We assessed the cost-effectiveness of adding a quadrivalent (6/11/16/18) human papillomavirus (HPV) vaccine to the current screening programme in the UK compared to screening alone.A Markov model of the natural history of HPV infection incorporating screening and vaccination was developed. A vaccine that prevents 98% of HPV 6, 11, 16 and 18-associated disease, with a lifetime duration and 85% coverage, in conjunction with current screening was considered.Vaccination with screening, compared to screening alone, was associated with an incremental cost-effectiveness ratio of £21,059 per quality adjusted life year (QALY) and £34,687 per life year saved (LYS). More than 400 cases of cervical cancer, 6700 cases of cervical intraepithelial neoplasia and 4750 cases of genital warts could be avoided per 100,000 vaccinated girls. Results were sensitive to assumptions about the need for a booster, the duration of vaccine efficacy and discount rate.These analyses suggest that adding a quadrivalent HPV vaccine to current screening in the UK could be a cost-effective method for further reducing the burden of cervical cancer. Despite a well-organised screening programme in the UK, and a marked decrease in cervical cancer incidence since 1988, there were 3,181 new cervical cancer cases and 1,529 deaths reported in 2002. In 2003, the National Health Service Cervical Screening Programme modified its recommendations by increasing the age to begin screening from 20 years to 25 years combined with a more frequent screening interval (every 3 years in women aged 25 to 49 years and 5 years for women between 50 and 64).®, Merck, Sharpe and Dohme (MSD), Whitehouse Station, New Jersey, USA) has been granted a marketing authorisation in the European Union [Invasive carcinoma of the cervix is preceded by premalignant lesions. These precancerous lesions are defined as cervical intraepithelial neoplasia (CIN), and classified as low grade CIN 1) or high grade CIN 2 or CIN 3) according to severity. Prevention of cervical cancer has been based on early detection of these precancerous lesions using conventional Pap smear tests or, more recently, liquid-based cytology (LBC) tests. However, with the knowledge that infection with oncogenic human papillomavirus (HPV) is necessary for the development of cervical cancer [ or high  or CIN 3an Union .The quadrivalent vaccine showed >90% efficacy in preventing pre-cancerous high grade lesions due to these two HPV types ,6. This Genital warts are the most common sexually transmitted infection in the UK. In 2004, 79,678 first attack cases of genital warts were reported in Genitourinary Medicine (GUM) clinics . ResultsWe examined the potential effectiveness and cost-effectiveness of a quadrivalent vaccine targeted at HPV types 6, 11, 16 and 18, administered to a cohort of girls aged 12 through a school-based vaccination programme in conjunction with the current screening programme in the UK over a lifetime period.We adapted a previously published and validated state-transition Markov model of HPV infection and cervical cancer ,13 to esThe model was calibrated to produce prevalence curves for HPV infection ,15, cervFor the base case, women aged 25 to 49 years were assumed to be screened every 3 years; women aged 50 to 64 years were screened every 5 years consistent with current National Guidelines . DiffereVaccination to prevent infection with HPV types 6, 11, 16 and 18 was assumed to be 98% effective, using the recent results from the FUTURE II trial to determine efficacy of the vaccine in preventing CIN 2–3 , but varWe modelled a reduction of approximately 35% for CIN 1, 55% for CIN 2 and 3 and 70% for cervical cancer . This reflects the percentage of cervical cancer and CIN 1–3 attributable to HPV types 6, 11, 16 and 18 ,35. MoreCosts for screening, diagnosis and treatment for cervical cancer as well as for diagnosing and treating warts were obtained from previously published studies ,38 and aUtilities for calculating quality-adjusted life expectancy were based on ongoing studies and are summarized in Table Health outcomes and costs are discounted at 3.5% annually for the base case. Results are presented as average lifetime costs, average life-expectancy, life year saved (LYS), Quality adjusted life year (QALY) and incremental cost-effectiveness ratios (ICERs). Strategies that were more costly and less effective, or less cost-effective than adjacent strategies were considered "dominated."The predicted age-specific annual incidence of invasive cervical cancer in the UK population is similar to the observed data in the UK Figure . The modWhen vaccination is added to screening, under base case assumptions, the lifetime risk of cancer is reduced from 0.71 to 0.29%. Considering a cohort of 100,000 women in the UK, the model estimates that around 418 cervical cancers, 127 deaths, 2,554 CIN 1, 1,683 CIN 2, 2,479 CIN 3 and 4,798 genital warts could be avoided , screening only is associated with an ICER of £11,156 per QALY. Compared to screening, vaccination combined with screening had an incremental cost-effectiveness ratio (ICER) of £21,059 per QALY and £34,687 per LYS . As shown, a combination of lower coverage, lower efficacy and short duration has an important effect on the ICER of vaccination and screening compared to screening only.Finally, as shown, the vaccine remains cost effective (with QALYs as the outcome) if the age of screening can be delayed or a less frequent screening interval used.These results suggest that adding vaccination to the current screening programme in the UK, to prevent infection with HPV types 6, 11, 16 and 18 is potentially cost-effective. The key parameters that affect this conclusion are the duration of vaccine efficacy and whether a booster is needed to achieve a duration that is sufficiently long to provide protection during the years of peak HPV incidence . Our findings are consistent with previous analyses performed in the US that show that duration affects the cost-effectiveness of screening and vaccination compared to screening only ,30. HoweTo date, all cost-effectiveness analyses of HPV vaccination show that duration of efficacy will be a key to determining how cost-effective the vaccine will be. The need for a long duration of vaccine efficacy is consistent with our understanding of the natural history of HPV infection: progression to cervical cancer can take more than 10 years . CurrentWe did not use a quadrivalent type-specific model for this analysis. There is a need for population-based data that accounts for the distribution of these specific types within CIN from the UK. In addition, data on the impact of the vaccine on the overall reduction in CIN (as opposed to the type-specific reduction in CIN reported to date) due to these specific types, in previously unexposed girls, is also needed, to confirm the pooled estimates reported in the literature. Work is currently underway to refine existing models, including the one used here, to more accurately reflect the expected type-specific reduction in CIN and cancer when girls are vaccinated using data from the UK .The use of QALYs is important since it allows us to incorporate, among other things, feelings of anxiety and embarrassment due to abnormal Pap test results as well as genital warts. However, the utilities used were derived from a study conducted among college-aged students in the US . AlthougOur model is conservative in that it does not take into account the impact of the vaccine on herd immunity. Prior analyses in the US with transmission models that accounted for herd immunity effects ,44 suggeAlthough the vaccine has recently been approved for use in the UK, its use is not mandatory . In addiOther limitations include lack of a probabilistic sensitivity analysis and the fact that the model provides a conservative estimate of the true value of a quadrivalent HPV vaccine targeted at HPV types 6, 11, 16 and 18, in terms of health benefits as it does not take into consideration the potential reduction of adenocarcinoma, vulvar and vaginal intraepithelial neoplasia, vulval and vaginal cancers, as well as laryngeal papillomatosis associated with the vaccine HPV types . In termIn conclusion, our results suggest that adding a quadrivalent vaccine to the current screening programme in the UK is potentially cost-effective. In order to more accurately quantify the effect that the vaccine will have, future models will need to account for the actual reduction in CIN and cancer based on data from the vaccine trials conducted in the UK, as well as to incorporate herd immunity effects.For this project, Dr. Kulasingam and Dr Myers have been supported by a grant from SP-MSD. Dr. Kulasingam and Dr Myers have been supported by grants from Merck and CSL-Australia. Dr. Kulasingam has been a consultant for SP-MSD and CSL-New Zealand. Nathalie Largeron is an employee of SP-MSD. Steve Bernard is a former employee of SP-MSD. Dr. Ruanne Barnabas has been a consultant for SP-MSD.SLK was responsible for the adaptation of a previously developed model used for this analysis, the analysis of the data, interpretation of results and drafting of the manuscript. SB was responsible for providing data and reviewing the manuscript. RVB was responsible for providing data and reviewing the manuscript. NL was responsible for providing data, reviewing the manuscript and providing independent confirmation of the cost-effectiveness results. EM was responsible for developing the original model used for this analysis, providing supervision for the conduct of the study and reviewing the manuscript. All authors have read and approved the final manuscript.


