There are more than 40 human papillomaviruses (HPVs) belonging to the alpha genus that cause sexually transmitted infections; these infections are among the most frequent and can lead to condylomas and anogenital intra-epithelial neoplasia. At least 18 of these viruses are causative agents of anogenital carcinomas. We evaluated the performance of a resequencing microarray for the detection and genotyping of alpha HPV of clinical significance using cloned HPV DNA. To reduce the number of HPV genotypes tiled on microarray, we used reconstructed ancestral sequences (RASs) as they are more closely related to the various genotypes than the current genotypes are among themselves. The performance of this approach was tested by genotyping with a set of 40 cervical smears already genotyped using the commercial PapilloCheck kit. The results of the two tests were concordant for 70% (28/40) of the samples and compatible for 30% (12/40). Our findings indicate that RASs were able to detect and identify one or several HPV in clinical samples. Associating RASs with homonym sequences improved the genotyping of HPV present in cases of multiple infection. In conclusion, we demonstrate the diagnostic potential of resequencing technology for genotyping of HPV, and illustrate its value both for epidemiological studies and for monitoring the distribution of HPV in the post-vaccination era. More than 170 human papillomavirus (HPVs) have been characterized and they are classified into the alpha, beta, gamma, mu, and nu genera The need for effective screening for cervical cancer is now recognized worldwide and testing for HPVs is a potentially powerful approach to the detection of cervical or anogenital lesions. Other than the hybrid capture II and the Cervista detection tests Papillomaviridae. Most of them are based on long oligonucleotide probes and virus identification is based only on hybridization patterns. This approach leads to a lack of information for pathogen identification at the single-pair resolution level Several low or high-density microarrays have been developed and successfully used for the detection of several viral families such as Enterobacteriaceae rpoB sequences used as models The aim of this study was to develop a multiplex bioassay method, based on RMA technology, for the detection of mucosal HPV genotypes of public health importance. Because of the wide genetic diversity of HPVs and to reduce the number of genotypes tiled on the microarray, we inferred reconstructed ancestral sequences (RASs); these sequences are more closely related to different genotypes than such genotypes are to each other. The advantages of using reconstructed ancestral sequences for RMAs has been described and illustrated using pathogen identification with A third generation resequencing microarray, named VirID v3.0, containing 724 viral sequences distributed into 27 families and 85 genera , was useL1 ORF of 38 alpha-HPV; there were neither insertions nor deletions in this stretch. A neighbor-joining tree was obtained using software BioNumerics v5.10 . Ancestral sequences were reconstructed by maximum likelihood using the PAML v4 software RASs were designed based on phylogenetic analysis of nucleotide sequences of a portion of 416 bases in the Viral DNA was obtained from cloned HPV DNA and from cervical smears provided by the National Reference Laboratory for Human Papillomavirus at Institut Pasteur (Paris). The cloned HPV DNA used for validation assays was amplified and purified as previously described Liquid-based cytology (LBC) samples were collected from women attending organized cervical screening in 16 pilot sites in France. After completion of cytology, residual LBC samples were anonymized and sent to the French HPV Reference Laboratory for genotyping. The residual material would otherwise have been discarded. According to French regulations of biomedical research, a written or verbal informed consent and an ethical approval are not required for such studies. However, the study was given favorable ethical opinion by the Comité de Recherche Clinique at the Institut Pasteur (Avis n° 200-51). The collection of human samples was declared to the French Research Ministry  in accordance with French regulations.Amplified DNA was fragmented and labeled using the GeneChip Resequencing Assay Kit . After overnight hybridization at 45°C, the RMA was washed, stained and scanned according to Affymetrix’ instructions. The raw image file (.DAT), obtained after the scan, was converted to a fluorescence intensity file (.CEL). Bases were called by the GeneChip Sequence Analysis Software (GSEQ v 4.1) which uses a derivative of the ABACUS base-calling algorithm GAACCGAAACCGGTTAGTATAA 3′; HPV16-rev 5′ ATGTATAGTTGTTTGCAGCTCTGT 3′; HPV18-for 5′ GGACCGAAAACGGTGTATATAA 3′; HPV18-rev 5′ CAGTGAAGTGTTCAGTTCGGT 3′) and 15 pmol of a probe  in a final reaction volume of 20 µl, using a LC Taqman Master kit . The LightCycler qPCR assay was performed as described by Schmitz et al. Quantitative PCR tests for HPV16 and HPV18 were performed using DNA obtained from cervical smears (5 ul) or amplified products (1/1000 dilution). The DNA was mixed with 100 pmol of each primer . This test involves PCR amplification with fluorescent primers (CY5 fluorophore) specific for the HPV E1 gene and the cellular housekeeping gene ADAT1. The amplification products are then hybridized to a DNA chip containing sequences for 18 high-risk genotypes  and six low-risk genotypes . The PapilloCheck test was used according to the recommendations of the supplier.genus sequences. Sequences at nodes at different phylogenetic depths on the tree were chosen as RASs (The third generation of the “VirID” RMA (v3.0) was used for the detection and identification of a broad panel of HPV sequences. Thirty-eight HPV sequences were selected and tiled on the microarray, including 32 from the α genus  and covering all the nine families found in cervical lesions and condylomas. The other six sequences belonged to the γ genus . Reconstructed Ancestral Sequences (RAS) were designed through phylogenetic analysis of selected α  as RASs ; we checOf the 38 HPV sequences deposited on the chip, 11 α-HPV sequences and one γ-HPV sequence were tested using cloned HPV genomes : all theBecause several sequences were tiled on the chip, we tested whether each HPV genotype could be detected following hybridization either to its homonymous sequence, or to related sequences belonging to the same or different species. Various cloned HPVs were tested, alone or in mixtures with HPVs belonging to the same or different species  and 3. HWe wanted to determine whether all HPVs belonging to a single species could be detected with a RAS specific for this species. Within any family, the RAS is more closely related to the sequences of the various HPVs than the sequences of the HPVs are to each other (data not shown). We tested HPV53, 56 and 66, all members of species alpha 6, and call rates of 59.1 to 75.7 were obtained with RAS-811 . These vWe next investigated whether HPVs that belong to different species could be identified with RASs. We tested a mixture of HPV16, 51, 66 and 108 (mixture A) and one of HPV6, 18, 42 and 53 (mixture B) with reconstructed RASs tiled on the microarray . HPV16 bWe assessed whether this RMA method could be used to identify HPVs in clinical samples. A set of 40 DNAs extracted from cervical smears with normal (n = 3) and abnormal , 1 Atypical squamous cells cannot exclude HSIL (ASC-H), 11 Low-grade Squamous Intraepithelial Lesion (LSIL) and 17 High-grade Squamous Intraepithelial Lesion (HSIL)) cytology were provided by the French HPV Reference Laboratory . For eacThe PapilloCheck genotyping kit can detect and identify 18 HR HPV  and six LR HPV . The two methods PapilloCheck genotyping and RMA genotyping with homonymous sequences gave concordant results for 28/40 (70%) of the samples, and compatible results (one or two HPV missed by one or other method) for 12/40 (30%) of the samples. A value of k = 0.36 was obtained with the kappa test in interrogation of agreement between these two tests. PapilloCheck did not detect some HPVs found by RMAs ; the RMAs did not detect some strains identified by PapilloCheck . RMAs allowed two closely related HPVs, HPV44 and 55, to be distinguished and genotyped, although they are indistinguishable by PapilloCheck (samples n° 2012-1620 and 2012-1649). No discordant result was observed .The RMA method with homonymous sequences appeared to be highly sensitive: HPV16 was detected in a sample in which there was only 1 copy per 1000 cells (sample 2012-266). Eighteen HPVs not identifiable with the PapilloCheck genotyping kit were detected by RMAs in 14 samples  .Multiple broad-spectrum PCR methods have been developed for the detection of the alpha-HPV genus and most are based on HPV sequences in the L1 open reading frame. Here, we show that resequencing microarray (RMA) technology