Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma is becoming more common. We examined prevalence and risk factors for oral HPV among men who have sex with men (MSM) and compared sampling and transport methods.In 2010, 500 MSM (249 HIV-positive) attending Melbourne Sexual Health Centre answered a questionnaire, swabbed their mouth and throat and collected a gargled oral rinse sample. Half the oral rinse was transported absorbed in a tampon (to enable postage). HPV was detected by polymerase chain reaction, and genotyped by Roche Linear Array®. Men with HPV 16 or 18 were retested after six months.Any HPV genotype was detected in 19%  15–25%) of HIV-infected men and 7% (95% CI 4–11%) of HIV-negative men (p<0.001), and HPV 16 was detected in 4.4% (95% CI 2–8%) of HIV-infected men and 0.8% (0.1–2.8%) of HIV-negative men. Oral HPV was associated with: current smoking (adjusted odds ratio (aOR) 2.2 (95%CI: 1.2–3.9)), time since tooth-brushing  and number of lifetime tongue-kissing partners aOR 3.2 95%CI: (1.2–8.4) for 26–100 partners and 4.9 95%CI: (1.9–12.5) for>100 partners. Lifetime oral-penile sex partner numbers were significantly associated in a separate model: aOR 2.8(1.2–6.3) for 26–100 partners and 3.2(1.4–7.2) for>100 partners. HPV 16 and 18 persisted in 10 of 12 men after a median six months. Sensitivities of sampling methods compared to all methods combined were: oral rinse 97%, tampon-absorbed oral rinse 69%, swab 32%.Oral HPV was associated with HIV infection, smoking, recent tooth-brushing, and more lifetime tongue-kissing and oral sex partners. The liquid oral rinse sample was more sensitive than a tampon-absorbed oral rinse or a self-collected swab. Oncogenic human papillomavirus (HPV), principally genotype 16, is now a recognised cause of a substantial proportion of oropharyngeal squamous cell carcinoma (SCC) and this proportion appears to be increasing. This increase in HPV-associated oropharyngeal SCC has led to a search for predictors of oral HPV infection in individuals without cancer. Multiple studies indicate that the presence of oral HPV DNA is associated with higher numbers of sexual partners, smoking and HIV infection To better understand the epidemiology of oral HPV infection population-based studies are needed. Population-based studies would be facilitated by self-collected oral samples that could be sent by post to laboratories. Gargled oral rinses have been reported as more sensitive samples for HPV DNA detection than tonsillar and oral mucosal brushings, We performed a study with two aims. The first was to compare HPV detection from three sampling and transport methods: oral rinse samples, oral rinse samples absorbed in a tampon and a self-collected mouth and throat-mouth swab. The latter two can be mailed. We also assessed the impact of other factors that may affect HPV detection, such as eating, drinking and tooth-brushing.Our second aim was to clarify the relative importance of lifetime compared to recent sexual partners and then look at different sexual practices in both time periods. We examined these questions in a population of men who have sex with men (MSM) attending a sexual health centre because they were likely to be at increased risk of oral HPV and do not seem to be protected by the HPV vaccination program which targets women. This was a cross-sectional study of HIV-positive and HIV-negative MSM attending Melbourne Sexual Health Centre, Victoria, Australia, between 2 March 2010 and 17 June 2010. In the clinic, participants completed a written questionnaire about risk factors including sexual history, smoking, alcohol-consumption and genital warts. The questionnaire also asked about factors that may affect detection, including how long prior to specimen collection they last ate, drank or brushed their teeth. The sexual history covered their number of sexual partners over different time periods  for tongue-kissing, oral-penile sex, and oral-anal sex. Each of these sexual practices was defined as involving only the participant’s mouth. Participants were also asked how many days since they had each type of sex, the proportion of lifetime male oral sex partners who ejaculated in their mouths, and the proportion who used condoms during oral sex with the study participant.After providing written informed consent, participants were shown a video describing how to swab their own gums, mouth and throat with a flocked swab . This was agitated in RNA stabilization reagent  and transported to the laboratory for processing. Participants also gargled 20 ml saline for ten seconds and expelled it into a container . A researcher then divided the oral rinse sample into equal halves: one was absorbed by a tampon  while the other was left in the original container. In the laboratory, the tampon was squeezed firmly to retrieve as much absorbed saline as possible. The order of collecting the oral rinse and throat swab samples from participants was alternated weekly.DNA was extracted using MagNA Pure LC . A 20 µl aliquot of extracted DNA was amplified in a PGMY09/11-based HPV consensus PCR assay Negative and positive controls were processed with each run, and lack of signal in the negative control was used to monitor possible carryover.Men who had HPV 16 or 18 detected on any sample were referred for oral examination and retesting for oral HPV using a gargled oral rinse and a swab taken by an oral medicine specialist, six months later. A subset of 37 HIV positive men had anal swabs in a subsequent study of anal cancer screening, performed 16 months after this study. This study recruited from the same HIV-positive MSM population with the exception that only men aged ≥35 years were eligible. These swabs were tested for HPV by the same method and these data are included for comparison.Sample sensitivity was calculated by comparing the number of positive specimens by each method to the total positive by any method and McNemar’s test was applied. HPV prevalence estimates and 95% confidence intervals (CI) were calculated using exact methods. Unadjusted and adjusted odds ratios and 95%CI were calculated to investigate associations with HPV by logistic regression using Stata 11.2 .The questionnaire recorded number of sexual partners over three overlapping time intervals: the last 2 weeks, 12 months or lifetime. In order to select the most appropriate of these for the multivariate model we created three variables of non-overlapping time periods: variable 1) number of partners in the last 2 weeks; variable 2) number of partners in the last 12 months minus the number of partners in the last 2 weeks; and variable 3) number of partners over the lifetime minus the number of partners in the last 12 months. This was done for each of the sexual practices: tongue-kissing, oral-penile sex and oral-anal sex.A separate model was generated for each sexual practice which included only the above three variables. This measured the association with oral HPV of each non-overlapping time period, adjusted for the other time periods.Other risk-factors associated with HPV at P≤0.05 in the crude analysis were included in the adjusted models. Using a stepwise forward approach, variables were removed from the model as they became non-significant, with the exception of age. Because of the strong correlation between number of partners for oral-penile sex and tongue-kissing and the strong association of each with HPV, two separate logistic regression models are presented.With an overall HPV prevalence of 13%, power of 80% and significance of 5%, a sample size of 500 would allow us to detect an odds ratio of 2.2 for a risk factor present in 30% of controls.This research was approved by the Alfred Health Human Ethics Committee.We recruited 500 MSM of whom 249 were HIV-positive and 251 HIV-negative. Of those approached to participate, 94% of HIV-positive men and 97% of HIV negative men agreed to participate. The median age of participants was 37 years, intraquartile range (IQR 27–45 years).Sixty five men  had at least one HPV type: 13  had HPV 16, 2  had HPV 18, 21  had more than one genotype detected (range 2–7 types), and 21  had at least one of the vaccine-preventable genotypes . Of the 251 HIV negative men, 17 (7% 95% CI: 4–11%) had at least one HPV type compared to 48 (19% 95%CI: 15–25%) of the 249 HIV-positive men, p<0.001 .Of the 65 samples positive on any of the three sampling methods, the number positive for each method was: oral rinse 63 , tampon-absorbed oral rinse 45  and swab 21  . The ordTable three shows the crude odds ratios for the three sexual behaviours  over the three non-overlapping time periods  and only the number of sexual partners more than a year ago was significantly associated with HPV detection.Table three also shows, for each sexual practice, the adjusted odds ratios when all three time periods are included in the model. Only the number of partners more than a year ago was significantly associated with HPV detection, and only for tongue-kissing and oral-penile sex .Table four shows crude odds ratios for factors significantly associated with HPV detection. These were HIV infection, older age, more recent brushing of teeth, current smoking, ever having anogenital warts, and ejaculation occurring more commonly with oral sex .HPV detection was not associated with: the number of days since last oral-penile sex (P = 0.04) or tongue-kissing (P = 0.4), hours since last ate (P = 0.4) or drank (P = 0.5), whether or not condoms were used for half or more oral-penile sex partners (P = 0.9), current or nadir CD4 T cell count (P>0.3 for both) or HIV viral load (P = 0.5) (if HIV infected. Data not shown).Table four shows the adjusted odds ratios in two logistic regression models. The first model includes lifetime number of tongue-kissing partners, and the second includes lifetime number of oral-penile sex partners. These two practices were strongly correlated (chi-squared p<0.001). In both models, HPV detection was significantly associated with current smoking, HIV infection, more recent brushing of teeth and the lifetime number of sexual partners for either tongue-kissing or oral-penile sex.Twelve of the 13 men with HPV 16 or 18 were retested after a median of six months  and 10 of the 12 (83%) remained positive for the same genotype.Of the 249 HIV positive men, 37 were involved in a subsequent ongoing study of HIV-positive MSM aged ≥35 years  where anal samples were taken a mean of 16 months after the oral samples. Of these 37 men 35, 95%(95% CI 87%–100%) tested positive for anal HPV. Of the 13 with anal HPV 16, 35%(95% CI 19%–51%), only one had oral HPV 16 detected .In our study of sampling and transport methods, oral rinse samples were significantly more sensitive than self-collected swabs and absorbing the oral rinse into a tampon for postage resulted in a significantly lower rate of oral HPV detection. HPV detection was more likely in those who had recently brushed their teeth and in current smokers. A higher number of lifetime sexual partners for tongue kissing and oral-penile sex, also predicted oral HPV detection. However only a minority of men with high numbers of oral sex partners were HPV positive, and prevalence in the mouth was much lower than prevalence in the anus, which was high in this and other studies of MSM. The finding that recent tooth-brushing increases HPV detection also suggests that current sampling techniques may be improved by prior epithelial abrasion, similar to that used for anogenital HPV detection in men. Reports differ on whether oral HPV is significantly associated with recent or lifetime numbers of sexual partners We found that HPV detection rose with age, consistent with most other studies Our study had a number of limitations. Firstly, it was cross-sectional and the associations are subject to the limitations of this design, such as unmeasured or incomplete adjustment for confounding. Secondly, the participants were from one sexual health service and therefore it may not be reasonable to generalise these findings to populations with a lower sexual risk. Finally, the reported number of sexual partners may have been affected by recall or social desirability bias.Future studies could examine the effect of brushing prior to obtaining oral rinse samples to enhance HPV detection. We found a high level of HPV persistence in the small number of men tested after six months. Larger longitudinal studies are required to confirm this and to establish the age of acquisition of oral HPV to inform future vaccination policies.


