Recently, substantial evidence has been mounting that Human Papillomavirus (HPV) infection is playing an increasing important role in oral cancer. Because of the attention and clamor surrounding oral HPV infection and related cancers, as well as the use of HPV prophylactic vaccines, in this invited perspective the authors raise some questions and review some controversial issues on HPV infection and its role in HNC, with a particular focus on oral squamous cell carcinoma.Head & Neck Cancer (HNC) represents the sixth most common malignancy worldwide and it is historically linked to well-known behavioural risk factors, The problematic definition and classification of HNC will be discussed, together with the characteristics of oral infection with oncogenic HPV types, the frequency of HPV DNA detection in HNC, the location of HPV-related tumours, the severity and prognosis of HPV-positive HNC, the diagnosis of oral HPV infection, common routes of oral infection and the likelihood of oro-genital HPV transmission, the prevention of HPV infection and novel therapeutic approaches. HNC) and particularly in oral squamous cell carcinoma (OSCC), as well as the diagnosis of oral HPV infection and its oro-genital transmission.Taking into consideration the attention and clamor surrounding Human Papilloma Virus (HPV) infection and related cancers, HPV vaccine controversies, as well as the recent Nobel Prize awarded to Prof. zur Hausen for his research on HPV and cervical cancer, authors believe it opportune, in this invited perspective, to raise various questions and review some of the most controversial issues related to HPV and its role in Head & Neck Cancer . However, this term defines a heterogeneous group of malignant tumours, involving several different several sub-sites. The majority of HN malignancies are squamous cell carcinomas (SCC) and they originate from the epithelium which lines the upper aero digestive tract, i.e., the oral cavity, the pharynx and the larynx. Arising in a multistep process resulting from the gathering of genetic and epigenetic defects and clonal spreading out of given cell populations [orldwide , and it ulations , HN maliHPVs are epitheliotropic oncogenic DNA viruses with more than 120 identified genotypes: the so-called high-risk (HR) HPV, like HPV 16 and 18, have been definitively recognised as being strongly associated with ano-genital  cancers. In patients with high-grade neoplasia and cancer, HR HPV DNA can usually be found integrated into host DNA. The oncogenic potential of HR HPV is attributable to its ability to insert specific DNA fragments (early genes E6 and E7) into the host cellular genome. As a result of this integration, various key functions of tumour suppressor factors (the p53 and pRb pathways) are abrogated, leading to defects in apoptosis, DNA repair mechanisms, cell cycle regulation and, finally, to cellular immortalization, thus inducing and maintaining the malignant phenotype.in vitro [The HPV involvement in HN carcinogenesis was first proposed in 1983 by Syrjanen et al. ; 3) the in vitro ; and 4) in vitro ,7.Whilst occurring in a lower percentage than in cervical mucosa, HR HPV E6/E7 transcripts and/or viral integration have also been detected in HNC  and, it In addition to these logical statements, it is timely to raise the following questions and shed light on these controversies:i) the site of the mucosa examined; and ii) technical issues (or the HPV molecular assay employed).A very wide range of viral prevalence (0%–100%), in addition to the presence of HR HPV in oral normal mucosa ,13, has HNC, HNSCC or oral cancer in place of the specific OSCC, oro-pharyngeal or laryngeal carcinoma. This issue plays a critical role since specific epithelial areas of the upper aero-digestive tract (such as the squamous-columnar junction at the level of the tonsillar crypts and the glottides) display greatest susceptibility to HPV due to the easy exposure of the basal cells, and this is also the case with the meta-plastic epithelial area in the cervix. Hence, it is critical to group HNSCC together as a single entity with the consequent difficulty of comparing the data. Recent epidemiological and molecular data have indicated the involvement of HR HPV in a given subset of HNSCC [A great deal of confusion is created by the use of the generic term of HNSCC -19  would seem more efficient than sucrose, glucose and saline, in terms of DNA yield, quality and stability [in situ hybridization should be avoided as they lead to lower HPV rates, compared with those obtained by using PCR assays [When performing the molecular detection of HPV DNA from oral samples, attention should be focused on the low productivity of oral HPV infections. Indeed, studies based on polymerase chain reaction (PCR) assay have shown that, when compared to those from HPV-positive cervical specimens, HPV-DNA positive oral samples from both normal oral mucosa ,48 and c samples . However samples ,52. Of ttability . As far R assays ,55.As is the case with the most validated sampling procedures, it should be kept in mind that there are several other variables which may affect the efficiency and reliability of HPV detection in oral samples. For instance, the method of DNA purification also has a potentially large impact upon the ability to detect HPV DNA by PCR amplification . Additioin utero, as a transplacental or ascending infection [Even though modes of HPV transmission in the head and neck mucosal districts have not been fully resolved, theories have proposed multiple pathways for HPV transmission, including perinatal transmission, auto-infection from oral-genital contact by hand and sexual transmission by oral-genital contact. The perinatal transmission of HPV to neonates at birth has been detected in several studies which have demonstrated that recurrent respiratory papillomatosis is associated with the perinatal transmission of HPV. While the possibility of auto-infection among women with cervical HPV infection is still a matter of debate ,58-60, onfection ,65. Caesnfection  and HPV nfection .Can oropharyngeal cancer now be placed in the category of virally-mediated cancers?", like, for instance, HPV-related cervical, anal, vulvar, and penile cancers, or the Epstein Barr virus-associated nasopharyngeal cancers. Whilst it is premature to imagine an HPV vaccine which could protect women and men against HNC, the possibility that various oral, oro-pharyngeal, and laryngeal cancers might be prevented by HPV vaccination is certainly a hope, and this would bring forth significant implications from a public health perspective.Although the use of condoms has proven efficient in the prevention of most sexually-transmitted infections, the effectiveness for prevention against HPV infection is not as clear, and a significant variability (from 0% to 80%) in the condom protection of HPV infection has been reported . Alternain vitro studies on antisense HPV RNA transcripts, the transcripts of the E6 and E7 genes of HPV type 16 were introduced  into human cervical cancer cells harbouring HPV 16; the effects of expression of these genes on cell and tumour growth were then analysed. It was found that, when E6 and E7 protein expression is suppressed, p53 and Rb protein expression increase, the Ad5CMV-HPV 16 AS-infected cells undergo apoptosis, and cell growth and tumourigenicity are greatly suppressed. However, even though gene therapy may prove to be beneficial in the treatment of cervical cancer and other HPV-induced diseases, a further understanding of the viral life cycle and the mechanisms underlying HPV-induced oncogenesis is necessary before this method could be employed for clinical use in humans.HPV vaccines should eventually reduce the impact of these viruses on human health. However, vaccines may not be useful for the treatment of existing disease, and it is necessary to develop effective therapies targeting those individuals who are already infected or are currently excluded from the first phase of a prophylactic vaccination program. Given the strong relationship between the expression of HPV E6 and E7 and cervical cancer carcinogenesis, many approaches have been directed against these oncogenes, for example, gene therapy for HPV-positive cervical cancers . The difThe authors declare that they have no competing interests.All authors read and approved the final manuscript.