was feasible and effective for genotyping human papillomaviruses, and is thus a potentially valuable tool for epidemiology. We have demonstrated that the use of homonymous HPV sequences and reconstructed ancestral sequences (RASs), either separately or combined, allows precise molecular identification of different HPV genotypes. We validated the method first with cloned HPV DNA and subsequently for detection of HPVs in clinical samples. The RMA method used involved treatment of cellular DNA with Plasmid-Safe DNase and the universal Phi 29-based amplification; this methodology was highly sensitive for the genotyping of HPVs in clinical samples, with a detection limit of one copy of HPV16 per 1000 cells.This RMA method allows the detection and characterization of HPV genotypes other than those tiled on the microarray. Indeed, the call rate values were a function of the percentage of divergence between the tiled sequence and the sequence present in the sample. We found that there was no substantial loss of detection signal when the tiled sequence diverged by up to 10–15% from the sequence of the pathogen in the sample. Moreover, the RASs were designed to minimizing the divergence between the tiled and the viral sequences potentially present in clinical samples. Indeed, the use of RASs allowed an improvement in the detection of several HPVs. Nevertheless, the nucleotide diversity of HPV sequences is substantial and not a single RAS was able to detect all the different HPVs present in cases of multiple infections.We compared this RMA method to a commercial HPV genotyping kit. It must be stressed that clinical samples with more than one HPV were intentionally selected to compare the two methods. Some HPV types identified in clinical samples by the RMA method were not identified by the PapilloCheck test. This discrepancy is in part explained by the number of HPV genotypes, limited to 24, which can be identified by the PapilloCheck test. The HPVs detected by RMAs but not by PapilloCheck tests include both LR HPVs  and HR HPVs . Also, HR HPV35, 73, and 82, found in some clinical samples by RMAs, were not detected by the PapilloCheck test. Conversely, HPV33, 39, 52, 56, and 70 identifiable in some samples by the PapilloCheck kit were not found by the RMA method. This presumably reflects different sensitivities of the two tests for some HPV genotypes.Our study has some limitations. First, due to the substantial genetic diversity of HPVs with more than 170 HPV genotypes already identified The cost of a high-density microarray could appear as a limit because this technology required the synthesis of a physical mask. However, the final cost can decrease if the demand increases, as may be the case in the context of a clinical diagnostic use. Moreover, the use of a RMA of the smallest size only dedicated to detect and characterize HPVs will help to reduce the global cost of assay. Adding of supplementary homonyms and reconstructed ancestral sequences will allow developing countries to obtain a better tool for the screening of HPVs. Based on the sequences obtained, RMAs may be useful for molecular epidemiological studies of HPV infection, particularly in geographical areas where the distribution of circulating genotypes has not yet been investigated. It can be used to identify variants of HPV genotypes that differ from the prototype until 10–15%. RMA-based tests may thus be informative about the prevalence and distribution of diverse HPV genotypes in the post-vaccination era.Table S1Content description of VirID V3.0 RMA.(DOCX)Click here for additional data file.Table S2RASs tiled on the resequencing microarray.(DOCX)Click here for additional data file.


MAGI-3 gene and one SNP (rs8031627) in the SMAD3 gene were associated with HR-HPV clearance (p<10−6). A variant (rs1633038) in HLA-G were also significantly associated in African American. Results from this study support associations of immune-related genes, having potential biological mechanism, with differential cervical HR-HPV infection outcomes.Persistent high-risk human papillomavirus (HR-HPV) is a necessary and causal factor of cervical cancer. Most women naturally clear HPV infections; however, the biological mechanisms related to HPV pathogenesis have not been clearly elucidated. Host genetic factors that specifically regulate immune response could play an important role. All HIV-positive women in the HIV Epidemiology Research Study (HERS) with a HR-HPV infection