In Canada, two vaccines that have demonstrated high efficacy against infection with human papillomavirus (HPV) types −16 and −18 are available. The HPV-6/11/16/18 vaccine provides protection against genital warts (GW) while the HPV-16/18 vaccine may provide better protection against other oncogenic HPV types. In this analysis, the estimated clinical and economic benefit of each of these vaccines was compared in the Canadian setting.A Markov model of the natural history of HPV infection among women, cervical cancer (CC) and GW was used to estimate the impact of vaccinating a cohort of 100,000 12-year-old females on lifetime outcomes and healthcare system costs . A budget impact model was used to estimate the impact of each vaccine by province.In the base case, vaccination with the HPV-16/18 vaccine was predicted to prevent 48 additional CC cases, and 16 additional CC deaths, while vaccination with the HPV-6/11/16/18 vaccine was predicted to prevent 6,933 additional GW cases. Vaccination with the HPV-16/18 vaccine was estimated to save 1 additional discounted quality adjusted life year (QALY) at an overall lower lifetime cost to the healthcare system compared to the HPV-6/11/16/18 vaccine (assuming vaccine price parity). In sensitivity analyses, the HPV-6/11/16/18 vaccine was associated with greater QALYs saved when the cross-protection efficacy of the HPV-16/18 vaccine was reduced, or the burden of GW due to HPV-6/11 was increased. In most scenarios with price parity, the lifetime healthcare cost of the strategy with the HPV-16/18 vaccine was predicted to be lower than the HPV-6/11/16/18 vaccine. In the probabilistic sensitivity analyses, the HPV-16/18 vaccine provided more QALY benefit than the HPV-6/11/16/18 vaccine in 49.2% of scenarios, with lower relative lifetime costs in 83.5% of scenarios.Overall, the predicted lifetime healthcare costs and QALYs saved by implementing each of the vaccines are similar. Vaccination with the HPV-16/18 vaccine is expected to be associated with reduced CC disease morbidity and mortality compared to vaccination with the HPV-6/11/16/18 vaccine. Differences in these outcomes depend on the extent of cervical disease prevented by cross-protection and the burden of GW caused by HPV-6/11. Fifteen of the approximately 40 human papillomavirus (HPV) genotypes that infect the human anogenital area are known to be oncogenic . While mIn Canada, secondary prevention of cervical cancer through opportunistic and organized screening has reduced the burden of disease relative to other countries . HoweverGardasil®, developed by Merck) has been licensed since July 2006, and a HPV-16/18 AS04-adjuvanted vaccine , was approved in February 2010. Both vaccines provide excellent protection against HPV-16 and −18 and their related cervical outcomes. The HPV-6/11/16/18 vaccine is a quadrivalent vaccine that also provides protection against non-oncogenic HPV types −6 and −11 which can cause genital warts and CIN1. The HPV-16/18 AS04-adjuvanted vaccine has a proprietary AS04 adjuvant system which has been shown to enhance humoral and B cell responses compared to the same antigens adjuvanted with aluminium [Two HPV vaccines are currently available in Canada: a HPV-6/11/16/18 vaccine  cross-protection against CIN2+ caused by HPV-31/45 and 68.4%  efficacy against CIN2+ caused by the 10 most common non-vaccine oncogenic HPV types  in an HPV-naïve population ,26. In aEconomic modeling studies have consistently predicted that the adoption of HPV vaccination programs to prevent cervical cancer are economically attractive. In Canada, published analyses have found that HPV vaccination plus screening for cervical cancer is cost-effective compared to use of a screening program alone, with incremental cost-effectiveness ratios (ICERs) varying from approximately $18,000 – $32,000 per quality adjusted life year (QALY) gained in the base case analyses -33. OnlyA few published economic analyses have directly compared the HPV-16/18 AS04-adjuvanted and HPV-6/11/16/18 vaccines resulting in discordant conclusions. Those comparisons that did not consider the additional benefit of cross-protection concluded that the HPV-6/11/16/18 vaccine provides better value for money than the HPV-16/18 AS04-adjuvanted vaccine due to the additional costs saved and QALY gained by preventing genital warts attributed to HPV-6 and −11 -37. The Calculation of the incremental costs and benefits of a new intervention relative to existing health care strategies allows determination of efficiency or value for money. Decision makers are also concerned about the intervention’s impact on their budget because a new technology may be efficient but not affordable. It is also important for decision makers to understand the absolute clinical impact of a new intervention within their population. Budget impact models are often constructed to help quantify the number of people eligible for a new intervention as well as the costs of that new intervention in a particular jurisdiction .The first objective of this analysis was to estimate the relative clinical and economic benefit of the HPV-16/18 AS04-adjuvanted vaccine compared with the HPV-6/11/16/18 vaccine in the Canadian setting while considering cross-protection efficacy. The second objective was to look at the clinical and cost impact of implementing each vaccine within each of the 10 Canadian provinces.This study used a previously published static Markov model that reproduced the natural history of oncogenic HPV with a one year cycle length ,32. Prev® Excel 2007 and simulates the effect of adding vaccination to the current screening program, where two cohorts of 100,000 12-year-old females were followed over a lifetime, one cohort vaccinated with the HPV-16/18 AS04-adjuvanted vaccine and the other with the HPV-6/11/16/18 vaccine. The model was previously calibrated to reproduce Canadian cervical cancer incidence and mortality, while keeping transition probabilities within pre-determined ranges [The model was developed in Microsoftd ranges ,32. The d ranges  and publd ranges .Table A 98% vaccine efficacy against HPV types 16 and 18 was used based on the latest results from each vaccine’s clinical trials ,50,62-66The lifetime number of CIN lesions, cervical cancer cases, cervical cancer related deaths, genital warts cases, QALYs, and costs were determined for each cohort. An ICER (cost per QALY gained) was calculated to compare the costs and outcomes of the two vaccines. A discount rate of 3% was applied to both costs and outcomes.A number of sensitivity analyses were conducted by varying inputs assumed to impact the relative value of the two vaccines. The HPV-6/11/16/18 vaccine’s price per vaccine dose was varied to determine the point at which both vaccines would be predicted to have equivalent lifetime costs to the health care system. In a series of two-way sensitivity analyses, the efficacy of each vaccine against CIN2+ outcomes associated with non-vaccine oncogenic types was varied using the confidence intervals from the clinical trials. In addition, alternative WHO data for the continent of North America was used to populate the distribution of HPV types within cervical health states ,69. The Multivariate probabilistic sensitivity analyses were conducted to explore the combined effect of parameter uncertainty using @Risk software . Distributions were assigned to transition probabilities, vaccine effectiveness, proportion of outcomes  attributed to each HPV type, screening effectiveness, costs, utilities using normal distribution (limited from 0–1 for transition probabilities) when confidence intervals were available, otherwise, a uniform distribution was assigned ranging from 25% below and above base case value Table . In totaIn order to customize the model to each of the Canadian provinces, a budget impact module that calculated the size of the target population and the cost of vaccination was constructed. It was linked to the cost-effectiveness model in order to predict lifetime costs and outcomes. The target population was based on the number of females in the assumed target age group for each province Table  in the 2The total annual cost from the perspective of the budget holder  was calculated using the cost of vaccine purchase and administration. The net impact was calculated by subtracting the cost of providing all vaccinated females with the HPV-6/11/16/18 vaccine from the cost of providing all with the HPV-16/18 AS04-adjuvanted vaccine. The lifetime health care system costs associated with the HPV-16/18 AS04-adjuvanted vaccine plus cervical cancer screening were compared to those associated with the HPV-6/11/16/18 vaccine plus cervical cancer screening. The total cost of each strategy included the health care costs incurred by those receiving vaccination plus cervical cancer screening as well as those receiving cervical cancer screening only. The lifetime number of cervical cancer events experienced in the cohort of the assumed target population, including both vaccinated and unvaccinated individuals, was also calculated for each vaccine.The results of the base case cost-effectiveness analysis for a cohort of 100,000 Canadian females across a lifetime time horizon are summarized in Table The estimated clinical and cost impact of vaccinating with the HPV-16/18 AS04-adjuvanted vaccine and the HPV-6/11/16/18 vaccine using base case assumptions for each province is shown in Table The deterministic sensitivity analyses conducted with the cost-effectiveness model illustrate the importance of various inputs to the predicted relative clinical and economic impact of the two vaccines. Table Additional deterministic sensitivity analyses show the impact of reducing the price per dose of the HPV-6/11/16/18 vaccine, changing the assumed HPV distribution or altering the burden associated in genital warts  is increased. In all scenarios, except where the cross-protection benefit of the HPV-16/18 AS04-adjuvanted vaccine is assumed to be lower than the HPV-6/11/16/18 vaccine, vaccination with the HPV-16/18 AS04-adjuvanted vaccine is associated with an overall lower lifetime cost to the health care system when the vaccines are priced at parity. In the probabilistic sensitivity analyses, the HPV-16/18 AS04-adjuvanted vaccine provides more QALY benefit than