Cervical samples were collected by trained health professionals, and HPV detection was performed in a central laboratory by Linear Array.Of the 5268 women, 33.00% (95% CI 31.07–34.92) had multiple infections. At least one type of high-risk HPV was present in 85.50% of all multiple infections. All HPV types were detected more frequently in association with other types than alone. Young individuals who were single or in a casual relationship and those who had more than one sexual partner in the past year were more likely to have multiple infections.In this work, a high rate of multiple HPV infections among unvaccinated young adults tended to increase due to certain risk factors. Such data can provide insight for decision makers in the development of public policies regarding HPV prevention. Understanding the characteristics of multiple infections is critical in the era of HPV multivalent vaccines for the prevention of cervical carcinomas. Therefore, in this cross-sectional study, we aimed to investigate the pattern of multiple HPV infections and associated factors in 5,268 sexually active unvaccinated women (16–25 years old) who access the Brazilian public health care system. Cervical samples were collected by trained health professionals, and HPV detection was performed in a central laboratory by Linear Array. A total of 33.00% (95% CI 31.07–34.92) had multiple infections (60.43% of the HPV-positive sample). The number of HPV types in a multiple infection ranged from 2 to 14 different types. The viral types more frequently identified were HPV 16 and 52. All HPV types were detected more frequently in association with other types than alone. The incidence of multiple infections was 1.29 times higher in single than in married or cohabitating participants. Women who had two or more partners in the last year also had higher rates of multiple infections than those who had fewer than two sexual partners. In conclusion, a high prevalence of multiple infections prior to the national HPV immunization program was observed, especially with the increase in less safe behavior factors. Cervical cancer kills approximately 300,000 women yearly, particularly middle-aged women and those living in lower-resource settings . In BrazThe first HPV studies seldom detected multiple infections. This is likely due to the characteristics of early diagnostic tests. The shift toward highly sensitive assays has, however, affected HPV findings in the most recent epidemiological studies in a way that cannot be ignored . NotablyIndeed, because some studies have demonstrated higher rates of abnormal cervical cytology in women with multiple-type infections , 11, 12,Understanding the characteristics of multiple infections is critical in the era of HPV multivalent vaccines. To contribute to this issue, this study was developed with the objective of investigating the pattern of multiple HPV infections and associated factors in young women who access the Brazilian public health care system.Prevalence of Human Papillomavirus in Brazil). This is the first nationwide study aimed at determining the prevalence of HPV infections (and their types) among sexually active women aged 16–25 years  had multiple infections , ranging in number of types from 2 to 14. The majority had two HPV types, but 11.13% had four or more HPV types , HPV 52 , HPV 6 , and HPV 58 . Individually, all HPV types were detected more frequently in association with other types than alone . The prevalence of multiple infections was 65.28% and 53.76% among women younger and older than 22 years, respectively  , 31.3% iHPV 16 was the most frequent genotype found in multiple infections, which is in accordance with previous studies involving sexually active young adults , 30–32. Our results are consistent with the literature, which demonstrates inverse associations of a higher prevalence of multiple HPV with younger age . High evDespite knowing that the prevalence of multiple HPV infections is high, it remains unclear whether this is a higher risk factor for the persistence of HPV and for cervical lesions than single infections , 8. WentRegardless of whether HPV types in multiple infections are randomly chosen, understanding the characteristics of these types of infections and the groups of risk factors is critical in the era of HPV vaccination. In Brazil, the quadrivalent vaccine  has been used in the Public Health System as a primary measure for cervical cancer prevention since 2014 . InitialSome limitations of this study should be noted. Although we recruited participants in different settings, this was a cross-sectional study with a convenience sample. However, the sociodemographic and behavioral characteristics are similar to those of the young Brazilian population, allowing inferences for the entire population in this age range. Additionally, we do not have information on HPV immunogenicity and were not able to assess concurrent versus acquired infections, considering that immune responses following concurrent acquisition of multiple HPV infections could be different from natural acquired infections.In conclusion, we observed a high prevalence of multiple infections, especially HR-HPV, prior to the national HPV immunization program. This prevalence tended to increase with a higher number of less safe behavioral factors. Such data can provide insight for decision makers in the development of public policies regarding HPV prevention.


Pulmonary fibroelastotic remodelling occurs within a broad spectrum of diseases with vastly divergent outcomes. So far, no comprehensive terminology has been established to adequately address and distinguish histomorphological and clinical entities. We aimed to describe the range of fibroelastotic changes and define stringent histological criteria. Furthermore, we wanted to clarify the corresponding terminology in order to distinguish clinically relevant variants of pulmonary fibroelastotic remodelling. We revisited pulmonary specimens with fibroelastotic remodelling sampled during the last ten years at a large European lung transplant centre. Consensus-based definitions of specific variants of fibroelastotic changes were developed on the basis of well-defined cases and applied. Systematic evaluation was performed in a steps-wise algorithm, first identifying the fulcrum of the respective lesions, and then assessing the morphological changes, their distribution and the features of the adjacent parenchyma. We defined typical alveolar fibro-elastosis as collagenous effacement of the alveolar spaces with accompanying hyper-elastosis of the remodelled and paucicellular alveolar walls, independent of the underlying disease in 45 cases. Clinically, this pattern could be seen in (idiopathic) pleuroparenchymal fibro-elastosis, interstitial lung disease with concomitant alveolar fibro-elastosis, following hematopoietic stem cell and lung transplantation, autoimmune disease, radio-/chemotherapy, and pulmonary apical caps. Novel in-transit and activity stages of fibroelastotic remodelling were identified. For the first time, we present a comprehensive definition of fibroelastotic remodelling, its anatomic distribution, and clinical associations, thereby providing a basis for stringent patient stratification and prediction of outcome. Fibroelastotic remodelling (FER) is a common morphological injury pattern occurring in a number of different clinical settings ,2,3. WitWe identified pulmonary specimens with FER in the archives of the Institute of Pathology at Hannover Medical School, sampled within the last ten years. To avoid conflicting terms we used stringent definitions for clinical and histological nomenclature see .For histologic evaluation, sections with approximately 1 µm thickness were cut from the formalin-fixed and paraffin-embedded archival tissue blocks and stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and elastic van Gieson (EvG) stains.To define a basis for systematic evaluation, 14 PPFE and PPFE-like as well as 12 PAC cases were systematically reviewed to develop a reference catalogue of morphological features. Subsequently, all included cases included were systematically evaluated and the observed morphological features methodically catalogued.We defined typical AFE as i. collagenous effacement of the alveolar spaces with ii. accompanying hyperelastosis of the remodelled and paucicellular alveolar walls characterized by fourfold thickening of the elastic layer when compared with normal alveoli and iii. at least 4 connected alveoli showing these changes.The systematic evaluation was performed by two trained pulmonary pathologists (P.B. and C.W. or I.M.) in four steps on a dual-observer transmitted light microscope (Olympus BX43) equipped with 2×–40× objective lenses. First, areas of typical AFE were identified, their size  estimated and the morphological features within the typical AFE regions assessed. In steps two and three, morphological features directly adjacent to the typical AFE and their respective distribution in the anatomical compartments of the lung were evaluated. Finally, the cases were re-evaluated for pathological changes in the lung but not in direct spatial association with the AFE. The consensus of both observers was recorded for future analysis. In case of disagreement cases were discussed with two consulting pulmonary pathologists (D.J. and F.L.). The study was in accordance with the regulation of the ethics committee of the Hannover Medical School (ethics vote no. 2050−2013).The last available in vivo CT before resection was assessed by a single trained thoracic radiologist to assess if radiologic changes were completely conclusive with the histological changes, were partly conclusive or inconclusive. n = 2), after radio-/chemotherapy (n = 2), chronic allograft dysfunction (CLAD) after LTX (n = 12) and following HSCT (n = 7). Two cases had AFE pattern as a minor companion pattern in other ILD.We evaluated a total of 45 cases. In detail, the PPFE collective consisted of 31 cases. Of these, 6 were iPPFE and 25 had PPFE-like pulmonary fibrosis due to other causes: with underlying AID  and 2 patients had undergone primary resection due to an unclear pulmonary apical mass.The patients were between 9 and 61 years old See . DetaileAfter a review of representative AFE cases, we could identify different