and at least one follow-up biannual visit were included in the study. Cervicovaginal lavage samples were tested for HPV using type-specific HPV hybridization assays. Type-specific HPV clearance was defined as two consecutive HPV-negative tests after a positive test. DNA from participants was genotyped for 196,524 variants within 186 known immune related loci using the custom ImmunoChip microarray. To assess the influence of each single-nucleotide polymorphism (SNP) with HR-HPV clearance, the Cox proportional hazards model with the Wei-Lin-Weissfeld approach was used, adjusting for CD4+ count, low risk HPV (LR-HPV) co-infection, and relevant confounders. Three analytical models were performed: race-specific (African Americans (n = 258), European Americans (n = 87), Hispanics (n = 55), race-adjusted combined analysis, and meta-analysis of pooled independent race-specific analyses. Women were followed for a median time of 1,617 days. Overall, three SNPs  in the  HPV DNA is present in 99.7% of all cervical cancer, the third most common cancer in women worldwide, with HPV types 16, 18, 31 and 45 being the most predominant HR-HPV types DRB1*13 and DRB1*1501-DQB1*06) have been shown to be associated with cervical cancer The factors that lead to the development of a persistent HPV infection in some women, but not others, remain unclear. The role of host genetics that regulate biological mechanisms of immune response may contribute to the differential responses to infection and HPV clearance among women. To our knowledge, most research on host genetics including genome-wide association studies (GWAS) have focused on cervical cancer as the outcome Written consent was obtained from all participants  and the study protocols for the parent study and this sub-study was approved by institutional review boards (IRB) at all sponsoring organizations and conformed to human-experimentation guidelines set forth by the United States Department of Health and Human Services. All protocols including the genetic work was finally reviewed and approved by the University of Alabama at Birmingham IRB.Participants from the HIV Epidemiology Research Study (HERS) cohort were included in this study HPV testing was performed on all HERS women at enrollment and every 6 months thereafter using cervicovaginal lavage samples. Viral DNA fragments from cervicovaginal lavage were amplified by using the consensus primers MY09/11 and HMB01 and were hybridized for a consensus probe and for 26 HPV types HR-HPV: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, and 82 and LR-HPV: 6, 11, 40, 42, 53, 54, 55, 83, and 84 using a chemiluminescent dot-blot format The analysis was performed in HIV-positive women in HERS who had no cervical treatment and were not pregnant during the follow-up. HIV-positive women with prevalent or incident HR-HPV infection who had peripheral blood mononuclear cells (PBMC) available at the baseline visit and at least one follow-up visit were included in the genetic analysis. In this study, the analyses focused on clearance of HR-HPV infections since these types are most likely to be associated with risk of pre-cancer and cancer of cervix and the models were adjusted for LR-HPV infections in order to account for the correlation between HPV subtypes. For the analysis, type-specific HPV status was assumed to remain unchanged across single missing visits. For women who tested negative for HPV at their last study visit, results were censored at that visit. Type-specific HPV clearance was defined as two consecutive HPV-negative tests to avoid the possibility of false-negative test results.Genomic DNA was extracted from stored PBMC and genotyped using the Human ImmunoChip, an iSelect HD custom genotyping array . The ImmunoChip is a custom SNP microarray developed by a consortium of specialists in the fields of immunology and inflammation. The microarray chip contains densely spaced 196,524 SNP variants ), previously reported from GWAS and candidate gene studies of major autoimmune and inflammatory diseases Data were analyzed using the Genome Studio Genotyping Module . The National Center for Biotechnology Information (NCBI) build 36 (hg18) map was used and the normalized probe intensities were extracted for all samples that passed standard laboratory quality-control (QC) thresholds. The Immunochip contains 763 duplicate SNPs and these were checked for concordance. Additionally, 174 genotyping assays failed which left 195,587 SNPs. All