression and HPV-16 and/or 18 for every histological diagnosis was made using Fischer Exact Test.The association between SOD2 expression and HPV types in CIN3, SCC and ADC samples, in an interdiagnosis comparison, was analyzed. For this purpose, binary logistic regression was used to estimate the probability of a binary response  taking as predictor variables the SOD2 expression and HPV types. The Cochran Mantel-Haenszel test was used to confirm the strong SOD2 expression as a predictor variable for the different stages of neoplastic lesions, controlling for presence of HPV 16 and/or 18.


We investigate the associations between religious practice and human papillomavirus (HPV) vaccine-related awareness, knowledge, and receipt among young women in Utah.We surveyed 326 insured women aged 18–26 by mail. Fisher's Exact Tests and multivariable logistic regression models were used to evaluate the relations between religious practice and HPV vaccine-related outcomes. Data collection occurred January-December 2013; analyses were conducted June-September 2015.Multivariable analyses reveal that when controlling for age, educational attainment, and marital status, participants who practiced an organized religion were significantly less likely to have heard of HPV , to have heard of the HPV vaccine , to know how HPV is spread , to have received a provider recommendation for the HPV vaccine , and to have received at least one  or all three  doses of the HPV vaccine. Bivariate analyses produce parallel results.Results indicate that religious young women in Utah are not only under-vaccinated, but are also under-informed about HPV and the HPV vaccine. These results suggest that suboptimal vaccine coverage among religious young women may present a serious health risk for the community. Strategies for educational interventions targeted to this population are discussed. The Centers for Disease Control and Prevention (CDC) recommends a 3-dose human papillomavirus (HPV) vaccine as a routine vaccination for girls aged 11–12 and as a catch-up vaccination for adolescents and young women aged 13–26 . Urgent th birthday, with 20% of women having their first sexual experience between the ages of 18 and 20 [Efforts to improve HPV vaccine uptake have largely focused on adolescents and their parents; however, young women may face a more immediate risk of HPV infection than adolescents, and thus merit serious attention, as well. National survey data indicate that 74% of women are sexually active before their 208 and 20 . MoreoveIt is clearly vital to study the barriers and facilitators to HPV vaccination for young women. Although the research concerning adolescents and their parents is informative, there are substantial differences between parents making vaccination decisions for their children and young adults making vaccination decisions for themselves. Perhaps most importantly, young adults are not uncertain about their own sexual activity in the way that parents may be about their teens’. Indeed, in the few studies examining the HPV vaccination of young women, sexual history was found to be significantly related to vaccination status, intention to vaccinate, and the belief that the vaccine is important , 9.Among the relevant factors that may inform young women’s thinking about the HPV vaccine is their religious beliefs. The connection between HPV and sexual activity has been a major source of religious controversy surrounding the HPV vaccine. Various religious groups have voiced concern that the HPV vaccine will promote sexual disinhibition among teens , 11. AddSuch investigation may be even more salient in religious communities where young women receive information and recommendations from religious health care providers and where community discourse implicates religion directly and indirectly. Utah is a good example of such a community. In Utah, nearly 80% of residents practice an organized religion, with a high density of individuals who identify as members of The Church of Jesus Christ of Latter Day Saints . FurtherIn the present study, we assessed the relations between religion and HPV vaccine-related awareness, knowledge, and receipt among young women in Utah. To the best of our knowledge, there has been no study to date in this community that addresses, in-depth, the association of religion and these HPV vaccine-related outcomes. Our study represents an essential conversation-starter for Utah patients, providers, and policy-makers, and our findings may also be informative for demographically similar communities.This study is part of a larger project for which we surveyed insured women aged 18–26 from University of Utah clinics by mail about their attitudes regarding health and vaccines, as well as about their HPV vaccine-related awareness, knowledge, beliefs, and receipt. Preliminary findings revealed a significant association between religious practice and HPV vaccine receipt ; the preWe recruited participants through the Utah Primary Care Research Network. We identified young women aged 18–26 who had visited a University of Utah Community Clinic in the preceding 12 months . Two groups of 1,000 were created; both comprised an outcome-dependent enriched sample of women who had at least one documented dose of the HPV vaccine , and a random sample of women with no documented doses of the HPV vaccine .https://osf.io/ajtcw/).Data collection was conducted in two waves. In the first wave, introduction letters with opt-out information were mailed, followed by the study surveys. The response rate in this wave was below our targets, so we revised methodology using the Tailored Design Method for the data collection among the second group , as descThe survey  evaluateFor this study, the predictor of interest was religion, assessed by two questions: “Do you practice an organized religion?” and “Does religion guide your daily decisions?”