the HPV-6/11/16/18 vaccine in 49.2% of scenarios, while it has lower relative lifetime costs in 83.5% of scenarios. The budget impact model analyses illustrate how the clinical and cost differences between the vaccines may manifest using estimated target populations in each of the Canadian provinces.Two other publications have compared the costs and benefits of the HPV-16/18 AS04-adjuvanted vaccine and the HPV-6/11/16/18 vaccine while considering the current cross-protection efficacy data from clinical trials. Capri and colleagues used a population model and estimated that the additional cross-protection benefit associated with the HPV-16/18 AS04-adjuvanted vaccine would prevent an additional 295 cases of cervical cancer in the Italian population compared with the HPV-6/11/16/18 vaccine . They alIn a third publication, Demarteau and Standaert conducted analyses using the model in this publication adapted to France, Ireland, and Italy to determine the impact of cross-protection on the relative cost-effectiveness of a hypothetical bivalent and a hypothetical quadrivalent vaccine. The bivalent vaccine was assumed to prevent 95% of HPV types −16 and −18 infection while the quadrivalent prevent 95% of HPV type −16, -18, -6, and −11 infections. Both vaccines were assumed to provide lifetime protection against HPV-16, while efficacy against HPV-18 was assumed to wane after 10 years. With the quadrivalent vaccine, efficacy against HPV-6 and −11 was also assumed to wane after 10 years. The discounted incremental cost per QALY of the bivalent vaccine compared to the quadrivalent vaccine fell below a cost-effectiveness threshold of the gross domestic product (GDP) when the bivalent provided an additional 22% in cross-protection efficacy in France, 48% in Ireland, and 43% in Italy. The WHO suggests that interventions with an incremental cost per QALY below this threshold be considered as highly cost-effective .This analysis is subject to limitations common to all decision analytic models in that it combines data from numerous sources, requires structural and data assumptions, and can be subject to certain biases. Given the uncertainty in epidemiological data, a number of simplifying assumptions were made. Co-infection with multiple oncogenic HPV types for example is not explicitly modeled. Patterns of HPV infection and cervical cancer screening practices were modeled based on average Canadian data and may therefore vary from actual practices in each of the provinces. The health economic model that generates estimates of lifetime costs and clinical impact in this analysis is a static Markov model reproducing disease burden in women, and as such does not account for the benefits associated with herd immunity of female vaccination on men. In terms of cervical cancer outcomes, the impact of vaccinating females may therefore be underestimated for both vaccines. We did not directly model outcomes in men who may benefit from a reduction in transmission of HPV-6/11 and an associated reduction in genital warts. We have attempted to simulate the impact of this in a number of sensitivity analyses, however, this indirect benefit can only be estimated with a dynamic model.In this analysis, it was assumed that both vaccines provided lifetime protection, but the true duration of protection of the vaccines is not yet known. In a comparative clinical immunogenicity/safety study of the HPV-16/18 AS04-adjuvanted vaccine and the HPV-6/11/16/18 vaccine, the HPV-16/18 AS04-adjuvanted vaccine induced superior neutralizing antibody response compared to the HPV-6/11/16/18 vaccine for both HPV-16 and −18 in 18–45 year old women up to 24 months after first injection ,89. AlthHPV infection may cause cancers at other sites in women, including anal, vaginal, vulvar, and head and neck cancers, and these outcomes were not modeled in this analysis. Jit and colleagues  demonstrBased on our model, implementation of an HPV-immunization program in Canada using the HPV-16/18 AS04-adjuvanted vaccine is expected to be associated with a lower lifetime cost and a similar number of QALYs saved compared to the HPV-6/11/16/18 vaccine under base case assumptions. Overall, the HPV-16/18 AS04-adjuvanted vaccine reduced additional cervical cancer disease morbidity and mortality compared with the HPV-6/11/16/18 vaccine but had no impact on the morbidity associated with genital warts. From an economic perspective, the lifetime relative costs and QALYs saved by implementing each of the vaccines will depend on the assumptions about the extent of cervical disease caused by HPV types prevented by cross-protection, and the burden of genital warts caused by HPV-6/11. The results of this analysis may therefore differ in countries with a different portion of cervical disease attributed to HPV types impacted by cross-protection or a higher genital warts burden.Cervarix® is a registered trade mark of the GlaxoSmithKline group of companies. Gardasil® is a registered trade mark of Merck and Co., Inc.AIS: Adenocarcinoma in situ; CC: Cervical cancer; CAD: Canadian dollars; CIC: Canadian Immunization Committee; CI: Confidence interval; CIN: Cervical intraepithelial neoplasia; CP: Cross-protection; GDP: Gross domestic product; HPV: Human papillomavirus; ICER: Incremental cost-effectiveness ratio; Onc: Oncogenic; PEI: Prince Edward Island; QALY: Quality adjusted life year; SD: Standard deviation; WHO: World Health Organization.MK, DL, JH are employees of OptumInsight, and OptumInsight was contracted by GlaxoSmithKline to collaborate on the research project and manuscript production. ND is an employee of GlaxoSmithKline Biologicals. AA was an employee of GlaxoSmithKline and is now an employee of Abbott Laboratories.MK led the design of the analyses, the design of the budget impact analysis and drafted the manuscript. DL participated in the design of the study, performed the analysis, and assisted in drafting the manuscript. JH helped design and build the budget impact model, performed all of these analyses. ND conceived of the study, and participated in its design and coordination. ND created the original cost-effectiveness original model and AA populated the model with Canadian inputs. ND oversaw the probabilistic sensitivity analyses. All authors reviewed and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/872/prepub


To evaluate the pattern of co-infection of human papillomavirus (HPV) types in both sexes in Sweden.Cell samples from genital swabs, first-void urine, and genital swabs immersed in first-void urine were collected in the present cross-sectional High Throughput HPV Monitoring study. Overall, 31,717 samples from women and 9,949 from men (mean age 25) were tested for 16 HPV types using mass spectrometry. Multilevel logistic regression was used to estimate the expected number of multiple infections with specific HPV types, adjusted for age, type of sample, and accounting for correlations between HPV types due to unobserved risk factors using sample-level random effects. Bonferroni correction was used to allow for multiple comparisons (120).Observed-to-expected ratio for any multiple infections was slightly above unity in both sexes, but, for most 2-type combinations, there was no evidence of significant departure from expected numbers. HPV6/18 was found more often and HPV51/68 and 6/68 less often than expected. However, HPV68 tended to be generally underrepresented in co-infections, suggesting a sub-optimal performance of our testing method for this HPV type.We found no evidence for positive or negative clustering between HPV types included in the current prophylactic vaccines and other untargeted oncogenic types, in either sex. Persistent infection with human papillomavirus (HPV), a common sexually transmitted infection, is considered a necessary cause of cervical cancer and is also associated with other anogenital cancers in men and women Current prophylactic HPV vaccines target the two most oncogenic types, HPV16 and 18, and one of the two vaccines also targets two non-oncogenic types, HPV6 and 11. A possibility exists that the decrease of certain HPV types through vaccination could lead to a shift in the distribution of HPV genotypes, with an increase (replacement) or decrease ; venereology/urology clinics; and primary care units. Samples collected for Chlamydia trachomatis testing were also analyzed for HPV DNA. All samples were anonymized and age and sex are the only information available for study participants. The Regional Ethical Review Board for Southern Sweden approved the study and decided that informed consent for HPV testing was not required, since samples were anonymized. We cannot, therefore, identify which samples are repeat tests on the same individuals and the total number of participants is not known exactly. Although the study size is reported in terms of samples, not individuals, it is estimated that the number of unique individuals is 78% of the number of samples The HT-HPV study was a survey of HPV prevalence among young sexually active women and men in Southern Sweden. The aim of the HT-HPV study was to establish baseline data to monitor the input of HPV vaccination. The study was based on participants in the Chlamydia trachomatis testing is performed on samples from various sites of the body, the present analysis is restricted to urogenital samples, with samples taken from the rectum, eye, pharynx and other sites being excluded. These restrictions leave a total of 31,717 samples from women, of which 13,347 were genital swabs immersed in first-void urine, 10,624 first-void urine and 7,746 genital swabs . From men, 9,949 samples were available, of which were 9,360 first-void urine, 535 urethral swabs and 54 genital swabs immersed in first-void urine.Overall, 44,146 urine and swab samples were collected  between March and October 2008 using the multi-Collect Specimen Collection Kit and following the manufacturer’s instructions . The present analysis is restricted to individuals aged 12–45 years. In addition, as Chlamydia trachomatis testing using the Abbott m2000sp system according to the manufacturer’s instructions , the residual DNA was tested for HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, using polymerase chain reaction (PCR) followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS), as