SNPs were checked for completeness (by SNP and by subject), rare variants, and deviation from Hardy-Weinberg Equilibrium (HWE) within each ethnic group separately. First, a large proportion of SNP assays failing on an individual may indicate poor quality of DNA sample and thus to avoid aberrant genotype calling, the threshold was set to 90% coverage. All African Americans and European Americans samples met this criterion, but 2 Hispanic samples did not and thus were removed from the analyses. Second, missing genotype data from a large number of individuals indicate poor assay quality for that SNP, and there were 5171 SNPs in African Americans, 5889 SNPS in European Americans and 6131 SNPs in Hispanics that were missing in more than 10% of individuals and were removed from the analysis. Third, there were 71,488 SNPs in African Americans, 73,017 in European Americans, and 68,264 in Hispanics that had a minor allele frequency less than 5% (rare variants) that were also removed in the analysis of each ethnic group, respectively. Fourth, there were 1234 SNPs in African Americans, 442 SNPs in European Americans, and 347 SNPs in Hispanics that deviated from HWE , and thus were also removed from the analyses. Further, kinship between the individuals was also assessed in KING software −7, 5.37×10−7, and 5.16×10−7, respectively. Additionally, among the SNPs common in all three ethnic groups, there were 80,510 effective numbers of tests .The same QC methods mentioned above for each ethnic group separately were then applied to the combined analysis which contained all three ethnic groups. For the combined analysis of all three distinct race (described below), there were 5190 SNPs removed because of low genotyping calls and an additional 66,536 rare variants (<0.05%) across any one ethnic group and were removed; thus, 123,861 SNPs were available for analysis. Since SNPs in the ImmunoChip are mostly in the fine-mapped regions with high linkage disequilibrium (LD), correction for multiple testing was performed based on the effective number of tests which resulted after pairwise correlations between markers, as previously described Cox proportional hazard models with the Wei-Lin-Weisfeld (WLW) extension To test for potential confounding effects of population stratification in our study cohort, principal component (PC) analysis was performed. Self-reported race  was confirmed using clustering as implemented in EIGENSTRAT, and all individuals were confirmed with no apparent population outliers All SNP analyses were conducted using an additive genetic model, adjusting for CD4+ count, which served as a marker for immune status as well as a surrogate for HIV treatment. CD4+ count was adjusted for in the model at all visits when an individual was infected with HPV, which allowed CD4+ count to serve as a time-varying covariate. LR-HPV infections were adjusted for in the model when an individual was co-infected with any HR-HPV type, which also allowed LR-HPV to serve as a time-varying covariate when infection time overlapped. Quantile-quantile (Q-Q) plots of p-values were constructed to evaluate deviations from the expected test statistic distribution. ImmunoChip based Manhattan plots were generated to visualize the results.3 [interquartile range (IQR): 262.3–546.1 cells/mm3]. Women were followed for a median time of 1,617 days (range 324–1729 days). Among the 400 women, there were 1052 HR-HPV infections, and 668 (64%) cleared during follow up. The most common HR-HPV types were HPV18 (n = 99), HPV16 (n = 88), HPV51 (n = 88), HPV58 (n = 83), and HPV52 (n = 79). Of the 668 infections that cleared during follow up, the average time to clearance was 457.9 days (median 322 days). Among the HR-HPV infections that cleared during follow-up, the average CD4+ count was 385.1 cells/mm3 (median 336.7 cells/mm3). Among the HR-HPV infections that persisted during follow-up, the average CD4+ count was 251.1 cells/mm3 (median 212.9 cells/mm3).The average age at baseline of the 400 HIV-positive women included in the study was 34 years and their median baseline CD4+ count was 426.3 cells/mmMAGI3 gene were associated with HPV clearance. HIV-positive women infected with HR-HPV that have minor allele A for SNP rs1112085, located on chromosome 1, had a HPV clearance rate 1.51 times (p = 1.14×10−07) higher than those with the G wild type allele, controlling for CD4+ count, LR-HPV infection(s), 10 PCs, and genotyping facility , while no association