HIV-1-positive patients clear the human papillomavirus (HPV) infection less frequently than HIV-1-negative. Datasets for estimating HPV clearance probability often have irregular measurements of HPV status and risk factors. A new transitional probability-based model for estimation of probability of HPV clearance was developed to fully incorporate information on HIV-1-related clinical data, such as CD4 counts, HIV-1 viral load (VL), highly active antiretroviral therapy (HAART), and risk factors (measured quarterly), and HPV infection status .3, p<0.05; and 28.03±1.47% at CD4 level 500 cells/mm3). HIV-1 VL was a significant predictor for clearance of low-risk HPV infections (p<0.05). HAART (with protease inhibitor) was significant predictor of probability of HPV16 clearance (p<0.05). HPV16/16-like and HPV18/18-like groups showed heterogeneity (p<0.05) in terms of how CD4 counts, HIV VL, and HAART affected probability of clearance of each HPV infection.Data from 266 HIV-1-positive and 134 at-risk HIV-1-negative adolescent females from the Reaching for Excellence in Adolescent Care and Health (REACH) cohort were used in this study. First, the associations were evaluated using the Cox proportional hazard model, and the variables that demonstrated significant effects on HPV clearance were included in transitional probability models. The new model established the efficacy of CD4 cell counts as a main clearance predictor for all type-specific HPV phylogenetic groups. The 3-month probability of HPV clearance in HIV-1-infected patients significantly increased with increasing CD4 counts for HPV16/16-like (p<0.001), HPV18/18-like (p<0.001), HPV56/56-like (p = 0.05), and low-risk HPV (p<0.001) phylogenetic groups, with the lowest probability found for HPV16/16-like infections (21.60±1.81% at CD4 level 200 cells/mmThis new model predicts the 3-month probability of HPV infection clearance based on CD4 cell counts and other HIV-1-related clinical measurements. The Reaching for Excellence in Adolescent Care and Health (REACH) dataset used in this study planned for measurement of HIV-1 status every 3 months and HPV status every 6 months. As in other cohorts, this study had missing and irregular visit measurements: only 82% of time intervals between measurements of HPV statuses were performed as scheduled , while other HPV tests were done at 3-, 9-, 12-, or other-month intervals. Analyzing these data with standard techniques would require multiple assumptions about the definition of HPV clearance and censoring time for each individual for time-dependent predictors; as a result, part of the dataset would not be utilized. We have developed transition probability-based models and applied an HPV/HIV co-infected cohort to estimate 3-month HPV clearance probabilities while maximizing all available data for both HIV and HPV in the estimations.Data on 266 HIV-1-positive and 134 high-risk HIV-1-negative adolescent females from the REACH cohort were analyzed. The REACH study design and methods of quarterly follow up with HIV-1 testing, immunophenotyping, HIV-1 RNA viral load (VL) and collection of biological specimens, demographic and behavioral factors, and other clinical data, along with incidence and prevalence of HPV infections, have been previously described All the participants of the study provided written informed consent in the parent study at the Adolescent Medicine HIV/AIDS Research Network for the REACH project, and the UAB Institutional Review Board approved this sub-study. The parent study and this sub-study conformed to the procedures for informed consent  approved by institutional review boards at all sponsoring organizations and to human-experimentation guidelines set forth by the United States Department of Health and Human Services.Cox proportional hazard regression was used to test which covariates have significant effects on probability of HPV clearance for each of four phylogenetic HPV groups. First, an univariable regression analysis was used for a wide spectrum of variables including demographic characteristics, clinical exams, antiretroviral therapy, behavioral factors, and coinfections  or varies from two to five, the likelihood function could be generalized by summing over all intermediate states. For example, for transitions with three missed HPV statuses see , the conTo test the different hypotheses regarding the possible effects of different potential predictors on probability of HPV clearance, a two-stage approach was designed. The two-stage approach complemented the advantages of methods used at each stage such as nonparametric estimates of hazard ratios in the Cox model and opportunities for evaluating 3-month probabilities for models (1) – (3). The comparison of the results obtained from both approaches allows for validating the properties of the new method. At the first stage, the effects were evaluated using the Cox proportional hazard model, and then the variables that demonstrated significant effects on HPV clearance in the Cox analysis were included in the logistic type models (substantively specified below). Note, the results can be obtained only under specific assumptions that are necessary to identify the times of HPV incidence and clearance/censoring. Moreover, the specific assumptions also are required to decide how to treat missing data on unknown HPV status. There is no consensus in literature about the choice of specific assumptions in these situations. Based on the published studies, our set of assumptions included: 1) time of HPV incidence was 90 days before the first exam with positive HPV status; 2) patient was removed when a time period between measurements exceeded 450 days; 3) when no time period between measurements exceeded 450 days, the clearance time was the time of the first (of the two subsequent) negative exams, ignoring any missing visits; 4) when the last exam was positive, the censoring time was the time of this exam plus 180 days; and 5) when the last exam was negative (after the positive), the censoring time was the time of this exam.The approach used at the second stage ) does not require these assumptions. All logistic-type models described the probability of a 3-month HPV clearance for different predictors using the base equation 2; their parameters were estimated using the likelihood described in equation 1 and generalized in the style of equation 3. The CD4 count parameter was selected to be tested by the model as a main predictor of HPV clearance in HIV-1-positive patients based on multiple studies M2 —includes the effect of the presence of HIV-1 infection on intercept of M3 —extends the M2 model by including the possible interference of the effects of HIV-1 seropositivity and CD4 count on HPV clearance M4 —in HIV-1-infected patients, it introduces CD4 count as a squared parameter in M5 —in HIV-1-positive patients, the effect of CD4 count is described as a piecewise-linear interpolation with an arbitrary set 3), low (200–499 cells/mm3), and normal range of CD4 counts.where M6 —includes the logarithm of HIV-1 VL (M7 —investigates the effects of HAART that include PIs on the intercept β (which describes the effects of predictor of clearance) and the clearance probability for a certain value of predictor, the latter can be used as a model parameter instead of β. Estimation of this model using the Proc NLP allows for evaluating its standard error.The nonlinear optimization techniques as implemented in PROC NLP in SAS 9.2  were used for the likelihood maximization in all these models to estimate a 3-month probability of HPV clearance. For the majority of calculations, the intercept and the effects of the predictors were chosen as model parameters and estimated with the standard errors (SEs). Because of the functional relation between parameter The main characteristics of studied patients are listed in Trichomonas vaginalis or Neisseria gonorrhea. CD4 count was a consistent predictor for clearance of HPV16/16-like, 18/18-like, and low-risk groups. For these HPV groups, a significantly higher probability of HPV clearance was at CD4 levels higher than 500 cells/mm3 (compared with those at <500 cells/mm3); the difference was especially pronounced for CD4 categorical cutout at 200 cells/mm3. For HIV-1 VL, significant effect on HPV clearance  was observed only without CD4 count as a second parameter . While being on HAART was a significant predictor of HPV16/16-like clearance only, being on HAART with PI significantly increased probability of clearance of 16/16-like, 18/18-like, and low-risk HPV infection. Also, being infected with Chlamydia trachomatis could be a positive predictor for low-risk HPV clearance.The results of the Cox proportional hazard analysis for HIV-1-infected adolescent females are shown in 3 changed for HPV16/16-like infection from 1.68 to 1.86, and from 2.53 to 3.07 (still remaining significant) when 365 days were used in assumptions #2 and #3 instead of 450 days. Another example is the changes of the HAART effect from 1.77 to 2.03 for HPV16/16-like infection, which remained highly significant while using 270 days instead of 450 in these assumptions.Sensitivity analysis was performed to check the stability of HRs estimated by the Cox model. In many cases, the HRs essentially shifted when assumptions changed: e.g., the effect of CD4 being >200 cells/mm11β  was 1.15 (0.27) for HPV16/16-like (p<0.001), 1.58 (0.36) for HPV18/18-like (p<0.001), 0.72 (0.38) for HPV56/56-like (p = 0.059), and 1.5 (0.41) for low-risk HPVs (p<0.001). HPV16/16-like infection was least likely to clear at low CD4 cell count (<200 cells/mm3) than other HPV groups (see 3 (see The results obtained from the basic model and from the models describing the effects of HIV-1 VL and HAART (with PIs) are shown in oups see : a probaoups see . The intee 3 see . The effee 3 see . A minoree 3 see .3, 26.63(3.19)% and 28.66(4.0)% at CD4 level 400 cells/mm3, and 34.12(3.49)% and 42.12(6.24)% at CD4 level 600 cells/mm3, for HPV16 and HPV18, respectively). The effect of HIV-1 VL was significant for clearance of HPV58 (16-like group) and HPV59 (18-like group), and the effect of HAART (with PIs) was significant for HPV16 clearance. In average, HPV67 had a higher probability to be cleared than the other 16/16-like HPV types: e.g., 64.11 (15.72)% vs. 25.76 (1.48)% at CD4 level 400 cells/mm3 (p<0.05). Likewise, HPV18 had the lowest probability to be cleared (18.16 (4.04)%), and HPV26 the highest (42.12 (8.39)%) than other 18/18-like HPV types at CD4 level 200 cells/mm3 compared to the group at average (27.40 (2.38)%), p<0.05.When two oncogenic groups of HPV infections—HPV16/16-like and HPV18/18-like—were analyzed for each HPV type separately, both groups showed heterogeneity in terms of how probability of type-specific HPV clearance was affected by CD4 counts, HIV VL, and HAART with PI see : while l3) using Kaplan-Meier curves and Cox proportional hazards models The longitudinal studies of HPV infection are important for determining the covariates and outcomes associated with HPV persistence, which leads to the development of cancer. Traditionally, the rate of HPV clearance are usually compared in HIV-1-positive patient subgroups based on baseline CD4 counts  on clearance of HPV16/16-like and HPV18/18-like infections were observed. However, while in the Cox model, being HIV-1-infected had a minor effect only on HPV56/56-like clearance, in the transitional probability model, this factor was a significant predictor of clearance of HPV16/16-like, HPV56/56-like, and low-risk HPVs.In immunodeficient patients, the mechanisms by which immune deficiency increases the risk of persistence of HPV infection are still poorly understood: some alterations in dendritic antigen-presenting cells, Langerhans cells, and macrophages function, as well as a deficient cytotoxic lymphocyte response to E6 and E7 proteins, might be the contributing factors 3) were considered, the multivariable regression showed high significance of HIV status as an independent predictor of HPV clearance event .HIV infection, independent of CD4 count, has also been suggested to be a predictor of persistence of HPV infection in HIV-1-positive women. This may imply an alternate mechanism besides CD4, e.g., via alteration of the cytokine response to HPV infection in the cervical mucus Currently, prognostic importance of high HIV-1 VL for HPV clearance is not clear, but it likely increases the risk of persistence of HPV infection at low CD4 cell counts C. trachomatis increasing probability of clearance of low-risk HPV falls in with the results from animal studies about potential role of interferon-γ as local “protector” against other  infections C. trachomatis could have effect on oncogenic HPV types The observation on The results of this study have several limitations. While the prevalence and incidence of HPV infections among HIV-1-positive adolescents in the REACH study is high In summary, our new model estimates a probability for HPV clearance of type-specific HPV groups at a 3-month period by coordinating uneven time scales of measurements on biannual HPV status and other quarterly HIV-1-related clinical data and risk factors.Table S1HPV type-specific characteristics obtained from the M1 – M7 models tested in the REACH cohort of HIV-1-positive and HIV-1-negative adolescent girls enrolled in 1996-2000.(DOC)Click here for additional data file.