. The outcomes of interest included HPV vaccine-related awareness , knowledge , and receipt .Fisher’s Exact Tests and multivariable logistic regression models were used to determine differences in demographics and vaccination rates between participants from the two waves. No statistically significant differences were found, so the participants from both waves were pooled for analyses.We used Fisher’s Exact Tests to compare distributions between participants who reported practicing vs. not practicing an organized religion on sociodemographic characteristics and on HPV vaccine-related awareness, knowledge, and receipt. We used multivariable logistic regression models to assess the association of religion with HPV vaccine-related outcomes while controlling for potentially confounding variables . For these tests, we calculated adjusted odds ratios (aOR) and 95% confidence intervals (CIs). We repeated these analyses (bivariate and multivariable) among participants reporting religious practice to compare those who reported that religion does vs. does not guide their daily decisions. For all tests, statistical significance was defined as p<0.05. Analyses were performed using SAS software, version 9.4.Participants were fairly evenly distributed by age, were mostly White , had mostly graduated or completed some college , and were mostly unmarried  . There wReligious practice was significantly associated with HPV vaccine-related awareness and knowledge , specifiNotably, HPV vaccine-related awareness and knowledge was quite high among all participants, including those reporting religious practice . Nearly Multivariable analyses revealed that when controlling for age, educational attainment, and marital status, religious practice was still significantly associated with certain HPV vaccine-related awareness and knowledge outcomes . CompareMultivariable analyses showed that religious practice was significantly related to HPV vaccine receipt, as well . CompareOf the 148 respondents who reported practicing an organized religion, 103 (69.6%) responded that religion guides their daily decisions, and 41 (27.7%) said it did not (n = 4 missing response). Analyses to compare religious participants who reported that religion does vs. does not guide their daily decisions yielded few significant results (data not shown). The sole demographic difference between these participants was educational attainment (p = 0.0039). We found no significant relation in either bivariate or multivariable analyses for any HPV vaccine-related outcome.Though the HPV vaccine has been recommended for girls and young women for a decade , coveragOur findings indicate that participants who practiced an organized religion were significantly less likely to have received a recommendation for, and to be vaccinated with, the HPV vaccine, even when controlling for age, education, and marital status. These findings reinforce results of previous studies that demonstrated significant associations between religion and the HPV vaccination and vaccination intention of young women –23. HoweHowever, our findings also reveal that religious participants were significantly less likely than their non-religious counterparts to be aware of or knowledgeable about the HPV vaccine. Religious participants were less likely to have heard of HPV and the HPV vaccine, and were also less likely to know how HPV is transmitted. These findings are novel in regards to this population; only one previous study has suggested that religious affiliation significantly relates to awareness and knowledge of HPV . In lighFinally, unlike the more straightforward question regarding whether participants practice an organized religion, the more subjective question about whether religion guides daily decisions yielded no significant findings. This may be because religious individuals are unaware of the extent to which religion informs their daily decisions, or because they do not think of decisions about HPV vaccination as “daily.” But perhaps the workings of religion do not occur via the subjective sense of being guided by it. Perhaps religious individuals, regardless of whether or not they feel that their religion guides their daily decisions, tend to choose doctors who endorse the same religious beliefs, participate in social networks with people of the same religion, and access media created by and for people of the same religion. Thus, whether or not they acknowledge that religion actively guides their daily decisions, the mere and actual practice of an organized religion may structure their lives in ways that significantly affect their HPV vaccine-related decisions and HPV vaccine-related outcomes.In sum, our findings document the significantly lower HPV vaccine-related awareness, knowledge, and receipt among young women who practice an organized religion compared to those who do not. The results signal the need for interventions geared to, at the very least, educating religious young women in Utah about HPV and the HPV vaccine. Such educational interventions are critical to ensure that a decision to not vaccinate is based on full and accurate information, thereby representing a low health risk. To create and enact such interventions will require the participation of a variety of stakeholders, including religious young women, their health care providers, church leaders, policy makers, and public health officials. Notably, our findings also suggest that it will be important to reach out broadly to religious young women, including those who may not feel as though religion guides their daily decisions. This could be accomplished by tailoring materials for religious audiences both explicitly  and implicitly , and by targeting