HPVE4 was associated with CIN1/normal (p = 0.06).•The dual biomarker approach may be useful to risk stratify cervical disease especially in resource-poor settings. While human papillomavirus (HPV) DNA testing offers high sensitivity for the detection of significant cervical disease, its specificity is suboptimal given the high prevalence of transient HPV infections (CIN1 or less). Biomarkers to identify those suffering from low grade disease from those with high grade disease could save healthcare costs and reduce patient anxiety.The objective of the present work was to develop and test an immunohistochemistry (IHC)-based dual viral and cellular biomarker strategy which was applicable to liquid based cytology (LBC) samples.We developed a novel IHC assay for detection of HPV E4 and cellular minichromosome maintenance (MCM) proteins in routinely taken cervical LBC samples using cytospin-prepared slides. The assay was applied to a prospective cohort of Scottish women referred to a colposcopy clinic due to preceding cytological abnormalities. The performance of the biomarkers for detection of clinically insignificant (CIN1 or less) versus significant disease was determined.A total of 81 women were recruited representing 64 cases of <=CIN1 and 28 of CIN2 + . Biomarker performance relative to histopathology outcomes showed high levels of MCM detection was significantly associated with CIN2+ (p = 0.03) while E4 was detected more frequently in <=CIN1 (p = 0.06).Combined detection of a host proliferation marker and a marker of viral gene expression could allow triage of cases of clinically insignificant disease prior to colposcopy. However, there was overlap between distributions of MCM levels in CIN2+ and <=CIN1 suggesting that additional biomarkers would be required for improved specificity. Combined with cytospin-prepared slides this approach could provide a means of risk stratification of disease in low resource settings. ClinicaThe infectious HPV life cycle is tightly linked to the differentiation process of the host epithelium . HPV infMost HPV infections are cleared by the immune system. However, if an infection becomes persistent in the epithelium, the host and viral genomes undergo alterations that make progression to preneoplastic and neoplastic disease more likely. Key among these changes due to persistence is increased expression of HPV E6 and E7 proteins leading to increased levels of cell cycle proteins such as p16, Ki67 and MCM. ,6,7. The2Biomarker-based risk-stratification of HPV infection is a key priority for development given the increasing move to cervical screening programmes based on primary HPV testing. A single biomarker may be less optimal for disease stratification compared to a biomarker combination/matrix. A matrix which incorporates both a viral and a cellular target may confer sensitivity and specificity respectively. Here, we describe an initial investigation into the performance of a dual biomarker based on IHC-based detection of E4 and MCM in routinely taken liquid based cytology (LBC) to determine/stratify underlying disease.33.1LBC samples were collected with informed consent from a cohort of 81 women referred to a colposcopy clinic in the west of Scotland. LBC samples were allocated a study number in the clinic unrelated to any patient identifiers, stored at 4 °C and transferred to the research laboratory.3.2Disease was defined by histopathology results: with significant/high grade defined as histology-confirmed CIN2+, insignificant/no disease defined as histology confirmed <=CIN1 or no clinically indicated biopsy due to no abnormality detected on colposcopy.3.3HPV genotyping was with the Optiplex HPV Genotyping Assay  according to manufacturer’s instructions. This PCR based test with luminex technology can resolve 24 HPV types  including all HR-HPV types as defined by the International Agency on Cancer.3.42 humidified incubator. Transfection with E4 expression plasmid pMV11 was with Lipofectamine 2000 according to the manufacturer’s instructions.HeLa cells were grown in Dulbecco’s modified eagles medium (Invitrogen), 10% foetal calf serum (Invitrogen) and penicillin (50 U/ml)/ streptomycin (50 μg/ml) (Invitrogen), at 37 °C in a 5% CO3.5LBC samples (100 μl) in PreservCyt Solution (Hologic) were deposited on Klinipath plus slides using a Shandon 3 Cytospin . Slides were fixed for 10 min in 10% buffered formalin and washed twice in buffer TBS-T  Tween-20). Thinprep slides were prepared using the ThinPrep T2000 (Hologic) using 20 ml LBC samples in PreservCyt Solution (Hologic). Slides were briefly placed in 95% (v/v) ethanol and treated with Cytofix (Cellpath), immersed in 50% (v/v) ethanol for 10 min and fixed for 10 min in 10% buffered formalin, followed by washing in TBS-T.3.6Heat-induced epitope retrieval was carried out in 10 mM sodium citrate buffer, pH 6.0 using a Menarini Access Retrieval Unit, at 110⁰C on full pressure for 10 min. Slides were loaded on to a Dako Autostainer, rinsed in TBS-T, blocked in Dako Real TM peroxidise-blocking solution for 5 min followed by washing in TBS-T. Slides were incubated for 30 min in primary antibodies diluted in Dako universal diluent ) and3.7At least 3 photographs of stained cells were taken across each slide. Images were input into Photoshop. One individual determined total cell numbers in each field of view by counting haematoxylin-stained nuclei, then MCM or E4 positive cells were counted by two different individuals independently blind to the clinical data. Averages of the two counts were taken for further analysis. Most samples contained intact cells and some cell fragments. Cytobrush acquisition of samples can damage cells. We counted staining of only intact cells because E4 is a cytoplasmic stain while MCM is a nuclear stain and we wished to be able to compare levels of E4 and MCM-stained cells in the sample cohort. Some LBC samples contained clumps of material that non-specifically trapped antibody. Therefore, samples were filtered using a cell strainer size 70 μm (Corning CLS431751 SIGM).3.8Thinprep slides were stained using the Roche CINtec®Plus system on the Roche Ultra immunohistochemistry platform (Roche MTM Laboratories) from residual LBC material. CINtec PLUS interpretation was blind to other results. Samples with one or more cervical epithelial cells that simultaneously showed brown cytoplasmic immunostaining (p16) and red nuclear immunostaining (Ki-67) were classified as positive regardless of the morphologic appearance of the cells. Slides with excessive background staining were considered not evaluable and excluded from analysis.3.9Basic descriptive statistics stratified by grade of disease were produced and the distribution of both MCM and E4 values were visualised in boxplots. Differences in E4 and MCM levels by disease grade were evaluated using logistic regression were the outcome variable was high-grade vs low-grade/normal disease. Finally MCM and E4 were considered together by examining the correlation between the measures and examining if this differed by disease group. Both MCM and E4 were considered together in a multivariable logistic regression model. This approach was used to access the association between biomarker and disease grade because the numbers in each disease grade were quite low for splitting into training and test data sets for building and testing a prediction model.4The aim of the study was to determine the performance of E4, a biomarker of transient HPV infection (≤CIN1), in combination with MCM2, a biomarker of significant cervical disease in a cohort of patients who had an abnormal Pap smear test result, were called to attend a colposcopy clinic, and tested positive for HPV.4.1To test specificity of the antibodies for cellular MCM and viral E4 proteins, HeLa cells, and HeLa cells transfected with an expression plasmid for HPV16 E4 were suspended in PreservCyt, stained with E4 and MCM antibodies and visualised by immunofluorescence microscopy. 4.2We compared cytospin centrifugation to the ThinPrep method (T2000) to determine the best slide preparation for IHC analysis using an initial cohort of 20 LBC samples. The same antigen retrieval protocol was used for each slide type. Cytospin performed better than Thinprep because adherence of LBC cells on the ThinPrep-prepared slides was poor following the antigen retrieval protocol required for efficient immunostaining with the selected biomarkers. For cytospin-prepared slides, only 100 μl of each LBC sample was required thus ensuring that multiple biomarkers could be tested on a single sample. Finally, cytospin deposits two cell populations on a single slide meaning that an antibody-negative control could be included on each slide A. Cells 4.381 patients referred to a colposcopy clinic in the West of Scotland as a consequence of preceding cytological abnormalities were consented to the study and an LBC sample was taken in the clinic from each patient for the study. The average age of the cohort was 31 years (range 20–60 years). HPV was detected in 78/81 (96.3%) samples with HR-HPV detected in 66/81 (81.5%) samples (Supplementary Table 1). One third of all study participants were infected with HR-HPV16 and/or 18 . Following histopathological examination, there were 18 cases of CIN2+, 40 cases of CIN1 and 22 were designated “normal” or “nil” or “no biopsy”. All CIN2+, 38/40 CIN1 and all “normal” cases were positive for HR-HPV. Only HR-HPV positive samples were included in the analysis.4.4Of the 81 clinical samples, 73 and 71 respectively had sufficient intact cells to allow MCM and E4 antibody staining. All 73 samples examined were positive for MCM (90.1%). Of the 71 samples that had sufficient cell numbers, 45 (63.4%) were positive for E4. The percentage of cells within a sample positive for MCM ranged from 3 to 86.7% with an average of 27.5%. MCM will normally be expressed in any cell undergoing cell cycle, e.g. in epithelial stem cells or in cells during wound closure. Therefore, there may always be some MCM expression in cervical tissue so we decided not to count as positive any sample with any MCM detection. An arbitrary limit of >20% of cells stained within a cell population was chosen as “high” (or positive) MCM because this was just below the average value and on the upward shoulder of a normal distribution plot of MCM values  or high E4/lowMCM (12.7%) staining. Of the remainder, 7.3% had high E4/high MCM and 21.8 had low E4/low MCM . MCM pos4.6The ability of MCM detection in cytospin-prepared slides to identify significant disease was compared to the detection of p16/Ki67 using CINtec. Of the 73 MCM-positive samples, 12 were excluded from analysis due to either insufficient cells or unsatisfactory results. Sensitivity was 1.00 while specificity was 0.404. Data from the remaining 61 LBCs showed an accuracy of 0.639 comparing cytospin MCM detection with CINTec positivity. MCM detection showed a sensitivity of 0.651 and a specificity of 0.611 at detecting high grade disease.5Since its introduction, cervical screening via cytology has reduced incidence of cervical cancer . Howeverp16/Ki67 are robust biomarkers of high grade disease and have been assessed as a triage of primary HPV positivity in primary screening . Like p1LBCs are a suitable biospecimen for high-throughput contexts. However, cell numbers in individual samples can vary widely and the sample can contain HPV-infected and uninfected cells. Moreover, the cells in the sample can represent a range of disease stages if these are present across an individual cervix. Consequently, percentage positivity for either biomarker assessed in the present study likely gives only an estimate of their levels in-situ. Our choice of cut-off values for MCM and E4 could be inappropriate given the small number of samples tested. It will be useful in a future study with greater numbers of samples to compare data with and without cut-off values. Despite these caveats, as predicted by the previous tissue-based study, high MCM detection was associated with CIN2+ and E4 association with <=CIN1 was close to significance. E4 expression levels can be very variable in CIN1 lesions  and, as in situ HPV DNA staining could indicate how many LBC cells in a sample are infected so MCM or E4 positivity could be calculated on HPV-positive cells only. Excellent antibodies against MCM are commercially available. The pan-E4 antibody is available from DDL Diagnostics Laboratory, The Netherlands [In the developed world, a matrix test could be used as an adjunct to HPV DNA testing to determine HPV activity in a lesion and predict the probability of disease progression in primary screening, low-grade triage or test of cure contexts. Approximately 70% of women who are HR-HPV positive at primary screen are cytology negative and this group represents a management challenge. A biomarker approach  which could mitigate against the subjectivity of cytological observation and allow separation of low-grade disease from significant CIN would prove useful. In future, chromogenic herlands . It woulherlands , which rherlands .6In this “proof of principle” study we have shown that MCM and E4 are a promising biomarker matrix for the separation of disease grade in routinely taken cervical samples. MCM can identify CIN2+ when used in the cytospin technique. Although E4 was detected in LBC cells, its usefulness as a biomarker of clinically insignificant disease requires further investigation in a larger LBC cohort. The cytospin approach could prove useful in low and middle income settings lacking infrastructure for standardised cytology and high-throughput HPV testing.SVG designed and directed the study and wrote the paper. AS, KC, ES and MD acquired the data. SVG, AS, KK and JP analysed the data. JD and HG helped conceive the approach. KC, JD and MD helped draft the article. All authors approved the final submitted version of the manuscript.Medical Research Council Confidence in Concept MC_PC_13063 award.This work was a sub-project awarded to SVG funded by a Southeast Scotland Research Ethics Committee.REC reference 12/SS/0108 was granted by the SVG, AS, MD, ES, JP, KK, LS, JD and HG declare no conflict of interest. KC’s institution has received grant monies and/or consumables from the following companies in the last three years: Hologic, Cepheid, Qiagen, Euroimmun, LifeRiver, Genefirst and SelfScreen.