Human papillomavirus (HPV) types 16 and 18 are the high-risk, sexually transmitted infectious causes of most cervical intraepithelial neoplasias (CIN) or cancers. While efficacious vaccines to reduce the sexual acquisition of these high-risk HPVs have recently been introduced, no virus-targeted therapies exist for those already exposed and infected. Considering the oncogenic role of the transforming (E6 and E7) genes of high-risk HPVs in the slow pathogenesis of cervical cancer, we hypothesize that timely disruption or abolition of HPV genome expression within pre-cancerous lesions identified at screening may reverse neoplasia. We aimed to derive model zinc finger nucleases (ZFNs) for mutagenesis of the genomes of two high-risk HPV (types 16 & 18).HpV/E7). In the absence of contextually paired three-zinc-finger arrays (pZFAs) that bind DNA corresponding to the genomic context of the E6 gene of either HPV type, we derived the DNA binding domains of another set of 9 & 14 contextually unpaired E6 gene-binding ZFAs (sZFAE6) to aid the future quest for paired ZFAs to target E6 gene sequences in both HPV types studied (pZFAE6). This paper presents models for (i) synthesis of hybrid ZFNs that cleave within the genomic DNA of either HPV type, by linking the gene sequences of the DNA-cleavage domain of the FokI endonuclease FN to the gene sequences of a member of the paired-HPV-binding ZFAs (pZFAHpV/E7 + FN), and (ii) delivery of the same into precancerous lesions using HPV-derived viral plasmids or vectors.Using ZiFiT software and the complete genomes of HPV types16 and 18, we computationally generated the consensus amino acid sequences of the DNA-binding domains  of (i) 296 & 327 contextually unpaired (or single) three zinc-finger arrays (sZFAs) and (ii) 9 & 13 contextually paired (left and right) three- zinc-finger arrays (pZFAs) that bind genomic DNA of HPV-types 16 and 18 respectively, inclusive of the E7 gene (s/pZFAWith further optimization, these model ZFNs offer the opportunity to induce target-mutagenesis and gene-therapeutic reversal of cervical neoplasia associated with HPV types 16 & 18. Productive infection with HPVs only occurs within the stratified epithelium of the skin or mucous membranes[, the E7 oncoprotein induces neoplasia by binding to and acting on multiple functional partners, notably retinoblastoma protein (pRB) and the class I histone deacetylases HDAC1 and HDAC2[Human papillomaviruses (HPVs) are circular double stranded (ds) deoxyribonucleic acids (DNA) viruses of the genus embranes. Althougembranes,3. Infecembranes. In someembranes. Cervicaembranes,2. High-embranes. Degradaembranes,7. On thnd HDAC2-10. Specnd HDAC2. E7 alsond HDAC2. While mThe slow neoplastic process following infection with high-risk HPVs, which usually takes 15–20 years, provides many opportunities for early detection and treatment of the pre-cancerous lesion. Specifically, progression to invasive cancer can almost always be prevented when standard prevention strategies are applied. In most countries, cervical screening using a Papanicolaou (Pap) test or liquiOverall, the picture described above underscores the relevance of investing in development of specifically HPV-targeted treatments as a complementary public health strategy for controlling the global incidence of cervical cancer. More recently, Lin et al. have proin-situ safety. Our failure to address this priority milestone limited the use of those REase-therapeutic precursors to modeling and the extracellular space[N) of the Flavobacterium okeanokoites restriction endonuclease FokI[, with the intent of disrupting incoming viral genomes, we recently identified DNA binding domains of ZFNs to use as gene-therapeutics against proviral HIV DNA and HSV-II[Bacteria endowed with the restriction modification (R-M) system exhibit amazing resistance to tropism by the xenogeneic DNA of bacteria-infecting viruses, bacteriophages. RM systems can be said to be a primitive form of the many restriction factors that have recently been found to possess innate antiviral properties-23. I2 previousar space. The lonease FokI. ZFNs haease FokI-32. On tease FokI, using eease FokI demonstrd HSV-II,32.in-situ.In the light of the above literature, we hypothesized that target mutagenesis of those high-risk forms of HPVs using ZFNs may possess potential for genetic therapy and reversal of cervical cancer The aim of this study was to identify and model DNA-binding domains of zinc finger arrays (ZFAs) as precursors for (i) synthesizing ZFNs for target mutagenesis of two high-risk HPV (types 16 & 18) –genomes, and development of a novel gene-therapeutic armament for reversing the primary oncogenic processes leading to cervical neoplasia.Context-Dependent Assembly (CoDA) inherent in the ZiFiT-ZFA software of the zinc finger consortium[HpV16 and sZFAHpV18 respectively, or simply sZFAHpV. In principle, the FASTA formats of each virus type’s complete genomic DNA sequences  were individually fed into the user-interface preset to assemble three finger arrays. Although the HPV genomes do not contain intronic sequences, the exon/intron case-sensitivity algorithm was turned to its ON-mode throughout these experiments; this algorithm is necessary to distinguish between intron and exon sequences by denoting exons in uppercase and introns in lowercase. Overall, for either virus type, we derived DNA-binding domains of unpaired or single three-ZFAs (sZFAs) capable of specifically recognizing and binding to consecutive 9 bp sequences across the entire genome  and late pA (AL). The early region of a papillomavirus genome occupies over 50% of the genome from its 5′ end and encodes six common open reading frames  that translate their respective proteins. Two additional ORFs, E3 and E8, were assigned to this region initially. However, only the E8 ORF in bovine papilloma virus-1 and HPV-31 have so far been proven to encode a spliced E8^E2C fusion protein, which functions as a negative regulator of viral transcription and replication. The late region of every papillomavirus genome, covering almost 40% of the genome, lies downstream of the early region and encodes L1 and L2 ORFs for translation of a major (L1) and a minor (L2) capsid protein. The long control region (LCR), a segment of about 850 bp (10% of the HPV genome), has no protein-coding function, but bears the origin of replication as well as multiple transcription factor binding sites that are important in regulation of RNA polymerase II-initiated transcription from viral early as well as late promoters. On the basis of this genome structure, those ZFAs binding to the 5′ end 20% would fall under the early region, while the 3′ end 20% falls both under the late and LCR region. For HPV type 18, on the other hand, despite an occurrence of ZFAs with DNA-binding domains able to target 9 bp sequences distributed randomly across almost all its genome, the highest incidence of ZFAs with HPV-type 18 genomic DNA binding-domains was found to be situated within the last 1,571 bp  of the complete genome (genomic context: 0.8-1.00)  residues[HpV16 and sZFAHpV18 databases, in order to yield a contextually unpaired multi-finger array capable of recognizing a longer and thereby preferentially unique sequence in any target double-stranded genomic DNA, aside from the host. As already shown in Additional fileFirst, using nsortium-36 and tresidues-40. Resiresidues-41. SeveContext-Dependent Assembly (CoDA) inherent in the ZiFiT-ZFN software of the zinc finger consortium[paired three zinc-finger arrays targeting HPV types 16 and 18 genomic DNA, respectively , 0.75 , and across 0.85 to 0.90 within the HPV type 16 genomic DNA context were derived. These genomic contextual regions approximately correspond to sequences between the early region’s hypothetical protein HpV16gp5 (context 3332-3619 bp) and the late region’s major L1 capsid protein  , 0.25 , 0.45 , 0.65 , 0.75  and 0.85  respectively within the HPV type 18 genomic DNA context were derived. These regions correspond to the genomic contexts of the genes E7, E1, E2, E3, E4, L2 and L1, respectively .Second, using nsortium-36 and t, in the absence of any paired ZFA (pZFAs) that are precursors for engineering ZFNs targeting within the genomic context corresponding to the E6 transforming gene of either HPV type 16 (>gi|310698439:83-559|) or 18 (>gi|9626069:105–581), we focused our efforts on assembling another two databases of 9  that target this early gene variant from HPV types 16 and 18, respectively. These sZFA databases offer us a basis for creating novel E6-targeting pZFAs precursors of ZFNs, by (i) sequential pairing of those neighboring sZFAs lying within the context of a desired pZFA target, and (ii) in-vitro optimization[Lastlymization. Alternamization.FokI endonuclease-FN to gene sequences coding for members of the paired-HPV-binding ZFAs (pZFAHpV/E7 + FN) and (ii) delivery of the same into precancerous lesions using HPV-derived viral plasmids or vectors:The following text describes the generation of models for (i) synthesis of hybrid ZFNs to cleave within either HPV type’s genomic DNA by linking the gene sequences of the DNA-cleavage domain of the Fok I endonuclease-FN (derived from Flavobacterium okeanokoites and belonging to the type IIS class) and members of a pair of ZFAs (pZFAs)  with ability to cleave the genomic DNAs of HPV types 16 and 18, respectively  are molecularly cloned into ZFN expression vectors in-vitro using unique XbaI/NotI restriction sites[HpV + FN complex to be inserted into alternative sites of