EAV-HP insertion that promotes the expression of SLCO1B3 gene in the uterus (shell gland) of the oviduct in chicken. In this study, the genetic map location of the blue eggshell gene was refined by linkage analysis in an 2F chicken population, and four candidate genes within the refined interval were subsequently tested for their expression levels in the shell gland of the uterus from blue-shelled and non-blue-shelled hens. SLCO1B3 gene was found to be the only one expressed in the uterus of blue-shelled hens but not in that of non-blue-shelled hens. Results from a pyrosequencing analysis showed that only the allele of SLCO1B3 from blue-shelled chickens was expressed in the uterus of heterozygous hens (O*LC/O*N). SLCO1B3 gene belongs to the organic anion transporting polypeptide (OATP) family; and the OATPs, functioning as membrane transporters, have been reported for the transportation of amphipathic organic compounds, including bile salt in mammals. We subsequently resequenced the whole genomic region of SLCO1B3 and discovered an EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion was found closely associated with blue eggshell phenotype following complete Mendelian segregation. In situ hybridization also demonstrated that the blue eggshell is associated with ectopic expression of SLCO1B3 in shell glands of uterus. Our finding strongly suggests that the EAV-HP insertion is the causative mutation for the blue eggshell phenotype. The insertion was also found in another Chinese blue-shelled breed and an American blue-shelled breed. In addition, we found that the insertion site in the blue-shelled chickens from Araucana is different from that in Chinese breeds, which implied independent integration events in the blue-shelled chickens from the two continents, providing a parallel evolutionary example at the molecular level.The genetic determination of eggshell coloration has not been determined in birds. Here we report that the blue eggshell is caused by an  EAV-HP insertion in the 5′ flanking region of SLCO1B3. The EAV-HP insertion in chicken is a derived mutation in domestic chickens. The genetic determination of blue eggshell in other birds requires further investigation. We also found that the EAV-HP insertions in the chickens from China and America were separate integration events, which presents us with a parallel molecular evolution example driven by artificial selection.The eggshell color of birds is of wide interest, but the molecular basis remained unknown until our discovery, reported here. The blue eggshell is found not only in wild birds but also in domestic fowls. In this study, we identified that blue eggshell in chickens from different geographical regions is caused by a ∼4.2 kb  Avian eggshell coloration is the result of crypsis or mimetism and plays important roles in filtering solar radiation and strengthening the eggshell Brown and white are the two major eggshell colors in chickens. Protoporphyrin-IX, biliverdin, and biliverdin zinc chelate are the main pigments of the eggshell O) O have been performed with O affirmatively mapped to the short arm of chromosome 1 ev1 and P which was identified as SRY (sex determining region Y)-box 5 (SOX5) ev1, two single nucleotide polymorphisms (SNPs) (rs15297163 and rs15297165) were found to be highly associated with the blue eggshell phenotype O gene was defined in an 2F resource population O was further refined to the vicinity of ss244244378 by linkage and association analysis O to be fine mapped to two small regions  Blue eggshell color exhibits an autosomal dominant inheritance and eggs laid by homozygotes are a darker blue than those from heterozygotes . In 1933SLCO1B3 coding a membrane transporter OATP1B3 which is responsible for transporting amphipathic organic compounds including bile salt.In the present study we found that the blue eggshell phenotype in chickens is caused by a retrovirus insertion in the 5′ flanking region of 2F resource population segregating for the O gene to refine the location of chicken blue shell gene in the present study. Eight molecular markers in the candidate region were used for linkage analysis (O gene was mapped in the region between marker L4 and L5 which were the closest flanking markers to O with recombination rate being both 0.02 (LOD = 15.84)   . Fifteenssembly)  and no rSLCO1C1, SLCO1B3, LOC418189 and SLCO1A2) were found in the ∼120 kb interval by a MapView search (http://www.ncbi.nlm.nih.gov/mapview/)  and 4 brown-shelled (O*N/O*N) Dongxiang chickens, 6 blue-shelled (3 O*LC/O*LC and 3 O*LC/O*N) and 6 brown-shelled (O*N/O*N) Lushi chickens, and 24 chickens from three brown-shelled and one white-shelled breeds  by real-time PCR. All blue-shelled chickens expressed the gene in the uterus while non-blue-shelled chickens did not  . The utechickens , thus we did not . In addiividuals . Fluosceled hens . These rg.67334934 G>T) in exon 5 of SLCO1B3 gene by sequencing the coding region and the SNP presented complete association with the blue eggshell phenotype in the Dongxiang chicken by genotyping it in Dongxiang blue-shelled and brown-shelled chickens. With six heterozygous individuals produced by mating a homozygous Dongxiang blue-shelled male with a White Leghorn female, the allelic expression of SLCO1B3 gene was demonstrated by RT-PCR analysis and pyrosequencing. More than 95% of the transcripts expressed in the uterus originated from the T allele corresponding to the blue-shell allele  of SLCO1B3 were taken for genotyping in 353 chickens from 3 blue-shelled breeds  and 9 non-blue-shelled breeds. However, none of the SNPs was found to be in complete linkage disequilibrium with blue eggshell (We sequenced the genomic region of eggshell .SLCO1B3 by 5′ RACE in a blue-shelled (O*LC/O*LC) and a brown-shelled (O*N/O*N) Dongxiang chicken and an extra 24 bps were found at the beginning of 5′UTR end in blue-shelled Dongxiang chicken (O*LC/O*LC) and 5 brown-shelled (O*N/O*N) Dongxiang chickens. A ∼4.2 kb insertion adjacent to 5′UTR containing the extra 24 bps was found in the blue-shelled but not in the brown-shelled chickens. The sequence of the ∼4.2 kb insertion (GenBank accession number: JF837512) represents an incomplete retrovirus and shows 95.8% identity with the sequence of the avian EAV-HP retrovirus (EMBL accession number: AJ238124) gag, pol and env flanked by long terminal repeat (LTR), which are arranged in the order of 5′LTR-gag-pol-env-LTR3′ pol gene and part of gag and env  of  chicken . We furt and env . The ret and env  at Chr1:tivities .EAV-HP insertion was performed in 705 chickens from 12 worldwide breeds and the 2F resource population . Functionally, the OATPs serve as membrane transporters that mediate a wide range of sodium-independent transport of amphipathic organic compounds, such as some endobiotic compounds of bile salts, eicosanoids, sterioids, thyroid hormones and some xenobiotic compounds of anionic oligopeptides, organic dyes, toxins, and drugs SLCO1B3 codes a membrane transporter OATP1B3 which is considered a liver-specific transporter and is highly expressed in liver where it transports a wide range of substrates including bile salts SLCO1B3 is a plausible candidate gene responsible for changes in bilirubin levels in humans SLCO1B3 in uterus could enhance transportation of biliverdin to eggshell. In this study, we found that SLCO1B3 is exclusively expressed in shell gland of uterus of blue-shelled chickens rather than in that of brown- or white-shelled chickens, which supports that the gene plays a pivotal role for coloration of blue eggs.By linkage analysis, we fine mapped the cis-acting elements ERV) on hosts is extensive. It can unfavorably influence certain production