The aim of this study was to determine the prevalence of human papillomavirus (HPV) and 15 species that cause sexually transmitted infections (STIs) in negative cytology. In addition, we compared the diagnostic performance of multiplex polymerase chain reaction (PCR) with widely available techniques used to detect HPV.We recruited 235 women of reproductive age who had negative cytology findings in a liquid-based cervical smear. STIs were identified by multiplex PCR, and HPV genotypes by multiplex PCR, hybrid capture 2, and DNA microaray; discordant results were analyzed by direct sequencing.Gardnerella vaginalis, Ureaplasma urealyticum. The incidence of HPV in negative cytology was 23.3%. Low-risk HPV infection was significantly correlated with Chalmaydia trachomatis, and high-risk HPV infection was significantly correlated with Group β streptococcus. The analytical sensitivities of the multiplex PCR and DNA microarray were higher than 80%, and the analytical specificity was nearly 100% for all tests.Approximately 96.6% of patients with negative cytology results were positive for pathogens that cause STIs. The pathogens most frequently detected were Multiplex PCR yielded results that most of patients with negative cytology were positive for pathogens that cause STIs, and were more similar to that of DNA microarray, than that of hybrid capture 2 in terms of analytical sensitivity and prediction value of HPV infection. Sexually transmitted diseases (STDs) are fairly common in people of reproductive age; more than 50% of new STD infections occur in adolescents. However, only 50% to 80% of reportable STDs that occur in the United States are actually reported . Testingtrichomoniasis or candidiasis, most inflammatory agents are only rarely recognized under the microscope and usually produce nonspecific cytopathic effects. For example, bacterial vaginosis (BV) is a leading cause of abnormal vaginal discharge as a sequel of gynecologic conditions, including endometritis, pelvic inflammatory disease (PID), post-surgical abortion, post-hysterectomy infection, and cervical intraepithelial neoplasia [Lactobacillus spp., logarithmically-increased Gardnerella vaginalis, and the presence of potentially pathogenic bacteria, including Bacteroides, Peptococcus, Mobiliuncus, Ureaplasma urealyticum, and Mycoplasma spp. Mycoplasma spp. and U. urealyticum are most frequently detected in the vaginal tract [Mycoplasma are the only cell wall-deficient, membrane-bound organisms capable of independent self-replication: they include Mycoplasma hominis, M. genitalium, M. primatum, M. spermatophilum, and M. penetrans [With the exception of a few organisms that cause sexually transmitted infections (STIs) such as eoplasia . Howevereoplasia ,5. The pal tract , and areal tract -5. The menetrans ,8.Despite the fact that many pathogens are not detected by Gram stain, Gram-stained vaginal secretions are still accepted as the diagnostic gold-standard, rather than the culture of secretions. With the advent of advanced techniques such as those based on liquid-based cytology -13, it iHigh-throughput technology including hybrid capture 2 (HC2) and DNA microarray has been currently adopted to detect HPV . HC2 is A total of 235 married and sexually active women  were recruited by the Department of cytopathology of Yonsei University College of Medicine for this study. Specimens collected from cervical liquid-based cytology medium were negative for intraepithelial lesions. Participants had no clinical STD symptoms that required a clinical visit, no history of STDs, and no abnormal Papanicolau tests (Pap smear) results. We also excluded patients with SIL or carcinoma, postmenopausal women, or those with previous operative or therapeutic history related to gynecologic disease. The study protocol was approved by the local institutional review board, and written informed consent was obtained from all participants.® STI Master ACE Detection  or Seeplex® HPV4A ACE Screening (Seegene) within 1 month of collection. PreservCyt transport medium containing endocervical cells was vortexed vigorously, and 1-mL samples were transferred into 1.5-mL polypropylene tubes, each containing 1 mL of PreservCyt transport medium. The tubes were centrifuged at 13,000 × g for 15 min at 20°C. Supernatants were discarded, and each cellular pellet was suspended in 200 μL of PBS. DNA was purified from these samples using the QIAamp DNA Mini Kit according to the manufacturers' instructions . The quality and quantity of purified DNA was measured by spectrophotometry.Cervix swab samples were collected from the posterior fornix and lavaged with 5 mL of sterile phosphate-buffered saline (PBS pH 7.4). PreservCyt specimens were tested by Seeplex® STI Master Panel 1, 2, 3, and HPV4A ACE Screening. Panel 1 comprised of Neisseria gonorrhoeae (NG), Mycoplasma hominis (MH), Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), and M. genitalium (MG). Panel 2 comprised Trichomonas vaginalis (TV), Gardenerella vaginalis (GV), Bacteroides fragilis (BF), Mobiluncus curtisii (MC), and M. mulieris (MM). Panel 3 comprised Candida glabrata (CG), C. tropicalis (CT1), C. parapsilosis (CP), Group β- streptococcus (GBS), and C. albicans (CA). Primers were designed such amplicon sizes differed sufficiently to be distinguished from each other; they ranged from 212 bp (UU) to 635 bp (CG) and the internal control was 981 bp. Optimized multiplex PCR was performed in 20 μL reactions containing DNA template, primer mixture , 2× Master mix  and 30 μg/mL of 8-methoxypsoralen (MOP), which prevents contaminating DNAs from being amplified. PCR amplification was performed in an Applied Biosystem 9700 thermal cycler  with the following conditions: 94°C for 15 min, followed by 40 cycles of 94°C for 30 sec, 63°C for 1.5 min (or 60 °C for 1.5 min for HPV4A ACE Screening), and 72°C for 1.5 min; and final extension at 72 °C for 10 min. A DNA plasmid was added to the PCR reaction mixtures as an internal control to be co-amplified with the target DNAs collected from the clinical specimens. Sterile deionized water was included as a negative control in each batch of PCR reactions. Target organisms, target genes, accession numbers, and expected amplicon sizes are summarized in Additional File Four primer sets were tested by STI multiplex PCR: Seeplex® HPV4A ACE screening kit for 14 HR-HPV types  and 5 LR-HPV-types . In addition individual genotyping of HPV-16 and HPV-18 was performed with different multiplex primer mixtures.HPV was detected with the SeeplexHC2 HPV test  was performed according to the manufacturer's instructions to detect HPV, starting from 4 mL of liquid cytology specimens. The HC2-13 HR probe cocktail detects HPV types-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Relative light unit/cut-off ratio between 1 and 2.5 relative light units was used as the threshold for a positive result.We used an HPV genotyping DNA microarray  with multiple oligonucleotide probes of L1 sequence of 26 types of HPV . L1 consensus PCR products were hybridized to the probes on the microarray, and HPV genotypes were identified with a fluorescence scanner  with a 532 nm laser for excitation of Cy3.® HPV4A ACE screening kit results were resolved by sequencing with the PGMY09/PGMY11 primer sets. PCR reactions (20 μL) were performed with 2 μL template DNA, 1 μM of each primer, and 2x Master mix . PCR amplification was carried out with an Applied Biosystem 9700 thermal cycler (Perkin-Elmer) with the following parameters: 94°C for 15 min followed by 40 cycles of 94°C for 30 sec, 55°C for 1 min, and 72°C for 1 min, and a final extension at 72°C for 10 min. The entire volume of each PCR reaction was run on a 2% agarose/1× Tris-acetate-EDTA gel containing ethidium bromide. The 465-bp bands amplified by the PGMY09/PGMY11 primers were excised and transferred to 1.5-mL tubes. DNA was extracted with the QiaexII gel extraction system  and eluted with 20 μL Tris-EDTA buffer. The products were ligated into the pGEM-T Easy vector , transformed into chemically-competent JM109 cells, and plated onto two Luria-Bertani (LB) plates containing ampicillin, isopropyl-D-thiogalactopyranoside, and 5-bromo-4-chloro-3-indolyl-D-galactopyranoside. DNA was extracted from the white colonies with the Qiaprep Spin kit (Qiagen) and sequenced with the ABI Big Dye Terminator v3.1 cycle sequencing kit and an ABI 377 sequencer . Sequences were trimmed to exclude the amplification primer and vector sequences, and the resulting fragments were analyzed by BLAST http://www.ncbi.nlm.nih.gov/blast. The top hit for each sequence was listed as the genotype.Discrepant results among the HC2, DNA microarray, and SeeplexThe agreement rate of kappa value with respect to a quantitative measure of agreement between assay methods was calculated. The sensitivity of the HPV multiplex PCR, HC2 or DNA microarray relative to DNA direct sequencing is the proportion of positive samples in each assay among sequencing positive samples, and the specificity is the proportion of negative samples in each assay among those negative samples in direct sequencing. The positive predictive value is the proportion of positive samples in DNA direct sequencing, among those positive samples in each assay, and the negative predictive value is the proportion of negative samples in sequencing among those negative in each assay.GV found in 88.1% of the samples (207/235), UU in 63.0% (148/235), and GBS in 26.4% (62/235). The most frequently co-infected organism was observed in MH infections, which was commonly observed with UU (p = 0.000), MM (p = 0.000), MC (p = 0.000), and CT (p = 0.009). In addition, GV and MC (p = 0.014), MC and MM (p = 0.003), MG and CA (p = 0.006), and TV and GBS (p = 0.011) were similarly detected in the same samples.The overall prevalence of STIs in cervicovaginal samples of negative cytology, which was not clinically presented with BV, was 97.9% (230/235). Most patients of 84.9% (202/230) were co-infected by more than two microbes, whereas twenty-eight specimens were infected by a single agent Fig. . Table 1The incidence of HPV in negative cytology varied according to the assay used: multiplex-PCR, 24.7% (58/235); HC2, 21.3% (50/235); HPV chip, 22.6% (53/235). The HPV types which were most often detected included HPV-16  and HPV-18 . Less commonly detected HPV types included 51, 58, and 33 in the HR group, and 70 and 6 in the LR group. Eighteen LR-HPV types were detected by PCR (31.0%), eight by DNA microarray (15.1%), and 11 by direct sequencing. Infection with multiple genotypes was detected in 13 specimens (22.3%) by PCR and 14 specimens (26.4%) by DNA microarray.Overall, there are good agreements between methods to detect HPV, and multiplex PCR showed the highest agreement ratio with relation to direct sequencing .Because HC2 detects only HR group, 11 specimens with LR-HPV infection were excluded in the analytic sensitivity/specificity. The results of 25 of 235 specimens (10.6%) were discordant among methods; therefore these specimens were analyzed by direct sequencing. DNA chip produced more false negative results than HPV multiplex PCR and HC2 did , whereas those with HR-HPV were frequently detected with GBS .Specimens positive for LR-HPV infection were frequently co-infected with Reports of the incidence of STIs in asymptomatic women are not currently available, because of few attempts to recognize STI species despite advanced techniques based on liquid-based cytology -13.N. gonorrhea and C.trachomatis [G. vaginalis, U. urealyticum, Group β streptococcus, and M. hominis. Microbes detected in less than 2% of the specimens were CT-1, NG, and C. parapsilosis (CP) [We found that the overwhelming majority of patients with negative cytologic findings were positive for at least one STI (97.9%); many were positive for multiple infectious agents (84.9%). These results are surprising in that we did not observe high rates of infection with microbes that are typically widespread, such as chomatis ,13. In mMH was often detected in the same sample with other microbes, such as UU, M. mulieris (MM), M. curtisii (MC), and CT. However, co-infection of multiple mycoplasma strains has rarely