patterns within typical AFE and a range of common changes in direct proximity to the AFE lesions .Within areas of typical AFE, the pattern of intra-alveolar fibrosis/fibrotic obliteration was classified as coarse fibrillary if broad hyalinised bundles of collagenous fibres  were present and as fine fibrillary if delicate, mostly curled fibres were demonstrable. The presence of anthracophages and lymphoid aggregates was noted as well as an increase in cellularity with diffuse infiltration of lymphocytes or the presence of an increased number of mesenchymal cells such as (myo) fibroblasts in the obliterated alveolar lumen see .In the majority of cases (67%) both coarse and fine fibrillary fibrosis could be detected, in the other cases either only fine fibrillary or only coarse fibrillary fibrosis (20% and 13% respectively) were detectable. Features found regularly in areas of AFE were aggregates of lymphatic cells (73%), often at the leading edge of the remodelling process see . These aWe observed and catalogued several morphological patterns in direct spatial association with regions of typical AFE. Besides structurally intact lung parenchyma, various forms of FER with either fibro-elastic expansion of alveolar septa, incomplete alveolar fibrosis, or irregularly distributed collagenous and elastic fibers could be observed (See In the majority of cases (93%) fibroelastic expansion of alveolar septa could be observed at the border of typical AFE besides a direct and abrupt transition to structurally intact alveolar parenchyma (91%). In 84% of cases, areas of incomplete fibroelastosis could be detected. Frequently, pleural fibrosis could be observed adjacent to typical AFE (79%).Patterns spatially associated with AFE were mostly similar in all cases examined, with the exception of obliterative airway remodelling , which was observed in all cases of CLAD after LTX, and fibrotic pulmonary remodelling following HSCT. BO was also present in half of the PPFE cases but not present in APC.The compartmental anatomical distribution of the delimitable changes was categorized as subpleural, parabronchial, para-arterial and paraseptal, when AFE was found in association with the respective anatomical structures. Areas of AFE in the parenchyma not associated with the anatomical structures lined out above were classified as “centrolobular” see .In PAC, the AFE pattern was always found in direct spatial association with the visceral pleura (in not-PAC cases in 93%) and only extended to other compartments in a minority of cases. In contrast to PAC, PPFE and PPFE-like disease showed AFE affection of the para-arterial (97%), paraseptal (85%) and parabronchial (74%) compartments.Other typical histologic features of ILD such as architectural distortion, myogenic metaplasia or an NSIP pattern are rarely found in direct association with AFE lesions, even if present within the same lung.Further radiological information was available in 38 (84%) patients. Of these 38 CT scans, 26 (68%) confirmed a main pattern of alveolar fibro-elastosis, in 10 (26%) patients this was a minor pattern and in 2 (5%) remaining patients, there was no radiologic evidence for AFE  remodelled parenchyma see .Anthracophages, however, were frequently encountered in PAC, PPFE and PPFE-like cases, especially in older patients, pointing towards entrapment of otherwise innocent bystanders in the fibrotic process, unlike in other ILD, where exposure to small particles is known to be a causative agent.The presence of FF in AFE has been pointed out in several studies. Frankel et al. reported them to be rare (4). Kusagaya et al.  describeIncomplete AFE is very common in close spatial proximity to AFE lesions and should possibly be interpreted as an equivalent to typical AFE in the context of the clinical setting. So far it is unclear whether incomplete AFE represents an incomplete transitional state or a premature consolidation. Emphysematous and even inconspicuous alveolar parenchyma can be often observed in direct proximity to AFE lesions, which typically expand with a “pushing border” aspect into the adjacent lung. Pleural fibrosis is common and can easily be recognized. However, it is present in only about 79% of cases and should not be considered as a mandatory criterion for establishing the diagnosis.Pulmonary arterial sclerosis is common in AFE lesions or in their close proximity both in patients with PPFE, PPFE-like disease and PAC. Some authors have suggested pulmonary arteriolosclerosis and the resulting ventilation-perfusion disparity as a trigger of AFE-type fibrosis .BO is commonly found in close proximity to AFE lesions in CLAD and following HSCT, where it has long been recognized as a defining feature of the disease. In the context of PPFE-like disease, our recent study on fibrotic airway remodelling points towards shared pathways in BO and AFE development .AFOP and intraalveolar macrophage aggregates can be observed in some cases. These features have been proposed to represent a transitory step in the formation of AFE . The ratIn our study, AFE pattern fibrosis was most commonly found in the subpleural compartment, compatible with current literature . Howeveri. Cardinal features of AFE are collagenous effacement of the alveolar spaces with accompanying hyperelastosis of remodelled alveolar walls and ii. pleural fibrosis does not represent a condition sine qua non for the diagnosis. iii. Incomplete AFE has to be considered an equivalent lesion to typical AFE, provided an appropriate clinical setting. iv. FF are not a typical feature of AFE and should raise suspicion of a concomitant lung injury pattern, such as UIP. AFE is commonly distributed along the visceral pleura, the bronchovascular bundles and the paraseptal parenchyma, and compartimental involvement can give an indication towards the identification of the underlying disease. vi. The previously proposed, step-wise progression model of AFE from initial fibrinous exudation, over macrophage-rich, insufficient resolution to fully developed AFE has possibly to be complemented by active and inactive AFE, according to the intra-alveolar cellularity in the remodelled alveolar spaces.The exact definition of what makes up AFE is important, not only to help to identify the underlying diseases and therefore specific treatment options, but also to stratify patients and predict their individual outcome. Moreover, the systematic application of exact histological criteria is needed as a basis for all morpho-molecular studies in order to gain further insights into the mechanisms of pulmonary FER.


To assess psychosexual distress over a 12‐month period among women receiving different human papillomavirus (HPV) and cytology results in the context of the English HPV primary screening pilot.Longitudinal, between‐group study.Five sites in England where primary HPV testing was piloted.n = 1133) who had taken part in the NHS Cervical Screening Programme.Women aged 24–65 years  and 6 and 12 months later. Data were analysed using linear regression models to compare psychosexual outcomes between groups receiving six possible combinations of HPV and cytology screening results, including a control group with normal cytology and no HPV test.Psychosexual distress, assessed using six items from the Psychosocial Effects of Abnormal Pap Smears Questionnaire (PEAPS‐Q).P < 0.001). At baseline, mean psychosexual distress score (possible range: 1–5) was significantly higher among women with HPV and normal cytology , HPV and abnormal cytology  and persistent HPV  compared with the control group . At the 6 and 12 month follow ups the pattern of results were similar, but coefficients were smaller.At all time points, there was an association between screening result group and psychosexual distress (all Our findings suggest receiving an HPV‐positive result can cause psychosexual distress, particularly in the short‐term. Developing interventions to minimise the psychosexual burden of testing HPV‐positive will be essential to avoid unnecessary harm to the millions of women taking part in cervical screening.Receiving an HPV‐positive result following primary HPV testing can cause psychosexual distress, particularly in the short‐term. Receiving an HPV‐positive result following primary HPV testing can cause psychosexual distress, particularly in the short‐term. Several countries, including England, have introduced primary human papillomavirus (HPV) testing for cervical screening because of its higher sensitivity for identifying high‐grade precancerous disease compared with cytology‐based testing.Psychosexual functioning includes feelings, worries and concerns that relate to, or impact on, sexual behaviour or sexual relationships. A systematic review of 25 studiesPrevious studies exploring psychosexual functioning following HPV testing have all been carried out in co‐testing contexts in England, and never in the context of HPV primary screening.Evaluating psychosexual distress following receipt of different HPV and cytology results will help to establish whether receiving particular results causes concern or has an adverse effect on women's relationships. Understanding the time‐points at which the impact is greatest could inform decisions about the timing of interventions. The aim of this study was to assess psychosexual distress following primary HPV testing among women receiving different HPV and cytology results, at three time‐points over a year.Data were collected as part of the Psychological Impact of Primary Screening for HPV (PIPS) study (details reported elsewheren = 1154) returned a questionnaire booklet at baseline. Participants returning a questionnaire >90 days after date of identification and those who were aged >65 years and ineligible to take part in the study were excluded (n = 21). Of the remaining 1133 participants, 1132 consented to receive follow‐up questionnaires, 67% (n = 768) returned a questionnaire booklet at 6 months and 47% (n = 542) at 12 months. Altogether, 40.3% (n = 456) returned questionnaire booklets at baseline, 6 months and 12 months. Women were included in the analyses if they returned a questionnaire at one or more time‐points. Please see Figure Of the 5488 women who were invited to take part in the study, 21%  and those who were HPV‐positive with abnormal cytology . In