What is the risk of second primary human papillomavirus (HPV)–associated cancer among survivors of HPV-associated cancers?In this cohort study of 113 272 survivors of index HPV-associated cancers, the incidence of most types of second primary HPV-associated cancers  was high and has increased over the last 4 decades.Persistent HPV infection at multiple sites might lead to second primary HPV-associated cancers, suggesting the need for increased screening for the detection of HPV-associated precancerous and early cancerous lesions among survivors of HPV-associated cancers. In the last 4 decades, survival among patients with human papillomavirus (HPV)–associated cancers has improved, while the incidence of these cancers has increased among younger cohorts. Among survivors of HPV-associated cancers, persistent HPV infection may remain a risk factor for preventable HPV-associated second primary cancers (HPV-SPCs).To investigate the risk of HPV-SPCs among survivors of HPV-associated index cancers and to test the hypothesis that the HPV-SPC risk among these persons has increased over the last 4 decades.A retrospective cohort study of 9 cancer registries of the Surveillance, Epidemiology, and End Results (SEER) database was conducted to identify patients with HPV-associated  cancers diagnosed from January 1, 1973, through December 31, 2014. The dates of analysis were July 1, 2017, to January 31, 2018.The HPV-SPC risk was quantified by calculating standard incidence ratios (SIRs) and excess absolute risks (EARs) per 10 000 person-years at risk (PYR). The HPV-SPC risk by time was estimated using Poisson regression.From 113 272  survivors of HPV-associated cancers, 1397 women and 1098 men developed HPV-SPCs. The SIRs for HPV-SPCs were 6.2  among women and 15.8  among men. The EARs were 18.2 per 10 000 PYR for women and 53.5 per 10 000 PYR for men. Among both women and men, those who had index oropharyngeal cancers had the highest HPV-SPC risk . Women who had index cervical cancers and men who had index anal cancers had the lowest HPV-SPC risk . Both women and men who had index HPV-associated cancers of any kind had a significantly higher risk of oropharyngeal HPV-SPCs. Over the last 4 decades, the risk of developing most types of HPV-SPCs after index cervical, vaginal, and vulvar cancers increased.According to this study, the HPV-SPC risk among survivors of HPV-associated cancers is significant, implying that persistent HPV infection at multiple sites may be associated with HPV-SPCs. These findings have the potential to inform surveillance recommendations for survivors of HPV-associated cancers. This registry-based cohort study using data from the Surveillance, Epidemiology, and End Results (SEER) database investigates the risk of human papillomavirus (HPV)–associated second primary cancers among survivors of HPV-associated index cancers and tests the hypothesis that the risk of HPV-associated second primary cancer has increased over the last 4 decades. As of January 2016, there were more than 15 million cancer survivors in the United States, representing almost 5% of the US population, and this number is projected by 2020 to increase to 18 million.6 Because of increased survivorship, the population at risk of developing second primary cancers (SPCs) has also increased,7 making SPCs the leading causes of morbidity, mortality, and economic burden among cancer survivors.11Substantial advances in cancer diagnosis and management have been made in the last few decades, leading to steady improvements in the survival rates of several patient populations with cancer. With advances in treatment technology that have led to longer survival durations among an aging, at-risk population,15 However, with the exception of cervical carcinoma, screening recommendations for survivors of most types of HPV-associated cancers are lacking, potentially putting individuals at risk of developing aggressive, difficult-to-treat HPV-SPCs before the disease is diagnosed. Studying the association between HPV-associated index primary cancers and HPV-SPCs occurring at various anatomic sites could (1) inform and guide surveillance recommendations for and the clinical management of survivors of such cancers, (2) highlight the need for improvement in screening technology for the detection of specific HPV-associated precancerous or early cancerous lesions among survivors of HPV-associated cancers, and (3) help us understand interactions between HPV infections occurring at various anatomic sites and their role in HPV-associated carcinogenesis.Most HPV-associated cancers are preventable if precancerous lesions are detected and adequately managed. Even among survivors of HPV-associated cancers who develop invasive HPV-associated SPCs (HPV-SPCs), early detection can provide a survival benefit.In the present study, our first objective was to investigate the HPV-SPC risk among survivors of index HPV-associated cancers substratified by cancer site. Our second objective was to characterize the HPV-SPC risk by calendar year, testing the hypothesis that the HPV-SPC risk among survivors of HPV-associated cancers is increasing.16 The present study was conducted in compliance with the National Cancer Institute’s SEER limited-use data agreement.We performed a retrospective cohort study using data from the Surveillance, Epidemiology, and End Results (SEER) Program database. The database includes 18 cancer registries, which represent approximately 28% of the US population. Individuals with cancer in the SEER database are also linked to any subsequent incident cancer diagnoses. Our analysis included data from 9 registries—Atlanta (Georgia), Connecticut, Detroit (Michigan), Hawaii, Iowa, New Mexico, San Francisco–Oakland , Seattle–Puget Sound (Washington), and Utah—that have been contributing to the SEER data since 1973, which enabled us to assess trends in cancer incidence over the years.20 which were defined as invasive cancers located at specific anatomic sites and comprising specific cell types in which HPV DNA is frequently found; these cancers comprised squamous cell carcinomas (SCCs) of the vagina, vulva, oropharynx, anus, and penis and all carcinomas of the cervix, including adenocarcinomas and SCCs. Oropharyngeal cancers included cancers of the base of the tongue; pharyngeal tonsils, anterior and posterior tonsillar pillars, and glossotonsillar sulci; anterior surface of the soft palate and uvula; and lateral and posterior pharyngeal walls. All cancers were malignant, classified by anatomic site using the International Classification of Diseases for Oncology, Third Edition (ICD-O-3), and confirmed histologically. Detailed ICD-O-3 site codes, histology codes, and additional restrictions that were used to define index cancers and SPCs are listed in eTable 1 in the We identified survivors of HPV-associated cancers diagnosed from January 1, 1973, through December 31, 2014. We used the Centers for Disease Control and Prevention criteria to identify HPV-associated cancers,STROBE) guidelines.21 The dates of analysis were July 1, 2017, to January 31, 2018, and the University of Florida institutional review board approved this study as exempt.The analysis followed the Strengthening the Reporting of Observational Studies in Epidemiology  were calculated as the ratio of observed HPV-SPC cases to those expected in the general population. The expected numbers of cases were estimated based on reference rates stratified by race/ethnicity, sex, age interval, and calendar year in the 9 SEER registries. We tested the statistical significance assuming that the observed number of second cancers approximated a Poisson distribution and that no variation was associated with the expected number of cases. We then used Byar approximation to calculate 95% CIs for the SIRs.23 The SIRs whose 95% CI excluded 1.0 were considered statistically significant (2-sided P < .05). Excess absolute risks (EARs), which represent the absolute number of additional subsequent cancers attributed to index HPV-associated cancers, were calculated as the excess number of second cancers  among patients with index HPV-associated cancer per 10 000 person-years at risk (PYR). Finally, we investigated the cumulative risk of developing HPV-SPCs using Fine and Gray competing risk proportional hazards models.26Participants were followed up starting 2 months after the diagnosis of an index primary cancer until HPV-SPC diagnosis, death, last follow-up, or the end of the study period , whichever occurred first. To investigate the HPV-SPC risk, we used standardized incidence ratios (SIRs), which were originally described by Schoenberg and Myers27 All analyses were stratified by sex. The SIRs were calculated using SEER*Stat . Additional analyses were performed using SAS .We also analyzed the HPV-SPC risk by calendar year and latency interval. Trends in the SIRs by calendar year and latency interval were calculated by fitting Poisson regression models for each cancer type.Because differentiating between cancer recurrences and SPCs at the same site can be difficult, we also performed some sensitivity analyses. First, we evaluated the overall risk of developing HPV-SPCs at sites different from that of the original cancer. Second, in our estimation of the HPV-SPC risk, we included only those HPV-SPCs diagnosed more than 1 year after the diagnosis of the index cancer. Third, to investigate whether the risk of HPV-SPCs is in excess because of HPV infection, we estimated the SIR for HPV-SPC among survivors of non–HPV-associated cancers .The cohort included 113 272 survivors of HPV-associated cancers (73 085 women and 40 187 men) who were followed up for 837 131 person-years (644 691 person-years for women and 192 440 person-years for men). Of these survivors, 5469 (4.8%) were lost to follow-up, leaving 107 803 (69 949 women and 37 854 men) to be included in our analysis  and 1098 among men —when the analyzed follow-up period began 2 months after the index cancer diagnosis . The SIRAmong women, the HPV-SPC incidence was highest after index oropharyngeal cancers  and lowest after index cervical cancers  . Among mWe then evaluated the HPV-SPC incidence by the index cancer type among women . We founP = .04 for trend), whereas their risk of vulvar SPC after index cervical cancer decreased over this same period . Among women, the risk of vulvar SPC at the index cancer site increased from the 1980s  to the 2010s  (P < .001 for trend); the risk of oropharyngeal SPC at the index cancer site increased from the 1970s  to the 2010s  (P < .001 for trend); and the risk of anal SPC at the index cancer site increased from the 1990s  to the 2010s  (P = .004 for trend). As the follow-up interval increased, the risk of vaginal and vulvar SPCs after index cervical cancer, the risk of cervical SPC after index vulvar cancer, and the risk of vulvar SPC after index anal cancer all decreased. Men’s risk of developing anal SPC at the index cancer site increased from the 1990s  to the 2010s  (P < .009 for trend), and their risk of penile SPC at the index cancer site increased from the 1980s  to the 2010s  (P < .001 for trend).We then evaluated the HPV-SPC risk by year of HPV-SPC diagnosis and by latency interval for women  and men The results of our study show that survivors of HPV-associated cancers are at an increased risk of developing HPV-SPCs, many of which may be preventable. Among both women and men, the risk of developing HPC-SPC at the index cancer site was higher than that of developing HPC-SPC at a non–index cancer site; however, our subsite-specific analysis showed that among women with index vaginal, vulvar, and anal cancers the risk for all HPV-SPCs remained high. In contrast, the HPV-SPC risk was lowest among women with index cervical cancer. Among men, the oropharyngeal SPC risk was elevated after index penile and anal cancers. We found a persistently elevated risk of SPCs across latency intervals, which suggests that surveillance bias is an unlikely reason for elevated risk. Given the large difference in the HPV-SPC risk between survivors of HPV-associated cancers and that of non–HPV-associated cancers, it is likely that persistent HPV infection contributes to the development of HPV-SPCs among survivors of HPV-associated cancers.28 associations between anogenital and oropharyngeal cancers,29 and an increased risk of SPCs among cervical cancer survivors.33 Our study confirms these findings and adds to a growing body of evidence34 supporting a role of HPV in the development of SPCs. To our knowledge, our study is the first to show that the risk for most types of HPV-SPCs remains high after index vaginal and vulvar cancers among women, after index penile cancer among men, and after index anal cancer among women and men. This highlights the need to investigate the efficacy of secondary and tertiary prevention methods, which may include, for example, (1) screening for prevention (using cytology)35 or early detection 37 of anal cancer among women with gynecological precancers or cancers38 and (2) the role of adjuvant39 or therapeutic40 HPV vaccination for prevention of SPCs (by decreasing the risk of index HPV-associated cancers).43Previous studies found a high risk of anal cancer among women with