In the stroma nearby the tumor mass we were able to demonstrate the presence of virus DNA in the stromal compartment, supporting its potential to be transferred from epithelial cells to the stromal ones. Thus, this case report favors the notion that human papillomavirus DNA can be vehiculated by exosomes in the blood of neoplastic patients and that it can be transferred, at least in vitro, to normal and neoplastic cells. Furthermore, we showed the presence of viral DNA and RNA in pluripotent stem cells of non-tumor tissue, suggesting that after viral integration (as demonstrated by p16 and RNA in situ hybridization positivity), stem cells might have been activated into cancer stem cells inducing neoplastic transformation of normal tissue through the inactivation of p53, p21, and Rb. It is conceivable that the virus has elicited its oncogenic effect in this specific site and not elsewhere, despite its wide anatomical distribution in the patient, for a local condition of immune suppression, as demonstrated by the increase of T-regulatory (CD4/CD25/FOXP3 positive) and T-exhausted (CD8/PD-1positive) lymphocytes and the M2 polarization (high CD163/CD68 ratio) of macrophages in the neoplastic microenvironment. It is noteworthy that our findings depicted a static picture of a long-lasting dynamic process that might evolve in the development of tumors in other anatomical sites.Here we present the case of a patient affected by rectal squamous cell carcinoma in which we demonstrated the presence of Human Papillomavirus (HPV) by a variety of techniques. Collectively, the virus was detected not only in the tumor but also in some regional lymph nodes and in non-neoplastic mucosa of the upper tract of large bowel. By contrast, it was not identifiable in its common sites of entry, namely oral and ano-genital region. We also found HPV DNA in the plasma-derived exosome. Next, by  Human papillomavirus (HPV) DNA has been detected in a variety of cancer, including colorectal cancer (CRC) –4. ThrouWhile there is a strong association between HPV and squamous cell carcinoma of some sites , the putative role of the virus in colorectal carcinogenesis still remains controversial . Detectiper 1,000 CRC; its diagnosis is based on the restrictive criteria given by Williams et al. , and RNA in situ hybridization (ISH) for E6/E7 viral oncogenes mRNA in neoplastic and non-neoplastic tissues, including lymph nodes and sites far from the rectum. We also found HPV DNA in the plasma-derived from the patient, and by in vitro studies, we confirmed the capability of HPV DNA positive exosomes to transfer its DNA to recipient cells.Here we present the case of a patient affected by rectal SCC in which we demonstrated HPV by immunohistochemistry, polymerase chain reaction (PCR), chromogenic A 53 years-old caucasian man presented to our hospital complaining of sub-occlusive syndrome that lasted for 1 week. Family and medical history was unremarkable. Physical examination including digital rectal examination was negative. Colonoscopy showed a 5.0 × 4.5 × 4 cm lesion, 10 cm from the anal verge. The lesion was ulcerated and bleeding, it narrows the lumen and renders difficult to be overcome. Computed tomography (CT)-scan identified a heteroplastic and locally advanced lesion with enlarged lymph nodes (the greatest measuring 13 mm in maximum diameter) along the sigmoid chain. Neither pulmonary, hepatic, splenic localizations nor pleural or peritoneal effusions were identified. An anterior resection of the rectum and regional lymph nodes was performed without colostomy. Postoperatively, the patient underwent adjuvant radiotherapy of 45 Gy delivered to the pelvis in 25 fractions over 5 weeks. To date, after 60 months follow-up, the patient is alive and in good conditions, with no evidence of recurrence. Ethics approval for this study was obtained from the Institutional Review Board at the University of Siena  and written informed consent was obtained from the patient for the publication of this case report and any potentially-identifying information/images.Grossly, an exophytic ulcerative mass with predominantly intraluminal growth and narrowing of the lumen was identified. The lesion measured 5.0 × 4.0 × 3.5 cm and extended throughout the muscular layer but without mesorectal involvement in the extraperitoneal tract of the specimen. On cut section, the mass had a relatively homogeneous appearance with focal areas of necrosis and hemorrhages. Microscopic examination demonstrated a moderately differentiated squamous cell carcinoma with basaloid features and koilocyte-like cells , inset. E6/E7 mRNA transcripts   and RNA ISH for , Italy) .By IHC for p16, a useful surrogate biomarker of HPV integration and E7 over-expression  and in spunctated” pattern with distinct multiple dot-like intranuclear signals by CISH  as well as at distant sites (ascending/descending colon). It is reasonable that HPV penetrated in the host by one of the known site of entry. Following, the virus disappeared from the original site thanks to clearing mechanisms (that are highly active during the early phase of HPV infection) and it was no more detectable there (Previous studies on cervical, oropharingeal, and breast diseases demonstrated the presence of circulating HPV DNA in the blood of affected patients , 32–35. le there –16. It ile there  charactele there –19, 37 ale there . Notablyle there , 40. It le there , non-canle there –44. Due Although important studies have been made for the understanding of HPV tumorigenicity in non-genital cancers, significant questions still remain. Our case-study seems to highlight the direct relationship of HPV with the pathogenesis of a squamous cell CRC; however, further studies including case control and cohort studies are necessary to evidence the causal link between HPV infection and colon cancer. The presence of HPV DNA positive plasma-derived exosomes in our patient shed important light on poorly understood mechanisms of HPV DNA horizontal transfer and on unconventional route of HPV spreading and carcinogenesis. It is noteworthy that HPV-induced cancerogenesis has a long-lasting history and our findings depicted only a static picture of a dynamic process for which we cannot exclude the development of cancers in other anatomic sites of the patient after viral spreading and DNA integration.MRA, MC, and SL conceived and developed the study. LM, SDC, AC, AG, and BJR performed experiments. MC, MRA, SL, and SDC analyzed data. RV, ALF, TC, and VN collected and provided patient samples. MC, SL, MRA, and PT wrote the paper. PT provided his experience in fruitful discussion. All the authors discussed the results and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Long-term results of the HPV vaccination programs in Australia and Scotland have shown a tremendous impact on the reduction of HPV infection rates and precancerous diseases. Both countries started mass vaccination ten years  and eight years (Scotland) ago and achieved a vaccination coverage of more than 80 %. Within 20 to 30 years a reduction in cervical cancer by more than 75 % is expected. Furthermore, there will be a reduction in other HPV related cancers like vaginal, vulva, perineal, anal and oropharyngeal cancers. In order to be successful, a high vaccination coverage is needed. In Belgium, the vaccination was introduced in 2010 in the Flemish community and in 2011 in the French community. In the first vaccinated cohorts the coverage in Flemish and French Communities was respectively 84% (2010) and 29% (2012-2013).The latest data suggest that the Flemish Community (Flanders Region) attained a coverage of 91 % while the French Community  attained a coverage of around 36 %. The regional difference in coverage offers a real-life case. The worst-case scenario could end up with proportionally one half of country having more HPV related cancers than the other half. Currently efforts are performed to increase the coverage rates in both regions and consequently decreasing this difference.Additionally, the updated recommendations regarding the HPV vaccination by the Belgian NITAG  stated that the HPV vaccination should be gender neutral. This could stimulate the vaccination program and increase the coverage. The coverage rate in Flanders is among the highest in the world and the rate in the French Community is increasing. Efforts should be continued in order to maintain trust and increase the coverage rate. The current universal HPV (human papillomavirus) vaccination programs in Australia and Scotland have reduced HPV 16 and HPV 18 infections by 89 % and high-grade lesions by 85% seven to ten years after the start . At the Within one country this offers the opportunity of an experiment of nature regarding the impact of HPV vaccination on cervical cancer rates and other HPV related diseases. There is a huge difference in HPV vaccination coverage in the industrialized world depending on many factors; in particular the way the program is implemented . A surveThe HPV vaccination program, with the bivalent and quadrivalent have the potential to reduce cervical cancer by more than 75% . Based oThe reduction figures could be jeopardized by suboptimal level of coverage. There are different factors and issues that can explain the successes and failures of vaccination programs in general, and HPV immunization programs in particular: the cost of the vaccine, the political will, the support of professional organization, the attitude of parents, adolescents and health care workers, the media, just to name a few, seem to play a decisive role . The difThe HPV vaccines are contracted through a public tender by each of the communities. It also includes a cold chain monitored transport and a direct delivery to the vaccinators. The vaccines are offered free of charge to the target group of young adolescent girls; thus, the cost is not an issue. The coverage of the universal HPV vaccination program however is a success in one half of the country and remains a challenge in the other half.The HPV vaccines Gardasil® and Cervarix® were introduced in Belgium, respectively in September 2006 and October 2007. They could be bought on prescription in the pharmacies, with different co- payment systems . In BelgDepending on the region in Belgium, however, the coverage rate is a success or remains a challenge. In the first vaccinated cohorts (three doses) the coverage in Flemish (2010) and French (2011) communities was respectively 84% (2010) and 29% (2012-2013). The data of 2016 revealed that in the Flemish region the coverage rate for a completed schedule in 16-year-old girls vaccinated in 2012 (at age of 12-13 years) was 91 % . These FFlanders has a “tradition” of adolescent vaccination mostly through the SHS and additionally through treating physicians, with a clearly defined mandate for school doctors for vaccination within the SHS tasks since 2000. The system of SHS is more or less a continuation of the vaccination program of the Well-Baby’s Clinics in Flanders (Kind en Gezin). A reason for this difference is that in the French Community vaccination against HPV through SHS is less well implemented than in Flanders  .The school-based programs are free of charge and pretty similar in both regions. In case the vaccines are administered by another physician there is a co-payment for the consultations; all vaccinating physicians are informed on HPV vaccination and all vaccinating physicians can order HPV vaccines beforehand, so the vaccines are available “on demand”. In the Flemish region there is a strict approach, population-wide for all girls in the cohort of first year of secondary school. The French SHS sends also an invitation for HPV vaccination (that can be done by their GP or by the SHS if offered) to all girls entering second year of secondary school.In Flanders a lot of attention was paid to transparent communication. All adolescents, in the Flemish community, entering the first class of secondary school receive a leaflet on vaccination together with the link to a