GnRH-I), neuropeptide Y (NPY), dopamine D2 receptor (DRD2), vasoactive intestinal polypeptide (VIP), VIP receptor-1 (VIPR-1), and prolactin (PRL), were selected as candidates for influencing AFE. Additionally, the region between ADL0201 and MCW0241 of chromosome Z was chosen as the candidate QTL region according to some QTL databases. The objective of the present study was to investigate the effects of mutations in candidate genes and the QTL region on chicken AFE.The age at first egg (AFE), an important indicator for sexual maturation in female chickens, is controlled by polygenes. Based on our knowledge of reproductive physiology, 6 genes including gonadotrophin releasing hormone-I  with AFE. Bioinformatics analysis indicated that T32742394C and T32742468C were located in the intron region of the SH3-domain GRB2-like 2 (SH3GL2) gene, which appeared to be associated in the endocytosis and development of the oocyte.Marker-trait association analysis of 8 mutations in those 6 genes in a Chinese native population found a highly significant association (P < 0.01) between G840327C of the GnRH-I gene and the haplotypes of T32742394C-T32742468C of the SH3GL2 gene were associated with the chicken AFE.This study found that  Although environmental factors such as body weight, body composition as well as age are critical for the onset of sexual maturity -4, but t strains -9. AlthoGnRH-I and NPY genes were chosen as candidates for chicken AFE in this study. On the other hand, some workers reported that dopamine (DA) might be one of the putative neurotransmitters responsible for the activity of GnRH and had both stimulatory and inhibitory influences on gonadotropin release in birds [DRD2), could be a candidate gene for the control of AFE. Kadarmideen found that prolactin (PRL) was one of the major regulators of GnRH and GnRH receptors and thus validated its important role in mammalian reproduction and sexual maturity [VIP and VIP receptor-1 (VIPR-1), as well as PRL were chosen as candidates to analyze the genetic effects on chicken AFE.AFE is controlled by polygenes -12 and tin birds -21. Thusmaturity . In aviamaturity ,24. Simimaturity ,26. Thushttp://www.animalgenome.org/cgi-bin/QTLdb/GG/index, http://chicken.genomics.org.cn/, and http://www.thearkdb.org/arkdb/, several novel QTL affecting AFE were identified on chicken chromosomes 1, 3 and Z [ADL0201 and MCW0241 of chromosome Z [To date, a number of QTL studies have been conducted in order to identify QTL affecting chicken AFE -29. On t 3 and Z -32. Tuis 3 and Z . AnotherGnRH-I, NPY, DRD2, VIP, VIPR-I, and PRL genes were chosen for marker-trait association analysis in a Chinese native chicken population. Based on two-tailed χ2 test method, 4 single nucleotide polymorphisms (SNPs) of the candidate QTL region were selected to analyze their associations with AFE.The objective of this study was to identify variations that showed an association with chicken AFE in the candidate genes and the candidate QTL region. In this study, a total of 8 mutations in the The Ningdu Sanhuang (NDH) chicken (a Chinese native chicken population from Jiangxi province) possesses markedly early maturity. Generally, NDH males first crow at 80 d of age and the AFE of females is about 133 d of age when reared on the floor. The population used for this study was NDH female chickens from a half-sib population of Guangdong Wens Foodstuff Company Ltd, Guangdong, China, which had been kept as a closed breeding population for five generations. A total of 1, 310 NDH female individuals from one hatch were used in association analysis. Chickens were exposed to light continuously during the first 3 days post hatch, and then to a 16 h light/8 h dark cycle. All individuals were fed ad libitum to 77 d of age with diet for 2, 837 kcal of ME/kg, and then changed to be fed with diet for 2, 907 kcal of ME/kg. From 90 d to 300 d of age, all chickens were shifted into individual laying cages and the AFE trait was recorded. The average value of AFE in this population was 124.3 ± 16.8 d. At 300 d of age, the blood samples of chickens were collected from a vein under the wing of each bird and the genomic DNA was extracted. All animal procedures were performed in accordance with Law of the People's Republic of China on Animal Protection.2 test in the QTL region. The mean AFE values were 92.5 ± 1.3 d in the early group and 166.4 ± 5.9 d in the late group.According to the AFE record, 24 birds with the earliest AFE and 24 individuals with the latest AFE out of 1, 310 NDH females were chosen for 2-tailed χGnRH-I, NPY, DRD2, VIP, VIPR-I, and PRL genes and (2) those located within a QTL region between ADL0201 and MCW0241 (32.17 ~ 34.26 Mb) on chromosome Z.The SNPs used for genotyping were obtained from two resources: (1) the variations of candidate genes for chicken AFE including the http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=snp and earlier reports [http://www.biologysoft.com/; BioTools, Alberta, Canada).Eight SNPs from the 6 candidate genes were selected on the basis of information in the dbSNP database  reports -36. Eighhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=snp and 5  from an earlier scan (data not shown) were selected from the candidate QTL region for 2-tailed test  in a final volume of 25 μL. The PCR were carried out on an Eppendorf Mastercycler  under the following conditions: 94°C for 4 min; 35 cycles of 94°C for 30 s, n°C .Genotyping assays of polymorphisms used for the association analysis were based on the presence or the absence of a restriction site in the PCR-amplified DNA fragments. Genotyping of M8 was performed directly by electrophoresis in a 3.5% agarose gel after PCR amplification. The genotypes of the other sites for marker-trait association analysis were determined by PCR-RFLP method: 7 μL of PCR product was digested with the corresponding restriction enzyme according to the manufacturer's protocol. Digestions were performed overnight at 37°C . Subsequently, the PCR products were separated by electrophoresis in a 2.5% agarose gel and the genotypes were determined with a TFM-40 Ultraviolet Transilluminator .2 test and a Bonferoni corrected P-value threshold (P = 0.05/12 = 0.0042) was employed.Comparisons of genotypes between the early and the late AFE groups were evaluated by a 2-tailed χhttp://www.broad.mit.edu/mpg/haploview/. Haplotypes were constructed with PHASE 2.0 software http://www.stat.washington.edu/stephens/software.html on the basis of the haplotype structure.The haplotype structure was analyzed with the Haploview v 3.32 software Association analysis of polymorphisms or haplotypes with AFE was performed with the GLM procedures of SAS 8.0 software  using the following model:Y is a trait observation, μ is the overall population mean, G is the effect of genotype or haplotype, S is the fixed effect of sire, and e is the residual error. Multiple comparisons were performed with least squares means by Fisher's least-significance difference method. The values were presented as least square means ± standard error means. The results for the 12 markers (8 in the candidate genes and 4 in the QTL region) were assessed by a Bonferoni 5% significance threshold of P = 0.05/12 = 0.0042 and a Bonferoni 1% great significance threshold of P = 0.01/12 = 0.00083, conservatively assuming the 12 tests to be independent. For the haplotype block in the QTL region, the level of statistically significant difference was set at P < 0.05.Where http://www.ensembl.org/Gallus_gallus/index.html and http://www.ncbi.nlm.nih.gov/mapview/.Gene mapping of the sites associated with AFE in the candidate QTL region and analysis of their function were completed by the use of 2 bioinformatic web sites of G840327C of the chicken GnRH-I gene and AFE. In addition, the mean AFE value of birds with the CC genotype was great significantly higher (P < 0.01) than that of birds with the GC or GG genotype. These effects remained great significant (P < 0.00083) even after Bonferroni correction for multiple testing. Significant associations (P < 0.05) were detected between AFE and 3 SNPs including C31394761T of the NPY gene, T5841629C of the DRD2 gene, and A1661691G of the VIPR-1 gene. Allele C of C31394761T and allele A of A1661691G were positive for chicken AFE. However, all these significant effects disappeared when results were corrected by Bonferroni method for multiple testing , H2 , and H3 , were observed in this block. A total of 1, 295 NDH chickens with 6 diplotypes, including 114 of H1H1, 249 of H1H2, 14 of H1H3, 853 of H2H2, 60 of H2H3, and 5 of H3H3, were used in the association analysis. However, no significant association (P = 0.3133) was detected between the haplotypes and chicken AFE.M5, M6 and M7 were all in the chicken T32742394C in the early and late AFE groups and differences in AFE between individuals with different alleles of C33379782T, although these differences were not significant  with chicken AFE, and birds with the C genotype had earlier AFE than those with the T genotype. However, these significant effects disappeared after Bonferroni correction. No significant association with AFE was found for the other SNPs.Based on the results of the 2-tailed χT32742394C and T32742468C was observed in the NDH population for the 4 SNPs in the QTL region of chromosome Z. Four haplotypes with frequencies higher than 1%, H1 , H2 , H3 , and H4 , were found in this block. A total of 1, 296 chickens with 7 diplotypes, including 432 of H1H1, 1 of H1H2, 1 of H1H4, 385 of H2H2, 1 of H2H4, 26 of H3H3, and 450 of H4H4, were used in association analysis and significant association (P < 0.05) of the haplotypes with AFE was observed  gene. T32742468C, which formed the haplotype block with T32742394C and then affected the AFE value, is also located in intron 1 of the chicken SH3GL2 gene.The roles of most of the 22 functional genes within the QTL region are not known. Based on database Ensembl GnRH-I, NPY, DRD2, VIP, VIPR-1 and PRL were selected to undertake association analysis in an NDH population and 3 significant effects (P < 0.05) on chicken AFE were observed in single analysis: for C31394761T of the NPY gene, T5841629C of the DRD2 gene, and A1661691G of the VIPR-1 gene. However, like most of previous studies, those 3 significant effects disappeared after Bonferroni correction for multiple testing. Bonferroni correction, adjusting the Type 1 error by the total number of tests, is a simple method to control the false discovery rate based on the assumption that all the tests are independent [NPY gene transcription start site had a significant dominant effect on broiler AFE, and heterozygous individuals had an earlier AFE. Although a different mutation site was analyzed in this study, the NPY gene was also proved to be important for chicken AFE. In an earlier study, no significant association was observed between any mutations in the coding region of the dopamine D1 receptor (DRD1) gene and AFE [DRD2, was chosen as the candidate for AFE in this study and the results showed that it was related with this trait. Additionally, an investigation completed by our groups in a different NDH population demonstrated that A1661691G of the VIPR-1 gene was associated with AFE [GnRH-I gene. The association between G840327C of the GnRH-I gene and AFE was consistently significant even with Bonferroni correction for multiple testing. In human, GnRH-I were mainly expressed in the hypothalamus and various compartments of ovary [GnRH-I gene might cause normosmic idiopathic hypogonadotropic hypogonadism in subjects displaying delayed puberty [GnRH-I mRNA signal was significantly increased in sexually mature birds and the ovary mass was correlated with the brain GnRH-I mRNA level [GnRH-1 gene is associated with AFE in NDH chickens.Recently, there were many studies seeking correlations between markers of candidate genes and chicken AFE -39. Althependent . Howeverependent -44. Dunn and AFE . Therefowith AFE , which w puberty ,49. In zNA level . In the 2 test method. Subsequently, T32742394C of chromosome Z was found to be associated with AFE, although the significant effect disappeared after Bonferroni correction. Results of haplotype analysis also showed that it was related to AFE. In the same way, Sutter [T+619C of the DRD2 gene associated with duration of chicken broodiness. Through bioinformatics analysis, T32742394C and T32742468C were found to be located in the intron region of the SH3GL2 gene. SH3GL2, also known as SH3P4 or endophilin I, is a gene encoding the 353 amino acids of a protein that belongs to the endophilin family. Earlier studies showed that SH3GL2 was correlated with the regulation of synaptic vesicle endocytosis in mammals [SH3GL2 gene is associated with AFE in NDH chickens and the haplotypes of T32742394C-T32742468C might play an important role in AFE. However, the genetic effects of the SH3GL2 gene on AFE require further study.In the present study, four variations among many sites in the candidate QTL region were chosen for the following association analysis through two-tailed χ, Sutter  proved t, Sutter  identifi mammals ,53. In a mammals . TherefoG840327C of the GnRH-I gene and the haplotypes of T32742394C-T32742468C of the SH3GL2 gene were associated with the chicken AFE. These results can provide some insight into the genetics of chicken AFE.In conclusion, the main genetic effects of genes were analyzed in this study and the results demonstrated that the SNP HX carried out the SNP genotyping of the Z chromosome, analyzed the data and drafted the manuscript. HZ and QW contributed to the SNP genotyping of the candidate genes. DZ and MZ contributed to materials collection. CL, LS and LY participated in the data analyses. QN and XZ contributed to the design of the study, the supervision of the study and the revision of this manuscript. All authors read and approved the final manuscript.