addition, two groups of women who had initially tested positive for HPV and were attending their 12‐month follow‐up appointment were recruited: those who were still found to have HPV (HPV persistent), and those who tested HPV‐negative at the follow‐up appointment (HPV cleared). A group of women who had taken part in cytology‐based screening and had received a normal result were recruited as a control group. Throughout this paper, when we refer to screening result, we mean one of the five groups we recruited based on combinations of HPV and cytology test results that women would receive in the screening programme.The primary outcome measures for the PIPS study (anxiety and distress) are reported elsewhere.n = 898). The potential range was 1–5, with higher scores indicating greater distress. Only women who had responded to all six psychosexual items were included in these analyses: 79% (n = 898) at baseline, 76% at 6 months (n = 581) and 78% at 12 months (n = 418). As the aim of the study was to assess the prevalence and magnitude of psychosexual distress following HPV testing, we excluded women who answered ‘not applicable’ to one or more questions .Overall psychosexual distress was calculated as the mean of all six items, as they showed good internal reliability , educational attainment  and relationship status (current partner versus no partner) were collected. Age and Index of Multiple Deprivation (IMD) quintile  and STATA SE v15 .We used linear regression models to explore the association between screening result group and psychosexual distress cross‐sectionally at baseline, 6 and 12 months.We also used conditional change linear regression models to examine changes in psychosexual distress by screening result group between baseline and 6 and 12 months. Using this approach, the baseline psychosexual distress score is controlled for, so the regression coefficients indicate how the screening result group is associated with changes in psychosexual distress over time.In all models, we adjusted for baseline demographic characteristics  and applied weights to adjust for the possibility that the approached sample may not have been representative of the screening population in the HPV testing pilot sites (details described elsewhere).We also explored between‐group differences for each individual PEAPS‐Q item at baseline, 6 and 12 months. All women who had responded to an item, regardless of whether they were excluded from the overall psychosexual distress analyses, were included in the individual item analyses. In the original PEAPS‐Q development paper, Bennetts et al.Characteristics of the 1088 women who responded to at least one psychosexual item at baseline are shown in Table P‐values for the relation between psychosexual distress and result group cross‐sectionally at baseline, 6 and 12 months are presented in Table Adjusted and weighted beta coefficients  and P < 0.001). Compared with the control group, psychosexual distress was higher among women in the HPV‐positive, normal cytology group (by 1.15 points), the HPV‐positive, abnormal cytology group (by 1.01 points), the HPV persistent group (by 0.91 points) and the HPV cleared group . There was no significant difference between the control group and the HPV‐negative group (P = 0.974) . The pattern of results was similar to that seen at baseline, although coefficients were smaller. Psychosexual distress remained highest and significantly different from the control group (P < 0.001) in all three HPV‐positive groups. Compared with the control group, psychosexual distress was higher among women in the HPV‐positive, normal cytology group (by 0.68 points at 6 months and 0.81 points at 12 months), the HPV‐positive, abnormal cytology group (by 0.64 points at 6 months and 0.50 points at 12 months) and the HPV persistent group (by 0.68 points at 6 months and 0.69 points at 12 months). For the HPV cleared group, psychosexual distress was not significantly higher than the control group at 6 months (P = 0.076) but was at 12 months . There was no significant difference between the control group and the HPV‐negative group at 6 months (P = 0.767) or 12 months (P = 0.931).At the 6 and 12 month follow up, the association between result group and psychosexual distress remained significant  or 12 months (P = 0.227) or the HPV‐negative group at 6 months (P = 0.767) or 12 months (P = 0.931).Adjusted and weighted beta coefficients  and The overall percentage of participants who were categorised as ‘distressed’ for each item at baseline, 6 and 12 months is presented in Table At baseline, the percentage who were distressed was lowest among the control group (range: 0–2.9%) and the HPV‐negative group (range: 0–1.4%) and highest among the three HPV‐positive groups . Overall, the percentage classed as distressed decreased over time for all items. At all three time‐points, distress was most prevalent for the two items assessing worry about infectivity.Women testing positive for HPV at cervical screening reported higher psychosexual distress than those receiving a normal cytology result with no HPV test. The differences were observed immediately after screening and were attenuated but remained significant 6 and 12 months later. HPV‐negative women who had tested positive 12 months previously (‘HPV cleared’) also had higher psychosexual distress immediately after their HPV‐negative result and 12 months later. Our findings suggest that psychosexual distress declines over time among HPV‐positive women in the first 6 months.This is the first longitudinal study to explore psychosexual distress following routine primary HPV screening among women with different HPV and cytology results. It is also the first study to include a group of women who had previously tested HPV‐positive and were found to have cleared the infection 12 months later. The main limitation of the study was the low response rate, which ranged by screening result group from 16% in those not tested for HPV to 28% in those with persistent HPV. In addition, a third of women who participated at baseline did not complete the 6‐month follow up, and a further 20% did not complete the 12‐month follow up. We have no psychosexual functioning data for the women who did not respond, so we cannot rule out the possibility that response to the survey was systematically associated with psychosexual distress. However, we were able to weight our data to the screening population in the HPV testing pilot sites for age and IMD, helping to improve representativeness with respect to demographic characteristics.This study consisted predominantly of women of white ethnicity, which reflects the screening population in the UK. Previous research suggests that the stigma of testing HPV‐positive may be greater among some minority ethnic groups.Our study was conducted in the context of the English HPV primary screening pilot. Although carried out in a different setting, our findings are similar to those by Hsu et al.,The percentage of women classified as distressed for each individual item at baseline ranged from 9 to 17%. Distress was more prevalent than reported by Bennetts et al.,The most commonly endorsed items at all three time‐points concerned infectivity, with around 25% of women who were HPV‐positive indicating infectivity concerns at baseline. This finding is consistent with a synthesis of qualitative research exploring the psychosexual impact of testing HPV‐positive.At baseline, psychosexual distress was highest among women in the HPV‐positive with normal cytology group. Testing HPV‐positive with normal cytology is a new result created by the primary HPV screening pathway, and because knowledge of HPV can be lowAt 12 months, psychosexual distress was still highest among women in the HPV‐positive with normal cytology group. However, there were smaller reductions in psychosexual distress between baseline and 12 months in the HPV‐positive with abnormal cytology group than in the HPV‐positive with normal cytology group. It is possible that women in the HPV‐positive with normal cytology group who returned the 12‐month questionnaire were the most concerned (responder bias), which is why, cross‐sectionally, psychosexual distress was highest in this group.Compared with women not tested for HPV, the HPV cleared group had significantly higher psychosexual distress at baseline and this remained significantly higher 12 months later. Although the mean psychosexual distress score was not as high in the HPV cleared group as the three HPV‐positive groups, this suggests that women who had previously tested HPV‐positive may still have residual psychosexual concerns, despite an HPV‐negative result. A qualitative studyOur findings suggest that receiving an HPV‐positive result can lead to elevated psychosexual distress, particularly in the short‐term. It should be noted that the differences between the three HPV‐positive groups and the control group were small at baseline  and smaller still at follow up (<1 point difference).For most women, it is unlikely that testing HPV‐positive would have a meaningful impact on psychosexual functioning. There is no established ‘normal’ range for the PEAPS‐Q, so it is difficult to determine whether these differences are clinically significant. Although we are unable to determine the number of women who are likely to present with psychosexual concerns requiring clinical services , the study suggests that there are women who have concerns therefore efforts to address these at a population level are important. As the individual psychosexual items suggest concerns about infectivity are relatively common, simple interventions such as including information about this in results letters and leaflets for women who test HPV‐positive should be considered.It is possible that women may have additional psychosexual concerns not captured by the items we used. Future research should use qualitative methodology to explore the full range of psychosexual questions and concerns among women taking part in HPV‐based cervical screening. This additional insight may help to ensure that screening information materials and results letters meet the needs of women with different HPV and cytology results.This study suggests that testing HPV‐positive can result in elevated psychosexual distress, particularly in the short‐term. It is reassuring