Zinc Finger Consortia plasmids capable of recognizing target sites with a 7 bp spacer[in-vivo by two paired zinc finger arrays (pZFAs) each fused to a nuclease domain, two members of a pair (pZFAs) are sub-cloned. Dimerization of the FokI nuclease occurs in-vivo when both members of the pZFA bind their target sequence, thereby ensuring that the two FokI nucleases attach to the target DNA in a particular configuration in order to introduce a double strand break (DSB). Following actual synthesis, it may be necessary to incorporate further improvements, say by (i) modular analysis to add one, two or even three other ZFA on to our currently three-paired ZFAs (pZFAs) so as to enhance specificity and avoid off-target genome toxicity[In-vitro assembly and testing for efficacy of the desired target-cleavage need to be done pre-clinically say by (ii) using either a bacteria-one hybrid (B1H) or yeast one-hybrid (Y1B) system, so as to inform and select the best ZFNs to use in-vivo[First, using an approach similar to that described by Kim et al. (1996), employing the gene sequences of the DNA-cleavage domain of the on sites-36,38-41p spacer,34. In ptoxicity-36. In-ve in-vivo. Elsewhee in-vivo.Second, an exact approach toward gene delivery and transduction of the ‘pre-cancerous lesion’ cells with these ZFHpV + FN complexes in-vivo is theoretically proposed. Specifically, we propose the use of human papillomavirus plasmids capable of episomal replication in human cell lines, to deliver the diploid or paired copy of the high-risk HPV-specific ZFN genotype, pZFHpV + FN. The pZFHpV + FN genotype may be sub-cloned using similar PCR-primers to the ones proposed above for consortia plasmids, with tailored modifications to HPV plasmids or PsV. Sverdrup et al.[HpV + FN (see results of modeling above) may suffice. Graham et al.[HpV + FN genotype[p et al. have prem et al., using sgenotype.The National Center for Biotechnology Information (NCBI) HPV type 16 and 18 genome databases used are available at the following URLs:http://www.ncbi.nlm.nih.gov/genome?term=NC_001526.2• http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=333761&lvl=3&lin=f&keep=1&srchmode=1&unlock• http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx.The two Zinc Finger Consortium’s CoDA-ZiFiT software used are available at the following URL:in-vivo gene-delivery and transduction plasmids or vectors, models of ZFNs synthesized from these pZFA may offer us the means for targeted HPV mutagenesis and therapeutic reversal of the primary oncogenic processes driving cervical neoplasia. Considering the oncogenic role of the transforming genes (E6/HpVgp1 or E7/HpVgp2) of high-risk types of HPVs in the slow pathogenesis of cervical cancer, therefore, we hypothesized that timely in-situ disruption or abolition of HPV genome expression within detected high-risk lesions may reverse cervical neoplasia. To this end, we identified DNA-binding domains of ZFAs for engineering ZFNs for targeted mutagenesis of these two high-risk HPV genomes as precursors for developing a novel gene-therapeutic armament for reversing the primary oncogenic processes leading to cervical neoplasia. As shown in Additional fileeither virus type studied, we focused our additional efforts on the E6 transforming gene, resulting in another set of two databases of 9 and 14 single ZFAs (sZFAs) that bind to sequences of this early gene from both types of HPV studied   that are precursors for engineering ZFNs to target and cleave the genomic DNA of the two high-risk HPV types most associated with cervical cancer. With the appropriate Figures  and6., any two of the modular ZFNs cleaving at the extreme 5′ and 3′ ends of a viral genome could further be modified and optimized[ in-vitro and in-vivo, say by using HeLa cell lines or humanized mouse models[HpV) as magnetic drivers for novel HPV-DNA targeting therapeutics such as transcriptional repressors or the proteosomal/histone deacetylase (HDAC) inhibitors discussed by Lin et al.[Many previous studies have explored the therapeutic potential of ZFN technology against DNA viruses that infect humans, primarily through editing host genomes-30, secoptimized to non-se models,47-49. Tn et al.. in-vitro studies. This can be attributed to the limited resource capacity of our laboratory. However, these same methods[ in-vitro using either a bacterial or yeast hybrid system as described in the modeling results in section d of our results need to be conducted to determine the efficacy of the model ZFNHpV before their trial-use in the clinic. Experiments such as those recently conducted by Wilen et al.[, it is unlikely that the entire genomes of these high-risk HPVs will be excised through frame-shift mutations. Therefore, the already error-prone NHEJ-repair of the DSBs introduced in the HPV genomes could facilitate rare and random yet possibly effective recombinations that may yield either wild-type or mutant HPVs still capable of infecting and transforming cervical epithelia to cancer. This further underlines the need for the above proposed safety and efficacy studies. Fourthly, it must be asked how these modeled HPV-plasmids carrying and transducing ZFNs targeting HPV types 16 and 18 will be used in the clinic. Our translational projection or proposition is to achieve this via direct subdermal or intra-epithelial injection into those sites identified by currently existing screening protocols as pre-cancerous[HpV). Also, the delivery plasmids or vectors are potentially prone to attack by cells of the host immune-system, and concomitant local or systemic immunosuppressive  therapy might be needed to avoid this. Lastly, recent unbiased genome-wide analyses have shown that ZFNs may exhibit off-target effects in-vivo that might not be predicted by in-silico approaches such as the ones employed here[This study has a number of limitations. First, the work has been limited to sequence analyses and is not accompanied by methods-36 have  methods-44,46. Hn et al. for the ancerous-15. If iancerous. In otheancerous,51, but yed here,49.HpV) and their model ZFNHpV for targeting two high-risk HPVs (types 16 and 18). With the appropriate optimization, modification of these modeled ZFNHpV and in-vivo delivery HPV PsV encapsidation plasmids or vectors, a translational possibility of therapeutically reversing the primary HPV-induced oncogenic processes driving cervical neoplasia is proposed.In conclusion, we present databases of paired ZFAs targeting HPV-genomic-DNA (pZFAWM is Chief Scientific Officer at Restrizymes Biotherapeutics (U) Ltd, and member of the steering committee of the Young and early careers’ investigators (YECI) of the Global HIV Vaccine Enterprise.WM concieved the idea for this study, conducted the experiments, analysed the data, and wrote the final manuscript.List of zinc finger arrays targeting HPV type 16 genomic DNA. This file offers a detailed list and loci of action of zinc finger arrays that specifically bind to 9 bp nucleotide sequences within the genomic DNA context of HPV type 16.Click here for fileList of zinc finger arrays binding to HPV type 18 genomic DNA. This file offers a detailed list and loci of action of zinc finger arrays that specifically bind to 9 bp nucleotide sequences within the genomic DNA context of HPV type 18.Click here for fileList of zinc finger nucleases cleaving HPV type 16 genomic DNA. This file offers a detailed list and loci of action of zinc finger nucleases that target and cleave >18 bp  sequences within the genomic DNA context of HPV type 16.Click here for fileList of zinc finger nucleases cleaving HPV type 18 genomic DNA. This file offers a detailed list and loci of action of zinc finger nucleases that target and cleave >18 bp  sequences within the genomic DNA context of HPV type 18. Click here for file


Chronic low back pain (CLBP) is prevalent among military veterans and often leads to functional limitations, psychological symptoms, lower quality of life, and higher health care costs. An increasing proportion of US veterans are women, and women veterans may have different healthcare needs than men veterans. The purpose of this study was to assess the impact of a yoga intervention on women and men with CLBP.Veterans Affairs (VA) patients with CLBP were referred by primary care providers to a clinical yoga program. Research participants completed a brief battery of questionnaires before their first yoga class and again 10 weeks later in a single group, pre-post study design. Questionnaires included measures of pain , depression (CESD-10), energy/fatigue, and health-related quality of life (SF-12). Yoga attendance and home practice of yoga were also measured. Repeated measures ANOVAs were used to analyze group differences over time while controlling for baseline differences.The 53 participants who completed both assessments had a mean age of 53 years, were well educated, 41% non-White, 49% married, and had varying employment status. Women participants had significantly larger decreases in depression (p=.046) and pain “on average” (p=.050), but larger increases in energy (p=.034) and SF-12 Mental Health (p=.044) than men who participated. The groups did not differ significantly on yoga attendance or home practice of yoga.Our results suggest that women veterans may benefit more than men veterans from yoga interventions for chronic back pain. Conclusions are tentative because of the small sample size and quasi-experimental study design. A more rigorous study is being designed to answer these research questions more definitively.