been reported [reported .Because HPV and STIs rely on the same route of infection, this increased risk of HPV infection in patients with STIs was expected. Studies of women visiting STD clinics have reported a greater prevalence of HPV infection in this group than in the general population ,17. In oCT, whereas those with HR-HPV were coinfected with GBS. The fact that CT associates with LR-HPV rather than HR-HPV suggests that CT is less likely to contribute to cervical intraepithelial neoplasia or squamous cell carcinoma. Previous reports suggested that coinfection with CT and HPV is common in ASCUS and could contribute to the development of intraepithelial lesions [CT may influence the clearance of HPV lesions [CT infection with HPV persistence is not a coincidence [GBS and HR-HPV in negative cytology (including RCC and ASCUS). However, it remains uncertain that GBS may be an independent factor or cofactor for HPV in the development of cervical intraepithelial neoplasm. For identification of its cofactor, novel immune evasion strategies and the analysis of their functions in the context of viral and bacterial infections should be required in further studies.Specimens infected with LR-HPV were frequently co-infected with  lesions -24. It i lesions ,19, and ncidence . LikewisWe compared high-throughput HPV detection methods in the present study, and found that discrepancies with direct sequencing were observed only in 10.6%. The microarray (DNA chip) method also resulted in a significant number of false negative results. Analysis of 25discordant samples showed that multiplex PCR-HPV method exhibited the highest sensitivity, positive predictive value, and accuracy. However, each HPV detection method covered different genotypes, and different typing or grouping methods were used to present the data. For example, the relatively low sensitivity of HC2 may be due to its inability to detect LR genotypes, and its lower positive predictive values and accuracy may be due to limitations in detecting individual genotypes, and thus, multiple infections.GV, UU, GBS, and MH. Most HPV infections were not associated with any specific STI pathogen; however, LR-HPV infection correlated with CT infection, and HR-HPV with GBS infection. In terms of sensitivity and prediction value, multiplex PCR yielded data most similar to that of DNA microarray.In conclusion, asymptomatic women of reproductive age with negative cytologic findings had a high frequency of infection with The authors declare that they have no competing interests.HSS and SMN summarized, analyzed data and equally drafted entire manuscript. ARP performed statistical analysis of given data. YNL & KSK participated in cytology screening and sample preparation with performance of HC2/DNA microarray. JKK & HJC performed multiplex PCR. NHC designed and participated in DNA direct sequencing, and revised manuscript. All authors read and approved the final manuscript.Two hundred thirty-eight asymptomatic women of reproductive age with negative cytology had high frequency of sexually-transmitted infection (96.6%), and 24.4% had HPV.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/284/prepubSummarized data of target organisms, target genes, accession numbers, and the expected amplicon sizes. Primers were designed such amplicon sizes differed sufficiently to be distinguished from each other; they ranged from 212 bp (UU) to 635 bp (CG) and the internal control was 981 bp.Click here for file


Prediction of future high-grade cervical lesions is crucial for effectiveness and cost. We performed a case control study in a retrospective cohort of women with at least two cervical smears, all but the last one being negative, from the organized cervical screening programme in Florence, Italy. We searched for high-risk HPV in all previous, archival, smears from cases  grade II or worse) and in one previous smear from each control  from last smear). We applied polymerase chain reaction (PCR), and the b-globin gene was used as a DNA preservation marker. High-risk HPV was identified in 71/92 (77.17%) previous smears from 79 cases and 17/332 controls (5.12%). The odds ratio (OR) was 63.76 (95% CI 30.57–132.96). Among cases the proportion of HPV-positive smears declined slightly with increasing latency. Among cases, HPV was found in 81.24% (95% CI 69.93–88.96%) of smears with latency < 4 years and in 67.80% (95% CI 47.72–82.93%) of those taken at longer intervals, up to 6 years. These findings suggest that testing for high-risk HPV allows predicting 80% of CINII/III 3 years before the cytological diagnosis and two thirds 6 years before. They also suggest that testing women negative for high-risk HPV at longer interval and strictly following-up women who are HPV positive could be an effective strategy for cervical cancer screening. © 2000 Cancer Research Campaign


Information on risk factors was obtained by personal interview. Using PCR-based assays on exfoliated cervical cells and biopsy specimens, HPV DNA was detected in 95.3% of women with squamous cell carcinoma and in 92.0% of women with adenocarcinoma/adenosquamous carcinoma compared with 17.7% in control women. The age-adjusted odds ratio was 116.0 (95% Cl = 48.6–276.0) for squamous cell carcinoma and 51.4 (95% Cl = 11.4–232.0) for adenocarcinoma/adenosquamous carcinoma. The commonest types in women with cervical cancer were HPV 16, 18, 31, 52 and 35. The association with the various HPV types was equally strong for the two most common types (HPV 16 and 18) as for the other less common types. In addition to HPV, long-term use of oral contraceptives and smoking were associated with an increased risk. HPV is the main cause of both squamous cell carcinoma and adenocarcinoma in Peruvian women. © 2001 Cancer Research Campaign


In this study, we investigated the presence of E6/E7 transcripts of seven common high-risk human papillomavirus (HPV) types in 190 cervical biopsies. The RNA-based real-time nucleic acid sequence-based amplification assay (NASBA) and type-specific PCR, both detecting HPV 16, 18, 31, 33, 45, 52, and 58, as well as consensus PCR, were performed on all 190 biopsies. High accordance between type-specific and consensus PCR confirms that the HPV types included in this study are the most common types present in cervical dysplasia. Furthermore, we see a clear increase in the incidence of HPV, both DNA and RNA, along with the histological severity of dysplasia. HPV RNA was detected in all but two PCR-positive cases, confirming that the virus exerts E6/E7 mRNA expression in cases of high-grade dysplasia. Out of 19 women given a normal or borderline diagnosis at conisation, only four were found HPV positive, which may suggest that unnecessary conisations can possibly be reduced by introducing HPV testing into the preoperative routine assessment. Human papillomavirus (HPV) infections are associated with the development of cervical neoplasia and are a necessary factor in the aetiology of cervical carcinoma . The mosIn this study, we investigated the presence of high-risk HPV E6/E7 transcripts in cervical biopsies from patients undergoing conisation. The analysis was performed on a biopsy taken from the ectocervix/transformation zone at the time of conisation, and the pathological diagnosis of this particular biopsy was not always representative for the existing lesion that was later revealed in the cone. This strategy gave us access to a uniformly preserved material with varying degrees of squamous cell dysplasia, yet from cones that in most cases harboured a CIN III lesion. For RNA detection, the RNA-based real-time nucleic acid sequence-based amplification assay (NASBA) was used, focusing on the reportedly most common oncogenic HPV types 16, 18, 31, 33, 45, 52, and 58 .The cervical carcinoma cell lines SiHa (squamous cell carcinoma), CaSki (squamous cell carcinoma), and HeLa (adenocarcinoma), used as positive controls, were obtained from the American Type Culture Collection, USA. The SiHa and HeLa cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM), CaSki cells in RPMI medium, supplemented with 10% fetal bovine serum (FBS), 2 mThe study was performed on 190 consecutive biopsies, taken from 190 women admitted to Østfold County Hospital for treatment of CIN during the period October 1999 to October 2001. The mean age at diagnosis was 37.4 years (range 22–74 years). Biopsies were frozen at −70°C immediately after collection. All women were subjected to subsequent conisation.Initially, routine cytological grading was performed according to local routine guidelines. No review cytology was performed. After a high-grade cytology report, or an equivocal cytological smear, the patients were referred to colposcopy and biopsy . Iodine painting of the portio was used as a means of visualising dysplasia. Multiple biopsies were taken from the external os and from any colposcopically pathological lesion. In addition, the cervical canal was scraped. If these biopsies  as previously described -specific A protein (U1A mRNA) , 0.01% Tween, 20 mM (NH4)2SO4, 1.5 mM MgCl2, 0.2 mM dNTPs, 50 pmol of Gp5+ and Gp6+ primers, and 1.0 U of Taq DNA polymerase . Human papillomavirus DNA was amplified by using a thermo cycler heat block from MWG, Ebersberg, Germany (Primus 96 HPL block). The first DNA denaturation was carried out for 2 min at 94°C, followed by 40 cycles of denaturation for 1 min at 94°C, annealing for 2 min at 40°C, and extension for 1.5 min at 72°C, prior to a final extension for 4 min at 72°C. Genotyping of HPV was performed by using PCR type-specific primers against HPV types 16, 18, 31, 33, 45, 52, and 58 , utilising 25 pmol of each primer. The PCR conditions were 35 cycles of denaturation for 0.5 min at 94°C, annealing 0.5 min at 57°C, and extension for 1 min at 72°C. For a cellular control, we used primers against the human β-globin gene (The same nucleic acid extracts and amounts as used in the NASBA reaction were used for HPV DNA PCR. The L1 consensus primers Gp5+/Gp6+ (Samples that tested HPV DNA positive with the consensus primers yet negative with type-specific PCR primers were sequenced in order to determine the HPV type. Sequencing was performed at the University of Oslo (Norway) by the MegaBACE Sequence Analyzer  according to in-house protocols. Sequence comparisons were performed using BLAST.n=14) were found to contain HPV RNA. Transcripts were also found in two out of seven cases of borderline samples, in two out of 11 cases not showing dysplasia, and in three samples being histologically inadequate. Among the 88 nonrepresentative samples, we detected HPV RNA in 31 samples (35%). Results are presented in Figure 1Human papillomavirus E6/E7 transcripts from HPV 16, 18, 31, 33, 45, 52, and 58 were detected in 88 out of 190 (46%) biopsies, of which 83 out of 88 (94%) represented HPV 16, 18, 31, 33, and 45. Of the 37 samples diagnosed as CIN III (Histology 2b) 34 cases (92%) showed E6/E7 expression, only representing HPV 16, 18, 31, 33, and 45. In addition, we found oncogenic expression in 16 out of 30 cases (53%) with CIN II. No CIN I cases , one sample as HPV 51 (nonrepresentative), and one sample as HPV 67 (CIN II).To our knowledge, this is the first study performing HPV mRNA analysis for several HPV types on a large number of histologically confirmed lesions. By comparing the RNA technology NASBA with conventional PCR of HPV DNA, this study permitted us to evaluate HPVs oncogenic activity in different grades of dysplasia. With a few exceptions, all patients included in the study were originally given a high-grade CIN diagnosis (CIN II/III) by cytological examination and/or by the first histological examination (Biopsy 1). However, the RNA analysis was performed on a separate biopsy (Biopsy 2), taken from the ectocervix/transformation zone at conisation. Only one biopsy was taken, and because local anaesthesia made it difficult to locate the lesion by colposcopy, this biopsy was not always representative for an existing CIN II/III lesion that was later revealed in the cone.Biopsy 2 was examined twice histologically, in order to grade any dysplasia and to define samples not representative for the transformation zone, that is, samples containing only tissue from portio, external to the os (Histology 2a vs 2b, In the NASBA reaction, as well as in the type-specific PCR, we tested for the reportedly most common oncogenic HPV types 16, 18, 31, 33, 45, 52, and 58 . This saHuman papillomavirus expression was not restricted to the CIN lesions. We also found oncogenic expression in two of 11 cases not showing dysplasia (Histology 2b, In addition to type-specific PCR, we also tested the biopsies for HPV DNA using HPV consensus primers . We madeThe problem of inter- and intraobserver variability in cytology and histopathology paves the way for additional objective strategies in detecting cervical cancer precursors. Moreover, current management protocols may in certain cases lead to overtreatment . Almost In conclusion, this study shows that HPV DNA and RNA detection achieve a similar result in biopsies of severe dysplasia, strengthening the case that HPV E6/E7 gene expression is necessary for oncogenesis in this region . In gene