that psychosexual distress decreased over time; however, even at the 12‐month follow up there were small differences between the control group (who were not tested for HPV) and women who were HPV‐positive or had cleared a previous HPV infection. Developing interventions to minimise the psychosexual burden of testing positive for HPV will be essential to avoiding unnecessary harm to the millions of women taking part in cervical screening.KB received grants from the Medical Research Council (MRC) during the conduct of the study. JW, AF and LM received grants from Cancer Research UK during the conduct of the study. EM received grants from the National Institute for Health Research (NIHR) during the conduct of the study. Public Health England (PHE) commissioned the project and paid salary costs for EM during the data collection period as well as all research costs. GDG and HK have nothing to disclose. Completed disclosure of interest forms are available to view online as supporting information.JW, AF, HK and LM conceived the study. JW, EM, AF and LM developed the measures. KB and GDG conducted the analyses. KB drafted the paper. All authors contributed to the final version of the manuscript.Health Research Authority approval was obtained on 26 September 2016 and approval from London‐Surrey NHS Research Ethics Committee (REC) on 30 August 2016 (Research Ethics Committee reference: 16/LO/0902). Section 251 approval was also obtained from the Confidentiality Advisory Group (CAG) for use of patient name and address without consent for the purposes of participant approach on 24 August 2016 .The study was funded by Public Health England (PHE). KB is funded by a Medical Research Council Studentship (MR/N013867/1). JW and LM are funded by Cancer Research UK (C7492/A17219). AF is also funded by Cancer Research UK (C49896/A17429). EM was funded by PHE from 3 January 2016 to 30 September 2017. EM was funded by the National Institute of Health Research (NIHR) from 1 October 2017 (DRF‐2017‐10‐105). The views expressed in this paper are not necessarily those of the NHS or the Department of Health.We would like to thank the NHS clinical laboratory managers and staff at the HPV primary screening pilot sites who helped us gain HRA approvals and recruit participants . We thank Ruth Stubbs and Karen Denton at Public Health England for facilitating the public facing aspects of the study and helping with HRA approvals. Thank you to Matejka Rebolj and Christopher Matthews at King’s College London who provided us with population‐level screening data to allow us to weight our sample, as well as providing helpful guidance at several points in the study. Thanks to Lauren Rockliffe who helped with participant recruitment and data entry. Finally, thank you to the women who kindly gave up their time to participate.Figure S1. An overview of recruitment and responseClick here for additional data file.Table S1. Percentage ‘distressed’¹ for individual psychosexual questions by screening result group at 6‐month follow upClick here for additional data file.Table S2. Percentage ‘distressed’¹ for individual psychosexual questions by screening result group at 12‐month follow‐upClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


The World Health Organization announced an ambitious call for cervical cancer elimination worldwide. With existing prevention and treatment modalities, cervical cancer elimination is now within reach for high-income countries. Despite limited financing and capacity constraints in low-and-middle-income countries (LMICs), prevention and control efforts can be supported through integrated services and new technologies. We conducted this scoping review to outline a roadmap toward cervical cancer elimination in LMICs and highlight evidence-based interventions and research priorities to accelerate cervical cancer elimination. We reviewed and synthesized literature from 2010 to 2020 on primary and secondary cervical cancer prevention strategies. In addition, we conducted expert interviews with gynecologic and infectious disease providers, researchers, and LMIC health officials. Using these data, we developed a logic model to summarize the current state of science and identified evidence gaps and priority research questions for each prevention strategy. The logic model for cervical cancer elimination maps the needs for improved collaboration between policy makers, production and supply, healthcare systems, providers, health workers, and communities. The model articulates responsibilities for stakeholders and visualizes processes to increase access to and coverage of prevention methods. We discuss the challenges of contextual factors and highlight innovation needs. Effective prevention methods include HPV vaccination, screening using visual inspection and HPV testing, and thermocoagulation. However, vaccine coverage remains low in LMICs. New strategies, including single-dose vaccination could enhance impact. Loss to follow-up and treatment delays could be addressed by improved same-day screen-and-treat technologies. We provide a practical framework to guide cervical cancer elimination in LMICs. The scoping review highlights existing and innovative strategies, unmet needs, and collaborations required to achieve elimination across implementation contexts. Globally, there are more than half a million new cervical cancer cases and more than a quarter-million cervical cancer-related deaths each year . Due to However, the timeline for cervical cancer elimination is significantly longer in low-and-middle-income countries (LMICs) . This loThe science, technology, and implementation of cervical cancer prevention and treatment are changing rapidly, with effective and scalable strategies on the horizon. A recent modeling analysis of 78 LMICs demonstrated the importance of successful implementation and scale-up by predicting that the triple-intervention would reduce cervical cancer mortality of women ages 30–69 years by 33.9%  by 2030 and almost 99% by 2120 . The purOur team conducted a scoping review of the literature on primary and secondary cervical cancer prevention strategies using methods adapted from Arksey and O'Malley . A scopiWe chose to review articles published beginning in year 2010, because while the HPV vaccine was introduced to the world in 2006, it was not until 2010 that it became incorporated into the national immunization program in LMIC settings, starting with Bhutan in 2010 and Rwanda in 2011 . We usedTwo authors (MS and GL) screened the search results for relevant articles and extracted data independently. All authors provided feedback on study selection, data extraction, and synthesis, which informed further search and interpretation of the findings. Our search for primary prevention strategies focused on the effect of HPV vaccination, voluntary medical male circumcision (VMMC), tobacco cessation, condom use as mentioned by the WHO , and vagWe divided secondary prevention into screening and treatment strategies. For screening, we compared cytology, HPV tests, and visual inspection with acetic acid (VIA). As the WHO recommends screening with a high-performance test equivalent to or better than HPV testing, we reviewed implementation challenges of HPV testing in low-resource settings. We recognize HPV-based screening may be considered a primary prevention strategy as it can detect individuals in whom cervical cancer has not occurred. However, for the purpose of our review and following the WHO guidelines, we reviewed this screening method under secondary prevention. For immediate treatment of precancerous lesions, we focused on cryotherapy, thermal ablation, and loop electrosurgical excision procedure (LEEP). We identified emerging screening and triage options in LMICs as HPV self-sampling, oncogenesis biomarkers, optical techniques, such as portable colposcopes and automated visual evaluation, and therapeutic vaccines. We also dedicated a section to prevention of cervical cancer among women living with HIV to highlight the differences in HPV acquisition and progression in this population.The logic model summarizes and describes the process flow for cervical cancer elimination . Step onHPV vaccines, which have the potential to prevent 90% of cervical cancer cases , are by Further, the cost-effectiveness of vaccination strategies is dependent on the vaccine price, which is likely to drop significantly as new vaccines being developed in India and China increase HPV vaccine supply over the next ten years . The ecoVMMC for HIV-negative men has been shown to reduce penile HPV viral load of incident infections and the persistence of prevalent HPV infections, which likely reduces male-to-female HPV transmission , 42. EveHPV incidence is inversely associated with the frequency of condom use . In a loCurrent smokers were 1.6 times more likely than never smokers to have prevalent hrHPV infection than never smokers (95% CI: 1.2–2.1) and 1.4 times more likely to have newly detected hrHPV infection (95% CI: 1.0 −1.9) . This inVaginal dysbiosis, including but not limited to bacterial vaginosis, is positively correlated with prevalent HPV infection and cervical intraepithelial neoplasia (CIN) among women with and without HIV , 47–49. We prioritized implementation and scale-up of HPV vaccination in primary prevention opportunities, as it directly addresses the known causal agent of most cervical cancer. Several barriers specific to LMICs have been identified, such as costs associated with the vaccine and service delivery, political commitment, challenges specific to the target population of adolescent girls, and global vaccine shortage . In addiSuccessful implementation and scale-up of HPV vaccine programs depends on achieving and maintaining high rates of vaccine uptake , which rHPV vaccine introduction efforts have faced more challenges than the pneumococcal conjugate, rotavirus and inactivated polio vaccines, which can be readily integrated into existing infant immunization schedules. While low school attendance in some settings limits school-based routine vaccination, most LMICs have well-established infant immunization programs , 60. TheClinicalTrials.gov)  .For all scenarios of expanded HPV vaccine coverage, clear communication addressing vaccine hesitancy and increasing parents', caregivers', and