website .The websThe accessibility and the organization play an important role in the vaccination coverage. Within the Flemish SHS (CLB) there is a strict organization. The Flemish Scientific Society for Youth Health care (Vlaamse Wetenschappelijke Vereniging voor Jeugdgezondheidszorg (VWVJ)) makes guidelines in order of the Flemish Goverment . These gThe immunization calendars in SHS  , 6th year primary school , 2nd year secondary school (HPV), and 4th year secondary school (dTpa). However, for several reasons notably (but not only) financial reasons, the vaccination offer by SHS is not always complete.Additionally, the role of the media is completely different. In Flanders the local TV and radio stations do not have a negative view on HPV vaccination. The French speaking population in Belgium on the otherhand, is influenced by the French media and it is well known that France is one of the most anti-vaccine countries in the world. Recently this appears to be changing. The trigger for this was the introduction of the mandatory vaccination against 11 sicknesses 2018) in France. Since then multiple articles were published in the media to inform the public in an untestable but scientific way . At the moment efforts are done to convince all professionals in the French community to use this system. For all vaccines ordered in the system it is mandatory to register the given vaccinations. First of all, the vaccinators can access this database to see if someone is vaccinated or not. And secondly the HPV vaccination records in this database will allow in the future the evaluation of the impact of the HPV vaccination on cervical cancer in Belgium, if data- linking between databases is possible. Furthermore, if data from both databases are linked to data from a screening and pathology database one can find out which types are responsible for cervical cancer in the vaccinated cohort, if ever, and compare with the non-vaccinated cohort. Therefore you need a common identifier which exists in Belgium - everybody has a personal national registry number. The lack of data on previous vaccination is one of the reasons why vaccination is postponed or not done. For the SHS this is a real problem as they don’t know if the individual should be vaccinated or not and it takes a lot of time and effort to find the answer  if any. The impact of a vaccination program depends on the coverage rate. Even within a country with the resources for mass vaccination program uptake seems to vary. The success of a vaccination program depends on the support of all stakeholders: the public acceptance, the political will, and the support of the public and healthcare professionals are essential. General barriers are 1) vaccine costs and reimbursement including insurance, (2) storage and/ or monitoring of vaccines, (3) vaccine knowledge, (4) provider and patient attitudes toward vaccination, (5) provider and patient concerns about vaccine safety, (6) obtaining appropriate consent from adolescents’ parents or from themselves and (7) missed opportunities for adolescent immunization (The electronic immunization ordering system linked to a database or register is a major benefit. In Flanders the Well-baby Clinics and SHS work with the electronic immunization ordering system Vaccinnet which is linked to a vaccination database. While in the French Community all the SHS are using an electronic ordering system which is linked to a vaccination register (nization .In Belgium most of these items were addressed successfully yet there was a huge difference in coverage rate between the two regions. Missed opportunities for adolescent immunization is still an issue in the French part of the country mostly due to the fact that not all SHS offer HPV vaccination.As soon as we will link data from disease and immunization database, we will be able to measure the impact of HPV vaccination on cervical cancer rates and other HPV related diseases in both parts of the country. The result difference will reflect the impact of the coverage rate.In the recent scientific advisory report on HPV vaccination, the Superior Health Council of Belgium recommends gender neutral prophylactic HPV vaccination in a two dose schedule for 9 to 14 years and in a three dose schedule for 15 – 26 years old . If thisThe availability of a HPV vaccine in a country is not a guarantee of success, not even when it is offered free of charge. The situation in both communities in Belgium illustrates the role different factors may play in different ways: differences in awareness, spill over effects of the media in neighbouring country, different penetration of vaccination by SHS in each part of the country. Understanding the role of these factors and tailoring the implementation of the HPV programme to the needs of each community is key, in addition to efforts to raise knowledge and awareness among the respective health care providers and parents in each part of the country.


Cervical cancer is the fourth most frequently occurring cancer in women around the world and can affect them during their reproductive years. Since the development of the Papanicolaou (Pap) test, screening has been essential in identifying cervical cancer at a treatable stage. With the identification of the human papillomavirus (HPV) as the causative agent of essentially all cervical cancer cases, HPV molecular screening tests and HPV vaccines for primary prevention against the virus have been developed. Accordingly, comparative studies were designed to assess the performance of cervical cancer screening methods in order to devise the best screening strategy possible. This review critically assesses the current cervical cancer screening methods as well as the implementation of HPV vaccination in Europe. The most recent European Guidelines and recommendations for organized population-based programs with HPV testing as the primary screening method are also presented. Lastly, the current landscape of cervical cancer screening programs is assessed for both European Union member states and some associated countries, in regard to the transition towards population-based screening programs with primary HPV testing. Cancer of the cervix uteri, more commonly known as cervical cancer, is an important public health concern. It was reported as the fourth most frequently occurring gynecological cancer, with an estimated worldwide incidence of 528,000 cases and 266,000 deaths in 2012 [The incidence and mortality of cervical cancer, however, have been declining in developed countries due to the discovery of the Pap test in the 1940s, which enabled the prompt identification of morphological changes in the cervical epithelium . The useCervical cancer screening was revolutionized in the early 1980s by the discovery of human papillomaviruses (HPV) as the single causative agents of the disease. In 1983, HPV type 16 (HPV16) was first identified in DNA from a biopsy sample of invasive cancer of the cervix, and in the following years, HPVs were reported as the main causative agents of cervical cancer ,11,12,13This review focuses on the available tests and strategies, which are currently employed for screening and prevention of HPV infection and cervical cancer. Furthermore, in accordance with recommendations specified in the recent European Guidelines, important aspects of screening programs necessary for the success and efficiency of such systems are highlighted. Finally, the current landscape of cervical cancer screening programs of member states of the European Union (E.U.) and some associated countries is reviewed.Testing to identify anomalies in the cervix can be dated as far back as the early 19th century, when anatomists and pathologists of the time observed and studied the cytological changes derived from cervical and other genital neoplasms, as well as the woman’s menstrual cycle . In the With the invention of the Pap test in the 1940s, by George N. Papanicolaou and H.F. Traut, cervical cytology gained a robust and low-complexity method of screening for cervical cancer . This prDespite its widespread use as the primary cervical cancer screening method, the Pap test has some important limitations. The staining procedure of the conventional Pap test requires a considerable amount of time (20–30 min) and consumables . The smeConsequently, to address the shortcomings of the Pap test, a number of derivative methods were developed, such as the UltraFast staining technique, the short-duration Papanicolaou stain, the REAP stain and the Enviro-Pap method ,37,38,39Another alternative method developed to address the shortcomings of the conventional Pap smear is liquid-based cytology (LBC). ThinPrep® Pap test  was the first LBC technique to be approved by the United States Food and Drug Administration (FDA) . This meVisual inspection by acetic acid (VIA) or with Lugol’s iodine (VILI) are two inexpensive screening methods frequently used in low-resource settings, with VIA being more widely used. These techniques are based on the fact that upon the application of acetic acid or Lugol’s iodine directly to the cervix, precancerous cervical lesions become discernible to the naked eye by both clinicians and nonclinicians. Although not perfect, both VIA and VILI have been reported to have acceptable specificity and sensitivity in low-resource settings ,48,49.Cervical cytology has undoubtedly played an important role in cervical cancer screening and continues to do so. However, it has inherited the limitation of being a morphological method requiring subjective interpretation by well-trained cytologists . DespiteIn contrast to screening methods based on cytology, HPV testing does not rely on morphological interpretation and is based on the detection of HPV DNA, HPV mRNA or other viral markers. In the last two decades, HPV testing has become in several countries an invaluable part of clinical guidelines for cervical carcinoma screening, triage and follow-up after treatment . As a geTaking into consideration these characteristics of HPV testing, when designing an HPV test to be used for agreed clinical indications, the ultimate sensitivity for the detection of precancerous lesions by inclusion of HPV types that are rarely associated with cervical cancer, must be carefully weighed against the potentially dramatic loss of clinical specificity when a particular HPV type  is frequent in low-grade disease or in women without disease. In addition, it should always be taken into consideration that absolute reassurance following a negative cervical cancer screening test result is not achievable at any analytic sensitivity, because of a myriad of factors that are independent of the actual screening test performance, including operator error and poor cervical sampling. Thus, a cervical cancer screening program should adopt an HPV test for use as screening tool, only if it has been validated by demonstrating reproducible and consistently high sensitivity for CIN2+ and CIN3+ lesions, as well as minimal detection of clinically irrelevant, transient HPV infections ,55. TherSeveral comprehensive inventories of commercially available HPV tests were published in the last decade ,57,58. AAs a multitude of hrHPV tests are available, regular evaluation updates are essential to ensure their suitability for primary cervical cancer screening. A recent systematic review , listed Since the performance of an HPV test may vary depending on the sample collection procedures and medium, regulatory approval in some settings requires validation of performance based on the choice of sample collection medium. Importantly, the validation of a pre-approved assay for use with a specific medium is a simpler process than de novo clinical validation of an HPV assay. It can be expected that several previously approved tests will eventually be validated for use with the most commonly used collection media .It is worth mentioning that although we have an increasing understanding of which HPV tests are valid for HPV-based primary cervical cancer screening, given an