Human papillomavirus (HPV) tests are crucial diagnostic tools for the prevention of neoplastic lesions of the uterine cervix. However most commercial methods are designed to detect high-risk (HR) HPV types and a limited selection of low-risk ones, thus missing a fair number of intermediate/low-risk types. As a result, many HPV infections remain undiagnosed, generating distrust in virological diagnosis among gynaecologists, who continue to rely preferentially on cytological and colposcopic findings.In this study, we tested 6,335 consecutive clinical samples, most of them from Italian patients with cytological abnormalities. The samples, collected in 2000–2007, were analyzed using PCR amplification of a 173–206 bp (depending on HPV type) conserved region in the L1 open reading frame, restriction endonuclease analysis and, where required, sequence analysis for type determination. Analysis of a smaller male sample and long term follow-up of a few female subjects was also performed. A total of 2,161 samples tested positive for HPV DNA (32.1%); 21.3% of them were mixed infections. Overall, 59 known and 2 unknown HPV types were detected. Their relative prevalence was calculated; notably, types not clearly identifiable using the most common commercial method accounted for 36% of infections. Clinical findings associated with the underdiagnosed types ranged from H-SIL to low-grade abnormalities, although none of these infections resulted in invasive cancer.Given the high prevalence of some underdiagnosed HPV types in the population  and their frequent association with cytological abnormalities, techniques capable of detecting and typing them would prove extremely useful. Human papillomavirus (HPV) infections of the genital tract are highly prevalent in the population worldwide; indeed, it is estimated that the great majority of sexually active individuals become infected with one or more of these viruses in their lifetimes. Most conditions are transient and are cleared without consequences. However, in a small proportion of cases, infections associated with the so-called high-risk (HR) HPV types can persist in typical lesions with a high viral load for years, and a fraction of such lesions eventually progress to invasive malignancies. Although many cofactors normally concur in this process, the HPV type implicated  and the inability of the immune system to clear the infection are by far the most important determinants of HPV-related diseases (for review see: ref ). RecentWe investigated the distribution of underdiagnosed HPV types in genital infections, using a molecular strategy capable of detecting a large number of known (and possibly unknown) HPV types, and calculated their relative prevalence and their role in the arising of cytological abnormalities and dysplastic lesions.th most frequent type. According to the technique used, 21% of all fully typed infections were mixed, 17% were double, 3% were triple and less than 0.2% were quadruple or more. However, the latter figure might be inaccurate, because some multiple infections were untypeable and the precise number of coinfecting viruses could not be determined. In Figure A total of 6,029 female genital samples (including 47 biopsies) received from 7 different clinical centres in 2001–2007 were analyzed for HPV DNA; 2,110 (33.8%) tested positive for HPV DNA. A total of 54 different restriction patterns were detected, identifying up to 62 different known types; of these, 46 unique restriction patterns identified single types, while 8 restriction patterns identified two different types each. Interestingly, most of the latter patterns identified couples of HPV types phylogenetically related within the same species , while in two cases they identified pairs made up of two more distantly related types: 27/39, a low-risk cutaneous/mucosal and a high-risk type and 40/63, a low risk mucosal and a cutaneous type. In the case of mixed infections, the individual co-infecting types were mostly resolved by the analyses of the mixed restriction patterns, up to four different types. In cases where restriction analysis of the amplified product resulted in a clear but unknown pattern, the amplification product was sequenced to achieve a definitive result, mostly showing the presence polymorhisms in the restrictions sites. Most of these variants are extremely rare, while common variants were detected for HPV type 66 and 54. Nonetheless, some mucosal HPV types were never detected in our samples, including HPV32, HPV71, HPV73, HPV86 and the most recently characterized types from HPV97 to HPV106 . A few exclusively cutaneous types, i.e. types 1, 4, 5, 8, 19, 22, 50, 75, and 76, were single findings. The source of these samples is unknown, possibly they derive from warts in the groin area. The frequency distribution of the types detected in the present study is summarized in Figure in situ (CIS) at histological analysis of the surgical piece.Positive samples for which cytological information was available were subdivided into grade of abnormality (ASCUS through H-SIL) and type prevalence was calculated. As shown in Figure Finally, in 4 subjects who underwent surgical excision without prior virological testing, but whose lesion was indeed histologically classified as CINIII/CIS, the follow-up genital swabs tested positive only for HC2-undetectable viruses (HPV53 or HPV87) suggesting that these viruses might have been implicated in the pathology observed. However, the analysis of their available formalin-fixed surgical samples revealed HPV16 infection in 3 cases and HPV31 in one, indicating that HC2 undetectable viruses were co- or subsequent infections. This underlines the importance of HPV testing and, in particular, of typing prior to excision, in order to perform the correct prognostic evaluation and a reliable subsequent follow-up.Of all the samples analyzed, 306 were genital swabs from male subjects. Clinical information on the reasons leading to HPV DNA testing was available for a minority of these samples, the main two causes being an HPV DNA-positive female sexual partner and detection of condylomata acuminata. Overall, 51/306 samples were positive for HPV DNA (16.5%). Of note, 83/255 (33%) of the samples collected at the glans penis were positive for HPV DNA, as opposed to 4 of the 51 urethral samples (7.8%). This suggests that the urethra is not a major site of replication for most HPV types and that it may not be the ideal site for sampling in male subjects . DatTo gain further insights into the discrepancy observed in type prevalence between the genders, we next tried to identify stable couples where both partners were positive for HPV DNA. We were able to document 26 such couples. Type concordance (including mixed infections with at least one type in common) was very low: 8/26 (30.8%); in all other cases the partners harboured different HPV types, confirming a high turnover of HPV infections in the population. In addition, no correlation could be found between persistence in the female partner after treatment and the presence of same virus in the male partner. The latter finding, despite the small sample, argues against the systematic investigation of male partners, which has lately become fashionable. Moreover, the usually obvious nature (and the low incidence) of dangerous HPV-related lesions in males does not justify widespread virological testing, even in partners of HPV-positive women.The extended observation period by the same method enabled some longitudinal analysis. Although with different schedules, depending on the different clinical centres or practitioners, a significant number of patients were followed-up after the initial sampling. Of these, 157 showed HPV DNA persistence (presence of HPV DNA of any type) in at least 2 consecutive samples, in the absence of treatment. Persistence >2 years was observed in 30 perfectly immunocompetent individuals; in 10 of them the same type infection persisted in the absence of treatment. The HPV types identified in these 10 cases were 31, 52, 53, 54, 56, 61, 66, 84. HPV66 persisted longest (6 years) in one patient, without cytologically (ASCUS) and colposcopically alarming lesions; to our knowledge the virus has not yet been cleared. These data confirm the notion  that cercandHPV87 candHPV87andHPV87  found a andHPV87  to the HAlthough the present study is limited in terms of the geographical distribution and selection criteria of the sample population, and might not have the solidity of a formal epidemiological study, the sheer number of samples and the extended period in which they have been collected provide firm support for the notion that ideal assays for the diagnosis of HPV infection should detect and type reliably the large majority of HPV types affecting the genital mucosa. Sensitivity to low copy numbers is less important than specificity and range of types identifiable. The most promising technology, capable of providing the necessary information with relative ease of execution and good potential for standardization, is based on macro- or micro-arrays. Current assays using such flexible molecular methods are still limited in terms of number of probes and specificity , therefoGenital samples (swabs and biopsies) from Italian female and male patients were referred to our laboratory for a range of HPV-related genital conditions including cytological abnormalities, colposcopically detected lesions and condylomata. Known HIV-positive or transplant recipient patients were excluded from this study. Overall, 6,335 consecutive clinical samples received in 2000–2007 were evaluated for the presence of HPV DNA. The samples included a small number of fixed bioptic or surgical specimens.RsaI and Tru91 restriction endonucleases, directly applied to an aliquot of the amplification reaction. To resolve cases with similar restriction patterns , the amplified product was also digested with AflIII restriction endonuclease. In a limited number of samples restriction analysis did not yield a satisfactory result, due to (in order of frequency): 1) faint bands in the presence of non-specific products, 2) multiple infections with many different types, 3) unknown restriction pattern due to polymorphisms (mostly subsequently resolved by sequencing the amplified product).The technique for DNA extraction and amplification from clinical samples has been described elsewhere . BrieflyIn some of the cases yielding unidentifiable restriction patterns, the amplified product was sequenced by the cycle sequencing technique, followed by detection on an ABI 3100 capillary electrophoresis automated sequence analyzer . Sequences were subsequently analyzed using the NCBI BLAST tool and phylogenetic analysis (Phylip package) after multiple alignment  of HPV sequences available from public databases.SM, PB and KM performed some of the sampling and all the restriction and sequencing analyses, SM and MC conceived the study and organized the data, AC selected specific clinical cases, provided clinical data and performed colposcopy and, in a few cases, cytology, SS performed histological analysis. SM drafted the manuscript, with helpful advise by MC and PB.