adolescents' acceptance of an HPV vaccine are critical. Unfortunately, literature on vaccine hesitancy from LMICs is scarce . In theiAccording to the WHO/United Nations International Children's Emergency Fund's joint reporting from 2015 to 2017, factors related to religion, culture, gender, and socioeconomic status were becoming more prominent in vaccine hesitancy in low-income countries, and risk-benefit (scientific evidence) factors in lower-middle-income countries . After tDo voluntary male circumcision, condoms, smoking cessation, and treatment for bacterial vaginosis reduce persistent HPV infection, and to what extent?How can monitoring and evaluation provide evidence to support best practices for HPV vaccination programs?How can we address vaccine hesitancy and the anti-vaccination movement to increase HPV vaccine coverage?What are the safety, efficacy, durability, and acceptability of an HPV vaccine administered in infancy?What are the efficacy and durability of one-dose HPV vaccination in routine and catch-up vaccination?How can we obtain LMIC context-specific estimates of the impact of prevention strategies and make them more reliable and approachable for policy makers?The WHO's cervical cancer elimination targets are to screen 70% of women with a high-performance test by age 35, and again by age 45 . Self-reScreening for cervical cancer is currently conducted by cytology (Papanicolaou/Pap smear screening), HPV DNA testing, or VIA. VIA is currently the predominant cervical cancer screening method in many LMICs due to its relatively low cost and ease of execution compared to cytology or HPV testing . VIA canWhile cytology-based screening is used widely in high-income settings, it requires established healthcare infrastructure, repeat client visits to diagnose precancer, training of pathologists and clinicians, and a robust quality assurance program. Cytology-based screening also has lower sensitivity and specificity compared to HPV DNA testing . For theThe wide range of sensitivity and specificity of subjective tests, such as cytology and VIA leads to variation in the number of women referred to triaged for treatment. For example, estimates of sensitivity to detect CIN2-3 varied between 40.7 and 73.7% using cervical cytology and 21.9–73.6% using VIA in one study conducted in India, Nicaragua and Uganda . VariatiHPV DNA testing has superior sensitivity compared to cytology and VIA in detecting CIN2-3  and has Large-scale randomized controlled trials have demonstrated the efficacy of HPV testing in reducing cervical cancer incidence, and that HPV testing provides 60–70% greater protection against invasive cervical cancer than cytology . DespiteThe WHO endorses self-sampled HPV tests as an additional approach to cervical cancer screening for individuals aged 30–60 years . In a meSelf-sampling as an alternative method of screening can overcome barriers, such as access to the health facility and fear associated with pelvic examination, opening new possibilities of reaching underscreened women . AcceptaThe international consensus is to treat CIN2-3/HSIL by ablation or excision except during pregnancy . CryotheThe cervical lesion must be small enough to be covered by the equipment and fully visible with no extension into the endocervix or onto the vaginal wall to be eligible for ablative therapy , 128. An2, is expensive and difficult to procure and transport   .LEEP is recommended for treating CIN2-3 if there is a medical contraindication for ablative therapy  or if thTreatment complication rates differ by the technique. The most common adverse events associated with treatment are bleeding and infection at the cervix, which occur in <5% of treated women , 133–135Point-of-care cervical cancer screening tests that facilitate same-day treatment would minimize loss to follow-up and improve continuity of care. While careHPV meets some of the REASSURED criteria  , 137, anink4a and E6/E7 oncoproteins  ongoing study in Nigeria noted that the squamocolumnar junction where cancers arise was not fully visible for almost 64.6% of women by age 49 using enhanced visual assessment   . Challen Israel) .Identification of women with a precancerous lesion necessitates appropriate linkage to care and treatment to prevent cancer . While tTo optimize screen-and-treat methods, portable treatment tools, such as battery-operated cryotherapy instruments are under development (see NCT03084081) , 162. ThPhotodynamic therapy has shown promising results in the treatment of CIN , 164. ThThe development of therapeutic vaccines against hrHPV could help women who are already infected by stopping progression, triggering regression of lesions, and preventing recurrence of disease . CurrentIn summary, the priority for secondary prevention is the optimization and scale-up of single-visit screen-and-treat modalities. Self-sampling for HPV testing offers several advantages to optimize screening coverage. Subsequent molecular oncogenic evaluation has the potential to detect lesions that are most likely to progress to cancer while reducing overtreatment. Further development is needed to simplify the testing procedure and reduce costs. The feasibility of integrating cervical cancer screening into the existing healthcare system is being explored , 169. FoIs the scale-up of current point-of-care HPV tests and HPV self-sampling effective, feasible, and cost-effective, and how can delivery models adapt sustainably to incorporate them?What is the most efficient and safe model of task-shifting for providing cervical cancer screening, cervical biopsy and treatment of pre-invasive disease?What is the optimal triage algorithm and follow-up for hrHPV positive women and those without visible precancerous lesions when biopsy is unavailable?Are there reliable biomarkers to predict persistent infection with hrHPV?Would self-visualization of the cervix as a screening tool be feasible, reliable, and acceptable?What are the strategies for surveillance of HPV positive women with negative oncogenic biomarkers?+ cell count <200 cells/ul), the risk for cervical cancer is 8-fold higher in women living with HIV compared to HIV-negative women  as the outcome are still needed. The duration of protection in vaccinated adolescent girls and young women living with HIV is unknown. Extending the age of vaccination to include infants and older women could accelerate cervical cancer elimination in HIV-positive populations, although the efficiency and cost-effectiveness of such strategies depend on the prevalence of HPV infection at older ages and the duration of protection afforded by the vaccines. Context-specific modeling work would be valuable to evaluate these outcomes and inform the implementation of effective vaccination programs in settings with high HIV prevalence. In addition, the vaccine efficacy and durability of a reduced dose schedule (two or one doses) among adolescents and young adults living with HIV needs to be determined. The OPTIMO Trial, which aims to see if fewer doses can be used for children/adolescents living with HIV, will begin soon (see NCT04265950) .Prevention strategies, such as VMMC can both help prevent HIV and reduce cases of cervical cancer. A modeling study of HIV prevention and HPV control in Tanzania predicts that VMMC will lower cervical cancer incidence and mortality rates by 28 and 26%, respectively, by 2070 .The American Society for Clinical Oncology and the WHO recommend screening sexually active women living with HIV for HPV or cervical abnormalities as soon as they are diagnosed with HIV and rescreening within 3 years if they are HPV-negative and free of cervical lesions . In the HIV presents challenges to accurate screening for women. There are more false-positive rates with VIA among women living with HIV than women without HIV , 200, liTreatment failure and recurrence is more common among women living with HIV than in the general population . A meta-Cervical cancer prevention services can be integrated into existing health infrastructure as women already engage in health care throughout their lifetime  schedule in women living with HIV?What are innovative ways of effectively integrating cervical cancer prevention and treatment into HIV care?What is the optimal treatment method of precancerous lesions among women living with HIV?What are the optimal screening modalities and intervals for women living with HIV on antiretroviral therapy?Global cervical cancer elimination is achievable with an increase in HPV vaccine uptake and coverage, implementation of screening and treatment strategies and emerging technologies, and development of innovative delivery approaches. Our scoping review designed a roadmap that prioritizes expanding HPV vaccination and collaborating with global organizations to allocate resources needed to eliminate cervical cancer. The focus of the concerted effort must be (1) scaling up evidence-based interventions, including the application of implementation science and measurement of population-level impact, and (2) filling the gaps through research and harnessing emerging innovations that are simple, effective, and affordable for all settings .Lessons can be learned from the global response to HIV, which built the infrastructure that allowed for the scale-up of HIV prevention and treatment services. Since the global initiative to eliminate mother-to-child transmission of HIV was announced in 2011 , 80% of Following the framework of the logic model, key domestic and global stakeholders should work together to prioritize funding to procure vaccines and strengthen healthcare systems. Healthcare practitioners and communities should be engaged at every step of discussion and programmatic planning in order to build capacity and ensure successful implementation . Global While reviewing tertiary prevention strategies  is beyond the scope of this article, their importance in low-resource settings cannot be overlooked. Screening implies the capacity and ethical responsibility for health agencies to make the treatment of cervical cancer available. The regions with the highest prevalence of cervical cancer have the lowest availability of skilled personnel and treatment facilities for diagnosis, surgery, chemotherapy and radiation , 220. ThThe limitations of this review result from its narrative