traits, i.e. egg production, egg weight and body weight Drosophila melanogasterERV could alter splicing patterns of transcript to produce variants such as the recessive white mutation in the chickens c-myc in B cell and aromatase in the extrogonadal tissues by LTR EAV-HP inserted in 5′ flanking region of SLCO1B3 in reverse orientation. The LTR of EAV-HP could induce expression of the downstream gene (SLCO1B3) by its bidirectional promoter activity SLCO1B3 transcripts from the blue-shell allele containing the 24 bp EAV-HP partial sequence implies that the expression of SLCO1B3 in blue-shelled chickens is closely related to the insertion.Regulatory mutations demonstrate an important role for phenotypic diversity which may be explained by SLCO1B3 is not expressed in the uterus of blue-shelled and non-blue-shelled ducks, and the EAV-HP insertion was not found in the homologous region in duck .2F resource family was constructed by crossing homozygous blue-shelled Dongxiang (O*LC/O*LC) males, which has been verified by a test cross, and brown-shelled Dongxiang (O*N/O*N) females. All 1F hens laid blue shelled eggs and individual egg color phenotypes were recorded for all 146 2F hens.A three-generation O*LC/O*N) hens were produced by mating one homozygous Dongxiang blue-shelled (O*LC/O*LC) male and one White Leghorn (O*N/O*N) female, and the progeny were used for pyrosequencing analysis.Six heterozygous  2007–0023.DNA was extracted from blood using standard phenol/chloroform method. RNA was extracted from the liver and uterus. All the tissue samples for RNA isolation were collected at 3 to 5 hours before the next expected oviposition.2F resource family was used for linkage analysis. A set of 8 markers covering the region anchored by GWAS and SOX5, ev1 were used in the linkage analysis (http://genome.ucsc.edu/cgi-bin/hgGateway. Fifteen SNP markers (L9–L23) between L4 and L5 were added to narrow the mapping region. Primers and genotyping methods for all markers were present in The analysis . Marker SLCO1C1, SLCO1B3, LOC418189 and SLCO1A2) and housekeeping gene GAPDH using Primer 5.0 for RT-PCR , followed by synthesis of cDNA from 2 µg of RNA using M-MLV reverse transcriptase . Five pairs of primer were designed for the four candidate genes , heterozygous blue-shelled , and brown-shelled  Dongxiang chickens, homozygous blue-shelled , heterozygous blue-shelled , and brown-shelled  Lushi chickens, and 4 non-blue-shelled breeds including White Leghorn O*N/O*N, n = 6), Beijing You , Silkies , Tibetan chicken  by real-time PCR with a Bio-Rad CFX96 instrument . Samples were run in triplicates using RealMasterMix (SYBR Green I) . PCR amplifications were carried out in a 20 µL reaction volume containing 1.2 pmol of each primer, 9 µL of 2.5×working concentration RealMasterMix and 1 µL of cDNA in following cycling conditions: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 58°C for 10 s, 68°C for 10 s. GAPDH is used as endogenous reference gene to normalizing amounts of input cDNA, heterozygous blue-shelled Dongxiang (O*LC/O*N) group was designed as a calibrator. Fold change of every group related to the calibrator was calculated as described in Livak et al.Expression of g. 67334934 G>T) in exon 5 was amplified with forward (CATGTTGCGAGGAATTGGTG) and reverse (TTCCTTAGCAAAATCGTCAAGATA) primers. The relative expression of the two allele (O*LC or O*N) transcripts in heterzygotes was scored by analyzing the SNP (g. 67334934 G>T) by pyrosequencing. A pyro-seq primer (CGTCAAGATAAGAGATGCC) was used as the sequencing primer and all steps were performed according to manufacturer's protocol. All samples were analyzed in triplicates.Tissues (the uterus and liver) were collected from the six blue-eggshell heterozygotes. Total RNA was extracted from the uterus and liver with trizol . The RNA quality was controlled using NanoVue plus spectrophotometer . The first-strand cDNA synthesis used M-MLV  with the 18 hexamers. A fragment containing the SNP  for 24 hours at room temperature. Fixed uterus was embedded in irrigation solution PBS for six hours to eliminate 4% paraformaldehyde. Then slides were dehydrated in increasing concentrations of ethanol  followed by transparentizing in two clearing agents respectively of xylene for 15 minutes each. After transparentizing, the slides were pretreated by the mixture of xylene and low melting paraffin for 30 minutes then were directly transferred into pure melting paraffin (58°C) twice for 3 hours each.5′-AACTCTGGCTGAACGCATCT-3′ were labeled by 6-FAM and were synthesized from mRNA of SLCO1B3 (XM416418.2) by Boxing Bio-engineering Limited Company . The in situ hybridization was then carried out according to the instruction of the FISH Detection Kit . Imaging was performed using a fluorescence microscope equipped with vision software.The cDNA probe SLCO1B3 was resequenced using a panel of ten birds from 5 blue-shelled (O*LC/O*LC) and 5 brown-shelled (O*N/O*N) Dongxiang chickens. Seventeen primer pairs used to generate overlapping PCR amplicons ranging from approximately 800 bp to 2000 bp in size were listed in Taq DNA polymerase , and 50 ng genomic DNA. All purified PCR products were directly sequenced in both directions using the same primers. The sequences were assembled and analyzed for polymorphisms using the ChromasPro 1.5 or BLAST program in UCSC (http://genome.ucsc.edu/cgi-bin/hgBlat?command=start).Twenty-four kilobases fragment (GenBank accession No. JN020139) covering the whole SLCO1B3 gene, RACE experiments were performed on 2 µg total RNA extracted from the uterus of a homozygous blue-shelled (O*LC/O*LC) and a brown-shelled (O*N/O*N) Dongxiang chicken using 5′ and 3′-Full RACE Kit , according to the manufacturer's instructions. 5′ and 3′ UTR of SLCO1B3 gene transcripts were amplified by nested PCR with gene specific (2+ plus), 2.5 U of LA Taq , 20 mM of each dNTP and 1–2 µL of cDNA or 1st PCR product. RACE products were cloned to pMD-18 vector , and then sequenced in both directions.In order to analyze the 5′ and 3′ untranslated regions (UTR) of the specific  and adapspecific  for the 2+ plus), 2.5 U of LA Taq , 20 mM of each dNTP, 20 pmol of each primer and 50 ng genomic DNA. The PCR condition was as follow: 94°C for 3 min followed by 33 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 5 min, and a final extension at 72°C for 10 min. The PCR product was completely sequenced using the other three pairs of bridging primers Click here for additional data file.Figure S2SLCO1B3 in white-shelled and blue-shelled ducks by RT-PCR.Expression analysis for (TIF)Click here for additional data file.Figure S3EAV-HP insertion. (A) primers information for diagnostic genotyping test of EAV-HP insertion. (B) results for diagnostic genotyping test of EAV-HP insertion in blue-shelled and non-blue-shelled Dongxiang chickens. Single 425 bp band represents homozygous blue-shelled chickens, single 340 bp band corresponds to non-blue-shelled chickens, and two amplifications are heterozygous chickens.Diagnostic genotyping test of (TIF)Click here for additional data file.Table S1Information of markers used in linkage analysis.(DOCX)Click here for additional data file.Table S2Two point linkage analysis with markers in L4–L5 interval.(DOCX)Click here for additional data file.Table S3Haplotype frequency analysis of all the breeds or populations.(DOCX)Click here for additional data file.Table S4Primers used in expression analysis, 5′ and 3′ RACE of candidate genes.(DOCX)Click here for additional data file.Table S5SLCO1B3 and EAV-HP.Primer sequences used in resequencing of (DOCX)Click here for additional data file.Table S6SLCO1B3 gene.Information of SNPs in (DOCX)Click here for additional data file.


