The paradigm of preventing human papilloma virus (HPV) infection through currently approved vaccines, namely, Gardasil, manufactured by Merck and Co., Inc.  and Cervarix, manufactured by GlaxoSmithKline  holds tremendous promise for the developing countries in decreasing the burden of HPV infection and its sequelae, such as cervical cancer, genital warts and anogenital cancers. Effective screening programs that have reduced the burden of this killer disease in the developed countries are still lacking in India, despite the high incidence of cervical cancer and the implementation of the National Cancer Control Programme since 1975. The recent breakthrough in the global war against cervical cancer will provide new insight for meeting the future challenge of the prevention of cervical cancer in India. Cervical cancer one of the leading cancers among women, which affects approximately 490,000 women each year, resulting in approximately 270,000 deaths worldwide. Approxim4This article reviews the available information on prophylactic vaccines against HPV infection and their relevance in the Indian scenario.HPVs belong to the family papillomaviridae. The genome of HPV consists of small 8-kb pairs) double-stranded circular DNA viruses that infect squamous epithelium of the genital tract, anal and perianal regions and mucosal epithelium of the larynx. Low-risk7-kb pairs7Although HPV-related cancers are found to be more common among women, increasing numbers of HPV-related carcinomas of the anal mucosa are also being reported in men who have sexual intercourse with men. At birthThe pathogenesis of cervical cancer is initiated by HPV infection of the cervical epithelium during sexual intercourse. The initial morphological changes in the cervical epithelia, pathologically classified as cervical intraepithelial neoplasia (CIN1), are associated with continuous viral replication and virus shedding. In most infected women, the infection resolves spontaneously in less than two years; however, 3–10% of women, in whom these infections do not resolve, become persistent HPV carriers and constitutes a high-risk group in whom these infections progress to the cancer of the cervix.Two distinct types of vaccines, one manufactured by Merck and Co., Inc. , namely, Gardasil, and the other manufactured by GlaxoSmithKline (GSK) (Philadelphia), namely, Cervarix, are available for the prevention of the HPV infection.–17 Each In June 2006, the U.S. Food and Drug Administration (FDA) has approved a quadrivalent HPV recombinant vaccine developed by Merck and marketed under the trade name Gardasil, and in September 2006, it was recommended for immunization of girls of age 9–26 years and women. It is the first and only vaccine to prevent cervical cancer, vulvar and vaginal precancers caused by HPV types 16 and 18 and genital warts caused by HPV types 6 and 11. In July 2007, the European Committee for Human Medicinal Products recommended GlaxoSmithKline's experimental HPV vaccine Cervarix for sale and marketing in the European Union and will soon receive the final regulatory approval.With regard to efficacy, both the vaccines have demonstrated remarkably similar results in the clinical trials and offered 100% protection for the type-specific lesions.18The HPV vaccine is licensed for use among women and girls in the age group of 9–26 years. Ideally, females should be vaccinated before their sexual debut because the vaccine is most effective in women who have not yet acquired any HPV type infection. Vaccine schedule requires three doses to be administered over a period of six months . The dose of the quadrivalent vaccine is 0.5 ml to be administered intramuscularly in the deltoid muscle. The vaccine should be shaken well before use, stored at 2°C–8°C and should not be frozen. The price of three dose series of Merck's quadrivalent vaccine is approximately $360 in the US. Undoubtedly, the price will be one of the greatest barriers to the introduction of this vaccine in the developing countries. Drastic revision of the price will be required to introduce this vaccine in the poorest parts of the world to facilitate its timely utilization.This will only be possible with subsidies provided from the Global Alliance for Vaccines and Immunization (GAVI) and assistance from agencies such as the World Health Organization (WHO), the Alliance for Cervical Cancer Prevention (ACCP), the International Agency for Research on Cancer (IARC), the Bill and Melinda Gates foundation and Program for Appropriate Technology in Health (PATH), who are ready to provide technical and financial support to developing countries; thus, this vaccine can be brought to India.The decision of the Indian Government to join the clinical trials of the HPV vaccine holds some promise for the women to fight this battle against the malignant HPV infection. The ICMR signed an agreement with Merck and Co., and clinical trials for HPV vaccine are under study to observe its efficacy and safety in the Indian population. The Institute for Cytology and Preventive Oncology (ICPO) has signed a Memorandum of Understanding (MOU) with Merck and has taken the initiative to develop the vaccine against HPV.A big challenge for us is to determine how to include HPV vaccine in a Cervical Cancer Control Programme, or what should be the mechanism to deliver the vaccine in Indian scenario?Many critical questions will be raised by the clinicians, public health professionals and public at large, and these will have to be addressed before the vaccine is made available in India.A major decisive question to be addressed is that how will the vaccine affect the tests used to screen Cervical Cancer? Will it be a replacement for the standard screening tests?We all know well from our past experience that large-scale routine screening programmes, followed by treatment of the affected is difficult to achieve in our country; however, a cost-effective vaccine can act as another line of defense, provided the women who need it the most may be able to receive it. Unfortunately, the number of women receiving three or more antenatal checkups is quite disappointing (16.9% in Bihar and 26.3% in Uttar Pradesh) in some of the Empowered Action Group (EAG) states according to the National Family Health Survey (NFHS III); this wilMoreover, the currently available HPV vaccines do not protect against all types of HPV; they provide protection against only the two most common high-risk types of HPV (16 and 18) which cause approximately 70% of cervical cancers. Females who are vaccinated could subsequently be infected with one of the less common types of HPV that the vaccine does not protect against. Furthermore, it is possible that sexually active women before vaccination could have been infected with a vaccine type HPV before vaccination.Another tricky question is that who is eligible to be vaccinated primarily: women or girls?This question needs to be answered in a very subtle manner because of religious and cultural taboos in our country.Therefore, the core part of a good public health policy against cervical cancer should be vaccination and screening program complementing each other. The availability of cervical cancer vaccine will enable to reduce the cost burden for cervical cancer screening programs. It is time that the government takes steps to place cervical cancer screening, HPV vaccination and Information Education and Communication (IEC) for cancer control on its high agenda to reduce the increased burden of this disease and its consequences.


Human papillomavirus (HPV) vaccination has been approved in more than 90 countries and is being implemented in many of these. In the UK, vaccination for girls aged 12–13 with catch-up for girls up to age 18 was introduced in 2008, using the bivalent GSK vaccine (Cervarix).We modelled the proportion of abnormal smears, cervical intraepithelial neoplasia grade 3 (CIN3) and invasive cancer, which will be prevented in women aged 20–29 in the UK as a result of HPV vaccination.It will take many years for the full benefit of vaccination to be achieved. The earliest effects will be seen in women aged 20–29. With 80% coverage in women aged 12–13, we project an eventual 63% reduction in invasive cancer, a 51% reduction in CIN3 and a 27% reduction in cytological abnormalities before age 30. The full effect in this age group will not be seen until 2025, although half of the benefit will be seen by 2019 in England, where screening starts at age 25. However in Scotland and Wales, where screening starts at age 20, 50% of the benefit for CIN3 and abnormal smears (but not cancer) will be seen earlier.Substantial reductions in disease can be anticipated by vaccination, but most of the benefit will not be apparent for at least another decade. High vaccine coverage is the key factor for achieving these benefits. High-risk human papillomavirus (HPV) types have been found in more than 95% of women with invasive cervical cancer  followedTo date two vaccines that protect against HPVs 16 and 18 are commercially available, Gardasil  and Cervarix , for intramuscular use in three doses over 6 months; Gardasil also protects against HPVs 6 and 11, which cause genital warts. The vaccines also differ by the type of adjuvant used. Vaccination after infection appears to have no therapeutic value for either vaccine. Several randomised trials have been reported for each vaccine. The phase III trials focused on persistent infection and cervical intraepithelial neoplasia (CIN) associated with the relevant HPV types  afforded by vaccination before infection .The relative protection in older girls compared with a girl vaccinated at age 13 . For vacThe likelihood of being vaccinated. This is expressed relative to the coverage in girls aged 12–13 who would be offered vaccination as part of a school-based programme .In our model, the relative protection afforded by vaccination depends on the product of four factors:Factors 1 and 2 are taken to be independent of age (under 30) and calendar year. Factors 3 and 4 are determined at the time of vaccination and carried forward as the woman ages. We calculate the number of events expected in single year age groups and then present the (average) rate in combined age groups .The HPV vaccine programme in the UK is primarily school based. Provisional data submitted by Primary Care Trusts in England by the 30 April 2009 report that 86.2% of girls aged 12–13 have had their first HPV jab and 82.6% have received their second jab . CoveragOn the basis of these experiences, our base case assumption is that coverage will be 80% at age 12–13 and somewhat lower at older ages , but we Data from the vaccine trials indicate that vaccination does not convey protection once the subject has been exposed to the virus  will be insufficient over the next 