previously described DNA was extracted for Among all types detected by MS-analysis, HPV68 was the type with the lowest probability to be detected (91%) when samples contained 200 copies per reaction. The detection probability was 26% when HPV68 was present in low copy numbers, i.e., 20 copies/reaction or in presence of other HPV types (detection probability = 17%) . Furthermore, the ME primer for HPV68 showed some cross-reactivity with HPV70 and consequently HPV68-positive samples were retested by using a Luminex-based detection with probes for HPV68A (GenBank accession number DQ080079), HPV68B , and HPV70 for confirmation of the results The present analysis focused on 16 HPV types, including all 13 oncogenic types, i.e., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 A total of 31,717 samples from women  and 9,949 from men  were included in the present analysis. Type-specific prevalence for women and men was described in detail elsewhere In appendix , we presIn women, the proportion of HPV types involved in multiple infections ranged between 58.4% for HPV11 to 70.0% for HPV33 . In men,Our large cross-sectional study of patterns of HPV types in multiple infections shows that, although HPV types often cluster together due to the common route of transmission, 2-type co-infections tend to occur at random in both sexes.Our findings are thus consistent with previous studies from different areas of the world that showed, in both sexes, high frequency of multiple HPV infections but no significant excess or deficit of any 2-type combination The evaluation of HPV type patterns in multiple infections requires large studies on account of the need for multiple testing. Adequate statistical power is especially important when assessing the significance level of negative associations that involve rare HPV types. In the present study, there was no evidence of systematic clustering for most HPV types. Two of the three statistically significant associations in women involved HPV68, with either HPV6 or 51. However, HPV68 revealed a general tendency to be underrepresented in 2-type infections, although in most instances negative associations did not reach any of the two chosen levels of significance. The same behaviour was not observed for any other HPV type, including HPV6 that was also involved in two significant clusters: one more frequent and the other less frequent than expected. The most likely explanation of HPV68 pattern in multiple infections is a diagnostic artefact, on account of a less-than-optimal performance of the MS diagnostic method to detect HPV68, particularly where HPV68 is present in low copy numbers and in the presence of other HPV types.Confirmatory testing of HPV68-positive samples with Luminex was performed to overcome cross-reactivity of HPV68 with 70 in the MS method. This step had the potential to improve the specificity but not the sensitivity of HPV68 testing in our study. Restriction to the original MS-based testing data did not make any difference (data not shown).The excess of co-infections involving HPV6 and 18, two HPV types that are both targeted by current vaccines and that belong to different alpha species , has no clear explanation, but we cannot exclude that it could be due to an extreme play of chance. An excess of co-infections, also emerged for genetically similar types HPV56 and 66, both belonging to alpha-6 species, but it was not statistically significant after Bonferroni correction.Chlamydia trachomatis testing programme cannot be ruled out.Overall HPV prevalence in our study was higher in women (38.5%) compared to men (11.1%) HPV testing in urine is affected by methods of collection, storage, DNA extraction, and the conservation medium used to avoid DNA degradation Genital swabs included in our study were collected in large majority from the cervix in women and the urethra in men and, therefore, our findings in swabs are likely to reflect the patterns of HPV co-infections in these genital sites. The comparison of findings in samples from women that included genital swabs with urine samples showed that, despite lower HPV-positivity in urine, the patterns of multiple infections with HPV types were consistent in the two sample types.Information on sexual behaviour was not available in the HT-HPV study. Therefore, the inclusion of sample-level random effects was particularly useful in order to allow for unobservable risk factors common to all HPV types. Sample-level random effects, in fact, reduced substantially the O/E ratio for multiple HPV infections in both sexes. Sample-level random effects keep into account unobserved risk factors but do not allow to distinguish the effect of sexual behaviour from that of other risk factors, e.g., immunological susceptibility. The small excess of multiple HPV infections, present even after the inclusion of random effects, has also been observed in previous studies in both genders A further limitation of the present study is that a proportion of samples, estimated to be around 22%, were repeat tests on the same individuals. As samples were anonymized, we could not identify repeat samples and allow for correlated measures, but we assumed that they were randomly distributed across subjects. Should that not be the case, we, however, expect that the use of random effects will prevent point estimates of O/E ratios from being substantially affected by the presence of repeated samples. Credible intervals, however, may be smaller than in the case of independent tests but this limitation should, if anything, lead to a larger number of significantly increased 2-type co-infections.In conclusion, understanding whether certain HPV types have a tendency to cluster together in unvaccinated populations is important to evaluate the potential for type-replacement and cross-protection in HPV vaccine recipients and to understand better the natural history of the infection. Our study confirms in both sexes the lack of evidence of negative and positive clustering between types included in the current prophylactic HPV vaccines and other untargeted oncogenic types. In the present analysis, HPV6/68 and HPV51/68 were observed significantly less often than expected, but that was part of a general tendency of HPV68 to be underrepresented in combination with all other types. Lack of multiple infections involving HPV68 was likely to be due to a less-than-optimal performance of the MS primer for this type. The elimination of some HPV types by vaccination is thus unlikely to have any major effects on the occurrence of other HPV types.Table S1Observed-to-expected ratio of multiple human papillomavirus infections, according to two models: in urine samples only (a); genital swabs only and genital swabs immersed in first-void urine (b), in women, Sweden. HPV: human papillomavirus; O: observed; E: expected; CI: credible interval. aControlling for age and type-specific HPV prevalence; bAs a plus sample random effects.(DOC)Click here for additional data file.


Pap test, and especially HPV DNA test, identify a large group of women who do not have any clinically relevant lesions, i.e., CIN2+ , but who are at greater risk of getting lesions in the future. The follow up of these women needs new biomarkers with prognostic value. The objective of this study is to evaluate the prognostic value of E6/E7 mRNA over-expression assay  for 5 HR-HPV types  for progression to CIN2+ after a negative colposcopy. This prospective study, conducted at four Italian centres, enrolled 673 women with either a negative colposcopy or a negative or CIN1 histology. The clinical end-point was histological confirmation of CIN2+. Women were classified at baseline according to mRNA results and managed according to local colposcopy protocols. At least one conclusive follow-up test was obtained for 347 women . Only seven CIN2+ were detected during follow up, three among the 82 women positive for mRNA at baseline, two among the 250 negative , and two among the 12 with an invalid test. Absolute CIN2+ risk was 6.7/1,000 person/years in the whole cohort. The absolute CIN2+ risk was 18.4/1,000 person/years and 3.6/1,000 person/years in mRNA-positive and mRNA-negative women, respectively. In conclusion, E6/E7 mRNA over-expression appears to be a good candidate as a prognostic biomarker to manage HR-HPV DNA-positive women with negative colposcopy or histology, particularly in order to decrease follow-up intensity in those who are negative. Cervical cancer screening is one of the most effective interventions ever implemented in medicine These women are difficult to manage since infections and cytological modifications can take a long time to clear and regress Biomarkers are needed to identify those HPV infections at risk of developing into a high-grade lesion in a short amount of time, i.e., less than the normal screening interval. The most promising targets for such novel biomarkers are the E6/E7 viral oncogene expression and its molecular consequences Commercially available robust assays for HPV mRNA detection can be performed by reflex after liquid-based cytology or HPV-DNA test In this study, we measured the prognostic value – the ability to identify women who will develop CIN2 or 3 during follow up – of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) in women with negative colposcopy or CIN1 histology.This prospective study collected data from four Italian gynaecological prevention clinics: a research hospital , two teaching hospitals , and one cervical cancer screening centre . All are public institutions and virtually all the tests are paid for by the National Health Care System. The Regina Elena National Cancer Institute Ethics Committee confirmed that the study did not require IRB approval, only IRB notification, based on Italian legislation . Patient consent was not required, based on the Italian personal data protection code (legislative decree no. 196 dated June 30 2003). The patients were managed strictly following the clinical indications related to their pathology and according to the National Guide Lines and good clinical practice. They did not receive different care on the basis of study findings and study results did not affect patient treatments. All data was analyzed anonymously.We recruited all patients who met the