Objectives. In Belgium, very few studies have focused on cervical high-risk human papillomaviruses (hrHPV) prevalence and the relationship between HPV and cervical cytological abnormalities. The aim of this study was to investigate hrHPV prevalence and its relationship with cytological screening and histological results in the French-speaking community in Belgium . Methods. A total of 58,265 liquid-based cytology tests were performed during this period. All cases of ASC-US, ASC-H, LSIL, and HSIL were tested by Hybrid Capture 2 for hrHPV screening. Results. The prevalence of cytological abnormalities was 3.1% for ASC-US, 0.3% for ASC-H, 1.5% for LSIL, and 0.3% for HSIL. The frequency of hrHPV infection was 47% in ASC-US, 90% in ASC-H, 86% in LSIL, and 98.4% in HSIL. CIN 2+ lesions were found in 12.2% of smears with an ASC-US result, in 54% of smears with an ASC-H result, in 12.5% of smears with a LSIL result, and in 89.3% of smears with a HSIL result. The ASC/SIL ratio was 1.9%. Conclusions. This study provides a good representation of cytological abnormalities and HPV status in patients living in Belgium's French-speaking community. The prevalence in our study was similar to that derived from meta-analyses of European studies. Our ASC/SIL ratio was 1.9%, being within the lower and upper limits proposed in the literature, which tends to prove the good quality diagnosis of cervical smears in our laboratory. Invasive cervical cancer (ICC) is the second most common female cancer worldwide and the third most common in Belgium , 2. In BThe study protocol received approval from Erasme Hospital's institutional ethics and research review boards. A computer search was carried out from the Department of Pathology's archives from January 2007 to January 2012 to identify all women with cervical cytology. A total of 58,265 liquid-based cytology (LBC) tests were performed during this period. All smears were carried out in the Erasme hospital by gynecologists on women living either in Wallonia or in Brussels. For cervical cytological testing, a clinician used an Ayres spatula to collect cells from the transformation zone and a cytobrush to collect cells from the endocervical canal. Both the spatula and brush were rinsed directly in vials that contained 20 mL of cytological fluid  for processing and production of a thin-layer cytological slide . Of these, 3,090 cases were ASC-US, ASC-H, LSIL, or HSIL lesions. Patients atypical glandular cells and adenocarcinoma on Pap smear were not included in this study. A colposcopy was carried out as soon as the ASC-US, ASC-H, LSIL, and HSIL lesions were identified from the smears. A biopsy was carried out during colposcopy on fresh, clinically suspect areas, on leukoplakic areas (acetic acid-positive areas), and iodine-negative areas (after application of Lugol's iodine). 2011 International Federation of Cervical Pathology and Colposcopy Colposcopic Terminology of the Cervix  was used to coloscopic classification. All ASC-US, ASC-H, LSIL, and HSIL smears and all biopsies were reviewed by two pathologists . The reports were based on WHO criteria for classification of histological diagnosis of CIN. All ASC-US, ASC-H, LSIL, and HSIL cases were tested by Hybrid Capture 2  for research of hrHPV (hc2). Normal cytology cases were not tested by hc2. A quantity of 4 mL of samples in PreservCyt was processed in the sample conversion kit  and tested with hc2 according to the manufacturer's recommendations, using the specific HPV RNA probe cocktail for carcinogenic HR-HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Presence or absence of HPV DNA in the specimen was defined according to its strength in relative light units (RLU) compared to 1 pg/mL HPV16 DNA-positive control (CO). The sample was considered positive when the ratio of RLU/CO was ≥1. The RLU/CO ratios also provided an estimate of the amount of HPV DNA in the specimens . The ASC/SIL ratio was obtained by dividing the sum of all ASC cases by the sum of all SIL cases. SIL for this purpose included low-grade SIL, high-grade SIL, and carcinoma. Atypical glandular cells and adenocarcinoma were not included.The prevalence of cytological abnormalities in the population with microscopically interpretable samples was 3.1% for ASC-US, 0.3% for ASC-H, 1.5% for LSIL, and 0.3% for HSIL. The frequency of hrHPV infection was 47% in ASC-US, 90% in ASC-H, 86% in LSIL, and 98.4% in HSIL . ColposcOverall, 89.8% of patients with CIN 1, 94.9% of patients with CIN 2, 100% of patients with CIN 3, and 100% of patients with invasive squamous cell carcinoma were hrHPV positive. As the main objective of screening was to highlight CIN 2+ lesions in the general population, we calculated the percentage of CIN 2+ lesions in relation to cytological lesions. In ASC-US, ASC-H, LSIL, and HSIL HPV positive lesions, the percentage of CIN 2+ was 15.4%, 55%, 18.2%, and 98.6%, respectively.In ASC-US HPV negative lesions, the percentage of CIN 2+ was 2.4%. In HPV positive lesions, the percentage of CIN 2+ was 55%. Finally, the ASC/SIL ratio in our laboratory was 1.9%.We examined the prevalence of hrHPV infection in squamous intraepithelial lesions of the cervix in French-speaking Belgian women , correlating it with cytological data. These data may assist the public health authorities in planning prophylactic and therapeutic strategies to prevent cervical cancer. One goal of the study was to assess the prevalence of HPV infection in our population, stratified by cytological abnormalities. While some of our patients were from Wallonia, the majority of patients were from the Brussels region, which also had the highest mortality rates of cervical cancer.The cytological diagnosis of ASC-US is undesirable but unfortunately inevitable, and results from the morphological variability of squamous cells in different physiological and pathological conditions . AlthougSignificance of the ASC/SIL Ratio. To avoid potential misuse of the ASC interpretation, it is recommended that the frequency of ASC interpretation should be no greater than 5% of the patient population [pulation . In our pulation , 27. Thepulation . An ASC/pulation . We agreThis study gives a good representation of cervical abnormalities and HPV status of women living in Belgium's French-speaking community . The results of our study are very similar to those of other studies of the same type, particularly in France, our neighboring country. It allows us to situate Belgium in the European Union with regard to the distribution of cervical lesions. We hope that it will also allow other laboratories, in Belgium and elsewhere, to compare their data with ours and provide useful information to public authorities regarding this disease.


Human papillomavirus (HPV) infections are related to the genesis of various benign lesions and some malignant tumors, but no clear relationship has been identified so far between the subtypes of HPV and skin tag. The present case-control study was designed to detect the existence of low risk and high risk HPV types in lesions of 50 patients with skin tag (case group) and normal skin around the melanocytic nevus of 30 patients (control group), using PCR. All of the samples were negative for HPV subtypes, except two samples in control group which were positive for high risk HPV. There was no significant relationship between the HPV subtypes and skin tag.There is no association between skin tag and low risk and high risk human papillomaviruses.  Skin tag is a common disease in the societies which has unknown causes. It usually involves flexural area such as axilla, groin, and neck and in rare cases genital and anus area. However, the most common site of involvement is neck .Skin tag is more common in elderly people, especially in the menopause women and it could be accompanied with seborrheic keratosis, melanocytic nevus and neurofibroma. Histological findings show a normal or hyperplastic epidermis overlying a fibrovascular connective tissue core. However, diagnosis is made mainly clinical; and histology examination is only for confirmation. Different treatment protocols are available for skin tag such as cryotherapy (using liquid nitrogen -196 centigrade degree), cutter, and surgery , 3.HPV or human papillomavirus is a 50-55 nm diameters DNA virus with which involves squamous epithelium and causes cell proliferation. HPV is contagious and transmission is possible. Infected person will have skin problems after an incubation period. Study results have shown a relationship between HPV and skin tag, which will imply the viral cause for skin tag , 5. TherTM Genomic DNA Isolation Kit from Tissue) according to the manufacturer’s instructions .This study was performed on skin tag of patients with clinical and pathological diagnosis of skin tag in axilla, groin and neck areas. Fifty patients with skin tag and thirty specimens from normal skin near melanocitic nevus were prospectively included in the present study. After reconfirmation of histological diagnosis, the specimens were cut and chapped. DNA was then extracted from these tissue samples using commercial DNA isolation kit from tissue  300 bp target as described before .. Then the presence of high risk HPV was also investigated by PCR amplification using general-purpose HPV primers  was tested by PCR Kit  which could detect several type of low risk HPV  according to the manufacturer’s instructionsP-value was <0.05.5'-GAAAAATAAACTGTAAATCATATTC-3') which amplify conserved sequences in the HPV-L1 region as previously described -9. The aP= 0298). Mann-Whitney test showed no significant difference regarding the age of case and control group (P= 0.14). High risk HPV was detected in only two samples which belonged to the control group (P= 0.138) (P= 0.628). All the samples in both groups were negative for low risk HPV  neck, thirteen samples (26%) axilla and eleven samples (22%) groin. In control group twenty samples (66.7%) were taken from face, six samples (20%) from trunk and four samples (13.3%) from arm. Chi-Square test showed no significant difference regarding the sex of the case and control group  . Moreoverisk HPV . SimilarA high number of HPV infection exists in tropical parts of world, therefore there is the possibility that continuous contact with sun ray reinforces HPV infection. Ultra violet light has many effects on dermis immunity including reducing the number of Langerhas cells in epidermis and decreasing their antigen production ability, increasing the production of IL-10 and prostaglandin E2 by keratonocytes, and increasing the serum level of IL-4 . It may Skin tag or fibroepitheial polyps are developed in areas of skin which are prone to rubbing. These areas are probably entrance routes of virus. HPV DNA and mechanical rubbing are cofactors and have role in pathogenesis of skin tag . In the in 2 samples of control group, concluding that there is no significant relation between HPV and skin tag. The negative results of HPV PCR could be due to one of the following reasons:The lack of any link between HPV and skin tag.Technical errorsSince in the present study, sensitive and specific methods were used to detect the tissue HPVs, therefore the negative results cannot be due to technical errors because: PCR method used in this study is sensitive. A positive control in all HPV PCRs was used and it showed the expected fragment size using DNA size marker. Also, the negative control was negative by the HPV PCR.The quality of each DNA sample was verified by amplification of portions of human β-actin gene. Successful amplification of the β-actin gene fragment indicated the integrity of the DNA samples and the absence of PCR inhibitors -8. et al, HPV DNA was positive in 77% of skin tags and had significant difference with samples taken from around skin tag (53% positive) and control group (20% positive). Therefore, they concluded that HPV has an important role on the pathogenesis and progress of skin tag  . in DianIn the present study PCR was done after pathology confirmation and definite diagnosis of skin tag, but in the reported studies pathology confirmation have not been done and diagnosis was made only on the basis of clinical appearance. Control group could also be a reason for incompliance. In the present study, healthy skin around the melanocitic nevus was used as control samples, while in other studies the healthy skin around skin tag was considered as control group. Since HPV infection could be subclinical, it could justify the positive results in our control group. Considering the fact that HPV proliferates in keratinicytes of basal layer of dermis, and is transmitted to cells of upper epiderm layer, it is usually associated with the epidermal proliferations such as wart, while skin tag is a benign connective tissue stromal tumor . This faThere was no significant relationship between low risk/high risk virus infection, age and gender of patients, which is in agreement with reported studies results.Based on the results of the present study, no precautions are necessary when contacting skin tags like those considered when contacting wart or premalignant diseases. Considering the fact that high risk HPV was positive around melanocitic nevus, the researchers suggest a further study be conducted on the relation between melanocitic nevus and high risk HPV. According this study results, there is no association between skin tag and low risk and high risk human papillomaviruses.