20 years to provide any material herd immunity.Both vaccines have reported information regarding cross-protection .However it is still premature to estimate the magnitude of the effect of cross-protection on cancer incidence precisely.The percentage of all cervical cancers attributable to HPV 16/18 in Europe has been estimated at 73%  of cervical cancer in women aged 20–29 being preventable by vaccination , however these estimates were based on only 26 women with CIN3 or worse. Estimates for CIN2 or worse are based on 143 cases and indicate a vaccine efficacy of 70.2% (96.1% CI 54.7–80.9). Another relevant clinical end point is how many women are treated for cervical disease. The PATRICIA trial observed a 68.8% (96.1% CI 50.0–81.2%) reduction in the number of cervical excision procedures .Results for women aged 20–29 from the ARTISTIC trial report that 28% of borderline or mild and 60% of moderate or worse cytology outcomes were attributable to HPV 16/18 . Thus, wDirect observation from the PATRICIA trial yielded a more moderate 26.3% (96.1% CI 14.7–36.4) reduction in referrals to colposcopy associated with vaccination (in situ) were obtained from the 2003 Cancer Statistics Registrations Bulletin for England . For CIN3 we used single year of age data from South East England for 1990–2005 .In the absence of vaccination, the rate of invasive cancer for women aged 20–29 is taken to be 7.2 per 100 000 per year, the rate for CIN3 in women aged 20–24 is taken to be 260 per 100 000 per year and 322 per 100 000 per year in women aged 25–29 .Using the Cervical Screening Programme Statistical Bulletin 2002–2003 , we founFor invasive cancer in women aged 20–29, we project a 63% reduction in rates by 2025 with 80% vaccine coverage and no cross-protection, based on an estimated 79% reduction in vaccinated women. If only 70% coverage is achieved, the reduction will be more moderate (55%). The reduction in rates taking into account cross-protection is only slightly higher. These rates are plotted against calendar year in We project that the CIN3 rates can be reduced by 51% with 80% coverage and no cross-protection, and by 56% taking into account the cross-protection observed in the PATRICIA trial. The reduction in rates of CIN3 due to vaccination is plotted against calendar year in Without cross-protection, and assuming screening begins at age 25, we project that cytological abnormalities can be reduced by 27% with 80% coverage. If screening were to begin at age 20, we estimate that 34% of these abnormalities would be prevented. However, using more direct estimates from the PATRICIA trial, with 80% coverage a more moderate 21% reduction is projected. Reductions of cytological abnormalities as a function of calendar time are plotted in It is clear that vaccination will eventually have a substantial impact on the rates of invasive cervical cancer and its precursors. However the relative impact on abnormal cytology is much less than that observed for cervical cancer and CIN3, due to the larger impact of other HPV types on this end point. The first effects will be seen in younger women, but even then it will take 10 or more years to be appreciable, especially if screening starts at age 25. In England we will not observe the projected benefit of vaccination on precursor lesions in women aged 20–24, as the screening programme only invites women over age 25. In fact (regardless of vaccination) we are likely to observe an increase in the number of abnormal smears and CIN3 detected during the first round of screening (at age 25) compared to historical data, due to prevalent lesions that might have been detected and removed if screening started earlier. However the effect should disappear by the second round of screening.One of the main uncertainties in modelling the impact of vaccination is coverage. We have assumed coverage of 80% as this seems likely for a school-based programme, but estimates based on 70% coverage are also given. We have assumed that vaccination offers lifetime protection, but the current analysis only requires protection for the next 18 years (i.e. up to 2025). Data on the duration of protection offered by HPV vaccines are only available up to 7.3 years after vaccination . These sAlso, it is not clear how much efficacy will be lost if only two doses of the vaccine are received and also how many women will fall into this category. Preliminary data on antibody titres suggest two doses may be as effective as three (Our primary model has assumed no cross-protection, but we have also evaluated a model reflecting findings on cross-protection in the PATRICIA trial. Both models take into account that previous infection with one HPV type does not weaken the protection afforded by the vaccine to other types. We used CIN3 as one of our main end points. Another relevant clinical end point would be the reduction in women treated for cervical disease, as provided for in the PATRICIA trial. However the KC65 returns used to produce screening statistics for colposcopy episodes provide only data on treatment at first appointment. Because we have been unable to estimate the baseline number of women who get treated annually in England by age, it was therefore impossible to make this calculation.Another major unknown is whether vaccinated women will reduce their attendance for screening. This is an important issue as screening and vaccination have benefits of similar magnitude, so the benefit of vaccination on cervical cancer incidence could be fully negated if vaccinated women choose not to be screened. Furthermore, based on associations with deprivation, it is likely that girls who are not vaccinated will, as women, be less likely to be screened. Although it is possible that some cultural or religious subgroups whose lifestyle puts them at low risk will choose not to participate in either programme, it seems likely that overall women who miss out on both programmes will have a greater disease burden than would be predicted by assuming that non-compliance to one intervention is independent of non-compliance for the other. In general non-screened women have a much higher risk than screened women so reaching this group remains a priority. Screening by self-sampling based on an HPV test may help to minimise the disease burden in this group, but developing methods to achieve a high vaccine coverage in deprived groups is also important. These issues will also be important for evaluating the joint cost-effectiveness of screening and vaccination programmes (On a more positive note, it is likely that less frequent screening using HPV testing will become the norm, and the substantially greater level and duration of protection for this form of testing may reduce the disease burden further (


To determine whether HPR-HPV DNA detection in primary routine screening could represent a sensitive and reliable technique for the detection of high-grade squamous intraepithelial lesions (HGSIL), laboratory analysis using 2 cytologic techniques , HPV testing with Hybrid Capture II assay (HC-II), followed by colposcopic examination of women with abnormal cervical finding and/or persistent HR-HPV infection, was conducted in 7932 women who had routine cervical examination. The sensitivity of HPV testing for detecting a histologically proven HGSIL was 100%, higher than that of conventional (68.1%) and liquid-based (87.8%) cytology. The low specificities of 85.6% and 87.3% of HPV testing slightly increased to 88.4% and 90.1% if HPV testing was reserved for woman >30 years old. The quantitative approach provided by the HC-II assay for the assessment of the viral load was not reliable for predicting HGSIL in normal smears. HR-HPV testing could be proposed in primary screening in association with cytology. With conventional cytology it significantly improves the detection of HGSIL. With the use of the same cervical scrape for HPV testing and liquid-based cytology, HR-HPV testing would allow to select positive samples treated in a second time for cytology which gives a good specificity. © 2001 Cancer Research Campaign


Human papillomavirus (HPV) is widespread and can cause latent infection in basal cells, with low HPV DNA copy-number insufficient for transmission of infection; can cause subclinical infection that is active but without clinical signs; or can cause clinical infection leading to benign, potentially malignant or malignant lesions. The HPV cycle is influenced by the stage of maturation of the infected keratinocytes, and the production of virions is restricted to the post-mitotic suprabasal epithelial cells where all the virus genes are expressed.Low-risk HPV genotypes are associated with the development of benign oral lesions, whereas high-risk HPV genotypes are implicated in the development of malignant epithelial neoplasms. The rôle of high-risk HPV as a causative agent in epithelial malignancy is different at different anatomical sites: it is almost invariably implicated in squamous cell carcinoma of the uterine cervix, fairly frequently implicated in squamous cell carcinoma of the oropharynx, and it is seldom implicated in squamous cell carcinoma of the mouth. Human papilloma viruses (HPV) are members of the papillomaviridae family that infect epithelial cells exclusively. After gaining entry into the cells of the epithelial basal layer, replication of the virus occurs in the nuclei of the infected cells, and the production of mature virions occurs in the suprabasal epithelial cell layers . HPV infHPV infection is the most common of all sexually transmitted diseases. It is estimated that two thirds of those who have had sexual contact with HPV-infected persons, will become infected . Oral HPThe clinical manifestations and the microscopical features of HPV-associated lesions vary with the anatomical site affected and with the genotype of the HPV . The varImmunosuppressed people are at significantly greater risk of developing HPV infection and of experiencing a more aggressive course of infection than immunocompetent people. The immune system plays an important rôle in controlling HPV infection ,10, but More than 100 types of HPV have been identified, differing in the regulatory sequences and coding potential of their genomes ,10,12. BIn a small minority of women with persistent vulvo-vaginal infection with high-risk HPV genotypes, cervical SCC may develop as long as 12-15 years after the initial infection ,15,19-24Low-risk HPV genotypes only rarely have been implicated in malignant transformation . In the High-risk HPV genotypes, in particular HPV-16 and -18 are prevalent in potentially malignant oral epithelial lesions -29 and iAll types of HPV have the same general organization of the genome: double-stranded circular DNA that carries early (E) open reading frames (ORFs) encoding for