approach. Compared to systematic reviews or meta-analyses, narrative reviews are characterized by subjective study selection. In addition, due to the broad nature of the scoping review, we did not compile an exhaustive list of potentially relevant, innovative strategies and technologies. However, this paper provides an overview of the current landscape of science around cervical cancer elimination and guides the formulation of pertinent questions that deserve further exploration.The effort to eliminate cervical cancer must focus on sustainable and continuous access to prevention strategies. Large scale demonstration projects have been successfully implemented across resource settings for HPV vaccination and screen-and-treat using HPV testing and thermal ablation. Building on the lessons learned, we propose a demonstration project that combines the above-recommended strategies and provides a comprehensive cervical cancer prevention continuum to show that cervical cancer elimination can be achieved at the local level within LMICs. With a strong evidence base and effective implementation established, strategies can be scaled up more broadly. By strategically and skillfully putting scientific advances to practice, global cervical cancer elimination can be achieved.MS and GL: methodology, data curation, writing—original draft, and writing—review and editing. NM, PG, and JW: conceptualization and writing—review and editing. DR, CB, LE, and LP: writing—review and editing. RB: conceptualization, writing—review and editing, and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We conducted a community‐randomized trial (NCTBLINDED) in Finland to assess gender‐neutral and girls‐only vaccination strategies with the AS04‐adjuvanted human papillomavirus (HPV)‐16/18 (AS04‐HPV‐16/18)vaccine.Girls and boys (12−15 years) were invited. We randomized 33 communities (1:1:1 ratio): Arm A: 90% of randomly selected girls and boys received AS04‐HPV‐16/18 vaccine and 10% received hepatitis B vaccine (HBV); Arm B: 90% of randomly selected girls received AS04‐HPV‐16/18 vaccine, 10% of girls received HBV, and all boys received HBV; Arm C: all participants received HBV. Effectiveness measurements against prevalence of HPV‐16/18 cervical infection were estimated in girls at 18.5 years. The main measures were: (1) overall effectiveness comparing Arms A or B, regardless of vaccination status, vs Arm C; (2) total effectiveness comparing AS04‐HPV‐16/18 vaccinated girls in pooled Arms A/B vs Arm C; (3) indirect effectiveness (herd effect) comparing girls receiving HBV or unvaccinated in Arm A vs Arm C. Co‐primary objectives were overall effectiveness following gender‐neutral or girls‐only vaccination.P = 0.232) with gender‐neutral vaccination. Following girls‐only vaccination, overall effectiveness was 49.6% . Total effectiveness was over 90% regardless of vaccination strategy. No herd effect was found. Immunogenicity of the AS04‐HPV‐16/18 vaccine was high in both sexes.Of 80,272 adolescents invited, 34,412 were enrolled. Overall effectiveness was 23.8%  in Finland failed to show indirect benefit for girls by vaccinating boys. Several factors might have prevented to show herd effect such as randomization bias. However, the study confirms the high total effectiveness of the vaccine in girls. The first vaccines launched were the bivalent HPV‐16/18 vaccine adjuvanted with the Adjuvant System AS04 (AS04‐HPV‐16/18 vaccine) and the quadrivalent HPV‐6/11/16/18 vaccine, followed more recently by a nonvalent vaccine. Clinical trials have shown that the vaccines protect against the risk of HPV infection and cervical pre‐cancer in young women,Organized HPV vaccination programs in girls and young women were implemented in many countries, primarily high‐income and upper‐middle‐income settings, shortly after the vaccines became available.Prior to implementation of an organized vaccination program in Finland, we began a community‐randomized trial to evaluate the effectiveness of two vaccination strategies using the AS04‐HPV‐16/18 vaccine: gender‐neutral vaccination vs girls‐only vaccination.Figure 2This phase III/IV, investigator‐initiated cluster randomized trial (NCTBLINDED) took place in Finland between October 2007 and December 2014. Its co‐primary objectives were to evaluate the overall effectiveness of two HPV vaccination strategies i.e., vaccinating both girls and boys , and vaccinating girls only.The study was conducted in accordance with good clinical practice (GCP) and all applicable regulatory requirements including the Declaration of Helsinki, and was approved by the ethics committee of the Pirkanmaa hospital district in 2007 and 2011. Participants <15 years of age provided written informed assent and their parents or legal representatives provided written informed consent. In line with local regulations, participants ≥15 years of age provided written informed consent. Methodology and study characteristics have been published previously.2.1Communities were eligible to participate if they had 1000−9000 male and female adolescent inhabitants from the 1992−1995 birth cohort and were geographically distinct (at least 50 km apart or 35 km apart in the Helsinki metropolitan area). These minimum distances were imposed to minimize inter‐community transmission of HPV. We included 33 communities, stratified according to historical HPV‐16/18 seroprevalence rate in pregnant women under 23 years of age, estimated from the Finnish Maternity Cohort:All male and female adolescents living in the study communities and born between 1992 and 1995 (12–15 years of age at the time of first vaccination)were eligible to participate. Girls had to be pre‐menarche or using adequate contraception before and during the vaccination period. Participants with acute disease at the time of enrollment and girls who were pregnant at the time of vaccination were excluded.2.22.2.1According to census information, 86,083 adolescents born between 1992 and 1995 resided in the 33 communities. Of these, we invited 80,272 native Finnish or Swedish speakers by letter to participate during two school years (2007−2008 and 2008−2009). Six thousand immigrants born in the same period who were non‐native Finnish or Swedish speakers were also eligible to participate, but were not invited by letter.We randomized the 33 communities in a 1:1:1 ratio into three study arms: Arm A: 90% of randomly selected girls and boys received the AS04‐HPV‐16/18 vaccine and 10% of girls and boys received hepatitis B vaccine (HBV); Arm B: 90% of randomly selected girls received the AS04‐HPV‐16/18 vaccine, 10% of girls received HBV, and all boys received HBV; Arm C: all girls and boys received HBV Figure . RandomiThe study was open‐label for investigators. Participants knew which study arm their community was assigned to, but girls and boys in Arm A and girls in Arm B did not know whether they received AS04‐HPV‐16/18 or HBV. The study was open‐label for boys in Arm B and all participants in Arm C.2.2.2The trial was divided into two phases: the immunization phase during which participants between 12 and 15 years of age (Visits 1−4), between Q4/2007 and Q1/2010, were vaccinated at all municipal junior high schools; and the effectiveness evaluation phase during which the impact of vaccination was evaluated in girls when they reached 18.5 years of age (Visit 5), between Q3/2010 and Q4/2014  and the HBV (Engerix B) were manufactured and supplied by GSK. AS04 is a GSK proprietary Adjuvant System containing 3‐O‐desacyl‐4′‐monophosphoryl lipid A  adsorbed on aluminum salt (500 µg Al3+). Blood samples were drawn from participants included in the immunogenicity subset at pre‐vaccination, at Month 7, and when participants reached 18.5 years of age (Visit 5). The immunogenicity subset was a convenience sample of boys and girls from pre‐selected communities in Arm A.During the immunization phase, school nurses administered the study vaccines intramuscularly in a three‐dose schedule . Both the AS04‐HPV‐16/18 vaccine  assay and multiplex type‐specific (MPTS) PCR Luminex assay were used to test cervical samples for DNA of 14 oncogenic types  and 11 non‐oncogenic types , as previously described.Antibody responses against HPV‐16 and HPV‐18 were determined by enzyme‐linked immunosorbent assay (ELISA) in the immunogenicity subset. Seropositivity was defined as an antibody titer greater than the assay cut‐off value. To increase precision, at Visit 5, the assay cut‐off value was changed from 8 ELISA Units (EL.U)/ml to 19 EL.U/ml for HPV‐16 and from 7 EL.U/ml to 18 EL.U/ml for HPV‐18.2.3Several vaccine effectiveness measurements against the prevalence of HPV‐16/18 cervical infection were estimated in girls at 18.5 years of age  were to evaluate: (1) overall vaccine effectiveness against HPV‐16/18 cervical infection regardless of vaccination strategy (pooled Arms A and B vs Arm C); (2) total vaccine effectiveness against HPV‐16/18 cervical infection following vaccination of girls and boys, girls only, and regardless of vaccination strategy ; (3) overall vaccine effectiveness against cervical infection with individual vaccine and non‐vaccine HPV types. Evaluation of immunogenicity of the AS04‐HPV‐16/18 vaccine was also a secondary objective.2.42.4.1We expected to enroll on average 650 participants per community per year. Approximately 11 communities were required in each study arm to allow statistically powered evaluation of the two overall effectiveness confirmatory objectives  and the total effectiveness confirmatory objective (at least 80% power) under the assumptions of a two‐sided alpha of 0.05, vaccine coverage of 70%, HPV prevalence of 5.2%, 6.7%, and 12.6% in Arms A, B, and C, respectively, 15% coefficient of variation, and loss to follow‐up of 15%.2.4.2The invited cohort comprised all individuals born in the 33 communities between 1992 and 1995, and invited to participate in the trial. Effectiveness was evaluated from data available in the enrolled cohort, which comprised all study participants who enrolled, including those whose participation was limited to completion of a behavioral questionnaire. Only participants with measured endpoints were included for analysis of that endpoint. Immunogenicity was evaluated in the according‐to‐protocol cohort for immunogenicity, which comprised participants in the