Accumulated evidence from epidemiological studies and more recently from randomized controlled trials suggests that male circumcision (MC) may substantially protect against genital HPV infection in men. The purpose of this study was to assess the association between MC and genital HPV infection in men in a large multinational study.A total of 4072 healthy men ages 18–70 years were enrolled in a study conducted in Brazil, Mexico, and the United States. Enrollment samples combining exfoliated cells from the coronal sulcus, glans penis, shaft, and scrotum were analyzed for the presence and genotyping of HPV DNA by PCR and linear array methods. Prevalence ratios (PR) were used to estimate associations between MC and HPV detection adjusting for potential confounders.MC was not associated with overall prevalence of any HPV, oncogenic HPV types or unclassified HPV types. However, MC was negatively associated with non-oncogenic HPV infections , in particular for HPV types 11, 40, 61, 71, and 81. HPV 16, 51, 62, and 84 were the most frequently identified genotypes regardless of MC status.This study shows no overall association between MC and genital HPV infections in men, except for certain non-oncogenic HPV types for which a weak association was found. However, the lack of association with MC might be due to the lack of anatomic site specific HPV data, for example the glans penis, the area expected to be most likely protected by MC. HIM Study)[HIM Study.Genital human papillomavirus (HPV) infection is one of the most common sexually transmitted infections (STI) in the world. GenitalM Study). In the HIM Study is a multinational longitudinal study of HPV in men. Between June 2005 and September 2009, 4072 men who completed both the pre-enrollment and the enrollment visits formed the study population for the present analysis. Details of the cohort have been previously described[The escribed,22. In bAt the enrollment visit, men completed an 88-item Computer-Assisted Self-Interview (CASI) that has been demonstrated to elicit reliable responses. The CASThree different saline pre-wetted Dacron  swabs were obtained from the coronal sulcus/glans penis, penile shaft, and scrotum, and placed in 450 μl of Specimen Transportation Medium, and combined into one sample before DNA extraction. Among uncircumcised men, the foreskin was sampled at the time of collection of the coronal sulcus/glans penis sample. We have previously shown the validity and high sampling reproducibility of these three anatomical sites in the assessment of HPV DNA status,25. All DNA was extracted with the Media Kit  by a robotic system according to the manufacturer’s instructions. DNA was stored at 4°C until use. HPV testing of the combined DNA extract was undertaken by use of PCR for amplification of a fragment of the HPV L1 gene. Specimens were tested for the presence of HPV by amplification of 30 ng of DNA with the PGMY09/11 L1 consensus primer system. HPV genBefore genotyping, amplification products were run on 2% agarose gels to visualise a 450 bp band corresponding to HPV amplification for identification of samples that might have an HPV type other than the 37 types analyzed in the genotyping assay. Samples in which HPV was amplified on PCR, but did not hybridise with a specific HPV type during genotyping were categorized as unclassified infections. The 13 HPV types that were classified as oncogenic were: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. The non-oncogenic HPV types were: 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 66, 67, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6108. SamplesThe present analysis included four classifications of HPV detection. A participant was considered positive for “Any HPV type” if he tested HPV positive by PCR or by genotyping for any HPV genotype. The category of “Oncogenic HPV” included men who were positive for only oncogenic genotypes as well as those who were positive for both oncogenic and non-oncogenic types. The “Non-oncogenic HPV” category consisted of those participants who tested positive for non-oncogenic HPV genotypes only. The “Unclassified HPV infections” included men with samples that tested positive for HPV by PCR but negative for all of the 37 genotypes tested.Differences in the distribution of socio-demographic characteristics, tobacco consumption and sexual behavior characteristics by circumcision status were compared using Pearson’s chi-square test. Prevalence ratios (PRs) and 95% confidence intervals (CIs) were estimated using univariate and multivariable modified Poisson regression with robust variance,30. SepaTo explore whether sexual behavior modified the association between MC and HPV detection, we stratified the analysis by lifetime number of female sexual partners, and formally tested the interaction between lifetime number of female sexual partners and MC by including in the model both the interaction and the main-effect terms. Statistical analyses were conducted using SAS software, version 9.2  and R 2.13.0 (R Development Core Team). All statistical tests were two sided with a significance threshold of 0.05.Socio-demographic characteristics of the 3969 participants by circumcision status are summarized in Table The univariate and multivariable associations of MC with each of the four pre-defined individual outcomes  are presented in Table As shown in Table Table HIM Study, anti-HPV 16 serum antibody status at enrollment was not associated with the risk of a subsequent genital HPV 16 infection[Our study found that MC was not associated with overall prevalence of genital HPV for any HPV, oncogenic HPV infections, or unclassified HPV infections. However, MC was negatively associated with non-oncogenic HPV infections. After adjustment for potential confounders and stratification by lifetime number of female sexual partners, the protective association between MC and non-oncogenic HPV detection was particularly evident in men reporting 1 to 4 lifetime female sexual partners. The lack of an effect of MC among men with a greater number of lifetime sexual partners might be explained by immunity to HPV acquired over time by men who were repeatedly exposed to HPV infections. Other snfection.A preliminary analysis of the HIM study that included the first 1158 men who completed both the pre-enrollment and the enrollment visits, showed that MC was negatively associated with the detection of both non-oncogenic and oncogenic HPV types. HoweverThe study of the effects of MC on genital HPV infections poses inherent complexities. Inconsistency of results across studies may be due to the genital site selected for HPV sampling or by the procedure used to collect exfoliated cells. Most studies sampled only a single anatomical site or a pooled combination of a few sites for the detection of HPV. It has been suggested that sampling of the penile shaft, the coronal sulcus/glans penis, and the scrotum is necessary for optimal overall genital HPV detection. In a USTaken together, results from these studies consistently show that the protective effect of MC may be restricted to infections occurring at the distal part of the penis, including the urethra, the coronal sulcus, and the glans. Since MC is limited to the removal of the foreskin, it seems unlikely that MC has any effect on infections occurring in the rest of the penis or the scrotum. Indeed the use in our study of an aggregate outcome that combined infections in more than one anatomical site greatly limited our ability to identify a true protective effect in specific anatomical subsites, possibly resulting in an overall underestimation of a MC protective effect.It is interesting to note that this anatomical site-specific pattern of HPV infections is also observed for genital warts in at least two clinical studies,36. One HIM Study showed that the median time to clearance was not substantially different between non-oncogenic and oncogenic HPV types[It is unclear why MC would be associated with a reduced detection of non-oncogenic HPV types but not of oncogenic HPV types. One possible explanation is that MC infers a weak protective effect capable of preventing only less virulent HPV infections such as those caused by non-oncogenic types. Indeed, some studies provide evidence that non-oncogenic HPV types have a shorter persistence than oncogenic HPV types,38,39. HPV types.In this study, the distribution of the most common HPV types was not different by MC status. However, for less common types, such as HPV types 11, 40, 61, 71 and 81, we did find a lower prevalence in circumcised men than in uncircumcised men. Four studies have reported on the type-specific HPV distribution by MC status,32,34,41The mechanism by which circumcision might protect against HPV infection remains unclear. Removal of the foreskin could minimize the chance of acquisition of new infections or could result in an increased clearance of preexisting infections,42. AddiIn conclusion, this study shows no association between MC and an overall prevalence of genital HPV infections in men but MC is associated with a reduced prevalence of genital non-oncogenic HPV types. However, the lack of association with MC might be due to the analysis could not asses specific associations in the glans penis, the area expected to be most likely protected by removal of the foreskin.HPV: Human papillomavirus; STI: Sexually transmitted infections; MC: Male circumcision; RCT: Randomized controlled trials; HIM Study: HPV in men study; CASI: Computer-Assisted Self-Interview; PRs: Prevalence ratios; CIs: Confidence intervals.G.A. My research unit is involved in vaccine trials organized by GlaxoSmithKline and Merck/Sanofi Pasteur MSD. Travel fund to conferences/symposia/meetings are occasionally granted by either GlaxoSmithKline, Sanofi Pasteur MSD or Qiagen. A.R.G. and L.L.V. are on the speakers’ bureau for Merck and are members of its advisory board. A.G.N. has received research funding from Merck & Co. X.C. Research Grants ; Speakers Bureau ; Steering Committee . F.X.B. Advisory Board ; Speakers Bureau (GlaxoSmithKline); Research Grants . X.C. and F.X.B. My research unit is involved in vaccine trials organized by GlaxoSmithKline and Merck/Sanofi Pasteur MSD. Travel fund to conferences/symposia/meetings and honorarium are occasionally granted by either GlaxoSmithKline, Merck, Sanofi Pasteur MSD, MTM or Qiagen. All other authors declare that they have no competing interests.ARG, LLV, and EL were the principal investigators, conceived the study, wrote the protocols, assured funding, identified clinical investigators and study personnel, and made critical revision of the manuscript. GA was responsible for conduct analysis, interpretation of data, and drafting and revision of the manuscript. WF, and MRP made the statistical analyses, and made substantial comments to the manuscript. MA trained and supervised study clinical staff, and made substantial contributions to the manuscript. DS wrote the HPV assays protocols, performed the HPV DNA detection and genotyping of the samples validating the techniques with other referent HPV laboratories and made substantial comments to the manuscript. JS and MQ coordinated the field work in terms of study implementation and data collection, and made substantial comments to the manuscript. AGN, BL, XC, and FXB participated in the interpretation of data, and critical revision of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/18/prepub