Several assays are used to measure type-specific serological responses to human papillomavirus (HPV), including the bead-based glutathione S-transferase (GST)-L1 multiplex serology assay and virus-like particle (VLP)-based ELISA. We evaluated the high-throughput GST-L1, which is increasingly used in epidemiologic research, as a measure of cumulative HPV infection and future immune protection among HPV-unvaccinated women.We tested enrollment sera from participants in the control arm of the Costa Rica Vaccine Trial (n = 488) for HPV16 and HPV18 using GST-L1, VLP-ELISA, and two assays that measure neutralizing antibodies (cLIA and SEAP-NA). With statistical adjustment for sampling, we compared GST-L1 serostatus to established HPV seropositivity correlates and incident cervical HPV infection using odds ratios. We further compared GST-L1 to VLP-ELISA using pair-wise agreement statistics and by defining alternate assay cutoffs.Odds of HPV16 GST-L1 seropositivity increased with enrollment age  and lifetime number of sexual partners , with similar results for HPV18. GST-L1 seropositivity did not indicate protection from incident infection over 4 years of follow-up . Seroprevalence by GST-L1  was 5.0% and 5.2%, compared to 19.4% and 23.8% by VLP-ELISA, giving positive agreement of 39.2% and 20.8%. Lowering GST-L1 seropositivity cutoffs improved GST-L1/VLP-ELISA positive agreement to 68.6% (HPV16) and 61.5% (HPV18).Our data support GST-L1 as a marker of cumulative HPV infection, but not immune protection. At lower seropositivity cutoffs, GST-L1 better approximates VLP-ELISA. Persistent infection with oncogenic types of human papillomavirus (HPV) is a necessary cause of virtually all cervical cancers  and someMeasurement of HPV infection is complex. HPV DNA testing using exfoliated cervical cells is the reference standard for identifying current cervical infection, but most infections revert to DNA negativity within 1–2 years . Thus, HSerological responses to HPV L1 commonly feature as exposures and stratifying variables in epidemiological studies, and are measures of immunogenicity that serve as presumptive correlates of protection in vaccine trials . SeveralThe GST-L1 assay can detect antibodies to up to 100 different antigens simultaneously, has been scaled up for large studies, requires a small specimen volume, and offers a low cost alternative to other assays . IncreasIn this study, in the context of naturally acquired HPV infection and immunity, we evaluated whether the GST-L1 assay measures cumulative HPV16/18 infection and/or future immune protection, and directly compared GST-L1 to VLP-ELISA, cLIA, and SEAP-NA.Our study population was sampled from the control (HPV-unvaccinated) arm of the Costa Rica Vaccine Trial (CVT), which has been described in detail . The conAll women in the CVT control arm were tested at enrollment for HPV16/18 DNA infection at the cervix and for HPV16/18 serum antibodies using VLP-ELISA. Using these results, we sampled 500 women from the CVT control arm using a two-stage stratified random sampling scheme  sample, and the more laborious and costly SEAP-NA for HPV16 on the Stage 1 sample only (N = 388). We assessed assay reproducibility as measured by the intraclass correlation coefficient (ICC) and coefficient of variation among seropositives (CV) for GST-L1, cLIA, and SEAP-NA using 25 blind duplicate specimens included in each batch. We excluded twelve women with failed assays, giving a final sample of 488 women.We tested cervical samples collected annually from each woman for 4 years for HPV16/18 DNA, and used the results to determine HPV16/18 incident infection status. Detection of HPV16 DNA at any visit for a woman who was HPV16 DNA negative at enrollment constituted an incident infection, and the analogous HPV18 variable was similarly constructed. Though we refer to newly detected infections as incident, we cannot distinguish between a true incident infection, a reactivated latent infection, and a missed prevalent infection.Antibodies to L1 proteins of HPV16 and HPV18 were detected simultaneously by the GST-L1 multiplex assay as previously described ,30. DiluThe VLP-ELISA was performed at GSK Biologicals as previously described . BrieflyThe multiplex cLIA was performed and cutoffs were determined at PPD Vaccines and Biologicals as previously described ,33. LaboThe HPV16 SEAP-NA was performed in duplicate as previously described at the HPV Immunology Laboratory, SAIC-Frederick, Inc. ,34. The Detection of HPV DNA at the cervix and genotyping was conducted at DDL Diagnostic Laboratory as previously described ,36. Brie16/18). First, we increased the weights for the 369 women selected in Stage 1 who were part of P16/18, as they were not eligible for selection in Stage 2. Second, we decreased the weights for women selected in either stage who were part of P16/18, as their IPWs were calculated to represent the same population. In other words, without this adjustment, the population P16/18 is represented twice. Our final weights allow the sample of 488 women to represent the 3,058 women in the CVT control arm who were HPV16 DNA negative, HPV18 DNA negative, or both at enrollment. We used these weights in all analyses with the exception of HPV16 SEAP-NA analyses, which included only the Stage 1 sample and therefore required standard IPWs.We developed a method to calculate sampling weights, which has been described in detail by Li et al. (under revision). Briefly, we first calculated standard inverse probability weights (IPWs) for women selected in both stages by dividing the eligible population in each cell by the number of individuals selected Figure . We thenWe performed each analysis separately for HPV16 and HPV18. Type-specific DNA negativity at enrollment was required for inclusion in the analysis for each HPV type, resulting in inclusion of 467 women in the HPV16 analysis  and 477 women in the HPV18 analysis . We first examined distributions of GST-L1 antibody reactivity  among all subjects, among GST-L1 seropositives, and among women with and without an incident type-specific infection over follow-up.To assess the utility of GST-L1 as a marker of cumulative HPV infection, we calculated odds ratios for GST-L1 seropositivity based on two established correlates of HPV seropositivity, age at enrollment (an HPV seropositivity correlate among younger women) and lifetime number of sexual partners at enrollment (hereafter referred to as “number of partners”) ,38. To dTo address whether GST-L1 indicates immune protection from future HPV infection, we calculated odds ratios for incident cervical HPV infection based on GST-L1 seropositivity at the laboratory cutoff and quintiles of GST-L1 antibody reactivity, with adjustment for number of partners.We examined agreement between GST-L1 and VLP-ELISA, cLIA, and SEAP-NA using percent agreement, positive agreement , and kapWe conducted analyses in R version 2.15.1  and Stata 11 . Statistical significance was assessed at α = 0.05.For GST-L1, ICCs based on our data were 99.3% for HPV16 and 96.9% for HPV18, and internal laboratory data gave CVs of 13.4% for HPV16 and 13.7% for HPV18. For VLP-ELISA, extensive testing was performed for the CVT and overall reproducibility data were calculated; the VLP-ELISA has a mean CV of 12.3% in the CVT . For cLIFor HPV16, GST-L1 antibody reactivity ranged from 0 to 5979 MFI; ranges among subgroups defined by seropositivity and incident infection status are shown in Table Seropositivity by GST-L1 at enrollment did not indicate lower risk of incident HPV16 infection over follow-up with adjustment for number of partners . Examining quintiles of GST-L1 antibody reactivity did not alter (data not shown).At the laboratory suggested cutoffs, overall agreement between GST-L1 and VLP-ELISA was 77.1%, and positive agreement was 20.8% Table . About oPositive agreement between GST-L1 and cLIA was low .As a marker of cumulative HPV infection, the GST-L1 assay measures many different HPV types in a high-throughput manner at relatively low cost. However, our data suggest GST-L1 is not appropriate for use as a marker of immune protection from future cervical HPV infection, and that its comparability to cLIA and SEAP-NA is limited. The GST-L1 more closely approximates the commonly used VLP-ELISA at a lower cutoff, and may be an appropriate choice for studies aiming to assess population-level patterns in the epidemiology of cumulative infection with many HPV types.cLIA: Competitive Luminex immunoassay; CV: Coefficient of variation; CVT: Costa Rica Vaccine Trial; ELISA: Enzyme-linked immunosorbent assay; GST: Glutathione S-transferase; HPV: Human papillomavirus; ICC: Intraclass correlation coefficient; MFI: Median fluorescence intensity; OD: Optical density; SEAP-NA: Secreted alkaline phosphatase neutralization assay; VLP: Virus-like particle.One or more of the authors is employed by a commercial company. These include: Mark Esser, MedImmune; Katie Matys, PPD Vaccines and Biologics; Wim Quint and Leen-Jan van Doorn, DDL Diagnostic Laboratory. John Schiller and Douglas Lowy report that they are named inventors on U.S. Government-owned HPV vaccine patents that are licensed to GlaxoSmithKline and Merck and for which the National Cancer Institute receives licensing fees. They are entitled to limited royalties as specified by federal law. The other authors declare that they have no conflicts of interest.Conception and design: HAR, YL, MP, AG, M. Schiffman, SW, RH, AH, TW, M. Safaeian. Acquisition of data: CP, AG, ACR, M. Schiffman, SW, PG, JS, DL, RH, LAP, AH, M. Safaeian. Serological assays: MP, TJK, ME, KM, WQ, LJvD, LAP, TW. Statistical analysis: HAR, YL, AG, M. Safaeian. Interpretation of data: HAR, YL, MP, M. Schiffman, SW, TJK, DL, LAP, AH, TW, M. Safaeian. Drafting and revision of manuscript: HAR, YL, CP, MP, AG, ACR, M. Schiffman, SW, TJK, PG, JS, DL, ME, KM, WQ, LJvD, RH, LAP, AH, TW, M. Safaeian. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/120/prepub