immunogenicity subset who met all eligibility criteria, complied with all procedures defined in the protocol , and had results available for antibodies against either HPV‐16 or HPV‐18.2.4.3Vaccine effectiveness was computed as one minus the odds ratio of the prevalence of infection between the investigational arm(s) and the control arm. The analysis of overall effectiveness aimed to infer effectiveness in the invited cohort. Because the distribution of vaccination status (HPV/hepatitis B/no vaccination) in the invited cohort was different from the distribution in the enrolled cohort, the analysis of overall effectiveness used a weighted average of prevalence from unvaccinated and vaccinated female participants for the estimation of prevalence in the invited cohort in each arm. The weight was one for vaccinated female participants while, for unvaccinated female participants, it was the ratio of the percentage of evaluable participants  among those receiving AS04‐HPV‐16/18 or HBV over the percentage of female individuals invited but not vaccinated from pooled Arms A, B, and C. No weighted average was used for the analysis of total or indirect effectiveness since these analyses were not based on a mix of AS04‐HPV‐16/18 vaccinated or unvaccinated participants.P‐value for the null hypothesis of no effectiveness were computed using the general inverse variance approach. The primary analysis of indirect effectiveness was done by logistic regression. The model included arm, birth cohort, and the interaction birth cohort × arm as fixed effects and community as random effect. Because it was expected that indirect effectiveness in Arm A would increase in each subsequent birth cohort as the size of the vaccinated male population increased, the demonstration of indirect effectiveness was based on a statistically significant effect in the 1995 birth cohort.The primary analysis of overall and total effectiveness was done using the Mantel Haenszel test adjusted for clustering and stratified by the historical seroprevalence used in the randomization. The 95% confidence interval (CI) on effectiveness and the two‐sided P‐value based on the Mantel‐Haenszel test was <5%. The subsequent objectives were met if all previous objectives in the hierarchy had been reached and the associated two‐sided P‐value was <5%.We used a hierarchical procedure to evaluate the multiple confirmatory objectives; objectives were evaluated in the order shown earlier. The first objective was met if the two‐sided For the confirmatory objectives, the primary analysis was complemented by two sensitivity analyses: (1) Pearson chi‐squared test adjusted for clustering, without stratification; (2) multivariable logistic regression including community as random effect. The multivariable analysis was adjusted for the following covariates at the community level: seroprevalence used in the randomization, urban vs semi‐urban location, percentage of smokers at 15 years of age ; and for the following covariates at the participant level: birth cohort  and quarter of birth (Q1–Q2 vs Q3–Q4). We also did a post‐hoc analysis for the co‐primary objectives using a Mantel Haenszel test stratified by area type (urban vs semi‐urban) instead of by historical seroprevalence.2.4.4Seropositivity rates with exact 95% CIs and geometric mean titers (GMTs) with 95% CIs were calculated for HPV‐16 and HPV‐18. GMTs were calculated by taking the anti‐log of the mean of the log titer transformations. Antibody levels below the assay cut‐off value were assigned an arbitrary value of half the cut‐off for the purpose of the calculation of GMT.3In total, of the 80,272 adolescents invited, 34,412 were enrolled (enrolled cohort), including 22,444 girls and 11,968 boysP = 0.232) following vaccination of girls and boys  and 49.6%  following vaccination of girls only  (Table P = 0.064). As the first confirmatory objective was not met, in accordance with the hierarchical analysis, none of the other confirmatory objectives was considered to be met.The prevalence of HPV‐16/18 in cervical samples from the AS04‐HPV‐16/18 vaccinated girls was 0.6% and 0.9% in Arms A and B, respectively Table . In the P = 0.050) following vaccination of girls and boys, and 45.4%  following vaccination of girls only . We estimated total vaccine effectiveness of up to 93.8% following vaccination of girls and boys, girls only, and regardless of vaccination strategy   was 37.4%  Figure . In addi) Figure .Immunogenicity of the AS04‐HPV‐16/18 vaccine was similar in girls and boys Figure . All ini4The study was designed to evaluate population‐based effectiveness with different HPV vaccination strategies. The first confirmatory objective of overall vaccine effectiveness against cervical HPV‐16/18 infection in young women following vaccination of girls and boys was not demonstrated with statistically significance . Thus, none of the other confirmatory objectives could be considered to be met. However, we found evidence of overall vaccine effectiveness against cervical HPV‐16/18 infection following vaccination of girls only . This was a counter‐intuitive finding, as there seems no plausible reason why including boys in the vaccination program would result in poorer vaccine effectiveness. Indeed, we had expected that overall effectiveness would have been higher in the per protocol analysis with a gender‐neutral vaccination strategy.A possible explanation for the unexpected result was a failure of the randomization algorithm accounting for historical seroprevalence to allocate comparable communities to each study arm. Indeed, we observed almost 50% higher prevalence of HPV‐16/18 among unvaccinated young women in the study arm using gender‐neutral vaccination than in the girls‐only arm or the control arm. This does suggest a randomization bias that was detrimental to outcomes following gender‐neutral vaccination. We noted that area type (urban vs semi‐urban) appeared to be a better prognostic factor for HPV‐16/18 infection rates than the historical HPV‐16/18 seroprevalence. We therefore did a post‐hoc analysis using area type as a stratification factor in the analysis instead of historical seroprevalence. This analysis narrowed the gap between the overall vaccine effectiveness in the gender‐neutral arm vs the girls‐only arm, and CIs of the two effectiveness estimates overlapped considerably , respectively.In addition, there might have been other assumptions that were incorrect and beyond our control, e.g., there might have been more interaction between the study population and other birth cohorts than was anticipated, in which case we would need to have vaccinated a broader cohort (across more birth years) than we did to see an effect between the arms. Since the study was open at the community level (participants knew which study arm their community was randomized to), there was a concern as to whether vaccine effectiveness could be affected by changes in sexual behaviors between different arms. However, there was no evidence of this in the data from the two behavioral questionnaires collected during the course of the study. Other studies have also reported that HPV vaccination does not result in increased sexual activity or risk‐taking sexual behavior among young men and women.Chlamydia trachomatis prevalence,C. trachomatis prevalence  also helps in rectifying the randomization bias. These and the outlier‐free  analyses revealed a significantly stronger herd effect against HPV types 18/31/33 with the gender‐neutral vaccination strategy than with the girls‐only strategy.The apparent lack of herd effect or benefit in vaccinating boys in the per protocol analysis was unexpected, given the wealth of evidence from real‐life studies and models demonstrating herd effect benefits of HPV vaccination.We did identify substantial total vaccine effectiveness against HPV‐16/18 regardless of the vaccination strategy used. These findings are in line with those of clinical trials of the AS04‐HPV‐16/18 vaccine, which have shown similar levels of vaccine efficacy against HPV‐16/18 infection in young women, as well as protection against pre‐cancer endpoints.At 18.5 years of age (3.5−6.5 years after first vaccination), anti‐HPV‐16 and anti‐HPV‐18 antibody titers remained above the level of antibodies observed following clearance of a natural infection in the PATRICIA study .Strengths of the study have been described previously,To our knowledge, this is the only community randomized trial to evaluate different HPV vaccination strategies. Several countries have already implemented vaccination of boys based on modeling of indirect benefit to girls and potential direct benefit to boys for anal, penile, and oropharyngeal cancer. This study contributes a substantial amount of safety and immunogenicity data in boys vaccinated with the AS04‐HPV‐16/18 vaccine. However, even with this large sample size and the unique setting of Finland, with suitable infrastructure in place, we were unable to demonstrate overall effectiveness in the per protocol analysis. This illustrates the limitations of a controlled, community‐randomized trial for demonstrating indirect effectiveness. Such limitations were comprehensively explored and addressed using birth cohort and outlier‐free approaches in the ancillary study analyses.In conclusion, conduct and interpretation of community randomized trial outcomes is not easy, and in this study was most likely confounded by factors such as different levels of baseline HPV prevalence between study arms despite randomization and the impact of urban vs semi‐urban residence. Thus, a direct comparison between the arms in this study did not reliably illustrate the potential of different vaccination strategies. Although the primary objective of the study was not met in the per protocol analysis, additional work done through the ancillary study and the independent community‐wise pre‐ and post‐vaccination HPV seroprevalence analyses has shed further light on the puzzling per protocol study results. The present study also confirms the high total effectiveness of the vaccine as indicated in clinical trials as well as its cross‐protective benefit, and adds important safety and immunogenicity data in boys and girls.The authors have no conflicts of interest to disclose.Supplementary MaterialClick here for additional data file.