Human Papilloma Virus (HPV) vaccine has undergone successful trials and has recently been approved for use for the primary prevention of cervical cancer. The aim of this study was to determine knowledge and attitudes towards HPV vaccination.Semi-structured interview and questionnaire delivered in a street survey. Standardised HPV-related statements used to measure HPV knowledge and attitudes to vaccination. The setting was three different areas of Birmingham, to target a mix of social class and ethnicity. The sample population was composed of 16–54 year olds.A total of 420 participants were recruited. Poor knowledge of HPV and its links with cervical cancer were observed. 81% had a knowledge score of zero. Knowledge about HPV was associated with different ethnic group and socio-economic group. The majority (88%) of participants were in favour of vaccination, with 83.6% indicating that they would allow a child under their care to be vaccinated.Initial responses to the proposed HPV vaccination within the UK public are favourable. However, knowledge levels are poor and media and health professional promotion are required to raise awareness. Since the 1970's it has been known that one of the causes of cervical cancer is through infection with Human Papilloma Virus (HPV) . This brth October 2007, the Department of Health (England) announced the Government immunisation programme would commence in 2009 with HPV vaccination of girls aged 12–13 years . If the respondent answered "No" to the first of these questions ("Have you heard of HPV?"), then the remaining HPV knowledge questions were not asked. For the purposes of modelling, the knowledge score was reduced to a binary variable (no knowledge: knowledge score = 0 vs. some knowledge: knowledge score >0).The six questions asked comprised:1) Have you heard about Human Papilloma Virus?2) Did you know the Human Papilloma Virus can be transmitted through sexual intercourse?3) Have you heard about the HPV vaccination?4) Did you know it is a vaccination which protects women against cervical cancer?5) Did you know that the government is considering offering HPV vaccination to school girls aged 10–12?6) Did you know a lot of women have already acquired one or more HPV type(s) covered by the vaccine?"Do you think that the introduction of HPV vaccination is a good idea?" A Likert scale ranging from strongly agree (score: 1) to strongly disagree (score: 5) was used. For the purposes of modelling, the attitude score was reduced to a binary variable . The interview ended with some questions relating to how much people were prepared to pay for vaccination and whether boys should also be vaccinated.Subsequent to the provision of standardised information  relating to HPV, attitudes to HPV vaccination were assessed using the question: To facilitate modelling, independent variables were dichotomised to: manual (3M-5) vs. non-manual (1-3N), older (age 45+) vs. younger (age <45), white vs. non-white .A parent was defined as anyone with children regardless of age of the child. Parents were asked to imagine when their child was the target age (10–12 years) and to answer the questions relating to attitudes to vaccination accordingly.Data were not normally distributed. The Mann Whitney U test  and Kruskal-Wallis test  were used to test for differences between groups in knowledge and attitude scores. Mean knowledge scores are reported in the text, all groups of interest having a median score of zero. Binary logistic regression (forward stepwise method) was conducted, using SPPS v14, to explore the effect of variables demonstrated to be significantly associated with the outcomes of interest  in the univariate analyses.Ethical approval for this study was provided by South Birmingham Research Ethics Committee in November 2006 (ref: S/2006/113).One hundred and forty people in each of the three locations were recruited, generating a total sample of 420 Table . Of the Three hundred and forty  of the study participants had a knowledge score of 0 . Only 25 (5.9%) participants had a score of 4 or more. The proportion of participants with a Knowledge scores of 0–6 varied by subgroup  (Table Significant differences in knowledge scores were observed between ethnic groups (scores >0: 23% of white and 8.3% of non-white category); social class ; with increasing age , and by gender  . Predictors of having no knowledge were non white ethnicity  and being a manual worker . Older age (45+ years) resulted in a decreased probability of no knowledge . After including ethnicity, social class and age band in the model, gender was no longer a significant factor.Three hundred and seventy  participants agreed or strongly agreed with HPV vaccination, 6% had no opinion and 6% disagreed or strongly disagreed. The median score was 2.0 ; participants tending to report that they agreed with vaccination. The mean response for attitude for the white ethnic group was 1.88 Table  whereas,Logistic regression modelling explored the effect of age group and ethnicity on attitude (positive attitude to vaccination vs. other). The only significant predictor of not having a positive attitude to vaccination was non- white ethnicity .Participants were asked whether they were parents or not but this was not associated with either the knowledge score or attitude response.A large proportion (70.5%) of the study population would want the vaccination to be free. The majority (59.3%) of participants thought that the proposed age group (10–12 years) was acceptable, whereas 34.3% disagreed.The majority of study participants believed that boys should be vaccinated as well as girls (91.2%).The majority of participants (83.6%), both parents and non-parents. reported that they would give consent for their child to have the vaccination.This study assessed knowledge and attitudes towards HPV vaccination at a time before the UK Government announced its plans for a vaccination programme. Participants aged 16–54 were recruited from three different areas of Birmingham. The majority of participants had little awareness of HPV or HPV vaccination and 81% had a knowledge score of zero. Nevertheless, after the provision of brief HPV related information messages  reported concern at the proposed target age group (10–12 years old). Most participants that disagreed with this target group reported that they felt the age group was too young. This survey was undertaken at when the likely age-group for vaccination was believed to be 10–12 years. The recent announcement that the target age group has been increased to 12–13 years may result in increased acceptability. This supports the findings of previous studies in both the USA  and UK  which haThe small group of the public (6%) who expressed no opinion towards the vaccination could be due to the fact that they had no or very little knowledge about the vaccination and didn't feel informed enough to form an opinion. Increased awareness and relating to vaccination and the reasons for vaccinating girls at age 12–13 years may address this.The vast majority (83.6%) of participants (parents and non-parents) would consent for their child to be vaccinated. This implies a high uptake of the vaccination within the UK population, although reported action does not always reflect actual behaviour.Limitations of this study include the possibility of sampling bias. As with most cross-sectional surveys, this street survey was only completed by a self-selected group of individuals who may not be representative of the general population. Similarly, the language barrier may have affected uptake ratio in the ethnic minority population, however, the vast majority of the people approached were fluent in English even if it was not their first language. It is probable that those not confident in written English were enabled to participate by the use of a semi-structured interview rather than a postal questionnaire.Pilot work identified that the initial approach to the public was important; initial reactions often being that the interviewers were attempting to sell a product. Therefore, in the actual survey, all participants were approached by medical students who stated that this was a University research project.People who failed to take part in the study might differ in their attitudes towards HPV vaccination. Unfortunately, due to the nature of the street survey (purposive sampling) refusal rates were not collected and it was not possible to establish the characteristics of non-participants. It is probable that those with no knowledge of HPV would be less inclined to participate. Knowledge of HPV and the proposed vaccination programme may therefore be over-estimated and social desirability bias may mean that reported acceptability of vaccination is over-reported.This study involved researcher/participant discussion about a Sexually Transmitted Infection (STI). STI's are a sensitive subject especially within different cultures. Questioning people's attitudes towards HPV could be potentially embarrassing for the participant and may have deterred honest responses. To minimise the embarrassment experienced by participants a very simple knowledge score comprising only six questions was used; this may not have been as sensitive as more detailed surveys .This study supports the findings of previous studies in respect to the public's knowledge and attitudes to HPV and the vaccination. The majority of the existing research has assessed the attitudes and knowledge of adolescents and their parents, especially mothers -18. ThisThe study confirms that there is still a lack of information or access to information about HPV and that more needs to be done to raise awareness of HPV and HPV vaccination especially amongst the minority ethnic groups and the lower social classes. Nevertheless, if the vaccination is introduced it appears that a significant number of people would be agreeable to vaccination after the provision of the quite simple messages.There is a need for the health care service and other agencies to play a more active role in publicising, educating and informing patients on HPV and potential value of HPV vaccination.Further work is required to look at the attitudes of health professionals in the UK towards HPV vaccination.In conclusion, the success of the newly proposed HPV vaccination will depend on public attitudes and acceptance. This study demonstrates that most people state that they would accept the vaccination if it were offered, but more information is needed to increase knowledge. Despite the general view being positive, many people expressed a wish to know more about vaccination. Possible side effects, efficacy, costs and the age group were highlighted as areas of concern.The authors declare that they have no competing interests.CDW wrote the first draft of the introduction and literature review, AG and AS the first draft of the methods and MS, JP and SW undertook the analyses. Abstract, results, discussion and other sections were equally contributed to by all medical students. SW and JP supervised the project. SW had the idea for the study, and is the guarantor. All the authors contributed to the final version of the manuscript and have approved this version.The pre-publication history for this paper can be accessed here:


Human Papillomavirus (HPV) vaccines have been considered potentially cost-effective for the reduction of cervical cancer burden in developing countries; their effectiveness in a public health setting continues to be researched. We conducted an HPV prevalence survey among Colombian women with invasive cancer. Paraffin-embedded biopsies were obtained from one high-risk and one low-middle-risk regions. GP5+/GP6+ L1 primers, RLB assays, and E7 type specific PCR were used for HPV-DNA detection. 217 cases were analyzed with 97.7% HPV detection rate. HPV-16/18 prevalence was 63.1%; HPV-18 had lower occurrence in the high-risk population (13.8% versus 9.6%) allowing for the participation of less common HPV types; HPV-45 was present mainly in women under 50 and age-specific HPV type prevalence revealed significant differences. Multiple high-risk infections appeared in 16.6% of cases and represent a chance of replacement. Age-specific HPV prevalence and multiple high-risk infections might influence vaccine impact. Both factors highlight the role of HPVs other than 16/18, which should be considered in cost-effectiveness analyses for potential vaccine impact. Cervical cancer continues to be the major cause of cancer mortality among women in developing countries . VirtualThe new HPV vaccines are designed to prevent HPV 16 and 18 infections which are the cause of about 70% of invasive cervical cancer cases worldwide. Thus, they are considered to be one of the most cost-effective interventions for cervical cancer control, particularly in developing countries where cytology-based screening programs have not been successful .HPV vaccine efficacy is 93%–100% for reducing CIN/2-3 lesions associated with HPV 16 and 18 types , 5; consAlthough various cost-effectiveness analyses, including Latin American countries, have been conducted based on available HPV prevalence information, there are some concerns about the inclusion of HPV vaccines in public health programs. The factor with the greatest influence on HPV 16/18 vaccine cost-effectiveness is price per vaccinated girl ; anotherDue to ethical restrictions, the burden of cancer related to specific HPV types should be estimated based on prevalence surveys. The most important surveys have been summarized in global meta-analyses providing relevant information for designing HPV vaccines as well as for assessing their impact in all regions of the world. Some limitations on meta-analysis and original studies are the differences in PCR technologies used to determine HPV infection and the analysis of multiple infections .The abovementioned factors are crucial when considering second generation vaccines with broader coverage of HPV types and the potential benefits of cross-protection with current vaccines , 10. In The study protocol was approved by the Ethics Committee of the National Cancer Institute of Colombia and each participating institution provided further approval.The cases were retrieved from 18 clinical centers in two Colombian cities categorized as high risk (Barranquilla) and low-middle risk (Bogota) according to incidence and mortality from cervical cancer , 12. TheClinical records were reviewed and patients with a history of HIV or any other immunodeficiency condition were excluded as well as patients who had undergone chemotherapy or radiotherapy prior to specimen collection.Tissues from paraffin blocks were processed at the National Cancer Institute of Colombia and all cases were reviewed by an expert pathologist who verified histopathological diagnosis, assigned diagnostic groups, and graded according to standard histological criteria .μm sections of formalin-fixed paraffinembedded tissue were sliced from each block and placed in sterile 1.5 mL Ependorf tubes with 1 mL octane. The sections were obtained after deep cutting into the block at least twice, and microtome blade and histotechnician gloves were changed for each case.After histopathological review, ten 5–10/μL acetone and the tubes dried at 55°C. The samples were incubated in digestion buffer (100 μL of 200 μg/mL Proteinase K) for 3 hours at 55°C and 12 additional hours at 37°C. Gentle centrifugation was carried out and the enzyme inactivated for 10 minutes at 95°C. 10 μL of the sample were used for PCR analysis after additional gentle centrifugation.Paraffin was removed with consecutive rounds of octane extraction followed by 100% ethanol washes. After high-speed centrifugation of the tissues, samples were added with 10 β-globin PCR with BPCO3 and BPCO5 primer combination [β-globin negative, new sections were obtained from the paraffin block and the whole process was repeated once.To assess the quality of the target DNA, all samples were prescreened using a 209-base-pair amplifying bination . If a spHPV-DNA detection was performed by a standard GP5+/GP6+ PCR based assay, which allows for the detection of a broad spectrum of genital HPV types . SubsequμL of the PCR products were added to 150 μL 2X SSPE/0.1% SDS, and the PCR products were denatured for 10 minutes at 99°C and cooled in ice. The membrane (with the oligonucleotide probes) was incubated for 5 minutes at room temperature in 2X SSPE/0.1% SDS and placed in a miniblotter, in a way that the slots were perpendicular to the line pattern of the applied oligonucleotides. The slots were filled with the diluted PCR products (160 μL) and hybridized for 60 minutes at appropriate temperature. The samples were removed by aspiration and the membrane was washed twice in 2X SSPE/0.5% SDS for 10 minutes at 51°C.Detection of 37 individual HPV types was achieved using a Reverse Line Blot assay (RLB), with a previously described system . BrieflySubsequently the membrane was incubated with 1 : 4000 diluted peroxidase labelled streptavidin conjugate in 2X SSPE/0.5% SDS  for 1 hour and washed in 2X SSPE/0.5%. For chemiluminescent detection of hybridized DNA, the membrane was incubated for 2 minutes in 20 mL of ECL detection liquid  and exposed to a film  for 30 minutes. Films were then developed. For repeated use, the membranes were stripped and stored at 4°C.β-globin positive and HPV negative cases with the GP5+/GP6+ PCR-EIA. PCR primers and conditions were described previously [HPV E7 type-specific PCR for 14 high-risk HPV types (HR-HPV) was used to analyze eviously , 17. PCRBased on available data on incidence and number of cases for the two regions included in the study , indepenAs described by Särndall et al. , prevalet-test and the Wilcoxon rank-sum test. The distribution of noncontinuous variables was analyzed with the chi-square test and Fisher's exact test.The differences among continuous variables were analyzed with the Student's β-globin negative and DNA-HPV was not found in 5 cases (2%), leaving 217 cases for final analysis .After reviewing inclusion criteria, biological specimens from 268 cases were collected in both cities. 20 cases were excluded . 26 (10.5%) out of 248 cases were The overall mean age was 51.5, with no significant variation between squamous cell carcinoma (53.2) and adenocarcinoma (51.3). Squamous cell carcinoma (SCC) corresponded to 86% and adenocarcinoma (ACC) to 11% of cases, with no significant differences between cities .P = .04), allowing for a higher participation of less common HPV types.HPV-16 was the most common type , followed by HPV-18 (13.8% and 9.6%), HPV-45 (9.7% and 6.4%), HPV-31 (7.3% and 4.2%), and HPV-58 (6.5% and 4.2%). For the low-middle-risk city (Bogota) the first 5 abovementioned HPV types were observed in 87.7% of cases, while in the high-risk city these types corresponded to only 65.9% of cases (P > .05), and HPV-44 (the unique specific LR-HPV identified in a single infection) was observed only in SCC.HPV types found in SCC had a similar distribution to other published results from around the globe since most of the cases corresponded to the SCC histologic category , and less common types (HPV 58 and 33) occurred mostly after age 50 and even over 60 (P > .05) , HPV-45 (14.3%), HPV-18 (10.1%), and HPV-31 (5.4%); for women 50-59, the most common types were HPV-16 (55.3%), HPV-18 (21.8%), HPV-58 (8%), and HPV-56 (3.4%); for women over 60, the most common types were HPV-16 (37.9%), HPV-58 (11.9%), HPV-33 (10.7%), and HPV-31 (9.8%). When considering age distribution for a given HPV type, HPVs 16 and 18 distributed evenly across age groups; most HPV-45 infections occurred before age 50  .Our study is the largest on the subject in Colombia and one of the few published in Latin America. Previous information from Colombia included 125 cases in two studies with no ACC .Several reports indicate that ACC has increased as a result of successful cervical cancer screening programs; however, the distribution of histological types in our report is similar to other studies in the region , and theβ-globin positive samples, and overall 99% HR-HPV detection among HPV positives. The high detection rates assure avoiding underestimation of HPV prevalence particularly for HR-HPV where all cases reported a specific type.Our data show good sensitivity for the combined techniques of HPV detection, with better performance than in previous studies from Colombia, and similar to that from other regional reports and from developed nations , 21. HPVThe largest previous Colombian survey included 87 cases with a restricted 72.4% HPV detection rate, in which only five HR-HPV types were analyzed using MY09/11 (exclusive of HPV-45 which played an important role in our study) . A studyIn our study, type specific prevalence was similar to that found in aggregative analysis, where HPVs 16/18 are the most frequent . As in tAs regards ADC, HPV-18 was significantly more prevalent than in SCC (34.3%) , and theWhen we turn our attention to the participation of multiple HPV types in a single case, we find out that it correlates with population risk and is coherent with the higher prevalence of multiple infections in developing countries: Africa 12.4%, South/Central America 11%, Asia 7.8%, Europe 6.2%, and North America 5.1% .The existence of multiple infections increases the possibility of HPV replacement following vaccination and should be taken into account as a potential decliner of vaccine impact over time, mainly in developing nations where population risk and number of HPV types are higher. This issue is independent from that of assigning a specific HPV type to a specific lesion which is the main concern of vaccine efficacy in clinical trials .Multiple infections have been dealt with differently in several studies: most estimates assume unique multiple-infection several times for different HPV types, resulting in overestimation of type specific prevalence , 20. In Even so, existing information on multiple infections lacks consistency making it difficult to use for vaccine assessment: differences in PCR technique sensitivity are greater for detecting multiple infections, several reports do not present data on multiple infections, and studies reporting data on the topic use different structures. Thus, detailed information on multiple HR-HPV infections ought to be more relevant for developing countries, and this is particularly important for HPV-18. The 47% of multiple infections among HPV-18 in our study  is higheIn Colombia, as in other Latin American countries, a double peak of HPV prevalence and incidence among women with normal cytology has been described: after an initial high HR-HPV incidence in young women, there is a decline that goes up again around menopause . In our Differential age-specific type prevalence might have different effects on global vaccine impact according to age-specific cervical cancer incidence. Thus, in Colombia less than 50% of invasive cancer occurs under age 50 which would imply a slightly lower impact of HPV 16/18 vaccines when the estimations are based on age-specific HPV prevalence . On the Age-specific prevalence reveals an important role for HPV types other than HPV 16/18. The higher percentage of HPV-45 in young women  has beenIn summary, we obtained similar results as those found in previous Latin American surveys, but we observed two relevant factors with the potential to influence the impact of HPV vaccines. Our data findings on multiple infections and age-specific prevalence are consistent with the scarce data on these topics and have further brought to light the role of HPVs other than 16/18. If the impacts of the HPV-18 vaccination were to diminish due to replacement in multiple infections, cross-protection for HPV-45 would be an asset . In addiAlthough if no cross-protection against HPV-45 could be fully demonstrated, our data highlight the necessity for well-designed future generation vaccines. Immune interference when adding HPV types might have negative effects on vaccine efficacy or duration of protection , 28. TheOur results challenge the discussion on HPV vaccine impact in developing nations where factors such as lower vaccine coverage and screening compliance will decrease population effect of vaccination. Hence, we would encourage the use of our data for assessing vaccine cost-effectiveness in Colombia and other Latin American countries. Yet, reports on multiple infections must attain a more standardized structure in future studies and findings on single LR-HPV infections need to be better understood, where differences between phylogenetic and epidemiologic classifications may play a part.This is the largest study on the topic in Colombia and Latin America. With an excellent PCR technique sensitivity for HPV detection, key issues previously narrowly analyzed such as multiple infections, age-specific prevalence, and the role of HPVs other than 16/18, are demonstrated as relevant factors influencing HPV vaccine impact among high-risk cervical cancer populations (developing countries). The data are useful to improve cost-effectiveness analyses and to discuss second generation HPV vaccines.