religious individuals directly  or indirectly . Interventions focused on medical providers who serve religious patients may also be effective—for example, through requirements to provide standardized HPV vaccine education.Participants were mostly white and were all insured, limiting the generalizability of our findings. Given that this study took place in a densely and fairly homogeneous religious population, the results may not generalize to more diverse communities. It is also worth noting that our sample exhibited lower rates of religious practice than the overall Utah population; therefore, our findings may not fully represent the population from which we sampled. The study may also suffer from response bias; we had a limited sample size, and the response rate for the survey was poor, especially in the first wave of data collection. We also received more responses from participants who were vaccinated vs. unvaccinated, suggesting a voluntary response bias. Additionally, the cross-sectional study design prohibits the evaluation of causal relationships. We did not assess specific religious affiliation (only religious practice), so we cannot be certain as to which religious groups our results may apply. Moreover, we did not assess the sexual history of participants, which could have been a significant confounding variable within this analysis.This study is, to the best of our knowledge, the first to explore the relations between religion and HPV vaccine-related awareness, knowledge, and receipt among young women in Utah. Not only was HPV vaccination significantly lower among religious individuals, so was HPV vaccine-related awareness and knowledge. These results must inform educational interventions to raise HPV vaccine-related awareness and knowledge among religious young women in Utah, including those for whom religion does not guide daily decisions. These interventions will require the participation of a variety of stakeholders, and may take the form of church-based education groups, targeted media campaigns, or provider-focused efforts.S1 File(PDF)Click here for additional data file.


After introduction of vaccination, some prevalences of nonvaccine types changed, without clear evidence for type replacement. We analyzed human papillomavirus (HPV) prevalences during prevaccination and postvaccination periods to consider possible changes in nonvaccine HPV genotypes after introduction of vaccines that confer protection against 2 high-risk types, HPV16 and HPV18. Our meta-analysis included 9 studies with data for 13,886 girls and women ≤19 years of age and 23,340 women 20–24 years of age. We found evidence of cross-protection for HPV31 among the younger age group after vaccine introduction but little evidence for reductions of HPV33 and HPV45. For the group this same age group, we also found slight increases in 2 nonvaccine high-risk HPV types (HPV39 and HPV52) and in 2 possible high-risk types (HPV53 and HPV73). However, results between age groups and vaccines used were inconsistent, and the increases had possible alternative explanations; consequently, these data provided no clear evidence for type replacement. Continued monitoring of these HPV genotypes is important. We excluded studies comparing HPV infection in vaccinated persons with HPV infection in unvaccinated persons as part of an individually randomized trial because such studies would not measure population-level effects. Similarly, we excluded studies in which HPV infection was compared only between unvaccinated and vaccinated persons in the postvaccination period. We also excluded studies in which only a small proportion (<2%) of the postvaccination study population was vaccinated . One author (D.M.) initially reviewed titles and abstracts of studies for eligibility; we reviewed in full those studies that appeared to address changes in HPV prevalence after introduction of HPV vaccination programs. We also compared search results with those identified in a recent related review  of sample collection, study setting and population, sample size, specimen type, assay used for HPV DNA testing, HPV genotypes included in the assay, demographic and sexual behavior data collected, and the measure of effect (and method used to determine any effect). For the postvaccination period, we also extracted data on the method used to ascertain estimated vaccination coverage.In addition, we assessed the potential bias in each study by considering the comparability of the study populations in the prevaccination versus postvaccination periods ; the extent of adjustment for potential confounders; the suitability of the specimen type to assess HPV DNA infection; the suitability of the assay used for accurate HPV DNA testing (and whether the suitability of assays differed between the prevaccination and postvaccination periods); and the method used to estimate HPV vaccination coverage. To assess external validity, we considered whether the study samples were population based. Each of these factors was scored as either low risk or high risk.When published data on HPV prevalence and prevalence ratios (PRs) for individual high-risk HPV types were unavailable, we contacted authors to request the HPV type-specific prevalences during the prevaccination and postvaccination periods and the PRs for the 2 periods for each nonvaccine high-risk HPV type. We requested PRs adjusted for demographic and sexual behavior data or the unadjusted PRs if data on confounders were unavailable; we calculated unadjusted PRs if authors provided raw data. By using data from a previously conducted validation study, 1 study included adjusted odds ratios rather than PRs to adjust for the change in assay used during the prevaccination and postvaccination periods (<19 and 20–24 years of age) because of expected