was detected between this SNP and HR-HPV clearance among Hispanics . In the pooled analysis using the meta-analysis approach, the same minor allele was associated with higher clearance rates . Likewise, a SNP (rs8031627), located on chromosome 15 in the SMAD3 gene, was also significantly associated with higher clearance rates in the race-adjusted analysis  and the pooled analysis . When analyzed among the separate races, the SNP (rs8031627) was significantly associated with clearance in Hispanics  and African Americans ; a similar hazard ratio was seen in European Americans, but it was not statistically significant .The Manhattan plots  summarizfacility . When asfacility , in AfriHLA-G gene region, was significantly associated with higher clearance rates  in African Americans, although this was not observed in Hispanics or European Americans separately. Several other SNPs presented in A SNP (rs1633038) located on chromosome 6 in the MAGI3 gene region: rs1112085, rs11102637, and rs12030900 (in LD). The minor alleles for these SNPs in LD were associated with faster time (days) to clearance. MAGI-3 is part of the membrane-associated guanylate kinases (MAGUK) family of proteins that have inverted domain structure, and are part of the PDZ domain-containing proteins, which are localized between epithelial cells in vivo studies have shown that HR-HPV E6 proteins target MAGI-1,-2,-3 proteins for degradation We report several variants in immune related genes that are associated with clearance of HR-HPV infection in African Americans, Hispanics, and European Americans HIV-positive women after accounting for the effects of CD4+ count, other LR-HPV co-infection(s), population stratification, and genotyping facility. In particular, CD4+ has been a major factor with HPV persistence/clearance among HIV patients; thus it was adjusted as a time-varying covariate. Also, none of the top hits in SMAD3 gene was significantly associated with higher clearance rates in the adjusted analysis. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways including TGF-β signals in vivo to block SMAD3 by binding to its target sequence of DNA, which then also inhibits TFG-β for inhibiting DNA synthesis Another interesting finding was that the minor allele for SNP rs8031627 located on chromosome 15 in the −7) in that those that were homozygous for the minor allele had faster clearance compared to heterozygous and homozygous for the major allele. This SNP is located in chromosome 6 near the HLA-G gene region. HLA-G is a nonclassical HLA class Ib molecule that regulates the immune response through interaction with surface receptors on natural killer, T and antigen-presenting cells Interestingly, the most significant association with time to clearance of HR-HPV in the African Americans analysis was with the SNP rs1633038 located on chromosome 6. The minor allele for SNP rs1633038 was associated with faster time (days) to clearance 1.9 times , which reduced the statistical power to detect significant findings after adjusting for multiple genetic testing, especially when analyzing the three races separately. However, we were able to observe several significant SNPs in the separate analyses for African Americans and European Americans. The ImmunoChip was designed for use in European populations and could be less informative for other ethnic groups if the disease-associated variants are not shared between them Of note, the Q-Q plot  A–D had MAGI-3, SMAD3 and HLA-G or other biologically plausible genes, potentially involved with HPV clearance.This analysis assessing SNPs in immune related genes and their associations with HR-HPV clearance brought forth hypotheses regarding several significant SNPs and gene regions. While these SNPs are associated with HPV clearance, future studies could examine if they are also associated with progression of pre-cancer neoplasia in larger cohorts with adequate events during follow-up periods. The variants in the current ImmunoChip are based on a consortium of genes involved in autoimmune diseases, and do not represent the comprehensive genes involved in human immunity. However, future research is needed to validate these associations and finemap the gene regions (which are not as dense in ImmunoChip for most regions) to identify one or multiple rare variants in LD with the functionally associated SNPs in ImmunoChip, specifically with Figure S110 P-value, the Y-axes display the observed −log10 P-value.Quantile-quantile (Q-Q) plot showing the association P-values of single nucleotide polymorphisms (SNPs) in the ImmunoChip with the time to clearance of HR-HPV. The X-axes display the expected −log(TIF)Click here for additional data file.Table S1Results of Cox Proportional Hazard Ratios (HR) associated with time to clearance of high-risk (HR-HPV) HPV infection for all the SNPs in the ImmunoChip in the race-adjusted analysis (Adujsted), individual race-specific analysis , and pooled analysis  of the three races separately.(TXT)Click here for additional data file.