gynecological cancers,44 using these assays to screen healthy populations for index oropharyngeal cancers may not be efficient because oropharyngeal cancer is rare in the general population.44 However, using HPV detection methods to screen individuals who have already been treated for an HPV-associated cancer for oropharyngeal SPC might be an important public health opportunity. Another prevention method worth investigating in the secondary screening setting is ultrasound imaging,49 which has been shown to be able to identify cervical metastases in patients with early-stage oropharynx cancers.Both women and men had an elevated risk of oropharyngeal SPC after any index cancer. Population-level screening for index oropharyngeal cancers is not feasible owing to our current inability to detect precancerous lesions. Furthermore, despite the high specificity  and moderate sensitivity  of oral HPV detection methods  for classifying HPV-positive oropharyngeal cancers,52 Future studies of HPV infection persistence occurring at all anatomic sites among survivors of HPV-associated cancers are crucial to understanding the role of HPV in SPC carcinogenesis. Future economic evaluations are also needed to determine the value of continued surveillance tailored to the HPV-SPC risk.Survivors of cervical cancer had the lowest HPV-SPC risk. One reason for this may be that the widespread implementation of cervical cancer screening and improvements in cervical cancer diagnosis, by enabling the detection and adequate management of precancerous lesions, provided greater protection against subsequent HPV-SPCs. We also found a decreasing trend in the risk of vulvar and oropharyngeal SPCs after index cervical cancer. A hypothesis for this could be that the prevention of index cervical cancer through the detection and management of cervical intraepithelial neoplasia might eradicate cervical HPV infection, thereby preventing SPCs at other sites, given a strong association between HPV infection occurring at oral, anal, and genital sites.23 Limitations specific to the SEER registry include that SPC at the same site is difficult to differentiate from the recurrent disease; therefore, it is likely that a small percentage of recurrence in adjacent anatomic sites  could theoretically be misclassified as SPC. Even in clinical practice, these concerns are inevitable. To address this limitation, we conducted rigorous sensitivity analyses by increasing the latency interval to 1 year in our estimation of the HPV-SPC risk. In an extreme case scenario, we also estimated the risk of developing any HPV-SPC different than the index HPV-associated cancers, yet the risk was significantly greater. Another limitation of our study is that our classification of HPV-associated cancers was based on histologic types and not the actual assessment of individual tumor for the presence of HPV DNA; therefore, there is a possibility of misclassification, particularly for anatomic sites that have lower rates of HPV detection in tumors, such as the vulva (68.8%) and penis (63.3%).18The principal strengths of our study included its large sample size, almost complete follow-up data, and high-quality control of the SEER program.Human papillomavirus infection may increase the HPV-SPC risk among survivors of HPV-associated cancers. Our findings suggest that HPV-SPC rates after index cervical cancer—the only HPV-associated cancer that has secondary prevention and surveillance recommendations—have declined in the last 4 decades. Vigilant monitoring and screening tailored to the HPV-SPC risks posed by specific HPV-associated cancers are crucial to decrease the mortality and economic burden of survivors of HPV-associated cancers and improve their quality of life.


This study was performed to evaluate the use of high-risk HPV (hrHPV) viral load in screening tests for cervical cancer to predict persistent infection and presence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+).We followed women between 30 and 60 years of age who performed self-sampling of vaginal fluid and subsequently a hrHPV test. Women who were hrHPV positive in their screening test repeated the hrHPV test 3–6 months later and were included in the present study.p = 5.33e-03). This was also true for sum of viral load for all hrHPV types in the primary screening test (p = 3.88e-07). 48% of women with persistent HPV16 infection and CIN2+ had an increase in HPV16 titer in the follow-up test, as compared to only 20% of women with persistent infection but without CIN2+ lesions. For the sum of all hrHPV types, 41% of women with persistent infection and CIN2+ had an increase in titer as compared to 26% of women without CIN2 + .Our results show that women with a persistent HPV16 infection had higher HPV viral load in their primary screening test than women with transient infections (The results show that hrHPV viral load in the primary screening HPV test is associated with the presence of CIN2+ and could be used in triaging hrHPV positive women for different follow-up strategies or recall times. Serial testing of hrHPV viral load has the potential to distinguish women with CIN2+ lesions from women with persistent infection but without CIN2+ lesions. Human papilloma virus (HPV) genital infection is associated with the development of cervical cancer. There are over 200 identified HPV types and 12 HPV types  are considered to be high-risk types (hrHPV) since they have substantial oncogenic properties [Cytology has been the most commonly used screening method to identify cervical lesions but due to its low sensitivity it is currently being replaced with hrHPV testing. HrHPV testing has a higher sensitivity than cytology but does not provide as high specificity. The specificity of hrHPV testing can be increased by triage with cytology or by repeating the HPV test in 4–6 months , 3. AbouAn association between hrHPV viral load in cervical samples and severity of prevalent cervical disease was first described in 1999  and replMost HPV infections are transient and a study of women 16–29 years of age has shown that 42% of HPV16 infections and 56% of HPV18 infections clear within one year . Both foThe ability to predict risk of future cervical disease using hrHPV viral load was first shown in a retrospective nested case-control study –16. ThesHrHPV viral load has also been studied for triaging of hrHPV positive women for cytology, colposcopy and clinical management, and high hrHPV viral load has shown a specificity of 96.4% and a sensitivity of 88% for distinguishing between women with high- and low-grade abnormal cervical cytology . Using hHere we have used a cohort of women derived from two previous randomized studies, to investigate the ability to use the individual and combined viral load of 12 hrHPV types in the primary screening sample to predict persistence of hrHPV infection and risk of CIN2 + .The data was derived from women between 30 and 60 years of age participating in two randomized intervention studies that were conducted in Uppsala County, Sweden, between 2013 and 2015 . The firThe procedure for self-sampling of vaginal fluid was based on using a silicon brush and the indicating FTA elute micro card™ and regular mail both for distribution of the sampling kit and return of the sample, as previously described . The womHPV testing was performed using the clinically validated, real-time PCR-based, hpVIR test , 26. ThiGynecological examinations and colposcopies with biopsies were performed at the Clinic of Obstetrics and Gynecology, and histology at the clinic of Pathology and Cytology, both at Uppsala University Hospital, Uppsala. The outcome was the number of women with CIN2+ based on histology according to the SNOMED classification code diagnosed during the 18 months follow-up period from date of invitation. We also included squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma as outcomes.p-values were adjusted for multiple testing using Bonferroni correction, and q-values< 0.05 considered significant. Odds ratios (OR) with 95% confidence intervals were calculated with the “oddsratio” function from the “epitools”-package (version 0.5–10) [p-values were calculated with Fishers exact test. Receiver operating characteristic (ROC) curves were generated for women with transient infections and women with persistent infections and CIN2+ lesions. A general linear model was built using 50% of the samples for a 5-fold cross-validation training using the “caret”-package (version 6.0–78) in R [Statistical calculations were performed and figures generated using R version 3.4.3 . Signifi 0.5–10) , and p-v78) in R . The rem78) in R .All tests were performed for both hrHPV copy number, representing the amount of virus per sample (and PCR) and hrHPV titer, i.e. hrHPV copy number normalized by HMBS copy number, representing the amount of HPV per number of cells in the sample.The study was approved by the Regional Ethics Committee in Uppsala (Dnr. 2012/099).This study included women from two previous randomized studies whom had a positive hrHPV screening test and a follow-up hrHPV test in 4–6 month based on a second self-sample , 23. FroThe women were divided into three groups depending on hrHPV status and histology; I) transient infections (i.e. those that cleared the infection in the 4–6 months between the first and second hrHPV test), II) persistent infections (those with two consecutive positive hrHPV tests) but no CIN2+ lesion within the 18 months follow-up period, and III) women with persistent infection and CIN2 + .p-value = 1.42E-02 and 5.33E-03)    Fig. , Table 2Considering all hrHPV types, i.e. total hrHPV viral load in the sample, the copy number and titer in the baseline test was higher in women with persistent infection with or without CIN2+, in comparison to women with transient infection   with best point sensitivity SE = 0.77 and specificity SP = 0.60 . For total hrHPV titer, 41% (79/191) of women with persistent infection and CIN2+ had an increase in hrHPV titer, as compared to 26% (64/248) of women with persistent infection but without CIN2+ .As an indicator of viral load changes between serially collected samples, we compared the number of women that showed an increase of HPV titer between the baseline and follow-up hrHPV test, in women with CIN2+ and without CIN2+. An increase of titer was defined as an increase of 0.5 hrHPV copies per cell between the two sampling time points. For HPV16, 48% (45/93) of women with persistent infection and CIN2+ had an increase in HPV16 titer between the screening and follow-up test, as compared to 20% (13/64) of women with persistent infection but no CIN2+. The results were significantly different using two-sided Binomial test  and the presence of CIN2+. Based on HPV16 titer, women in the three highest copy number percentiles have OR = 2.4–3.9 for persistent infection and OR = 3.0–3.4 for CIN2+ presence within 18 months as compared to women in the lowest percentile. The results indicate that triaging based on hrHPV titer in the screening test could be used to stratify hrHPV positive women into those at high risk of CIN2+ presence and those likely to clear their infection. Women with high hrHPV titer could be directed for immediate clinical follow-up (colposcopy) without repeating the hrHPV test, while those with low titer could perform a second self-sampling and HPV test prior to clinical investigation.Since the relationship between hrHPV viral load and CIN2+ risk differs between hrHPV types, an HPV test is required that is able to identify individual or specific groups of hrHPVs. Also, the test must enable quantification of hrHPV copy number and titer, such as the clinically validated hpVir test . The mosSeveral previous studies have shown the potential of using serial measurements of HPV titer to predict the outcome of the infection, and suggested that only two measurements would be sufficient –13. We fFurther studies are needed to establish the optimal titer threshold for stratification of HPV positive women to alternative clinical strategies. Hybrid Capture 2 RLU values, which is a semi-quantitative estimate, shows an association with grade of CIN . In resoSome limitations of our study should be recognized. The short time span between the baseline and the follow-up hrHPV tests, due to the medical practice at the time point of the study, imply that we measure short-term persistence of an infection, as a proxy for persistent infection. The short time between the baseline and the follow-up test leads to an overestimate of the number of persistent infections, which would reduce the statistical power to detect an association between titer and persistence of infection as compared to using a longer time interval.We found that triaging with hrHPV viral load could be used to identify women with higher risk of persistent hrHPV infection and presence of CIN2+. Also, for HPV16 infection, HPV tests that identify individual HPV types and can quantify viral load can be used for triage with HPV positivity and viral load. Triaging on the hrHPV viral load could be used to identify women that are likely to clear their infection without intervention, and therefore only need to recalled within a suitable time limit to check their HPV status.