Persistent infections with human papillomavirus (HPV) are a necessary cause of cervical cancer and are responsible for important morbidity in men and women. Since 2007, HPV vaccination has been recommended and funded for all girls aged 12 to 17 in Germany. A previously published cost-effectiveness analysis, using a static model, showed that a quadrivalent HPV vaccination programme for 12-year-old girls in Germany would be cost effective. Here we present the results from a dynamic transmission model that can be used to evaluate the impact and cost-effectiveness of different vaccination schemas.We adapted a HPV dynamic transmission model, which has been used in other countries, to the German context. The model was used to compare a cervical cancer screening only strategy with a strategy of combining vaccination of females aged 12–17 years old and cervical cancer screening, based on the current recommendations in Germany. In addition, the impact of increasing vaccination coverage in this cohort of females aged 12–17 years old was evaluated in sensitivity analysis.The results from this analysis show that the current quadrivalent HPV vaccination programme of females ages 12 to 17 in Germany is cost-effective with an ICER of 5,525€/QALY . The incremental cost-effectiveness ratio (ICER) increased to 10,293€/QALY when the vaccine effects on HPV6/11 diseases were excluded. At steady state, the model predicted that vaccinating girls aged 12 to 17 could reduce the number of HPV 6/11/16/18-related cervical cancers by 65% and genital warts among women and men by 70% and 48%, respectively. The impact on HPV-related disease incidence and costs avoided would occur relatively soon after initiating the vaccine programme, with much of the early impact being due to the prevention of HPV6/11-related genital warts.These results show that the current quadrivalent HPV vaccination and cervical cancer screening programmes in Germany will substantially reduce the incidence of cervical cancer, cervical intraepithelial neoplasia (CIN) and genital warts. The evaluated vaccination strategies were all found to be cost-effective. Future analyses should include more HPV-related diseases. Persistent infections with human papillomavirus (HPV) are a necessary cause of cervical cancer and are responsible for important morbidity in both men and women,2. HPV aIn Germany, it is estimated that there are nearly 5,500 new cases of cervical cancer and 1,500 cervical cancer deaths every year,10. The Cervical cancer is one of the target cancers covered by the statutory German cancer screening programme. All German women are eligible to receive an annual cervical examination including a Papanicolaou (Pap) smear, beginning at 20 years old. The annual uptake has been estimated to be about 50% of the eligible population.Since 2007, HPV vaccination has been recommended and funded for all girls aged 12 to 17 in Germany. Two proA previously published cost-effectiveness analysis, using a static model, showed that a quadrivalent HPV vaccination programme for 12-year-old girls in Germany would be cost effective, with an incremental cost-effectiveness ratio (ICER) of 10,530€/QALY . HoweverWe adapted a HPV dynamic transmission model that has already been used in the United States (US), United Kingdom (UK), Mexico and Norway to the German context-22. The The model outcome parameters were driven by five categories of input parameters: demographic data; sexual behaviour; cervical cancer screening; natural history; and treatment patterns.There were three steps in the adaptation process:1. Assessment of the transferability of the model structure to the German setting2. Review of the input parameters to fit the German setting3. Manual calibration of the model to fit the epidemiology data observed in GermanyThere is no evidence that the course of disease, from HPV infection passing through the various disease stages up to cervical cancer death, is different in Germany from that in the US or any other country. The original model also considers treatment probabilities instead of explicit treatment strategies. This makes the model easily transferable to other countries. Furthermore, the screening module in the original model is based on yearly routine cervical cancer screening for women over 20 years old which is consistent with the recommendation in Germany. Thus, wA literature search was undertaken to identify German-specific values for the input parameters; this search was supplemented by advice from experts and cancer registry analyses, when necessary.The demographic data was obtained from the German federal statistical office; the total population aged 12 and over in Germany on 31 December 2008 was estimated to be 73 234 448 people. No GermThe annual cervical cancer screening rates and the sensitivity and specificity of Pap smear tests and colposcopy were based on data from a German health technology assessment report Table10]..10].Based on clinical trial data, the prophylactic efficacy of the quadrivalent HPV vaccine for preventing incident HPV 6/11/16/18 infections was assumed to be 90%. We assuThe model was used to compare a cervical cancer screening only strategy with a strategy of combining vaccination of females aged 12–17 years old and cervical cancer screening, based on the current recommendations in Germany. The vaccination coverage rates were set to reflect the current situation in Germany for the first years of vaccination. In the base case, annual rates by age were set to reach a total cumulative coverage rate of 45% and 55% for the 12–14 and 15–17 respectively  and genital warts were based on German cost studies  in both males and females. The potential cross-protection effect of the vaccine against HPV types not included in the vaccine was not evaluated as these types are included in the model.The model also provided an estimation of the quality-adjusted life years (QALYs), total costs and the incremental cost-effectiveness ratio (ICER).Univariate sensitivity analyses were performed to identify which of the following parameters influenced the results most strongly:1 Vaccine duration of protection (20 years)2 Disease management costs (by +/− 20%);3 Disease utility scores (+/− 20% and cancer survivor utility set to 1)4 Discount rates at 0% and 5%;5 No prevention of HPV6/11 diseases;6 Increased vaccine coverage for women aged 12–17 years ; 4,507 cases of HPV16/18 related cervical cancer  and 1,101 cervical cancer deaths   and greater (87% and 81% reduction in CIN2/3 and CIN1 in the long term) reductions.The impact on genital warts is seen within a short time because of their more rapid disease progression. After less than five years, it is estimated that the incidence of HPV6/11 related genital warts will be reduced by 25% Figure. After 1Overall, the vaccination strategy would avoid 29% and 61% of the total discounted HPV6/11/16/18-related diseases costs at 15 years and long term, respectively. Due to the nature of the diseases, the discounted annual HPV 6/11/16/18-costs avoided show their peak for genital warts after 10 years, for CIN after 30 years and, finally, for cervical cancer after 40 years. In the first five years of vaccination, 96% of the total HPV-related diseases costs avoided would be attributable to the prevention of HPV6/11-related diseases, due to the generally shorter latency period for these diseases. This relative proportion would decrease to 44% after 50 years. Over 100 years, 57% of the total cumulated discounted costs avoided would be linked to prevention of genital warts.The ICER for vaccination of 12–17 year-old girls in Germany was estimated at 5,525€/QALY and 10,205€/LYG (life-years gained) Table, which iThe results were most sensitive to discount rates, duration of vaccine protection and utility scores but remained below the NICE threshold of £30,000/QALY so that HPV vaccination in the German setting can be considered to be cost-effective Figure. ExcludiThe results from this analysis show that the current HPV vaccination programme of females ages 12 to 17 in Germany is cost-effective with an ICER of 5,525€/QALY. Excluding the vaccine effect on HPV6/11 diseases was considered, the ICER increased to 10,293€/QALY. While this remains cost-effective using the NICE threshold, it emphasises the added value of protection against HPV6/11 diseases. Increasing vaccine coverage in the recommended population would lead to larger and earlier reductions with only a small impact on the ICER .At steady state, the model predicted that vaccinating girls aged 12 to 17 could reduce the number of HPV 6/11/16/18-related cervical cancers by 65% and genital warts among women and men by 70% and 48% respectively. The impact on HPV-related disease incidence and costs avoided was found to occur relatively soon after initiating the vaccine programme, with much of the early impact being due to the prevention of HPV6/11-related genital warts. There is consistent and growing evidence from several studies that the effects predicted by this model are being observed in the real world. In Australia, in 2009, 65% of Australian females eligible for free vaccination  had been vaccinated. A studyOur model also predicts a reduction in cervical disease outcomes, which has been reported in two studies, so far. In a case–control study in New York, girls aged 11 to 21 who had received at least one HPV vaccination prior to the first Pap smear were reported to have a statistically significant lower risk of having an abnormal Pap smear result . ResultsOur results are consistent with those predicted using a static model for Germany which found an ICER of 10,530€/QALY for preventing cervical cancer and genital warts, althougThe limitations of this model have been described in detail elsewhere but here we will briefly discuss some potential limitations specific to this analysis. FirstlyAdditionally, we did not include potential vaccination impact on non-vaccine types in our model, as the model did not include HPV types non-included in the vaccines. There is uncertainty about the accuracy of the observed cross-protection as it is not always easy to determine the causal HPV type in a lesion. It is aAnother limitation is that we did not perform probabilistic sensitivity analyses. Nevertheless, results from the sensitivity analysis confirm the findings of many other models and showed that results were sensitive to assumptions about the duration of protection and discount rates.Lastly, only cervical diseases and genital warts were included in the present model. However, HPV has been found in significant numbers of vulvar, vaginal and anal cancers and the quadrivalent vaccine has demonstrated high efficacy in preventing these lesion,17,42. HThe results from this model show that the current quadrivalent HPV vaccination programme, in addition to the screening programme in Germany, will provide public health benefits by substantially reducing the incidence of cervical cancer, CIN