P<0.001) and prediagnostic HPV18 seropositivity correlated with HPV18 DNA in the tumour (P<0.03). The link between prediagnostic seropositivity and type of viral DNA in the cancer implies that the carcinogenic effect of infection with these viruses is dependent on persistent presence of type-specific viral DNA.Several prospective studies with invasive carcinoma as endpoint have supported Human Papillomavirus as a cause of cervical carcinoma. However, the largest study used seroepidemiology and did not analyse presence of Human Papillomavirus DNA in the subsequent tumour. Linkage of serum bank registries and cancer registries had identified 196 women with a registered cervical carcinoma after donation of a serum sample. For the present study, biopsies for 127 cases could be located, verified to contain invasive carcinoma and be amplified by PCR. Three control women who had remained alive and without cervical carcinoma during an equal length of follow-up had been matched to each of the case women and tested for HPV antibodies. Presence of Human Papillomavirus DNA in the tumours was analysed by general primer and type specific PCR. HPV16-seropositive women had a relative risk of 4.4 (95% CI: 2.2–8.8) to develop cervical carcinoma carrying HPV16 DNA. By contrast, there was no excess risk for Human Papillomavirus 16-seropositive women to develop cervical carcinoma devoid of HPV16 DNA. Prediagnostic HPV16 seropositivity was strongly correlated with later HPV16 DNA positivity of the tumour  37, 175–180. doi:10.1038/sj.bjc.6600454www.bjcancer.comCancer Research UK© 2002  The most important risk factor for development of cervical carcinoma is infection with the oncogenic types of human papillomavirus (HPV). A series of cross-sectional case-control studies, based both on HPV DNA testing of tumours  for screening for congenital infections. Enrolment started in 1983 and is still ongoing. In 1993, the bank contained 710 000 samples obtained from 390 000 women (stored at −25°C) (The Västerbotten project (Sweden) was initiated in a county in northern Sweden in 1986 and is still ongoing. In 1993, it contained blood samples obtained from about 15 000 female residents (stored at −80°C). Each year all residents of ages 30, 40, 50, and 60 years are invited by letter to participate in a health-promoting project, including the donation of biologic samples for medical research. The response rate is usually about 65%  found no statistically significant evidence on dependence of lag time (in situ), six cases that could not be amplified by PCR in the HLA test, five cases with an empty control slide and one case where the PCR detection for HPV was done on incorrect material. The registry linkage is the same as in the previous study , 18, or 33 or of bovine papillomavirus (BPV) type 1 generated in insect cells by use of recombinant baculoviruses were the kind gift of Dr John T Schiller, National Cancer Institute, USA and Dr Martin Sapp, University of Mainz, Germany. The detection of human serum antibodies was done in the standard enzyme-linked immunosorbent assay method employing a monoclonal antibody against human immunoglobulin G (IgG) (γ chain) and a goat anti-mouse IgG horseradish peroxidase conjugate  histologic typing of female genital tract tumours . When poDNA was extracted from paraffin-embedded tissue as described by 2O as a negative control in each PCR assay. The quality of the DNA extracts was tested by amplification of HLA-DQ1 with the primers GH26/GH27, giving a PCR product of 250 bp. PCR products were controlled by gel electrophoresis (10% polyacrylamide), followed by transillumination with UV-light. Specimens not giving a DQA1 product, but giving a Gp5+/6+ product, went to further examination, while specimens not giving products were excluded from the HPV–PCR.HPV–PCR was performed with the L1 consensus primers Gp5+/6+, giving a PCR product of 150 bp  and their 95% confidence intervals (CI) were estimated by conditional logistic regression  with theHPV16 seropositive women had an increased risk to develop invasive cervical carcinoma compared to HPV16 seronegative women , which was a finding similar to our previous report from the same material . For squamous cell carcinoma the corresponding figures for HPV16 DNA positivity were 26 of 29 HPV16 seropositive, and 42 of 76 HPV16 seronegative women, respectively (P<0.001).Among 29 HPV16 seropositive women who developed invasive cervical carcinoma, 26 had HPV16 DNA positive tumours whereas 46 of 96 HPV16 seronegative women developing carcinoma had HPV16 DNA positive tumours (P=0.027).HPV18 DNA positivity was found in six of 18 HPV18 seropositive women with invasive cervical carcinoma and in 14 of 110 HPV18 seronegative women  were excluded. Twelve of these (7.8%) were excluded because the diagnosis of invasive cervical carcinoma was not confirmed. This is in line with what is shown by others  chance, (ii) cross-induction of HPV33-specific antibodies by infection with the closely related HPV16 and (iii) a biological interaction whereby previous HPV33 infection promotes the oncogenicity of HPV 16. The fact that a similar tendency for HPV33 to increase the cervical carcinoma risk together with HPV16 has been noted before (HPV serology has high specificity for sexually transmitted HPV types, since HPV seropositivity is rare among virginal or monogamous women (There were a few cases with no HPV DNA in tumour. This could represent false negative HPV DNA tests due to old tissue samples or samples of bad quality. Since most of the HPV infections are transient (In conclusion, this study advances previous prospective epidemiological studies of HPV and cervical carcinoma by providing improved case definition with reclassified histological diagnoses and by providing a within-study evaluation of the sensitivity of HPV serology, enabling correction of the relative risk estimates for misclassification. Furthermore, the finding that prediagnostic seropositivity only increases risk for development of carcinomas with the same type of HPV DNA in the tumour further supports the view that the role of HPV infection in cervical carcinoma is mediated via persistent type-specific HPV infection.


Of the screened women 89.5% should test negative for human papillomavirus and be reported as such in the final cytologic diagnosis. In conclusion, preliminary results suggest that this diagnostic pathway has the potential to improve primary cervical cancer screening and cost-effectiveness. By using a combination of testing methods to focus screening and clinical attention to cases at risk, it would be possible to lengthen screening intervals for 90% of women and to archive without further review all low-score/human papillomavirus-negative slides, representing 50% of the screening workload.Primary screening for cervical cancer precursors has considerably evolved with the introduction of new technology to improve the early detection of disease. The objective of this study was to elaborate a diagnostic pathway integrating liquid-based and computer-assisted cytology and human papillomavirus DNA testing to focus screening on women at risk which may be more cost-effective for the healthcare system. A single laboratory analysis was conducted during a 5-month period using liquid-based cytology followed by human papillomavirus DNA testing for women with an abnormal result or with previous abnormal cytology. Human papillomavirus prevalence was estimated by testing 909 consecutive unselected samples. All slides were then rescreened using automated cytologic testing and triaged into a high- or low-score group according to computer results. Of the 8676 slides scanned, 352 had a test result of atypical squamous cells of undetermined significance or worse. Two hundred and ninety-seven (84.3%) samples with an atypical squamous cells of undetermined significance or worse result and 100% of those with detection of high-grade squamous intraepithelial lesions and carcinomas (HSIL+) were triaged into the high-score group. The combination of instrument scores and human papillomavirus results indicated that 51.0% of high score/human papillomavirus-positive cases should be considered as ASCUS+, while 99.6% of low-score/human papillomavirus negative cases remained negative in the final cytologic diagnosis, representing 49.0% of all casesBritish Journal of Cancer (2002) 86, 382–388. DOI: 10.1038/sj/bjc/6600073www.bjcancer.comThe Cancer Research Campaign© 2002  Concern about the reliability of cervicovaginal cytology as a screening technique has been repeatedly voiced by both the public and health professionals in Europe and the United States. New technologies such as liquid-based cytology, automated computer-assisted screening, and human papillomavirus (HPV) DNA testing have enabled significant progress to be made in this important area of women's health.Liquid-based, thin-layer cytology reduces sampling and preparation errors of the conventional smear. It has demonstrated improved specimen adequacy and sensitivity for the detection of cervical cancer and its cytologic precursors, squamous intraepithelial lesions (SILs) . ScreeniThe AutoCyte PREP system  is an automated screening tool for thin-layer processing. Independent studies have shown it to be significantly more effective for the detection of both low-grade squamous intraepithelial lesions (LSILs) and more severe diagnoses  addresses both sources of error. It has shown both superior sensitivity and specificity in the identification of all cervical abnormalities including atypical squamous cells of undetermined signficance (ASCUS) and LSILs alone or higher, when compared to current laboratory practice (Detection of high-risk HPV (HR-HPV) types by the Hybrid Capture II system  is of diagnostic and prognostic value. Recent studies show that HR-HPV types are strongly associated with SIL severity and cervical cancer . No selection criteria were applied to the cohort. Liquid-based samples were obtained from 33 gynaecologists of the region for routine screening purposes. A subset of 10% of specimens was created to determine the prevalence of HPV infection in the study population. Table 1Cohort study participants were 8676 women who had a routine cervical Pap examination in Geneva, Switzerland, between September 2000 and January 2001, a population already studied with a low incidence of cervical cancer  for final diagnosis. Final case diagnoses were classified according to the Bethesda nomenclature system and were considered as ‘reference diagnoses’.Following preparation of the AutocytePrep slides, the remaining samples were used, according to our usual procedure, to conduct HPV DNA detection in patients reported positive for ASCUS or atypical glandular cells of undetermined significance (AGUS), or worse. Patients with normal results but with a previous history of abnormal cytology were also tested. The prevalence of HPV infection in the study population was estimated by testing a subset of 10% of unselected consecutive cases.g for 10±1 min. The supernatant was decanted and the residual removed by blotting the inverted tube on clean absorbent paper. Specimen Transport Medium  (200 μl) was added to each cervical cell pellet and the tube vortexed for 15 s to resuspend the pellet. Denaturation reagent of 100 μl was then added to produce single-stranded DNA reacting with 13 full-length RNA probes recognizing HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, vortexed for 5 s, and incubated in a 65±2°C water bath for 90±5 min. The denatured specimen was then immediately tested by using the Hybrid Capture II assay according to manufacturer's instructions. A test was considered positive for the presence of HR-HPV-DNA if the relative light unit (RLU) obtained from the luminometer equalled to or exceeded the mean of the three positive control values . A RLU measurement <1.0 pg ml−1 indicated either the absence of the 13 HR-HPV types or HPV DNA levels below the threshold of detection.HPV testing was performed on a routine basis using the Hybrid Capture II assay. Each centrifuge tube contained 0.8 ml of residual cervical cells in water and was centrifuged at 800±15 AutocytePrep slides were then subjected to the AutoPap, an automated primary screener , step IIAll