following inclusion criteria: they had an mRNA test; they were > = 18 years of age; they had a colposcopic assessment within five months from mRNA test, with negative result without biopsy or a negative or CIN1 biopsy. Women were referred for colposcopy because of an abnormal Pap test or a positive HPV test.Exclusion criteria were: treatment for cervical lesions in the previous five years or a history of any type of cancer; having a CIN2 or more severe cervical neoplasia (CIN2+) at baseline colposcopy-guided biopsy; undergoing a surgical or ablative treatment during baseline colposcopy.Enrolment lasted from January 2004 to December 2006. All subjects were followed up until 30 June 2011. Recruitment started from the date of collection of the cervical sample for the mRNA test. Tests performed more than 5 months after baseline assessment colposcopy were considered as follow-up tests in order to rule out that repeated colposcopies were due to technical reasons and to be sure of not including prevalent lesions.Follow up was performed according to local colposcopy protocols. Negativity for CIN2+ at a given time was defined by a negative cytology or a negative HR-HPV DNA test. In the event of ASC-US or more severe cytology or a positive HR-HPV DNA test, being negative for CIN2+ was determined by means of a negative colposcopy or a colposcopy-guided biopsy that was histologically negative or CIN1. When the last follow up episode is a positive cytology or HPV not followed by colposcopy, the episode was considered not conclusive and the follow up was censored at the last conclusive episode. Women were considered positive for CIN2+ only with a histology-confirmed result. Women with surgical or ablative treatment, without histological confirmation of CIN2 or more severe diagnosis, were censored.Cervical samples were taken by cytobrush  and plastic Ayre’s spatula (Hologic) according to the manufacturer’s instructions and stored in 20 ml of PreservCyt solution (Hologic) at 4°C until use. Liquid-based cytology was prepared using the ThinPrep 2000 System following the manufacturer’s instructions (Hologic). The cytological specimens were reported using the 2001 Bethesda reporting system Five ml of PreservCyt solution were used for the detection of E6/E7 mRNA of HPV types 16, 18, 31, 33, and 45 within 14 days of sample collection by means of the PreTect HPV-Proofer Kit (referred to as the mRNA test)  according to the manufacturer’s instructions. mRNA was extracted using the RNeasy Mini Kit . The PreTect HPV-Proofer utilizes an isothermal nucleic acid sequence-based amplification (NASBA), which amplifies mRNA in a DNA background, detecting and genotyping HPV transcripts in the same reaction. The amplified products were detected in real time using fluorescent-labelled molecular beacon probes directed against full-length E6/E7 mRNA. Accumulated mRNA fluorescent profiles were analyzed and ascribed positive/negative status, for each type included, by the supplied PreTect analysis software. Human U1 small ribonucleoprotein (U1A mRNA) was used as an RNA integrity/adequacy internal control. When the U1A amplification was not detected, the test result was qualified as invalid.Testing for HR-HPV DNA was performed by the HC2 test (QIAGEN), a semi-quantitative signal-amplified hybridization assay for the chemiluminescent detection of the 13 most common HR-HPV types , according to manufacturer’s instructions. Before performing the HC2 test, 4 ml of PreservCyt solution were processed with the HC2 Sample Conversion Kit (QIAGEN). The positive cut-off (CO) value was considered the mean of the positive control samples. The results were considered positive when the ratio between the relative-light units of the sample (RLU) and the chosen positive CO (RLU/CO) was 1.00 or higher. The repeat zone between 1.00 and 2.50 was not used.All statistical analyses were performed with STATA software version 11. The difference in CIN2+ incidence between positives and negatives to the mRNA test at baseline, as well as progression to CIN1 and CIN1 persistence, were calculated with the Log Rank test.The mean age of the enrolled women was 35.6 years (SD = 10.0); the range was 18–71 years, with 96% between 21 and 65 years. One hundred sixty-three (24.6%) women were negative for HR-HPV DNA, only three of whom had a positive mRNA test. No CIN2+ occurred in the 81 who had at least one follow-up test.vs HR-HPV DNA negative p = 0.152)  .vs 3.7, 95% CI 0.5–13), although ASC-H or H-SIL cytology significantly predicted the risk of CIN2+ .The 7 CIN2+ had the following cytological results at baseline: two Negative for Intraepithelial Lesion or Malignancy (NILM), two Low Grade Squamous Intraepithelial Lesions (L-SIL), one Atypical Squamous Cells cannot exclude High Grade Squamous Intraepithelial Lesion (ASC-H), and two High Grade Squamous Intraepithelial Lesions (H-SIL) . BaselinMoreover, as shown in Although the association between E6/E7 mRNA overexpression and CIN severity has been demonstrated in several studies The risk of developing a CIN2+ is five times higher in mRNA-positive than in mRNA-negative women. The latter make up about three quarters of our sample and have a risk of 3.7/1,000 person/year, only slightly higher than those in the general population H-SIL and ASC-H cytology identified a small group of women at very high risk, whereas there were no differences between negative and ASC-US or L-SIL cytology.It must be noted that HR-HPV DNA test is more sensitive than mRNA test and no CIN2+ occurred in the two-year average follow up of the 81 HR-HPV DNA-negative women. Most of the guidelines recommend that the HR-HPV DNA-negative women with negative or CIN1 colposcopy should be referred to normal screening interval A lower sensitivity and higher specificity of PreTect HPV-Proofer assay compared to HPV-DNA tests and to other E6–E7 mRNA tests, have already been reported It must be highlighted that two CIN2+ lesions occurred during the follow up of the 27 women with invalid mRNA result, making this group at high risk. Previous studies observed that women with invalid mRNA result were not a random sample of the average tested population Neither the progression from negative to CIN1 histology nor the persistence of CIN1 was associated with mRNA positivity at baseline. This observation, even though limited by the very low statistical power of this study, confirms the idea that CIN1 should not be considered as a screening target, it should not be treated Despite the relatively high number of eligible subjects, we had a high proportion of loss to follow up and very low risk of progression to CIN2+ in HR-HPV DNA-positive women compared to what has been found in other cohort studies There are many possible explanations for the low risk of progression found in our cohort: 1) the study subjects came from a highly screened population, with very low prevalence of high grade lesions; 2) during follow up, in our study the colposcopy and biopsy rate were quite low compared to previous studies High-grade cytology (H-SIL and ASC-H) is still the strongest predictor of developing a colposcopically detectable CIN2+ and the follow up after negative colposcopy should be intensive for women with this cytology. On the other hand, in well-established screening programmes, women with low-grade cytology may be retested after longer intervals than currently performed. E6/E7 mRNA overexpression for HPV types 16,18, 31, 33, and 45 seems to be a good candidate as a prognostic biomarker to determine the intensity of follow up in HR-HPV DNA-positive women after a negative colposcopy or histology, even though more research is needed.


Claspin is a nuclear protein involved in DNA replication and damage response and is a key mediator for the S-phase checkpoint. Claspin expression is significantly high in several human solid tumors. Furthermore, high levels of claspin have been found in cervical cancer cell lines. Nevertheless, no data are available regarding claspin expression in cervical tissues.In order to investigate whether claspin immunoreactivity is related to the lesion severity and High-Risk (HR) HPV infection, we analyzed claspin expression by immunohistochemistry in a series of cervical biopsies which represent the steps occurring during cervical carcinogenesis . All patients also had a cervico-vaginal sample for HPV testing, collected immediately before the colposcopy-guided biopsy. The HR-HPV DNA detection was performed by the HR-HPV Hybrid Capture 2 test. HPV genotyping was performed using the Linear Array HPV Genotyping Test.2trend < 0.0001). In fact, the normal tissues displayed either no or faint claspin immunoreactivity, whereas a moderate/high positivity was observed in 16% of the CIN1, 76% of the CIN2, 87.5% of the CIN3 and 93.3% of the cancers. Moreover, we found a statistically significant correlation between claspin expression and HR-HPV infection (pχ2 < 0.0001), irrespective of the genotype. Finally, we demonstrated the feasibility of claspin immunostaining in cervical cytology.Our results evidenced a constant and significant increase of the rate of claspin positivity from the normal tissues to carcinomas (pχin vivo claspin expression is significantly related to HR-HPV infection and lesion grade both in histological and cytological samples. Therefore, the analysis of claspin expression could be clinically relevant in the diagnosis of HPV-related cervical lesions, in particular when applied to cervico-vaginal cytology. Moreover, giving information on the proliferation rate of each lesion, claspin immunostaining may contribute to the evaluation of progression risk, thus being helpful in patient management. Nevertheless, only large prospective studies may clarify the true clinical usefulness of claspin expression in distinguishing lesions with different progression potential.Our findings indicate that  In addition, the cell lines expressing the HPV-E7 oncoprotein also showed an increased claspin turnover [Claspin is a nuclear protein involved in both DNA replication and damage response and is a key mediator for the S-phase checkpoint. Its main function is to facilitate phosphorylation of the effector kinase Chk1 by the sensor kinase ATR in response to replication stresses . Inhibitturnover .Therefore, in addition to viral markers, such