non-structural regulatory proteins, and late (L) ORFs encoding for capsid proteins. Noncoding upstream regulatory region (URR) encompassing the origin of replication, the E6/E7 gene promoter, and enhancers and silencers, is located between the early and the late regions Figure  2,3].,3.2,3].Early ORFs encode for E1, E2, E4, E5, E6 and E7 proteins. E1 and E2 proteins bind to viral origin of replication, recruit cellular proteins that mediate viral DNA replication, and regulate viral gene expression ,7. E1 anThe integration of HPV DNA within the cellular genome that is evident in some HPV-associated malignancies disrupts the E2 ORF leading to loss of E2 repressing function , thus peE3 ORF does not encode for proteins . The funLittle is known about the action of E6 and E7 proteins of low-risk HPV genotypes during latent and subclinical HPV infection ,3. DurinE6 and E7 of low-risk HPV probably play a rôle in the pathogenesis of HPV-associated benign tumours and only rarely are implicated in malignant transformation. It is unclear what properties E6 and E7 of high-risk HPV genotypes possess that the E6 and E7 of low-risk HPV genotypes do not possess, that enable them to induce malignant transformation of some epithelial cells infected with high-risk HPV. One possibility is that E6 and E7 proteins of high-risk HPV genotypes have a greater affinity for the regulatory factors of the cell cycle, thus causing an exaggerated dysregulation of mechanisms of cell growth and of cell survival .E6 and E7 are not expressed in cells of the basal layer with low-risk HPV infection , and it In high-risk HPV infection associated with intraepithelial neoplasia or with invasive malignancy, E6 and E7 oncoproteins are expressed in progenitor basal layer keratinocytes mediating basal cell proliferation and genomic instability, making the infected cells susceptible to additional genetic alterations leading to cell transformation and possibly to the subsequent development of SCC . The bioE8 ORF has been identified only in bovine papillomavirus . L1 ORF The cells of the epithelium comprise two populations of cells, the progenitor and the maturing. The former cells are based in the basal and parabasal layers, have the capacity to proliferate by mitotic division following which some of the newly divided (amplifying) cells enter a maturation process and gradually rise through the several morphologically distinct cell layers to the surface, while some remain to maintain the integrity of the basal layer. In the mouth this process of maturation follows two main patterns: keratinization, and non-keratinization .Some authors refer to this process of maturation as differentiation ,3,10. ThWhether the cells that give rise to new progenitor cells and those that give rise to cells destined to go through the process of maturation are in fact distinct sub-populations, or whether the fates of all cells resulting from mitoses in the basal cell layer are determined by changing local microenvironment, is uncertain.The HPV cycle is influenced by the stage of maturation of the infected keratinocyte in the squamous epithelium ,3,10. ThHPV infection starts when the viruses enter epithelial basal cells which are referred to as the target cells of the virus ,44. OnceIn cases of high-risk HPV infection, E6 and E7 may also be expressed in HPV-infected basal cells, and the epithelium may then enter a proliferative phase characterized by an increasing number of HPV-infected basal cells , culminaWhy HPV can infect basal cells in the first instance but not the cells of other epithelial layers is not known; but this is probably related to specific surface receptors confined to basal cells . The attIn latent HPV infection the affected epithelium looks both clinically and histologically normal because the gene expression of the latent genes is restricted to E1 and E2 . The virAs the epithelial cells mature, the HPV cycle progresses to productive replication ,43. In HMultiplication of the viral genome, synthesis of early gene proteins  and late gene proteins , assembly of virions, and release of virions from exfoliated epithelial cells occur at progressive stages of maturation of postmitotic HPV-infected epithelial cells. It is suggested that specific cellular factors associated with epithelial cell maturation activate late viral promoter located within E7 ORF resulting in expression of high levels of E1 and E2 viral replication proteins which activate late viral gene expression. This increase in E1 and E2 expression leads to HPV amplification resulting in an increase in viral genome numbers parallel with the stages of epithelial cell maturation. Eventually, mediated by L1 and L2 proteins, the virus escapes from the shedding epithelial cells ,39.Unlike the early promoter, the late promoter is not suppressed by high levels of E2 . The ampHPV-induced benign lesions are characterized by proliferation of cells of all epithelial layers . This prMicroscopic examination of high-risk HPV-induced high-grade intraepithelial neoplasia shows greatly increased numbers of mitotic figures and features of cellular atypia, and extension of these multiplying atypical cells into the suprabasal layer of the epithelium as the outcome of expression of high-risk HPV E6/E7 oncoproteins in the basal cells .In persons who have been exposed to HPV, several HPV types have been detected in their apparently normal oral mucosa, and in certain specific benign, potentially malignant and malignant oral lesions, in the aetiology of which they appear to be implicated. In infected persons, in clinically and histologically normal looking oral mucosa, the HPV exists in a latent form as a low-copy number episome in basal epithelial cells . In HPV-Oral HPV infection is acquired primarily by sexual transmission, or less frequently by non-sexual direct transmission, by mother to child transmission or by autoinoculation. Persons who practise oral-genital sex, those who have had a number of sexual partners, and those who are immunocompromised are at greater risk of acquiring oral HPV infection ,14,47-49Many of the low-risk genotypes and uncommonly, high-risk types have been found in the HPV-associated benign oral lesions, oral squamous cell papilloma, verruca vulgaris (common wart), condyloma acuminatum and focal epithelial hyperplasia (Heck disease) ,18,50,51Although certain HPV types have been implicated in the aetiology of the different oral warts, there is considerable overlapping of viral types among the lesions, and not infrequently viral types not generally associated with a particular lesion, can be identified. Therefore viral typing, which one would expect to be a diagnostic differentiator of oral warts cannot always be relied upon.The reported rates of HPV DNA detection in potentially malignant  and malignant (squamous cell carcinoma) oral lesions range from 0 to 100% ,55. ThisHigh-risk HPV genotypes, in particular HPV-16 and -18, have been reported to be the most prevalent in oral squamous cell carcinoma ,31,56-58In a meta-analysis of data from 94 studies of 4580 specimens, Miller and Johnston (2001) determined that the likelihood of HPV to be detected in normal oral mucosa, in non-dysplastic leukoplakia, in dysplastic oral leukoplakia and in other precancerous intra-epithelial oral neoplasms, and in oral squamous cell carcinoma is likely to be about 10%, 20%, 26% and 47% respectively. This suggests that HPV may have an oncogenic or a co-oncogenic rôle in some HPV-cytopositive potentially malignant and malignant oral epithelial neoplasms .INK4A, and infrequent loss of heterozygosity (LoH) at chromosomal loci 3p, 9p and 17p [With HPV-16 cytopostive oropharyngeal SCC as a model of HPV-mediated epithelial cancerization, a causal association between HPV and oral SCC is likely if the cells of HPV-cytopositive oral SCC express transcriptionally active E6/E7 mRNA, frequently demonstrate viral integration within the cellular genome, show high viral load (> 1 copy per cell), show unmutated p53 gene, reduced expression of Rb proteins, overexpression of p16 and 17p ,60-66.INK4A, of p53 and of Rb proteins is not unequivocally associated with the presence of high-risk HPV DNA [As the cells of HPV-cytopositive oral squamous cell carcinoma and potentially malignant lesions are typically characterised by infrequent expression of transcriptionally active E6/E7 mRNA, by infrequent viral integration within the cellular genome, by low HPV viral load and by frequent LoH at chromosomal loci 3p, 9p and 7p ,33,66; a HPV DNA , there wIt is possible that HPV super-infection of initially transformed oral keratinocytes may promote progression of transformation in an additive or synergistic manner, through mechanisms as yet unknown , or thatAlthough it is clear that there is some link between HPV and potentially malignant and malignant epithelial oral lesions, the precise rôle of the virus in the aetiopathogenesis of these oral lesions is unknown.HPV is epitheliotropic, and it may be present in a latent form, a subclinical form or in a form which in association with other ill-defined factors can induce benign or malignant epithelial neoplasms. Figure While some viral proteins are expressed in HPV-infected basal cells independently of the cell maturation process regulating early viral DNA transcription from early promotor prior to production of virions, the productive phase of the viral cycle is dependent on specific cellular factors associated with the progressive stages of maturation of post-mitotic cells as the cells gradually rise trough the layers of epithelium to the surface, and exfoliate.Persistent high-risk HPV infection in a subset of subjects with HPV-cytopositive oropharyngeal epithelium induces genomic instability in these cells, making them susceptible to additional genetic alterations leading to cell transformation and to subsequent development of squamous cell carcinoma. In these oropharyngeal squamous cell carcinomas there is a causal association between HPV infection and the malignancy. In contrast, the nature of the link between high-risk HPV genotypes and potentially malignant and malignant oral epithelial lesions is not known.Low-risk HPV infection of oral epithelium is associated with squamous cell papilloma, verruca vulgaris (common wart), condyloma acuminatum and focal epithelial hyperplasia (Heck disease); and possibly with the potentially malignant oral epithelial lesions, leukoplakia and erythroplakia.The authors declare that they have no competing interests.LF and RAGK contributed to the literature review. LF, JL and NHW contributed to the conception of the article. LF, JL, NHW and RAG contributed to the manuscript preparation. Each author reviewed the paper for content and contributed to the manuscript. All authors read and approved the final manuscript.