Racial and ethnic minorities are disproportionately affected by human papillomavirus (HPV)-related cancer, many of which could have been prevented with vaccination. Yet, the initiation and completion rates of HPV vaccination remain low among these populations. Given the importance of social media platforms for health communication, we examined US-based HPV images on Twitter. We explored inconsistencies between the demographics represented in HPV images and the populations that experience the greatest burden of HPV-related disease.The objective of our study was to observe whether HPV images on Twitter reflect the actual burden of disease by select demographics and determine to what extent Twitter accounts utilized images that reflect the burden of disease in their health communication messages.We identified 456 image tweets about HPV that contained faces posted by US users between November 11, 2014 and August 8, 2016. We identified images containing at least one human face and utilized Face++ software to automatically extract the gender, age, and race of each face. We manually annotated the source accounts of these tweets into 3 types as follows: government , organizations , and individual  and topics  to examine how images varied by message source.Findings reflected the racial demographics of the US population but not the disease burden . Gender disparities were evident in the image faces; 71.70% (874/1219) represented female faces, whereas only 27.89% (340/1219) represented male faces. Among the 11-26 years age group recommended to receive HPV vaccine, HPV images contained more female-only faces  than males ; the remainder of images included both male and female faces . Gender and racial disparities were present across different image sources. Faces from government sources were more likely to depict females (n=44) compared with males (n=16). Of male faces, 80% (12/15) of youth and 100% (1/1) of adults were white. News organization sources depicted high proportions of white faces . Face++ identified fewer faces compared with manual annotation because of limitations with detecting multiple, small, or blurry faces. Nonetheless, Face++ achieved a high degree of accuracy with respect to gender, race, and age compared with manual annotation.This study reveals critical differences between the demographics reflected in HPV images and the actual burden of disease. Racial minorities are less likely to appear in HPV images despite higher rates of HPV incidence. Health communication efforts need to represent populations at risk better if we seek to reduce disparities in HPV infection. Approximately 23,300 women and 16,500 men develop human papillomavirus (HPV)-related cancer annually in the United States, many of which could have been prevented with the HPV vaccination [National rates of the HPV vaccination remain suboptimal with only 60.4% of adolescent girls aged 13-17 years) initiating and 49.5% completing the series and 56.0% of adolescent boys initiating and 43.4% completing—well below Healthy People 2020 objective to increase HPV 3-doses vaccination series completion for adolescents aged 13-15 years to 80% by 2020. CDC findings indicate that these rates are comparably low for childhood vaccinations, for example, tetanus, diphtheria, and pertussis (88.0%), measles, mumps, and rubella , and hepatitis B (91.4%) -17 years. Gelman Targeted and tailored communication methods can be utilized to enhance the HPV vaccination uptake for high-risk groups . TargeteSocial media can be utilized for health promotion for minorities. Twitter has been used extensively to study vaccine narratives, including those related to HPV, measles, and influenza -22. GiveGiven the importance of social media platforms for health communication, we examined HPV vaccine messages. Specifically, we focused on image tweets, which tend to receive more shares than nonimage tweets ,29. PrevThis study’s implications have the potential to inform the development of more culturally relevant messaging, aligning health promotion imagery salient to intended audiences of those disproportionately affected by HPV-related negative health outcomes. Public health agencies can utilize findings to improve health communication approaches on social media to reduce disparities of HPV-associated disease for all racial and ethnic groups.Hence, this study aims to observe whether the demographics reflected in HPV images on Twitter reflect the populations suffering from the actual burden of disease by the gender and race and determine to what extent individual users, governmental users, and organizational accounts utilized images that reflect the populations bearing the burden of disease in their health communication messages.F1=0.93) [We constructed a corpus of tweets relevant to HPV following the approach described in a study by Chen et al . Figure F1=0.93) . The timF1=0.93) . We then++ [We next used Face++ , a Web-bIn addition, we obtained the locations of these image tweets using the CARMEN geolocation tool . CARMEN We obtained 3 face attributes, gender , age (an integer), and perceived race , of HPV images from Face++. Face++ has been widely used for face detection, recognition, and face attribute identification in social images like Twitter -38 and oExact age estimation is a difficult task for both machines and humans. Face++ has a mean absolute error of 11.0 for exact age estimation  but a muTo further validate the performance of Face++, we manually annotated the perceived gender, race, and age group attributes of all visible faces in each HPV image. The 2 annotators first worked independently and then resolved discrepancies by consensus. This process also helped us to gain insights from the HPV images as well as challenges of classifying the race, which is further detailed in the Discussion section.We performed a mixed-methods analysis for the demographics reflected in the 3 face attributes. Based on the gender of faces, we first categorized images into 3 groups as follows: images that only have female faces, those that only have male faces, and those that have faces of both genders. We then compared the race and age distribution across these 3 groups. Categories chosen for the race were consistent with the current CDC standards for data collection and included Asian or Pacific Islander, black, and white individuals . Owing tOur analysis focused on images containing youths (recommended HPV vaccine recipients) or adults . In addition, we examined the association between facial attributes and the source of the image tweet; for example, do government users or users with a health focus tend to post faces that reflect the actual burden of HPV diseases? To answer such questions, we manually examined  all the source profiles of our HPV dataset . We categorized these sources into 3 types, namely, government , nongovernment organizations , and individuals. We further stratified sources into health-related  or news-related categories. We then compared facial attributes across the resulting source categories.Manual annotation identified more faces (1219) than Face++ (999). The discrepancy is primarily caused by images with multiple small, blurry, or nonfront-facing faces. Such faces are still distinguishable by humans but are rather difficult for automatic tools like Face++ to recognize. Because the subject of an image is usually the clearest and largest face, we believe Face++ has detected most important faces in our dataset see .For gender, Face++ mistakenly labeled 27 female faces as male when considering manual annotation as ground truth. Of these 27, 12 (44.4%) were actually black women. For the race, Face++ identified 100% of black faces but had difficulty in differentiating white and Asian faces. Our human annotators acknowledged the same difficulty, leading them to label the race of 27 faces as ambiguous. Regarding age, we identified youth and adult age categories to have the greatest discrepancy between Face++ and manual annotation. From manual annotation, we acknowledge the challenge in distinguishing between age cutoffs . As such, the actual performance of Face++ on age has an accuracy of >85.1%. Overall, Face++ is reliable in detecting important faces and recognizing the facial properties  for our HPV images.Considering manual annotation is more accurate than Face++, we only detail the results of manual annotation in the following tables. Our results from manual annotation and adoption of Face++ were broadly consistent, highlighting the potential for the automated face analysis utilization in public health research see . We firsLooking at the race , the majFinally, we focused on youth and adult age faces and examined the gender and racial breakdown for the 2 age groups  and adult age groups , more frequently than other sources . FurtherThis study examined the content of HPV Twitter images. Despite reflecting demographics similar to US Census data, our results show a distinct difference between the demographics reflected in Twitter images and the actual burden of disease by the race and gender. Additional Census comparison figures are included in the First, our analyses demonstrate that male faces are significantly underrepresented in HPV images (9.3%), particularly for youth, the recommended group for the HPV vaccination—34.3% girls versus 6.0% boys ; this isOf note, images that included both females and males were usually represented by groups of youth , whereas for female-only and male-only faces, images were typically youth accompanied by adults who were more likely to be male (65.5%). From manual annotation, annotators noted that adults were typically represented by parents or health care providers. From the limited number of male faces in the sample , male adults were twice as likely to be represented  compared with male youth , highlighting the insufficient communication efforts to target male youth for the HPV vaccination. Third, our results showed that the frequency of Twitter images posted from individual, organization, and government sources also reflected discrepancies in the minority representation. Individual sources shared more images with females (n=117) than males (n=37), suggesting the majority of users perceive HPV as an exclusively female issue. Organization users  depict almost exclusively white faces.The influence of Web-based news organizations is significant, especially given that 62% of American individuals access the internet for health information . AlthougThese findings are distinct from government images, which could be attributed to the differing incentives and roles of the media related to promoting vaccination compared with the government. In addition to having a lower presence tweeting black HPV images, government sources, which were typically local, state, and federal public health agencies, also had an uneven distribution of gender and racial representation within aforementioned images. Males represented in the images were overwhelmingly white—80% (12/15) of youth and 100% (1/1) of adults. Government HPV images with both genders included predominantly racially ambiguous youth , which may reflect an intentional choice to reach a more diverse audience, especially given the increasing number of multiracial youth in the Census . HoweverIn health communication, there is frequently an emphasis on the power of culturally sensitive communication, defined by Betsch et al as “the deliberate and evidence-informed adaptation of health communication to the recipients’ cultural background to increase knowledge and improve preparation for medical decision making and to enhance the persuasiveness of messages in health promotion” ,51. The Our findings demonstrate discordance between HPV images on Twitter and those at the greatest risk of HPV-associated cancer. Twitter can be harnessed to disseminate HPV messages aimed at racial minorities who are more likely to be Twitter users . Public There are limitations to this observational study. In this study, we also collected data for specific time points that yielded a relatively small sample of Twitter images, which speaks to the generalizability of findings. However, we found that characterizing the race, ethnicity, and age of faces was particularly challenging with both automated image analysis algorithms and manual annotation. Nearly half of the errors produced by Face++ were related to the misclassification of black female faces. Consistent with these results, Buolamwini and Gebru evaluated commercial gender classification algorithms and found that darker females have highest misclassification error rates (20.8%-34.7%) than darker males (0.7%-12.0%), lighter females (1.7%-7.1%), and lighter males (0.0%-0.8%) . These fSelf-identification is considered the gold standard for all race and ethnicity variables. In this instance, self-identification was not possible, and the race was ascribed by researchers engaged in manual annotation and through algorithms designed for this purpose. Unfortunately, the automated image analysis and, to a lesser extent, the manual annotation are dependent on the stereotypical phenotypic expressions of racialized features. The increasing population of mixed-race individuals, the complexity of the race and ethnicity with Latino populations, and indeed, the recognition that the race is only a social construct make an accurate determination of racial categories difficult. Furthermore, similar constraints made it difficult to consider any nonbinary expressions of gender. Thus, the potential for error of categorizing faces by specific age and race and gender needs to be considered in this study and future research.This study provides insights into racial and gender differences in HPV images on Twitter. Findings can inform imagery-driven health communication strategies to increase the vaccine uptake to mitigate negative health outcomes, particularly within the context of social media. Culturally sensitive communication, which would include increased representation of minorities in images, may enhance the salience of HPV messaging to populations disproportionately affected by HPV-related health outcomes.