The association between human papillomavirus (HPV) infection and non-melanoma skin cancers (NMSCs) such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) is not yet fully understood. We analysed the prevalence and spectrum of cutaneous beta-HPV types and mucosal/genital HPV types in paired biopsies  obtained from 50 BCC immunocompetent patients. A small group of SCC patients (n=9) was also included. We also evaluated some previously postulated risk factors for HPV infection in NMSC patients.All biopsies were negative for mucosal/genital HPV types. Overall, beta-HPV DNA was detected more often in SCC compared to BCC patients . The frequency of infection increased with the patient’s age [OR=4.88 (95% CI 1.29-18.39)]. There was no significant correlation between beta-HPV positivity and sex, skin type and UV exposure. The prevalence of beta-HPV species 1 types was significantly higher than those belonging to other beta-HPV species in biopsies from BCC (p=0.022) but not from SCC subjects (p=0.091). There was no significant difference in the overall prevalence of beta-HPV infection and the number of viral types between tumour lesions and perilesional skin. BCC samples were significantly more likely to be infected with beta-HPV species 1 types compared to perilesional skin (p=0.036) and showed a higher frequency of mixed infections (p=0.028).These findings demonstrate that beta-HPV types belonging to species 1 are the most common HPV types detected in the skin of BCC patients. Moreover beta-1-HPV types and mixed infections are significantly more frequent in tumour samples than in healthy perilesional skin. Our results suggest that beta-1-HPVs as well as co-infection with more than one viral type could be important in NMSC and in particular in BCC.Further studies aimed to compare the biological activity of viral types in tumours and in healthy skin  are necessary to understand the role of HPV infection in skin cancer. Human papillomaviruses (HPVs) are highly prevalent in the human population -3 and arThe oncogenic role of some mucosal HPVs has been demonstrated for a number of epithelial cancers of the anogenital and upper respiratory tracts  while thThe majority of studies on the role of HPV in NMSCs, so far, have focused on squamous cell carcinomas whereas there are a limited number of reports regarding HPV infection in basal cell carcinomas. BCC is the most common skin cancer and, in recent years, its incidence has increased worldwide ,19. The In the present study we analysed paired biopsies (tumour and adjacent skin) obtained from 50 BCC immunocompetent patients. A small group of SCC patients (n=9) was also included. The comparison between HPV infection pattern in tumour and in perilesional skin obtained from the same subject, and thus characterized by the same variables that influence skin cancer risk, seems to be a very good approach for assessing a possible correlation between HPV and tumours. We also evaluated some previously postulated risk factors for HPV infection in NMSC patients.Overall beta-HPV infection was detected in 69 out of 118 (58%) samples. The infection was found to be more prevalent in samples from SCC patients [14 out of 18 (78%)] in comparison to BCC patients [55 out of 100 (55%)], however this difference did not reach statistical significance (p=0.058) (Table Forty-four out of 59 (75%) patients harboured beta-HPV DNA in tumour and/or in perilesional skin. Thirty-five out of 50 (70%) BCC patients and 9 out of 9 (100%) SCC patients resulted positive at tumour and/or at perilesional skin. There was no statistically significant difference with regard to HPV prevalence between SCC and BCC patients (p=0.056).In the analysed samples we detected beta-HPV types belonging to species 1 , species 2 , species 3 (HPV49), species 4 (HPV92), and some previously described putative unclassified types . The most prevalent types , all belonging to the beta-HPV species 1, were present in 18%, 10%, 15%, and 10% of total samples respectively. HPV types 5, 8 and 93 were found in both tested groups whereas HPV24 was present in samples from BCC patients only.Viral types within beta-HPV species 1 were significantly more common, found in 60 out of 118 (51%) samples, in comparison to those belonging to other beta-HPV species which were detected in 40 out of 118 (34%) biopsies (p=0.006) Table . Among bForty-four out of 118 (37%) samples harboured more than one genotype. There was no statistically significant difference with regard to the prevalence of mixed infections between samples from BCC and SCC patients   (Table Beta-HPV DNA was detected more often in tumours [39 out of 59 (66%)] than in perilesional skin [30 out of 59 51%)], however this difference did not reach statistical significance (p=0.067). Thirty-one out of 50 (62%) BCC samples and 24 out of 50 (48%) corresponding perilesional skin biopsies tested beta-HPV positive and among samples obtained from SCC patients 8 out of 9 (89%) tumours and 6 out of 9 (67%) paired perilesional skin resulted beta-HPV positive  were HPV negative, 19 (32%) patients were positive only at tumour or at perilesional skin while 25 (42%) had beta-HPV DNA in both biopsies. In 2 patients the viral types in tumours were different from those detected in perilesional skin, 3 patients had identical HPV types in both biopsies and in 20 patients one or more identical HPV types were detected in tumours and in corresponding healthy skin.Among the most prevalent types, HPV5, 8 and 93 were uniformly distributed between tumours and perilesional skin samples while HPV24 was twice as frequent in BCC than in corresponding perilesional skin. No particular beta-HPV types were significantly associated tumours or with healthy tissue Figure .Thirty-six out of 59 (61%) tumours tested positive for beta-1-HPV types compared with 23 out of 59 (39%) tumours positive for other beta-HPV types. This difference was statistically significant (p=0.013) (Table Infections with multiple HPV types was found to be significantly more common in tumours [28 out of 59 (47%)] than in perilesional skin [16 out of 59 (27%)] (p=0.018). Mixed infections were detected in 22 out of 50 (44%) BCC samples compared with 12 out of 50 (24%) of corresponding perilesional skin biopsies. This difference was statistically significant (p=0.028) . The comparison of HPV5 DNA level in tumour and perilesional skin obtained from 3 patients revealed a 20–50 times higher viral load in tumours than in perilesional skin. On the other hand, HPV24 DNA level was 20–60 times higher in perilesional skin than in tumour samples obtained from 4 patients  was observed in the SCC group, but this did not reach statistical significance. All samples were negative for the high risk and low risk HPV DNA screening and genotyping test able to detect 28 main mucosal/genital HPV types. This could underscore the propensity of certain HPV types to infect and replicate preferentially in either mucosal or cutaneous epithelium and is in line with some previous studies, where the mucosal types were not found at all or only rarely either in tumour or normal skin samples ,23,24. OWe observed that increasing age was associated with a higher prevalence of beta-HPV infection in our studied population. This observation is in line with some data reported in the literature  and may We found no significant differences in HPV prevalence between tumours and perilesional skin samples. However, viral DNA was detected more often in both BCC and SCC than in corresponding perilesional skin biopsies. These findings are in line with some previous studies in which the prevalence of HPV infection in both kinds of tumours was slightly higher than in adjacent perilesional ,26. The We found that beta-1-HPV types were significantly more common than types belonging to other beta-HPV species (p=0.006) in our studied population. This was also observed in the case of samples from BCC (p=0.022) but not from SCC patients. The most prevalent types  identified in samples from BCC patients accounted for 41% (54 out of 133) of total infections. HPV24 was never detected in SCC samples and the most prevalent types  represented 26% (9 out of 34) total infections. Some literature data indicate that beta-HPV species 2 is more likely to be identified in SCC than in perilesional healthy skin ,26. In aThe prevalence of mixed infections was found to be high since 44 out of 69 (64%) positive samples harboured more than one beta-HPV type. Multiple infections were more common in samples from SCC patients (56%) compared to samples from BCC patients (34%) although this difference was not statistically significant. Mixed infections were significantly higher in tumours than in perilesional skin (p=0.018) of the overall studied population and of BCC patients alone (p=0.028).Twenty-five out of 44 beta-HPV positive patients (57%) harboured HPV infection in both biopsies whereas 19 (43%) patients had only positive tumour or perilesional samples.There was no significant difference in the number of viral types between tumours and adjacent skin. We observed a high degree of overlap between the beta-HPV types in paired biopsies as the viral types found in tumours were completely different from those present in perilesional skin from the same patients in only in 2 out of 25 cases. Our results differ somewhat from the findings of a study of HPV prevalence and spectrum in SCC patients carried out by Plasmeijer and collaborators . In this. It cannot be excluded that different beta-HPVs may exhibit different rates of replication and/or different expression patterns in different types of lesions such as tumour and normal perilesional skin. A comparison of HPV DNA loads in NMSC and in corresponding adjacent skin described in previous studies [A number of studies report the prevalence and distribution of HPV types in NMSCs, in benign lesions and in perilesional skin but there are limited data regarding the rate of replication and expression of different beta-HPVs in different kinds of lesions studies ,29 did nINK4a and AKT/P13K pathway in BCC is often associated with the presence of beta-HPV species 2 suggesting, that in a subtype of BCC these viruses may exert a role in the carcinogenesis or in other, still undefined, biological property of these tumours [Further studies need to be performed to investigate the biological activity of viral types detected in paired biopsies. It can be useful to determinate viral load, viral gene expression and the interference of HPV infection with cellular gene expression in tumours and in healthy skin to understand the role of HPV infection in skin cancers. A recent study has demonstrated that the up-regulation of p16 tumours .In summary, the overall prevalence of beta-HPV was higher in SCC than in BCC patients. No mucosal/genital HPV types were detected. Beta-HPV types belonging to species 1 represented the most common infection detectable in NMSC, and in particular, in the BCC population. The most common types detected in samples from the BCC patients  accounted for 41% of total infections. The tumour lesions did not seem to differ significantly from perilesional skin with regard to the overall prevalence of beta-HPV infection and the number of viral types detected in paired biopsies. However beta-HPV types from species 1 were significantly more frequent in BCC samples which were also characterized by a higher frequency of multiple infections. Our results suggest that beta-1-HPVs as well as co-infection with more than one viral type could be important in NMSC and in particular in BCC.All procedures followed were approved by appropriate Ethics Committee related to our institutions . Research was carried out in accordance with Helsinki Declaration. All patients were recruited after written, informed, consent. Fifty-nine patients, 25 female and 34 male, referred to the Dermatology Clinic at the University of Florence for the removal of suspected NMSCs were studied. All patients were immunocompetent . None of the patients was receiving immunosuppressive or immunostimulating drug therapy.later RNA Stabilization Reagent (Qiagen) and conserved at −20°C. The remaining tumour was excised and sent for histopathological diagnosis.Punch biopsies were collected from both the tumour and perilesional skin  from each patient. The samples were immersed in RNAOverall, 50 BCC patients and 9 SCC patients were included in this study. The samples were classified on the basis of skin type . The leThe DNA was extracted from each sample using All Prep DNA/RNA Mini Kit (Qiagen) and stored at −70°C. The quality of isolated DNA was checked by PCR amplification of the human beta-globin gene .http://blas.ncbi.nlm.nih.gov//Blast.cgi).To maximize the reliability and sensitivity of analysis, the samples were tested by two different methods. First the two-step version of the nested FAP-PCR with degenerated primers was performed. This PCR yielded a 235-base pair fragment corresponding to part of an L1 gene and is able to amplify more than 20 known HPV types, mainly beta-HPVs . The PCRSubsequently HPV detection and genotyping were carried out using the RHA Kit Skin (beta) HPV . This method was designed for the amplification and typing of 25 beta-HPV types. Briefly, a broad spectrum PCR using biotinylated consensus primers was used to amplify a 117-base pair region from the E1 gene. The biotinylated amplicons were subsequently hybridized with specific oligonucleotide probes immobilized as parallel lines on membrane strips. The bands were then analyzed against a reference sheet.The necessary precautions to avoid contamination between samples were observed during all steps of sample handling and, in each PCR run, water samples and known HPV negative specimens were included as negative controls.All samples were analysed by INNO-LiPA HPV Genotyping Extra  according to the manufacturer’s instructions. INNO-LiPA HPV Genotyping Extra is a line probe assay, based on the reverse hybridization principle, designed for the identification of 28 different genotypes of the human papillomavirus by detection of specific sequences in the L1 region of the HPV genome. The assay covers all currently known high-risk HPV genotypes and probable high-risk HPV genotypes  as well as a number of low-risk HPV genotypes  and some additional types .Stata software was used to analyse the data by logistic regression analysis. Fisher’s exact test was used for analysis of the differences in distribution of HPV prevalence among groups. A P<0.05 was considered to be statistically significant.Type-specific real time PCR protocols described previously  were useBCC: Basal cell carcinoma; HPV: Human papillomavirus; NMSC: Non-melanoma skin cancer; SCC: Squamous cell carcinoma; AK: Actinic keratosis.The authors declare that they have no competing interests.KZ conceived and planned the study and wrote the manuscript, ER and FP carried out the sample extraction, amplification and typing, RA carried out the quantitative assay, SM took mucosal HPV detection and helped to draft the manuscript, AG and VDG provided clinical samples and helped to draft the manuscript. All authors have read and approved the final version of the manuscript.