internationally accepted and applied validation framework and published professional guidance, we do not have widely accepted equivalent metrics to judge the validity of HPV tests in other clinical settings, including post-treatment surveillance and the triage of low-grade abnormalities ,59. InteThe identification of HPV as the main etiological agent of cervical cancer, presented novel opportunities for the development of preventative modalities against cervical cancer . With thInitially, all three HPV vaccines had been approved for a 3-dose series in order to generate sufficient and long-lasting protective immunity . CurrentDespite their potency in providing protection against HPV infection, HPV vaccines are not therapeutic, as they are not effective in curing preexisting HPV infections . Hence, With extensive knowledge of the biology of cervical cancer and with an arsenal of screening and prevention tools, the disease can be detected at an early enough stage to be curable. As a concept, the fundamental principles of cervical cancer screening can be dated back as far as the 1940s, before organized screening programs took place . HoweverTo this end, the first edition of the European Guidelines for Quality Assurance in Cervical Cancer Screening, published in 1993 , designaIn contrast to organized population-based screening, opportunistic screening depends on the initiative of the individual woman and/or her doctor. This type of screening often results in high coverage only in certain parts of the population, which are screened frequently, while other parts of the population, usually with a lower socioeconomic status, exhibit lower coverage. This situation results in uneven coverage with heterogeneous quality, limited effectiveness, and reduced cost-effectiveness, as well as difficulty in monitoring the population . Thus, as the European Guidelines recommend, a program with an organized population-based nature may substantially improve the accessibility and equity of screening access while simultaneously improving effectiveness and cost-effectiveness ,81. The Three options that are currently in use for primary cervical cancer screening, are cytology, HPV testing, and cotesting . CytologThe development of clinically validated HPV tests, which are more accurate and sensitive than primary cytology testing, has recently caused a paradigm shift. According to the European Guidelines, as well as the World Health Organization (WHO), HPV testing is now proposed as the primary screening tool for cervical cancer ,88. HPV When deciding at what age to start HPV-based screening it is important to take into account the natural history of HPV infection in order to avoid unnecessary follow-up and/or overtreatment of women with only transient HPV infections ,94. ThusAt the same time, setting the age to stop screening is important. The European Guidelines suggest that primary HPV screening could stop at the same age recommended for cytology, that is, at 60–65 years of age, provided that the most recent screening test was negative . The reaCotesting combines the sensitivity of HPV testing with the specificity of cytology at the level of primary screening. Even though some non-European studies reported marginal superiority of cotesting over HPV-based screening alone, the European Guidelines recommend against cotesting at any given age because it is not substantially more effective than HPV testing and is considerably more costly ,83,89. Having established HPV testing as the recommended primary screening method, the age range, and the screening intervals of a negative test, it is important to specify the management of positive results from primary testing. Triaging women with a positive HPV primary test result can compensate for the lower specificity that characterizes HPV testing. In this regard, the European Guidelines recommend the performance of cytology as the main triage test in order to manage the increased number of screen positives identified by primary HPV testing, which would otherwise lead to an excessive number of referrals to colposcopy. Thus, only women with both an HPV-positive result and cytological abnormalities are immediately referred for colposcopy. If the primary HPV test employs partial genotyping for HPV16 and HPV18, then direct referral colposcopy (without cytology) is possible . The samTo increase the specificity and improve the detection of precancerous lesions, other techniques, in addition to cytology, that could potentially be used for the triaging of women after a positive HPV primary test result are: partial HPV genotyping (HPV16/18 or extended), p16-Ki67 immunostaining, HPV E6/E7 mRNA detection, and cellular and viral methylation assays. However, at this time, there are insufficient data to favor such methods over cytology for triaging in Europe. The use of partial HPV genotyping triage is based on the fact that there is substantial variation in risk depending on HPV type, but it is still a matter of debate as to which HPV types other than HPV16  it is worth implementing a routine risk-stratification algorithm . The p16HPV-positive, cytology triage-negative women are recommended to undergo a different path than women with triage-positive cytology and/or borderline cytological results. Cytology triage-negative women who are infected by hrHPV, are still at risk for persistent infection and thus, require repeat testing at shorter intervals than HPV-negative women . The opeFollowing the referral of a patient for colposcopy, the identification of high-grade cervical lesions may be diagnosed after biopsy (in approximately a quarter of referred women), followed by surgical treatment. Various treatments of high-grade cervical lesions are available, including cryotherapy, laser, loop electrosurgical excision procedure (LEEP) or large loop excision of the transformation zone (LLETZ) and cone biopsy, which are all characterized by an overall high success rate. However, treatment may fail with regard to residual or recurrent precancer, with 5–15% of treated women being diagnosed again with CIN2+ and therefore requiring additional therapy. Indeed, women once diagnosed with high-grade lesions are characterized by an increased lifetime risk of developing cervical cancer ,108. TheConsidering the pros and cons of all available cervical cancer screening tests, and despite the existence of evidence-based recommendations, it is clear that there is no “one size fits all” model for cervical cancer screening. There are various factors affecting the implementation of a screening program: the amount of healthcare funds available in each region/country, the preexisting medical and economic infrastructure, and the risk perception and tolerance of the society . Table 1Some of these countries which are still lacking organized national screening programs have made attempts to implement national and/or regional screening programs . In 2009As it can be observed from the data in Despite all the efforts to implement screening programs, their success depends primarily on sufficient population coverage. Unfortunately, many countries report suboptimal participation in screening programs ,232,315.Low coverage is directly affected by the targeted population, and accordingly, there have been numerous studies in various countries evaluating the awareness, perception, and knowledge of the population in regard to HPV, cervical cancer screening programs and vaccination programs ,320,321.Cervical cancer is an important health care problem in many parts of the world as well as in the E.U. It is a disease with a clearly defined natural history caused by essentially one etiological agent, and with long clinical latency. These characteristics of the disease enabled the development of acceptable and valid testing, such as the Pap test that was invented in the 1940s, to identify the precursor lesions, which can be treated in a safe, effective and acceptable way. This subsequently led to the establishment of routine cervical cancer screening in the 1960s. Primary prevention of cervical cancer was implemented more recently with the release of the first prophylactic HPV vaccine in 2006. Currently, the European Guidelines recommend organized population-based screening with primary HPV testing. However, this paradigm shift requires either the reformation of currently existing cytology-based organized programs or the implementation of new programs for countries still relying on opportunistic screening, which also mainly use cytology as a screening tool. The existing cytology-based screening programs are in many instances inefficient and costly because of the subjective nature of cytology, threatening to strain the public health budget of many countries, an effect that is expected to be exacerbated further as population HPV vaccination coverage increases . We are


Introduction: Although human papillomavirus (HPV) routine vaccination programmes have been implemented around the world and recommendations have been expanded to include other high-risk individuals, current recommendations often differ between countries in Europe, as well as worldwide.Aim: To find and summarise the best available evidence of HPV vaccination in high-risk patients aiding clinicians and public health workers in the day-to-day vaccine decisions relating to HPV in Spain.Methods: We conducted a systematic review of the immunogenicity, safety and efficacy/effectiveness of HPV vaccination in high-risk populations between January 2006 and June 2016. HPV vaccination recommendations were established with levels of evidence according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system.Results: A strong recommendation about HPV vaccination was made in the following groups: HIV infected patients aged 9–26 years; men who have sex with men aged 9–26 years; women with precancerous cervical lesions; patients with congenital bone marrow failure syndrome; women who have received a solid organ transplant or hematopoietic stem cell transplantation aged 9–26 years; and patients diagnosed with recurrent respiratory papillomatosis.Conclusions: Data concerning non-routine HPV vaccination in populations with a high risk of HPV infection and associated lesions were scarce. We have developed a document to evaluate and establish evidence-based guidelines on HPV vaccination in high-risk populations in Spain, based on best available scientific evidence. Human papillomavirus (HPV) is the main cause of uterine cervical cancer (UCC) and its precursor lesions ,2. HPV cThe estimated impact of newly diagnosed cases of HPV-related disease is high around the world: annually there are 30.9 million cases of cervical precancerous lesions, 32 million cases of genital condylomata and 630,000 cases of cancer  diagnosed ,13.HPV vaccination is an effective primary intervention to prevent HPV infection and its associated disease burden. Vaccination is used as part of a coordinated strategy with quality screening programmes and treatment services in cervical cancer prevention . Three HIn the first decade of HPV vaccine use, the main strategy worldwide was routine vaccination programmes for adolescent and young women. In Europe, according European Centre for Disease Control (ECDC) information reviewed in 2018, routine vaccination is included in the national immunisation schedule in the majority of countries  ,20. In ASupplementary material.In clinical practice, there is growing interest in expanding vaccine recommendations to patients at high-risk of HPV infection and development of related neoplasms. Currently, information on HPV vaccination for these high-risk populations is scarce and recommendations for selective vaccination differ between countries in Europe. Several reasons may explain the lack of guidelines for vaccination in high-risk populations, including: heterogeneity in the definition e.g. age, sex, disease characteristics and variations in the risk of infection, persistence or malignancy; limited published clinical trial data ; and limitations associated with establishing