HPV vaccines are available to men but there are few studies investigating the acceptability of HPV vaccines among men who have sex with men (MSM), a high risk group. We assessed the intention to take up HPV vaccines among MSM in Hong Kong and the associated factors related to cognitions on HPV and HPV vaccines, basing on the Health Belief Model (n = 542). The acceptability of HPV vaccines was 20% , 29.2% , 51.7%  and 79.1% . Adjusting for background variables, composite scores of perceived susceptibility, perceived severity, perceived barriers and cue to actions were significantly associated with acceptability of HPV vaccines conditional on specific efficacies and the market price. Acceptability of HPV vaccines was highly price sensitive. Future studies need to use conditional measures. Implementation and translational researches are warranted. HPV is highly infectious. It causes not only genital warts as a sexually transmitted disease (STD) but also cervical cancer among females and anal/penile cancer among males Recent studies have shown that HPV vaccines could effectively prevent genital warts, penile and anal cancers, and reduced high grade anal lesion (HGAIN) among males The U.S. Center for Diseases Control and Prevention (CDC) recommends men who have sex with men (MSM) of age 26 years or younger to take up HPV vaccines HPV and HIV infections are closely related to each other, whilst MSM in general greatly concern about HIV. The prevalence of genital and anal warts was 8.9% and 19.6% respectively among Australian HIV-negative homosexual men Six published studies have investigated acceptability of HPV vaccines among MSM. The prevalence of acceptability ranged from 36% to 86% Health promotions basing on health behavioral theories are more likely to be effective than non-theory-based interventions We therefore investigated the prevalence of intention to take up HPV vaccination in the next six months among MSM age 18–60 years old in Hong Kong under four scenarios (see Measures) and perceptions related to HPV and HPV vaccines derived from HBM. Associations between acceptability  and the HPV-related perceptions were also investigated.Inclusion criteria were: 1) Hong Kong Chinese men of 18 to 60 years old and 2) having had oral or anal intercourse with at least one man in the last six months. Participants were recruited from some gay venues (bars and saunas) frequently visited by gay people during September 2010 through January 2011. This method has been used in previous studies Participants were asked about their socio-demographic information, sexual orientation, utilization of HIV-related services, history of STD (symptoms and diagnosis), unprotected anal intercourse (UAI) with men and the number of male sex partners with anal intercourse in the last six months see . AcceptaQuestions asked included knowledge and HBM-related cognitions on HPV and HPV vaccines  and 3. SOdds ratios were firstly presented in the univariate analysis (ORu). A multiple stepwise regression model was then fit using the significant univariate background variables as candidates and multivariate odds ratios (ORm) were derived. Associations between the cognitive factors and the dependent variable  were assessed, adjusting for those background variables that were found to be significant in the multivariate analysis and adjusted odds ratios (AOR) were derived. Corresponding 95% confidence intervals (CI) of odds ratios were presented. SPSS version 16.0 was used for data analysis, with p values of <.05 taken as statistically significant.About half of the participants (53.4%) were 30 years old or younger; 63.5% had attained colleges; 12.7% were cohabitating with a man; 86.7% considered himself as a homosexual person; 85.4% had utilized HIV prevention services; 10.0% had had some STD symptoms in the past year. The prevalence of UAI (last six months) was 32.3% and the prevalence of multiple male sex partnerships (last six months) was 44.8% .The prevalence of acceptability was 20% , 29.2% , 51.7%  and 79.1% . Comparing participants of < = 26 and >26 years old, the differences in prevalence of acceptability of HPV vaccination under the aforementioned four scenarios were not found to be statistically significant. The prevalence of those who were < = 26 years old under the four scenarios was respectively 16.0%, 27.2%, 51.9% and 81.5%, whilst that of those who were >26 years old was respectively 18.9%, 30.0%, 51.6% and 78.2% .The prevalence of appropriate response for the other individual knowledge items ranged from 24.5% to 58.7% . There wThe prevalence on item responses corresponding to correct knowledge about HPV vaccines  was low and ranged only from 3.9% to 27.7% . The preTwo of the background variables that were listed in Furthermore, the adjusted analysis showed that four of the six HBM-related composite indicator variables (see Measures) on perceived susceptibility , perceived barriers , perceived cue to action , and perceived severity  were of statistical significance . In addiLike their counterparts in other countries e.g. , the samIt is known that the cost of the HPV vaccine is a strong determinant of HPV vaccination among females It was found that MSM did not know enough about HPV, as close to half of them possessed misconceptions about HPV  and 72.3% of them did not know about availability of HPV vaccination for men. Such knowledge may potentially affect perceived needs to adopt preventive behaviors as it is associated with perceived susceptibility and perceived severity. To promote the newly available HPV vaccines among MSM, we hence need to increase their knowledge about HPV and HPV vaccines , as we found that the knowledge levels were low and some of the HPV-related knowledge variables were significantly associated with acceptability of HPV vaccination.Four HBM-related composite indicator variables  were significantly associated with acceptability of HPV vaccines. It is important to remove perceived barriers. In this study, cognitive and psychological barriers  instead of logistical barriers (e.g. troublesome to take up the vaccines) were of statistical significance. It is known that worrying about side-effects is a common determinant of acceptability of new vaccines Perceived susceptibility was another statistically significant factor. Though high percentages of the participants perceived high infectivity of HPV and high prevalence of HPV among local MSM, only about 6% of them perceived a high or very high chance in contracting HPV in the future. This phenomenon of having high perceived risk for others but low perceived risk for oneself in contracting HIV/STD has been reported in previous studies Cue to action is strongly associated with vaccination behaviors Perceived benefit was not significantly associated with acceptability of HPV vaccines. The participants were briefed about the vaccines’ efficacies before they answered the questions on acceptability of HPV vaccines; questions on perceived benefits were asked after the questions on acceptability. The sequence may have reduced the strength of this association. One single item on perceived benefit was significant . Genital warts increase the risk of contracting other types of STD Promotion of HPV vaccines is warranted and the social context seems favorable for such campaigns. MSM in China, including Hong Kong, tend to be young and well educated. In general, younger age and higher education levels are associated with adoption of preventive behaviors Our sampling method has some limitations. The sample may be subjected to selection bias since no sampling frame existed and convenience sampling involving multiple sources was conducted. We did not recruit participants from the Internet though about half of the local MSM have recruited male sex partners from this source In sum, HPV vaccination among MSM is an important public health initiative and promotion is warranted. Such campaigns need to focus on cognitions and knowledge related to HPV and HPV vaccines. To recapitulate, the studies have important implications for designing HPV vaccination programs. Such programs should start with media campaigns promoting knowledge about HPV and HPV vaccination among MSM. The Health Belief Model can be used as a framework to guide design of such programs as all except one of the constructs (self-efficacy) were found to be significantly associated with acceptability of HPV vaccination in this study. Enhanced risk perception would increase perceived susceptibility of HPV infection. Increased perceived severity can be achieved by emphasizing the causation between HPV and both penile/anal cancers and genital warts. Such programs need to remove both financial and psychological barriers. Discounted low-cost or free HPV vaccination schemes should be provided to high risk MSM such as male sex workers and HIV-infected MSM. Psychological barriers also need to be removed, possibly by collaborating with MSM-friendly NGO and empathetic clinicians. Media campaigns tailored to accessing MSM and new approaches involving health professionals and peers should be used as cues to actions to regularly remind MSM to take up HPV vaccination. In social marketing, marketing mix includes manipulation of product, price, place and promotion to facilitate behavioral change