as E6/E7 mRNA or protein detection ,10, cellin vivo and, as a consequence, if it could be used as a putative biomarker of dysplasia and/or HR-HPV infection. To this end, we firstly analyzed claspin expression by western blotting in a series of both HPV negative and positive non tumorigenic and tumorigenic cell lines. Furthermore, we determined immunohistochemical claspin distribution, in parallel with HR-HPV DNA status, in a retrospective series of human cervical biopsies which encompass a representative spectrum of dysplastic and neoplastic cervical lesions, aiming at investigating whether claspin expression correlated with the grade of the lesions and HR-HPV infection in vivo.In the present study, we addressed the question of whether the expression of claspin is associated to human uterine cervix transformation Finally, in the knowledge that identifying molecular alterations in cytological specimens might improve the detection of cervical lesions at higher risk of transformation in both primary screening and triage settings, we evaluated claspin distribution in a subset of available cytological samples, including negative, low and high grade squamous intraepithelial lesions, collected in parallel with the cervical biopsies.The human cell lines NHEK, HK-168, SiHa, CaSki, and HeLa were used. They were kindly provided by Dr. Federico De Marco . NHEK is a HPV negative human keratinocyte diploid cell line; HK-168 is a non tumorigenic cell line derived from primary human keratinocytes transfected with HPV16 and containing about 1–2 viral copies/aploid genome . SiHa, CNHEK and HK-168 were cultured in a K-SMC  definite medium while SiHa, CaSki and HeLa were cultured in DMEM supplemented with 10% of fetal bovine serum and antibiotics .Analysis with rabbit polyclonal antibody against claspin  was performed according to a published protocol . Brieflya priori power calculation based on data from our laboratory regarding Ki67 reactivity in cervical pathology (data not shown). In fact, given that Ki67 is a well known proliferation marker which is widely applied in solid tumors [This study was reviewed by the Ethics Committee of the Regina Elena Cancer Institute. We selected 80 cases from the Regina Elena Cancer Institute files, which had been collected between June 2006 and July 2009, submitted to a colposcopy-guided cervical biopsy immediately after a cervico-vaginal sampling for HPV testing. All histological slides were independently reviewed by two experienced pathologists and an adjudicated final diagnosis was established. Of the 80 formalin fixed biopsies, 9 had no relevant lesions , 19 were diagnosed as Cervical Intraepithelial Neoplasia (CIN) 1, 21 as CIN2, 16 as CIN3 and 15 as invasive Squamous Cell Carcinomas (SCC). For the purpose of our study, the WNL and CIN1 diagnoses were referred to as CIN2-, while CIN2, CIN3 and SCC were referred to as CIN2+. Therefore, on the basis of this classification, we analyzed 28 CIN2- and 52 CIN2+ cases. This sample size was adequate to detect a significant difference at 1% level with a statistical power of 90%, according to an Of the 80 cervico-vaginal specimens collected in PreservCyt  for HPV testing, 19 had a residual sample which was sufficient to obtain thin layer slides which were adequate for both morphological and immunocytochemical analyses. The thin layer slides were prepared using the ThinPrep 2000 System (Hologic), following the manufacturer’s instructions. Papanicolau slides were interpreted independently from all other findings, and classified according to the Bethesda 2001 guidelines , by two The immunohistochemistry (IHC) performed to detect claspin expression was carried out on 3 μm thick sections cut from formalin-fixed paraffin embedded blocks. We used a monoclonal antibody which was kindly provided by Professor T. Halazonetis (University of Geneva Switzerland), and directed against the amino-acidic residues 785–1056 of the full-length protein as previously described [The immunocytochemical analysis of the 19 available cervico-vaginal samples was performed following the above described procedure, after fixing Thin-Prep slides in 10% buffered formalin for 20 minutes followed by water rinsing. Immunostaining was considered positive when at least one cell showed nuclear reactivity either among the superficial and intermediate typical cells or among the clearly atypical cells, independently of all other findings.The HR-HPV DNA detection was performed on cytological samples by the HR-HPV Hybrid Capture 2 (HC2) test , following the manufacturer’s recommendations. Before the HC2 test, 2 mL of each sample were processed using the HC2 Sample Conversion Kit (Qiagen). The HR-HPV HC2 assay detects the most common 13 HR-HPV types in cervical cancer: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. The HPV genotyping test was performed by the PCR based Linear Array HPV Genotyping kit , utilizing 250 μL of the residual liquid sample and following the manufacturer’s instructions. This assay is able to detect 37 high, intermediate and low risk HPV types . Only samples that were found positive to at least one of the 13 genotypes recognized by the HC2 test were considered to be HPV DNA positive by PCR. Moreover, because of the higher oncogenic potential displayed by HPV 16 and 18 types [Data were analyzed with SPSS statistical software version 17.0. . The associations between variables of interest were performed by the non-parametric Pearson Chi-Square test and the Kruskall-Wallis test, when appropriate. Analyses for trend were also carried out by using the Chi-square test for trend. A p-value < 0.05 was considered to be statistically significant. To evaluate the sensitivity and specificity for the presence of a CIN2+ lesion, the cases showing negative or low claspin expression were considered as negative whereas the cases with moderate or high claspin immunoreactivity were regarded to as positive.In order to investigate the correlation between claspin expression and uterine cervix transformation, we examined the expression of claspin by western blotting both in normal human keratinocytes (NHEK) and in a series of cell lines that resembled cervical malignancy at different stages of tumorigenesis . We found no claspin expression in the normal cell line and in the HK-168 cell line which has a basal/parabasal keratinocyte phenotype resembling the CIN2 stage , while iIn order to investigate whether results obtained on the cell lines could be reproduced in human cervical tissue samples, and whether claspin expression could be linked to the severity of the lesions, we analyzed this biomarker by IHC Figure  in a sertrend2 < 0.0001). In addition, claspin showed a high sensitivity for CIN2+ lesion (84.6%), and an even higher specificity (89.3%) (data not shown). However, due to the limited and nonrandomly selected series of cases, the accuracy indicator values should be interpreted with caution.Afterwards, according to the four-score categories of reactivity described in the Materials and Methods section, we compared the proportion of the negative/low positive and the moderate/high positive cases within each histological category Figure . The 9 Wχ2) <0.0001). In fact, 46 out of the 47 moderate/high claspin positive cases were HR-HPV positive (97.8%). In addition, 44 out of the 46 claspin positive/HR-HPV positive cases (95.6%) were CIN2+, whereas among the 23 claspin negative/HR-HPV positive cases we found only 8 CIN2+ lesions (34.7%). Therefore, among the HR-HPV positive cases, immunoreactivity for claspin seems to be associated with presence of a CIN2+ lesion.In order to investigate the correlation between claspin expression and HR-HPV infection, we compared the IHC findings with the HC2 results  ,22. Therd so far . The autFocusing on cervical cytology, we found that claspin positivity was associated with HR-HPV positivity and consistently increased with the severity of cellular abnormalities. Immunoreactivity was mainly found among cases with a histologically confirmed diagnosis. These findings, although preliminary, may have relevant clinical implications, mainly in the management of HPV positive cases. In fact, the immunocytochemical detection of this biomarker may represent a novel and useful tool which is capable of identifying those HR-HPV infections associated to a lesion. In fact, HR-HPV testing, despite its high sensitivity, is limited by a low specificity in detecting high grade lesions, since the majority of HPV infections are transient and not linked to histologically confirmed cervical dysplasia .in vivo claspin expression is significantly related to HR-HPV infection and lesion grade both in histological and cytological samples. Therefore, the analysis of claspin expression could be clinically relevant in the diagnosis of HPV-related cervical lesions, in particular when applied to cervico-vaginal cytology. Moreover, giving information on the proliferation rate of each lesion, claspin immunostaining may contribute to the evaluation of progression risk, thus being helpful in patient management. Nevertheless, only large prospective studies may clarify the true clinical usefulness of claspin expression in distinguishing lesions with different progression potential.Our findings indicate that HR-HPV, High risk human papillomavirus; WNL, Within normal limits; CIN, Cervical intraepithelial neoplasia; SCC, invasive squamous cell carcinomas; NILM, Negative for intraepithelial lesion or malignancy; L-SIL, Low-grade intraepithelial lesions; H-SIL, High-grade intraepithelial lesions; IHC, Immunohistochemistry; HPF, High power field; HC2, Hybrid capture 2.The Authors declare that they have no competing interests.MB conceived of the study, wrote the manuscript and performed literature search and review; AM performed Western blotting analyses and revised the manuscript; AV and MGD evaluated the cytological and molecular findings and addressed relationships between molecular and pathological findings; FR performed immunohistochemical analysis and HPV testing; IT performed the statistical analyses; MC and EP performed histological analyses; GV helped in the enrollment of cases; MM participated in study design, supervised all the activities, and helped write the manuscript. All authors read and approved the final manuscript.