Significantly lower bacterial abundances and higher inorganic nitrogen species  were measured in aquarium water when biofouling organisms were present within a 7-year old inlet pipe feeding a tropical reef running seawater aquaria system, compared with aquarium water fed by a new, biofouling-free inlet pipe. These water quality changes are indicative of the feeding activity and waste production of the suspension- and filter-feeding communities found in the old pipe, which included sponges, bivalves, barnacles, and ascidians. To illustrate the physiological consequences of these water quality changes on a model organism kept in the aquaria system, we investigated the influence of the presence and absence of the biofouling community on the functioning of the filter-feeding sponge Halisarca caerulea, by determining its choanocyte (filter cell) proliferation rates. We found a 34% increase in choanocyte proliferation rates following the replacement of the inlet pipe . This indicates that the physiological functioning of the sponge was compromised due to suboptimal food conditions within the aquarium resulting from the presence of the biofouling organisms in the inlet pipe. This study has implications for the husbandry and performance of experiments with marine organisms in running seawater aquaria systems. Inlet pipes should be checked regularly, and replaced if necessary, in order to avoid excessive biofouling and to approach in situ water quality.Marine organism are often kept, cultured, and experimented on in running seawater aquaria. However, surprisingly little attention is given to the nutrient composition of the water flowing through these systems, which is generally assumed to equal  In the first series of experiments, the proliferation rate of H. caerulea filter cells (choanocytes), i.e., the percentage of proliferative choanocytes after 6 h, was estimated to be 46.6 ± 2.6% (mean ± 95%-CI) under steady-state (negligible growth) conditions (5 per mL) than in water samples taken at the reef entrance of the inlet pipe (8.8 × 105 per mL). Bacterial numbers are a good proxy indicating the food availability to sponges. The natural diet of these filter-feeding organisms mainly consists of bacterio- and phyto-plankton  were assessed in the reef water flowing along the length of the inlet pipe and in the flow-through aquarium fed by the inlet pipe. Subsequently, the presence and distribution of biofouling communities inside the 7-year old pipe was investigated. After replacing the old inlet pipe, the aforementioned water quality assessments were repeated, and the choanocyte proliferation rates for our model organism Fieldwork was performed under the research permit (#2012/48584) issued by the Curaçaoan Ministry of Health, Environment and Nature (GMN) to the CARMABI foundation.−1) from 10 m water depth at the reef slope through a 100-m long polypropylene pipe (5 cm inner Ø). The first 60 m of the pipe lies underwater, whereas the last 40 m, including the pump, is located above-water, partially underground. Water flow is regulated separately for each flow-through aquarium. The last time the old inlet pipe of the running seawater aquaria system had been replaced was in 2006. The new inlet pipe (only the first 60 m of the underwater section) was replaced on April 6th, 2013.The running seawater aquaria system is located on the Southern Caribbean island of Curaçao at the CARMABI research station . The land-based facility consists of 18 glass flow-through aquaria ranging in volume from 50 to 160 L. Seawater is pumped  at 15–30 m water depths by SCUBA between February and March 2013, 1–2 months before the replacement of the old pipe. Pieces of sponge were chiseled from the reef framework and the attached substrate was cleared of other organisms. All sponges were trimmed to a size of approximately 25 cm2 and subsequently kept in 100-L running seawater aquaria, with a flow