To evaluate the long-term cost-effectiveness of different strategies for human papillomavirus (HPV) DNA testing combined with Pap smear for cervical cancer screening in Taiwan.This study adopts a perspective of Department of Health in cost-effectiveness analysis to compare a no-screening strategy with nine different screening strategies. These strategies comprise three screening tools , and three screening intervals . Outcomes are life expectancy, quality-adjusted life years (QALYs), lifetime costs, and incremental cost-effectiveness ratios (ICERs). Probabilistic sensitivity analyses (PSAs) were conducted to assess parameter uncertainty.When three times gross domestic product per capita is used as the decision threshold, all nine screening strategies were cost-effective compared with the no-screening strategy. Compared with the current screening strategy , HPV DNA testing followed by Pap smear triage every 5 years and every 3 years were cost-effective. Results of PSA also indicated that a HPV DNA testing followed by Pap smear triage every 5 or every 3 years achieved the highest expected net benefits.Possible economic advantages are associated with extending the cervical cancer screening interval from one Pap smear annually to HPV DNA testing followed by Pap smear triage every 5 years with an ICER $1 247 000 per QALY gained, especially in a country with a publicly financed health-care system. Cervical cancer is one of the most common cancers in Taiwan, currently ranked as the fifth leading cancer causing death for females  DNA testing with Pap smear triage and repeat HPV DNA testing for those who test positive in HPV DNA testing with negative Pap smear results were the most effective cervical cancer screening strategy  adopts the perspective of Department of Health. A decision analytic model was developed using The natural history model for cervical cancer was adopted from literature . During simulation, individuals may remain in the normal state or progress to the HPV infection state in the next year (or cycle). Individuals with HPV infection may remain in the HPV infection state, regress to the normal state, or progress towards the CIN I state. The CIN I patients may remain in the CIN I state, regress to the normal state, or progress towards the CIN II or III state. The CIN II or III patients may remain in the CIN II or III state, regress to the normal state, or progress towards the persistent CIN II or III state. From there, patients may remain in the persistent CIN II or III state, regress to the normal state, or progress towards the cervical cancer state. When individuals reach the cervical cancer state, we assume no regression is possible; that is, they can only progress to a cured cancer state when alive at 5 years after diagnosis or to the cervical cancer death state. In this model, a female in any state can die because of other causes.This study was designed to compare a no-screening strategy with nine different screening strategies, which varied using three different screening algorithms – Pap smear alone, HPV DNA testing followed by Pap smear triage, and HPV DNA testing combined with Pap smear, with three screening intervals – annually, every 3 years, and every 5 years. We assume the ceiling age for screening is 69 years.When a female is invited to undergo cervical cancer screening, she is stratified into the never-screened or regularly screened categories . AccordiIn the regular screening strata, a female may or may not be willing to participate in an appropriate year. If a female chooses to undergo screening, the following three combinations of screening tools were evaluated: (1) Pap smear as the primary screening test followed by colposcopy-guided biopsy for positive Pap smear results ; (2) HPV DNA testing as the primary screening test, using a Pap smear as a triage strategy for those who test positive for high-risk HPV types in HPV DNA testing ; and, (3) HPV DNA testing in combination with Pap smear as the primary screening test, using a Pap smear as a follow-up screening strategy for those who have positive Pap smear results or test positive for high-risk HPV types .Experts and existing guidelines were consulted before making the following assumptions for management of females with different screening results: (1) women with negative primary screening results are asked to undergo regular screening; (2) when a woman had a positive final screening result, she is urged to undergo a colposcopy-guided biopsy. All women with a confirmed histology of CIN I, CIN II or III and cancer are assumed to be treated with the therapy recommended by the Taiwan Cooperative Oncology Group . These pWe further assume that compliance with follow-up and treatment is 100% in the model.Parameters of HPV infection rates and disease transitions were obtained from the study by The annual probabilities of dying from cervical cancer for patients were obtained from survival rates published by the Age-specific other-cause mortality was obtained from the 2007 Life Table for Taiwan's female population . For exaet al conducted a randomised trial comparing the sensitivity and specificity of the HPV DNA testing and Pap smear for females aged 30–69 years in Canada (n=10 154). This study used Pap smear sensitivity for CIN II or III of 55.4% (95% CI=33.6–77.2%), and a specificity of 96.8% (95% CI=96.3–97.3%). Arbyn et al conducted meta-analyses of clinical applications of the HPV DNA testing. Their analytical results indicated that sensitivity of the HPV DNA testing (89.5% 85.1–93.1%) or the combination of the HPV DNA testing with Pap smear (99.2% 97.4–100.0%) are higher than Pap smear alone in other studies for detecting CIN II cases or worse. The specificity was 87.5% (95% CI = 85.0–89.9%) for the HPV DNA testing and 87.3% (95% CI=87.3–90.4%) for the combination test.Test sensitivity was defined as a positive screening result for patients with CIN I or worse in this model. Test specificity was defined as a negative result for females in the normal state. Screening test characteristics used were obtained from and The health utility data for the base case and ranges were adopted from a CEA evaluating HPV vaccination programs in Taiwan  Pap smear sampling, (2) pelvic examination, and (3) Pap smear cytological examination. Health-care providers are reimbursed NT$430 by the NHI system for these services . AdditioFor the HPV-Pap and combination strategies, patients who had a positive primary screening test underwent another Pap smear test. The procedures for a repeated Pap smear are (1) vaginal irrigation, (2) pelvic examination, (3) obtaining Pap smear sample, and (4) Pap smear cytological examination. Health-care providers are reimbursed NT$440 by the NHI system for these services . Those wDirect medical costs for precancerous lesions and cervical cancer treatments in Taiwan were obtained from the study by Considering uncertainty is crucial in a decision analytical study. To better understand the impact on the results for different levels of participation, along with the purpose to present the validity of the model results, a scenario sensitivity analysis was performed by approximating the highest coverage rate at 100% for all screening intervals. Probabilistic sensitivity analyses (PSAs) were conducted to assess parameter uncertainty. The ranges of parameters were mostly from published studies . A 50% iProbability distributions were defined for the following six sets of model parameters: (1) screening rate; (2) disease transition probabilities in the natural history model; (3) survival rate for cervical cancer; (4) test characteristics; (5) utility; and, (6) costs. A beta distribution was fitted for screening rate, the natural history model, survival rate for cervical cancer, test characteristics, and utility, those parameters were between 0 and 1. A gamma distribution can be used to represent uncertainty in cost data, which was constrained to be non-negative and characterised in the interval of 0 to positive infinity  constructed from cost-effectiveness acceptability curves (CEACs) for the different strategies. The CEACs were calculated from net monetary benefit over a range of willingness-to-pay (WTP) thresholds for each strategy, and produced by plotting the probability of being cost-effective for a strategy under different WTP thresholds  probability of recurrent cervical lesions than those who have negative test results. However, we did not have good evidence on this difference. Thus, we assumed that women treated for CIN I, CIN II or III were cured and returned to a normal state and acquired a new HPV infection at similar rates to those without previous abnormal results. The model assumed that women must have been infected with HPV to develop further lesions or diseases. This study did not consider disease progression without previous HPV infection.Second, while the natural history model was based on Taiwan calibrated data sources from studies, most transition probabilities were obtained from literature with no age stratification for all Pap, HPV-Pap, and combination strategies. The model also used the same cervical cancer survival rate for all ages, which is not the case in reality. This may add to model uncertainties and parameter uncertainties in analytical results; however, sensitivity analyses demonstrate the robustness of study results.Third, this study did not consider women age <30 years who have already infected with high-risk HPV. According to a cohort study in Taiwan, the prevalence of HPV infection was 9.09% in women age <30 years (Finally, this study also assumed that all women with negative primary screening results were asked to undergo regular screening until 69 years of age. Although the recent scientific evidence suggests that women aged 50 or above with negative HPV test results will have a lower risk to develop cervical cancer, it is the general consensus in Taiwan that aged women should also be covered in screening programs. It will be of value, however, to explore the cost-effectiveness of polices that considered the strategies for different age groups.In conclusion, this study demonstrates that, compared with a no-screening or an annual Pap strategy, using the HPV DNA testing in the cervical screening program in Taiwan is cost-effective. This study also identified as cost-effective for extending the cervical cancer screening interval from Pap annually to HPV-Pap strategy every 3 or 5 years had ICERs of NT$1 303 000 or NT$1 247 000 per QALY gained, respectively. Considering the potential economic advantages, HPV-Pap strategy every 5 years would seem to be more attractive, especially in Taiwan, a country with a publicly financed health-care system.