the results were entered into a Microsoft Access database  for analysis and elaboration of the screening and management diagnostic pathway . Trends From September 2000 to January 2001, a total of 9665 specimens were collected and initial screening was conducted using the AutoPap system. Of these, 8688 (89.9%) qualified for scanning and 977 (10.1%) were classified as PR and were excluded from the study because rerun was impossible. Nine hundred and sixty-five were cytologically negative and 12 were positive . HPV DNA testing could not be performed for 12 positive cases  because the residual sample was considered to be unsatisfactory or the clinician did not propose an HPV test. Finally, 8676 cases were included in the study.The age-group distribution of the subset of 909 cases to estimate the prevalence of HPV was similar to the entire study population . A prevaTable 2P<0.001.An association was found between HPV positivity and cytologic diagnosis. Sixty-six out of 868 (7.6%) of cytologically-negative cases without a past history of abnormal cytology were HPV-positive. In contrast, 21.7% (63 out of 290) of cytologically-negative cases with a past history of abnormal cytology were HPV-positive. Finally, 35.9% (46 out of 128) of ASCUS, 83.7% (150 out of 179) of LSIL and 95.5% (43 out of 45) of HSIL+ were HPV-positive. Chi-square for trend were 575, HR-HPV positivity and automated scoring were not closely related. This is not surprising since the Autopap system uses a statistical classifier algorithm to assign a numerical score to each slide. In the presence of benign cellular changes sharing some morphologic similarities with significant cervical abnormalities, the slide is triaged into the high-score group. These slides may be HR-HPV negative. On the other hand, HPV positivity is found in samples without cervical abnormalities; in these cases, the slide may be triaged into the low-score group.Although organized screening programs based on the Papanicolaou (Pap) smear have been very successful in reducing mortality, a major problem has emerged. Cervical cancer has not been eradicated and its incidence has remained virtually constant for several years. In Switzerland, cervical cancer is still among the 10 leading causes of cancer with approximately 400 new cases and 100 deaths annually, mostly occurring in women over 65-years-old . The majHowever, false-negative results from Pap smears do occur, leading to deaths in routinely screened women. Results of one study showed that 14% of women with an invasive cervical cancer or HSIL had received a negative smear result within the 2 years prior to diagnosis. Approximately two-thirds of false-negative smears were related to sampling errors, and the remaining third were due to screening and/or interpretative errors, mainly due to the small number of diagnostic cells present in a suboptimal smear .Studies carried out elsewhere have also confirmed that the Pap smear is not a highly effective primary screening test in the detection of cervical cancer and its precursors . It was It has also been demonstrated that false-negative rates of less than 5% are unlikely to be achieved  and may It is reasonable to expect that the ultimate way forward in the next 5–10 years will be the use of standardized liquid-based cytologic preparations, automated screening, HPV DNA testing, early detection with molecular markers, or completely novel methods. These technologies can provide improved sensitivity and specificity but at increased cost. For this reason, the way in which we use them will define the future of cervical cancer prevention. We believe that they should be considered as complementary diagnostic tools and not as competing methods. Their combination has been shown to increase sensitivity and specificity  could then be archived without further review since virtually all of them are negative in the final cytologic diagnosis. In 0.4% of these slides, we found some minor grade abnormalities (eight ASCUS and eight LSIL) corresponding to a false-negative rate of 5.2%. This rate compares favourably with the false-negative rate for ASCUS and LSIL reported in routine practice. The detected abnormalities are considered to be related to a transient HPV infection or an inflammation and will regress spontaneously. It is also highly unlikely that an underlying HSIL+ lesion will be missed since all HSIL+ will be triaged into the high-score group. In our study, 95.5% of these cases were HPV-positive.The largest volume of patients with abnormal cytology requiring focused clinical attention and triage concerns those identified with ASCUS and LSIL. In the context of an ASCUS cytologic diagnosis, the triage by HPV DNA testing is considered as a sensitive method for the detection of an underlying HSIL . Failure to detect the accepted rates would suggest that the Bethesda classification criteria are incorrectly applied.The ability of this diagnostic pathway to identify cases with a high probability of abnormality and those that can be archived without further review will assist cytotechnologists to screen more accurately slides designated for review. In addition, the tedious part of manual screening and associated tiredness that occurs when predominantly normal slides are screened will be significantly reduced since attention is focused to cases at risk.A problem that may emerge with the introduction of the new approach is the long-term surveillance of the HPV-positive/ cytology negative cases. We estimate that it will concern approximately 7% of the screened women in our population. The clinician caring for the patient may be concerned by the sexual transmission of the HPV infection or of leaving untreated an underlying significant lesion, and this may lead to an increase in the number of colposcopies. We consider that the availability of more accurate information on this topic to women and the clinician would ensure that both would be more at ease with an annual follow-up only. It should be emphasized that HPV is a very common virus and for most women the infection is transient and very unlikely to progress to cancer. Two HPV tests 8 to 12 months apart are sufficient to allow clearance of infection. Only persistence of certain types is a risk factor and this risk can be eliminated by participating in regular recommended cervical screening programmes. The major concern of our cervical cancer screening approach is the impact on the cost-effectiveness of this combination of new technologies. We are presently conducting a study to evaluate this aspect in detail. Substantial savings are expected to arise from the extended screening interval, the reduced rate of recalls, the 50% rate of archived slides requiring no further review from the cost-effective oriented quality control and from the reduced screening time of those slides requiring manual review. Savings in terms of absence from work and travel for unnecessary repeated tests and treatments, as well as in terms of creating unnecessary anxiety to women, need also to be evaluated. The issue of the age at which to start combined screening tests is another important component of a successful and cost-effective system (In conclusion, our study indicates that a diagnostic pathway combining new technologies is realistic and has the potential to improve cervical cancer screening. This approach could be cost-effective for the patient and clinical practice as it focuses screening and clinical attention to cases at risk, thus allowing to effectively test and treat women for significant cervical abnormalities. Healthcare costs could be reduced by lengthening screening intervals to 90% of women and by archiving without further review 50% of the slides. However, additional clinical studies are necessary to validate these preliminary results and to assess the cost-impact of introducing new technologies.


In the last few years much attention has been focused on the implementation of human papillomavirus detection in population based screening programmes to identify women at risk for cervical cancer. Short-term fluctuations in prevalence of human papillomavirus were investigated within a single menstrual cycle. The highest prevalence was found at the follicular phase (55%), whereas the cumulative prevalence was 75%.British Journal of Cancer (2002) 87, 373–376. doi:10.1038/sj.bjc.6600485www.bjcancer.comCancer Research UK© 2002  Epidemiological and molecular studies over the past two decades have convincingly demonstrated that certain types of human papillomavirus (HPV) are etiologically related to the development of most cases of cervical cancer .On a weekly basis at four visits during their fertility screening cycle, cervical swabs were taken for HPV detection. The first cervical sample was taken in the menstrual period (2nd or 3rd day of the cycle), the second sample between the 7th and 11th day of the cycle, the third sample around ovulation (between the 12th and 15th day) and the fourth sample in the luteal phase . Ovulation was ascertained by ultrasound examination. Cytological classification was done by an experienced pathologist.10-PCR). HPV genotyping was performed via a reverse hybridisation line probe assay (LiPA), capable of detecting and genotyping 25 different HPV types simultaneously . All women except one were non-smokers. The use of oral contraceptives was discontinued by all, at least 1 year before attending our fertility clinic. The mean age of sexarche was 18.6±4.5 years. Nine women had been monogamous since their sexarche, while the other 11 women were at least monogamous for the past 2 years. The mean frequency of sexual intercourse was 2.3 weekly.Chlamydia serum antibodies were present in four patients. Urine samples from these four women, obtained to test for an active chlamydia infection, were all negative.Three patients (15%) had a history of abnormal cervical smears. Two of them had ASCUS in 1986 (Table 1At the end of the study 15 (75%) women appeared to be positive for HPV at least one time within one menstrual cycle. Single point measurements of HPV ranged from 20 to 55% .In the follicular phase (7th to 11th day) a higher rate of HPV positive samples was found compared to the other phases, which was significantly different according to the chi-square test (Fifteen different HPV genotypes were detected of which 73.3% were high risk. Three (15%) patients harboured HPV 16 in their lower genital tract, whereas five (25%) women tested positive for HPV 18 for at least one time within the menstrual cycle . ChlamydFive women in the HPV positive group (33.3%) contained multiple HPV genotypes in one or more of their cervical smears. In one patient seven different oncogenic HPVs were detected of which type 56 and 66 were consistently present . Whereas others found a significantly higher prevalence of HPV in the luteal phase of the cycle (The high prevalence of HPV in the follicular phase of our study group is remarkable. A possible explanation for this finding is the physiological widening of the cervical canal in the follicular phase, resulting in the collection of more or different clinical material from the deeper layers of the endocervix. This could also be an explanation for the predominance of HPV 18 in the follicular phase. After all, in a recent study of the prevalence of HPV in cervical adenocarcinomas it was demonstrated that HPV 18 was the most prevalent followed by HPV 16 (per se could have led to a population based bias in HPV prevalence. However, the cervical HPV prevalence in a group of infertile women undergoing ovarian stimulation with gonadotrophins varied between 6.8 and 7.8 %, which was similar to 8.4% in the control group (It could be argued that fertility problems In women with normal cytological cervical smears, a cumulative prevalence of HPV as high as 75% in one menstrual cycle has never been reported. This result suggests that single point detection of HPV underestimates the true prevalence of HPV in a population. Sequential scrapes appear to be necessary to determine the true HPV status in individual cases.We conclude from our study that detection of HPV on single sample basis may lead to false negative results. These results may have important implications for population based HPV screening.