Persistent high-risk HPV (hrHPV) infection is higher among women living with HIV/AIDS thus increasing their risk for cervical cancer. We evaluated the virological and immunological correlates of cervical dysplasia in HIV-infected women.A cohort of 220 consenting women attending the antiretroviral clinic of the Federal Medical Centre, Keffi, Nigeria was tested for cervical human papilloma virus (HPV) infection using PCR. The prevalent HPV genotypes were determined by DNA sequencing. CD4+T count and type specific HPV was correlated with cervical cytology. Descriptive and inferential statistical analysis of the data was done using the statistical package for social sciences (SPSS) version 20  for analysis after validation3. A statistically significant association was observed between cervical premalignant lesions and CD4+ count  as well as hrHPV infections .Overall HPV prevalence was 54.1% while the hrHPV prevalence was 35.9%. Premalignant and malignant lesions were observed among participants with CD4+T counts between 200-300/mmRisk stratification with HPV screening among HIV-infected women will help in early case management of cervical precancerous lesions. Human papilloma virus (HPV) is the most common sexually transmitted pathogen in humans and has been implicated in the aetiology of cervical, oropharyngeal and other anogenital cancers ,2. More The study was carried out at the antiretroviral clinic of Federal Medical Centre, Keffi in Nigeria between August 2016 and May 201® Cervex-Brush® cell sampling device  and liquid based cytological processing and preservative reagent  were used for specimen collection and transport. Specimen was obtained by inserting the cytobrush into the cervical canal and rotating it four times to collect all the cervical epithelial cells which adhered to the flat sides of the bristles. The brush was then transferred into the vial containing preservative fluid.RoversCervical smears obtained from each participant was stained using the standard papanicolaou's method in order to assess the presence of cervical premalignant/malignant lesions which was rated according to the 2001 Bethesda system of classification by a cytopathologist.HPV testing was done at the DNA Labs, Kaduna, Nigeria. Gel extraction prior to DNA purification for sequencing was done using QIAquick Gel Extraction Kit . Briefly, HPV DNA was extracted from exfoliated cervical cells using proteinase K-based digestion protocol. Cells were incubated with proteinase K solution (100 μg/ml) for three hours at 55°C. DNA was then further purified by spin column chromatography.® HIV  and Determine® HIV-1/2 . Indeterminate results were subjected to enzyme-linked immunosorbent assay to confirm HIV status.The extracted DNA was amplified for HPV using a nested PCR approach with GP5+/GP6+ and PGMY 09/11 primer sets (MY/GP(+) [Descriptive and inferential statistical analysis of the data was done using the statistical package for social sciences (SPSS) version 20  for analysis after validation. Categorical variables were summarized as proportions and further analyzed using Chi square test to assess significance of association between the variables. Logistic regression analysis was done between CD4+T count and cervical premalignant lesions and relationship expressed in odd ratio (OR).A total of 220 HIV-positive women were enrolled. Majority of the participants, 79 (35.9%) were in the age group 30-40 years followed by the age groups; <30 years; 56 (25.5%) and 41-50 years 47 (21.7%) while only 2 (0.9%) were within the age group 61-65 years. Most of the participants, 133 (60.5%) were married while others, 87 (39.5%) were either single or divorced. Most of the participants were Christians, 149 (69.9%) and predominantly from monogamous family setting, 138 (65.4%). Many of the participants, 69 (31.4%) had only primary education, while 67 (30.5%) had no formal education. More than half of the participants, 115 (52.3%) were unemployed and were housewives, while 66 (30%) were self-employed and the remaining 33 (15%) were employed either in the public or private sector. Majority of the participants 215 (97.7%) were already on HAART while the others were treatment naive.Overall, HPV DNA was detected in 119 (54.1%) of the participants while high-risk HPV types were detected in 79 (35.9%) samples. HPV 35 was the most frequent 12 (20.5%), with multiple HPV infections seen in 25 (21.0%) samples. Other observed genotypes are listed in 3, there was no significant statistical association between CD4+T counts and hrHPV infection.CD4+T counts estimated within six months of sample collection were available for 193 (87.7%) of the participants. Although majority, 91 (47.2%) had CD4+ T values greater than 500 cells/mm2=46.800, P<0.001**)  cases with 54 (25.3%) showing premalignant/malignant cytology. The observed premalignant/malignant variants were as follows; invasive squamous cell carcinoma, 1 (0.47%), adenocarcinoma and adenocarcinoma in situ, 1 (0.47%). The others were CIN III, CIN II and CIN I which constituted 6 (2.8%), 23 (10.7%) and 18 (8.4%) respectively. There was a statistically significant association between hrHPV and cervical precancerous lesion (X0.001**) .3. Of the 16 cytology slides reported for participants with CD4+T count less than 200 cells/mm3, 3 (18.3%) were premalignant compared with the 22 slides reported for participants with CD4+T count between 200-300 cells/mm3, among which 16 (72.2%) were premalignant. Of the 90 slides from participants with CD4+T count greater than 500 cells/mm3, only 14 (15.6%) were premalignant. A significant statistical association was found between CD4+T count and cervical premalignant lesions in this study . The logistic regression model predicted correctly 68.4% of all cases of cervical cytology and also suggested that the CD4+T count is responsible for the 16.5% variation in cervical cytology. Participants whose CD4+T count were less than 200 cell/mm3 or ranged between 200 to 300 cell/mm3 or between 301 to 400 cells/mm3 were more likely to have premalignant lesions than HPV/inflammatory changes {(ODD ratio (OR)=1.266, significance=0.74),  and  respectively}. This finding implies that these individuals are 1.266 times, 14.657 times and 2,624 times more likely to have premalignant lesions rather than HPV/inflammatory changes in their respective CD4+ T count groups  were invasive squamous cell carcinomas and 1 (1.9%) was adenocarcinoma type of cervical cancer. This finding is consistent with reports from other sub-Saharan African countries where varied rates of 33%, 26.7%, 24.3%, 66.3% and 73.0% were reported from Zambia, Kenya, Rwanda, South Africa and Uganda respectively -25. Howe3 had more premalignant lesions and the risk of developing premalignant lesions decreased with increasing CD4+T counts. This finding conforms to similar reports from Kenya and China, where a significant association was observed between CD4+T count and cervical precancerous lesions [In the present study, the risk of cervical precancerous lesions was higher in patients with lower CD4+T counts and there was a statistical significant association between CD4+T and cervical precancerous lesion. Participants whose CD4+T counts ranged between 200-300 cells/mm lesions ,27. The  lesions ,29.The current study highlights high hrHPV prevalence of 35.9% and among HIV-infected women. Nineteen different HPV types were detected with the five most predominant types  being 16, 18, 31, 33 and 45. Our finding suggests that high risk HPV infection is an independent risk factor for cervical precancerous lesion. Additionally, HIV positive women with low CD4+T count are at a higher risk of cervical precancerous lesions. The high prevalence of precancerous lesions in the HIV-infected sub-population justifies the need for routine targeted screening and treatment of HIV-infected women to reduce morbidity and mortality from cervical cancer.Multiple studies have shown that HIV-positive women are significantly at a higher risk of developing cervical cancer;The incidence of cervical cancer in women with HIV compared with women without HIV is highest in low and middle-income countries;Majority of invasive cervical cancer are preventable with improved access to high-functioning HPV testing and other cervical cancer screening modalities would substantially decrease the burden of cervical cancer for women living with and without HIV.Levels of CD4+ count and hrHPV infection are important determinants of cervical premalignant lesions;The most prevalent HPV genotype (HPV 35), is not covered by the currently available quadrivalent and nonavalent HPV vaccines;The study therefore justified the need for routine targeted screening and treatment of HIV-infected women to reduce morbidity and mortality from cervical cancer.The authors declare no competing interests.