The Pap test has been used for cervical cancer screening for more than four decades. A human papillomavirus (HPV) vaccine has been approved for use in Canada and is commercially available now. These two preventive interventions should be considered simultaneously. General population support is an important factor for the successful combination of these interventions. The study had two objectives: 1) To assess practices, beliefs, and attitudes regarding Pap test screening and HPV immunization; 2) To identify socio-demographic factors for Pap screening and vaccine acceptability.In 2006, 500 adults were invited to participate in a telephone survey in the region of Quebec City , Canada. Some neutral and standardized information on Pap test and HPV was provided before soliciting opinions.471 adults (18–69 year-olds) answered the questionnaire, the mean age was 45 years, 67% were female, and 65% had college or university degree. Eighty-six percent of women had undergone at least one Pap-test in their life, 55% in the last year, and 15% from 1 to 3 years ago. Among screened women, the test had been performed in the last three years in 100% of 18–30 year-olds, but only in 67% of 60–69 year-olds (P < 0.0001). Only 15% of respondents had heard of HPV. Eighty-seven percent agreed that HPV vaccines could prevent cervical cancer, 73% that the vaccine has to be administered before the onset of sexual activity, 89% would recommend vaccination to their daughters and nieces. Among respondents < 25 years, 91% would agree to receive the vaccine if it is publicly funded, but only 72% would agree to pay $100/dose.There is an important heterogeneity in cervical cancer screening frequency and coverage. Despite low awareness of HPV infection, the majority of respondents would recommend or are ready to receive the HPV vaccine, but the cost could prevent its acceptability. Genital Human papillomavirus (HPV) infection is the most common sexually transmitted infection (STI). More than 50% of sexually active women have been infected with genital HPV at some time in their life -5. InfecCervical cancer screening and treatment have been in place for more than four decades. There is now a new approach: vaccination against two of the most common disease-causing HPV genotypes. The HPV vaccines, if widely used, have the potential to prevent thousands of cases of cervical cancer worldwide, as well as to substantially reduce costs and emotional stress associated with abnormal screening test (Pap) results,10. The In previously surveyed populations, estimated vaccine acceptability was high -16. HoweIn preparation for the implementation of HPV vaccine in Canada, we measured acceptability, attitudes, beliefs and practices related to cervical cancer screening and vaccination, in Canadian adults.We hypothesize that regular cancer screening, a positive attitude toward vaccines in general and the HPV vaccine in particular, as well as perceived personal risk for cervical cancer and seriousness of its consequences would be associated with a higher HPV immunization acceptance and vaccine up-take.This HPV survey was conducted as a part of a study on a number of personal health-related beliefs, attitudes and practices, including smoking, nutrition, exercise, cardiac disease and cervical cancer prevention.Survey population included adult females and males aged 18–69 years. The potential participants were recruited during February and March 2006 in 11 private outpatient clinics in the region of Quebec City. At the moment of the survey, 84 (of the 500 in the region) family physicians were practicing in these clinics. Most Canadian family physicians (78%) practice at least part of their time in private outpatient clinics. Two speThe questionnaire was built using elements of the Health Belief Model . It was Proportions were compared by using chi-square or Fisher exact test. Trends were evaluated with Cochran-Armitage test. All tests of significance were two-tailed and significance was set at 5% level. SAS Institute software (version 9.1) was used for data statistical analysis.The Research Ethics Board Committee of Laval University Hospital Centre approved the research protocol.Eleven out of 12 solicited clinics agreed to participate in the study and 685 potential participants were initially recruited . Eight individuals refused to respond to the questionnaire and 27 telephone numbers were invalid. As predetermined by the study protocol, no further telephone calls were made when 500 interviews were completed. Among those interviewed, 471 (94.2%) were age eligible and their answers were included in the analysis. The participants' average age was 44.8 years and 67% were female. Sixty five percent of participants had college (32%) or university (33%) degree.Fifty-seven percent of women were afraid of developing cervical cancer sometime in their life, and 93% thought cervical cancer has serious consequences. Cervical cancer related anxiety and perceived seriousness did not vary by age group or level of schooling.Eighty-six percent of participating women had had at least one Pap test during their life Table . Among aOnly 15% of respondents had heard about HPV before the interview. The proportion increased from 7% in those with lower than college level of schooling, to 14% and 24% in those with college and university schooling level, respectively (P for trend = 0.0006). No significant differences by sex (P = 0.7) or age group (P = 0.1) were observed on this issue. However, the proportion of women who had heard about HPV was significantly higher among those with at least one Pap test (17%) than those with none (4%) (P = 0.02).Ninety percent of respondents agreed with the statement that vaccines in general are effective against infections, but only 47% strongly agreed. A significantly higher proportion 63%) of 60–69 year-olds strongly agreed with vaccine usefulness compared to 34% of 18–29 year-olds (P < 0.0001) (Table % of 60–6Nineteen percent of respondents under 30 years versus 47% among 60–69 years-old (P < 0.001) perceived HPV vaccination as an incentive for earlier onset of sexual activity Table . Ninety Despite low awareness of HPV infection, our findings suggest that most young women would accept a vaccine that protects against cervical cancer, especially if it is free of charge and recommended by a physician. The need to pay $100 per dose decreases by half young women's vaccine acceptance. These results show that a potential risk for inequitable preventive care may emerge if HPV vaccines are by individual patient purchase. The Canadian National Advisory Committee on Immunization recommenThe proportion of respondents who have heard about HPV (15%) has not changed from what was reported in Ontario adolescents (13%). These fOverall, opinions on vaccine usefulness in the prevention of infectious diseases are positive. However, as in a recent USA survey -23, a diCertain results present a challenge concerning the HPV vaccine use and its potential consequences. Willingness to recommend the vaccine to daughters/nieces was similar to that obtained in UK parents (80%), and wasLimitations of this study include the non-random sample of participants recruited in outpatient clinics, the absence of knowledge on non participant characteristics and the limited recruitment area. Since HPV vaccines will be administered in a primary care setting, we concur with the other investigators, that itThe large sample size, high rate of participation, recruitment in both urban and rural areas, and results obtained both in women and men make this survey results useful for planning public health interventions regarding cervical cancer screening and eventual HPV vaccine implementation.Larger periodic nationwide studies on acceptability, knowledge and attitudes about prevention issues will be needed.The author(s) declare that they have no competing interests.CS conceived the study, coordinated the data collection and helped to draft the manuscript. BD participated in the design of the study and helped to draft the manuscript. VG participated in the design of the study and wrote the first draft of the manuscript. FL participated in the design of the study and helped to draft the manuscript. MO performed the statistical analysis and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Human papillomavirus (HPV) infection of the mouth and oropharynx can be acquired by a variety of sexual and social forms of transmission. HPV-16 genotype is present in many oral and oropharyngeal squamous cell carcinomata. It has an essential aetiologic role in the development of oropharyngeal squamous cell carcinoma in a subset of subjects who are typically younger, are more engaged with high-risk sexual behaviour, have higher HPV-16 serum antibody titer, use less tobacco and have better survival rates than in subjects with HPV-cytonegative oropharyngeal squamous cell carcinoma. In this subset of subjects the HPV-cytopositive carcinomatous cells have a distinct molecular profile.In contrast to HPV-cytopositive oropharyngeal squamous cell carcinoma, the causal association between HPV-16 and other high-risk HPV genotypes and squamous cell carcinoma of the oral mucosa is weak, and the nature of the association is unclear.It is likely that routine administration of HPV vaccination against high-risk HPV genotypes before the start of sexual activity will bring about a reduction in the incidence of HPV-mediated oral and oropharyngeal squamous cell carcinoma.This article focuses on aspects of HPV infection of the mouth and the oropharynx with emphasis on the link between HPV and squamous cell carcinoma, and on the limitations of the available diagnostic tests in identifying a cause-and-effect relationship of HPV with squamous cell carcinoma of the mouth and oropharynx. Human papillomaviruses have been categorized by their genotypes into low-risk and high-risk types according to the risk of that virus causing squamous cell carcinoma of the uterine cervix . InfectiHPV infection of the mouth and of the oropharynx, like HPV infection of the uterine cervix, is associated with high-risk sexual behaviour, in particular with orogenital sex; and high-risk HPV genotypes, in particular HPV-16, are present in many oral and oropharyngeal SCC where in some cases they probably play an essential aetiological role . PersonsIn order to prove a causal relationship between HPV and SCC of the mouth and oropharynx, as has been proven in the case of SCC of the cervix uteri, there should be evidence that in a significant number of cases of apparently normal oral or oropharyngeal epithelium infected with HPV, in time SCC will develop. The demonstration of HPV DNA, even of high-risk HPV oncogenes in squamous cell carcinoma is not in itself sufficient evidence of oncogenesis by the HPV in that context. HPV may well have been either present but a non-participant during the oncogenesis, or have been superimposed upon the malignancy.On the other hand, absence of HPV DNA from any carcinoma does not exclude the theoretical possibility of its having played some role in the initiation of the malignancy since HPV infections are frequently transient . In suchThe local viral load and viral distribution, the clonality of HPV infection, the mechanisms of HPV oncogene transcription, and the specific site of viral integration are all factors critical to the understanding of HPV oncogenesis; and the testing for these factors is as complex and