and genital warts. The vaccination strategies evaluated in this study were all found to be cost-effective. Future modelling analyses should include more HPV-related diseases.CIN: Cervical intraepithelial neoplasia; CIS: Carcinoma in situ; HPV: Human papillomavirus; ICER: Incremental cost-effectiveness ratio; LYG: Life year gained; NICE: National Institute for Health and Clinical Excellence; Pap: Papanicolaou test; QALY: Quality-adjusted life year; RRP: Recurrent respiratory papillomatosis; UK: United Kingdom; US: United States.DS and OS are employees at Friedrich-Alexander-University Erlangen-Nürnberg, which received an unrestricted grant from Sanofi Pasteur MSD.VR is employed by Sanofi Pasteur MSD. The authors have no other relevant affiliations or financial involvement with any organisation or entity with any financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.All authors satisfied the ICMJE authorship criteria. In addition, DS conducted the acquisition of data, suggested analysis and interpretation of results and participated in designing the manuscript. VR gave support in the acquisition of data and validated the results. OS conceived the study, participated in its design and coordination. All authors reviewed and approved the final manuscript.Table S1. Sexual parameters. Table S2: Estimated cervical cancer mortality rates by age and disease state. Table S3: Treatment patterns for each CIN/CIS stage and for genital warts. Table S4: Percentage of the German female population that undergo hysterectomy annually by age group. Table S5: Health utility values by age and gender for individuals without cervical disease or genital warts.Click here for file


Objective. To evaluate oral cavity and pharynx cancer (OCPC) patterns by gender. Methods. We used Surveillance, Epidemiology, and End Results program data for 71,446 cases diagnosed during 1975–2008 to classify OCPC by anatomic subsite as potentially HPV-related or not, with oral tongue cancer considered a separate category. Results. Total OCPC rates among men were 2–4 times those among women. Among whites, total OCPC rates rose in the younger age groups due to substantial increases in successive birth cohorts for HPV-related cancers, more rapid among men than women, and oral tongue cancers, more rapid among women than men. Among blacks, total OCPC rates declined among cohorts born since 1930 reflecting the strong downward trends for HPV-unrelated sites. Among Hispanics and Asians, HPV-unrelated cancer rates generally declined, and oral tongue cancer rates appeared to be converging among young men and women. Conclusions. Decreases in total OCPC incidence reflect reductions in smoking and alcohol drinking. Rising HPV-related cancers among white men may reflect changing sexual practices. Reasons for the increasing young oral tongue cancer rates are unknown, but the narrowing of the gender differences provides a clue. In the United States, incidence rates of oral cavity and pharynx cancer (OCPC) have been decreasing at about 1% per year over the past decade, with some differences according to race, sex, and subsite . TobaccoData from population-based registries in the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) program , 18 wereData from the original nine SEER registries (SEER 9) for cases diagnosed during the 34-year period from January 1, 1975, through December 31, 2008, were available for whites and blacks. These nine registries are located in the metropolitan areas of Atlanta, Detroit, San-Francisco-Oakland, and Seattle-Puget Sound and the states of Connecticut, Hawaii, Iowa, New Mexico, and Utah. For cases diagnosed during 1992–2008, data were available for the racial groups of whites, blacks, Asian/PIs, and AI/ANs and whether of Hispanic origin in the SEER 13, which includes the SEER 9 plus four additional areas, the California metropolitan areas of San Jose-Monterey and Los Angeles, rural Georgia, and Alaska Natives in Alaska. In the recent years 2004–2008, the SEER 9 and 13 registries accounted for about 10% and 14% of the US population, respectively . IncidenTemporal trends in age-adjusted incidence rates using five- or six-year time periods 1975–79, 1980–85, 1986–91, 1992–97, 1998–2003, and 2004–2008 were plotted according to year of diagnosis by race/ethnicity, subsite category, and sex. Temporal trends in age-specific rates were plotted according to year of birth to facilitate visual comparison of rates. The year of birth was derived by subtracting age at diagnosis  from calendar year of diagnosis ; the cohorts are referred to by their approximate midyear of birth. Only rates based on at least 10 cases were presented. Temporal trends were plotted so that a slope of 10 degrees represented a change of 1% per year  . EstimatThere were 27,767 cases  of OCPC diagnosed among whites and blacks during 1975–1991 in the SEER 9 and 43,679 cases  among whites (non-Hispanic), blacks, Hispanics (white), Asian/PIs, and AI/ANs during 1992–2008 in the SEER 13 . Total OTrends in the age-adjusted OCPC incidence rates by race/ethnicity (whites (non-Hispanic), blacks, Hispanics (white), and Asian/PIs), subsite category, and gender in the SEER 13 during 1992–2008 are presented in HPV-related rates among white men rose 88% from 3.4 in 1975–1979 to 6.4 in 2004–2008, more rapidly in recent years than earlier, while HPV-unrelated rates declined 50% from 7.2 in 1980–1985 to 3.6 in 2004–2008 . Among t The MF IRRs among whites doubled from 2.4 to 4.8 for HPV-related cancers declined modestly from 2.4 to 2.1 for HPV-unrelated cancers, and moderately from 2.0 to 1.5 for oral tongue cancers. Among blacks, the decrease in MF IRR for HPV-unrelated cancers was quite rapid, from 4.0 to 2.8.Among whites, total rates rose in the youngest age group among men and women due to substantial increases in successive birth cohorts for HPV-related cancers, more rapid among men than women, and oral tongue cancers, more rapid among women than men . The HPVAmong blacks, total rates declined among cohorts born since at least 1930 reflecting the strong downward trends among men and women especially for HPV-unrelated sites . Black mAmong Hispanics and Asians, total OCPC trends were not consistent across the genders or age groups. However, rates of HPV-unrelated cancer generally declined across successive birth cohorts for both genders. Oral tongue cancer rates appeared to be converging among young men and women of both groups.The MF IRRs for total among whites remained about 2 among those born prior to 1940, after which they rose to 2.9–3.8 among those born during the 1950s and then declined to 2.3 among those born around 1970 . MF IRRs In this study, we analyzed temporal trends of total OCP, presumed HPV-related, presumed HPV-unrelated, and oral tongue cancer among US men and women of white, black, Hispanic, Asian, and AI/AN ethnicity. Our study, which highlights changing patterns by gender, is based on the same categorization of anatomic subsites as presumed HPV-related or not used in two recent publications , 12, and Similar to other studies, we found overall OCPC incidence rates among men to be 2–4 times those among women of all race/ethnicities, and in recent decades, they varied from 4 or greater for HPV-related sites to 2-3 for HPV-unrelated sites, and 1–3 for oral tongue cancer. The MF IRR for HPV-related sites doubled among whites from 2.4 to 4.8 over the study period, due especially to rapidly rising rates of HPV-related OCPC among white men, particularly those born since 1940. Consistent decreases in the MF IRRs for HPV-unrelated sites were observed for whites (from 2.4 to 2.1) and blacks (from 4.0 to 2.8), due to rapidly declining rates especially among black men born since 1940. The IRRs for oral tongue cancer generally declined among all ethnic groups due especially to rising rates among younger white, Hispanic, and Asian women. Total OCPC trends varied markedly over the period 1985–2008. The age-adjusted rates for HPV-related cancers rose 3% per year among white males, for HPV-unrelated cancers declined 3% per year among whites of both genders and black females and 5% per year among black males, and for oral tongue cancer rose 1% per year among white females. The declines seen in HPV-unrelated cancer for all race/ethnic-sex groups are consistent with a pathway mainly driven by alcohol and tobacco  that paAmong white males, the much smaller declines in the incidence of total OCPC appear to be driven by the rising incidence of HPV-related cancers, in contrast to the declines in HPV-unrelated cancers –29. A nuHPV is a very common sexually transmitted infection in the United States . Data frSimilar to other recent studies of oral tongue cancer , 37, we  A limitation of our descriptive study is that HPV status is based on a categorization by anatomic subsite and thus likely resulted in some misclassification of actual HPV status. It has been suggested that HPV-related base of the tongue cancer may have been misclassified as oral tongue , 16; how In summary, our large numbers based on SEER 13 allowed us to plot race-, sex-, and age-specific incidence rates of OCPC by HPV category and year of birth and to carry out a detailed analysis of oral tongue cancers. The significant decreases in total OCPC incidence seen for most race/sex groups reflect reductions in smoking prevalence due to public health efforts aimed at cessation and lowering initiation rates as well as decreases in alcohol consumption. The increasing rates of cancer of the oral tongue especially among young men and women is of growing concern and suggest the need for dedicated research into the epidemiology of these understudied tumors; the narrowing of the gender differences apparent across all the racial/ethnic groups provides a clue worthy of pursuit. The much smaller decline in total OCPC incidence and recent increases among white men born since the mid-1940s appear related to the rapidly rising rates of HPV-related cancers and may reflect changes in sexual practices since the mid-1960s. The potential impact of HPV vaccination recently approved for both girls and boys on these trends is unknown, but changes are likely many years in the future. Dentists and physicians need to be aware of the notable and significant rise in rates of cancers of the oral tongue and HPV-related cancers of the oropharynx, especially among young men and women, and ensure that these new at-risk groups receive routine oral cancer screening.