Thirteen human papillomavirus (HPV) genotypes have been judged to be carcinogenic or probably carcinogenic, and the cause of virtually all cervical cancer worldwide. Other HPV genotypes could possibly be involved. Although the inclusion of possibly carcinogenic HPV genotypes may hurt test specificity, it may indirectly increase the reassurance following a negative HPV test . The future of cervical cancer screening in low-resource setting, however, may include once-in-a-lifetime, low-cost and rapid HPV testing. However, the tradeoff of more false positives for greater reassurance may not be acceptable if the local infrastructure cannot manage the screen positives. Now is the time for the community of scientists, doctors, and public health advocates to use the data presented at the 100th International Agency for Research on Cancer monograph meeting to rationally decide the target HPV genotypes for the next generation of HPV tests for use in high-resource and low-resource settings. The implications of including possibly HPV genotypes on HPV test performance, also for guidance on the use of these tests for cervical cancer prevention programs, are discussed. Based on large case series, meta-analyses, and laboratory data of mechanistic studies, twelve HPV genotypes  were deemed carcinogenic and HPV68 was considered probably carcinogenic.Periodically the International Agency for Research on Cancer (IARC) convenes a meeting of cancer experts to review and update the evidence regarding potential carcinogens. In February of 2009, the meeting for the 100 [et al.  provideset al. [One of the important outcomes of the meeting was the downgrading of HPV66 from the category of probably carcinogenic (Group 2A), as judged in 2005, to possibly carcinogenic (Group 2B), which also includes HPV genotypes HPV26, HPV53, HPV67, HPV70, HPV73, and HPV82. As explained by Schiffman et al. ,5, these and several other assays from other companies are being evaluated in clinical trials. Most have included HPV66 in their panel of targeted HPV genotypes.Temporarily categorizing HPV66 as probably carcinogenic during the four-year interval meetings impacted the development of new clinical HPV assay. In 2005, there was only one FDA-approved HPV DNA test, hybrid capture 2 , Gaithersburg, Maryland, USA), which targets the 13 certain and probably carcinogenic HPV genotypes as a pool of HPV genotypes. In 2009, one additional test, Cervista , received FDA approval What is the impact of including HPV66 in clinical tests? The lifecycle of these tests, from development to validation, is 10 years or more. As a consequence, there will be a generation of HPV tests  that include HPV66 and in theory will be slightly less specific than what might otherwise be achieved. Thus, more women will test positive for HPV when used in primary screening or as triage/reflex test for equivocal  cytology.Worldwide, the prevalence of HPV66 estimated to be 0.4% (95%CI = 0.3–0.4%) although there are some regional variations in its prevalence . Therefoin situ methods if they ever prove to be sufficiently sensitive, and genotype-specific detection of HPV E6/E7 transcripts or protein [Future meetings will no doubt try to resolve whether these borderline carcinogenic HPV genotypes  are cancer causing or not. Integrative epidemiologic and further laboratory approaches will be needed for clarification. New investigative tools, such as micro-dissection of the lesion and HPV genotyping or  protein ,12 may hth IARC monograph meeting. Experts then could come to a consensus about what is an acceptable tradeoff in sensitivity versus specificity vis-à-vis which HPV genotypes to include in clinical assays, accepting the notion that no test will achieve perfect sensitivity and that poor specificity has important implications, including potential unnecessary treatment, which can cause negative reproductive outcomes [In the end, perhaps resolving this issue of which of the borderline HPV genotypes are carcinogenic does not matter except for academic albeit important biological reasons: to understand the nature of HPV carcinogenicity and its relationship to viral genetics and phylogenetics . We alreoutcomes ,16.th IARC monograph [As a first approximation, a simple version of this ROC-like analysis approach based on previously reported data ,17 is shonograph  is needeThe inclusion or exclusion of borderline carcinogenic HPV genotypes in a clinical assay for carcinogenic HPV becomes moot if robust strategies for management (risk stratification) of test positives become available. That is, there would be no significant penalty to include some of these borderline HPV genotypes if there was downstream, specific method of triaging HPV-positive women to separate those in need of immediate colposcopy versus those who could be monitored by repeat screening in a year or two. The key is good stratification of risk for cervical precancer and cancer, and consistent, appropriate management of that risk . Some apAlthough the inclusion of possibly carcinogenic HPV genotypes may hurt test specificity, it may indirectly increase the reassurance following a negative HPV test i.e. the negative predictive value of an HPV test for cervical precancer and cancer. Because non-carcinogenic and carcinogenic HPV infections alike are sexually transmitted, identification of women with borderline carcinogenic or non-carcinogenic HPV identifies a subset of higher-risk women who are more likely to subsequently acquire a new carcinogenic HPV infection. That is, women who are HPV positive for any HPV type at any given time point represent a sub-group that engages in more risky behavior, such as having more sexual partners, or have a partner(s) who is more likely to do so.The future of cervical cancer screening in low-resource setting may include once-in-a-lifetime, low-cost and rapid HPV testing ,26. The Other prevention programs may adopt screen and treat approaches, in which all screen positives undergo some treatment. For example, HPV-positive women could then be evaluated by visual inspection with acetic acid to decide the best treatment option: chemotherapy or palliative care for women with obvious cancer, surgical lesion excision  for large precancerous lesions that are not amenable to cryotherapy, and cryotherapy for all other HPV-positive women. Such an algorithm would only be applied to populations of older women, more than 10–15 years past the population median age of sexual debut, to minimize the over-treatment of mostly transient HPV infections in reproductive-age women. In general, because HPV-positive women will not undergo any further characterizations such as colposcopy or a secondary test, there should be a greater emphasis on specificity i.e. inclusion of only the certain carcinogenic HPV genotypes. However, there is some evidence to suggest that cryotherapy provides a secondary benefit of reducing the likelihood of acquiring new HPV infections post-treatment , which mth IARC monograph meeting to rationally decide the target HPV genotypes for the next generation of HPV tests for use in high-resource and low-resource settings. And provide guidance on the use of these tests for cervical cancer prevention programs. However, the adoption of any cervical cancer screening and prevention program will depend on its social and cultural acceptance as well as available resources and the political willpower to implement it.In the end, it seems unlikely that companies that develop HPV tests will tailor their tests to individual country needs. Now is the time for the community of scientists, doctors, and public health advocates to use the data presented at the 100


While infections with human papillomavirus (HPV) are highly prevalent among sexually active young women in Uganda, information on incidence, clearance and their associated risk factors is sparse. To estimate the incidence, prevalence and determinants of HPV infections, we conducted a prospective follow-up study among 1,275 women aged 12-24 years at the time of recruitment. Women answered a questionnaire and underwent a pelvic examination at each visit to collect exfoliated cervical cells. The presence of 42 HPV types was evaluated in exfoliated cervical cells by a polymerase chain based (PCR) assay (SPF10-DEIA LiPA).Three hundred and eighty (380) of 1,275 (29.8%) women were followed up for a median time of 18.5 months (inter-quartile range 9.7-26.6). Sixty-nine (69) women had incident HPV infections during 226 person-years of follow-up reflecting an incidence rate of 30.5 per 100 person-years. Incident HPV infections were marginally associated with HIV positivity . Clearance for HPV type-specific infections was frequent ranging between 42.3% and 100.0% for high- and 50% and 100% for low-risk types. Only 31.2% of women cleared all their infections. Clearance was associated with HIV negativity  but not with age at study entry, lifetime number of sexual partners and multiplicity of infections. The prevalence of low-grade squamous intraepithelial lesions (LSILs) was 53/365 (14.5%). None of the women had a high-grade cervical lesion (HSIL) or cancer. Twenty-two (22) of 150 (14.7%) HPV negative women at baseline developed incident LSIL during follow-up. The risk for LSIL appeared to be elevated among women with HPV 18-related types compared to women not infected with those types .Incident HPV infections and type-specific HPV clearance were frequent among our study population of young women. These results underscore the need to vaccinate pre-adolescent girls before initiation of sexual activity. Persistent infection with one or more high-risk HPV types is an important etiologic factor in the development of cervical cancer . High-riWomen were recruited and followed up between September 2002 and December 2006. A description of the study population at baseline is available elsewhere [During all visits, trained midwives explained the study aims and procedures, which included answering a questionnaire and having a pelvic examination to record visible abnormalities. In all visits after visual inspection of the vulva, a non-lubricated sterile speculum was inserted, and cervical exfoliated cells were collected. At the first visit at cohort enrolment, cervical exfoliated cells were collected with a sterile swab  which was then placed in a labelled 15 mL holding tubes containing 5 mL of phosphate buffered saline (PBS), pH 7.2; samples were kept temporarily at 4°C for an average of 6 hours and then transferred to a freezer for storage at 20°C until shipment to the laboratory for HPV analysis. PBS is not a suitable solution for preservation of cells for liquid based cytology and consequently no liquid based cytology could be done with baseline samples. However, during follow-up, cervical exfoliated cells were collected and stored in a vial containing PreservCyt solution  which is suitable both for liquid-based cytology and HPV testing. Briefly, for these follow-up samples, a broom style cytobrush  was inserted deep into the endocervical canal and rotated gently in a clockwise direction 5 times to collect cells from the endo- and ectocervix. The cytobrush containing cervical cellular material was then placed in a vial containing PreservCyt solution (Cytyc) and rinsed by pushing it to the bottom of the vial 10 times. The cytobrush was then discarded. The vials were closed and kept at room temperature until shipment in dry ice to DDL Diagnostic Laboratory, Voorburg, The Netherlands, for liquid based cytology and HPV testing. Cervical abnormalities in liquid-based cytology samples were read at the cytology department of the Slotervaart Hospital, Amsterdam, The Netherlands and classified according to the 2001 Bethesda Classification [Before removal of the speculum, both for the baseline and follow up visits, visual inspection with acetic acid (VIA) and with Lugol's Iodine (VILI) was performed. A urine sample and 4 ml of blood (in heparinised tubes) were collected for pregnancy, HIV and syphilis testing, respectively. Non-pregnant women with external genital warts were treated with 2% podophylline paint. The treatment of pregnant women was deferred until after delivery. Women with vaginal discharge and cervicitis received a one-week syndromic treatment and were requested to ask their sexual partners to receive treatment.Total DNA was isolated from 200 μl of the suspension containing the cervical cells by the MagNa Pure LC instrument , using the Total NA isolation kit . DNA was eluted in 100 μl of elution buffer, and 10 μl was used for each PCR reaction. Each run contained positive and negative controls to monitor the DNA isolation, PCR and HPV detection and genotyping procedures.The short PCR fragment (SPF) 10 primer set was used to amplify a broad spectrum of HPV genotypes, as described earlier ,12. BrieHIV-1 testing was performed at NTIHC following the National HIV Rapid Testing algorithm consisting of Determine rapid test  as screening test, Statpak rapid test  as the confirmatory test and Unigold  as tie breaker . For quaSyphilis testing was performed at NTIHC using a commercially available standard Rapid Plasma Reagin (RPR) 18 mm circle card test . Samples with reactive RPR test results were referred to