Objectives. Asystematic review of parental surveys about HPVand/or child HPV vaccination to understandparental knowledge, attitudes, and behaviourbefore and after FDA approval of thequadrivalent HPV vaccine and the bivalent HPVvaccine. Search Strategy.Searches were conducted using electronicdatabases limited to published studies between2001 and 2011. Findings. Thepercentage of parents who heard about HPV roseover time (from 60% in 2005 to 93% in2009), as did their appreciation for the HPVinfection and cervical cancer link (from 70% in2003 to 91% in 2011). During the FDAapproval, there was a stronger vaccine awarenessbut it has waned. The same pattern is seen withparents whose children received the HPV vaccine(peak at 84% in 2010 and now 36% in2011) or the intention to vaccinate (peak at80% in 2008 and now 41% in 2011).Conclusions. Parents had safetyconcerns and wanted more information theirphysician from to recommend and to confidently HPVvaccinate their children. Human papillomavirus (HPV) is the most common sexually transmitted infection in the world and is an established causative agent for cervical, anal, and penile cancers, as well as genital warts in both men and women , 2. It iAs a result of the approval of the HPV vaccines, recent health policy discussions have introduced the idea of adjusting the age of initial PAP smears from 18 years old  to 21 or 22 years old  . AdditioCurrently, policy is modelled on an 80% uptake by young women, which means when combined with vaccination, reducing the frequency of testing and increasing the age of initial PAP smear would be part of an efficient plan to reduce cervical cancer. However, actual uptake of the vaccines is relatively low and not consistent in all areas that the vaccine is offered. For example, in the province of Quebec where there is a passive consent strategy to school immunizations , there is an 80% vaccine uptake for grade-8 girls . HoweverThe purpose of this systematic paper is to compare the findings of previous studies that have examined parental knowledge, attitudes, and behaviours towards the HPV vaccine. Particular emphasis will be placed on changes within parental knowledge, attitudes, and behaviour following the availability of the HPV vaccines. Beyond identifying trends in uptake, the paper will also focus on factors that affect parental intentions to vaccinate their children against HPV. Based on previous studies regarding vaccination, these factors may include parental knowledge regarding cervical cancer and STIs ; perceived risk and severity of cervical cancer and genital warts; attitudes towards vaccines in general; issues concerning increase in sexual activity or promiscuity; availability of health insurance to cover vaccine costs. Additionally, a preliminary analysis of parental attitudes towards STI-prevention interventions versus anticancer interventions will be conducted to determine the policy implications of the two vaccines; one of which is part of an anticancer strategy as well as STI  prevention and one of which only targets cervical cancer prevention. Prior to conducting the literature search, a librarian was consulted for assistance in building a comprehensive search strategy. Relevant research studies were located through an extensive search of the electronic databases PubMed, Ovid MEDLINE, Embase, and Cumulative Index of Nursing and Allied Health Literature (CINAHL). Searches were limited to only those studies which were published between 2001 and 2011 in an effort to obtain the most recent articles regarding parental knowledge, attitudes, and behaviour before and after FDA approval of the quadrivalent HPV vaccine and the bivalent HPV vaccine.A preliminary hand search of the literature was completed in order to identify appropriate keywords and medical subject heading (MESH) terms. The terms that were selected to be used in this paper were “HPV”, “parent” and “vaccine,” with “parent-child relations,” “papillomavirus vaccine,” “parent or child parent relation,” and “wart virus vaccine” often being utilized as synonyms. Search terms were combined using the operators “AND” and “OR” to ensure that all relevant articles were located.n = 71), 254 articles were screened for inclusion in the paper  with one or more doses of the vaccine. Following this initial analysis, an analysis of factors affecting parental attitudes toward the vaccines was conducted.The literature search resulted in 53 studies that met inclusion criteria and were included in this systematic paper –63. All The total number of parents included in this study was 54,194 with a median study sample size of 506, and a mean study sample size of 1,022 . Six studies only reported “parents” and did not differentiate between mothers and fathers. Twenty-three studies (43%) reported mothers' responses only. Of those studies that reported both mothers' and fathers' attitudes, the majority of respondents were mothers with the average sample size of mothers at 82.3% (minimum: 47.7% and maximum: 95.1%).Studies posed a wide variety of research questions; in order to simplify analyses, each study's central question and results were grouped according to whether they were concerned with parental knowledge of HPV, parental behaviours toward the HPV vaccines, parental intent to vaccinate their children against HPV (attitudes), or a combination of the three factors . Of the 53 studies included, 73.6% (39/53) attempted to gauge parental knowledge of HPV and the HPV vaccine. This ranged from whether parents were aware of HPV to whether parents could correctly identify HPV as the causative agent of cervical cancer. Thirty-eight percent (20/53) of studies focused on parental behaviour . Finally, 92.5% (49/53) of studies focused on parental attitudes toward the vaccine (whether parents intend to vaccinate their children against HPV). Many studies also focused on factors affecting parental attitudes and behaviour regarding the HPV vaccine. These factors included perceived vaccine efficacy; vaccine safety; perceived threat of HPV. They will be discussed more thoroughly in the section regarding factors and barriers.Three primary knowledge questions were examined: whether parents had heard of HPV, whether they had heard of the HPV vaccine, and whether they could correctly identify the relationship between HPV and cervical cancer. Of the 53 studies, 19 studies (36%) asked parents whether they had heard of HPV prior to being included in the study . Parental awareness of HPV increased in 2008 and 2009. Of the 53 studies, 15 studies (28%) asked parents whether they had heard of the HPV vaccines prior to being included in the study . Parental awareness of the HPV vaccine spiked in 2007 with a mean percentage of 59% compared to 14% in 2006. Awareness continued to climb to 65% in 2008 and dropped off slightly to 47% by 2010. These years are of particular interest since they mark the introduction and availability of the quadrivalent HPV vaccine and the bivalent HPV vaccine. Of the 53 studies, 5 studies (9%) asked parents if they could identify the relationship between cervical cancer and HPV .It is important to note that only 5 studies asked parents to make the connection between HPV and cervical cancer. In the study in which data were collected most recently (2011), an average of 74% of parents could correctly identify the relationship between HPV and cervical cancer. With only 5 studies examining parental knowledge of the relationship between HPV and cervical cancer, it is difficult to make any connections between knowledge and the introduction and availability of the HPV vaccine.Of the 53 studies, 17 studies (32%) asked parents whether their child or children had already been vaccinated against HPV .Following the availability of the quadrivalent HPV vaccine in 2007, studies began asking whether parents had already vaccinated their children against HPV. The highest percentages of parents who had vaccinated their children against HPV occurred in 2009 and 2010. This is following the introduction and availability of the bivalent HPV vaccine in 2009.Of the 53 studies included, 30 studies (57%) asked parents whether they intend to vaccinate their child or children against HPV . The highest percentage of parents who intend to vaccinate their children (86%) occurs in studies where the data were collected in 2005, prior to the release of the first HPV vaccine. Intent increases in 2008 to 80% of parents from 67% in 2007 and then gradually decreases in 2009, 2010, and 2011.All three knowledge components have increased from pre-2007 studies to post-2007 studies. While levels of uptake pre-2007 and post-2007 cannot be compared, intent to vaccinate has decreased from pre-2007 to post-2007 .Of the 53 studies included, 81% made some mentioning of examining barriers to parental intent to vaccinate. Parental experiences and demographic characteristics were too mixed to show any clear pattern within the 53 studies. Cost factors were also mentioned, but were difficult to compare across studies.In 20 studies 37%), parents expressed concerns about vaccine safety and the potential side effects of the HPV vaccine %, parent, 60, 61.In 13 studies (25%), parents needed more information about HPV vaccination , 60, 62.In 16 studies 30%), parents mentioned a concern about cancer risk as increasing the likelihood of HPV vaccination. Parents who believed it was likely that their daughters might contract HPV , 50, 52,%, parentThere were 10 studies (19%) that mentioned the child's age affecting the parents' decision to vaccinate. Parents were less likely to vaccinate their children if they believed their children were too young or not sexually active , 44, 48.Parents were not concerned that their children would become sexually active if they were given the HPV vaccine in 13 studies (25%) , 60, 62,In 17 studies (32%), parents indicated that having their doctors recommend the vaccine increased the likelihood of HPV vaccination , 60, 62.Parents who had previously vaccinated their children against meningitis  or had aThis paper reviews the parental knowledge, attitudes, and behaviours toward having their daughters and sons vaccinated against cervical cancer. The parents in these studies were largely from a high resource background. The percentage of parents that participated in these surveys who had heard about HPV clearly rose over time (from 60% in 2005 to 93% in 2009). Parents' appreciation for the link between HPV infection and cervical cancer did rise (70% in 2003 to 91% in 2011). During the era of FDA approval of the vaccines, there appeared to be stronger awareness of the vaccines and this has waned with time. This same pattern is seen with the percentage of parents whose children had received the HPV vaccine (high of 84% in 2010 and now 36% in 2011). Unfortunately, this pattern is also seen with the intention to have a child vaccinated against HPV (peak at 80% in 2008 and now 41% in 2011). In terms of barriers against the vaccine, parents still have safety and side-effect questions and they want more information. Parents view the vaccine like the oral contraceptive pill; it is best to invest in it only when you become at risk . Parents who have high cancer worries and receive strong messages about HPV risks are more likely to advocate for the HPV vaccine. Parents look to their physicians to recommend the vaccine. The strengths of this study are that it involves information gathered from a large number of parents from several countries. It shows trends in knowledge, attitudes, and behaviours over a time period just preceding the FDA approval of the vaccine; during the approval phase when there were extensive educational campaigns both by the pharmaceutical companies, professional societies, and media, after the FDA approval. The limitations of this study was an inability to validate parental responses, for example, determining how many parents had their child vaccinated with at least one dose of the vaccine. It will be interesting to see if there are changes in parental attitudes as the types of information about HPV and the HPV vaccine continue to flood the literature. The information about the role of oncogenic HPV in more than cervical cancer is certainly evolving. We are just beginning to grasp the prevention implications of the HPV vaccine in the prevention of anal, oropharengeal, and a proportion of vulvovaginal, and penile cancers. The recent approval of the vaccine in young men may have an impact on decreasing condyloma transmission and having an impact on the rise of anal dysplasia/cancer in the male having sex with male population. As the cervical screening strategy moves toward primary HPV testing, this will also enhance education of the population. Although cost did not emerge as a significant barrier, as the vaccine prices continue to fall, it will be fascinating to see the impact on parental attitudes and behaviour. As public health looks at successful population-based prevention strategies, it will be interesting to look at parental attitudes toward passive consent versus active consent in school-based vaccination programs. Time will provide information on how durable the vaccine is and long-term sequelae; whether this will influence parental attitudes remains to be seen. In terms of future implications for policy, when the goal is to preserve the health of the population, certainly the passive consent approach, whether it is for vaccination or cervical screening, seems to be showing profound benefits. There is preliminary data that shows women who are vaccinated have less need for cervical precancer procedures like biopsies and treatment, however, how this will impact guidelines and availability of such services in the future remains to be seen.Initial awareness of the virus and the ability of the virus to cause cancer have increased in the time period under study. However, awareness of the vaccine, intent to vaccinate, rates of vaccination rose during the initial introduction of the HPV vaccine but have fallen in subsequent years. Surveys have confirmed that parents want more knowledge and reassurance from their physicians that the HPV vaccine is safe for their children to receive. Policy programs, aimed at increasing HPV vaccination rates as part of an overall HPV strategy to reduce the incidents of cancers and infections caused by the virus, will need to heed the parents' concerns and information needs to be effective.