An emerging subset of oropharyngeal squamous cell carcinomas (OPSCC) is caused by HPV. HPV-positive OPSCC has a better prognosis than HPV-negative OPSCC, but other prognostic markers for these two different diseases are scarce. Our aim was to evaluate serum levels and tumor expression of matrix metalloproteinase-8 (MMP-8) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and to assess their prognostic role in HPV-positive and HPV-negative OPSCC.A total of 90 consecutive OPSCC patients diagnosed and treated with curative intent at the Helsinki University Hospital between 2012 and 2016 were included. Serum samples were prospectively collected. An immunofluorometric assay and an enzyme-linked immunosorbent assay were used to determine MMP-8 and TIMP-1 serum concentrations, respectively. HPV status of the tumors was determined using a combination of HPV-DNA genotyping and p16-INK4a immunohistochemistry. The endpoints were overall survival (OS) and disease-free survival (DFS).p = 0.011) and DFS  among HPV-negative patients; this association was not observed in HPV-positive OPSCC. Although TIMP-1 was immunoexpressed in the majority of the tumor tissue samples, the level of immunoexpression was not associated with prognosis, nor did MMP-8 serum levels.High TIMP-1 serum levels were strongly and independently associated with poorer OS (adjusted HR 14.7, 95% CI 1.8–117.4, Our results indicate that serum TIMP-1 levels may serve as an independent prognostic marker for HPV-negative OPSCC patients. The incidence of oropharyngeal squamous cell carcinoma (OPSCC) is increasing in many countries due to infection with oncogenic HPV strains. Currently, more than a half of all newly diagnosed OPSCCs in Northern Europe and in North America are HPV related –4. HowevPrevious studies have indicated that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) exert an important role in tumor pathogenesis and patient survival in various cancers, including head and neck cancers –11. MMPsTo the best of our knowledge, the role of MMP-8 and TIMP-1 in OPSCC is unknown. To evaluate their role as prognostic factors, we studied serum levels of MMP-8 and TIMP-1 and their expression in OPSCC tumor tissue. Our specific aim was to study the association of MMP-8 and TIMP-1 with prognosis in HPV-positive and HPV-negative OPSCC.Patients with consecutive biopsy-proven OPSCC diagnosed and treated with curative intent during a 4-year time period between March 2012 and May 2016 at the Departments of Oncology and Otorhinolaryngology-Head and Neck Surgery at the Helsinki University Hospital, Helsinki, Finland were included. The inclusion criteria were tumor tissue availability for p16 and HPV DNA PCR status determination from each tumor and collected serum samples at the time of diagnosis from each patient. A total of 90 OPSCC patients met the inclusion criteria.Clinical data included age on date of OPSCC diagnosis, sex, history of tobacco smoking and heavy use of alcohol, tumor-related factors, the date of treatment completion, and details on follow-up. All the data were collected from medical records and it is partly the same as in our previous reports , 36. TumFollow-up time was determined from the date of treatment completion to the date of last follow-up or death. Both follow-up time and follow-up protocol were adopted from our previous report .Sera were extracted from peripheral blood samples by centrifugation at 1600 g for 10 min and stored at − 70 °C. An immunofluorometric assay , 39 and ®  was used to detect 24 different HPV genotypes. The method detects following low-risk HPV genotypes: 6, 11, 42, 43, 44, and 70, and following high-risk HPV genotypes: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82. The method is described previously in more detail  743) and 762 pM , respectively. The mean and the median MMP-8 serum levels for HPV-negative patients were 844 pM (SD 699) and 658 pM , respectively.TIMP-1 serum levels were approximately 10 times higher than MMP-8 serum levels. The mean and the median TIMP-1 serum levels for HPV-positive patients were 8206 pM (SD 4291) and 7054 pM , respectively. The mean and the median TIMP-1 level for HPV-negative patients were 7869 pM (SD 3128) and 7271 pM , respectively. However, significant differences in TIMP-1 serum levels or in MMP-8 serum levels were not observed between HPV-positive and HPV-negative patients (Table p = 0.039) . Univariate analysis was first performed for the entire patient cohort. Univariate analysis revealed that differences in TIMP-1 serum levels, stage, age, HPV status, and smoking status were statistically significantly associated with OS. In contrast, differences in TIMP-1 tissue expression, MMP-8 tissue expression, or MMP-8 serum levels did not reach statistical significance (Table p = 0.011) among HPV-negative patients  among HPV-negative patients. TIMP-1 serum levels did not have any impact on DFS among HPV-positive patients.Additionally, a similar multivariate analysis was performed to evaluate differences in DFS. High TIMP-1 serum levels were independently associated with poorer DFS  and DFS (p = 0.010) when compared with patients with lower serum levels (≤ 7000 pM) by Kaplan–Meier method. Similar statistically significant associations were not found in HPV-positive patients. Survival curves drawn by Kaplan–Meier method are presented in Fig. A TIMP-1 serum cut-off value of 7000 pM was found to maximize Youden index. In addition, a TIMP-1 serum level of 7000 pM was close to the median serum level of both HPV-positive and HPV-negative patients. Consequently, it was chosen as an optimal cut-off concentration to discriminate patients into favourable and unfavorable survival groups for further Kaplan–Meier analyses. HPV-negative patients with high TIMP-1 serum levels (> 7000 pM) had significantly poorer OS (This prospective study provides new evidence on the potential of TIMP-1 serum levels to serve as an independent prognostic biomarker for OPSCC. TIMP-1 serum levels were found to be a significant independent prognostic marker for OS and DFS in HPV-negative OPSCC patients. Similar results regarding the prognostic value of TIMP-1 serum and plasma levels have also been found in various other cancers , 29, 30,The strengths of the present study were the prospective setting with a relatively long follow-up period and availability of both p16 and HPV-DNA status for all tumors. Regarding limitations, MMP-8 and TIMP-1 IHC were not available for all patients and the number of patients was relatively small, which limited more extensive statistical analyses.TIMP-1 has been reported to have two distinct functions. In addition to directly binding to various MMPs and inhibiting their function, TIMP-1 exerts a specific growth factor function by interacting with the cell surface molecule CD63 and thereby activates intracellular signaling through FAK leading to cell proliferation , 31, 32.Although, serum levels of MMP-8 were not associated with survival in the present study, we cannot formally exclude that the association between TIMP-1 and survival is somehow caused by TIMP-1 inhibiting the MMP-8 function. However, our observation that elevated TIMP-1 serum levels, but not MMP-8, are associated with poorer prognosis raises the possibility that the association between TIMP-1 serum concentration and survival is not mediated by the inhibition of MMPs, but instead by interacting with its cell surface receptor CD63 leading to FAK activation. FAK has a key role in immunoevasion and tumor growth and may be a possible target for immunotherapy , 47, 48.In the present study, multivariate analysis revealed that increased TIMP-1 serum levels were independently associated with poorer prognosis in patients with HPV-negative tumors. However, there was also a trend of increased TIMP-1 serum levels and poorer survival in patients with HPV-positive tumors. However, this trend was clearly weaker than in patients with HPV-negative tumors. This weak trend did not reach statistical significance in the present study but might show a stronger effect in a larger patient cohort. Nevertheless, increased TIMP-1 serum levels more strongly associated with poorer prognosis in patients with HPV-negative tumors than in those with HPV-positive tumors. It is possible that the oncogenic changes associated with HPV transformation are sufficiently strong leading to oncogenesis without TIMP-1 up-regulation. The oncogenic changes leading to TIMP-1 up-regulation may be more responsible for cancer progression in HPV-negative tumors. In addition, it is notable that on average HPV-negative tumors have more oncogenic mutations and the mutation profile is different when compared with HPV-positive tumors –51. AddiThe level of MMP-8 immunoexpression in inflammatory cells appeared to be higher in patients with a HPV-negative tumors than those with HPV-positive tumors. However, the difference was not statistically significant. The differences in the biological and pathophysiological backgrounds of HPV-positive and HPV-negative tumors may explain this phenomenon but a larger patient cohort may be necessary to reach statistical significance.p16 overexpression, which is characteristic for HPV-positive tumors, is an established independent prognostic factor for OPSCC . BesidesThis study provides new evidence for the potential of TIMP-1 serum levels as an independent prognostic biomarker for HPV-negative OPSCC patients. This should be studied in a larger cohort in a multi-center setting to confirm and validate these findings.