as multifaceted as the complexity of the process itself.In situ hybridization assays for HPV DNA can provide data on the presence of HPV in different cells, but have limited sensitivity for certain HPV genotypes and cannot demonstrate oncogene transcription. Viral oncogene expression can be demonstrated by the polymerase chain reaction (PCR) technique, but this does not provide information about the viral load and the distribution of HPV DNA [in situ hybridization tests can pinpoint the specific site of viral integration in the genome [in situ hybridization can detect HPV-infected cells with low viral loads, and can also elucidate the distribution of HPV DNA within the tumour [ HPV DNA . As PCR  HPV DNA ,23. Neite genome . PCR come tumour .Circumstantial evidence for the role of high-risk HPV types in the pathogenesis of SCC of the mouth and oropharynx can be found, firstly, in the presence of high-risk HPV genomic sequences and expression of transcriptionally active E6/E7 oncoproteins in the malignant cell nuclei of the tumour and of its metastases; secondly, in HPV DNA integration in the cellular genome; and thirdly, in the existence of substantial viral DNA copy-numbers ,11,12,24In relation to HPV viral load, although there is a clearly demonstrated association between increased HPV DNA copy-number  and increased risk of cervical cancer, this viral load is not a reliable predictor of HPV-induced progression to cervical cancer; and presumably, viral load will be no more reliable as a predictor of HPV-induced progression to oral and oropharyngeal cancer. Determination of viral load cannot discriminate between HPV infection of a few cells with a large number of HPV DNA copies each, and of many cells with a few DNA copies each; or between recent HPV infection and long-standing infection .Regarding HPV DNA integration into the cellular genome, although this molecular event is a strong indication of the oncogenic role of the virus, the presence of high HPV DNA copy-numbers and transcriptionally active (high risk) E6/E7 mRNA in HPV cytopositive SCC of the oropharynx is not necessarily dependant on viral integration and can occur when the virus is in an episomal form .Both oral and oropharyngeal HPV infection and oral and oropharyngeal SCC are associated with the practice of orogenital sex and with the high-risk sexual behaviour of cohabiting with many partners, particularly when started at a younger age ,17,19,27Although oral and oropharyngeal HPV infections are primarily sexually acquired, mouth to mouth contact between partners and between family members, autoinoculation, and vertical birth-transmission are also routes whereby HPV infection of oral and oropharyngeal sites can be established ,27,29.As oral and oropharyngeal subclinical HPV infection is not uncommon, it is possible that the epithelium may serve as a reservoir of the virus, and when activated the virus may play a role in HPV-associated oral and oropharyngeal SCC.In epidemiological studies, SCC of the head and neck is frequently treated as a homogeneous group, and the various component carcinomata  are not often separated out statistically. The reported rates of detection of HPV DNA in head and neck SCC range from 0 to 100% ,30. ThisUnderstanding of the role of HPV in the pathogenesis of oral and oropharyngeal SCC is further clouded by inconsistencies in the evidence brought about by differences in methods of tissue collection and preservation; by the use of molecular assays and HPV DNA probes with different specificities and sensitivities; by low viral load in these carcinomata; by lack of adequate controls; and by the inability to identify and assess the influences of other confounding factors ,16,23. HCoinfection with HPV-16 together with one or more other HPV types is not uncommon ,18. HPV-Serum antibodies against L1, E6 and E7 proteins of HPV-16 were detected in well over 60% of persons with oropharyngeal SCC . Since aOwing to the non-specificity of clinical sampling methods for HPV and to the confounding effect of benign HPV infection in the mouth or elsewhere, prediction of development of HPV-associated oral and oropharyngeal SCC can not yet be made ,15,34.INK4A [HPV-associated and non HPV-associated  oral and oropharyngeal SCC are different in cytogenetic profiles, clinical characteristics and courses of the disease ,12. WhilINK4A ,36. HPV-INK4A .It is not yet clear whether the use of tobacco/alcohol and HPV are, or are not synergistic in the aetiopathogenesis of oral and oropharyngeal SCC ,12, but HPV-16 has been shown to be causally associated primarily with HPV-cytopositive SCC of the palatal tonsils ,26,32,37INK4A, unmutated p53 gene, and decreased levels of pRb; and LoH at chromosomal loci 3p, 9p and 17p is uncommon [In these persons with HPV-cytopositive oropharyngeal SCC, the tumour cells have a distinct molecular profile . The celuncommon -37,39-41In contrast to cells of HPV-cytopositive SCC of the oropharynx as described above, the cells of HPV-cytopositive oral SCC are typically characterised by low viral load, and by infrequent viral integration and by expression of transcriptionally active E6/E7 mRNA ,42. A loHowever, it is possible that in some cases of HPV-cytopositive oral SCC that do not express E6/E7 mRNA, the virus has participated in the initial stages of transformation but phased out during later stages ; or thatOne must not overlook the fact that not all oral and oropharyngeal SCC are either HPV or tobacco/alcohol related. Some are idiopathic but the proportion of idiopathic to HPV and to tobacco-alcohol induced neoplasms remains undetermined.In view of the fact that HPV infection is most frequently sexually acquired and that HPV infection is implicated in the aetiology of oropharyngeal SCC, and to a lesser degree in the aetiology of oral SCC, anything that can be done to discourage early sexual activity and to encourage safe sexual practices may reduce the frequency of SCC in anogenital, oral and oropharyngeal sites.In addition to the encouragement of responsible sexual behaviour, the introduction of HPV vaccination as a public health measure against anogenital HPV infection, will most probably also have a favourable impact on the frequency of HPV-mediated oral and oropharyngeal SCC. The current quadrivalent vaccine against HPV types 6, 11, 16, and 18 consists of L1 protein of HPV which generates a high level of HPV genotype-specific neutralising antibodies ,45. The The quadrivalent vaccine is highly effective (98%) in preventing HPV-16 or HPV-18- related high-grade cervical intraepithelial neoplasia in a population of women aged 15 to 26 who had not been previously exposed to either HPV-16 or HPV-18; but, the vaccine is much less effective in women who have previously been exposed to these HPV types . It is cGenital HPV infection in men appears to be as common as it is in women, is also positively related to a history of sexual activity, but is generally asymptomatic and is therefore an important occult reservoir of the virus, contributing significantly to cervical disease in women. HPV-16 is associated with both penile carcinoma and male oral and oropharyngeal SCC. The conclusion must be that young men before starting sexual activity might also be protected from HPV infection and subsequent oral and oropharyngeal SCC by timeous prophylactic HPV vaccination; and moreover, their sexual partners can also benefit from this preventive measure ,50.• Oropharyngeal SCC to a higher degree, and to a lesser degree oral SCC, are associated with HPV infection.• Oral and oropharyngeal HPV infection and HPV-related oral and oropharyngeal SCC occur more frequently in persons who have had a number of sexual partners and in those who have practised oral sex.• Social mouth-to-mouth contact, autoinoculation and vertical birth-transmission are less frequent, but still important routes of transmission of HPV infection.• The importance of latent HPV infection in the oral and oropharyngeal mucosa as a reservoir of the virus, is undetermined.• Reliable markers for progression of high-risk HPV-infected epithelium to malignancy are not yet available.• It is unknown whether co-infection by more than one HPV genotype increases the risk of malignancy, and in the event that it does, whether that malignancy will be more aggressive than that following infection with a single HPV type.• A number of factors that may well prove to be important in HPV-induced carcinogenesis still remain uncertain:• the role of immunity;• variations in genetic profiles of host and virus;• the specific nature of, and the sequence of the cytogenetic alterations;• the influences inherent in specific anatomical sites on carcinogenesis.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.


To be really useful, a vaccine must be highly efficacious, safe, and must be accepted by and administered to the majority of the target population. Current prophylactic vaccines against human papillomavirus (HPV), based on L1 virus-like particles, have been extensively evaluated for both efficacy against severe precancerous lesions of the cervix uteri and safety  were such agents . AlthougInformation from over 30 000 invasive cervical cancers from five continents has now accumulated, and has not only confirmed the predominance of HPV16 (57%) and HPV18 (16%) in every studied population, but has also showed the similarity in the relative importance of other oncogenic types  . Immunisation against HPV16/18 in England has, therefore, the potential to prevent 56–65% of CIN3 and 73–77% of invasive cervical cancer. Finally, cross-protection against oncogenic types other than HPV16/18 may prevent an additional 3–4% of invasive cervical cancer.Howell-Jones et al also highlighted the high frequency of infection with oncogenic HPV types among women in England. Prevalence was 16% among all screened women, but rose to 29% among women aged 25–29 years, which is as high as that found in countries like Colombia  they are expensive vaccines; (2) they target adolescent girls for whom no delivery platform is readily available in any country; and (3) they are meant to prevent a cancer for which an effective secondary prevention strategy exists . Also imHigh-vaccine uptake was also reported in similar school-based programmes in Scotland , Wales , and Aus


The purpose of this study was to define a correlation between telomerase activity and human papillomavirus (HPV) in normal control tissue and in benign, premalignant and malignant cervical lesions. Telomerase activity was detectable in 33 out of 34 cases of squamous-cell carcinoma, five out of six cases of microinvasive carcinoma, 8 out of 20 cases and two out of six cases of high- and low-grade squamous intraepithelial lesions (SILs) respectively. The higher frequency of positive telomerase in invasive carcinoma compared with SILs was observed in both HPV-associated and non-associated groups. Whereas 92.6% of HPV-positive and 100% of HPV-negative invasive lesions expressed telomerase, only 50% of HPV-positive and 25% of HPV-negative SILs did. Interestingly, telomerase activity was also detectable in 13 out of 28 cases of benign lesions regardless of the presence of HPV. In conclusion, there may be two roles of telomerase in the cervix. The first one would present in benign lesions; the second is associated with cancer development and activated during the late stage of multistep carcinogenesis in both HPV-positive and -negative groups.