The need to review cervical cancer prevention strategies has been triggered by the availability of new prevention tools linked to human papillomavirus (HPV): vaccines and screening tests. To consider these innovations, information on HPV type distribution and natural history is necessary. This is a five-year follow-up study of gynecological high-risk (HR) HPV infection among a Chilean population-based cohort of women.A population-based random sample of 969 women from Santiago, Chile aged 17 years or older was enrolled in 2001 and revisited in 2006. At both visits they answered a survey on demographics and sexual history and provided a cervical sample for HPV DNA detection (GP5+/6+ primer-mediated PCR and Reverse line blot genotyping). Follow-up was completed by 576 (59.4%) women; 45 (4.6%) refused participation; most losses to follow-up were women who were unreachable, no longer eligible or had missing samples. HR-HPV prevalence increased by 43%. Incidence was highest in women < 20 years of age (19.4%) and lowest in women > 70 (0%); it was three times higher among women HR-HPV positive versus HPV negative at baseline . Type-specific persistence was 35.3%; it increased with age, from 0% in women < 30 years of age to 100% in women > 70. An enrollment Pap result ASCUS or worse was the only risk factor for being HR-HPV positive at both visits.HR-HPV prevalence increased in the study population. All HR-HPV infections in women < 30 years old cleared, supporting the current recommendation of HR-HPV screening for women > 30 years. Most research on the natural history of human papillomavirus (HPV) infection and its progression towards cancer has been conducted in developed areas. In Chile, a middle developing country, the only two population-based studies to date analyzed the prevalence of HPV at one point in time ,2. ChileIn 2001 we studied the prevalence of gynecological HPV among a population-based sample of 969 women 17 years of age and older, from a low socio-economic urban area of Santiago. Results and detailed description of enrollment and data collection procedures were previously reported [Participants attended a health center where a midwife collected cervical samples for HPV testing. An Ayre spatula was used to obtain exfoliated cervical cells and then placed in a tube with PBS; additionally, a spatula and cytobrush used for a Pap smear obtained at the same visit were washed in this tube. In both 2001 and 2006, HPV DNA testing was performed at the Department of Pathology of the VU University Medical Center in Amsterdam, the Netherlands. Specimens were tested for the presence of HPV DNA using a GP5+/6+ primer-mediated PCR and enzyme immune assay. Reverse line blot genotyping was used to type for 44 HPV genotypes, 14 HR-HPV  [P value < 0.2 were then entered in multivariate models. Sexual behavioral changes in the study period were explored as risk factors for HR-HPV incidence. Statistical analyses were performed using SPSS version 17 for Windows.Multivariate comparison of participants and non-participants in the follow-up examination was performed using logistic regression to identify predictors of participation. Prevalence of HR-HPV infection in 2001 and 2006 was compared, using prevalence rate ratios. Risk factors for HR-HPV incidence and persistence  were examined with univariate analysis; variables significant at Sociodemographic characteristics of the study sample are presented in Table The HR-HPV infections identified in the participants at baseline and follow-up are presented in Table n = 59), using women who did not acquire a HR-HPV (n = 517) as referent, were being younger than 30 years and not having a previous Pap test  tested positive for any HR-HPV (regardless of type) both years and 427 (74.1%) were HPV negative both years. In univariate analysis, baseline characteristics associated with non-type-specific HR-HPV persistence, using women who remained HR-HPV negative as referent, were low schooling  and an abnormal baseline Pap test ; however, only the later remained significant in multivariate analysis .(n = 18), in comparison with women who cleared a HR-HPV infection (n = 33), was observed with low schooling (Table HR-HPV type-specific persistence was 35.3%. All type-specific persistence occurred in women older than 30 years of age, increasing from 33.3% in women 31-40 years old to 100% in women over 70 years old. Additionally, a significant association with type-specific HR-HPV persistence Reasons for the increase in HR-HPV infection are unclear, especially since the increase in age of the study population should have produced a decline in the number of HR-HPV positive women. The same protocol for HPV testing was used in both surveys and therefore the increase in HPV prevalence could not be attributed to an increase in sensitivity of HPV testing, but rather to an increase in exposure to HPV. A possible explanation for this is a change in sexual behavior; national health surveys have shown an increasing lifetime number of sexual partners among Chilean women . In our The age distribution of new infections showed a first peak in the youngest and a second peak at age 45 to 55 years old at follow-up. This second peak has been previously described in other Latin American countries ; howeverHR-HPV type-specific persistence increased with age, which is in line with findings reported in other studies ,9. Our fWomen with type-specific HR-HPV persistence are at high risk of developing cervical cancer -15 and sA major strength of this study is that it is the first in Chile that follows a population-based cohort of women to learn about the natural history of HR-HPV infection in our population. Other strengths include the broad age range of enrolled women, the collection of comprehensive information on risk factors, and the performance of DNA testing at a world reference laboratory.In conclusion, HR-HPV prevalence in our study population increased during the follow-up period, presumably the result of changes in sexual behavior, which could lead to an increase in cervical cancer in this population. In order to strengthen our prevention programs, newly available tools such as HPV-based screening tests should be considered in Chile. In our study, all HR-HPV infections in women under 30 years old cleared, supporting the current recommendation of HR-HPV screening for women over 30 years of age.HPV: human papillomavirus; HR: high-risk; ASCUS: atypical squamous cells of undetermined significance; STD: sexually transmitted disease; OR: odds ratio; CI: confidence interval.The authors declare that they have no competing interests.CF was responsible for the study conception, design and implementation, and analysis and interpretation of data. FO carried out the field work and was responsible for data collection. KP coordinated the field work and AC coordinated the collection and preparation of samples at the laboratory. AD performed the statistical analyses. CF, VV, SF and PS were responsible for the analysis and interpretation of data. CF, VV and AD drafted the manuscript. All authors revised the manuscript critically and approved the final version for publication.


High-risk human papillomavirus (HPV) infections are the cause of nearly all cases of cervical cancer. Although the detection of HPV DNA has proved useful in cervical diagnosis, it does not necessarily predict disease presence or severity, and cannot conclusively identify the causative type when multiple HPVs are present. Such limitations may be addressed using complementary approaches such as cytology, laser capture microscopy, and/or the use of infection biomarkers. One such infection biomarker is the HPV E4 protein, which is expressed at high level in cells that are supporting (or have supported) viral genome amplification. Its distribution in lesions has suggested a role in disease staging. Here we have examined whether type-specific E4 antibodies may also allow the identification and/or confirmation of causal HPV-type. To do this, type-specific polyclonal and monoclonal antibodies against three E4 proteins  were generated and validated by ELISA and western blotting, and by immunohistochemistry (IHC) staining of epithelial rafts containing these individual HPV types. Type-specific detection of HPV and its associated disease was subsequently examined using formalin-fixed paraffin-embedded cervical intra-epithelial neoplasias ) and normal controls (n = 28). All koilocytotic CIN1 lesions showed type-specific E4 expression of their respective HPV types. Differences were noted amongst E4 expression patterns in CIN3. HPV-18 E4 was not detected in any of the 6 HPV-18 DNA-positive CIN3 lesions examined, whereas in HPV-16 and -58 CIN3, 28/37 (76%) and 5/9 (55.6%) expressed E4 respectively, usually in regions of epithelial differentiation. Our results demonstrate that type-specific E4 antibodies can be used to help establish causality, as may be required when multiple HPV types are detected. The unique characteristics of the E4 biomarker suggest a role in diagnosis and patient management particularly when used in combination. Human papillomavirus (HPV) DNA is found in nearly all cases of cervical cancer (>99.7%) and high-grade pre-cancers The viral E4 protein is abundantly expressed in infections caused by diverse HPV types, and as a viral biomarker, it can identify cells supporting vegetative viral genome amplification and virus assembly . The primary aim of the study was to establish a simple method for confirming HPV causality, as is required (for instance) when assessing vaccine efficacy. To do this, type-specific staining was carried out on 275 cervical biopsy specimens  and 28 normal cervical tissues)) of different disease grades and different HPV association in order to demonstrate the general utility of the approach  and 10–12 week old New Zealand white rabbits  were cared for in accordance with local and international animal welfare regulations and guidelines.53–60), rabbits were injected on Days 0, 7, 10 and 18 with an 8 amino acid peptide, (DSRRSSIV), conjugated using glutaraldehyde to keyhole limpet hemocyanin (KLH). For the anti-HPV-58 E4 polyclonal antibody (R58E423–30), rabbits were immunized using the same schedule, but with a nona-peptide (CTTKVHRGQ) containing an N-terminal cysteine residue that was conjugated to KLH using m-maleimidobenzoil-N-hydroxysuccinimide ester (MBS). Bleeding was performed on day 28. The rabbit polyclonal anti-HPV E4 antibody (RE4) was raised against the full length E4 protein prepared as a HPV18 maltose binding protein fusion (MBP-E4). The anti-HPV-16 E4 monoclonal (MoAb16E435–42) and polyclonal (M16E435–42) antibodies were prepared using a nona-peptide, CAPKKHRRL containing an N-terminal cysteine residue that was conjugated to ovalbumin using MBS. Female BALB/c mice were immunized 4 times subcutaneously in a 13-day period with conjugate combined with the GlaxoSmithKline (GSK) Biologicals Adjuvant System AS02A containing MPL  and QS21  in an o/w emulsion To prepare antibodies that can specifically identify the E4 proteins of HPV-16, -18 and -58, sequence alignments were first carried out, and short peptide sequences (8 to 9 amino acids in length) were chosen in regions of highest divergence. These peptides were synthesised chemically before being used as antigens to generate anti-peptide polyclonal antibodies (28 day immunization protocol carried out by Eurogentec). For the anti-HPV-18 E4 polyclonal antibody -E4 fusion proteins were produced according to the manufacturer’s instructions .