Schistosoma mansoni is one of the most common helminth infections affecting a large population of people in sub-Saharan Africa. This helminth infection is known to cause immunomodulation which has affected the efficacy of a number of vaccines. This study examined whether a chronic schistosoma infection has an effect on the immunogenicity of HPV vaccine which is currently administered to girls and women aged 9 to 24. Little is known about the immune responses of the HPV vaccine in individuals with chronic schistosomiasis.S. mansoni cercaria, and allowed to reach chronic stage (week 12 onwards), at week 13 and 14 post-infection, one group was treated with 80mg/kg of praziquantel (PZQ). Sixty four weeks post schistosoma infection, all groups received 2 doses of the Cervarix HPV vaccine a month apart. Specific immune responses to the HPV and parasite specific antigens were evaluated.This study was carried out at the Institute of Primate Research (IPR) and involved an Olive baboon model. The experimental animals were randomly placed into three groups (n = 3–4); Two groups were infected with S. mansoni infection elicited significantly reduced levels of HPV specific IgG antibodies 8 weeks after vaccination compared the PZQ treated and uninfected groups. There was no significant difference in cellular proliferation nor IL-4 and IFN-γ production in all groups.Animals with chronic S. mansoni infection results in reduction of protective HPV specific IgG antibodies in a Nonhuman Primate model, suggesting a compromised effect of the vaccine. Treatment of schistosomiasis infection with PZQ prior to HPV vaccination, however, reversed this effect supporting anti-helminthic treatment before vaccination.Chronic  In sub-Saharan Africa countries, vaccines are administered to people who may suffer from existing infections, especially helminth infections. These infections are known to modulate immune responses rendering some vaccines ineffective. The impact of helminth infections such as schistosomiasis on a recently introduced Human Papillomavirus (HPV) vaccine on infected or treated populations and the degree or duration has not been clearly elucidated. This study was set up to investigate whether a chronic schistosoma infection compromises the specific immune responses elicited by the HPV vaccine. It has been estimated that 527,624 women are diagnosed with cervical cancer each year and 266,672 die due to the complications caused by the disease worldwide. In Africa, the incidence of cervical cancer is high, approximately 99,038 cases were recorded in 2012 ), Karen, Nairobi, Kenya. This was according to institutional standards and guidelines for primate welfare and housing based on the International Guiding principles for Biomedical research Involving Animals development by the Council of International Organizations of Medical Sciences in 1985: Appendix A of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Scientific Purposes , Convention for International Trade in Endangered Species, the U.S. Guide for the Care and Use of Laboratory Animals and the European Primate Resources Network/Primate Vaccine Evaluation Network [PVEN]. These guidelines were also in compliance with the Association for Assessment and Accreditation for laboratory Animal Centers; and the Statement of Compliance with standards for Humane Care and use of Laboratory animals by Foreign Institutions issued by the National Institutes of Health (NIH)[This study involved a total of ten Olive baboons (lth (NIH).ad libitum access to water. They were fed daily with monkey cubes  and supplemented with fruits and vegetables [The baboons were housed in outdoor group cages to allow social interactions and only moved to individual cages during stool and blood sample collection. Both grouped and individual cages were designed to allow natural light and dark cycles and they had getables .Ten Male and female, sub-adult, Olive baboons weighing approximately 5 to 9Kgs were selected for the study. The animals first underwent screening to ensure that they were free from ectoparasites, protozoan and helminth infections, tuberculosis and Simian Immunodeficiency virus (SIV).S. mansoni cercaria as previously described [S. mansoni infection, Schisto-infected+HPV, Schisto/PZQ+HPV group, and an additional HPV vaccine only group (HPV-only n = 3) received 0.5ml Cervarix HPV vaccine containing a dose of 20μgHPV16 and20μgHPV18. The vaccine was administered via Intramuscular injection at the Vastas lateralis muscle of the right hind leg. The animals were monitored for 15 to 60 minutes for any side effects, such as reddening and swelling, at the point of vaccination. As it has been observed that 2 doses of the HPV bivalent vaccine is just as effective as 3 doses [The baboons were randomly placed in 3 groups. Two groups, Schisto-infected+HPV; (n = 3) and Schisto/PZQ+HP; (n = 4)were infected percutaneously with 500 escribed . At week 3 doses ,32 the aSchistosome infection in the Schisto-infected and Schisto/PZQ groups was monitored weekly  by examination of parasite eggs in the animals faeces using the Kato Katz method. By the time of vaccination, animals with an active schistosome infection (group) had an average of 179±74.9eggs/grams of faeces while those that received PZQ treatment (group) displayed a significant reduction, approximately 95.4% (26 ±6.675eggs/grams of faeces) (P<0.001), at week 15 post cercarial challenge. This ensured elimination of the schistosome infection in the Schisto/PZQ group prior to HPV vaccination .The animals were euthanized by intramuscular administration of Ketamine-HCL at a dose of 10mg/kg and Xylazine at a dose of 0.5mg/ml which sedated the animals followed by intravenous administration of 100mg/kg of Sodium pentobarbitone . Perfusion of the worms from the mesenteric vasculature and liver by the administration of citrated saline through the abdominal aorta was done as previously described . The recLevels of HPV-specific IgG antibodies present in sera were determined using Enzyme Linked Immunosorbent Assay (ELISA). Briefly, flat bottomed 96-well ELISA plates were coated with 1.25μg HPV16/18 diluted in 1X Phosphate Buffered Saline (PBS) at 50μl per well then incubated at 4°C overnight. The plates were washed with 0.05% PBS-Tween20 (wash buffer), blocked with 3% BSA in wash buffer and incubated at 37°C for 1 hour. The plates were washed 5 times and 50μl of serum samples diluted 1:200 in wash buffer was added in duplicate. For the blanks wells 50μl of wash buffer was included. This was followed by incubation at 37°C for 2 hours. The plates were washed five times and 50μl anti-monkey secondary antibody , diluted 1:2000 in wash buffer, was added per well and incubated for 1hour at 37°C. 50ul of 3,3,5,5-tetramethylbenzidine  was added per well and the color was allowed to develop for 30 minutes The plates were read using the ELISA reader (Biotek Elx808) at a wavelength of 630nm.6 cells) were plated in 48-well culture plates in 1 ml of complete culture media . 5μg/ml Schistosoma Egg Antigen  and 5μg/ml Soluble Worm Antigen Preparation  and HPV Ag  at a final concentration of 1.25μg/ml were added to the test wells. 5μg/ml of Concanavalin A  was included as positive control while medium only (no stimulant) cells acted as negative (background) control. The cultures were incubated at 37°C 5% CO2. Culture supernatant was collected at 48hours and 72hours and stored at -45°C.Whole blood was mixed in a dilution of 1:2 blood to Alsevers solution  followed by layering on Ficoll paque . The blood was centrifuged at 2000rpms for 20 minutes at 24°C. The buffy coat containing the Peripherial blood mononuclear cells was collected and washed twice with 1X PBS at 1500rpm for 10 minutes at 4°C. The isolated PBMCs were cultures to determine antigen specific T cell reactivity and cytokine production as previously described . Under s6 cells were stained with 5mM of 5(6)- Carboxyfluorescein diacetate N-Succinimidyl ester  and cultured in the presence of antigens and mitogens as mentioned in the PBMC cultures[Proliferation of PBMCs was performed as described previously 34, 35). 1 X10, 35. 1 XAntigen-specific IL-4 and IFN-γ were detected from culture supernatants at 48 and 72 hours respectively using Human IL-4 ELISA  and Monkey IFN-γ ELISA  kits, as indicated by the manufactures instructions and as previously described . Brieflywww.graphpad.com).Statistical level of significance was set at p<0.05.Antibody and proliferation data was normalized and analyzed using one-way ANOVA, followed by Tukey post hoc test for multiple comparison. Cytokine data on the other hand was analyzed using Kruskal Wallis, followed by Dunn’s post hoc analysis. Egg counts derived from Katokatz was analyzed using Wilcoxon matched-pairs sign rank test. All analysis were done using the GraphPad Prism software Version 7.00 for Windows . However, at this same time point, there was no significant difference between HPV-only and Schisto/PZQ+HPV (p = 0.8259). This indicates that HPV specific IgG antibody response is compromised during a chronic S. mansoni infection.  in optical density (OD) which corresponds to levels of HPV specific serum IgG antibodies present in the samples. Four weeks after the first dose of vaccine was administered, the HPV-only group had a significantly higher OD levels (1.042±0.084) compared to the Schisto/PZQ+HPV group (0.946±0.007) (p = 0.0306). The HPV specific IgG levels of animals of the HPV-only and Schisto/PZQ groups continued to increase 2 weeks after the 2nd vaccine dose was given. While these levels declined significantly (p = 0.0301) between 6fection. .low), specifically, the number of cells in subsequent generations. The CFSElow of the 3 groups over the 8 weeks were compared.Histograms  were anaPBMCs that were cultured in the absence of any stimulant (MED) showed the least number of cells that lost CFSE fluorescence intensity, thus indicating that very low cellular division occurred in these cells as compared to the PBMCs stimulated with HPV Ag, SEA, SWAP and CONA .low levels of PBMCs collected at the 8th week and cultured in the presence of HPV antigen were higher than those collected prior to vaccination for the Schisto-infected+HPV, Schisto/PZQ+HPV and HPV-only groups . PBMCs of Schisto/PZQ+HPV group and HPV-only group collected 4 weeks after the 1st dose of vaccination had higher CFSElow cell numbers compared to those of Schisto-infected+HPV thus indicating a slightly higher proliferation capacity in the Schisto/PZQ+HPV and HPV-only, however, this was not significantly different . However, PBMCs of the Schisto-infected+HPV collected at the 6th and 8th week had the highest levels of CFSElow cells though this was not significantly different when compared to the other groups.The CFSEth, 6th and 8th week had high levels of CFSElow cells. This trend was also observed in PBMCs cultured with SWAP with Schisto-infected+HPV and Schisto/PZQ+HPV having higher levels of CFSElow cells compared with the HPV-only group. Similar to what was observed in PBMCs cultured with HPV Ag, the PBMCs cultured with SEA, SWAP and CONA had higher CFSElow cell levels after the 8 weeks compared with Week 0 , while Schisto/PZQ+HPV showed an increase and produced the highest level (11.01±4.757pg/ml).Highest levels of IL-4 were observed in PBMCs that underwent non-specific stimulation by CONA while the lowest levels of IL-4 were observed in PBMCs cultured in the absence of stimulant (MED). Stimulation with the HPV Ag generally produced very low levels of IL-4 cytokines in PBMCs collected over the 8 weeks. Prior to vaccination (week 0), Schisto-infected+HPV produced the highest levels of IL-4 (5.89±3.021pg/ml) while the HPV-only group produced the least amount of IL-4 (1.653±1.169pg/ml). HPV-only group continued to produce low levels of IL-4 for the 8 weeks. Four weeks after the 1th week when there was a decrease to levels that were below the ELISA threshold. At this time point, Schisto-infected+HPV had the highest level of IL-4. At the 8th week, the 3 groups produced similar levels of IL-4, with HPV-only group having slightly higher levels.Peripheral Blood Mononuclear cells cultured in the presence of SEA illustrated relatively high levels of IL-4 cytokines in the 3 groups prior to vaccination, with the highest observed in Schisto/PZQ+HPV (23.26±10.89pg/ml). Schisto/PZQ+HPV maintained the highest IL-4 until the 6th week. Schisto/PZQ+HPV produced higher levels of IL-4 prior to and after vaccination, while the Schisto-infected+HPV group only showed an increase in IL-4 levels at the 8th week , Parasite specific stimulation with SEA and SWAP, and HPV antigen specific stimulation was quantified for the Schisto-Infected+HPV, Schisto/PZQ+HPV group and HPV-Only group using sandwich ELISA. Cells that were unstimulated (MED) were used as a negative