recommendations for patient groups not included in the product label. Some of these issues can be viewed in the A guideline on HPV vaccination in high-risk individuals, based on the review of current evidence by an independent and multidisciplinary expert panel, was recently released by the Spanish Association of Cervical Pathology and Colposcopy . This paIn October 2015, a steering committee was formed in Spain to compile this guideline after a rigorous review of the HPV-associated disease burden and a systematic review of studies investigating the immunogenicity, safety and efficacy of HPV vaccination in high-risk populations. This committee consisted of 10 Spanish professionals including gynaecologists, epidemiologists, paediatricians, infectologists, haematologists, gastroenterologists and preventive medicine specialists with recognised expertise in the area.This review included both randomised controlled trials (RCT) and observational studies  that assessed the effects of HPV vaccination on high-risk groups.Based on clinical expertise of the steering committee and published literature, the following groups were defined as individuals at high-risk of HPV infection and HPV-related disease and were included in the guideline on HPV vaccination in high-risk individuals .Box• HIV infected patients• MSM• Inflammatory bowel disease patients• Women with precancerous cervical lesions• Patients with congenital bone marrow failure syndrome, primary immunodeficiency or survivors of childhood neoplasia• Solid organ or haematopoietic stem cell transplant recipients• Patients with immunosuppressive or biological treatment• Patients with recurrent respiratory papillomatosisHIV: Human immunodeficiency virus; HPV: human papillomavirus; MSM: men who have sex with men.The interventions of interest were the bivalent (Cervarix), quadrivalent (Gardasil) and nonavalent (Gardasil 9) HPV vaccines. The outcomes of interest were the efficacy, effectiveness, immunogenicity and safety of HPV vaccines.Studies that did not report original data e.g. editorials or reviews were excluded.Supplementary material.An electronic search was carried out using PubMed to find studies published in English from January 2006 to June 2016. The search terms were a combination of the names and synonyms of the different risk groups, the HPV vaccines and the outcomes of interest. The details of the search strategies are presented in the All authors conducted unstructured electronic searches and reviewed their personal files for relevant published papers.Two authors (XMG and JARP) independently screened the titles and abstracts of the articles identified by the PubMed search and selected articles for full-text reading in accordance with the selection criteria. Full texts for the relevant references were independently evaluated by XMG and JARP to determine their inclusion. Reference lists of included articles were screened to consider further potentially eligible studies . Any disThe agreement between reviewers in the selection process was computed using Stata/SE 14.2 for Windows  and resulted in agreement of qualitative measures (Cohen’s Kappa = 0.98) .Panel subgroups reviewed the initial literature search, selected references, evaluated evidence, drafted recommendations and summarised the evidence for each section. The content of the guideline was decided by the consensus panel; the evidence evaluation process involved a systematic weighting of the quality of evidence and the grade of recommendation using the Infectious Diseases Society of America (IDSA) version of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system ,27.The developed guideline for Spain was reviewed and endorsed by several groups acknowledged at the end of the article.The prevalence of HPV infection in individuals with HIV infection is high -30. WithThree risk subgroups can be identified in the overall HIV-infected population: HIV-infected women, HIV-infected men who have sex exclusively with women (MSW) and HIV-infected MSM. While UCC screening programmes and the improvement in HIV disease control have led to a decrease in the incidence of UCC in HIV-infected women, the incidence of anal cancer in this population is 30 per 100,000 person-years  , and theSupplementary material Table S1).Nine papers of HPV vaccination in HIV population were selected -43. AvaiThe efficacy results obtained in RCT of MSM  cannot bIdeally, people with HIV who receive a HPV vaccine should be on antiretroviral treatment and have good immune-virological control at the time of vaccination.The European Acquired Immune Deficiency Syndrome (AIDS) Clinical Society guidelines recommend HPV vaccination in all individuals with HIV; however, they state that in situations where there is an established HPV infection, vaccine efficacy is questionable . The US The 2013 IDSA clinical practice guideline for vaccination of the immunocompromised host, recommends the administration of three-dose of quadrivalent HPV (qHPV) vaccine for females and males with HIV aged 11–26 years .HPV vaccination is recommended in both male and female individuals with HIV from 9–26 years . HIV-positive people aged 26 years and older could also benefit from HPV vaccination . The use of any of the available vaccines is indicated, although the quadrivalent or nonavalent vaccines are preferred given the high prevalence of anogenital warts in these patients.MSM have a high risk of HPV infection and HPV-related disease than MSW , with thSupplementary material Table S2). One study evaluated the immunogenicity, efficacy and safety of the qHPV vaccine against HPV in healthy males aged 16–26 years [Six papers of HPV vaccination in MSM population were selected  ; it alsoOne study assessed the immunogenicity and safety of the nonavalent vaccine vs placebo . This trThe Advisory Committee on Immunisation Practices (ACIP) recommends routine HPV vaccination for MSM aged 9–26 years, including transgender persons .HPV vaccination is recommended in MSM aged 9–26 years . MSM aged 26 years and older may also benefit from HPV vaccination . The use of any of the available vaccines is indicated, although the quadrivalent or nonavalent vaccines are preferred given the high prevalence of anogenital warts in MSM.Treatment of inflammatory bowel disease IBD (Crohn’s disease and ulcerative colitis) often includes immunosuppressants, which increase the risk of HPV persistence. Compared with patients not receiving immunosuppressive treatment, patients with IBD receiving azathioprine show a higher incidence of cutaneous warts and skin and genital herpes flares . AlthougControversy remains regarding to the increased risk of HPV infection and UCC in patients with IBD vs the general population. A study of 40 patients with IBD showed a higher incidence of abnormal cervical cytology in women with IBD compared with similar healthy controls , and a higher incidence of lesions . In thisSupplementary material Table S3). One study investigating the profile of the qHPV vaccine in patients with IBD showed an adequate immunogenicity and safety profile [One paper of HPV vaccination in IBD population was selected . Although vaccination can be administered at any time, it is preferable to vaccinate at the time of diagnosis and before the use of immunosuppressive or biological therapies, to ensure higher immunogenicity. There are no data supporting HPV vaccination in men with IBD.In Spain, a large-scale study estimate that the prevalence of HPV infection is 13.4% and 7.9% in women aged 26–45 and 46–65 years, respectively, compared with 28.8% in women aged 18–25 years . Rates oA substantial proportion of adult women become infected with HPV, particularly genotypes 16 and 18. The probability of persistence increases with age, which consequently increases the risk of precancerous lesions or UCC developing. Women with precancerous cervical lesions have a 5–25% risk of recurrence and an increased risk of developing UCC compared with the general population . This inSupplementary material Table S4). Precancerous lesions are generally diagnosed in women aged 25 years or older; data about HPV vaccination in this group of women are limited. However, a few studies focused on adult women and showed that both qHPV vaccine and the bivalent HPV vaccine are immunogenic, effective and safe in this age group [Three papers of HPV vaccination in women with HPV infection and precancerous cervical lesions were selected . Patients with precancerous cervical lesions who have not yet been treated, may benefit from HPV vaccination . Ideally, the vaccine should be administered early, either at diagnosis or before cervical conisation.Congenital bone marrow failure syndrome (CBMFS) is a group of heterogeneous diseases associated with inadequate blood cell production, constitutional malformations and predisposition to cancer. Patients with Fanconi’s anaemia and dyskeratosis congenita, both congenital bone marrow failure syndromes, are at risk of gynaecological and head and neck squamous cell carcinomas . The incCertain primary immunodeficiency conditions, such as epidermodysplasia verruciformis, hyper-IgE syndrome, idiopathic CD4 lymphopaenia, GATA2 deficiency and Warts-hypogammaglobulinaemia, infections and myelokathesis (WHIM) or Netherton’s syndrome, have the presence of cutaneous warts as one of the main clinical manifestations of the disease . PatientSurvivors of childhood, adolescent or young adulthood cancer e.g. Hematopoietic stem cell transplantation (HSCT) recipients, post-transplant survivors with chronic graft-vs-host disease receiving immunosuppressive therapy, individuals with a history of pelvic radiation and those with Hodgkin’s lymphoma, are also at risk of adverse outcomes associated with HPV infection . In one Supplementary material Table S5). Patients with Fanconi's anaemia and dyskeratosys congenita have a similar immune response to HPV vaccination to that of the general population [Two papers of HPV vaccination in CBMFS, primary immunodeficiency or survivors of childhood neoplasia were selected .HPV vaccination may benefit patients with primary immunodeficiency . The use of any of the available vaccines is indicated, although the quadrivalent or nonavalent vaccines are preferred given the high prevalence of anogenital warts in these patients.Survivors of childhood neoplasia can benefit from HPV vaccination .Patients who have undergone solid organ transplantation need life-long immunosuppressive therapy, which increases their risk of complications associated with persistent HPV infection. Studies have shown that immunosuppressed transplant recipients have an increased presence of HPV in the oral cavity, higher prevalence of genital warts , high onPatients who have undergone HSCT have an increased risk of developing solid organ cancer . In someSupplementary material Table S6). Epidemiological data show a high risk of gynaecological and other cancers in transplant recipients [Three papers of HPV vaccination in solid organ or haematopoietic stem cell transplantation were selected -106 (Supcipients . LimitedACIP recommends routine HPV vaccination for those aged 9–26 years for transplant patients .The 2013 Infectious Diseases Society of America (IDSA) clinical practice guideline for vaccination of the immunocompromised host recommends that physicians consider the administration of three doses of HPV vaccine 6–12 months after HSCT for female patients aged 11–26 years and qHPV vaccine for males aged 11–26 years. The HPV vaccine series should also be administered to solid organ transplant candidates aged 11–26 years .HPV vaccination is recommended in female transplant recipients aged 9–26 years . Female patients aged 26 years and older may also benefit from HPV vaccination, particularly those with chronic graft vs host disease following HSCT . If possible, vaccination should be provided before solid organ transplantation and preferably before the use of immunosuppressive or biological therapies to ensure high immunogenicity. Vaccination is not recommended within 