P = 0.001), the number of lifetime sexual partners , and years of sexual activity  as risk factors for HPV acquisition. Hence, our study revealed that oncogenic HPV infection is common in young asymptomatic women Portuguese women, with a history of 2–5 sexual partners and over 2 year of sexual activity. Moreover, these results demonstrate that HPV detection performed in self-collected samples may be important to appraise better preventive strategies and to monitorize the influence of vaccination programmes within different populations.This study aimed to characterize the HPV infection status in adolescents and young university women in Portugal. The distribution of HPV genotypes was evaluated by PCR DNA genotyping after self-sampling collection from 435 women of exfoliated cervical cells using a commercial kit. We observed an overall frequency of HPV infection of 11.5%. Furthermore, HPV DNA prevalence was 16.6% in those young women that self-declared as sexually active. The more frequently detected HPV types were 31, 16, 53, and 61. Statistical analysis identified median age (OR = 3.56;  Genital Human papillomavirus (HPV) is highly prevalent in sexually active women. This infection has been established as the etiological agents of genital warts and squamous intraepithelial lesions of the cervix, and nevertheless, only certain types of HPVs are able to induce cervical cancer development –3.Cervical cancer is one of the leading causes of mortality by cancer with approximately 495 000 women newly diagnosed each year [Adolescents and young-adult women are more vulnerable to HPV infection. These observations are based on biological/physiological differences in the cervical epithelium. While in adults the predominant cell type is squamous, in adolescents the predominant cell type is columnar and metaplastic . HoweverHPV molecular detection has been implemented in several countries and it was already proposed as the primary test for screening by replacing the Pap smear test –11. Wherin-house methodologies for HPV genotyping have been developed in hospitals and research centres, and they are financially advantageous to detect and characterize HPV in clinical specimens [Nevertheless, HPV genotyping may be considered a more important methodology than detection only, since it helps to identify the persistent infections which are predictive of HPV infection outcome . Several pecimens , 14.Despite the incidence and mortality rates of cervical cancer in Portugal, until now no national screening program was implemented and there is no sufficient epidemiological data for HPV infection, particularly in young women. Screening programs in young women are essential to understand not only the epidemiology of HPV but also behaviours and community practices and consequently prevent the propagation of infection. Adolescents should be educated regarding HPV and associated risk factors of infection. Hence, they should also be encouraged to obtain suitable gynecological cares after initiating sexual activity , 16. Several studies have reported lately that self-obtained samples from the anogenital tract were accurate and suitable for HPV-DNA testing and have shown a similar correlation to clinically obtained samples , 18. SelThe present study is the first to characterize the presence of HPV in adolescents and university women from northern region of Portugal using the self-sampling method. University Fernando Pessoa and all individuals included in the study gave their informed consent according to the Declaration of Helsinki, which in the case of women younger than 18 years was signed by parents/guardians, in accordance with the Portuguese legislation.  A population-based study was developed to obtain a profile of HPV infection status in the Northern region of Portugal. Between March and May 2010, female students aged from 14 to 30 years old, from eight high schools and two university institutions from northern region of Portugal, were invited to participate in the study. Ethics approval was obtained from the Ethics Committee of n = 277, 63.7%), age at first sexual activity (16.0 ± 1.67), number of years after first sexual intercourse (2.0 ± 2.06), and lifetime number of partners (This study was undertaken in 435 women (median age 17.0 ± 2.46 years old), who have answered an anonymous questionnaire for epidemiological characterization. The study population was characterized according to age, age of menarche (median age 12.0 ± 1.34), education level, HPV vaccination, sexual activity  from Digene , which contains a conical brush and a tube with transport media to stabilize the sample. The self-collection process consists to inserting a sterile brush into the vagina until their fingers reached their labia, and then rotating the brush five times in the same direction and remove which from the vagina and place which in UTM as suggested by the commercial kit instructions.All participants were volunteers and informed of the study through workshops developed specifically to explain the study and self-sampling procedure. Briefly, exfoliated cervical cells were collected by self-sampling using the commercial kit Sample processing included the concentration of cells by centrifugation, followed by DNA extraction using QiAmp DNA Blood mini Kit , according to manufacturer's instructions. DNA quality was assessed by measuring the absorbance at 260 nm using a UV/Visible spectrophotometer and its purity assessed by the ratio of the values of absorbance at 260/280 nm. 3 primer 5′-ACA CAA CTG TGT TCA TAG C-3′ and BGII primer 5′-GTC TCC TTA AAC CTG TCT TG-3′.To assess the validation of extraction method, the presence of genomic DNA was tested with a PCR protocol for amplification of beta-globin gene with the PCOμL solution with 1x Taq buffer, 4.0 mM MgCl2, 0.2 mM DNTP'S, 0.30 μM each primer, 1 U de Taq DNA Polimerase, and 0.2 ug of genomic DNA. The amplification conditions were as follows: denaturation of DNA template at 95°C for 3 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final extension step at 72°C for 10 min. The amplified fragment of 175 base pairs (bp) was analyzed by electrophoresis in 1.5% (w/v) agarose gels stained with ethidium bromide and visualized under UV light. The PCR reaction was performed in a 50 HPV DNA was detected through PCR using two different pairs of consensus primers: GP5+/6+, which amplify a region of 150 bp ; and MY0μL of reaction mixture with 1x PCR Buffer, 3.0 mM MgCl2, 0.2 mM DNTP'S, 0.30 μM of each primer, 1 U of Taq DNA polymerase, and 0.2 ug of genomic DNA. Thermal cycling was performed as follows: initial denaturation of DNA template at 95°C for 4 min, followed by 40 cycles of 94°C for 30 s, 44°C for 60 sec, 72°C for 90 s, and a final extension step at 72°C for 10 min. The amplified fragment was analyzed by electrophoresis in 1.5% (w/v) agarose gels stained with ethidium bromide and visualized under UV light. PCR amplification reaction with GP5+/6+ primers was carried in a 50 μL of reaction mixture with 1x PCR Buffer, 4.0 mM MgCl2, 0.2 mM DNTP'S, 0.40 μM of each primer, 1 unit of Taq DNA polymerase, and 0.2 ug of genomic DNA. The amplification conditions include an initial denaturation at 95°C during 3 min, followed by 40 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 1 min, and a final extension step at 72°C for 5 min. The amplified fragment was analyzed by electrophoresis in 1.5% (w/v) agarose gels stained with ethidium bromide and visualized under UV light.PCR amplification reaction with MY09/11 was performed in a 50 μL final volume reaction, using 5 μL of MY09/11 PCR product, 2 μL of 10x recommended restriction buffer, and 10 units of each restriction endonucleases: PstI , HaeIII , DdeI , and RsaI . Digestion was performed at 37°C for 5 hours. The restricted fragments were separated by electrophoresis on 3% agarose gels with ethidium bromide staining and visualized under ultraviolet light. The identification of HPV types was executed using the algorithm proposed by Nobre et al. in 2008 [HPV-positive cases by amplification with primers MY09/11 were typed by RFLP analysis as described by Nobre et al. in 2008 . Each reThe definition of HPV positivity was based on the result of MY09/11 and/or GP5+/6+ PCR amplification. Nonconcordant results by both primer sets were sent to sequentiate for confirmation of result. HPV genotyping was performed in MY09/11 positive cases and HPV genotypes were divided into four groups based on their oncogenic activity: high-risk types , probable high-risk types , low-risk types , and types of undetermined risk  , 23.2O) replacing template DNA in the reaction mix; and as positive control we used an HPV positive sample used in the Virology Service for diagnosis of HPV infection using the commercial kit hc2 High-Risk HPV DNA test ; (3) repetition of 100% of samples for validation of results; and (4) analysis of both PCR and RFLP results individually by three of the authors. Nonconcordant results were sent to sequentiate for confirmation of result.To ensure the quality of the study, the following quality criteria were defined: (1) blind study; (2) inclusion of negative and positive controls in all PCR reactions: as negative control we have used double distilled water  test was used to compare frequencies among groups. The OR (Odds Ratio) and its 95% Confidence interval were calculated as a measure of the association between the categorical variables  and HPV infection status.Statistical Analysis was performed using the computer software n = 277), where HPV infection had a prevalence of 16.6% (46/277)  . HPV infection was analyzed according to age, age of first menarche, age of first sexual intercourse, number of years after first sexual intercourse, number of sexual partners, educational level, and HPV vaccination status . When coWe observed that HPV was more frequent among university students (24.0%) compared with nonuniversity students (12.8%). However, within positive cases, the prevalence of high-risk HPV was higher for nonuniversity students in comparison to university students (47.9% versus 34.8%) . OverallP = 0.001), women with 2–5 sexual partners , and women who had initiated their sexual activity over 2 years .Statistical significant differences were found when comparing HPV distribution regarding education level, HPV vaccination, mean age, lifetime number of sexual partners, and number of years after first sexual intercourse . We founHPV infection is one of the most common sexually transmitted infections and a major public health concern, especially during adolescence , 25. MosDue to the worldwide distribution of HPV infection and to the high-incidence in undeveloped countries, many companies have attempted to developed low-cost tests with high efficiency to detect HPV. Self-sampling has been suggested as a useful tool for testing women for HPV infection mainly in low-resource populations or for people who have difficult access to health care . Self-saAlthough the number of samples was limited and further large-scale epidemiological studies are needed to assess the significance of less common genotypes, this study provides the first view on the spectrum of the most common mucosal HPV types in young Portuguese women. Epidemiologic studies refer to a prevalence of HPV infection from 2% to 44% in general population, with the highest prevalence in women aged 20 to 24 years old , 31. OurAccording to several studies, the behavioral risk factors associated with the HPV infection in adolescents and young adult women include early age of first sexual intercourse , 37–39. The number of lifetime sexual partners is considered to be one of the major risk factors for HPV infection , and in Present results showed differences comparing HPV distribution regarding education level and HPV vaccination. Since the median age of first sexual intercourse was of 16.0 years old, it is expected that university students have more years of sexual life than nonuniversity students. Therefore, they would be exposed more frequently to a HPV infection than nonuniversity students. Regarding HPV vaccination, since the National program of Vaccination in Portugal started to include the HPV vaccine 2 years ago for girls between 13 and 18 years old, it is expected that these girls would be protected from acquiring an HPV infection. Therefore our data is correlated to these evidences and vaccination may reduce efficiently the infection by HPV. Despite these important epidemiological data, characterization of HPV genotype frequencies among populations is considered to be even more imperative. HPV diagnostic assays such as hybrid capture second-generation (HC2) and PCR-based methods employing the consensus primers, MY09/11 and GP5+/6+, allow the detection of a large number of HPV types, mainly high-risk types . These pIn our study, we have used the genotyping method described by Nobre et al. in 2008, which allows a good discrimination of mucosal HPV types, including several HPV types that are underdiagnosed by current diagnostic methods. Our data showed that high-risk HPV types are the most frequent, with HPV31 and HPV16 having higher frequency. Surprisingly, HPV31 was found as the most frequent among our study. A recent meta-analysis shows that HPV31 may be the second or third most common type in European women with normal cytology , 42. DesRegarding our results, we must consider that after an open invitation to participate in the study, all participants were volunteers and informed of the study through workshops developed specifically to explain the study and self-sampling procedure. We cannot rule out the influence of two factors, that the attending students were more keen to know more about HPV and that subjects at higher risk of HPV infection are fear of being detected positive. However, we do think that the influence of those factors was reduced because knowing HPV results was optional, and only under the supervision of the gynecologist. Furthermore, our rate of participation was not conditioned by the cost of the test .Molecular pathology methodologies have been demonstrated as important in the improvement of screening, diagnostic, or monitoring cancer, and genetic- or viral-associated diseases. Furthermore, they provide valuable tools for the new paradigm in Medicine known as personalized medicine –46. The It is acceptable to consider that, to achieve the maximum efficacy in health prevention strategies, we must maximize the knowledge of some populations about their epidemiology, costumes, and concerns and that is particularly important in those populations where no screening or vaccination has already been developed –56. ThisIn conclusion, we have to refer that self-sampling may be a good alternative in substitution of the vaginal speculum examination by a clinician as a tool for primary screening of HPV infection in young asymptomatic women. This study contributes to a better understanding of the epidemiology of HPV infection among young Portuguese women. The exact knowledge of HPV profile in young women may be important to appraise the vaccination strategies within the different population.