Infection with Human Papillomavirus (HPV) is a necessary event in the multi-step process of cervical carcinogenesis. Little is known about the natural history of HPV infection among unscreened young adults. As prophylactic vaccines are being developed to prevent specifically HPV 16 and 18 infections, shifts in prevalence in the post vaccine era may be expected. This study provides a unique opportunity to gather baseline data before changes by nationwide vaccination occur.10 DEIA) were genotyped using the INNO-LiPA HPV genotyping assay. HPV point prevalence in this sample was 19%. Low and high risk HPV prevalence was 9.1% and 11.8%, respectively. A single HPV-type was detected in 14.9% of all women, while multiple types were found in 4.1%. HPV-types 16 (2.8%) and 18 (1.4%) were found concomitantly in only 3 women (0.1%). There was an increase in HPV prevalence till 22 years. Multivariate analysis showed that number of lifetime sexual partners was the most powerful predictor of HPV positivity, followed by type of relationship, frequency of sexual contact, age, and number of sexual partners over the past 6 months.This cross-sectional study is part of a large prospective epidemiologic study performed among 2065 unscreened women aged 18 to 29 years. Women returned a self-collected cervico-vaginal specimen and filled out a questionnaire. All HPV DNA-positive samples (by SPFThis study shows that factors independently associated with HPV prevalence are mainly related to sexual behaviour. Combination of these results with the relative low prevalence of HPV 16 and/or 18 may be promising for expanding the future target group for catch up vaccination. Furthermore, these results provide a basis for research on possible future shifts in HPV genotype prevalence, and enable a better estimate of the effect of HPV 16-18 vaccination on cervical cancer incidence. Upcoming mass vaccination with Human Papillomavirus (HPV) vaccines will most certainly change HPV epidemiology. Monitoring these changes on population level may prove crucial in assessing the effect of mass vaccination and overall HPV vaccine efficacy. In the Netherlands, girls aged 12 years will be vaccinated as of September 2009, and the catch-up vaccination (girls aged 13 to 16 years) will probably start in the first part of 2009.Until now, only a limited number of large studies have investigated HPV epidemiology in female adolescents and young female adults. Even fewer studies have investigated HPV epidemiology in relation to past en present sexual behaviour.Genital infection with the Human Papillomavirus (HPV) is the most common sexually transmitted infection (STI) among young sexually active women Estimates of HPV infections among asymptomatic women around the world range from 2% to 44% Additionally, little is known about risk factors for acquiring genital HPV in young female adults. Therefore, further assessment of risk factors like sexual behaviour is important. Knowledge of baseline, i.e. pre-vaccination, epidemiology of type specific HPV infections in relation to sexual behaviour is important in order to decide whether catch-up vaccination may be beneficial. After nationwide implementation of the prophylactic HPV vaccine, HPV epidemiology will most likely change due to expected decreases in HPV 16-18 prevalence and incidence, as well as possible changes in other types occurring due to cross protection of the vaccine. Due to these shifts, prevalence and incidence of other HPV types may increase and therefore may change the oncogenicity of these types.Therefore, this study, conducted before the nationwide introduction of HPV vaccines, provides a unique opportunity to determine baseline data on HPV prevalence in 18 to 29 year old women in the Netherlands. Additionally, no regular cervical cancer screening is performed in this age group, as the Cervical Screening Program starts at the age of 30 years. This study is part of a large prospective epidemiologic study conducted to study the dynamics of HPV infections, in particular HPV 16/18, and to get more insight in specific risk factors for acquiring genital HPV, like past and present sexual behaviour. These results provide a basis for understanding possible future shifts in genotypes, and presumably enable a better estimate of the effect of HPV 16/18 vaccination on cervical cancer incidence.This cross-sectional study is part of a large prospective epidemiologic study performed among 2065 unscreened women aged 18 to 29 years. Women were recruited between June and September 2007, using different advertisements, as well as active recruitment sites, and posters at general practices in the city regions of Arnhem, Nijmegen, and Den Bosch, the Netherlands. Furthermore, advertisement on the internet were used, which were accessible in the whole of the Netherlands. Of the 2297 women who responded to the advertisements, 2065 (89.9%) consented with the study, returned the cervico-vaginal swab specimens, and filled out the questionnaire. Written informed consent was obtained from all participants. This study was approved by the Local Medical Ethics Committee.tm, Tripath Imaging®, Inc., Burlington NC, U.S.A.), instructions how to perform the cervico-vaginal self-sample (written and in cartoon), and a return package consisting of a leak-proof seal bag, absorption sheet, and a reclosable plastic return envelope . In brief, participants were instructed to wash their hands before opening the brush cover, to hold the brush by the end of the handle, to insert the brush approximately 7 cm into the vagina (similar to inserting a tampon), to gently turn the brush 5 times, and to place the top of the brush in the collection tube. The collection tube was closed, and enclosed in the seal-bag. Finally, the collection tube was placed in the return envelope, together with the questionnaire, and sent to the Department of Obstetrics and Gynaecology for further processing and HPV assessment. The samples were stored at room temperature.All women were asked to fill out a questionnaire and to self-collect a cervico-vaginal sample in the privacy of their own home. Women received an explanatory letter, an informed consent form, a questionnaire, and a self-sample kit by mail. The self sample kit contained a collection device , a collection tube containing medium .10-LiPA HPV detection/genotyping assay, SPF10 system version 1, manufactured by Labo Biomedical Products bv, Rijswijk, The Netherlands). This assay amplifies a 65-bp fragment of the L1 open reading frame and allows detection of at least 43 different HPV types 10 PCR was performed with a final reaction volume of 50 μl containing 10 μl of the isolated DNA sample, 10 mmol/liter Tris-HCl (pH 9.0), 50 mmol/liter KCl, 2.0 mmol/liter MgCl2, 0.1% Triton X-100, 0.01% gelatin, 200 μmol/liter of each deoxynucleoside triphosphate, 15 pmol each of the forward and reverse primers tagged with biotin at the 5′end, and 1.5 U of AmpliTaq Gold (Perkin-Elmer). The mixture was incubated for 9 minutes at 94°C, 40 cycles of 45 s at 45°C, and 40 cycles of 45 s at 72°C, with a final extension of 5 minutes at 72°C. Each experiment was performed with a separate positive and negative PCR control. The presence of HPV DNA was determined by hybridization of SPF10 amplimers to a mixture of general HPV probes recognizing a broad range of HPV genotypes, in a microtiter plate format, as described previously Broad-spectrum HPV DNA amplification was performed using a short PCR fragment assay (SPF10 DEIA) were genotyped using the INNO-LiPA HPV genotyping assays.All HPV DNA-positive samples (by SPFThe 28 oligonucleotide probes that recognize 25 different types were tailed with poly(dT) and immobilized as parallel lines to membrane strips . The HPV genotyping assay was performed as described previously All women who completed the questionnaire and submitted a swab for HPV evaluation were included in the final analysis (n = 2065).The Chi-Square test was used to test associations between demographic variables or behavioural characteristics and HPV. Differences of medians of continuous variables between the groups were analysed using non-parametric tests (Mann-Whitney).In univariate and multivariate analysis, data of some variables were grouped due to small numbers and/or to gain a better overview. We grouped ethnicity into two groups: Dutch and not Dutch. Lifetime number of partners and number of partners in the past six months were divided into four categories, and frequency of sexual contact was grouped into five categories. Years of being sexually active  ranged from 0 to 23 years, the category “0” years consisted of women who became sexually active in the past year. Because of the small numbers, 0 and 1 year were combined as well as 13 to 23 years. Chlamydia, genital warts, Syphilis, Gonorrhea, Genital Herpes, and HIV were defined as STI. In further statistical analysis previous STI's were defined as yes or no.Variables found to be significantly related to HPV infection by univariate analyses were entered into a multiple logistic regression model with forward selection procedures to identify variables that contributed independently to the probability of HPV prevalence.Participants with missing data on variables included in the multivariate analysis were excluded. In all tests, p values <0.05 were regarded statistically significant.Statistical analyses were performed using SAS (8.0) and SPSS 14.1 .The age distribution and socio-demographic characteristics of the 2065 participants are summarised in Additionally, the mean age at first sexual contact was 16.7 years, and the mean sexual age  was 6.8 years. Women who were not sexually active yet were significantly more often living with their parents .Of the 2065 adequate specimens, 19% (n = 393) tested positive for one or more HPV genotypes. Age-specific prevalence is shown in The overall prevalence of hr-HPV types was 11.8% and of lr-HPV types 9.1%, including co-infections. Prevalence of both hr- and lr-HPV types showed an almost similar age-distribution see .A single HPV-type was detected in 14.9% of all women, while multiple types were found in 4.1% . We identified 25 different genotypes, most common types detected were HPV type 16 , HPV type 51 , and HPV type 52 . HPV types 18, 6, and 11, were detected in 1.4% (n = 28), 0.6% (n = 12), and 0.2% (n = 4), respectively see . In 3.5%When univariate analysis was restricted to sexually active women, factors significantly associated with HPV prevalence were increasing age, current smoking, number of partners in the past 6 months, and years of being sexually active  see . Sexual After logistic regression, age, smoking, number of sexual partners (lifetime and in past 6 months), type of relationship, living with parents, and sexual age were significantly associated with HPV prevalence. Additionally, HPV prevalence was lower among women without a previous STI (Odds Ratio (OR) 0.355, p<0.001, see The analysis was concluded by completing a multivariate regression analysis on all factors that showed a significant relation with HPV in the univariate analysis. The factors independently associated with a risk of being HPV positive were, with an exception for age, mainly related to sexual behaviour see . The num10-LIPA is used, which could make it difficult to discriminate between active (i.e. chronic productive infections) and latent infections because of its low threshold value. Therefore, results of this study, showing point prevalences of HPV infections, may be a mixture of latent and active or persistent infections.This is the first Dutch HPV epidemiological study conducted among unscreened women aged 18 to 29 years. The point prevalence of HPV DNA in this sample was 19%. Lr- and hr-HPV prevalence were 9.1% and 11.8%, respectively. Hr-HPV types 16 (2.8%) and 18 (1.4%) were found concomitantly in only 3 women (0.1%). These results are comparable with recent studies among young women, although different sampling methods were used Furthermore, multivariate analysis provided insight in the independent risk factors for prevalent HPV infection. The independent risk factors were all related to sexual behaviour, with the exception for age. We found that HPV prevalence increased with age. Studies often show HPV prevalence decreasing towards 30 years. This may be explained by the fact that the Dutch Cervical Screening Program starts at the age of 30 years, and therefore these women are unscreened. Furthermore, we did not study women above the age of 29, and we did not combine different age groups, which may provide another perspective by leveling the differences. Other explanations could be the techniques used or differences in the population studied. The use of contraceptive methods like condoms was influenced by type of relationship. Results of several studies on condom use have been inconsistent partly owing to the fact that different populations have been studied No correlation of educational level with HPV prevalence was seen. However, the overrepresentation of women attending university college possibly limits generalization of these findings, and may be influenced by the method of recruitment. Nevertheless, this study provides a unique sample with an equal distribution of women over the age groups of 18 to 29 years, using one of the last opportunities to gather baseline i.e. pre vaccine data. These baseline data enable future study on HPV dynamics. HPV epidemiology will most likely change after vaccination due to expected decreases in HPV 16-18 prevalence and incidence, as well as decreases in other types due to cross protection of the vaccine. These decreases in type specific prevalence and incidence may be substituted by increases in other HPV genotypes.The relative low point prevalence of HPV 16 and 18, and co-infection with both types in only 0.1%, combined with independent predictors of prevalent HPV infection, may be promising for future catch-up vaccination. These results suggest that it may be possible to expand the future target group for catch up vaccination by including women with a higher age or by targeting women with a low-risk profile.This study shows that sexual behaviour, especially the number of sexual partners as well as type of current relationship, remain the dominant and individual risk factors for HPV positivity i.e. HPV point prevalence. These unique baseline epidemiological data on HPV prevalence in combination with knowledge of sexual behaviour provide a basis for research on possible future shifts in HPV genotype prevalence, and enable a better estimate of the effect of nationwide HPV 16-18 vaccination on cervical cancer incidence.