Human papillomavirus (HPV) E6/E7 mRNA in situ hybridization (HPV E6/E7 RNAscope) appears to be a sensitive and specific technique in detecting transcriptionally active HPV. We aimed to examine the diagnostic utility of this technique in endocervical adenocarcinoma (ECA), to explore the prognostic factors for ECA patients and develop a clinically useful nomogram based on clinicopathological parameters to predict it.We retrospectively analyzed 200 patients with ECA who had undergone surgery at Sun Yat-sen University Cancer Center from 2010 and 2014. The diagnostic performance of HPV E6/E7 RNAscope were evaluated by receiver operating characteristic (ROC) curve. A prognostic nomogram model including HPV E6/E7 RNAscope was generated based on multivariate Cox regression analysis, then we compared the predictive accuracy of the prognostic model with FIGO staging and treatment using concordance index (C-index), time-dependent ROC (tdROC), and decision curve analysis (DCA).The sensitivity and specificity of HPV E6/E7 RNAscope for distinguishing HPV-associated adenocarcinoma (HPVA) from non-HPV-associated adenocarcinoma (NHPVA) in the whole cohort were 75.8% and 80%, respectively. According the univariate analysis and multivariate logistic regression analysis, age, lymphovascular invasion (LVI), lymph node involvement (LNI), and HPV E6/E7 RNAscope were valuable predictive factors for OS. These parameters were incorporated into the nomogram model (nomogram A) compared with FIGO stage and treatment. The C-index of nomogram A for predicting OS was 0.825, which was significantly higher than FIGO stage  and treatment . HPV E6/E7 RNAscope is highly specific for ECA, and the 4-variable nomogram showed more accurate prognostic outcomes in patients with ECA.The online version contains supplementary material available at 10.1186/s12935-021-02349-1. Endocervical adenocarcinoma (ECA) accounts for 15%–20% of all cervical carcinomas. Studies have shown that ECA is increasingly reported in young women , 4. The A new classification—the International Endocervical Adenocarcinoma Criteria and Classification (IECC) has replaced the 2014 WHO classification and categorizes ECA based on the morphological features linked to the etiology , thus grouping them into HPV-associated (HPVA) and non-HPVA (NHPVA) types . CompariIn the present studies, the following tests were used to detect HPV infection: HPV DNA, genotype, RNAscope, and immunochemistry (IHC) against p16 protein. Plasma HPV DNA levels can be determined using PCR and is a potential marker for cervical cancer . The othNumerous controversies surround the FIGO staging system, wherefore ECAs are morphologically stratified into A, B and C subgroups using the Silva based on their LVI or lymph node involvement (LNI) status . Tumors In the present study, we aim to validate the utility of the novel RNA scope method for detecting HPV RNA in tissue samples for proper initial risk stratification of patients. We also built a nomogram that will compliment FIGO staging for prognostic classification of EAC. We hope the link between these two modalities will bring new insights into the clinical care of EAC patients.The present retrospective study included 200 patients with pathologically diagnosed ECA who had been treated at the Sun Yat-sen University Cancer Center from January 2010 to December 2014. We enrolled patients with confirmed pathological diagnoses of ECA with available complete clinical data and test results. We excluded patients with systemic metastasis or with synchronous primaries and any patient with a prior cancer history. The last follow-up period was June 2020. The histological diagnoses were based on our proposal for a new ECA classification: the IECC 2017 , and patA tissue microarray consisting of the ECA and adjacent non-tumorous tissue was constructed using a tissue array instrument . For each patient, hematoxylin and eosin (H&E) stained slides were examined, and at least two areas from different regions were marked for sampling. A 1.0 mm diameter tissue core was punched from the marked fields and re-embedded. FFPE ECA sections were dewaxed in xylene and graded alcohols, hydrated, and washed in PBS. After pretreatment in a microwave oven, endogenous peroxidase was blocked using 3% hydrogen peroxide in methanol for 20 min. This was followed by avidin–biotin blocking using a kit . The slides were then incubated with antibodies for p16 , MLH1 , PMS2 , MSH2 , MSH6 , and Ki-67  overnight at 4 °C, washed in PBS, and incubated with biotinylated goat anti-rabbit/mouse antibodies . The slides were developed using diaminobenzidine (DAB) and counterstained with hematoxylin. Two independent, experienced pathologists evaluated the staining; and p16 was interpreted as positive if diffuse, block-like staining was found in all cores. No staining or patchy staining was interpreted as unfavorable. MSH2/MSH6/PMS2/MLH1 was interpreted as positive if ≥ 1% of the tumor cell nuclei were positive. Representative images were show in Additional file DNA load of High-risk-HPV (HR-HPV) in plasma sample was evaluated by Digene second generation hybrid capture (HC2) DNA test and in vitro acid hybridization assay with signal amplification using microplate chemiluminescence for HR-HPV DNA detection. A commercial kit detecting high risk was used. The alkaline solution was used to denature the double-stranded DNA, and the liberated single-strand DNA was combined with the RNA probe mix. RNA–DNA hybrids were then transferred to a capture microplate coated with goat polyclonal for immobilization. Subsequently, alkaline phosphatase-conjugated antibodies were added to the RNA–DNA hybrids. After washing, the chemiluminescent substrate was added for signal amplification. Results were analyzed by DIGENE DML 2000 Software. DNA load was expressed by the unit of Relative Light Units/Cutoff Value (RLU/CO), representing the ratio of the light emission of the sample to the average of three positive control samples. HR-HPV DNA load of 1.0 (pg/ml) or more was defined as HR-HPV DNA positive.Tissues were scraped by coverslip as per the labeled region on the H&E slide and transferred into a clean 1.5 ml centrifuge tube. DNA was extracted using a QIAGEN DNA FFPE Kit  according to the manufacturer's instructions and was quantified with a Nano-Drop2000 . HPV was detected by the Human papillomavirus Genotyping Test Kit  using the BioRad CFX96 real-time quantitative PCR instrument. The Roche Cobas 4800 system  was used to detect HPV and evaluate the presence of the following 14 types of HPV DNA: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.The stained slides of each specimen were examined using the RNAscope scoring system, as described in previous studies , 18. HigAmong the patients enrolled, the following variables were recorded: age, the modality of treatment (Surgery with or without chemotherapy/radiotherapy), HPV DNA, HPV subtype, HPV E6/E7 RNAscope, histological type, tumor size, differentiation, LVI, invasion level of the uterine cervix, LNI, parametrium invasion, surgical margin, MMR status, p16, Ki-67, and FIGO stage.http://www.R-project.org/). Continuous data were expressed as means ± standard deviations and were analyzed using Student’s t-test or Mann–Whitney U test, as appropriate based on the results of the Kolmogorov–Smirnov test. Categorical variables were presented using numbers (percentages) and were analyzed with Fisher’s exact test. Survival analysis was conducted utilizing the Kaplan–Meier curves. Univariate and multivariate Cox proportional hazard regression were used to explore the risk factor for OS. The best cutoff values were determined using X-tile [Statistical investigations were conducted using SPSS 19.0  and R 3.4.0 , HPV E6/E7 RNAscope (p = 0.006), tumor size (p = 0.005), FIGO stage (p < 0.001), histological type (p = 0.004), LVI (p < 0.001), invasion level of uterine cervix (p < 0.001), LNI (p < 0.001), parametrium invasion (p < 0.001), surgical margin (p = 0.006), chemotherapy (p = 0.009), radiotherapy (p < 0.001), p16 p = 0.039), and Ki-67 (p = 0.025). In a multivariate analysis, the following items remained independently prognostic: age , HPV E6/E7 RNAscope , LVI , and LNI . The results are shown in Table 9, and Ki, HPV E6/Two different nomograms that predict the OS of ECA patients were built and compared for efficiency. Nomogram A included age, HPV E6/E7 RNAscope, LVI, and LNI, while nomogram B included age, HPV E6/E7 RNAscope, LVI, LNI, FIGO stage, and treatment. The results of time-dependent ROC curve for OS showed that AUCs of nomogram A and FIGO stage were higher than treatment Fig. A. The reAll patients were divided into a low ≤ 15.75) and a high-risk group (> 15.75) for OS  the validity of the HPVA and NHPVA categories were supported by p16 immunophenotyping and HPV status; (2) HPV E6/E7 RNAscope is more sensitive and specific than p16 and HPV genotype in identifying HPVA; (3) we established a prognostic nomogram model based on ECA pathogenesis that adjusted for age, LVI, HPV E6/E7 RNAscope, and LNI, which had a greater predictive efficiency than the current conventional staging system; (4) in the future, we believe, based on our model, ECA patients could be divided into low and high-risk groups.HPV E6/E7 RNAscope measurement is not routinely available in most hospitals; hence the methodology cannot be extrapolated to standard practice. About 94% of adenocarcinomas are associated with HPV, particularly high risk strains like 16 and 18 , which iClinicopathological variables carry a prognostic significance and usually affect clinical management , 28. DepThe present study had several limitations. Firstly, there may have been selection bias in our patient cohort. Secondly, we lacked data on the impact of our nomogram on other prognostic endpoints, such as disease-free survival (DFS) prediction. Thirdly, in situ measurement of HPV RNA using RNAscope is not easily available and not globally standardized. Fourthly, our sample size was relatively small and from only one center. In order to validate our results, a study with a larger sample with multicenter data is required. Lastly, it remains unclear whether our nomogram can be applied to ECA patients with advanced-stage that is, stage III and IV.We generated new nomograms that incorporate HPV E6/E7 mRNA in situ hybridization and patients’ clinical characteristics in order to prognosticate the OS rate in ECA patients. Our simple and explicit nomograms have a good clinical application value, with a good diagnostic discernment and calibration ability. They may be a useful tool for assessing the prognosis and the management of ECA patients.Additional file 1: Figure S1. Representative images for clinicopathological features in paraffin-embedded endocervical adenocarcinoma (ECA) samples. Figure S2. The best cutoff values for all variables as determined by using X-tile in all cases of endocervical adenocarcinoma (ECA). Figure S3. Endocervical adenocarcinoma (ECA): proportions of combinations of positive human papillomavirus (HPV) tests and p16 immunohistochemistry (IHC). Figure S4. The positive rates of human papillomavirus (HPV) subtypes, mRNA, protein, and p16 protein in the subgroups of endocervical adenocarcinoma (ECA) cases.Additional file 2: Table S1. The positive proportion of factors examined in ECA samples. Table S2. Correlation of P16 IHC, HPV DNA, HPV genotype and HPV RNAscope. Table S3. HPV genotypes in different ECA histological types.