Cervical cytology and human papillomavirus (HPV) DNA co-testing is recommended as a screening method for detecting cervical lesions. However, for women who are HPV-positive but cytology-negative, the appropriate management and significance of HPV-58 infection remain unknown.This study of prevalent HPV detected at baseline with a median follow-up of 3.2 years evaluated the risk factors associated with cervical abnormalities and assessed the significance of HPV-58 infection. A total of 265 women were enrolled. All high-grade squamous intraepithelial lesions (HSIL) that were detected by cytology were confirmed by histology. Histological diagnoses of cervical intraepithelial neoplasia 2/3 were classified as HSIL. Women were classified into four groups according to the HPV genotype that was detected at their first visit: HPV-58 (n = 27), HPV-16 , ten other high risk (HR) types (n = 79), or low/undetermined risk types (n = 107).p<0.001). The 5-year cumulative incidence rates of HSIL were 34.0% (95% CI: 17.3–59.8%) in HPV-58 positive cases, 28.0% (95% CI: 13.8–51.6) in HPV-16 positive cases, 5.5% (95% CI: 2.1–14.0%) in one of the ten other types of HR-HPV positive cases, and 0% in women with low/undetermined risk HPV. When seen in women with HR-HPV (n = 158), persistent HPV infection was a significant factor associated with the development of HSIL . Women with HPV-58 had a higher risk  for the development of HSIL than women with HPV-16  in comparison with women with other types of HR-HPV.Of 265 women, 20 (7.5%) had HSIL on their follow-up examinations. There were significant differences in the cumulative incidence of HSIL between the four groups (HPV-58 has a high association with the development of HSIL in women who are HPV-positive and cytology-negative. The use of the Papanicolaou (Pap) smear as a screen for cervical cancer has significantly decreased the incidence of cervical cancer and its associated mortality in recent years. However, the Pap smear has a low sensitivity (<70%) for detecting high-grade squamous intraepithelial lesions (HSIL), which has raised a number of issues regarding the need for additional methods to improve detection HPV genotype-specific testing for HPV-16 and/or -18 (option 2) in HPV-positive and cytology-negative women may be an ineffective screening strategy for women in East Asia because there are significant differences in the distribution of HPV genotypes between Western and Eastern populations. Specifically, HPV-58, which is associated with a high risk of developing cervical dysplasia and cervical cancer, is rare worldwide but is commonly found in East Asia HPV-16 and/or -18 positivity is associated with a significant risk of developing HSIL in HPV-positive and cytology-negative women In this study, we evaluated the risk factors associated with cervical abnormalities in women with HPV-positive but cytology-negative results and assessed the significance of HPV-58 infection in women living in a country with a high prevalence of HPV-58 infection.This study was approved by the Institutional Review Board (IRB) of Asan Medical Center, Seoul, Korea. Given that this was a retrospective study that used registry data gathered by Asan Medical Center, the IRB waived the need for written informed consent. Data were anonymously analyzed. Our institute processed approximately 50,000 Pap tests per year, of which > 95% are SurePath liquid-based preparations. In total, 3246 women who were co-tested using cytology and HPV genotyping between December 2005 and September 2010 at Asan Medical Center were selected after searching our electronic pathology database. Of these 3246 patients who underwent co-testing, 377 (11.6%) were determined to be cytology-negative but HPV-positive. Among these 377 patients, 94 were excluded due to being lost to follow-up or having an inadequate follow-up period . However, the excluded women were not different in terms of certain characteristics, such as HR-HPV distribution, in comparison with the women enrolled in this study. Of the remaining 283 women, 18 women < 30 years of age were also excluded from the study. Finally, a total of 265 women who were HPV-positive but cytology-negative at the initial assessment with no previous cytologic abnormalities were included in the study. Women who did not undergo colposcopy, conization or hysterectomy were followed using cytology smears at 6–12 month intervals for > 1 year. All cervical smears were prepared using SurePath liquid-based preparations . Data collection was stopped when HSIL positivity was detected or when the patient underwent biopsy, conization, or hysterectomy during the follow-up period. The median follow-up period was 3.2 years (range: 0.04–6.26 years). The occurrence of low-grade squamous intraepithelial lesions (LSIL) and HSIL during the follow-up period was considered indicative of cervical abnormalities. Of a total of 265 women, all 20 women with HSIL, 30 of 62 women with LSIL, and 8 of 187 women with no cervical abnormality on follow-up cytology underwent further histological examination, such as colposcopic biopsy, conization, or hysterectomy. For this study, histological diagnoses of cervical intraepithelial neoplasia (CIN) 1 were classified as LSIL, and diagnoses of CIN 2 and 3 were classified as HSIL. Of 265 women included in this study, 193 were consistently assessed using repeated HPV testing for > 1 year as well as cytology. The remaining 72 women were assessed by cytology only during the follow-up period after the initial co-testing. Persistent HPV infection was assessed in the 193 women who had repeated HPV tests for > 1 year, and it was defined as the detection of the same HPV genotype as seen at baseline on ≥ 2 consecutive HPV tests performed for greater than 1 year. The presence of multiple infections was defined as the presence of multiple HPV types (≥ 2 genotypes) on the initial HPV test. To determine HPV-58 oncogenicity, the HPV genotypes detected at the first visit were grouped into four categories: HPV-16 positive, HPV-58 positive , the remaining ten other HR-types , and low/undetermined risk types. There were three women who were diagnosed with HPV-16 and HPV-58 who were classified as HPV-16 positive in order to identify the risk of HSIL development in women infected with HPV-58. The characteristics of the women with HPV-positive and cytology-negative results are summarized in st PCR, 2ul of PCR products were used for 2nd PCR. Ten microliters of the second PCR product was denatured for 5 minutes at 95°C and left in ice water for 5 minutes. The product was then mixed with the hybridization buffer (MyGene). This mixture was spotted on the DNA chip and subjected to a hybridization reaction for one hour in a hybridization chamber  at 43°C. After hybridization, the product was rinsed twice with buffer I , twice with buffer II (0.2× SSC), and once with buffer III (0.1× SSC) for 5 minutes each. Hybridized HPV DNA was identified using a model G4000 scanner . A pair of oligonucleotide probes for each of the 24 HPV types was attached to each DNA chip. When a pair of specific probes elicited a fluorescence response, the assay was said to be positive for that type of HPV. The DNA microchip used is capable of identifying specific HPV genotypes and multiple HPV infections. The HPV DNA microchip included 43 genotypes, including HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 82, 26, 53, 66, 67, 69, 70, 73, 6, 11, 34, 40, 42, 43, 44, 54, 55, 61, 62, 72, 81, 7, 10, 27, 30, 32, 57, 83, 84, and 91. All 43 subtypes of HPV are detectable on the chip. We considered HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 as types of HR-HPV HPV testing was performed using the MyHPV DNA microchip  in accordance with the manufacturer’s protocols. Briefly, samples for HPV testing were separately collected from Pap smears using the endocervical brushes that were supplied with the MyHPV Chip kit. DNA was isolated using an isolation kit (MyGene), and nested PCR was performed for gene amplification. For the first PCR, a mixture of two sets of primers—MY09  and MY11 —was used. For the second PCR, the HPV primers GP5+  and GP6+  were used. β-Globin PCR amplification was performed as the internal control. The PCR products were subjected to electrophoresis on a 3% agarose gel. First PCR products were 450 bp and second PCR products were 150 bp. β-Globin PCR products were 250 bp. After 1p-values < 0.05 were considered statistically significant. Statistical analyses were performed using Stata/IC statistical software version 12 .Curves of the cumulative LSIL and HSIL incidence rates were generated using the Kaplan-Meier method, and were compared using the log-rank test. Cox regression analysis was performed to evaluate the risk factors associated with HSIL, including age, persistent infection, multiple infection, and HPV genotype. The hazard ratios and 95% confidence intervals (CI) were assessed for each factor. All tests were two-sided, and A total of 265 women with a median age of 46 years (range: 30–71 years) who were HPV-positive but cytology-negative at baseline were included in the analysis. HR-HPV infection was detected in 158 (59.6%) women and low/undetermined risk HPV infection was detected in 107 (40.4%) women at their initial HPV tests. Of the 265 women analyzed, a single HPV type was detected in 190 (71.7%) women and ≥ 2 HPV types were detected in 79 (29.8%) women. Of these, 60 women had 2 types, 16 women had 3 types, 1 woman had 4 types, and 2 women had 5 types of HPV. HPV-16 was the most common genotype detected , either alone or in combination with other types, followed by HPV-70  and HPV-53 . The prevalence of HPV-18 was 7.5% (20/265). The prevalence of HPV-58, which is known to be a high prevalence genotype in East Asia, was present in 11.3% (30/265) of the study population. The prevalence of the remaining types of HPV included HPV-52 , HPV-66 , and HPV-54 . The prevalence of HPV-31, 33, 35, 39, 45, 51, 56, 59, 6, 11, 40, 42, 43, 44, 68, 69, 72, and 84 were less than 5%. The prevalence of each genotype is shown in During the follow-up period, LSIL was detected in 23.4% of women (62 of 265) and HSIL was detected in 7.5% of women (20 of 265). The cumulative incidence rates of LSIL and HSIL among the enrolled 265 women are shown in p = 0.049). Persistent HR-HPV infection also tended to show an increased risk of LSIL development, but was not statistically significant . Persistent infection was present in 84 (43.5%) of 193 women. Among them, 63 had persistent HR-HPV infection and the remaining 21 had low/undetermined risk-HPV infection. The remaining risk factors, including age , HPV type , and multiple infections (p = 0.759) were not significantly different in terms of the incidence of LSIL.Of the risk factors that were assessed, the development of LSIL during follow-up was associated with persistent LR-HPV infection  and persistent HR-HPV infection  were significantly associated with the development of HSIL. HSIL was also not detected in women with low/undetermined risk HPV, even in women with persistent low/undetermined risk HPV infection, and all cases of HSIL developed in women with HR-HPV infection. Older age (> 45 years old)  and multiple HPV infections  tended to be associated with a higher incidence of HSIL, but these relationships were not statistically significant.Of the risk factors that were assessed, HR-HPV  (p = 0.828), which suggests that one of the other ten types of HR-HPV is a major oncogenic type of HPV. The 5-year cumulative incidence rate of HSIL was 21.2% (95% CI: 10.2–40.9%) in HPV-16 and/or -18 positive cases, but 15.8% (95% CI: 8.9–27.2%) in women with the other ten types of HR-HPV.When seen in women with HR-HPV infection, persistent HPV infection was still a significant factor associated with the development of HSIL  . The 5-yp<0.001, p = 0.008) for the development of HSIL than women with HPV-16  in comparison with women with the other ten types of HR-HPV (p = 0.011) and HPV-58  were independently associated with HSIL development in women with HR-HPV infection . Thus persistent infection in this study is equivalent to either uncleared HPV infection or reinfection with the same genotype. Of the 63 women with persistent HR-HPV infection described in the present study, 15 (23.8%) developed HSIL during the follow-up period. However, of 63 HR-HPV positive cases with no persistent infection, only 1 (1.6%) developed HSIL during follow-up. Women with persistent infection but not HSIL may have repeated infections that are not persistent, or HPV is eliminated after ≥ 1 year, or these patients may only be receiving short-term follow-up examinations.HR-HPV status, especially HPV-16 and/or -18, is an important predictor of HSIL+ in women with negative cytology The high prevalence of HPV-58 has been reported in East Asia, including China , Korea (16%) and Japan (8%) In conclusion, the present results suggest that the detection of prevalent and persistent HPV infection might be a promising strategy for the management of patients who test positive for HR-HPV but demonstrate negative cytological results. Furthermore, the high association between HPV-58 and developing HSIL indicates that the identification of the HPV-58 genotype is important for the management of women who are HPV-positive and cytology-negative in a country with a high prevalence of HPV-58 infection.