The world age-standardised prevalence of high-risk HPV (hrHPV) infection among 5038 UK women aged 20–59 years, with a low-grade smear during 1999–2002, assessed for eligibility for TOMBOLA  was 34.2%. High-risk HPV prevalence decreased with increasing age, from 61% at ages 20–24 years to 14–15% in those over 50 years. The age-standardised prevalence was 15.1, 30.7 and 52.7%, respectively, in women with a current normal, borderline nuclear abnormalities (BNA) and mild smear. In overall multivariate analyses, tertiary education, previous pregnancy and childbirth were associated with reduced hrHPV infection risk. Risk of infection was increased in non-white women, women not married/cohabiting, hormonal contraceptives users and current smokers. In stratified analyses, current smear status and age remained associated with hrHPV infection. Data of this type are relevant to the debate on human papillomavirus (HPV) testing in screening and development of HPV vaccination programmes. Infection with human papillomavirus (HPV) is necessary for the development of cervical cancer  and are Human papillomavirus population prevalence mainly depends on patterns of sexual exchange , which vSubjects were women assessed for eligibility for TOMBOLA , a randomised controlled trial (RCT) of alternative management policies and HPV triage .Women aged 20–59 years, resident in Grampian, Tayside or Nottingham, with a low-grade smear ) taken routinely in the UK national cervical screening programmes (CSPs) during 01/10/1999–31/10/2003, with no previous treatment for cervical lesions, were eligible for TOMBOLA. Recruitment was in two phases: 01/10/1999–12/03/2001 and 13/03/2001–31/10/2003. During phase one, eligible women had no abnormal smears in the previous 3 years; during phase two, they had up to one BNA smear in the previous 3 years. In phase one, women with a BNA smear were invited to a recruitment clinic approximately 6 months later for a follow-up smear and a swab for HPV testing. Women with a mild smear during both phases, or a BNA smear during phase two, were invited to a recruitment clinic approximately 2 months later and a swab for HPV testing was taken. The swab, an endocervical sample, was taken with a cytobrush; this was immersed in 2 ml of sterile phosphate-buffered saline containing 0.05% thiomersal.n=5514) consented to participate; 27 were subsequently excluded because the smear was inadequate. Five thousand and seventy-four (92%) of the remainder provided an HPV sample.A total of 52% of eligible women attended a recruitment clinic, of whom 95% . Adequate samples underwent HPV PCR using GP5+/6+ consensus primers, followed by enzyme immunoassay for detection of 14 high-risk types  . Human and viral DNA were extracted using the Qiagen UK kit  (QIAamp® DNA Mini Kit) following optimisation of the manufacturer's protocol. Each batch included negative controls containing only elution buffer AE. Extracted DNA was amplified and quantitated by type-specific real-time polymerase chain reaction (PCR) for the housekeeping gene, human betaglobin. Samples without recordable DNA levels were considered inadequate . For other women, the ‘current’ smear was the smear that triggered the invitation to participate in TOMBOLA. Six smear status categories were defined: normal and one previous BNA; BNA and no previous abnormal; BNA and one previous BNA; mild and no previous abnormal; mild and one previous BNA; and moderate or severe dyskaryosis and one previous BNA.χ2-tests. High-risk HPV prevalence was age-standardised to the truncated (20–59 years) world standard population. Multivariate unconditional logistic regression models were constructed to assess factors associated with hrHPV+ve risk. Since age modified the relationship between smear status and hrHPV status, these factors were fitted by a single variable, which combined the six smear status categories with two age groups (20–29 and ⩾30 years). The global likelihood ratio test (LRT) was used to assess the impact of lifestyle and socio-demographic factors on age-and-smear status-adjusted risk estimates; factors with P<0.1 were retained in the final model. To explore sub-group heterogeneity, the modelling was repeated stratifying by age  and current smear . All multivariate models had adequate fit , non-white ethnic groups  or those with a previous abnormal smear . Since two of three centres did not take swabs from menstruating women, the proportion providing a sample varied by centre . It did not vary by tertiary education level .Younger women, white women and those with a single BNA or mild smear were less likely to provide an HPV sample than older women (P(interaction) 0.0004). The overall age-standardised prevalence was 34.2% (95% CI 32.6–35.8). For women with a current normal smear, it was 15.1% (95% CI 12.6–17.6), for those with a current BNA smear 30.7% (95% CI 8.6–32.8) and with a current mild smear 52.7% (95% CI 48.6–56.8).The crude hrHPV prevalence was 39.2%  37.8–40.5). Positivity declined with increasing age , and increased with increasing smear grade. For all grades, prevalence and risk were higher in younger (20–29 years) than older (⩾30 years) women, but not by a constant amount  and physical activity .In multivariate age-stratified analyses of women aged 20–29 years, age and smear status were significantly associated with infection. Tertiary education and having had children were also significant risk factors, with risk estimates similar to those in unstratified analyses. In women aged 30–59 years, age and smear status were significantly associated with infection, as were tertiary education, having had children, ethnicity and smoking; effect sizes were similar to those in In multivariate smear-stratified analyses of all smear groups, age was significantly associated with infection. Having a college/university degree reduced infection risk in women with a current normal  or BNA smear  but not in those with a mild smear. In all smear strata, divorced/separated/widowed women had higher risk than married/co-habiting women . In the BNA strata only, being single also increased risk . Having been pregnant was inversely associated with infection in those with a current normal  or BNA smear , but not in those with a mild smear. Having had children was associated with reduced risk in all smear strata, only reaching statistical significance in the current normal group . Hormonal contraceptive use was associated with increased risk in the current normal  and BNA  strata, but not among the mild group. Barrier contraceptive use, caesarean delivery, smoking, physical activity and ethnicity were unrelated to infection in all strata.Our study was large, population-based and nested in a pragmatic RCT within the UK national CSPs. Among study participants, the current BNA : mild smear ratio (1.8 : 1) was close to that reported for the CSP screening age group in 2004–2005 (1.9 : 1) , suggestWhile TOMBOLA participation was 52% overall, it was lower among younger women and those resident in more deprived areas , groups The treatment of lesions, and possibly also the act of taking a smear, can potentially clear cervical HPV infection  was similar to that among women with a BNA smear from the UK ARTISTIC trial (31%) . In the Our crude (16.0%) and age-standardised (15.1%) prevalences among women with a current normal smear were similar to the pooled estimate from 27 PCR-based studies of cytologically normal women mainly from North America and Europe (16.2%) . HoweverNotable geographical variations in age-specific HPV curves have recently been described (n=223), precluding multivariate analysis of individual groups. Crude infection frequencies  are consistent with sexual behaviour data and suggest that the raised risk may be limited to black women.In the United Kingdom, white, black-African and black-Caribbean women have higher numbers of lifetime sexual partners, lower median age at first heterosexual intercourse and higher incidence of (non-HPV) sexually transmitted infections than women from Indian or Pakistani ethnic groups (vs never users 1.23 (95% CI 0.99–1.51); OR current users vs never users 1.46 (95% CI 1.24–1.73)). This appears compatible with the postulated mechanisms by which current OC use influences infection, which include the enhancing effect of oestrogens on cervical ectopy, thereby permitting potential carcinogens (including HPV) easier access to the transformation zone (High-risk HPV infection risk was >50% higher in women who had used OCs or other hormonal contraceptives. The latter have been studied little previously. Among ALTS participants, no association was found with injectable contraceptives or Norplant , in seveion zone .Our observation that having been pregnant was associated with reduced hrHPV infection risk is consistent with the IARC HPV Prevalence Surveys Study Group analysis of >15 000 women . HoweverIdentifying differences in the relative importance of risk factors in sub-groups can be informative – as is evident from the few previous studies using this analytical approach (


The human papillomavirus test (HPV) test could improve the (cost−) effectiveness of cervical screening by selecting women with a very low risk for cervical cancer during a long period. An analysis of a longitudinal study suggests that women with a negative Pap smear and a negative HPV test have a strongly reduced risk of developing cervical abnormalities in the years following the test, and that HPV testing lengthens the detectable stage by 2–5 years, compared to Pap smear detection alone. One of the possible uses of the human papillomavirus test (HPV) is in primary cervical cancer screening in addition to or instead of the current Pap smear .Introduction of HPV screening should be based on established (cost-) effectiveness. The (cost-) effectiveness of HPV testing is primarily determined by the duration of the detectable preclinical stage , and the sensitivity and costs of HPV testing. To estimate preclinical duration and sensitivity, longitudinal studies on the association between HPV infection and the development of neoplasias are necessary. Several large longitudinal screening studies have started, but no longterm results have been reported yet, although smaller longitudinal studies have been published  in case The study population, and screening and follow-up results are described in The disease model used in this study is schematically presented in Figure 1In the model, we assumed a constant duration of the HPV infection, and an exponentially distributed duration of low-grade CIN with a mean of 6 years. The incidence rate of HPV infections in the age group considered (34–54 years) was set at five per 1000 woman years . Using tInitially, it was assumed that there were no diagnostic errors, that is, the results of the HPV test and the Pap smear as found by In Next, we dropped the perfect-test assumptions (100% sensitivity) of the HPV test and Pap smear separately, by assuming 50% sensitivity for detecting an HPV infection and 50% sensitivity for detecting CIN I+, respectively. Furthermore, we halved and doubled our assumptions on the incidence rate of HPV infections and the duration of low-grade CIN. The estimated range for the duration of HPV before progressing to CIN widened, from 2 to 5 years. Only where the HPV test was assumed to have a sensitivity of 50% for HPV infections that will progress to CIN does the cumulative incidence 10 years after a double-negative result become slightly higher than the incidence within 5 years after a negative Pap smear.Our analysis shows duration of the HPV infection before it will progress into CIN of 2–5 years. For Pap smear screening, the preclinical duration was the combined duration of CIN and microinvasive cervical cancer, a period estimated at 15 years on average . This situation has been represented by assuming a relatively short average duration of low-grade CIN of 2 years. Together with the assumption that this stage is exponentially distributed among women, this leads to a situation in which part of the women will develop CIN III shortly after having no neoplasia. Under these assumptions, the duration of the HPV infection before progressing to CIN is estimated to be relatively long, and the selection of low-risk women by adding HPV to cytology will be even better (incidence of CIN III 2.2 The end point of the model was CIN III as imposed by the data. Invasive cancer is the end point to be preferred as prevention of invasive cancer and therefore, death is aimed at by cervical cancer screening. This end point, however, does not yield sufficient power due to the low risk for invasive cancer in Pap smear screened women, unless extremely large and long-term trials are performed. The current estimate on the duration of HPV before developing CIN is a combined estimate for the duration of HPV for women who will have a regressive CIN III lesion and those who will progress to cervical cancer. To solve the uncertainty on the confounding of regressive CIN III lesions, this prospective analysis with CIN III as end point should be accompanied by archival studies, in which retrospectively the HPV status of smears preceding a diagnosis of cervical cancer, is assessed. Doubling the screening interval for double-negative women will result in cost savings, as half of the screening rounds can be omitted. If, for example, the effectiveness of 10-yearly combined screening is the same as 5-yearly screening using the Pap smear, the costs of adding the HPV test to the Pap smear must be lower than these savings to be at least a cost-equal alternative. For a full cost analysis, other costs and savings should also be taken into account, such as the costs of follow-up in HPV positive/cytologically negative women, and possible savings due to a decrease in the detection of regressive cervical lesions because of a longer screening interval.In conclusion, adding the HPV test to cytology in primary screening for cervical cancer results in an additional duration of the detectable preclinical stage of 2–5 years. Consequently, the screening interval for women with cytological and HPV-negative test results may be considerably lengthened. These results remain to be confirmed by the large longitudinal studies that are currently underway.