6 months post-transplantation. Patients undergoing HSCT should be vaccinated 6–12 months after transplantation, even if vaccinated beforehand.Immunosuppression is an important risk factor for cervical HPV infection . HPV hasSome studies have shown an increase in the prevalence of HPV infection, dysplasia and premalignant lesions in patients with SLE and those with kidney failure undergoing haemodialysis ,109. A sTNF blockers are widely used to treat chronic inflammatory diseases. TNF plays an essential part in host defence and the immune response. Although uncommon, cases of extensive cutaneous HPV lesions have been reported following TNF blocking therapy ,113,114.The high rates of HPV infection and associated lesions in immunocompromised individuals justify the need to consider vaccination in this population . AlthougSupplementary material Table S7) [Five papers of HPV vaccination in patients with immunosuppressive or biological treatment were selected. (able S7) -119. In able S7) . Lower sable S7) .Two studies evaluating the immunogenicity and safety of HPV vaccines in JIA patients have been published ,119. OneAt present, there are no published data from randomised clinical trials on the efficacy, immunogenicity or safety of HPV vaccination in patients receiving immunosuppressive and/or biological treatment.The evidence-based recommendations on vaccination in patients with autoimmune rheumatic diseases from the European League against Rheumatism specify that HPV vaccination should be considered in women with SLE aged less than 26 years .ACIP recommends routine HPV vaccination for immunosuppressed patients aged 9–26 years .The 2013 IDSA clinical practice guideline for vaccination of the immunocompromised host recommends HPV vaccination for females and males 11–26 years old with chronic inflammatory diseases on immunosuppressive medications .HPV vaccination is recommended in women receiving immunosuppressive and/or biological treatment aged 9–26 years, in particular, women with SLE or JIA . While the vaccine can be given at any time, administration before the use of immunosuppressive or biological therapies is preferable. There are no data supporting HPV vaccination in males receiving immunosuppressive or biological therapy.Recurrent respiratory papillomatosis (RRP) is characterised by the presence of multiple papilloma in the airway that cause obstructive symptoms such as shortness of breath, hoarseness or stridor . The disSupplementary material Table S8). No randomised clinical trials on the efficacy of HPV vaccine have been conducted in individuals with RRP. Several studies have investigated the effectiveness of the quadrivalent vaccine in both juvenile RRP [Supplementary material Table S8). Vaccination-induced partial or complete remission in a substantial proportion of the RRP patients, and no remarkable adverse effects were reported.Nine papers of HPV vaccination in patients with recurrent respiratory papillomatosis were selected . Given the relationship of RRP to HPV genotypes 6 and 11, the use of the quadrivalent or nonavalent vaccine is recommended. Despite scarce data, the potential benefit should outweigh the risk of vaccination; therefore compassionate use may be considered in juvenile patients who are under the age indicated in the summary of product characteristics for the vaccine.Data concerning non-routine HPV vaccination in populations with a high risk of HPV infection and associated lesions are scarce, and recommendations differ between countries. The evidence-based recommendations for HPV vaccination in high-risk populations conducted by an independent expert panel are summarised in Systematic HPV vaccination is considered the most effective intervention for controlling the infection and preventing HPV-related disease burden. Along with other interventions, such as information, screening and treatment, HPV vaccination is crucial in the prevention of uterine cervical cancer.In all high-risk populations, based on the summary and recommendations, the quality of evidence was found to be very low to moderate. A strong recommendation about HPV vaccination was made in HIV infected patients aged 9–26 years; men who have sex with men aged 9–26 years; women with precancerous cervical lesions; patients with congenital bone marrow failure syndrome; women who have received a solid organ transplant or hematopoietic stem cell transplantation aged 9–26 years; and patients diagnosed with recurrent respiratory papillomatosis. In the rest of high-risk categories with only a weak recommendation , the quality of evidence was low or very low. Further studies are needed.


Cervical cancer is a major public health concern in China. We report the end‐of‐study results of a phase II/III trial to assess the efficacy, immunogenicity, and safety of the AS04‐human papillomavirus (HPV)‐16/18 vaccine in Chinese women aged 18‐25 years followed for up to 72 months after first vaccination. Results of approximately 57 months following first vaccination have been previously reported.3 (control) at Months 0‐1‐6. Vaccine efficacy against HPV‐16/18 infection and cervical intraepithelial neoplasia (CIN), cross‐protective vaccine efficacy against infections and lesions associated with nonvaccine oncogenic HPV types, immunogenicity, and safety were assessed. Efficacy was assessed in the according‐to‐protocol efficacy (ATP‐E) cohort , total vaccinated cohort for efficacy  and TVC‐naïve .Healthy 18‐25‐year‐old women (N = 6051) were randomized (1:1) to receive three doses of AS04‐HPV‐16/18 vaccine or Al(OH)In initially HPV‐16/18 seronegative/DNA‐negative women, vaccine efficacy against HPV‐16/18‐associated CIN grade 2 or worse was 87.3%  in the ATP‐E, 88.7%  in the TVC‐E, and 100%  in the TVC‐naïve. Cross‐protective efficacy against incident infection with HPV‐31, HPV‐33 and HPV‐45 was 59.6% , 42.7% , and 54.8% , respectively (ATP‐E). At Month 72, >95% of initially seronegative women who received HPV vaccine in the ATP cohort for immunogenicity (N = 664) remained seropositive for anti‐HPV‐16/18 antibodies; anti‐HPV‐16 and anti‐HPV‐18 geometric mean titers were 678.1 EU/mL  and 343.7 EU/mL , respectively. Serious adverse events were infrequent . Three and zero women died in the control group and the vaccine group respectively. New onset autoimmune disease was reported in two women in the vaccine group and two in the control group.This is the first large‐scale randomized clinical trial of HPV vaccination in China. High and sustained vaccine efficacy against HPV‐16/18‐associated infection and cervical lesions was demonstrated up to Month 72. The vaccine had an acceptable safety profile. Combined with screening, prophylactic HPV vaccination could potentially reduce the high burden of HPV infection and cervical cancer in China.NCT00779766. Cervical cancer is one of the most aggressive forms of cancer that women can suffer from. The presence of human papillomavirus (HPV) is responsible for almost all cases of cervical cancer. For these reasons, the world health organization recommends that HPV vaccines should be included in national vaccination programs. From the first licensure of the first HPV vaccine in 2006 to the development of new HPV vaccines, the latter have been studied in clinical trials and demonstrated to decrease in the number of cervical cancers while remaining acceptably safe. Many developed countries have included HPV vaccination in their program. These countries reported a decrease of cervical cancer cases. Many countries from the less developed regions are still yet to introduce HPV vaccination. The novelty: In 2016, the Chinese authorities approved licensure of the first HPV vaccine for use in China to help prevent cervical cancer and based their decision on clinical trials conducted in China. We present here the final results of the key clinical trial of this HPV vaccine that involved around 6000 Chinese women over 6 years. The impact: Chinese women represent almost 10% of the worldwide population at risk of cervical cancer. Latest estimations from 2016 indicate that 111 000 new cervical cancer cases are reported every year in China. This number could be reduced with the HPV vaccination. In a cross‐sectional epidemiological survey across urban and rural areas of China, there was a trend towards earlier sexual debut (median 17 years) and riskier sexual behaviors in younger cohorts of Chinese women.Cervarix, GSK) is licensed in over 135 countries and was approved in China in July 2016 for use in females 9‐25 years of age. The licence was extended for use in females 9‐45 years of age in May 2018. The vaccine has been shown to offer protection against infection, cytological abnormalities and cervical intraepithelial neoplasia (CIN) associated with HPV‐16/18, including CIN grade 3 or worse, the closest surrogate endpoint to cervical cancer.The AS04‐HPV‐16/18 vaccine , which were supplied in identical prefilled syringes. A randomization list was generated by the study sponsor using a standard Statistical Analysis System (SAS) program. A randomization blocking scheme was used to ensure balance between treatments. Treatment allocation was performed at the investigator's site using a central randomization system on internet (SBIR). The randomization algorithm used a minimization process accounting for study center. Participants, investigators, and study staff were blinded to treatment allocation and HPV DNA and serology results throughout the whole study period. The study blinding was maintained until database freeze for this end‐of‐study analysis.2.210 primers amplifying a 65‐nucleotide region of the HPV L1 gene was used to test cervical samples and biopsy material for HPV DNA from most known HPV types. HPV‐positive specimens were typed by reverse hybridization line probe assay (LiPA) using 28 HPV‐specific hybridization probes, which allowed detection of 14 oncogenic and 11 nononcogenic HPV types. All HPV‐positive samples were also tested using HPV‐16 specific and HPV‐18 specific PCR.Cervical samples were obtained every 6 months for HPV DNA and cytology testing . A polymerase chain reaction (PCR) assay using specific SPFCervical cytology was performed using the ThinPrep Pap Test  and samples were evaluated according to the Bethesda 2001 classification system. CIN terminology was used for reporting of histologically defined cervical lesions.Antibody responses against HPV‐16 and HPV‐18 were determined using enzyme‐linked immunosorbent assay (ELISA)Serious adverse events (SAEs), new onset chronic diseases (NOCD), new onset autoimmune diseases (NOAD), medically significant conditions, pregnancy and pregnancy outcomes were recorded throughout follow‐up. Medically significant conditions were defined as: adverse events (AEs) prompting emergency room or physician visits that were not (1) related to common diseases or (2) routine visits for physical examination or vaccination, or SAEs that were not related to common diseases. Common diseases included: upper respiratory infections, sinusitis, pharyngitis, gastroenteritis, urinary tract infections, cervicovaginal yeast infections, menstrual cycle abnormalities and injury.2.3The primary endpoint was vaccine efficacy against a combined endpoint of 6‐month persistent infection (6MPI) with HPV‐16 and/or HPV‐18 (HPV‐16/18) and/or CIN grade 1 or worse (CIN1+) associated with HPV‐16/18. Secondary endpoints were incident infection, 6MPI, 12‐month persistent infection (12MPI), atypical squamous cells of undetermined significance or worse (ASC‐US+), CIN1+, and CIN grade 2 or worse (CIN2+), associated with HPV‐16/18 or with nonvaccine oncogenic HPV types individually or in combination. 