control. The responses against the stimulants of Schisto-infected+HPV and Schisto/PZQ+HPV were compared among the 3 groups over 8 weeks after the 2 doses of the HPV Vaccine was administrated .st dose of vaccine. Four weeks later, HPV-Only group produced the highest level of IFN-γ (7.49±2.67pg/ml) while the Schisto-infected+HPV group produced the lowest (1.59±1.5pg/ml) however this difference was not significant. After administration of the 2nd dose of the vaccine, there was an increase in IFN-γ levels observed in the 3 groups. At the 8th week, Schisto-infected+HPV and Schisto/PZQ+HPV groups’ IFN-γ continued to rise slightly while a slight decline in IFN-γ the HPV-only group was observed. However, this was not statistically significant when comparison among the three groups was done.Cells cultured in the presence of HPV Ag (1.65μg/ml) displayed a general increase of IFN-γ production after administration of the 1SEA and SWAP stimulation produced relatively low and constant IFN-γ levels throughout the 8 weeks. However the HPV-Only group (Week 4) saw relatively high IFN-γ levels when the PBMCs were stimulated with SEA, this could be due to one extreme value from one animal of the group (38.91pg/ml). The difference among the three groups was not statistically significant.st dose of vaccine. IFN-γ was produced in high variable levels in the 3 groups. (Non-specific stimulation with CONA (20μg/ml) also saw a general increase of IFN-γ levels after administration of the 1 groups. Protection against several diseases is highly dependent on the magnitude and quality of antibodies produced after vaccine administration. The HPV 16/18 bivalent vaccine induces high levels of protective antibody concentrations, both systemic and mucosal, in vaccinated individuals, . CirculaSchistosoma mansoni infection has on the levels of HPV specific IgG antibodies. During the study, substantial levels of HPV specific IgG antibodies were detected in the serum obtained from the 3 groups of animals that received 0.5ml of Ceravix vaccine. There was a considerable increase in HPV specific IgG antibody levels after the first dose of the vaccine was administered, with the HPV-Only group having the highest IgG antibody titers compared to the Schisto-infected+HPV and Schisto/PZQ+HPV group, throughout the 8 weeks. This increase in HPV specific IgG levels in serum after vaccination have been observed in several human clinical trial studies [In this study, we purposed to determine probable effects a chronic  studies ,36,39st vaccine dose (2 weeks after the 2nd dose) as opposed to the HPV-only group which showed a relatively high level of HPV specific IgG antibodies 4 weeks after the 1st vaccination and these levels rose further after the 2nd vaccination. The HPV specific IgG level titers were slightly higher in the group that underwent anti-helminth treatment compared to the group with the chronic S. mansoni infection. This finding is in accordance with a study that investigated the effect anti-helmithic treatment has on the efficacy of the influenza and Tetanus toxoid vaccine [In this study, a delayed increase in HPV-specific IgG levels was observed in the group that underwent anti-helminthic treatment prior to vaccination (Schisto/PZQ+HPV group), as the highest antibody titers were observed 6 weeks after the 1 vaccine ,40.th week a decrease in IgG levels was observed. This reduced humoral response has also been observed in helminth infected subjects administered with a number of vaccines [The study provided an indication that the study subjects with chronic schistosomiasis were capable of responding to the vaccines and produced HPV specific IgG antibodies, however, after the 6vaccines ,40–42.st dose would provide a strong first line of defense against HPV if the individual gets infected. However, according to this study’s findings, an S. mansoni infection may result in a reduction in HPV specific IgG antibodies after the 1st vaccine is administered and these levels may not rise even after the 2nd booster is given. Therefore protection against HPV may not be maintained for a long period, hence putting the individual at risk of HPV infection over time. While individuals that have previously undergone anti-helminthic treatment (such as PZQ) may experience a slight delay in the increase of HPV specific IgG antibody titer. However, elimination of the S. mansoni appears to result in improved humoral response compared to what could occur if the helminth infection remains chronic and untreated.This rapid increase in HPV specific antibodies titers after the 1Successful vaccinations should elicit effective lymphocyte responses such as proliferation. Previous studies have shown that vaccination with the HPV vaccine results in substantial levels of T lymphocyte proliferation ,43–46.low cells in response to the HPV Ag, especially 4 weeks after the 1st vaccination. While the Schisto-infected+HPV group only showed high proliferation 6 weeks after the 1st vaccination (2weeks after the booster). This increase in Proliferating CFSElow cells are due to the cells, especially the Lymphocytes, expanding and differentiating into effector cells as a reaction to antigen recognition, resulting into rapid cytokine production [th week and rose slightly at the 8th week, while the Schisto-infected+HPV group proliferation capacity continued to rise at the 8th week. The decline in proliferating CFSElow cells could be an indication of the cells entering homeostasis decline prior to the formation of memory cells [low cells were observed in Schisto-infected+HPV and Schisto/PZQ+HPV during stimulation with SEA and SWAP. This is expected as helminth infections would stimulate the production of protective cytokines, such as IL-4, which are responsible for the activation and expansion of the Th2 cells [In the current study, substantial cellular proliferation occurred upon specific and non-specific stimulation, with lower levels of proliferation observed in the PBMCs cultured in media alone (MED). PBMCs collected from Schisto/PZQ+HPV and HPV-Only groups showed higher Proliferating CFSEoduction ,48. The ry cells . This sury cells . High prh2 cells .S. mansoni infection may require 2 doses of the HPV bivalent vaccine to enable their lymphocytes to undergo effective proliferation.The data from this study implies that individuals with a chronic HPV vaccination has been shown to induce increased T-cell proliferation which result in increased cytokine production which is important in the stimulation and maintenance of the humoral responses which are required for effective HPV Vaccine response as well S. mansoni infection would restore the Th1-Th2 balance. During the study, ample amounts of IL-4 and IFN-γ cytokines were released into the culture supernatants upon specific and non-specific stimulation of the PBMCs collected from the 3 groups during the 8 weeks. As expected, it was observed that prior to vaccination higher amounts of IL-4 cytokines were produced in the subjects that had chronic schistosomiasis (Schisto-infected+HPV) and those that were previously exposed to the infection (Schisto/PZQ+HPV) while the control group (HPV-only) had slightly higher levels of IFN-γ than IL-4 prior to vaccination. Higher levels of IL-4 in these 2 groups is typical as this cytokine is involved in production of IgE which play a role in eosinophil-mediated defense against helminths [st vaccination. This high Th1 response discerned in this study is quite different from another study that showed higher levels of IL-4 and significantly lower IFN-γ levels in S. mansoni infected mice administered with the HIV-1C vaccine [This study aimed to determine whether schistosomiasis triggers a Th2 biased profile caused by the shift from Th1 to Th2 that it typical of helminth infections, as well as to determine if elimination the elminths ,49. High vaccine . In the nd vaccination is similar to the lack of bias seen in a study that aimed to investigate the effect of Schistosoma mansoni infection on the TB vaccine, MVA85A candidate vaccine [in vitro indicated by high levels of IFN-γ, IL-5 and IL-10, especially in individuals with strong antibody titers. It was reported that these strong antibody titres have a role in increasing T cell responses [S. mansoni infection also stimulated the production of proinflammatory cytokines [S. mansoni infection or previous exposure to schistosomiasis does not down-regulate Th1 cellular responses to HPV Vaccine.A stronger Th1 response after the 2 vaccine . Investiesponses ,44. The esponses  which isesponses . There iytokines . The finThe research had an important limitation in which the sample size of the baboons was narrowed to 10  due to the high maintenance costs for each animal. This small sample size could have contributed to the inconclusive cellular responses results. However from the data, there is no indication that Schistosomiasis reduces the HPV vaccine induced cellular responses. The second limitation is that, even though the baboons are physiologically, anatomically and genetically similar and disease progression and immune response to the disease are related, baboons are not humans, hence there will be differences.S. mansoni infection did interfere with the production of systemic HPV specific IgG antibodies, as the resulting humoral response was not as strong as it could have been if the subject did not have the infection or had undergone antihelminthic treatment prior to vaccination. There is also an indication that anti-helminthic treatment with PZQ prior to vaccination may be beneficial as it could improve humoral responses. This study implies that individuals with a chronic S. mansoni infection may require 2 doses of the HPV bivalent vaccine to enable their lymphocytes to undergo effective proliferation. The data also indicates that Schistosomiasis does not interfere with the cellular immunogenicity elicited by the HPV Vaccine. Policies for helminth treatment prior to HPV vaccination may be required so as to provide an efficient vaccine induced response.We can conclude that the S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


Cervical cancer is almost entirely preventable through vaccination and screening, yet remains one of the ‘gravest threats to women’s lives’ according to the World Health Organization. Specific high-risk subtypes of human papillomavirus (HR-HPV) are well-established as the primary cause of cervical cancer. Uganda has one of the highest cervical cancer incidence rates in the world  as a result of limited screening access and infrastructure. The integration of a self-collected cervical cancer screening program using HPV testing within existing community-based primary health care services could increase access to screening and reduce cervical cancer rates among Ugandan women.Using a pragmatic, sequential, cluster randomized trial design; we will compare the effectiveness of two cervical cancer screening models for self-collected HPV testing: 1) community health worker recruitment (door-to-door); and 2) community health meetings. In Mayuge district, Uganda, 31 villages are randomized to one of two treatment arms. Due to the nature of this trial, blinding is not possible. Women are eligible to participate if they have no previous history of hysterectomy or treatment for cervical cancer or pre-cancer and are aged 25–49 years old. All participants receive an integrated package of cervical cancer screening and education. Samples are tested for HPV using GeneXpert point of care testing. All women who test positive for HR-HPV types are referred to a designated health centre for follow-up inspection by Visual Inspection with Acetic acid (VIA) and treatment with thermal ablation. The primary outcome for the trial is the number of women who attend follow-up for VIA screening at a designated Health Centre after a positive HR-HPV test out of all women screened per arm. Secondary outcomes include: cervical cancer screening knowledge; patient-reported experience measures for self-collected cervical cancer screening; and HPV incidence.Results from this study will inform the national scale-up of cervical cancer screening in Uganda, aligning with the World Health Organization’s target of achieving cervical cancer elimination through the pillar of increased HPV screening coverage., ISRCTN12767014. Registered 14 May 2019, 10.1186/ISRCTN12767014; clinicaltrials.gov, NCT04000503; Registered 27 June 2019, https://clinicaltrials.gov/ct2/show/NCT04000503ISRCTNJanuary 8, 2020, version 1. Although cervical cancer is almost entirely preventable through vaccination and screening, it continues to be responsible for unnecessary deaths among women around the world. The World Health Organization (WHO) has called cervical cancer one of the ‘gravest threats to women’s lives’  as it isSpecific high-risk