RRP is a devastating disease in which papillomas in the airway cause hoarseness and breathing difficulty. The disease is caused by human papillomavirus (HPV) 6 or 11 and is very variable. Patients undergo multiple surgeries to maintain a patent airway and in order to communicate vocally. Several small studies have been published in which most have noted that HPV 11 is associated with a more aggressive course.Papilloma biopsies were taken from patients undergoing surgical treatment of RRP and were subjected to HPV typing. 118 patients with juvenile-onset RRP with at least 1 year of clinical data and infected with a single HPV type were analyzed. HPV 11 was encountered in 40% of the patients. By our definition, most of the patients in the sample (81%) had run an aggressive course. The odds of a patient with HPV 11 running an aggressive course were 3.9 times higher than that of patients with HPV 6 (Fisher's exact p = 0.017). However, clinical course was more closely associated with age of the patient (at diagnosis and at the time of the current surgery) than with HPV type. Patients with HPV 11 were diagnosed at a younger age (2.4y) than were those with HPV 6 (3.4y) (p = 0.014). Both by multiple linear regression and by multiple logistic regression HPV type was only weakly associated with metrics of disease course when simultaneously accounting for age.The course of RRP is variable and a quarter of the variability can be accounted for by the age of the patient. HPV 11 is more closely associated with a younger age at diagnosis than it is associated with an aggressive clinical course. These data suggest that there are factors other than HPV type and age of the patient that determine disease course. To our knowledge, this collection of specimens represents the largest of its type in the world. The current study brings the largest dataset yet developed to bear on the potential relationship between clinical course and HPV type. Equally importantly, and often neglected, we explore how much of the variability of the disease can be attributed to the type of HPV while simultaneously accounting for other variables such as age.Between January 2003 and April 2007 fresh papilloma biopsies were preserved in TRIzol® from 135 unique patients with RRP and were subjected to HPV typing. By using both allele specific PCR and RFLP typing on all cases we were able determine whether HPV 6 versus HPV 11 was present in the papilloma. Papillomas from 8 cases were positive for both HPV 6 and HPV 11 by both methodologies. Thus data was available on 127 unique patients who either had HPV 6 or HPV 11. Eight of the cases were excluded since we had less than one year of clinical course data . Of the remaining 119 unique cases only one had adult-onset RRP and therefore was excluded. Initial analysis was directed at determining whether or not HPV type and clinical course were associated or independent of each other. There was indeed an association between HPV 11 and a more aggressive course . “AggresRather than simply looking at the data dichotomously (aggressive vs. indolent) the total number of surgeries were analyzed as a count variable. Total number of surgeries thus far in the clinical course was log transformed since the untransformed variables were highly skewed. The geometric mean number of surgeries in the cases with HPV 6 was 16 and in the cases with HPV 11 it was 30  suggesting that subjects infected with HPV 11 tend to follow an aggressive course of the disease.The above data not only represents yet another set supporting the association between HPV 11 and aggressive course but it is also the largest data set to ever be analyzed. At this point one may conclude the case finally settled and resolve to use HPV typing as a clinical prognosticator. Yet that would be premature since other variables, and not just HPV type may be associated with clinical course.−7). On the other hand, HPV type was not related to the interval since the last surgery . . For the 6 cases with pulmonary involvement, the mean age of diagnosis(based upon square root transformed data) was 1.3 years for those affected and 3.1 years for those with no recorded pulmonary involvement . The range of age of diagnosis for those with pulmonary involvement was 0.5 to 2.6 years. In other words, all 6 cases fell below the 42nd percentile for the sample as a whole. While age of diagnosis has been associated with aggressiveness as measured by the frequency of surgery, Reeves et al. = 0.25). −5, square root of age at diagnosis = 0.015 and surgeon's style = 0.013. Fourteen observations were excluded either because the surgeon's style was missing or the patient had been enrolled at the time of the first surgical intervention and thus there was no duration between the last and the second last surgery. Similarly, mother's highest educational qualification and gross household income accounted for none of the variability. Even the anatomical component of the Derkay/Coltrera score at the time of enrollment did not account for the variability in the time since the last surgery given that the age variables and the surgeon's style had already been accounted for.It is quite conceivable that the duration between any two surgeries may be a function of more than just the current aggressiveness of the disease. Some surgeons schedule subsequent surgery as symptoms dictate, whereas other surgeons set a pre-determined duration based upon the agressiveness of the clinical course over the preceding year. Others have adopted a hybrid of the two methods. The variation in practice style was controlled for by coding the surgeon's response for each patient as 1 for “as required (prn)”, 2 for “hybrid” and 3 for “predetermined”. Adding the surgeon's style to the linear model accounted for an additional 7% of the variability . Age at diagnosis was added to the model and further accounted for some of the variability in a statistically significant manner  albeit that it only accounted for 6% of the variability. So while age at diagnosis was correlated with the duration since the preceding surgery, this correlation was very weak when contrasted against that of age at the time of enrollment in the study. Together, age at enrollment+age at diagnosis+surgeon's style of determining surgical interval accounted for 31% of the variability in the time since the preceding surgery. Adding HPV type accounted for none of the residual variability. Thus in the multiple linear regression equation where y = log (days between last and second last surgery), coefficients were calculated for 4 variables simultaneously; in other words it was not a step-wise hierarchical model where a subsequent term is allowed to explain the residual left over after a preceding term has already been entered into the model. The p-values for each of the estimated coefficients were as follows: HPV type = 0.95, square root of at age of enrollment = 6×10−7). Together, age at enrollment+age at diagnosis+surgeon's style of determining surgical interval accounted for 37% of the variability in the time since the preceding surgery. However, unlike for the variability in duration since preceding surgery, annual frequency variability was very highly significantly correlated with both age variables: age at time of measuring frequency p-value = 0.00023; age at time of diagnosis p-value = 0.00020. Adding HPV type once again accounted for none of the residual variability.Since the duration between surgery at the time of enrollment in the study and the preceding surgery utilizes information from only a small interval of the subject's clinical course it may be highly variable. Thus, a similar analysis was conducted using the annual frequency of surgeries around the time of the enrollment. The sampling period was a median of 1.8 years (range 0.9 to 2). As above, the age was transformed to the square root and the frequency of procedures in the time period surrounding enrollment was log transformed. Results were similar to that obtained above; a simple linear regression model declared that square root of the age at the time of enrollment accounted for 20% of the variability in the natural log of the annual frequency of surgeries around the time of enrollment . Logistic regression in which HPV-type was stipulated as the sole independent variable accounted for 5.6% of the deviance  and when age of diagnosis (transformed to square root) was the sole independent variable, it accounted for 7.4% of the deviance . When the two variables were handled simultaneously in a multiple logistic regression then 12% of the deviance was accounted for with the individual p-values being 0.057 for HPV Type and 0.017 for age of diagnosis.RRP is a devastating disease-sometimes because of the urgency of symptoms at presentation (such as respiratory distress), but far more commonly because of the chronicity of the disease and the multitude of surgeries undertaken. Amongst the many patients with RRP there is considerable variability in the clinical course. For many years investigators have explored the relationship between the clinical course, and whether the patient was infected by HPV type 6 or type 11, but many studies suffered from small sample size. Now, thanks to a large multi-center collaboration that was founded to explore the underlying genetic susceptibility to RRP, we have access to the largest dataset ever assembled. With confidence one can note that HPV 11 is associated with a more aggressive disease but that the clinical course is more closely associated with the age of the child which in turn is associated with HPV type.Younger children tend to have a more aggressive clinical course and so do children with HPV 11. However, HPV type is associated with both the age of the child and with the outcome, namely an aggressive clinical course. Once the age of the child is controlled for, then the relationship between HPV type and clinical course becomes one of borderline significance. We can speculate upon the association between the age of the child and frequency of surgery. Investigators have believed that the relationship is due to the relatively small airway of younger children demanding more frequent surgery to keep it clear. In our study we did not measure the diameter of the airway following papilloma removal. The closest approximation to such a value can be garnered from an article in which appropriate endotracheal tube diameter was discussed for each age Previous studies A possible criticism of our findings is that we have analyzed a biased sample of aggressive RRP cases based upon our acquisition process. The data described herein was acquired for the principal purpose of determining the underlying genetic susceptibility to RRP. The design is cross-sectional and acquires probands at any stage of their disease. Our enrollment mechanism was unlikely to reach cases diagnosed a long time ago who subsequently had an indolent course and went into remission before our study was initiated. On the contrary those who were diagnosed at a similar time but have endured an aggressive and protracted course would have been reached by our acquisition mechanism. Furthermore, we excluded 8 cases from analysis since the duration of their follow up had been shorter than a year. These factors combined, bias our sample to a generally more aggressive clinical course than the population of all incident cases tracked over their lifetime. Another weakness of this cross-sectional study is that data was not collected from each and every surgical intervention. Rather, physical findings were recorded only at the time