6MPI was defined as at least two positive HPV DNA PCR assays for the same viral genotype with no negative DNA sample between, over an interval of approximately 6 months; 12MPI was defined in the same way over an interval of approximately 12 months. CIN1+ was defined as CIN1, CIN2, CIN3, adenocarcinoma in situ (AIS) or invasive cervical cancer. CIN2+ excluded CIN1. Vaccine efficacy irrespective of HPV DNA in the lesion was evaluated as an exploratory endpoint in the end‐of‐study analysis. Immunogenicity and safety endpoints were followed throughout the entire study period.2.4We have previously published confirmatory event‐driven analyses reporting efficacy against 6MPI/CIN1+ (primary endpoint)The according‐to‐protocol cohort for efficacy (ATP‐E) was the primary efficacy analysis cohort, comprising women with available efficacy data, who met all eligibility criteria, received three doses of vaccine or control, complied with the protocol and had normal or low‐grade cytology  at baseline. Efficacy analyses were also conducted in the total vaccinated cohort for efficacy (TVC‐E), comprising women with available efficacy data, who received at least one dose of vaccine or control and had normal or low‐grade cytology at baseline, and in the TVC‐naïve, comprising women with available efficacy data, who received at least one dose of vaccine or control and at baseline were HPV DNA‐negative for all 14 oncogenic types tested, seronegative for HPV‐16 and HPV‐18 and had negative cytology. The primary analysis in the ATP‐E was performed in women DNA‐negative at baseline and Month 6 and seronegative at baseline for the HPV type considered in the analysis. In the TVC‐E, the analysis was conducted in women DNA‐negative and seronegative at baseline for the type considered in the analysis. Analyses in the ATP‐E and TVC‐E were also conducted in women DNA‐negative at baseline regardless of serostatus. All women in the TVC‐naïve were DNA‐negative for all oncogenic HPV types tested and seronegative at baseline for HPV‐16 and HPV‐18.Vaccine efficacy was calculated using a conditional exact method, which computes an exact confidence interval (CI) around the rate ratio (ratio of event rates in the vaccine vs control groups), taking into account follow‐up time within each group. Vaccine efficacy was defined as 1 minus the rate ratio. In the previously reported confirmatory analyses, statistically significant vaccine efficacy was defined as the lower limit of the 95% CI above zero.Immunogenicity was primarily analyzed in the ATP cohort for immunogenicity which included women in the immunogenicity subset with available immunogenicity data, who met all eligibility criteria, received three doses of vaccine or control and complied with the protocol. Women who acquired HPV‐16 or HPV‐18 infection during the study were excluded from the ATP cohort. Seropositivity 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 antilog 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. Safety was analyzed in the total vaccinated cohort (TVC) which included all women who received at least one dose of vaccine or control. The percentage of participants with an adverse event was calculated with exact 95% CIs. SAS version 9.2 was used for all statistical analysis.33.1The first woman was enrolled in October 2008. Final data were collected in February 2016. Of 6051 women enrolled in the initial study, 5430 consented to participate in the first extension up to month 48, and 4666 women consented to participate in the second extension up to month 72    in the ATP‐E cohort seronegative at baseline for HPV‐16/18, one case of 6MPI/CIN1+ associated with HPV‐16/18 was detected among 2497 women in the vaccine group and 17 cases among 2502 women in the control group.A key focus of the end‐of‐study analysis was efficacy against CIN2+. Vaccine efficacy against CIN2+ associated with HPV‐16/18 was 87.3%  in the ATP‐E in women DNA‐negative at months 0, 6 and seronegative at baseline for the corresponding type, 90.0%  in the ATP‐E in women DNA‐negative regardless of initial serostatus, 88.7%  in the TVC‐E in women DNA‐negative and seronegative for the corresponding type at baseline, 83.3%  in the TVC‐E in women DNA‐negative at baseline regardless of initial serostatus and 100%  in the TVC‐naïve in women DNA‐negative and seronegative for HPV‐16/18 at baseline (Table The first CIN2+ case in the vaccine group (case 1) was a woman who was also included in the ATP‐E cohort. The woman was identified with a CIN3 associated with HPV‐16 at Month 36. At baseline, she was seropositive for HPV‐18  and had normal cytology. She had an infection with HPV‐16, HPV‐51 and HPV‐52 at Month 12, and had ASC‐US associated with HPV‐16 and HPV‐52 at Month 36. Assessment of causality was difficult due to multiple oncogenic HPV infections detected in both the lesion and the preceding cytological specimens. The second woman with CIN2+ in the vaccine group (case 2) was included in the TVC‐E (but not in the ATP‐E), and was seropositive for HPV‐16 and had ASC‐US associated with HPV‐52 at baseline. She was diagnosed with CIN3 associated with HPV‐16 at Month 6; she was also identified with infection with HPV‐33 at Months 6, 12 and 18. No new CIN2+ cases were reported between the previous event‐triggered analysis3.2.2The analysis was performed in women DNA‐negative at baseline for the HPV type considered in the analysis, regardless of initial HPV‐16/18 serostatus. Women in the ATP‐E analysis were also DNA‐negative at Month 6 for the HPV type considered. Vaccine efficacy against virological endpoints associated with nonvaccine HPV types in the ATP‐E and TVC‐E is shown in Table In addition, vaccine efficacy was observed against ASC‐US+ associated with HPV‐31  and HPV‐33  in the ATP‐E; efficacy was similar in the TVC‐E but the lower limit of the 95% CI was below zero for HPV‐33 Table ; vaccine3.2.3Vaccine efficacy against CIN2+ irrespective of HPV DNA in the lesion was assessed in the TVC‐naïve. A total of 27 CIN2+ cases was observed at the end of the study, 11 in the vaccine group and 16 in the control group, resulting in a vaccine efficacy point estimate of 33.5% . None of the cases in the vaccine group was associated with HPV‐16/18 ; this has been previously shown in women who were HPV‐16/18 seropositive before vaccination.In the two CIN2+ cases associated with HPV‐16 in the vaccine group of the TVC‐E, infections with HPV‐16, HPV‐51 and HPV‐52 were detected in one woman, and with HPV‐16 and HPV‐33 in the other woman. Although both cases were assigned as CIN2+ associated with HPV‐16 according to the HPV type assignment algorithm,Evaluation of cross‐protective efficacy against nonvaccine HPV types is a challenge in HPV vaccine trials.Vaccine efficacy against incident infection was higher than vaccine efficacy against 6MPI or 12MPI for some nonvaccine HPV types such as HPV‐45 and HPV‐58. This may be explained by a phenomenon known as masking by which infections with some nonvaccine types may have been missed by the SPF10‐LiPA assay used to genotype HPV types in this trial when HPV16 is present.To explore the potential overall effect of HPV vaccination, vaccine efficacy against CIN2+ irrespective of HPV DNA in the lesion was evaluated in the TVC‐naïve, comprising women who at baseline were DNA‐negative for all 14 oncogenic HPV types tested, seronegative for HPV‐16 and HPV‐18 and had negative cytology. The analysis of the overall effect of vaccination was conducted in the TVC‐naïve because these women had no evidence of HPV infection at baseline; the HPV vaccine is a prophylactic vaccine intended to be administered to girls before HPV infection can be acquired via sexual activity. However, all subjects in the current study were enrolled after their sexual debut. Therefore, it is possible that some women included in the TVC‐naïve cohort were not truly naïve and may have preexisting HPV infection that was not detected due to constraints of sampling or the laboratory assays used. In the present study, vaccine efficacy against CIN2+ irrespective of HPV DNA in the lesion was 33.5% . This compares with vaccine efficacy of 64.9%  against CIN2+ irrespective of HPV DNA in the lesion in the TVC‐naïve population of the PATRICIA trial.The observed difference of the point estimates in overall efficacy in HPV‐naïve women between the current study and PATRICIA may be explained by several factors related to sample size, study design and local HPV epidemiology. The present study enrolled approximately 6000 young women  and was not powered to assess overall impact, which leads to a point estimate with a wide CI that includes the point estimate observed in PATRICIA.Median age at enrolment was 23 years in the current study compared with 20 years in the PATRICIA trial. In addition, due to ethical and cultural considerations in China, only women who were already sexually active before the start of the study were enrolled in the trial, and the majority of women were already married with stable sexual relationships. Likely linked to these enrolment criteria, the incidence rates of CIN2+ irrespective of HPV in the control group of the TVC‐naïve population were very low in the current study (0.20 per 100 person‐years), more than four times lower compared with the PATRICIA trial (0.84 per 100 person‐years).Interestingly, the incidence rate of CIN2+ irrespective of HPV types in the current study is similar to that observed in the VIVIANE trial (0.19 in 100 follow‐up years) that enrolled women 25 years and older.Another factor worth considering in the interpretation of overall vaccine impact is related to HPV prevalence and genotype distribution in different regions. While HPV‐16/18 are the most common oncogenic types accounting for cervical cancer in China,We observed high and sustained anti‐HPV‐16 and anti‐HPV‐18 antibody levels in Chinese women, in line with previous long‐term studies.Serious AEs and AEs of interest (eg autoimmune disorders) occurred at a similar frequency in women receiving the AS04‐HPV‐16/18 vaccine or control, and at a comparable rate to data reported in global studies of women in a similar age group.Strengths and limitations of the study have been discussed previously.A lay language summary contextualizing the results and potential clinical research relevance and impact is displayed in Figure 5This long‐term efficacy study showed that the AS04‐HPV‐16/18 vaccine protects young Chinese women against infection, abnormal cytology and CIN lesions associated with HPV‐16/18 up to 72 months after first vaccination. In addition, data from this study on cross‐protection against HPV‐31, HPV‐33 and HPV‐45 are generally consistent with data from other trials of the same vaccine, including the large, multinational PATRICIA trial.Cervarix is a trademark owned by or licensed to the GSK group of companies.The study protocol and informed consent were reviewed and approved by the ethics committees of the Center for Disease Control and Prevention (CDC) Jiangsu Province and the Cancer Foundation of China. Written informed consent was obtained from each participant prior to the performance of any study‐specific procedures.http://www.icmje.org/coi_disclosure.pdf and declare the following potential conflicts of interest: The institutions of Fang‐Hui Zhao, Shang‐Ying Hu, Ying Hong, Yue‐Mei Hu, Xun Zhang, Yi‐Ju Zhang, Qin‐Jing Pan, Wen‐Hua Zhang, Cheng‐Fu Zhang, Xiaoping Yang, Jia‐Xi Yu, Jiahong Zhu, Yejiang Zhu, Feng Chen, Qian Zhang, Hong Wang, Changrong Wang, Jun Bi, Shiyin Xue, Lingling Shen, Yan‐Shu Zhang, Feng‐Cai Zhu received grants/investigator fees from the GSK group of companies for the conduct of this study. Fang‐Hui Zhao, Feng‐Cai Zhu and Yue‐Mei Hu received support for travel to meetings related to the study from the GSK group of companies. Haiwen Tang, Naveen Karkada, Dan Bi and Frank Struyf are employees of the GSK group of companies. Haiwen Tang, Dan Bi, and Frank Struyf hold shares in the GSK group of companies. Pemmaraju Suryakiran and Yunkun He were employees of the GSK group of companies at the time the study was conducted.All authors have completed the Unified Competing Interest form at All authors participated in the design or implementation or analysis, and interpretation of the study; and the development of this manuscript. All authors had full access to the data and gave final approval before submission. Click here for additional data file.