established laboratories for confirmation before treatment was offered to the women concerned and their partners.All women were tested for pregnancy using a commercially available HCG dipstick pregnancy test , and the results were communicated immediately to each woman. By the end of the study 30 women were tested positive for pregnancy. The pregnant women were referred for free pre-natal counselling, evaluation and care within the same health care center. Only the pre-natal care services are being evaluated in our study, not the number of weeks of pregnancy or the decision if the pregnancy was or not to be kept.Women consented to participation in the study. The women younger than 18 years of age were treated according to the Uganda guidelines for conducting research on human beings that allows minors to assent for participation without a parent or legal guardian agreement. These guidelines are approved by the ethical review committees at the Faculty of Medicine, Makerere University, the Uganda National Council of Science and Technology, and the International Agency for Research on Cancer.We analyzed incidence and clearance of HPV infection for all women who had at least one follow-up visit. Any HPV type was defined as a combined outcome if any HPV type was present. We defined HPV 16-related types as HPV 16, 31, 33, 35, 52, and 58 and HPV 18-related types as HPV 18, 39, 45, 59 and 68. Single and multiple infections were defined without considering uncharacterized HPV types (HPV X). We computed relative risk (RR) for HPV incidence and clearance using Poisson regression dividing the follow-up time in 6-month windows for risk time and events.For incidence analysis, the risk time was measured for each specific HPV type or combined set of HPV types from the first time a woman tested negative until a positive test or the last visit.For each of the groups of infections  incidence was calculated for women free from all the infections in each group until infection of at least one of the HPV types in the group. Correspondingly clearance was calculated for all women infected with at least one of the HPV types in each group until the woman was free from infection of all the HPV types in each group. Thus, clearance will typically be a lot longer than for specific HPV types. This choice of groups of HPV types intend to measure an exposure assuming interchangeable risks within the group so a woman was not considered cleared from exposure of high-risk HPV if one high-risk infection was cleared while she was still infected with other high-risk type(s). Thus, clearance of any HPV was interpreted as being cleared from all HPV infections. Highly sexually active women as our study population would tend to have long clearance time even if each specific type is cleared fast.Women positive for HPV X at baseline who tested positive for a known HPV type at follow-up visit were considered to have developed a new infection in the analysis of incidence but were excluded from the analysis of clearance as persistence of HPV X could not be known. For each HPV type, we fitted Poisson models including a time covariate only and models adjusting for HIV status, age at baseline , positivity for genital warts or syphilis (yes/no), and lifetime number of sexual partners .We evaluated prevalent cytological abnormalities only on follow-up visits as the medium used to collect baseline specimens was not suitable for cytological analysis. Subjects were classified as normal or low-grade squamous intraepithelial lesions (LSILs). No high-grade squamous intraepithelial lesions or invasive cervical cancer cases were diagnosed. Among women with a baseline HPV negative sample with valid follow-up cytological sample we evaluated incident cytological abnormalities according to type-specific HPV infections at follow-up .All tests of statistical hypotheses were made on the two-sided 5% level of significance with corresponding 95% confidence intervals. All analyses were made using the SAS software version 9.2 (SAS Corp). Procedure genmod was used to fit the Poisson regression.st follow-up visit was 18.5 months, with inter - quartile range of 9.7 to 26.6 months  women had at least one follow-up visit. Compared to women with adequate follow-up, the women with inadequate follow-up were slightly younger at study entry (>18 years 85.5% vs. 90.3%), less likely to have genital warts (7.6% vs. 8.2%), less likely to be pregnant at time of examination (11.8% vs. 15.0%) but more likely to be HIV positive (6.2 vs. 5.8%) and more likely to have initiated sex below 15 years (15.0% vs.10%). There was no difference at baseline between women who were adequately followed up and those who were not with respect to HPV positivity (74.6% vs. 74.7%) and the number of lifetime sexual partners . The time between visits was highly variable. The median time to the 1Table Table Overall, 143 women with prevalent infections cleared their infections during 459 person-years of observation reflecting complete clearance for all HPV types of 31.2% (Table Incident HPV infections were common in both HIV positive (rates between 2.2 and 15.4 per 100 person-years) and HIV negative women (rates between 0.8 and 4.2 per 100 person-years) [See Additional file st and 2nd follow-up visit leaving 168 and 170 samples, respectively, available for cytological analysis. The prevalence of LSILs at 1st cytologic sample was 15/168 (8.9%) and declined to 9/170 (5.3%) at the 2nd visit. Evaluation incident LSIL was possible for 150/155 (96.8%) women who were HPV DNA negative at baseline. Twenty-two (22) of 150 (14.7%) developed LSILs between baseline and 1st follow-up (Table Three hundred seventy-eight (378) of 380 (99.5%) women with at least one follow-up visit provided a sample for liquid-based cytology. Twelve (12) samples were inadequate leaving 365 samples for cytological analysis; of these 365 samples, 53 14.5%) had LSILs. None of the women had HSIL or cancer. One hundred and seventy-three (173) women provided at least 2 samples for cytologic analysis of which, 5 and 3 samples were inadequate at 1.5% had LThe majority of women who were tested HIV positive during follow-up had declined HIV testing at enrolment; We suppose that the women already knew they were HIV positive therefore we chosen to not provide the analysis of HIV incidence during follow-up.et al. (2008) [The observed high incidence rate of HPV infections among the young women in our study is similar to finding described in previous studies -17. A wo. (2008)  in a pop. (2008) -21.Our findings differ from studies conducted among adult populations who found that the 12-month clearance for low-risk HPV types was more than for high-risk types but no clear difference between infections with high-risk types other than HPV 16 . The difet al. (2001) [st and 2nd visit in women who provided at least 2 adequate samples for cytologic analysis underscores the transient nature of HPV infection and associated cervical lesions among young women. Our findings therefore, imply that initiating screening for cervical cancer in young women is likely to detect transient low-grade cervical abnormalities. Current knowledge of the natural history of HPV infection and its associated cervical lesions suggest that conservative management, such as follow-up without aggressive surgical treatment may be indicated in young women with LSIL. Secondly, screening with HPV testing among young women is not useful as many women are likely to be HPV positive as shown by the cumulative HPV results.Despite the high incidence of HPV infections, the incidence of LSIL was relatively low (14.5%) which suggests that many young women who were infected with HPV had no cellular changes. Moscicki . (2001) , in a st. (2001)  and indeOur study has strengths and limitations. We used a highly sensitive HPV testing PCR assay for HPV detection to show a high rate of acquisition of new HPV infections in a population of young women in an area with high-risk of cervical cancer. The same PCR assay was used for HPV detection at baseline. Given the high sensitivity of the PCR assay, under detection of HPV infections was unlikely in our study. Thus, new infections were unlikely to be infections previously missed and cleared infections were unlikely to be due to errors in follow-up samples.Our study underscores the challenges of conducting follow-up studies in low resource countries where residence has no proper street address. Hence our study had significant loss to follow-up (70.2%) which could have introduced selection bias. Women were lost to follow-up because they could not be located, declined to have further specimens collected or were back in school at the time of scheduled visits. However, there was no difference between the women adequately followed up and those who were not regarding lifetime number of sexual partners, which is a strong and consistent risk factor for acquisition of HPV infections. Secondly, loss to follow-up was unlikely to be associated with the woman's HPV status because the woman was blinded of her HPV status throughout the follow-up period. We used prevalent rather than incident infections to assess clearance and as a result, some women could have lost an HPV infection between visits and this could have led to the underestimation of the clearance.In conclusion, we investigated incidence, clearance and potential risk factors of HPV infections among 380 young women followed up in Kampala, Uganda. Our study shows that incident HPV infections were frequently underscoring the importance of vaccination of young women before they become sexually active.The authors declare that they have no competing interests.CB & EW conceived the study. CB wrote the study protocols, coordinated the field work, designed data collection forms, including the questionnaires, clinical databases and wrote the manuscript. EW supervised CB throughout the study from its conception, preparation of study protocols, data and specimen collection, interpretation of the results and made substantial contributions to the manuscript. FWM & EKM supervised CB during fieldwork and made substantial comments to the manuscript. SS made the statistical analyses, produced the working and final tables, and wrote part of the methods section. BK, LJD, & WQ performed HPV detection and genotyping of the study samples, created the HPV database, wrote parts of the methods section and made substantial contributions to the manuscript. All authors read and approved the final manuscript.Table S1 - HPV incidence and clearance among HIV positive and HIV negative women, Kampala, Uganda. original data used to perform analysis of clearance by HPV type.Click here for file


N=54 cases, 50 are epithelial cancer, four are nonepithelial cancer) embedded in paraffin blocks were used. Controls are 30 nonmalignant ovarian tissue blocks. In situ hybridisation (ISH) and immunohistochemistry (IHC) were used to detect the presence of HPV-16 and p53 expression. In all, 52 or 36% of the epithelial ovarian tumours detected by ISH or IHC, respectively, were HPV-16 E6 positive. In contrast, only 6.7% of normal ovarian tissues were HPV-16 positive proved by ISH. Human papillomavirus-16 infection was significantly higher in cancer tissues compared to controls with an odds ratio of 16.7 . No significant correlation between HPV-16 infection and histological types of cancer was found (P>0.05). p53 gene expression was detected in 42% epithelial ovarian cancers. No correlation between p53 expression and HPV-16 infection was found. The results showed the presence of HPV-16 E6 in ovarian carcinoma, suggesting that HPV infection might play a role in ovarian carcinogenesis.Human papillomavirus is the causal factor for cervical cancer. However, the role of HPV infection in ovarian cancer is unclear. This study aimed to determine the presence of human papillomavirus-16 (HPV-16) in ovarian cancer tissues. Archived human ovarian cancer tissues ( Human papillomavirus (HPV) infection has been identified as the necessary cause of cervical cancer . OvarianThe oncogenic HPV product E6 targets p53 for degradation and therefore manipulates the host intracellular signal network in cervical cancers . Histological types for 50 epithelial cancers were 24 serous cystadenocarcinomas, 19 mucinous cystadenocarcinomas, five endometrioid adenocarcinomas, and two undifferentiated carcinomas. Histological types for four nonepithelial cancers were two thecomas, one endodermal sinus tumour, and one malignant mesothelioma. The control group consisted of 30 pathologically confirmed nonmalignant ovarian tissues collected from 30 women . Among the nonmalignant tissues, 24 were ovaries removed for uterine pathology, five were ovarian cysts, and one was polycystic ovarian syndrome. Specimens were paraffin-embedded and archived from 1996–2000 in Inner Mongolia Medical College affiliated hospital.