of enrollment and summary data pertaining to history were recorded at enrollment and 1 year later. An ideal metric of papilloma burden would have been a documentation of the full Derkay/Coltrera Staging Score for each individual from the time of diagnosis to the time of remission. The area under such a curve would be less subject to stochastic phenomena in each individual's course.Less than half of RRP surgeons send papillomas for HPV typing Given the variability of the clinical course it would be useful to be able to predict when a particular child would require subsequent surgery. The current study suggests that HPV type would be of no or little utility in making this determination. Age of the patient at the time and age of the patient at diagnosis would be more accurate predictors but even with these factors taken into effect we could only account for about one third of the variability.Malignant transformation of RRP is a devastating but rare complication that has been investigated by others. Malignancy was not specifically addressed by the incumbent study. Nevertheless, it is important for the reader to be aware that other groups have investigated the features of malignant transformation and have associated them with pulmonary involvement and with HPV 11 exclusively  The cohort described herein had a similar rate of co-infection compared to that which has already appeared in the literature. In the initial cohort of 135 cases  for whom we had fresh papilloma specimens, only 8 (6%) were deemed to harbor both HPV 6 and 11. Following exclusion of cases with insufficient clinical course data there were only 5 cases with co-infection; statistical analysis of such a small group would be of questionable meaningfulness. Furthermore, one would not know if both HPV types were leading to the phenotype since it is conceivable that one of the types had caused the disease and the other was merely present much in the same way that HPV 6 and 11 have been found in the mucosa of people without RRP. Our method of typing used two methods on each specimen . Co-infection rates between papers could be difficult to interpret since methodologies vary from study to study. The RRP Task Force and the Center for Genomic Sciences, Allegheny-Singer Research Institute in Pittsburgh, PA are currently collaborating to determine the underlying genetic susceptibility in RRP. A secondary objective is to determine what the genetic factors are that account for the variability in clinical course. One of the reasons for obtaining a papilloma specimen at the time of enrollment was to determine whether the patient was infected with HPV 6 or 11. We believed that HPV type would be an important factor to control for in trying to observe an association between aggressiveness and underlying genotype. Given the data contained in this report it appears as if HPV type may not be a significant variable, or if it is, then only weakly so. While it is not known what factor(s) account for the remaining two thirds of the variability in clinical course, a reasonable hypothesis would be that host genetic factors account for the remainder of the clinical variability. A genome wide search for those factors is underway.The RRP patients studied here were identified through an ongoing collaborative study on the genetics of RRP with the coordinating site located at Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, PA Information about the clinical course of each case was supplied by their otolaryngologist at the time of enrollment and one year later updated clinical information was supplied. Metrics of clinical course included total number of airway procedures (count), frequency of procedures (count per year), ever had involvement in the trachea or distally , ever had a tracheostomy . A clinical course was defined as “aggressive” if any of the following criteria were met: total procedures ≥10, procedure frequency ever ≥4 per year, ever had distal involvement, ever had a tracheostomy tube. If none of these criteria were met the patient was considered to be experiencing an indolent course. Numeric cuttoffs can appear arbitrary but the metrics used in this study are based on precedence set by other investigator groups and importantly, were decided upon a priori. Doyle et al, working in New Orleans and Derkay, reporting a national survey of otolaryngologists, used identical or similar cut offs for classifying aggressive versus benign For each patient the surgeon was asked about the manner in which he or she decided to perform the next surgical intervention. The exact wording on the forms was, “Some surgeons operate on patients according to pre-determined intervals. Others only return to the operating room on a prn (as required) basis. Some adopt a hybrid approach whereby only the next interval is pre-determined in response to what happened over the last one or two intervals. What approach are you closest to with respect to this patient over the past year? “The surgeon's response for each patient was coded as 1 for “as required (prn)”, 2 for “hybrid” and 3 for “pre-determined”.5′-GAAGAGCCAAGGACAGGTAC-3′) and PC04 (5′-CAACTTCATCCACGTTCACC-3′) primers which span a 268 bp segment of the β-globin gene During surgery to control the patient’s disease, two 1mm by 1mm papilloma biopsies were collected by the patient's attending physician and preserved in 700 µl of TRIzol® solution contained in a 1.5 ml microcentrifuge tube. The biopsy specimens were shipped to Allegheny-Singer Research Institute, Pittsburgh, PA, the specimen and data coordinating site. DNA was extracted from laryngeal biopsies according to the manufacturer's guidelines . To assess the quantity and quality of the isolated DNA, samples were tested by spectrophotometry using a ND-1000 spectrometer (NanoDrop) and by PCR using the GH20  and Restriction Fragment Polymorphism (RLFP) on all laryngeal specimens. These two HPV typing techniques were systematically used in our specimens to ensure reproducibility and accuracy of genenotyping results. In fact, like any other PCR-based genotyping, TS-PCR is very sensitive and can produce either false positives or false negatives, which is a potential explanation for the very high frequency of dual infection reported in some RRP studies. RLFP typing using a different set of PCR primers followed by specific endonuclease digestion was used to replicate TS-PCR results and confirm HPV genotype.http://hpv-web.lanl.gov/). These sequences were aligned using BLAST 2 SEQUENCES (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) to reveal highly conserved and discordant regions between the two types. Only areas of high DNA sequence discordance were used to design primers using Oligo, a primer analysis software V.6.65 . The sequences of primers that best met Oligo PCR primers design criteria and empirical annealing temperature were as follow; HPV 6-specific primers: forward primer (6930) 5′-ACCTAAAGGTCCTGTTTCGAGG-3′, reverse primer (6932) 5′-CAGCGACCCTTCCACGTAC-3′ and HPV 11: forward primer(6931) 5′-ATATACCCTTGGGAAGCTCTCC-3′, reverse primer(6933) 5′-TTGTTGGGGCAGATATAAGTATG-3′. These primers were blasted against public genomic databases to ensure they matched to HPV 6 and 11 with high probability scores. To empirically determine the optimal annealing temperature for each primer set, a gradient PCR was then carried out separately using each TS primer and HPV 6 and 11 cloned DNA as template DNA . Annealing temperatures used were 67°C and 68°C for both primers and AmpliTaq Gold® DNA Polymerase from Applied Biosystems . HPV positive and negative control reactions were included in all HPV TS-genotyping experiments. The HPV-positive controls consisted of HPV 6 and HPV 11 cloned genomic DNA. PCR products were subjected to electrophoresis on 1% agarose gels, and visualized by ethidium bromide staining  primers were designed in the E6 (HPV 6) and L2 (HPV 11) region. Prior to selecting the primers, HPV 6 and HPV 11 DNA sequences were downloaded from the public database at Los Alamos National Laboratory  following the manufacturer's protocol. The products of the digestion were run on 1% agarose gel and visualized by ethidium bromide staining (HPV6 and 11 consensus E7 and E1 primers Forward primer: staining .http://www.r-project.org/)). Boxplots and histograms of each variable were inspected. Several variables did not follow a Gaussian distribution and thus were either transformed by taking the natural logarithm or by taking the square root of the value. Age of diagnosis, age at enrollment, total number of procedures, frequency of procedures per year, time from last operation to time of current operation and gross household income were transformed. Initial analysis consisted of performing a Fisher exact test of independence for the two binomial variables: HPV type (6 vs 11) and clinical course (aggressive vs indolent). Secondarily, multiple logistic regression was then used to analyze two independent variables that had been repeatedly described in the literature; HPV type and the age of the subject were simultaneously related to the dependent (outcome) variable of clinical course (aggressive vs. indolent). Finally, in a post-hoc manner, the 8 independent variables described above were explored in models in which total number of surgeries, frequency of surgery in a one year period and time between last and second last surgery were modeled as continuous variables. The purpose of the models was to determine the fraction of the variability in the outcome that could be accounted for by the known independent predictor variables. Specifically, we sought the contribution of the HPV type in accounting for clinical course variability in the context of other known variables. No adjustments have been made for multiple testing.Prior to any subject enrollment the primary metric of clinical course was defined as the composite label “aggressive” or “indolent” as stated above under “clinical course”. While clinical course was considered a dependent outcome measure, the following variables were considered to be independent potential predictor variables: HPV type, age at diagnosis, age at enrollment, gender, race, educational status of mother, gross household income, surgeon's style. The dataset was initially inspected by conducting a descriptive statistical summary of all the variables in isolation (R statistical software (


To examine the relationship of dietary and serum vitamin A to the risk of cervical dysplasia, a case-control study was conducted in Miyagi, Japan. Cases were 137 women who were found by Papanicolaou test screening and histological examination provided by Miyagi Cancer Society between October 1987 and September 1988 to have cervical dysplasia. Controls were selected from participants of the general health examination provided by the Society and individually matched to cases on age and screening date. The consumption of retinol or carotene-rich foods during the past 7 days was assessed at interview. Information was also collected about other risk factors of cervical dysplasia, such as reproductive histories and sexual behaviour. The mean serum retinol levels were significantly lower among cases compared with controls, although dietary intake levels of retinol and carotene were not different between the two groups. When examined by tertile, the risk of cervical dysplasia was significantly higher among women in the highest tertile of dietary vitamin A level. An inverse association was observed between serum retinol level and risk of cervical dysplasia, although it did not achieve statistical significance.