lower rates of vaccination coverage and lower vaccine effectiveness in those vaccinated at older ages. Consequently, for each study, we requested data from authors for the same 2 age groups. One study included data for girls <13 years of age, so we requested data restricted to those 16–19 years of age . PRs within each subgroup were combined to obtain a summary PR by using a fixed-effects model if data were not shown to be heterogeneous; lack of heterogeneity was determined by a p value ,,–,,,,,<19 years of age and 23,340 women 20–24 years of age.After we eliminated duplications, we identified 4,648 unique articles in searches from all 4 databases . An init,,,The studies varied in methodologic quality on the basis of potential bias . Most st<19 years of age  but found little evidence of changed prevalences for HPV33 or HPV45 among this age group . Results were heterogeneous for HPV31, HPV33, and HPV45 in women 20–24 years of age; consequently, we did not calculate summary PRs   ; Table 2<19 years of age for HPV39 , HPV53 , and HPV73 . For women 20–24 years of age, evidence indicated increased prevalence for HPV39 .No consistent patterns appeared across the studies for other HPV vaccine types not used in the nonavalent vaccine  Figure 1>3 factors indicating high risk of bias) (>50%). For studies in settings that used the bivalent vaccine, we found evidence of increased prevalence between the prevaccination period and postvaccination periods among those <19 years of age for HPV52, HPV53, HPV56, and HPV70 ; by potential bias of the original study  ; and by <19 years of age, studies with a relatively low potential bias showed no evidence of increased prevalence for HPV52 or HPV39, although evidence existed when the studies were unstratified. For studies with relatively high potential bias, among this younger age group, evidence showed increased prevalences of HPV51 and HPV70, although these increases were not present in the unstratified analysis. In women 20–24 years of age, evidence showed decreased prevalence for HPV33 in those studies with a relatively low potential bias. No summary estimate was provided in the unstratified analysis because of heterogeneity of data. Studies with a relatively high potential bias showed evidence of increased prevalences of HPV52 and HPV58 among women 20–24 years of age. Among this older age group, evidence existed for decreased prevalence of HPV82 in those studies with both relatively high potential bias and relatively low potential bias, although those studies with relatively high potential bias had a larger decrease. Again, no summary estimate was provided in the unstratified analysis because of heterogeneity.Many of our analyses that were stratified by potential bias of the included studies had results similar to those in the unstratified analyses  Table 3.Vaccination coverage was high for the younger age group in all studies  Table 4.<19 years of age. Our main analysis showed increases in other nonvaccine HPV types , but these increases were inconsistent for the 2 age groups examined and the vaccines used.Comprehensive postvaccination surveillance should not only consider reductions of vaccine type–specific infection and associated disease but should also assess any other potential effects of reductions of targeted infections. We assessed changes in nonvaccine HPV types to determine evidence of cross-protection for individual HPV types and to investigate the potential concern that reductions in certain HPV types after the introduction of HPV vaccination in a population could create a niche that enables other nonvaccine high-risk HPV types to become more common . We found evidence of a reduction in the prevalence of HPV31 among girls and women A previous systematic review evaluated changes in high-risk HPV types combined and found evidence of a reduction in the prevalence of HPV types closely related to vaccine types  when they were considered as a single group   can lack sensitivity for detecting individual HPV types when multiple types are present, particularly if another HPV type with a higher viral load is present. In the postvaccination period, in the absence of HPV16 and HPV18, this lack of sensitivity could lead to an apparent but artificial increase in nonvaccine types because these types were underestimated in the prevaccine period because of the predominance of HPV16 or HPV18. Studies have shown this potential unmasking effect  vaccines. Conversely, type 1 errors can occur with multiple testing, so modest evidence for increases should be interpreted with caution.Given the low prevalence of some nonvaccine HPV types, assessing changes in prevalence for individual types since the introduction of HPV vaccination has been challenging. By combining data from several studies, we enhanced our power to consider changes in individual HPV types. However, even with data from 13,886 girls and women We decided against performing random-effects meta-analyses in the presence of between-study heterogeneity because, in most instances, inconsistency occurred in the direction of effect, making a summary estimate  uninformative , and some studies included populations with relatively low coverage, compared with nationally reported vaccination coverage for routine cohorts. Future studies should continue to monitor population-level prevalences of these HPV types. In particular, studies should consider populations vaccinated at young ages and having high vaccination coverage and, perhaps more important, should examine the absolute prevalence of cervical intraepithelial neoplasia 3 lesions attributed to each high-risk HPV type.Search strategies and additional details of a meta-analysis of changes in prevalences of nonvaccine human papillomavirus (HPV) types after introduction of HPV vaccination.