μm thickness were cut from formalin-fixed, paraffin-embedded blocks. Tissue sections were attached on APES-treated glass slides for in situ hybridisation (ISH) and immunohistochemistry (IHC).Sections of 5 in vitro transcription kit . Briefly, the in vitro transcription vector containing E6 gene was linearised by SalI digestion. The transcription was performed with T7 RNA polymerase in the presence of digoxin-labelled UTP and other unlabelled NTPs.The full-length HPV-16 E6 gene was kindly provided by Dr zur Hausen. A 483 bp E6 probe was labelled with digoxin using an N HCl. The tissue slides were then digested with proteinase K at 37°C for 15 min. The HPV-16 E6 probe was mixed with hybridisation solution containing 50% formamide, 4 × SSC, 5% dextran sulphate, 5 × Denhardt's solution and 200 mg ml−1 ssDNA. The tissue slides were incubated overnight at 42°C in 20 μl of the hybridisation solution with probe. The slides were washed in SSC. The E6 hybrid was detected by an alkaline phosphatase-conjugated antidigoxin antibody. The tissue slides were observed under a microscope. Pictures were taken for analyses. The HPV-16-positive oesophageal cancer was used as positive control  or anti-p53 antibody  at 4°C overnight. The biotinylated secondary antibody and HRP-labelled streptavidin were then added and incubated at 37°C for 30 min. The signal was developed in DAB–H2O2 solution. The slides were counterstained with haemotoxylin.Specimens were dewaxed and treated with PBS containing 3% hydrogen dioxide. Slides were incubated at 37χ2 test and odds ratio was used to determine prevalence of HPV-16 infection in epithelial ovarian cancers, nonepithelial ovarian cancers, and normal ovarian tissues.Statistical analysis was performed using P<0.01 OR=16.7 95% CI ∼3.2–71.4). Human papillomavirus-16 E6 was not detected in any of the nonepithelial ovarian cancers .A total of 21 or 42% of the epithelial ovarian cancers were p53 positive. The brown signal located uniquely within the nucleus represents p53 protein. No correlation has been found between HPV-16 infection and p53 positive (Table 3P>0.05). Owing to the sample size limitation, statistical comparison was only carried out between cystadenocarcinomas and mucinous cystadenocarcinomas.HPV-16 infection was not identified in any nonepithelial ovarian cancers, which are not the primary host for HPV. There was no correlation between HPV infection and histological types of ovarian cancers , P>0.05.Human papillomaviruses play a causal role in cervical cancer. Human papillomavirus infection is also detected in other cancers of the female lower genital tract, including cancers of vulva, vagina, and perineum (In addition to the host and pathogen genetic variation, the difference of the detection methods employed in the studies might also account for data discrepancy. It has been well documented in cervical cancer that the episomal viral DNA frequently integrates into the host genome as HPV-infected lesions progress to cervical cancer. During viral DNA integration, only E6 and E7 genes remained  in the hIn this study, we employed both ISH and IHC to detect HPV-16 E6 expression. Among the 26 HPV-16-positive cancer samples detected by ISH, 18 were confirmed positive by IHC. More importantly, none of ISH-negative samples were detected positive by IHC. These results indicate that IHC is an accurate method with less sensitivity.The prevalence of HPV-16 infection in ovarian cancers is much higher than in nonmalignant ovarian tissues, suggesting that HVP-16 infection may play a role in the development of ovarian cancer. However, large case–control studies need to be conducted before reaching a conclusion.Mutations of p53 are frequently detected in ovarian cancer (


High-Risk (HR) and Low-Risk HPV screening was performed using HPV PCR-EIA with genotyping of HR positives and data correlated with age, SDS and cytology. Overall HPV prevalence was 13.5% (9.3% age standardised) and the most frequent HR types were HPV 16, 31, 18 and 58. In HR HPV-positive cases 42.4% had a single HR type and they were predominant in women with severe cytological abnormalities. Here, 66% of all HR HPV cases were in women aged 30 years of age or less and SDS had no significant effect on HPV status. HPV prevalence increased significantly with degree of dyskarosis from 7% in cytology negative samples to 80% in samples with severe cytological abnormalities . Overall, 46% of HR HPV cases were positive for the two HR types targeted by the prophylactic vaccines (HPV 16 and HPV 18). The data presented represents the largest type-specific investigation of HPV prevalence in an unselected UK population.In this cross-sectional population-based study we determine human papillomavirus (HPV) prevalence in South Wales to provide comprehensive baseline data for future assessment of the impact of prophylactic HPV vaccination and to help inform future screening strategies. Liquid-based cytology samples from women attending routine cervical screening were collected ( Human papillomavirus (HPV) infection is the essential underlying cause of cervical cancer, found in 99.7% of invasive cervical carcinomas . WorldwiMultiple HR types occur in up to 43% of HR HPV-positive women and to date no association has been found between multiple HR types and progression of cervical neoplasia  and Gardasil , both active against HR HPV types 16 and 18 . Currentn=10 000; 20–65 years).We aimed to extend preliminary findings  screening samples were collected in collaboration with Cervical Screening Wales over a 5-month time period in 2004 (non-probability convenience sample). This method included all women attending for routine cervical screening within the catchment area of Llandough Hospital Cytology Laboratory and constitutes a representative sample of the screening population in South Wales. Age, cytology result and Social Deprivation Score (SDS) were provided for each specimen and inadequate cytology samples or those from colposcopy clinics were excluded. The study was approved by South Wales Local Research Ethics Committee.M Tris pH 7.4. 10 μl of recombinant proteinase K was added to a 100 μl cell suspension from each sample, incubated at 56°C for 2 h, followed by 100°C for 10 min. Samples were refrigerated (4°C) for 10 min, centrifuged at 13 000 r.p.m. for 10 min and the supernatant transferred into a 96-well plate. A PCR for the human β-globin gene was performed on every DNA sample to determine extraction efficiency as described earlier  and negative (water) DNA extraction, PCR and ELISA controls were included for every 94 samples. Re-analysis of 10% of all samples processed was undertaken for internal quality assessment purposes. Results were analysed in an automated Excel worksheet and raw data from each reading at each time point pasted into a specific position in the datasheet and results are then given as positive (1) or negative (0). The negative extraction control included in every 96-well plate serves as the background reading for which all the other results are compared. A positive result is equivalent to three times background.The LBC samples (Thinprep) were processed by the Cytology Laboratory according to the British Society of Clinical Cytology guidelines . Residuaβ-globin PCR positive; complete information available on age, cytology result and SDS; within the screening age group 20–65 years. The HR HPV prevalence was age-standardised using the European Standard Population and the age-standardised rate calculation ∑ Si ria/∑ Si  were calculated where appropriate. All HR HPV-positive cases were differentiated into those with a single HR type and those with multiple HR types.Statistical analysis was performed on samples that complied with the following inclusion criteria: ria/∑ Si . Social ria/∑ Si . The oveThe mean age was 38 years with 29.4% aged between 20–29 years, 25.8% 30–39 years, 23.4% 40–49 years, 15.8% 50–59 years and 5.6% over 60 years old. In this study population, 93% were cytology negative, 4.7% had borderline changes and 1.4, 0.5 and 0.52% had mild, moderate and severe dyskaryosis respectively. The distribution of samples according to SDS based on the LSOA ranking system was 9.5% from the 10% most deprived; 5.6% from the 10 to 20%; 7.4% from the 20 to 30%; 12.6% from the 30 to 50%; and 64.8% from the 50% least deprived LSOA in Wales.β-globin-positive DNA extraction controls and 96% reproducibility for HR and LR HPV-positive results. Here, 1015 HR HPV-positive and 209 LR HPV-positive cases were identified. In the 1015 HR HPV-positive cases there were 2007 HR infections and 157 were also positive for LR HPV. Overall HPV prevalence was 13.5%  with an age standardised HPV prevalence in the South Wales population of 9.3%.Of the 10 000 samples processed, 921 did not meet the inclusion criteria and full statistical analysis was performed on the remaining 9079 samples. Internal quality assessment indicated 93% reproducibility for n=1015) the most prevalent HR genotypes detected were 16 (31.4%), 31 (22.6%), 18 (21.7%) and 58 (19.8%). The proportion of HR HPV cases attributable to types included in currently licensed prophylactic HPV vaccines (HPV 16 and HPV 18) was 46% overall (n=471 out of 1015) and 70% (n=28 out of 40) for cases with severe cytological abnormalities. Here, 42%  of HR HPV-positive cases had an infection with a single HR type and in these women HPV 16 was the predominant genotype (n=143 out of 430). The remaining 58%  had multiple HR types and HPV 18 was the most predominant type detected (n=201 out of 585). The prevalence of each HPV genotype in cases with a single HR infection was compared with cases containing multiple HR types and no statistically significant differences were found for HPV 16, 51 or 66 . All other genotypes were statistically significantly more common in cases with multiple HR types compared with those with a single HR type .In HR HPV-positive cases ( HR type . In casen=262 out of 430) and cases with multiple HR types (n=411 out of 585) were found in women aged 30 years or less. Cases with multiple HR types were more common than single HR cases in all women upto 44 years and this was statistically significant in women aged 30 or less .Here, 66% of all HR and 57.6% of LR HPV cases were found in women aged 30 years or less and the prevalence of HR cases decreased progressively with age from 29.4% at 20–24 years to 1.4–2% at ages 50–65 years . In concn=8434), borderline (n=426), mild (n=126), moderate (n=43) and severe (n=50). There was a statistically significant increase in the proportion of HR HPV-positive cases from 7% (n=590 out of 8434) in cytology negative samples up to 80% (n=40 out of 50) with severe dyskaryosis (P<0.0001). There was a limited variation between total HR HPV prevalence in cases with borderline, mild, moderate and severe dyskaryosis because of the many cases with multiple HR types in all grades with abnormal cytology. In contrast, cases with a single HR type predominated in women with severe dyskaryosis and this was significant relative to their prevalence in negative, borderline, mild and moderate cytology samples . Severe cytological abnormalities were detected in 50 cases and 40 out of 50 were also HR HPV positive: 21 out of 40 cases contained a single HR HPV infection 14 of which were positive for HPV 16 and in the remaining 19 out of 40 cases with multiple HR types 11 were HPV 16 positive. Here, 25 of the 366 LR HPV cases detected in this study population had an abnormal cytology .HPV prevalence by cytology grade was calculated as a percentage of the total number of women in the study population classified as negative (Most of the women in the study were from the 50% least deprived LSOA (64.8%) and the lowest HR HPV prevalence was detected in the 50% least deprived LSOA in Wales (10.5%) with the highest in the 10% most deprived LSOA (13.3%) according to SDS. However, HR HPV cases were not significantly more prevalent in any one rank .This is the largest type-specific investigation of HPV infection in an unselected UK population to date, with an overall HPV prevalence of 13.5% (9.3% age standardised). This baseline HPV data was particularly robust for two reasons: (i) Samples were anonymous and therefore did not require consent allowing collection of consecutive samples from all women attending routine cervical screening; (ii) A two-tier typing procedure was followed in which HR-positive samples were identified and then validated with a second HPV PCR before typing for 14 individual HR HPV types, thus reducing the risk of false positive results. Stringent quality control procedures were also employed involving random retyping of 10% samples.Within Europe, the closest comparative study was in Scotland  of these women were aged 30 years or less and more recent studies have observed an increasing number of multiple infections within this age group (The highest HR HPV prevalence was found in women aged 30 years or less consistent with other findings (The use of an anonymous screening population avoids many potential sources of bias. However, it inevitably only includes people who attend for their smears and we do not have data on the HPV status of non-attendees. The reported correlation of increased cervical cancer incidence associated with social deprivation (We identified HPV 16, 31, 18 and 58 as the predominant HR types, which is broadly consistent with other studies . Our datIn summary, current prevalence of HPV infection in South Wales is 13.5% and half of all current HR HPV cases would be targeted by the prophylactic HPV vaccines.