BackgroundIn April 2018, the Irish cervical smear screening programme, “CervicalCheck”, came under intense scrutiny as the accuracy of hundreds of “negative” results were brought in question.AimThe goal of this brief report was to assess the impact of this real-life event on public information-seeking behaviour, using Google search anomalies as a proxy. Irish relative search volume data for several terms relating to cervical testing/cancer and human papillomavirus were extracted for a five-year period from February 2014 to January 2019 and analysed for the presence of anomalous spikes and shifts in the mean baseline.Resultsp < 0.05). This public interest preceded a mirroring increase in uptake of complimentary consultations offered by the Department of Health to the women concerned. Despite this service engagement and interest in cervical health, the relative search volumes for terms “human papillomavirus infection” and ”HPV vaccine” were just 78 and 51% of their maximum search volume for the five-year period.An unprecedented positive spike in searches relating to cervical testing/cancer was observed immediately after the CervicalCheck revelations, which remained anomalous for the month to follow (ConclusionsAnomaly analysis revealed an unprecedented spike in information-seeking behaviour following the CervicalCheck revelations. However, this was not associated with a comparable elevation in HPV interest. This suggests that more public education and promotion of the HPV vaccine is warranted, in the context of vastly reduced uptake in recent years. Finally, Google Trends data represents a free an open source means by which to assess information-seeking behaviour of the public in relation to health and disease. The cytological analysis has been carried out by two Irish and two US laboratories since 2008. Until recently, the programme appeared to be functioning well and, as such, garnered little attention. In April 2018, however, it was revealed that roughly 220 women with cervical cancer were never informed that their negative smear test results were inaccurate. Subsequent inquiry into the revelations revealed that, although the laboratories contracted to perform the sample processing and testing were safe, there existed serious failures in governance and structure of the screening programme, including a grave lack of transparency with in ongoing performance audits which led to harmful delays in many diagnoses. This revelation and the lack of publicly available information combined to create a media frenzy, which further fuelled the considerable public anxiety around the issue.Human papillomavirus (HPV) is found to be associated with virtually all cervical cancers and a significant proportion of sexually active individuals will come into contact with the virus at some stage . In 2010Google Trends search data has proven to be a valuable complementary tool in epidemiological and public health research in recent years. Most recently, Google search data was used to demonstrate the increase in sexual assault and sexual harassment-related information-seeking behaviour in the wake of the #MeToo movement .The present brief report aimed to utilise Irish Google search trends in the wake of the CervicalCheck revelations as a proxy for public interest. The authors predicted that Google searches relating to cervical testing and cancer would increase anomalously. In concert, we expected that the increased public fear would, in turn, lead to increased information-seeking behaviour relating to primary prevention of cervical cancer, that is, the HPV vaccine.Data acquisitionhttps://trends.google.com/trends/). Initially the term “cervical check” was investigated and several of the top related queries were then included in the analysis. These additional terms included “cervical smear test” and “cervical cancer”. In addition, trend data for “HPV vaccine” and “human papillomavirus infection” searches were assessed over the same time period. Google provides relative search volumes (RSV), which outline the percentage proportion of the highest search volume in the predetermined region and period. Although this limits the comparability of the data to other studies, it is entirely adequate for the purposes of the present study as it is focussed solely in the presence of positive peaks, or anomalies. Finally, General Practitioner (GP) complimentary consultation data in the aftermath of the CervicalCheck revelations was garnered from the CervicalCheck Steering Committee weekly reports to the Minister for Health [This study monitored the volume of Google searches relating to the cervical smear test and HPV in Ireland over a five-year period (02/02/2014-20/01/2019). Data were extracted from the Google Trends website in January 2019  and prepared for analysis. R packages “Anomalize” (v0.1.1) and “Tidyverse” (v1.2.1) were used for anomaly analysis and plotting, respectively 3 and pre. “AnomalThe simplified timeline of the CervicalCheck revelations is presented in Figure Until recently, trends in Google searches relating to the CervicalCheck screening programme remained minimal, with modest annual non-anomalous peaks in January of each year. In response to the CervicalCheck failure disclosure, the Irish public reacted by vastly increasing the relative rate of Google searches with the terms “cervical check” and “cervical cancer” (p < 0.05; Figure Due to the considerable media attention afforded to the anti-vaccination lobby groups in previous years, we observed a number of anomalous search weeks for “HPV vaccine” Figure  and “humThe CervicalCheck controversy was a public event which was high on emotion and low on reliable information. This, in turn, led to a media storm which propagated anxiety amongst the population, as is indicated by the results of this study. In January 2019, a high-profile victim publicly criticised the government’s management of the issue, suggesting that there was a loss of momentum and interest in reform. Indeed, the results of this study indicate that public attention for the issue dwindled relatively rapidly to baseline mean and did not reach comparable heights in the subsequent months, despite the publishing of an expert scoping inquiry into the failings of the CervicalCheck screening programme in September 2018 (Figure These data suggest that, although the CervicalCheck revelations triggered an increased information-seeking behaviour with regards to cervical testing and cancer, the Irish public did not demonstrate a heightened awareness of the primary prevention scheme for the main etiological contributor of the disease. The HPV vaccination programme has encountered challenging times in the past two years and, as a result, the HPV Vaccine Alliance was established in 2017 to present the facts about HPV vaccination in the factsheet and infographic-style outreach projects. Although efforts in educating the public appear to have stemmed the tide, the hangover from the anti-vaccination lobby groups is clear as uptake rates remain wholly inadequate. The present brief report suggests that vaccine advocacy groups should address the apparent understanding deficit and utilise high-profile publicity to reiterate the connection of HPV and cervical cancer to the public, in order to promote the national primary prevention scheme. This type of advocacy should be clear, factual and presented in a manner which does not detract from the suffering of those who were failed by the CervicalCheck programme. Importantly, Ireland has recently announced intentions to begin offering universal HPV vaccination for both males and females during the first year of secondary school education beginning in September 2019, representing a step forward with prospective protection which should not be understated. Previous knowledge and the conclusions of the present study are summarised in Figure Google Trends data is a publicly-available resource which appears to be a useful epidemiological and public health tool in assessing anomalous or seasonal information-seeking behaviour. The tool has recently been successfully applied to topics such as seasonality of several diseases, as well as public interest “cheap cigarettes” following the US states increases in cigarette taxation -11. ThesIn the wake of the CervicalCheck revelations, information-seeking behaviour regarding cervical testing and cancer was vastly increased in comparison to the previous five years. This anomalous spike in public interest immediately preceded a mirroring increase in uptake of complementary GP consultations offered by the Department for Health to concerned women. Despite this increase in cervical testing/cancer interest and healthcare engagement, we did not observe a comparable and concurrent increase in searches relating to HPV infection and vaccination. This indicates that the public currently may not conflate the two issues entirely and, therefore, further educational work is warranted to encourage primary prevention of HPV. Despite this, efforts are beginning to realise, with universal HPV vaccination being offered to secondary school attendees of both sexes in Ireland from September 2019 onwards. To our knowledge, this is the first study to use Google Trends data to examine public information-seeking behaviour in relation to cervical cancer testing and HPV, or the temporal association thereof.