subtypes of human papillomavirus (HR-HPV) are well-established as the cause of cervical cancer , 7. NearThe WHO recommends a screen-and-treat strategy to reduce incidence of cervical cancer. This strategy can include either screening with an HR-HPV test followed by visual inspection with acetic acid (VIA) and treatment with thermal ablation for women who test positive on both tests, or screening with an HR-HPV test and treatment for all HR-HPV positive cases . HR-HPV Self-collected HR-HPV testing is highly acceptable to women and care providers and has potential to be rapidly expanded through communities –15, partThe primary objective of this study is to compare cervical cancer screening follow-up completion using two implementation approaches for self-collected HR-HPV testing in a rural, low-resource setting: 1) community health workers recruiting women door-to-door and 2) community health meetings. Secondary objectives of the trial are to compare cost-effectiveness of each cervical cancer screening model measured by the incremental cost-effectiveness ratio (ICER) and reduction in lifetime cervical cancer risk for each study arm and to conduct a process evaluation guided by the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework  focused Advances in Screening and Prevention in Reproductive Cancers (ASPIRE) Mayuge is a pragmatic, sequential, two-arm cluster-randomized trial in Mayuge district, Eastern Uganda. The sequential nature of the study arms was chosen to prevent contamination between arms related to the enhanced community mobilization efforts included in arm 2. Ethics approval was granted from the University of British Columbia / Children’s and Women’s Health Centre of British Columbia Research Ethics Board (UBC C&W REB # H17–03332) and the Uganda Cancer Institute Research Ethics Committee (UCIREC REF-02-2018). All study participants have provided informed consent.Mayuge district in Eastern Uganda has an estimated population of 480,000 residents divided into 13 sub-counties . The majThe study involves task shifting self-collected CCS to CHWs, known locally as Village Health Teams (VHT). VHT are an existing element in Uganda’s community-based primary health care service model in rural areas and currently administer health education to communities through informal community health days. Topics for discussion at the community health days are determined through the demands of the local community or based on government defined priorities. For this trial, a total of 61 CHWs have completed a one-week training session guided by the WHO’s CHW training guidelines for CCS . TraininPrior to recruitment, all laboratory staff at the Kigandalo HCIV were trained on the use of the GeneXpert IV System for HPV sample analysis including clinical utility, reagents, sample collection, kit storage and handling, preparing the cartridge, quality controls, and results analysis. Concurrently, a group of 7 nurses who provide VIA screening at three designated health centres  were provided refresher training in cervical cancer screening, VIA, thermocoagulation, and quality monitoring.Women living in the Mayuge district are eligible to participate if they have no previous history of hysterectomy or treatment for cervical cancer or pre-cancer, are between the ages of 25 and 49 years, and have provided written informed consent. All women in the study villages who are approached and meet the inclusion criteria are invited to participate in a survey and self-collected HR-HPV testing. Women not meeting the inclusion criteria or those unable to provide informed consent are excluded. Any women who consent to the survey but decline self-collected CCS are referred to a HCIV in Kigandalo and are included in the study population but are not included in the primary analysis Fig. . For armThe ASPIRE Mayuge trial includes two sequential intervention arms. In both arms, participants are offered a package of self-collected cervical cancer screening and education that is integrated into community-based primary health care services, but interventions differ primarily by the model of delivery: door-to-door vs community health day. Participants who test positive for HR-HPV types follow the same pathway to care regardless of intervention allocation  described in Michie et al.’s Behaviour Change Techniques Taxonomy version 1(BCTTv1) . The BCTIn arm 1, participants receive one-to-one interaction with the CHW at their homes. A CHW goes door-to-door asking women to participate in a baseline survey, offers participants sexually transmitted infection (STI) and cervical cancer health education, and provides them with instructions on how to self-collect for CCS. The specimen is then dropped at a HCII or HCIII by the CHW and transported to a central laboratory at a local health centre for testing using GeneXpert point of care testing for HPV . SamplesIn arm 2, participants receive group interaction with a team of CHWs and other members of their community at a community health day meeting. Participants are asked to complete a baseline survey individually with a CHW. STI and cervical cancer health education and self-collected screening instructions are provided to participants in a group setting led by the CHW from their village. Participants choosing to complete self-collected CCS do so at a private location and a CHW drops the specimens at a HCIV at the end of the community health day. A follow-up community health day meeting is held to provide additional group education and CHWs deliver test results to participants in a one-on-one setting. Participants who test positive for HR-HPV are provided counselling and are invited to schedule their follow-up visit for VIA at an HCIII or HCIV.To describe the association between HPV and other STI, a small sub-sample of 100 participants from each arm are provided the opportunity to self-collect for chlamydia and gonorrhea testing in addition to HR-HPV testing. For those who test positive for either of the STIs, treatment is dispensed by a nurse at the Health Centre for the patient and their partner. Treatment is provided according to National guidelines in Uganda.Study outcomes were determined by applying RE-AIM framework  to interCervical cancer screening knowledge measured as the count of correct responses to knowledge questions per individual within 6 months of recruitment.Patient-reported experience measures for self-collected cervical cancer screening at intervention completion at the individual level within 6 months of recruitment.HPV incidence at baseline as a sensitivity analysis to test our assumption of equal distribution of HR-HPV among the study population.Secondary outcomes compared between arms include:A summary of objectives, outcomes and analysis plans can be found in Table A time line of study activities can be found in Table Sample size for the study was restricted to the 31 villages in the region available for randomization. Power was estimated using R statistical software across a range of plausible interclass correlation coefficients (ICCs) and absolute changes in follow-up rate from the baseline of 20% in one study arm. Power calculations assumed 15 clusters per study arm with an average size of 70 women per cluster. Power estimates were repeated using a cluster size of 50 women per village with negligible effect. Using these assumptions, we are adequately powered for a 20–30% absolute change in the outcome rate between the study arms, and up to 10% absolute change if low heterogeneity in clusters is found  using a 1:1 allocation. Stratification was performed to balance the clusters around the two sub-districts that each includes health centres where VIA follow-up is taking place. Blinding of study staff, outcome assessors, or participants is not possible due to the spatial and temporal nature of intervention arms.Primary trial data is captured through a survey of all participants at enrollment; a follow-up survey within 6 months of results; an audit of laboratory result logs; VIA screening logs; and referral and treatment logs at the participating facilities. All enrollment surveys are administered by a trained CHW in Lusoga or English based on the language of preference of the participant. The Core Plus module 1 (CPLUS1) from the Improving Data for Decision Making in Global Cervical Cancer Programs Toolkit-Part 2 (IDCCP)  is used After completion of CCS follow-up and within 6 months of baseline recruitment, an additional subgroup of 775 participants are recruited by a research assistant to complete a knowledge retention and patient-experience survey to better understand inhibitors and facilitators to self-collected CCS behaviour. The CCS knowledge and patient experience survey design was primarily informed by the IDCCP CPLUS1 module  and the A supplementary survey is distributed to approximately 300 men in the Mayuge district who attend the community health meetings for arm 2. The men’s survey was designed using the IDCCP CPLUS1 module  to captuCost and person-time data used for cost-effectiveness analysis is collected from the study’s financial records and by using customized survey tools completed at each health centre and through observations of both HPV and VIA screening during each study arm. An audit of the screening logs in each study designated health center is completed at the time of arm 1 recruitment start to estimate a cluster level measure of engagement in the VIA screening program prior to initiation of the study. Focus group discussions with participants who screen positive for HR-HPV and either complete or do not complete follow-up are conducted to evaluate the acceptability of screening and barriers and facilitators to engagement in care.A customized data management tool was developed using Research Electronic Data Capture (REDCap) software . Data isStatistical analysis will be performed in R Statistical software using the clusterPower package  with an Apparent prevalence of HPV and STIs will be estimated in the study regions using trial data. Population prevalence of CIN2+ will be estimated based on this apparent prevalence using the reported sensitivity and specificity of the GeneXpert HPV test for this disease status Table .Table 5Secondary outcomes compared between arms will be estimated in a similar manner. The effect of each study arm on HPV incidence is estimated using a multi-level logistic regression model as per the primary analysis. The effect of each study arm on CCS knowledge and patient experience will be estimated using a multi-level Poisson model, adjusted for the same individual and cluster level confounders with the addition of HPV status as an individual compounder and with person-time in follow-up as an offset.Planned secondary analyses include estimating the group by arm interaction of HIV and other STI status on effectiveness of each CCS model. This will be done by adding an interaction term for each comorbidity and arm in the primary model. Temporal trends in HPV incidence and follow-up will be presented graphically.Cost-effectiveness of the intervention will be estimated using a societal perspective and including all health system and out of pocket costs incurred for women participants in each arm. A decision tree model will be developed based on the patient pathway through care See Fig.  in each Process evaluation will be performed using a study specific logic model framework to define inputs, activities and outcomes See Fig. .. ProcesQualitative data will be analyzed using InVivo with initial open coding will be done based on the FGD guides to identify themes using a deductive approach.The ASPIRE Mayuge trial will provide evidence on the best model of delivery for integrated self-collected cervical cancer screening and follow-up in a rural low-resource setting. According to Mayuge District’s Development Plan II, a lack of skilled human resources and infrastructure are the main health system-related challenges faced by the district . To meetn = 43) reported ever being screened, 3.6% (n = 16) of whom were from Mayuge district [This approach aligns with the World Health Organization’s efforts towards achieving elimination within the twenty-first century through three key target areas: increased coverage of vaccination against human papillomavirus (HPV); increased screening coverage with an HPV test and appropriate management of women who have screened positive; and reduced mortality from cervical cancer . Ndejjo district . BarrierThe major challenges of implementation in real-world global health settings are unsustainable reliance on foreign donors, and the lack of infrastructure, commodities, and human resources . Models Additional file 1. Written Consent FormAdditional file 2. Verbal Consent Script: Baseline SurveyAdditional file 3. Verbal Consent Script: Follow-up Survey