Seropositivity was defined as five standard deviations above the mean optical density obtained for studied virgins (n=573). HPV-16, -18, -31, and -45 seroprevalence was 15, 15, 16, and 11%, respectively. Of women DNA-positive for HPV-16, -18, -31, or -45, seropositivity was 45, 34, 51, and 28%, respectively. Peak HPV seroprevalence occurred a decade after DNA prevalence; lifetime number of sexual partners was the key determinant of seropositivity independent of DNA status and age. DNA- and sero-positive women showed the highest risk for concurrent CIN3/cancer, followed by DNA-positive, sero-negative women.Human papillomavirus (HPV) seroprevalence and determinants of seropositivity were assessed in a 10 049-woman population-based cohort in Guanacaste, Costa Rica. Serologic responses based on VLP-based ELISA were obtained from the plasma collected at study enrollment in 1993/1994 for HPV-16 ( Currently, detection of cervical human papillomavirus (HPV) infection often relies on the identification of HPV DNA in exfoliated cervicovaginal cells. However, because most HPV infections (including oncogenic types) are transient and clear within 2 years, HPV DNA detection is limited to identifying current infections  found in the majority of cervical cancers worldwide (n=107) was considered as the reference standard of serious high-grade disease.This study was conducted in an on-going population-based cohort study of 10 049 women in Guanacaste, Costa Rica (n=9949), HPV-18 (n=9928), HPV-31 (n=9932), and HPV-45 (n=3019). Based on results from our on-going DNA testing for the cohort, serologic testing for HPV-45 was discontinued following 3040 assays due to the comparatively low DNA prevalence (<1%).Plasma samples collected at study enrollment were tested for anti-HPV L1 antibodies at the Johns Hopkins Medical Institutions by a VLP-based enzyme linked immunosorbent assay (ELISA) for HPV-16, -18, -31, and -45. Of the 10 077 women enrolled in the Costa Rican cohort, results were obtained for VLP serologic responses to HPV-16 (Trichoplusia ni (High Five™) cells  from recombinant baculoviruses expressing the L1 and L2 genes of HPV-16 or -31 or the L1 gene alone of HPV-18 or -45. Virus-like particles were purified from cell pellets by density-gradient ultracentrifugation and column chromatography techniques as described previously , pH 7.2. The plates were blocked at room temperature for 3 h with 0.5% (wt vol−1) polyvinyl alcohol, MW 30 000–70 000  in PBS (0.5% PVA). The blocking solution was replaced with PBS and the plates were stored at −20°C until use. Before use and following each incubation step, the plates were washed four times with wash solution (PBS-0.05% Tween 20) in an automatic plate washer . Using a MultiPROBE II robotic liquid handling system , plasma specimens were diluted 1 : 10 in 0.5% PVA and 10 μl of the diluted plasma was added to the antigen-coated plate containing 90 μl of 0.5% PVA and incubated at 37°C for 1 h. After extensive washing, antigen-bound immunoglobulin was detected with horseradish peroxidase-conjugated goat anti-human IgG, gamma chain specific , diluted 1 : 4000 in 0.5% PVA, 0.025% Tween 20, 0.8% (wt vol−1) polyvinylpyrrolidone, MA 360 000 (Sigma) in PBS. After 30 min at 37°C, colour development was initiated by the addition of 2,2′-azino-di-(3-ethylbenzthiazoline-6-sulphonate) hydrogen peroxide solution . The reaction was stopped after 20 min by addition of 1% dodecyl sulphate and absorbance was measured at 405 nm, with a reference wavelength of 490 nm, in an automated microtitre plate reader .HPV VLPs were prepared in Specimens were tested in duplicate on separate plates, with retesting of specimens with results exceeding a preset, acceptable coefficient of variation (CV) of 25% if one of the replicate optical density (OD) values was greater than the cut point for seropositivity (>0.05 OD units). Retesting by CV, however, did not apply to samples where both OD values were lower than the seropositivity cut point. Each batch comprised approximately 2000–3000 specimens, including both intrabatch and interbatch reliability repeat specimens. Each batch also included specimens from random samples of 200 of the 573 virgins in the study population, which were used to calculate an ELISA cutoff.n=9112), HPV-18 (n=9102), HPV-31 (n=9105), and HPV-45 (n=2774).Cervical cytologic specimens were tested for HPV DNA using L1 MY09/MY11 consensus primer methods (n=200). We used an iterative statistical approach that excluded outliers in the distribution of virgin test results until no remaining value was greater than two standard deviations above the mean optical density of the virgin specimens in that batch. Seropositivity for each HPV type was then defined as five standard deviations above the mean OD obtained for the concurrently tested virgins (minus virgin outliers). Alternative classifications of seropositivity using a three standard deviation cutpoint, or a receiver operating curve (ROC) approach contrasting the virgins to known type-specific DNA positives yielded the same conclusions.Serology results were dichotomized as antibody positive or negative. The cutoff was calculated independently for each test batch, by comparison with the distribution of the values obtained for the concurrently tested virgins in that batch . To assess possible antigenic crossreactivity between types, prevalence ORs were calculated for HPV seroreactivity and DNA status of the same or other HPV types . To further assess the type specificity of the serologic assay, we calculated prevalence ORs for seroreactivity of HPV-16, -18, -31, and -45 to DNA positivity of HPV-6, -11, -26, -33, -35, -39, -40, -42, -51, -52, -53, -54, -55, -56, -57, -58, -59, -61, -62, -66, -68, and -71.χ2. We included in our final multivariate model, variables statistically significantly associated with seropositivity in our univariate models: age , number of lifetime sexual partners , age at first intercourse , number of recent sexual partners , smoking , and OC use .To identify determinants of seropositivity, univariate associations for HPV-16, -18, and -31 seropositivity were assessed for the following behavioural and reproductive variables: smoking , alcohol intake, early age at first intercourse (defined as <16 years), number of sexual partners , number of pregnancies, number of live births, and use of oral contraceptives (OC). Study virgins were excluded from these analyses; we also do not report associations with HPV-45, due to the discontinuation of this assay. Odds ratio estimates with 95% confidence intervals were obtained to assess the magnitude and statistical significance of the associations between HPV seropositivity and HPV DNA positivity, and to identify determinants of HPV seropositivity; statistical significance was also obtained by To further characterise HPV exposure in the population, we assessed exposure using both serological and DNA-based measurements. We categorised exposure into four groups: (i) women both HPV sero- and DNA-negative, (ii) women sero-positive but DNA-negative, (iii) women sero-negative but DNA-positive, and (iv) women both sero- and DNA-positive. For each HPV type, we used logistic regression to assess the cross-sectional association ) between HPV exposure and CIN3/cancer, adjusted for age. All analyses were conducted on SAS 8.2 for Windows.The median age of the 9949 women for whom IgG antibodies to HPV VLPs were measured was 38 years (range: 18–97 years). Overall, seroprevalence for HPV-16, -18, -31, and -45 was 15, 15, 16, and 11%, respectively . Of all demographic and behaviuoral risk factors measured in univariate analyses, age, number of recent and past sexual partners, age at first intercourse, smoking, and OC use were statistically significantly associated with HPV seropositivity, and we therefore included them in our final multivariate model. As shown in As shown in We assessed the risk of CIN3/cancer associated with HPV exposure as determined by HPV DNA and serology measurements for HPV-16, -18, and -31. As shown in We also conducted analyses stratified by age to delineate clearly risk relationships among young women, because past and recent exposures are likely equivalent in this age group. For women less than 25 years of age, the highest level of risk for CIN3/cancer was observed for HPV-16 DNA-positive women (OR=41.9) rather than DNA- and sero-positive women (OR=26.2). Higher risk estimates in DNA-positive women less than 25 years of age were also demonstrated for HPV-18. No statistically significant associations were observed between women HPV seropositivity only and CIN3/cancer for women less than 25 years of age. Exclusion of women less than 25 years of age did not significantly change the results.Overall, the HPV seroprevalence of 15–16% in our population is consistent with the broad range (10–52%) found in previous reports (The differential peaks for HPV DNA- and sero-prevalence are of interest. It is well understood that high DNA positivity rates in young women are due to sexual activity. While HPV seroconversion can take up to 18 months , in GuanThe moderate overall agreement between HPV seropositivity and DNA positivity in our study is consistent with other studies (A number of studies have supported the use of HPV-16 VLP as a biomarker of past HPV exposure, with moderate associations between HPV seropositivity and cervical cancer (To our knowledge, this is the largest population-based seroprevalence study of the four major HPV types: HPV-16, -18, -31, and -45; subjects were representative of the adult female population of Guanacaste, Costa Rica. Although serology measurements underestimate cumulative exposure to HPV due to low seroconversion and possibly waning seropositivity over time, detection of serum VLP antibodies to HPV nevertheless provides an informative assessment of HPV exposure in a population. To understand the implications of HPV serology in the natural history of cervical cancer, however, will require prospective studies.