Organized screening based on Pap tests has substantially reduced deaths from cervical cancer in many countries, including Australia. However, the impact of the program depends upon the degree to which women participate. A new method of screening, testing for human papillomavirus (HPV) DNA to detect the virus that causes cervical cancer, has recently become available. Because women can collect their own samples for this test at home, it has the potential to overcome some of the barriers to Pap tests. The iPap trial will evaluate whether mailing an HPV self-sampling kit increases participation by never- and under-screened women within a cervical screening program.The iPap trial is a parallel randomized controlled, open label, trial. Participants will be Victorian women age 30–69 years, for whom there is either no record on the Victorian Cervical Cytology Registry (VCCR) of a Pap test (never-screened) or the last recorded Pap test was between five to fifteen years ago (under-screened). Enrolment information from the Victorian Electoral Commission will be linked to the VCCR to determine the never-screened women. Variables that will be used for record linkage include full name, address and date of birth. Never- and under-screened women will be randomly allocated to either receive an invitation letter with an HPV self-sampling kit or a reminder letter to attend for a Pap test, which is standard practice for women overdue for a test in Victoria. All resources have been focus group tested. The primary outcome will be the proportion of women who participate, by returning an HPV self-sampling kit for women in the self-sampling arm, and notification of a Pap test result to the Registry for women in the Pap test arm at 3 and 6 months after mailout. The most important secondary outcome is the proportion of test-positive women who undergo further investigations at 6 and 12 months after mailout of results.The iPap trial will provide strong evidence about whether HPV self-sampling could be used in Australia to improve participation in cervical screening for never-and under-screened women.ACTRN12613001104741; UTN: U1111-1148-3885ANZCTR Identifier:  Organized screening programs based on Pap tests have substantially reduced deaths from cervical cancer in resource rich settings, includiNumerous strategies have been used to improve participation in cervical screening, but most have had limited success, particularly in engaging ‘hard to reach’ groups. Common barriers include test-related issues such as pain, discomfort or embarrassment, or doctor-related issues including access, difficulty obtaining appointments or time constraints-9. ReminTesting for DNA of the human papillomavirus (HPV), the virus that causes cervical cancer, has been evaluated as a primary screening test over the last decade-15. EvidEight randomised controlled trials of HPV self-sampling evaluating whether it improves participation in screening have been reported from countries with organised screening programs Table 22-29].-29.22-29All trials found participation to be significantly higher for HPV self-sampling than for a reminder to attend for Pap testing. However, the actual participation proportion in both the intervention (range 10% to 39%) and the control arms (range 2% to 26%) varied widely, with an absolute difference in participation between trial arms ranging between 3% and 30%. The studies also found a high compliance with follow-up regimens: between 86% and 98% of women who tested positive to HPV DNA underwent appropriate follow-up investigations,24-28 exTo determine whether offering home-based HPV self-sampling increases participation in cervical screening, overall and separately for never- and under-screened women when compared to current practice of a reminder letter to attend for a Pap test.The main secondary objective is to estimate the proportion of women who have a positive HPV test who undergo appropriate further investigation, separately for never- and under-screened women. Other secondary objectives include documenting women’s experience with home-based HPV self-sampling, their willingness to participate in HPV self-sampling screening in future, and exploring reasons for non-participation.We will conduct a parallel, randomized controlled, open label trial. Women will be randomly allocated to either the HPV self-sampling arm and receive an invitation letter with a kit for home-based self-sampling or the current practice arm of a letter prompting them to attend a health practitioner  or nurse) to have a Pap test Figure . Women wTo determine which women have no history of a previous Pap smear on the Victorian Cervical Cytology Registry (VCCR) and are thus presumably never-screened, we will link the VCCR to the enrolment information on the Victorian Electoral Commission (VEC). Registration to vote is compulsory in Australia and use of the electoral roll for specific public health programs, such as cancer screening, is permitted under legislation. Women wThe trial is based at the Victorian Cytology Service Inc (VCS). VCS hosts the VCCR, which records almost all Pap tests in Victoria , and the VCS Pathology, which is a NATA  accredited laboratory that reports about half of those Pap test as well as HPV tests.In Australia, the National Cervical Screening Program (NCSP) currently recommends that all women aged 18–69 years, who have ever been sexually active (regardless of HPV vaccination status), should have Pap smears every two years if they have no symptoms or history suggestive of cervical pathology. The NCSParticipants will be Victorian residents, age 30–69 years, for whom there is either no record on the VCCR of a Pap test (never-screened) or the last recorded Pap test in the VCCR was between five and fifteen years ago (under-screened). Eligibility will be restricted to women 30 years of age or older given the low specificity of HPV DNA tests in younger women,32.Different exclusion criteria apply to women in the two screening groups and at different stages of the participant selection process Figure . For und‘The Pap test alternative: the HPV test and cervical cancer’, and the HPV self-sampling kit. The kit comprises a nylon-tipped flocked swab  for vaginal sampling enclosed in a plastic tube within a resealable plastic bag; an instruction sheet  on ‘How to take a vaginal sample and how to pack and post the sample’; a pathology information form; and a postage paid envelope to return the swab and the form. The form will ask for the woman’s country of birth, language spoken at home, whether she identifies as an Aboriginal or Torres Strait Islander woman, hysterectomy status, pregnancy status, Pap screening history, updates of her contact details, and the date she took her sample. Women are able to nominate a GP to receive a copy of the results to enable appropriate referral, follow-up and management should high-risk HPV be detected.Women allocated to the intervention (the HPV self-sampling) arm will be mailed an envelope containing an invitation letter, an information brochure on HPV and cervical cancer entitled Two to three weeks prior to receiving the kit, women will receive a pre-invitation letter informing them of the upcoming HPV self-sampling kit and the fact that the test is free, and a phone number for calling the Registry (or VCCR) with an option for cancellation of the kit. It will also allow women to call the Registry and update information such as a recent Pap test, correct contact details, hysterectomy, or pregnancy to identify women not eligible for the trial. A multilingual flyer included with the pre-invitation and the invitation letter will state that information is available on the website in the ten most common languages and that an interpreter service is available.All materials for use in the HPV self-sampling arm were focus group tested. Four focus groups were conducted in August 2013 with Victorian women who would be eligible for the trial, separated by their screening history (never- and under-screened) and age (30–49 and 50–69 years). The main aim was to obtain suggestions for refining all written materials sent with the HPV self-sampling kit with the intent of maximising response to the trial. The overall response to the iPap concept was very positive in the focus groups. The details of the focus group findings will be published elsewhere.Women in the HPV self-sampling arm will also receive a questionnaire a few weeks later. This will collect information about their experience with the HPV self-sampling  and their willingness to participate in HPV self-sampling screening in future. Those who did not return a completed HPV self-sampling kit will be asked to provide reasons for not participating.‘How Pap tests can help prevent cervical cancer, and the Pap test registry’. Also included will be a form to collect the similar information as for the HPV self-sampling arm, and a reply paid envelope for return of the form. The requested information will enable analysis of results by cultural background and Indigenous status, and also identify women not currently eligible for screening.Women allocated to the current practice arm will receive either a tailored invitation letter (never-screened) or a standard reminder letter (under-screened) to have a Pap test, as well as a Pap test brochure entitled All the kits received will be handled, processed and tested by VCS Pathology using the Cobas® 4800 HPV Test (Roche Diagnostics GmBH) according to the manufacturer’s instructions. The test is clinically validated and approved by the FDA. The test specifically identifies high-risk types HPV16 and HPV18 while concurrently detecting 12 other high-risk types  in a single pool at clinically relevant cut offs for detecting infection. This test allows for risk stratification and identification of women who are at the highest risk of cervical cancer (those who are 16/18 positive),35; a BeWomen will be sent a letter notifying them of their HPV result directly, with a copy to their nominated GP (if provided) within two weeks of testing. GP correspondence will detail the study, HPV result and recommended follow-up and management. The letter to the women will be accompanied by appropriate educational information on the meaning of the test result and the recommended follow-up or clinical management. Figure Women with high-risk HPV positive results but without a GP will be strongly recommended to contact a GP or arrange referral to a gynaecologist for further follow-up. In the case of HPV 16 and/or HPV18 positive results, women will be contacted by the VCS Liaison Physician to help them seek further medical advice. Follow-up of non-compliant women with positive results will be as per VCCR usual protocol for follow up of high grade cytology with shorter timelines and addition of phone calls. After 6 months, Medicare  will be contacted to supply any change of address details held for the woman for up to 2 years. There will be no follow-up of women who chose not to respond to the kit or the reminder letter, with the exception of a participation questionnaire sent to intervention non-responders. Women in the current practice arm with abnormal Pap test results will be managed as per NHMRC guidelines by their treating doctor or nurse. Because the women who participate in either trial arm will be part of the screening program, in other words their test results will be recorded on the VCCR; they will receive future reminders from VCCR at the appropriate time interval depending on their screening result.The primary outcome is the return of a completed HPV self-sampling kit or the notification of a Pap test result to the VCCR per trial arm. Women in the HPV self-sampling arm who attend for a Pap smear instead of self-sampling will be counted as ‘successes’. Outcomes will be measured at 3 and 6 months after the mailout of the kits or letters. The secondary outcome is whether women who have a positive high-risk HPV test undergo appropriate further investigation i.e. either have a Pap test or colposcopy depending on the type of high-risk HPV detected. This will be measured 6 and 12 months after women are informed of their results.The expected study duration is 36 months. Mailout of kits and letters will occur progressively in batches over about 4 months beginning March 2014. Results will be mailed out within two weeks of testing and follow-up monitored in the next 6–12 months of the mailout of the test positive letter. The questionnaire will be mailed to women, with differing timelines for responders and non-responders.The sample size was determined by the secondary objective (the proportion of women who have a positive HPV test who undergo appropriate further investigation). We will contact 16,000 women; 8,000 women never-screened and 8,000 women under-screened. 