The UK Human Papillomavirus (HPV) vaccination programme was introduced in 2008 for girls aged 12–13. The vaccine offers protection against HPV types 16 and 18, which together cause about 70% of cervical cancers. Vaccinated girls will receive future invitations to the NHS Cervical Screening Programme, to prevent cancers associated with HPV types not included in the vaccine, and in case of prior infection with HPV 16 or 18. Little is known about parents' and girls' understandings of the protection offered by the vaccine, or the need for future screening.Qualitative interviews with twenty-six parents, and nine girls aged 12–13 who were offered HPV vaccination through a Primary Care Trust (PCT) in the South-east of England, UK.Thirty-nine schools, and four general practices.Uncertainty about the level of protection offered by the HPV vaccine was evident among parents, and to a lesser extent among vaccination-aged girls. There was a lack of understanding among parents and girls that cervical screening would be required irrespective of vaccination status; some parental decisions to accept the vaccine were made on the misunderstanding that vaccination provided complete protection against cervical cancer.Sufficient awareness of the issues related to screening is necessary for informed decision-making about whether or not to accept the HPV vaccine. Clearer information is needed concerning the incomplete protection offered by the vaccine, and that cervical screening will still be required. Future invitations for cervical screening should stress the necessity to attend regardless of HPV vaccination status, to ensure that high levels of prevention of cervical cancer through screening are maintained. However, parental perspectives on the HPV vaccination, and the rationale on which decisions are made to accept or decline the vaccine are unclear, as research to date has largely examined planned rather than actual behaviour.7The Human Papillomavirus (HPV) vaccination programme was introduced in the UK in 2008 for all girls aged 12–13.8 Previous studies have reported low levels of knowledge and understanding of HPV, and the HPV vaccine, among parents and vaccinated girls11, and have shown that decisions are often made without sufficient awareness of relevant issues.12It is important that parents and girls make informed decisions about accepting the HPV vaccine based on accurate understandings of the advantages and disadvantages that vaccination offers, particularly the level of protection it provides against cervical cancer.15 There is concern, however, that not all parents and girls who accept the offer of the HPV vaccine will be aware of these issues, and may assume they are fully protected from cervical cancer.8 Such a misconception among vaccinated girls could lead to a reduction in future participation in cervical screening, and decreased protection against cervical cancer as a consequence.The NHS Cervical Screening Programme will continue to play an important role for vaccinated girls by protecting them against cervical cancers caused by the high-risk HPV types not included in the vaccine, and to help prevent cervical cancer in those already infected with HPV.16 The interview series included an exploration of the information needs of the first cohort of vaccination-aged girls and their parents, in relation to the decision to accept or decline the HPV vaccine. Uncertainties emerged surrounding the relationship between the level of protection the HPV vaccine provides against cervical cancer, and the need for future cervical screening. These issues were explored during subsequent interviews, and it is these exploratory findings that are reported in this paper.The study we report here is one component of a larger project which aims to develop evidence-based core HPV messages, relevant to the new testing and vaccination programmes, through the synthesis of systematic reviews, a series of qualitative interviews, and public surveys.Qualitative research aims to achieve detailed levels of understanding through in-depth interviews and systematic analysis, and is the method of choice when little is known about the issues under investigation. We adopted a qualitative methodology in this study as an individualized and discursive approach is well suited to the exploration of issues such as participant awareness, information needs and understandings in relation to acceptance of the HPV vaccine, and the investigation of individual values and barriers to uptake or decline in the real life setting.Qualitative data were collected from 37 parents whose 12–13-year-old daughters had been offered the HPV vaccine, and 44 girls aged 12–13 years who had been offered the HPV vaccine during the first wave of the HPV programme (September 2008) within one Primary Care Trust (PCT) in the South-east of England  to thank them for their participation in the study.One of the authors (LH) interviewed the parents between July 2009 and June 2010. The majority of interviews were conducted in the parents' home, and with the exception of two interviews which took place with both parents, were conducted solely with the mothers. The girls were interviewed by AC between October 2008 and April 2010. Thirty-eight girls chose to be interviewed in their own homes, two with their mothers present; and six girls chose small group discussions at their school.Semi-structured interview topic guides were used to explore parents' and girls' reasons for accepting or declining the HPV vaccination. The topic guide provided a flexible set of content areas  to direct the interview process, while allowing the participants to raise areas of relevance to them. Cervical screening as an area of uncertainty and relevance to the decision-making process originated from the parents, and, as the study progressed, subsequent interviews explored the understandings of the HPV vaccine in relation to cervical screening. The interviews lasted thirty minutes to one hour and were digitally audio-taped, transcribed verbatim, and anonymized. Ethical approval was granted by the Research Ethics Committee for Wales and written informed consent was obtained from the participants.18 The interviews were compared by selecting text which described similar or opposing experiences, both between interviews as well as in the context of each interview. A qualitative software package was used to help with the management of the data.19 LH and AC regularly discussed the coding and interpretation of the data to ensure a deep understanding, and to limit inconsistencies in the analytic process.The transcripts were reviewed to identify instances when parents or girls discussed cervical screening. Data was retrieved from twenty-six parent, and nine girl interviews Table . From anThe findings are presented around the three key themes which emerged during the analysis of parents' and girls' data, where cervical screening was discussed.1.The interviews revealed a range of understandings among parents about the level of protection the HPV vaccine offers against cervical cancer. At the time of making the decision as to whether or not their daughters should have the HPV vaccine, some parents believed the vaccine offered complete protection against cervical cancer.‘Well it's sort of like a vaccine against cervical cancer: that's what it seemed to come across as … in the press they seemed to try and make you think this is a cure … if they had these injections (they) won't have to worry about cervical cancer at all. It would be totally almost unheard of if you, if everybody had the injections.’ (ID:W9 – mother who consented to have her daughter vaccinated.)Other parents were at the opposite end of the spectrum, believing that the HPV vaccine offered only minimal protection against the disease.‘At the end of the day it's only against one form of cervical cancer isn't it? It's only a minor prevention really.’ (ID:07 – mother who declined to have her daughter vaccinated.)A number of parents were well informed, and aware of the limitations of the vaccine in the level of protection it afforded.‘I've had abnormal cells myself so I am aware of that  and I do get screened very regularly, so I was aware that this is an anti-cervical cancer vaccine, but I was also aware that it doesn't protect against all of the viruses.’ (ID:NP02 – mother who declined to have her daughter vaccinated.)Overall, the girls interviewed tended to have a greater understanding of the level of protection offered by the HPV vaccine, and realized that by completing the course of HPV vaccinations they would have a high, but not complete level of protection against developing cervical cancer.‘All I really know about how it will prevent me from getting cancer is it will reduce the risk of it by seventy percent – and that it's a lot better than having cancer.’ (ID:21 – 12-year-old girl who received the HPV vaccine.)2.It became clear that some parents had made the decision about whether their daughter would receive the HPV vaccine based on misconceptions about the need for cervical screening in the future. Several parents had believed that the vaccine would eliminate the need for cervical screening. The following mother perceived this to be a key benefit of being vaccinated, and had made her decision accordingly.‘It sounded very positive! (the HPV vaccine) … If it meant that people didn't have to have smear tests when they were older that was great! … I don't like smear tests!’ (ID:W10 – mother who consented to have her daughter vaccinated.)Another mother, who was unaware that the HPV vaccine offered limited protection, had also made her decision based on an inaccurate understanding. At the beginning of the discussion she had described the vaccine as ‘a miracle’, but when the incomplete protection offered by the HPV vaccine was described by the interviewer, she was surprised.‘I wasn't aware of that. So you still need belt and braces?’ (ID:W3 – mother who consented to have her daughter vaccinated.)The issue of whether or not cervical screening would be needed in the future was not part of the decision-making process for all parents. Some had greater levels of understanding than others, and were aware that cervical screening would still be necessary, whether or not the course of HPV vaccinations had been completed. This awareness was greater among parents who decided against accepting the vaccine for their daughter, possibly reflecting the additional information sought about HPV and the vaccine by those in this group. Rather than being information-based, for a number of mothers this realization came as a logical extension to knowing the vaccine only offered partial protection.‘Oh yes, you would still need to go for a smear I'm sure but I haven't had any information on that I don't think. But because you can … there's always the fluke types that people get illnesses even after they've had the jab, and there were other sorts of cervical cancer that aren't caused by HPV is what I've understood …’ (ID:NP01 – mother who declined to have her daughter vaccinated.)At the point of making the decision several parents remained uncertain as to whether vaccinated girls would need to attend cervical screening or not.‘In the future they still need cervical screening? I don't know if that is clear actually.’ (ID:07 – mother who consented to have her daughter vaccinated.)The few girls who had been vaccinated and who were aware of the cervical screening programme, usually through their mother's participation, were generally unclear of their need to attend screening in the future. The confusion surrounding screening is evident from the following quotation, which describes the belief that cervical screening will not be compulsory, but available for those who feel concerned.‘Even though you've had the injection … if you're still worried then they can say in there (the leaflet) “don't worry, have the injection but if you're still worried then you can have scans when you're older.” I think that would be quite good to put in there (the leaflet).’ (ID:54 – 12-year-old girl who received the HPV vaccine.)3.The vaccine does not protect against all of the other cancer-causing types, so it's vital that women still go for routine cervical screening tests when they are older’.21 The following parent, who consented to have her daughter vaccinated on the premise that she would not need to undergo cervical screening, only became aware of this misunderstanding when she read the Department of Health ‘Arm against cervical cancer’ leaflet at the time of interview.Several parents did not recall having received any information about the need for vaccinated girls to attend cervical screening in the future. The Department of Health information leaflet, provided to parents and girls with the invitation for HPV vaccination, states that cervical screening will still be required for vaccinated girls: ‘‘I think from reading this leaflet it sounds like you still do have that screening … I did think that actually (that screening would not be necessary), but I realize it's more about actually reducing the instances … it's not reducing the need to screen.’ (ID:W10 – mother who consented to have her daughter vaccinated.)Among parents who discussed cervical screening in the interviews, there was dissatisfaction with the delivery of the information about the HPV vaccine, and many felt they needed to conduct their own independent research on the topic.‘To my knowledge it's not advertised in school that they should get that (screening) done anyway, unless, at some point later we start getting leaflets to be aware of these things … I think a lot of the information is purely what you can find out as a family really.’ (ID:07 – mother who declined to have her daughter vaccinated.)Overall there was a profound lack of awareness among the girls interviewed concerning the NHS Cervical Screening Programme, with few having heard of smear tests, or cervical screening. During one interview clarification was sought from one girl about whether she had received any information about the need to attend cervical screening in the future.Interviewer: One of the messages about the vaccination is that cervical screening is still really important – to pick up that remaining 30% (of cancers) that you are still at risk of. But it doesn't sound as though anybody's said that to you?Girl: No. (ID:51 – 12-year-old girl who received the HPV vaccine.)Providing accurate and relevant information concerning the level of protection that the HPV vaccine offers is vital in ensuring that vaccinated girls realize the importance of cervical screening in offering protection against all cervical cancers. The following mother described the vaccine as offering a false sense of security, and the danger of vaccinated girls believing they are no longer at risk of developing cervical cancer.‘It gives you a sense of security … a false sense of security! I wouldn't be surprised if in that age group the number of people going for smears drops off because you think: “Well I've got protection you know!” You think you're vaccinated against everything – it's hard to remember always that it's that thirty percent!’ (ID:NP02 – mother who declined to have her daughter vaccinated.)This exploratory study has revealed inconsistencies among parents' and daughters' understandings of the HPV vaccine in two key areas: the degree of protection which the HPV vaccine offers against cervical cancer, and the need to attend cervical screening in the future irrespective of HPV vaccination status. The study findings also illustrated that the girls tended to have a greater understanding than their mothers of the level of protection the HPV vaccine offers against cervical cancer, but were broadly unaware of the NHS Cervical Screening Programme.21 That this key message is not being clearly communicated highlights the need for ongoing educational interventions, tailored to vaccinated girls and their parents, to clarify that they will not be completely protected from cervical cancer when vaccinated, therefore cervical screening at a later stage will remain important for the prevention of cervical cancer. Our finding that girls have little awareness of the NHS Cervical Screening Programme is unsurprising, given the age differences of girls involved in the vaccination programme, and those eligible for cervical screening. However, raising awareness of the role of screening in the prevention of cervical cancer among younger girls could be beneficial, but will certainly need to be reinforced at the time of invitation to screening, particularly in light of recent research highlighting the decline in the number of young women attending for cervical screening.22Some parents had accepted the offer of vaccination based on the belief that their daughters would not need to attend cervical screening in the future. The need for future attendance is clearly stated in the Department of Health leaflets provided to girls, and the discussion sheets designed for parents and girls.Parents' lack of awareness as to whether vaccinated girls would still need to attend for cervical screening suggests that parents are not always making fully informed decisions concerning the HPV vaccine. Despite the availability of the information relating to the continued need to participate in cervical screening, it is clearly not being conveyed in a consistent way. If parents are to make informed decisions they require clearer information, perhaps delivered in an alternative format to leaflets. Highlighting the key messages on the vaccination consent form, clearly and simply, may be one way to overcome the problem of written information not always being referred to, or understood.14 One study, conducted within the Australian vaccination programme, similarly indicated that the understandings of adolescents at the time of vaccination were unlikely to promote future participation in cervical screening.9 Together these findings clearly establish that future invitations for screening will need to stress the importance of attendance regardless of HPV vaccination status to ensure the future uptake of cervical screening is not adversely affected, risking a possible increase in the number of cases of cervical cancer. The findings of this current study are also important because they suggest parents' understandings of the need for their vaccinated daughters to attend future screening may play an important role in their decision to accept or decline the HPV vaccine. This has not been a factor previously identified as important to parents in relation to decision-making about acceptance of the HPV vaccine.23The findings of this study are supported by previous research, which notes the importance of educating members of the public to negate the danger that HPV vaccination programmes are viewed as a replacement for the existing NHS Cervical Screening Programme.One limitation of the study is the relatively small number of parents and girls among the participants in this study with whom cervical screening in relation to the HPV vaccine was discussed. The importance of these (mis)understandings emerged at a relatively late stage in the interview process, when there was limited scope for further exploration. However, the insights that have been gained are important. The understandings that parents and vaccination-aged girls have of the role of cervical screening in preventing cancers caused by the HPV types not covered by the vaccine, and whether these understandings affect decision-making about acceptance of the vaccine, need further investigation, particularly if revised information materials are to be widely accessible, understandable and relevant.A major strength of this study is that it explored the understandings and reactions of those making actual decisions about acceptance of the HPV vaccine, as opposed to the use of hypothetical scenarios and imagined reactions. We included in the sample both girls and parents who had, and had not, accepted the offer of the vaccine. It is now equally important to explore the uptake rates of cervical screening when the first cohort of girls offered the HPV vaccine becomes eligible for screening. While it will be several years before the younger cohort are invited, the older girls within the ‘catch-up’ programme are eligible now for screening within parts of the UK. An exploration of the impact of HPV vaccination on attendance at cervical screening would help to determine the relevance of these exploratory findings to practice.


Women testing positive for human papillomavirus (HPV) infection experience increased levels of anxiety that have been attributed to fears of stigmatization and developing cervical cancer. The objective of this study was to investigate the association between HPV infection and anxiety in women who were unaware they had been tested specifically for HPV, to determine if any anxiety experienced by HPV-positive women could be due to causes other than learning of test results.This study was nested within a randomised controlled trial of management of women with abnormal cervical cytology conducted in the United Kingdom with recruitment between 1999 and 2002. At baseline, prior to having a sample taken for HPV testing, the results of which were not disclosed, women were assessed for anxiety using the Hospital Anxiety and Depression Scale and asked about fears of developing cervical cancer (“cancer worries”); this assessment was repeated at 12, 18, 24, and 30 months of follow-up. Logistic regression and generalized estimating equations were used for the cross-sectional (baseline) and longitudinal analyses, respectively.Among the 2842 participants, there was no association between HPV status and anxiety among white women. Among non-white women, however, anxiety was less common among HPV-positive than HPV-negative women . Among non-smokers, cancer worry was more common in HPV-positive than HPV-negative women; the opposite association was observed among ex-smokers.Associations between HPV status and anxiety may be explained by factors other than learning of test results and may vary by ethnicity and lifestyle factors. Worldwide, an estimated 10% of women are infected with human papillomavirus (HPV) and with a lifetime risk of infection near 80%, HPV is considered to be one of the most common sexually transmitted infections Qualitative and quantitative studies have found that women testing positive for HPV experience anxiety, report feelings of stigmatization, and worry about disclosing test results to their partner, family, and friends Most studies reporting associations between HPV and anxiety have been conducted after women had received their test results and have focused on the positive test result as the only potential cause of anxiety The aim of this study was to determine if adverse psychological effects experienced by HPV-positive women could be due to causes other than learning of test results. We examined whether anxiety and cancer worries were more common in HPV-positive than in HPV-negative women using data from a large, population-based sample of women undergoing management for low-grade abnormal cervical cytology, who had undergone HPV testing but were not aware of their test result. We also assessed whether the association between HPV status and anxiety differed by lifestyle or sociodemographic risk factors for HPV and anxiety.Ethics approval was obtained from the joint research ethics committee of NHS Grampian and the University of Aberdeen, the Tayside committee on medical research ethics, and the Nottingham research ethics committee. All participating women provided written informed consent.The study population consisted of women who participated in TOMBOLA , a randomised controlled trial to determine the most effective and efficient management for women with low-grade abnormal cervical cytology  Anxiety was the primary outcome of interest assessed by means of the anxiety subscale of the Hospital Anxiety and Depression Scale (HADS) Cervical cancer worry was a secondary outcome of interest. Such worries are commonly reported in women undergoing follow-up for abnormal cervical cytology results Details of the HPV testing have been described elsewhere Information on sociodemographic characteristics was collected at study entry during the TOMBOLA recruitment appointment and consisted of questions concerning the participants' ethnicity, reproductive history, education, smoking, and physical activity.The study population was restricted to the 3258 women who completed at least half of the HADS anxiety subscale at baseline (recruitment) . Best subset regression was used to impute scores for women who had answered at least half, but not all, of the anxiety subscale questions at any time point . Women (n = 416) with baseline values missing for cancer worry (n = 36), HPV status (n = 351), or any covariates included in the multivariable analysis (n = 29) were excluded, leaving 2842 in the final analysis. Outcomes were dichotomized, with significant anxiety defined as a score of 8 or greater on the HADS anxiety subscale  Cross-sectional analyses were performed using logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) for associations at baseline between HPV status (exposure) and (a) anxiety or (b) cancer worry. Generalized estimating equations with an autoregressive correlation structure were used to take into account the longitudinal nature of the data when outcome measurements from baseline and 12, 18, 24, and 30 months of follow-up were included. To explore the impact of using a defined cut-off for the definition of significant anxiety, a sensitivity analysis was undertaken. The analyses were repeated treating HADS anxiety score as a continuous variable using linear regression and generalized estimating equations for the cross-sectional and longitudinal analyses, respectively. The analyses were also repeated without imputation for missing HADS anxiety scores. In each analysis, all variables aside from the outcomes (anxiety and cancer worry) were assumed to be time-invariant.Potential confounders or effect measure modifiers considered in the models included: trial centre , trial arm , age at recruitment smear , ethnicity , smoking , and neighbourhood deprivation  women were HPV-positive . Over haIn the cross-sectional analysis, no association was found between HPV status alone and anxiety . In the fully-adjusted model, an interaction term between HPV status and ethnicity remained (p = 0.03). Anxiety was equally likely in HPV-positive and HPV-negative white women , but was less likely in HPV-positive than HPV-negative non-white women . Similar results were found in the longitudinal analysis . When anResults from the sensitivity analyses with HADS anxiety score considered as a continuous variable were similar. No association was observed between HPV status and HADS anxiety score . Unlike the analysis with dichotomized HADS score, no interaction term remained in the final adjusted model. Although the interaction term between HPV status and ethnicity did not reach formal statistical significance (p = 0.13), there was a weak positive association between HPV status and HADS anxiety score in white women while in non-white women, HPV infection appeared to be associated with lower anxiety scores .Both the cross-sectional and longitudinal analyses suggested weak associations between HPV status and cancer worry, with cancer worry more likely among HPV-positive women than HPV-negative women . In the fully adjusted analysis, all interaction terms were removed from the model except that between HPV and smoking status . Both the cross-sectional and longitudinal analyses suggested that among non-smokers cancer worry was more common in HPV-positive women than HPV-negative women . The assDifferences in the association between anxiety and HPV status were observed by ethnicity, with anxiety less common in HPV-positive compared to HPV-negative non-white women, while cancer worry was more common among HPV-positive women, but only among non-smokers. In the total population no association between HPV status and anxiety was observed, in contrast to previously reported findings from the ARTISTIC trial A major strength of this study was that participants were assessed for anxiety prior to their HPV test and were not told specifically that they were receiving such a test, and were not told their results. As a result, receipt of positive test results can be excluded as an explanation for anxiety or cancer worry experienced by HPV-positive women. It is possible that a few women knew their HPV status from previous tests, but it seems unlikely that this would substantially affect results given the low awareness of HPV and its relation to cervical cancer at the time this study was conducted a priori hypotheses regarding effect measure modification by smoking or ethnicity; in particular, with a small number of non-white women in the study (n = 122), investigation of this association in populations with larger proportions of non-white women would be beneficial.Reasons for the observed differences in the associations by ethnicity and smoking status are not immediately obvious, but there are several possible explanations. Pre-existing or long-term anxiety share risk factors with HPV infection, and it is possible that these associations may differ between strata of these risk factors or exist only in certain segments of the population. Information on risk factors related to sexual behaviours which might also correlate with anxiety, such as number of sexual partners and age at first intercourse, were not available in this study to further investigate this hypothesis Given the transient nature of most HPV infections In the total study population, anxiety and cervical cancer worry were no more common in HPV-positive than HPV-negative women with low-grade abnormal cervical cytology who were unaware of their HPV status. Secondary analyses from this study suggest differences in associations between HPV status and anxiety and cervical cancer worry by ethnicity and smoking status. Explanations for these observed associations remain unclear and may be due to chance; however, it is possible that factors other than knowledge of test results, such as ethnicity and smoking status, may be important in explaining occurrence of anxiety and other adverse psychosocial outcomes in this population.