The natural history of infections with many human papillomavirus (HPV) types is poorly understood. Here, we describe for the first time the age- and sex-dependent antibody prevalence for 29 cutaneous and five mucosal HPV types from 15 species within five phylogenetic genera  in a general population. Sera from 1,797 German adults and children  between 1 and 82 years (median 37 years) were analysed for antibodies to the major capsid protein L1 by Luminex-based multiplex serology. The first substantial HPV antibody reactions observed already in children and young adults are those to cutaneous types of the genera nu (HPV 41) and mu . The antibody prevalence to mucosal high-risk types, most prominently HPV 16, was elevated after puberty in women but not in men and peaked between 25 and 34 years. Antibodies to beta and gamma papillomaviruses (PV) were rare in children and increased homogeneously with age, with prevalence peaks at 40 and 60 years in women and 50 and 70 years in men. Antibodies to cutaneous alpha PV showed a heterogeneous age distribution. In summary, these data suggest three major seroprevalence patterns for HPV of phylogenetically distinct genera: antibodies to mu and nu skin PV appear early in life, those to mucosal alpha PV in women after puberty, and antibodies to beta as well as to gamma skin PV accumulate later in life. Papillomaviruses (PV) are a large and highly diverse group of DNA viruses that infect cutaneous and mucosal epithelia of warm-blooded vertebrates. Of the more than 100 identified human PV (HPV) types, many cause benign lesions like warts and papillomas, and some also cervical, other anogenital, and oral cancers. For most HPV, transmission routes, pathogenesis, and time and duration of infection are only poorly understood. In the German general population, we investigated the prevalence of antibodies to the capsid proteins of 34 HPV types representative of all five PV genera  that contain HPV. We provide evidence for different age- and sex-dependent seroprevalence patterns of phylogenetically related HPV: antibodies to cutaneous mu and nu PV appear early in life, those to mucosal alpha PV after puberty, and those to beta and gamma skin PV accumulate in adulthood. Papillomaviruses (PV) are non-enveloped DNA viruses infecting cutaneous or mucosal epithelia of warm-blooded vertebrates. So far at least 118 distinct PV types, more than 100 of them isolated from humans, have been completely described PV within the same genus may or may not show similar biological and pathological characteristics. Thus, cutaneous HPV are found among the five genera alpha , beta (β), gamma (γ), mu (μ), and nu (ν), whereas the 48 HPV types infecting the mucosa belong exclusively to the genus alpha (α). HPV infections are widespread and can cause a variety of mostly benign tumours such as warts and condylomata. However, the infection with certain mucosal HPV types leads to malignant cell proliferation Besides the extensively studied HPV 16 and some closely related mucosal HR types, only little is known about the natural history of infections by other types. For both HR  and LR (e.g. HPV 6 and 11) mucosal HPV, transmission occurs mainly via sexual intercourse For mucosal HPV types, antibodies to the viral major capsid protein L1 have been shown to be markers for present and past infection. Thus serology provides a powerful epidemiological tool to investigate the wide variety of mucosal and cutaneous HPV types and their distribution in the population Seroepidemiological studies especially on cutaneous HPV infection are scarce and those performed in the past were restricted to a single or few HPV types. To our knowledge, mucosal HPV L1 antibody analyses have been performed so far for HR HPV types 16, 18, 31, 33, 39, 45, 52, 58, 59, 73, for LR HPV types 6 and 11 and for HPV types 13 and 32. The serologically best studied cutaneous types are HPV 1, 5, and 8 et al.HPV antibody analyses are complex because of the large number of HPV types, and conventional enzyme-linked immunosorbent assay (ELISA) methods allow analysing the reactivity of a serum sample to only one antigen per reaction. We recently developed multiplex serology Here we describe for the first time the age- and sex-dependent antibody prevalence for 29 cutaneous and five mucosal HPV types representing 15 species within five phylogenetic genera  in a general population.To obtain an unbiased (cut-off free) impression of HPV antibodies, the strength of the antibody reactions was plotted against the percentile for each HPV type and age and sex group. For all HPV types and in both sexes, antibody reactivity was lowest in children. The biggest difference in antibody reactivity was observed between children and young adults (15–24 years) against HPV 1, which is representative for the HPV types within the μ and ν genera. HPV 1 antibody reactivities were similar in adults aged 25–54 years and gradually declined thereafter.Antibody levels against HPV 16, which is representative for genital/mucosal HPV types within the α PV, changed with age in women but hardly in men. In comparison to children, antibody reactivities were substantially higher in young women and peaked in the 25–34 years age group.Antibody reactivity to HPV 8, which is representative for HPV types within the β and γ PV, became stronger with age in both genders. A first peak in antibody reactivity was observed in women at about 40 years and in men at about 50 years. Especially among men, a second peak was found in the oldest age group.These age-dependent antibody patterns were present for both strong and weak responses, down to reactions as low as 50 median fluorescence intensities (MFI).The analysis of the complex antibody patterns was simplified by the generation of seroprevalence values. HPV seroprevalence is defined here for all HPV types and age and sex strata as percent of sera reacting with a given HPV type above a cut-off value of 200 MFI.In all 1797 sera, the overall seroprevalence for any of the 34 HPV types analysed was 59.7% . HoweverHPV types 1 and 4 showed the highest type-specific seroprevalences  . Among cSeroprevalence patterns were similar for μ and ν PV, for high-risk mucosal α PV in females, and for β and γ PV, respectively . Most HPFor statistical analysis of the age- and sex-dependent seroprevalence patterns, age groups were combined to increase statistical power . Highly The strongest sex difference was observed for the mucosal high-risk HPV type 16. Seroprevalence in younger women was 5.8-fold and in older women still 2.4-fold higher than in men of the same age. For two other mucosal high-risk types, seroprevalence was also slightly elevated in younger women , however without statistical significance. Seroprevalence for cutaneous α, β and γ PV tended to be higher in younger women than in younger men (median 1.3-fold), but HPV 2 was the only individual type for which this difference was statistically significant. In contrast, women >34 years showed a lower seroprevalence for these genera than men (median 0.8-fold), with significant differences for the HPV types 75, 9, and 5.Antibody reactivity to more than one HPV type was frequent . While 2Multiple seropositvity to μ and ν PV showed a steep increase already in young adults (15–24 years) and thus was the earliest to occur in both genders. Multiple seropositivity to high-risk mucosal α PV in women (but not in men) first occurred and also peaked at 25 to 34 years. For cutaneous α PV, multiple seropositivity showed an isolated peak in the oldest age group in both genders. For β and γ PV, both genders showed two prevalence peaks, in women at around 35 and 60 years and in men some 5 to 10 years later.Fundamental data on the humoral immune response to HPV infections is scarce. Most serological analyses performed in the past were case-control studies that mainly focussed on mucosal HR HPV types highly prevalent in cervical cancer and mostly on women at productive age. Little is known about the age distribution of HPV infections, especially in children, men and in old-aged individuals, and for many HPV types no serological data is available at all. To understand the natural history of HPV infections, investigations of a broad range of HPV types belonging to different species and genera are needed. This is the first large study to analyse simultaneously antibody reactions to 34 HPV types in a general population. The cross-sectional data presented here allow a detailed and comprehensive assessment of the seroprevalence in the adult German general population by HPV type (and higher taxonomic order), sex and age. The data for the children originating from two hospitalized groups may be less representative for the German population. The conclusions of our study results might also be limited due to the use of unadjusted seroprevalence values. However, when age standardization was applied seroprevalence estimates changed only marginally.HPV of the same genus, with the exception of cutaneous α PV, showed similar antibody patterns and are therefore discussed as group. For comparisons with previously published prevalence data across different laboratories it is important to keep in mind that different assay formats and, probably even more important, different cut-off definitions may greatly influence absolute prevalence figures.Genus μ PV are associated with distinct common warts, HPV 1 with deep palmoplantar warts (myrmecia) and HPV 63 with cystic or punctate, mainly plantar warts In our study, seroprevalence for HPV 1 (17.0%) was the highest among all types analysed, antibodies to HPV 63 (10.6%) were less prevalent. These findings are in agreement with a study reporting HPV 1 as the most prevalent type in warts from Germany (27.3%) et al.et al.In both sexes, antibodies against μ and ν PV were the first to be seen. Seroprevalence peaked after the age of 14 and declined only slightly thereafter. In line with this observation, Hamsikova In our study, type-specific seroprevalences for HR mucosal HPV in adult women (>14 years) were highest for HPV 16 (10.9%) followed by HPV 33 (4.7%), 18 (4.6%), 58 (4.1%), and 52 (0.7%). DNA prevalences for HR types in German women without cervical abnormalities were concordantly also highest for HPV 16 (14.6%) followed by HPV 58 (2.7%), 52 (2.5%), 33 (2.2%), and 18 (1.7%) Antibodies to HR mucosal α PV were rare in children , which is consistent with seroprevalence rates for HPV 16 in children ranging from 1.5–7.6% reported by other studies HPV 16 serology in comparison to the