Cleavage of heparan sulphate by heparanase leads to disassembly of extracellular barriers, enabling local invasion and metastatic spread of the tumour, and releases heparan sulphate-bound growth factors from the extracellular depots. Heparanase is tightly implicated in head and neck cancer progression; yet, molecular mechanisms underlying transcriptional activation of the heparanase gene in HNSCC are largely unknown. We found that HPV16 oncogene E6 is capable of inducing overexpression of heparanase in HNSCC. Notably, radiation treatment dose-dependently suppresses E6-induced heparanase expression in vitro. Our results provide the first evidence for a functional involvement of HPV in heparanase induction in head and neck tumourigenesis and, given ongoing clinical testing of several heparanase-inhibiting compounds, offer important avenue for future therapeutic exploration in HNSCC, as well as other HPV-associated malignancies .High-risk human papillomavirus (HPV)-positive head and neck squamous cell carcinomas (HNSCCs) are highly invasive; however the identity of downstream effectors responsible for their aggressive phenotype remains underinvestigated. Here, we report that HPV-mediated up-regulation of heparanase enzyme can provide mechanistic explanation for augmented invasiveness of HPV-positive HNSCCs. Heparanase is the sole mammalian enzyme (endo-β- High-risk human papillomavirus (HPV) infection is an important mechanism underlying development of head and neck squamous cell carcinomas (HNSCCs). The incidence of HPV-positive oropharyngeal squamous cell carcinoma has been steadily increasing during the last decade and currently is almost equivalent to HPV-negative oropharyngeal tumours i.e. bFGF, VEGF, HGF) from the ECM depots Heparanase is a single mammalian endoglycosidase capable of degrading heparan sulphate (HS), the main polysaccharide component of the extracellular matrix (ECM) This resemblance, taken together with the established role of both HPV and heparanase in HNSCC tumourigenesis, prompted us to examine the effect of HPV16 oncogenes on heparanase expression in head and neck cancer.in vitro studies and xenograft models CAL-27 and SCC-25 human oral squamous carcinoma cells were a generous gift from Dr. I. Vlodavsky, Technion, Haifa, Israel. CAL-27 cell line was isolated from the tissue taken prior to treatment from a 56-year-old male  Total RNA was isolated from the cells using TRIzol , according to the manufacturer's instructions, and quantified by spectrophotometry. After oligo (dT)-primed reverse transcription of 500 ng total RNA, the resulting single-stranded cDNA was amplified using real-time quantitative PCR analysis with an automated rotor gene system RG-3000A . The PCR reaction mix (20 μl) was composed of 5 μl QPCR sybr master mix , 5 μl of diluted cDNA (each sample in a six-plicate) and a final concentration of 0.3 μM of each primer. PCR conditions were as follows: an initial denaturation step at 95°C for 10 min.; 40 cycles of denaturation at 94°C for 15 sec., hybridization at 58°C for 30 sec. and elongation at 72°C for 30 sec. Actin primers were used as an internal standard. The following primers were used: E6 – S: 5′-GCTAGCATGCACCAAAAGAGAACTGC-3′, AS: 5′-TCTAGATTACAGCTGGGTTTC-3′; E7 – S: 5′-CAGAGGAGGAGGATGAAATAGA-3′, AS: 5′-CGAATGTCTACGTGTGTGCTTT-3′; β-Actin – S: 5′-TCCCTGGAGAAGAGCTACG-3′, AS: 5′-GTAGTTTCGTGGATGCCACA-3′; Heparanase – S: 5′-CTGATGTGGAGGAGAAGTTTACG-3′, AS: 5′- GTTATACCCCTTGGAAGAGCA-3′.Immunoblot analysis and immunostaining were carried out with anti-heparanase monoclonal antibody 01385-126 Equal protein aliquots (60 μg) were subjected to SDS-PAGE (10% acrylamide) under reducing conditions. Proteins were transferred to a polyvinylidene difluoride membrane  and probed with the anti-heparanase monoclonal antibody 01385-126 2O2. Antigen unmasking was carried out by heating (20 min.) in a microwave oven in 10 mM Tris buffer containing 1 mM EDTA. Slides were incubated with primary antibodies diluted in CAS-Block (Invitrogen) or with CAS-Block alone, as a control. Appropriate secondary antibodies were then added and slides incubated at room temperature for 30 min. Controls without addition of primary antibody showed low or no background staining in all cases. Human papillomavirus status of the tumour specimens was determined by the presence of diffuse nuclear and cytoplasmic staining pattern of p16, which is regarded as a suitable surrogate marker for HPV positivity Formalin-fixed, oral squamous carcinoma tissues from 23 non-selected patients  were available from the Dept. of Pathology, Hadassah Medical Center, Jerusalem. Comparing TNM classification, average tumour staging in p16-positive patients was T3, while in p16-negative patients average staging was T2. There were no differences in lymph node involvement and metastatic spread. The use of these specimens and data in research were approved by the Ethics Committee of the Hadassah Medical Center. Paraffin-embedded slides were deparaffinized and incubated in 3% Ht-test, P < 0.05 were considered statistically significant. Results are presented as mean ± SD.Data were analysed by unpaired eV6e7) or with empty expressing vector pLXSN (Vo). Transfections were confirmed by RT-PCR analysis of E6 and E7 mRNA production using extracted cellular RNA (data not shown). Transiently transfected cells were then tested for heparanase expression at several time-points. As demonstrated in Figure eV6e7, as compared to Vo-transfected cells.As HPV-driven tumourigenesis is primarily associated with viral oncoproteins E6 and E7, we initially investigated whether expression of E6/E7 genes in HPV-negative OSCC cell lines affects heparanase expression. For this purpose, human OSCC cells Cal-27 and SCC-25 were transfected with plasmid vector encoding for HPV16 oncoproteins E6 and E7 (eV6), E7 (eV7) or with the empty vector (Vo). While transfection with E7 had no effect on heparanase expression . These findings, although limited by as small sample size, further support the role of HPV in induction of heparanase expression in HNSCC.To validate the relevance of our versus lack of expression in normal tissues of the same origin (including HNSCC) i.e. inactivating mutations in p53 Despite favourable prognosis under treatment Heparanase expression correlates with both invasiveness of HNSCC cell lines and the metastatic potential of head and neck tumours In summary, our results provide the first evidence for a functional involvement of HPV E6 in heparanase induction during head and neck tumourigenesis (most likely through p53-dependent mechanism) and may provide important avenue for future therapeutic exploration, relevant not only for HNSCC, but also for additional HPV-associated tumours, including cervical carcinoma (where heparanase overexpression is associated with aggressiveness and poor prognosis


Renal allograft recipients, as for HIV/AIDS patients, have a well-documented increased incidence of human papillomavirus (HPV)-related malignancies and preventive strategies should be specifically implemented. While in females the use of the Papanicolau test and HPV detection assay are used currently as a screening test for cervical cancer, no diagnostic procedures have been implemented to monitor HPV infection in males. The aim of this study was to test for HPV infection and to determine the spectrum of viral genotypes in urine samples of men with renal transplants.The study included 103 patients who underwent kidney transplantation between 1999 and 2008. HPV sequences were detected by nested PCR, using the broad-spectrum consensus- primer pairs MY09/MY11 and the new MGP system, and characterized by nucleotide sequence analysis.Overall, 49 (47.5%) samples were found positive for HPV sequences and the most common genotypes were HPV 16 (51.0%) and HPV 54 (10.2%) followed by HPV6, 53, 56, 58, 66, 11, 12, 20, 45, 62, and 71, in descending order of prevalence (Table The high prevalence of HPV 16 among renal allograft recipients suggests that an HPV-16-based preventive or therapeutic vaccine may be effective for prevention or treatment of HPV-related neoplasia in this group of immune compromised patients.