7,000 women will receive the HPV self-sampling kit within each screening group. Based on an estimate of 20% participation and prevalence of positive result for HPV test of 10%, there will be at least 140 women invited for a follow-up. With 140 women we will obtain a 95% confidence interval of +/− 5% points around an estimated follow-up proportion of 90%. Table The electoral roll from the VEC will constitute the sampling frame for never-screened women. The electoral roll will include female Victorian electors born between 1 January 1944 and 31 December 1983, excluding silent and overseas electors. A subset of women will be selected by simple random sampling (using a computer generated random number) and a check will be made against the VCCR CIS database to find a match. Women will be matched on name, address and date of birth and if no match is found, they are eligible for inclusion as never-screened women. If insufficient eligible women are found in the first random subset, another will be selected and the process repeated.Under-screened women will be identified directly from the VCCR CIS database after excluding women whose last Pap test was <5 years and ≥15 years ago, age <30 years or 70+ years, who have a prior history of cancer, hysterectomy, are deceased, or whose follow-up has ceased. A subset of women will then be selected by simple random sampling (using a computer generated random number) and further considered for: completeness of mailing address, or a change of address, most recent Pap test showing no high-grade abnormality and having not recently migrated or moved. The two-stage process is necessary because the second stage requires assessing episode level information. At this point, if all the above criteria are met, selected under-screened women are considered eligible. Under-screened women will then be grouped into strata  with approximately equal numbers in each stratum.Selected women will be randomly allocated to the two arms of the trial in a 7:1 (HPV self-sampling: current practice) ratio as per a computer generated randomisation schedule stratified by screening history and time since last Pap test (for under-screened only) within blocks or batches of fixed size. The batches will ensure a close balance of numbers in each arm at any time during the trial and will enable us to regulate the administrative work flow as well as the work flow in the laboratory. This will also enable us to monitor participation and contain expenditure if the response is higher than anticipated.The study ID and no other information will be used for the randomisation. The nature of the intervention and the randomization ratio precludes masking of participants and other study staff, including the data analyst.There are a number of ways in which participant information will be returned and collected. Women can post back their pathology information form, call the telephone centre and update details, or return the completed kit (with the form), or staff at VCCR process the return to sender generated during mailout. The data flow will go through several checkpoints of information matching and validation prior to entry, amendment of details or sample processing. Additionally, women in the self-sampling arm will return the questionnaire by post. These questionnaires will be anonymous, therefore when they are returned they cannot be linked to a specific woman.Within each stratum (never- and under-screened) the proportion of women in each arm participating (i.e. primary outcome) will be calculated as will the absolute difference in the proportion of participation between the HPV self-sampling and current practice arms and the corresponding 95% confidence interval and two-sided p-value. An intention-to-treat approach will be used for the analysis, with women who subsequently report they are adequately screened, are pregnant, or have had a hysterectomy, or return to sender, analysed within the arm to which they were randomised. We will also calculate adjusted participation proportions, in which women who report that they are pregnant, have had a hysterectomy, or that they are adequately screened, or return to sender, are removed from the denominators. Participation will be also be reported by age, socio-economic status (SES), cultural background, Indigenous status and time since last Pap test (for under-screened only) within each screening group and trial arm status. SES status classification is an area level variable assigned to women according to their postcode of residence based on the ABS Index of Relative Socioeconomic Disadvantage (ABS 2008) which is derived from Census information. Additionally, the distribution of age and SES will be compared between women who participate and those who don’t within each screening group and trial arm. Estimates and 95% confidence intervals of the proportion of women having appropriate further investigation (i.e. secondary outcome) in the overall and sub-groups of positive HPV test for each screening group will also be calculated. The responses to the different items  of the self-sampling follow-up questionnaire will be summarised and reported as the total number and proportion. We will also report the reasons for not returning a self-sampling kit.VCCR has strict data security arrangements, data privacy and data linkage policies and protocols in place to ensure the privacy and confidentiality of the personal information that it holds. Additional conditions surround the VEC data which include all staff involved signing confidentiality agreements, to ensure that the information remains confidential and protected. The information provided by the VEC has been allowed based on the public health benefit of this research and will not be used for any other purposes. The collating of the letters and/or letters and kits and inserting into envelopes is performed in a specifically designed and purpose built area with limited staff access.In accordance with VEC requirements, VCCR will fully destroy the information collected from VEC at three months from the date of release of the electoral roll from VEC, except for women selected for the study. The details of women selected for the study who do not participate will be deleted after 12 months. Year of birth and postcode will however be retained for statistical purposes. Notably when a never-screened woman returns the kit and consents to her information being recorded on the VCCR this information is no longer VEC data and becomes VCCR data. The final data set for analysis will be de-identified and published in an aggregate manner so confidentiality will be protected.The study was approved by the Human Research Ethics Committee (HREC) of the Victorian Department of Health. Informed consent has been waived for the study because it is primarily a trial of participation in screening, and we wish the results to be directly translatable to the current screening program. Asking women to consent to a trial of participation would potentially invalidate such results. However, women are otherwise fully informed about the testing and subsequent follow-up and once a woman returns the kit with a pathology information form it will represent her consent to test her sample and undergo further appropriate management and use of data. Correspondence sent to women includes a clear statement indicating the process under which VCCR obtained addresses from the electoral roll and participant information form includes statements about privacy of information. Additional information on how the data collected will be stored and utilized in the study is detailed in the brochure sent with the kit or the reminder letter. Provision has been offered for women to opt-off the study. VCCR will inform VEC of women who opt-off and who do not want to be contacted for health screening purposes in future. All materials were revised following focus groups and amended materials submitted for consideration by HREC.rd month post initial contact). The DSMB could halt or extend the study based on the primary end points. They will also provide clinical advice on detected high-risk HPV where a woman is non-compliant with follow-up, and assess VCCR compliance of follow-up reminders based on participants becoming part of the routine screening program.A Data Safety Monitoring Board (DSMB) has been appointed for the duration of the study. The DSMB will be responsible for the review of the primary outcome data. The DSMB will be provided with the full outcome data at six and twelve months post mail-out of the invitation letter. Following review of the data, the DSMB will report back to the Principal investigator with written details of any identified issues and/or recommendations. An interim analysis will also be performed on the primary end point when 50% of the mail-outs have occurred (anticipated to take place around the 3The success of the National Cervical Screening Program is limited by incomplete and unrepresentative participation. More than half of invasive cervical cancers presently occur in women who have never been screened and close to a quarter occur in women who are under-screened. Improving participation and reducing inequalities are important priorities for the cervical screening program and new strategies are needed to target the hardest to reach women. HPV self-sampling is a valid screening test that performs better than cervical cytology in detecting abnormalities and has the potential to overcome known barriers to screening, as trials have shown it can improve participation by hard-to-reach groups, although the participation rates varied widely between countries. Given the importance of the local context to screening participation, evidence from Australian trials is necessary to inform policy. None of the previous published trials have had sufficient power to evaluate participation of never-screened women separate to under-screened. This novel aspect of the study has great public health significance as these women are hardest to reach and have the highest rate of cervical cancer. A pragmatic trial of HPV self-sampling is timely and the findings will have direct relevance to the cervical screening program.MS is the Principal investigator of the COMPASS trial of primary HPV screening in Australia that has received funding contribution from Roche Molecular Systems USA. DG, SH, DW and JB are co-investigators on the COMPASS trial. No funding from Roche has been received for the purpose of the iPap trial. All other authors declare no other conflicts of interest.DG is the principal investigator of the study and is responsible for the overall conduct of the study. FS developed the first draft of the protocol that was further developed by DG, DE, FS, JAS, JB and MS. DG, DE, MS, JB, JAS and FS are primarily responsible for the design of the study, with input from all authors. FS is a PhD student doing her PhD on the topic and will be responsible for scientific coordination of the trial, statistical analysis and manuscript preparation, with oversight from DE, DG, JAS, JB and MS. KD is managing the operational coordination of the study. MS will oversee the laboratory testing for self-sampled HPV, reporting of results and ensure laboratory quality assurance. DW and SH will provide clinical advice on follow-up of women with positive results. RM was responsible for oversight of the focus groups. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/207/prepub