To compare costs and effectiveness of three strategies used against cervical cancer (CC) and genital warts: (i) Screening for CC; (ii) Bivalent Human Papillomavirus (HPV) 16/18 vaccine added to screening; (iii) Quadrivalent HPV 6/11/16/18 vaccine added to screening. A Markov model was designed in order to simulate the natural history of the disease from 12 years of age (vaccination) until death. Transition probabilities were selected or adjusted to match the HPV infection profile in Colombia. A systematic review was undertaken in order to derive efficacy values for the two vaccines as well as for the operational characteristics of the cytology test. The societal perspective was used. Effectiveness was measured in number of averted Disability Adjusted Life Years (DALYS). At commercial prices reported for 2010 the two vaccines were shown to be non-cost-effective alternatives when compared with the existing screening strategy. Sensitivity analyses showed that results are affected by the cost of vaccines and their efficacy values, making it difficult to determine with certainty which of the two vaccines has the best cost-effectiveness profile. To be ‘cost-effective’ vaccines should cost between 141 and 147 USD (Unite States Dollars) per vaccinated girl at the most. But at lower prices such as those recommended by WHO or the price of other vaccines in Colombia, HPV vaccination could be considered very cost-effective.HPV vaccination could be a convenient alternative for the prevention of CC in Colombia. However, the price of the vaccine should be lower for this vaccination strategy to be cost-effective. It is also important to take into consideration the willingness to pay, budgetary impact, and program implications, in order to determine the relevance of a vaccination program in this country, as well as which vaccine should be selected for use in the program.  CC is a significant health issue in Colombia. According to the National Cancer Institute every year there are 5,500 new cases of CC in the country . For 200The screening program implemented in 1991 has contributed to a decrease in mortality from this cause over the past 7 years. However, this decrease is less than originally expected.Epidemiological studies conducted in the 1990's provided evidence on the role of the HPV in the etiology of CC,4. This According to the World Health Organization (WHO), it is estimated that developing countries, with a high incidence of CC and difficulty implementing screening programs, are the ones that may derive the greatest benefit from vaccination against the human papillomavirus . For ColHence, an in-depth analysis of the issue is necessary, in order to evaluate the convenience of vaccination with one of the two types of vaccines available. Such analysis must be based on the evidence available so far and consider the unique characteristics of the national context such as the prevalence of infections by the different HPV types, the coverage of the vaccination programs, and the cost of the vaccine in the domestic market, among others.Consequently, the goal of this work is to compare the cost and the effectiveness of HPV vaccines in the Colombian context. This project was approved by the independent ethics committee of Universidad Nacional de Colombia – Medicine School. Since this project uses secondary data, no ethical concerns exist.A Markov model was developed in order to represent the natural history of CC and genital warts, based on prior models of the disease accessed through a systematic search in Pubmed-Medline, Embase and Lilacs. The model and the validity of the assumptions were then reviewed by gynecologists, gynecological oncologists and experts in decision-making models. The specialized TreeAge Pro 2011® software was used to build the model.The model was designed as described in The following assumptions were used for the sake of simplification:- All cases of genital warts are diagnosed and treated within the same year, and treatment efficacy is 100%. - Only healthy women may become infected with HPV 6/11 and, consequently, acquire genital warts. - The screening approach for all women who have access to it is called 1-1-3 program, which means that after two negative cytology test results, they wait three years for testing. Otherwise, they should come back the following year for another cytology test .- For cancer stages I and II, treatment will be given to those women who are identified by screening or who develop symptoms that prompt them to seek medical care. For cancer stages III and IV, considering that the probability of symptoms appearing is 0.6 and 0.9, the model was simplified by bypassing screening as a tool for diagnosing the disease. This model will measure costs in US Dollars and effectiveness in averted DALYS (Disability-Adjusted Life Years), in accordance with the WHO recommendation. DALYS aProbabilities of transition throughout the natural history of the disease were also obtained by means of a systematic search in three pre-selected databases, namely, Pubmed- MEDLINE, Lilacs and Embase. A key requirement was to collect transition probabilities specific to the Colombian population. After this search, three transition probabilities were found in studies performed in Colombia -18. ThenIt is known that prevalence of HPV types is distributed differently among countries. Also, the risk of causing CC is different between HPV types. Therefore, in order to represent the Colombian population as faithfully as possible, and consequently to assess the impact of vaccination in this country, it was considered important to obtain the probabilities for the local population that would discriminate for virus type and evaluate the efficacy profile of each vaccine not only for viral types 16/18, but also for all the other high-risk types.Given that the difficulty of finding such specific probabilities was foreseen from the beginning, the following strategy was suggested in order to adjust general probabilities found in the literature to the infection profiles of the Colombian population, based on the data supplied by the National Cancer Institute . LSIL/(HPV16/18). Given that this probability is not available in the literature specifically for the Colombian population, it was derived from the following equation: Consider, for example, the probability of progression to LSIL in women infected with HPV 16/18, where this probability may be PWhere:(LSIL/HPV)= Probability of progression to LSIL for women with infection caused by HPV of any type• P1= Proportion of women infected with HPV 16/18 among the total number of infected women• R2= Proportion of LSIL cases attributed to HPV 16/18• RThe probability of death from any cause was derived from the National Department of Statistics (DANE for its acronym in Spanish) mortality data for different ages above age 12 for the year 2008. Additionally, the coverage of Pap cytology testing in the domestic context was derived from the National Health and Demographics Survey conducted by Profamilia in 2010 and vaccination coverage was obtained from national records regarding immunization of children in 2010 . For UKIn addition, WHO’s proposed price of US$ 10-25 per vaccinated girl supports the idea of reducing the price . These pAs in previous studies, it was found that when screening coverage is reduced, the impact of vaccination is greater . In ColoAdditionally, it is important to mention that the model included the percentage of diagnosed women who underwent treatment, obtained from previous studies . AccordiOne weakness of this work is the fact that it is a predictive theoretical model based on data reported in the scientific literature and not on specific clinical findings pertaining to CC, the natural history of the disease, or the associated prevention and promotion programs. A pilot trial of vaccination would allow considering other variables that are outside the scope of this work. Additionally, it would be important to collect cost data not on the basis of a typical case but, better still, from a real-world study designed to follow these patients and to determine how much they and their families spend for the treatment of the disease. Finally, three additional analyses which were not performed for this study should be discussed. The first one is male vaccination. Previous analyses have found that vaccinating boys in addition to men is unlikely to be ‘cost-effective’ ,57, and Secondly, the effect of HPV vaccination in Human Immunodeficiency Virus (HIV) or Acquired Immunodeficiency Syndrome (AIDS) population was not included here. Even though epidemic in Colombia is considered as concentrated and low , this anOn the other hand, herd protection is suggested to be included in further analysis, since this consideration can influence the final results. For example, Elbasha et-al described that if herd protection is not taken into account, along with protection against genital warts, the ICER can increase from USD 3,000/QALY to USD 214,000/QALY . So, by Since 2009, the WHO has stated its position regarding HPV vaccination. It recommends including vaccination whenever the prevention of CC is a public health priority, as long as programs are viable, funding is guaranteed, and the cost-effectiveness profile is taken into consideration in the country, or at least in the region . This wo