HIV-infected women are at increased risk for developing cervical cancer. Women living in resource-limited countries are especially at risk due to poor access to cervical cancer screening and treatment. We evaluated three cervical cancer screening methods to detect cervical intraepithelial neoplasia grade 2 and above (CIN 2+) in HIV-infected women in South Africa; Pap smear, visual inspection with 5% acetic acid (VIA) and human papillomavirus detection (HPV).HIV-infected women aged 18–65 were recruited in Johannesburg. A cross-sectional study evaluating three screening methods for the detection of the histologically-defined gold standard CIN-2 + was performed. Women were screened for cervical abnormalities with the Digene HC2 assay (HPV), Pap smear and VIA. VIA was performed by clinic nurses, digital photographs taken and then later reviewed by specialist physicians. The sensitivity, specificity and predictive valves for CIN-2 + were calculated using maximum likelihood estimators.3. One third of women had a high grade lesion on cytology. VIA and HPV were positive in 45% and 61% of women respectively. Estimated sensitivity/specificity for HPV, Pap smear and VIA for CIN 2+ was 92%/51.4%, 75.8%/83.4% and 65.4/68.5% (nurse reading), respectively. Sensitivities were similar, and specificities appeared significantly lower for the HPV test, cytology and VIA among women with CD4 counts ≤200 cells/mm3 as compared to CD4 counts >350 cells/mm3.1,202 HIV-infected women participated, with a median age of 38 years and CD4 counts of 394 cells/mmAlthough HPV was the most sensitive screening method for detecting CIN 2+, it was less specific than conventional cytology and VIA with digital imaging review. Screening programs may need to be individualized in context of the resources and capacity in each area. Invasive cervical cancer is the third most common cancer among women worldwide Among HIV-infected women, there are no standard guidelines on the optimal methods to screen and treat for cervical cancer in resource-limited countries (RLC). Access to screening in RLC is limited due to financial and personal capacities. The implementation of a cervical cancer screening program with technically appropriate detection methods could reduce morbidity and mortality among HIV-infected women. While alternative screening methods have been evaluated for the detection of high-grade cervical intra-epithelial neoplasia (CIN 2/3) in Africa To determine optimal cervical cancer screening approaches for HIV-infected women, we aimed to compare the sensitivity and specificity of conventional Pap smear screening to that of HPV DNA and VIA testing for the detection of histologically confirmed high-grade cervical neoplasia grade 2 and above (CIN 2+) in 1,202 HIV infected women from Johannesburg, South Africa. We present results of the largest screening study in HIV positive women to date to determine the clinical performance of these three screening methods.The protocol was reviewed and approved by the Human Ethics Committee  of the University of the Witwatersrand and, for secondary data analyses, by the University of North Carolina.A total of 1,202 HIV-infected women (18–65 years of age) were recruited from an HIV treatment clinic located in a tertiary government hospital in Johannesburg South Africa. Women were ineligible to participate if they (i) were pregnant, (ii) had previously undergone a hysterectomy or treatment for cervical neoplasia or cancer, iii) were severely ill, or (iv) had signs and/or symptoms suggestive of a sexually transmitted disease (STD). Women were study-eligible following the treatment of any symptomatic STD. Women who were menstruating at study enrollment were asked to return within one to two weeks to participate.After an educational session was presented on cervical cancer screening in English or Zulu/Sesotho, health workers screened potential eligible women for exclusion criteria, explained study aims, and obtained written informed consent. A medical history was obtained through participant interviews to obtain information on socio-demographic characteristics, antiretroviral therapy status, and other lifestyle factors, including smoking and snuff  use, reproductive/menstrual characteristics, sexual history and history of contraceptive use.Each woman was screened for this cross-sectional study using three different methods: i) HPV DNA test (QIAGEN Hybrid Capture 2: HC2), ii) conventional Pap smear cytology, and iii) VIA. During a pelvic examination, the HPV sampling was first conducted using a Digene Cervical Sampler Hybrid Capture-2 (HC-2) brush and placed in standard transport media (STM) (QIAGEN Corporation). HPV DNA laboratory testing was conducted at the University of Cape Town using the Digene Hybird Capture-2 (HC-2) method (QIAGEN), and the HPV laboratory team was blinded to other study results. HPV DNA test results were not used for clinical management.For a conventional Pap smear diagnosis, cervical exfoliated cells were then collected using a Papette Cervical Cell Collector  and smeared onto cytology slides which were read and analyzed according to Bethesda 2001 guidelines After Pap smear sampling, VIA was preformed, by applying 5% acetic acid to the cervix followed by a three minute waiting period. Nurses were previously trained at a two week course in Lusaka, Zambia The cytology unit and the Anatomical Pathology Department are accredited by the South African National Accreditation System (SANAS) and undergo regular proficiency testing by the Royal College of Pathologist of Australasia Quality Assurance Programme (RCPA). Internally there is 100% second on minute review by another cytotechnologist of all negative Pap smears and senior cytotechnologist/pathologist review all positive (ASCUS+) cases. Also a there is a cytological/histology review process. Study cytology readings have previously undergone quality assurance by University of North Carolina with 80–85% concordance of results Discrepant results between cytology and histology resulted in a review of the Pap smear slide. If discrepancy was confirmed, then a repeat colposcopic biopsy was conducted if clinically indicated. For quality assurance (QA) of the VIA technique, the study gynecologist with a medical officer trained in colposcopy reviewed each digital picture and the initial VIA diagnosis of the nurse within two weeks of the VIA procedure. Medical staff was blinded to both the cytology and HPV results at the time of VIA interpretation. If the quality assurance team could not agree on interpretation of VIA results, the digital photos were sent to Professor Parham  for a final diagnosis. HPV testing QA was done per recommendation of the manufacture's guidelines. The final VIA reading used in the analysis was the reading done after review by the doctors at the QA meeting. However calculations using the nurse interpretation for CIN 2+ are also presented.As all women were not referred for cytology verification by colposcopy, using only the histology results to estimate sensitivity and specificity could lead to biased inference. To correct for this verification bias, we employed the maximum likelihood method proposed by Zhou et al. 3, 201–350 cells/mm3, 351–500 cells/mm3 and >500 cells/mm3), HIV viral load ≤400 copies/ml, 401–1000 copies/ml and >1000 copies/ml) and combination antiretroviral therapy (cART) (yes and no), were compared across categories using the standard Z test, assuming independent samples. Agreement between the VIA results by gynecologists and by nurses was measured by a kappa statistic No adjustment was made for multiple comparisons. All statistical analyses were conducted using SAS 9.2 . The Analysis of VIA for determining CIN 2+ was evaluated by looking at the gynecologists and the nurses VIA interpretation separately.For estimation of sensitivity and specificity for CIN-2+ or CIN-3+, Pap smear results were considered negative if the test result was negative, LSIL or ASCUS, and positive if the result was HSIL, ASC-H, or SCC. However additional sensitivity and specificity analyses were done evaluating the Pap smear results comparing negative to positive if the results were ASCUS, LSIL, HSIL, ASC-H or SCC. Estimated sensitivities and specificities of the screening tests, stratified by levels of CD4 counts (≤200 cells/mm3 (Interquartile range [IQR] 252.5–572). Approximately 75% (N = 872) of our population had abnormal Pap smears, of which a third (N = 399) of the overall the Pap smears results were high grade lesion by cytology. VIA and HPV were positive in 45% (N = 528) and 61% (N = 727) of participating women, respectively. There were two diagnosed cases of invasive cervical carcinoma. Of the 93.1% (N = 1111) women on cART, 82.9% had undetectable HIV viral loads (≤400 copies/ml).A total of 1,202 women were screened between November 2009 and August 2011. Of these, 9 were excluded . A total of 1,193 women (98.1% black African) were evaluated, with a median age of 38 years (IQR 32–43) and a median CD4 count of 394 cells/mmAs stand-alone tests, overall sensitivity for the detection of CIN-2+ was the highest for HPV testing (92%), followed by Pap smears (76%) and VIA at 65.4% (nurse interpretation)). However VIA sensitivity was increase to 76% with physician QA review. Specificity for CIN-2+ was the highest for Pap smears (83%), followed by VIA (68% for both doctor and nurse interpretation), and lowest for HPV testing (51%). When CIN3+ was used as the end point, sensitivities were higher, while specificities somewhat lower (∼10%) than observed for CIN-2. Pap smears had a notably higher sensitivity (95%) for CIN-3+ with a corresponding decrease in specificity (73%). Sensitivity for the detection of CIN-3+ increased by 0.7% for VIA, 6.0% for HPV, and 18.7% for cytology as compared to CIN-2+ .When the results of two screening methods were combined as either test positive, the sensitivity increased to above 89% for detecting CIN 2+ and greater than 97% for CIN 3+. However, there was a corresponding decrease in specificity . For CIN3 compared to CD4 counts >350 cells/mm3   . Compari14% of all participants had a discrepancy between cytology and histology results. Verification biopsies were done on 25% of all women with negative VIA and Pap smears. Only 3% (2/63) of these verification histology results were positive, resulting in two CIN 2 cases. Histological review of the follow-up Loop Electrical Excision Procedure (LEEP) results for these two CIN-2 cases revealed only minimal changes consistent with HPV infection. No neoplasia was found. There was substantial agreement between the VIA readings of the nurse and that of the doctor [kappa statistic = 0.69 (95% CI 0.64–0.73)].This analysis of just over 1,200 HIV-infected women represents, to our knowledge, the largest comparative screening study of HIV-infected women, comparing the screening performance of three cervical cancer screening methods to detect histological CIN 2+ endpoints. We observed a notably high rate of positive test results, including 33% HSIL, 61% HPV DNA positivity, and 45% VIA positivity. All three screening methods had sensitivities of >65% for determining CIN 2+ disease in HIV positive women, with variations in specificity for CIN-2 ranging from 83% for cytology to 51% for HPV testing. Test sensitivities were similar across strata of CD4 counts, while the specificities of all the screening methods decreased with immune suppression as measured by CD4 counts.3, as per the South African HIV treatment guidelines and thus had a prior history of significant immunosuppression The observed HSIL prevalence in this study (33%) is almost double than that previously reported from a different cohort of 1010 HIV-infected women from the same clinic (18%) Cytology based screening (via the Pap smear) is the only screening method that has been shown to reduce mortality in many places in the world including middle and lower resource countries such a Colombia, Chile and Vietnam At present, there are only three studies evaluating different screening methods for the detection of histological CIN 2+ in HIV infected women in Africa. None of these studies conducted had direct comparisons of these three screening methods. In Nigeria (N = 205), VIA was found to have a sensitivity and specificity of 76% (95% CI 52–91%) and 83% (95% CI 77.0–88.0%), respectively A very similar study to ours was performed with 303 HIV positive women in India, and showed similar sensitivity/specificity of VIA for CIN-2+ detection of 80/82% respectively The results of VIA in HIV negative women in two large meta-analyses showed that the range of sensitivity of VIA was relatively similar at 79%–80%. The range for specificity for VIA was slightly higher (85–92%) in HIV-negative than in HIV infected women The VIA sensitivity for CIN 2+ is within the range of the Pap smear sensitivity in this study, although the observed specificity is not as high as the Pap smear in HIV positive women. However for reasons that are unclear, the Pap smear had a much higher sensitivity then the VIA (which remained unchanged) for detecting CIN 3+. Sequential HPV testing as seen in High risk HPV DNA was present in 61% of participants in our study which is somewhat higher than that observed among 956 HIV-infected women from Cape Town (46%) South Africa has one of the highest HIV prevalence rates in the world In terms of study strengths, we implemented intense QA measures to ensure reliable visual inspection results, which included weekly meetings to review all digital photos by a team of study personnel. This helped ongoing education of the nursing staff and likely contributed to the good correlation between the nurses' and doctors' VIA readings. In addition to skills resources needed, the QA of VIA also required significant infrastructure . Such resources may not be available in other areas of South Africa or other RLC. However, this QA model to optimize VIA results was adapted from the program in Lusaka, Zambia. For rural sites in resource limited countries, this essential QA model could potentially be modified by sending pictures via “memory sticks”/CDs or through cell phones to skilled personnel. We have been able to achieve this model in rural South Africa.Study limitations relate to the study review and intense QA which might be difficult to successfully replicate in other public clinic and non-academic environments. Participant files were reviewed to ensure results and follow-up visits were achieved. In busy under-resourced clinics in RLC, these types of review and meetings may be extremely difficult to thoroughly implement. Further, most of our patients were on effective cART so precluding our ability to determine the effect of cART and HIV viral load on screening.Cervical cancer screening in HIV positive women is an urgent public health requirement which demands immediate attention and coordinated efforts of both national and local governments. Our study results indicate that all three screening modalities  are viable alternatives for consideration as screening options in different programmatic settings, which is important as a “one size fits all approach” may not to work. The decision of which screening modality to implement will be influenced by cost, patient population, availability of skilled human resource and laboratory capacity. Within this decision process, quality assurance needs to be considered at all stages of the program. Careful consideration and evaluation will be needed to determine for the best screening approach for a country and maybe for different geographical settings within a country.