other HR HPV was unique. Only for HPV 16, seroprevalence was significantly higher in women than in men both among younger and older adults, while seroprevalence for the other mucosal HR types showed only a non-significant increase in women. Although studies on HPV seroprevalence in men are rare, the lower seroprevalence for mucosal HPV types 6, 11, and 16 assessed by virus-like particle (VLP) serology is well known It has been shown that persistent infection is associated with higher seroprevalence The most heterogeneous antibody distribution with regard to both age and type was seen for cutaneous α PV, although they are closely sequence-related. However, due to the overall low seroprevalence, the power of this pattern analysis is low. While antibodies to HPV 3 were frequent in children and decreased with age, seroprevalences for HPV types 2, 57, 10, and 77 were low in children and increased with age. These findings may suggest different natural histories of the individual wart-associated α PV types. HPV 3 and 10 are associated with plane, HPV 2 and 57 with common, and HPV 27 with intermediate warts For β and γ PV, age distribution patterns were very homogeneous. Seroprevalence for these types was low in children and increased with age. In middle-aged women, seroprevalence tended to be higher than in men of the same age. In older adults, this sex ratio shifted to a higher seroprevalence for men. We observed two seroprevalence peaks, both occurring slightly earlier in women than in men. At present, we can only speculate about potential reasons for the sex-associated serological differences for β and γ PV. In men, body hair is more abundant, the use of chemicals on the skin (cosmetics) is probably reduced, and for the population studied here sun exposure for large parts of the body during outdoor work might have been more frequent.et al. found a statistically significant association of seropositivity with age and male sex for HPV 24 but not for HPV 5, 8, 15, 20 and 38 Serological studies showed elevated prevalences of antibodies to β PV, mainly HPV 5 and 8, in EV and in immunosuppressed patients, in patients with dermatological diseases like squamous cell carcinoma of the skin DNA prevalence data indicate an ubiquitous distribution of these types in the population. Cutaneous HPV mainly of genus β are found in normal skin and plucked hairs of different body sites from healthy individuals, with up to 96% overall DNA prevalence in adults For most of the analysed HPV types, seroprevalence peaked twice. A possible explanation for the second peak at ages beyond 55 years is a reactivation of latent infections perhaps by reduction of immune surveillance with increasing age followed by increasing viral load and antibody induction. For mucosal α PV in women, it might in addition be caused by changes in the genital epithelium associated with menopause. Alternatively, the seroprevalence in older age groups of this serum collection might be due to a cohort effect. Possibly varying behaviour in different birth cohorts might have influenced exposure, such as sexual behaviour for genital HPV. We speculate that also environmental factors, such as sun exposure or nutrition, may have influenced the extent of HPV replication and/or the functionality of the immune system. The sera of this study were collected in 1987 and 1988. It remains to be examined whether this age-dependent distribution will also be present in sera collected 20 years later.We observed a substantial frequency of multiple seropositive reactions, e.g. 48% of HPV 5 positive sera reacted also with HPV 8. Multiple infections with mucosal HPV One limitation of this study is the use of a uniform, arbitrarily defined cut-off for seroprevalence calculations. For assays of antibodies to sexually transmitted genital HPV, cut-off definitions can be based on seroreactivities in groups of virgins In our study all antigens were identically constructed L1 fusion proteins expressed in the same bacterial expression system. The full-length L1 fusion protein density on the beads for different HPV types was very similar, since MFI values obtained after staining of the carboxy (C)-terminal tag epitope with a monoclonal antibody varied less than two-fold. Thus, given the similar properties of the antigens the use of an uniform cut-off appears justified. To avoid false-positivity by low-level cross-reactivity and to increase type specificity of the seroprevalence values, a cut-off well above background levels was chosen. Thus, the cut-off is rather stringent and probably underestimates the true seroprevalence of cutaneous HPV infections.However, as shown in Direct comparison of GST-L1 fusion protein- and VLP-based ELISA with human sera has been performed for HPV 16 and 18, and showed good correlation In conclusion, the serological results presented here suggest different seroprevalence patterns of phylogenetically related HPV: Antibodies to cutaneous μ and ν PV appear late in childhood, those to mucosal high-risk HPV after puberty mainly in women, and seroprevalence for β and γ PV peaked late in life in both genders.The assays developed here allow further serological investigations to enlarge our still scarce knowledge on the natural history of especially cutaneous HPV infections. Interesting issues are e.g. effect of sun exposure on HPV prevalence, and the immune response in EV patients and patients with cutaneous warts in comparison to healthy people. In addition, seroepidemiological case-control studies might help to understand the potential role of cutaneous HPV in the development of non-melanoma skin cancer.E.coli Rosetta cells  as double fusion proteins with amino (N)-terminal GST and a C-terminal peptide (tag) consisting of the last 11 amino acids from the large T antigen of simian virus 40. The expression constructs for the L1 proteins of HPV types 16 and 18 have been described http://genome.dkfz-heidelberg.de/). Sequences of 27 of the 34 L1 constructs were identical with the published HPV nucleotide sequence. Seven of the 11 sequence variations in L1 of HPV 5, 8, 10, 16, 18, 41, and 57 resulted in amino acid changes  in  changes . Eight o changes , D90252,2 for 1 h at room temperature, lysates were cleared from insoluble components (e.g. cell membranes) by centrifugation . For all HPV types, the proportion of insoluble fusion protein was low. Supernatants were stored with 50% glycerol at −20°C.Fusion protein expression was induced at room temperature by 0.25 mM isopropyl-β-D-thiogalactoside (IPTG) and six h after induction bacteria were harvested. The pellet from a 1 liter culture was resuspended in 10 mL of 40 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 8.0, 2 mM DTT containing complete protease inhibitor cocktail , and lysed with a high pressure homogenizer . After incubation with 2 mM ATP and 5 mM MgClFusion protein expression was characterised by Coomassie-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described Verification and concentration of full-length GST-L1-tag proteins in the lysates was determined by GST-capture ELISA Anonymous sera from 1797 individuals  were analysed. These sera originated from three serum collections. The largest contribution is from the population-based German Nutrition Survey , and the age structure of our total study  is characteristic for the age structure of the total NVS population (p = 0.9629). This allows the conclusion that our study is likely to be representative of the German population.in situ through binding to the glutathione casein-coated fluorescence-labelled polystyrene beads. Each fusion protein was bound to a spectrally distinct bead set. A total lysate protein concentration of 1 mg/mL was used for all lysates, an at least five-fold excess to saturate GST fusion protein binding on the beads. Monoclonal anti-tag antibody binding to the different antigen-loaded bead sets varied less than two-fold, indicating similar full-length L1 fusion protein density for the different HPV types. Fusion protein-loaded bead sets were mixed. Sera were pre-incubated at 1∶50 dilution in PBS containing 1 mg/mL casein, 2 mg/mL lysate from bacteria expressing GST-tag alone to block antibodies directed against residual bacterial proteins and GST-tag, 0.5% polyvinylalcohol , 0.8% polyvinylpyrrolidone  and 2.5% Superchemiblock  to suppress unspecific binding of antibodies to the beads themselves Sera were analysed simultaneously for antibodies to 34 HPV types by multiplex serology as described The glutathione-casein coupled bead sets were loaded with their respective antigen in one batch. The efficiency of antigen loading on beads was quantified via the C-terminal tag as described 2) for the individual antigens ranged from 0.761 to 0.986 (median 0.963) for day 2 versus 1 and from 0.691 to 0.989 (median 0.947) for day 3 versus 1. The raw data of days 2 and 3 for each antigen were divided by the slopes of the regression lines of the QC data pairs of days 1 and 2 or days 1 and 3, respectively, to correct for inter-day variation.Study sera were analysed once on three consecutive days. A quality control panel (QC) of 46 sera was included each day resulting in three QC data sets to determine inter-day variation. For inter-day variations of raw MFI values, Pearson correlation coefficients  calculated from these data for the various antigens ranged from 13.0% to 23.4% with a median of 16.6%.Auto-fluorescence of each bead set and background reactions resulting from binding of secondary reagents to the antigen-loaded beads were determined in one well per plate without human serum. After correction for inter-day variation, mean background values  were subtracted from the raw MFI values and then antigen-specific reactivity was determined by subtraction of the MFI of GST-tag from the MFI of the specific antigen. Cut-off values to define seropositivity for all antigens were arbitrarily set to 200 MFI.Serological data were stratified by sex and age. Since sexual transmission is known to play a major role in mucosal HPV infection, the first age group encompassed children (14 years and younger), followed by age groups in 10 year intervals. The age distribution of our study and the German standard population as reported by Figure S1Double seropositivity in relation to amino acid sequence identity of the paired HPV L1 proteins(0.57 MB TIF)Click here for additional data file.Text S1Double seropositivity in relation to amino acid sequence identity of the paired HPV L1 proteins(0.03 MB DOC)Click here for additional data file.