(1.25 µg) purified recombinant MBP-E4 fusion proteins were denatured and run on a 10% NuPAGE™ Bis-Tris gel according to the manufacturer’s instructions . Proteins were then transferred to a polyvinylidene-difluoride membrane and detected using rabbit sera or monoclonal antibodies and standard protocols et al.The NIKS cell line  was cultured in the presence of J2 3T3 fibroblast feeders which were maintained at low passage in selected growth media. The transfection of NIKS cells, and the generation of stratified squamous epithelial rafts were performed according to Lambert Cervarix™) Colposcopic biopsies of CIN2 and 3 were mostly from HERACLES (EPI-108290), a GSK-funded retrospective, cross-sectional, European multicentre epidemiological study on HPV type distribution in women with CIN2 and 3 The diagnosis and grading of areas of CIN1, 2 and 3 were made at Quest Diagnostics  according to standard criteria on the haematoxylin and eosin (H&E) -stained sections All biopsy blocks were sectioned according to the sandwich cutting procedure, which ensured that PCR for HPV and E4 IHC were performed within a sandwich of histology diagnosis as described previously 35–42; concentrated supernatant, 2,111 mg/ml) or 100-fold . HPV anti-L1  and anti-MCM antibody  were used on some sections and were diluted 100-fold, while RE4 non-HPV type-specific antibodies (raised against the whole E4 protein) 35–42 were carried out using anti-rabbit or anti-mouse biotinylated antibody  followed by development using ABC kit (Vector) and TSA-reagent .HPV E4, MCM and L1 IHC was performed on raft or cervical biopsy sections according to standard procedures 53–60 or anti-HPV-58 E4 R58E423–30 rabbit polyclonal antibodies were subsequently used for E4 IHC with the anti-HPV-16 E4 MoAb16E435–42 mouse monoclonal antibody, followed by visualisation with a 150-fold diluted Alexa-488 (green) conjugated anti-mouse secondary antibody (Invitrogen). When sufficient sections were available for staining, tissue sections were also stained for E4 as a positive poly-reactive control using the previously developed anti-HPV E4 antibody (human Fab TVG405) Due to limited number of sections for each case , slides used for E4 IHC with anti-HPV-18 E4 R18E4The IHC staining and reading were done by three individuals  who where blind to both the HPVDNA data and the CIN diagnosis. Persons who did not work in the lab made decision of which antibodies to use for each case. The E4 staining pattern was very distinctive and intense, and in this study no disagreements were encountered with regard to scoring the presence or absence of the E4 protein in the lesion. Images were captured using an Axiovision microscope system (Zeiss).10) PCR-DNA ELISA (DEIA) immunoassay system was used for both whole tissue section (WTS) and laser capture micro-dissection (LCM) PCR 25) version 1 HPV genotyping system , which identifies 25 different HPV genotypes, 14 high-risk HPV types  and 11 low-risk HPV types . For available specimens with multiple HPV types as determined by WTS-PCR, LCM-PCR was performed as recently described HPV DNA genotyping was done according to the PCR algorithm described earlier 35–42 mouse polyclonal  raised against the HPV-16 E435–42 peptide, the R18E453–60 rabbit polyclonal antibody against the HPV-18 E453–60 peptide, and the R58E423–30 rabbit polyclonal antibody against the HPV-58 E423–30 peptide yielded highly potent ELISA responses  or rabbits . A useful anti-protein response could only be assessed for a particular peptide following immunization of both species.All the peptides from divergent regions of E4 proteins of HPV-16, -18 and -58 see  were immesponses  against s  was assessed by testing cross-reactivity with MBP-E4 fusion proteins prepared from a panel of 10 different HPV types  by ELISA and western blotting. As shown in 35–42 monoclonal and M16E435–42 polyclonal antibodies, the R18E453–60 polyclonal antibody, and the R58E423–30 polyclonal antibody, were highly specific for HPV-16, 18, and 58 E4 proteins respectively in both ELISA and western blot analyses, as predicted by sequence alignment  did not cross-react with the other types of HPV E4 tested (see 35–42 (M16E435–42 data not shown) and R18E453–60 produced similar patterns of staining as the TVG405 anti-E4 Ab .35–42, MoAb16E435–42, R58E423–30, and R18E453–60) were compared with TVG405 as well as with the RE4 non-HPV type-specific antibodies Six different epitope retrieval solutions were tested with two different heating methods (12 min microwave or 2 min autoclave) to optimise staining. The newly generated reagents  were damaged during the IHC process. In biopsy 41, HPV-16 and -18 were detected by WTS-PCR but only HPV-16 was identified by LCM-PCR in the lesional area. HPV16 E4 was also detected in this region, providing unambiguous confirmation that this HPV type was active in driving the CIN2-grade abnormality. The CIN2/3 and 8 of the CIN3 lesions assigned by WTS-PCR to HPV 16 DNA  in this region , although the positive E4 signal with R58E423–30 and absence of staining with TVG405 did reveal active HPV-58 expression in regions not sampled by LCM. The CIN1/2 biopsy (biopsy 19) was E4-negative by IHC with R18E453–60, and was typed as HPV-31 by LCM-PCR. Among 6 HPV-18 DNA CIN2 biopsies by WTS-PCR, 3 showed positive HPV-18 E4 staining, while 2 biopsies (biopsy 30 and 41) had no HPV-18 DNA by LCM-PCR. All 6 CIN3 biopsies typed as HPV-18 by WTS-PCR were negative for HPV-18-specific E4 expression. Among these, LCM-PCR confirmed biopsy 61 was positive for HPV 18 while biopsy 65 was negative. No other biopsy was positive with the type-specific HPV-18 antibody including those containing HPV-39 (biopsy 6) and -70 (biopsy 43), which are phylogenetically related to HPV-18 and members of the alpha 7 species 53–60 antibody for all 5 HPV18-associated CIN1 associated with HPV-18, and for the CIN2 and 3 that were tested.The antibody R18E423–30 showed positivity in 2/2 CIN1 lesions (of which one is a borderline CIN1) typed as HPV-58 by WTS-PCR. The borderline CIN1 (biopsy 5) was negative by LCM-PCR, which may be because a different area was sampled compared to that which was positive by E4 IHC. Only 3 CIN2 biopsies typed as HPV-58 were stained for HPV-58 E4 however 2 were positive, and this result was confirmed by LCM-PCR. Biopsy 34 was used to demonstrate the absence of cross–reactive staining with the type-specific 16 and 18 reagents. Five of the 9 CIN3 lesions typed as HPV-58 by WTS-PCR were positive for HPV-58 E4 , and which could reasonably be assigned a causal HPV type either by WTS-PCR (when only one type was present) or by LCM-PCR (when multiple types were present), are shown graphically in 35–42, R18E453–60 and R58E423–30) can be generated using a short-peptide approach, and that such reagents can be applied to formalin fixed paraffin-embedded clinical tissue sections to identify sites of active infection by specific HPV types. HPV-16, -18 and -58 were chosen for these ‘proof of principle’ studies because of their importance, and because they are representative of the various types of HPV that are associated with cervical cancer. Previously, a synthetic Fab (TVG405), was raised against the full length HPV-16 E4 protein 35–42, and the rabbit polyclonal antibodies R18E453–60 and R58E423–30 recognize their specific HPV targets, but do not react with the E4 proteins of even closely related HPV types, including HPV31, HPV 45 and HPV 33.Here we show that HPV type-specific anti-E4 antibodies  and L1 expression (genome packaging) were apparent using antibodies to MCM, HPV E4 and HPV L1, respectively, in rafts infected with HPV-16, 18 or 58. Subsequent staining with the anti-E4 peptide antibodies detected E4 in differentiating epithelial cells and accurately distinguished between HPV types. In this study, the HPV rafts served as an important tool for optimizing the staining procedures for the type-specific anti-E4 antibodies, and were used as positive controls when examining clinical tissue sections. The approach may be generally useful for the evaluation of HPV biomarkers, especially for HPV types that are found infrequently in the population.Since the HPV life cycle and E4 expression are linked to epithelial differentiation, a differentiating organotypic raft culture model was used here to allow us to recreate the productive cycle of individual HPV types The results of our work are broadly consistent with the previous more limited studies of E4 expression using poly-specific antibodies All of the established biomarkers in current use have some limitations. For example, antibodies to MCM and Ki-67, which are used as surrogate markers of E6 and E7 expression in HPV-associated CIN, will also identify cells that are proliferating during normal metaplasia or wound healing. These markers are also found in replication competent (but non-dividing) cells that are supporting viral genome amplification, even in low-grade lesions caused by low-risk HPV types. Similarly p16 may be detected in senescent cells in the upper layers of the epithelium in high-risk HPV-associated CIN, as well as in E6/E7-expressing cells of the basal and parabasal layers