rate of 3 L min−1. Aquarium water was at ambient seawater temperature (26–27 °C) and kept under natural light cycles . Semi-transparent black plastic sheets were used to imitate in situ cryptic  light conditions that sponges experienced  level 5–15 µmol photons m−2 s−1 during daylight hours). Prior to cell proliferation experiments, sponges acclimatized to aquarium conditions for a minimum of 1 week before and after the old pipe was replaced by the new pipe to ensure they fully recovered from the collection and transportation to the aquaria . Triplicate 20 mL water samples were taken to determine bacterial abundances and inorganic nutrient concentrations along the length of the old and new inlet pipes at 0 (a few cm inside the inlet pipe), 3, 6, 12, 24, and 60 m from the inlet pipe entrance  as well −1) and bacteria were counted using an epifluorescence microscope . At least ten fields (each 0.0025 mm2) of a counting grid were counted per slide, or up to a minimum of 200 bacteria, when ten fields were not sufficient.Ten mL of the water sample was fixed immediately with 0.57 mL 35% formaldehyde solution  for 1 h at 4 °C in the dark. Fixed samples were then filtered on 0.2 µm polycarbonate filters  with 0.45 µm cellulose nitrate support filters . The polycarbonate filters were air-dried and stored at −20 °C until further processing. Filters were mounted on microscope slides in DAPI-mix  and stored at −20 °C in 6 mL vials  until further processing. The concentrations of dissolved inorganic nutrients  were enclosed in incubation chambers (3 L) with magnetic stirring devices (−1 BrdU for 6 h (continuous labeling) in order to estimate choanocyte proliferation rates (2) was taken from each sponge and fixed in 4% paraformaldehyde in phosphate-buffered saline , rinsed in PBS, dehydrated through a graded series of ethanol and stored in 70% ethanol at 4 °C until further processing.Sponges  of BMM-embedded sponge tissue were cut on a pyramitome  using glass knives and collected on glass slides . BrdU immunohistochemistry was performed according to Alexander and colleagues ; 2015 usAfter water samples were taken from the old pipe and tissue samples were taken from the sponges in the flow-through aquarium, the 60 m underwater section of the pipe was removed and cut into 1-m pieces over its entire length. Each 1-m piece was then cut in half along its length and photographed to assess the presence of biofouling communities  and linear (nutrients) models. Differences in bacterial abundances and dissolved inorganic nutrients were also tested in the water flowing through aquaria fed by the old and new pipe, using linear models. Additionally, differences in choanocyte proliferation rates between sponges kept in flow-through aquaria fed by the old and new pipe were investigated with a linear model. The significance threshold was set at 0.05. All calculations were carried out in R  were 3.9 fold higher than in the flow-through aquarium (1.9 ± 0.1 × 105 mL−1)   of the rp values < 0.05, p = 0.77, p = 0.07 [bacteria], p = 0.12 , p = 0.48 [nitrate]). Bacterial abundance was significantly higher along the length of the new pipe compared to the old  and no longer changed along the length of the new pipe . However, bacterial abundance in the flow-through aquaria supplied by the new inlet pipe was lower . Still, bacterial abundance in the flow-through aquarium fed by the new pipe was 3.1 times higher compared to flow-through aquarium fed by the old pipe (1.9 ± 0.1 × 105 mL−1). Dissolved inorganic nitrogen  concentrations were lower in the new pipe compared to the old pipe  and did not change along the length of the new pipe . However, inorganic nitrogen concentrations were higher in the flow-through aquarium compared to the reef entrance of the new inlet pipe  than sponges after the installation of the new inlet pipe  within the aquarium water. These alterations are logical consequences of the biological activity of suspension- and filter-feeding biofoulers, which included sponges, bivalves, barnacles, and ascidians e.g., . Bacteri5 mL−1) where nevertheless still in the range of ambient in situ bacterial abundances measured for Curaçaoan reef waters (5–10 × 105 mL−1)  .H. caerulea following the replacement of the inlet pipe. The cause of this increase cannot be unequivocally linked to changes in our water quality proxies. However, to the best of our knowledge, the only parameter that was changed in this case study was the inlet pipe. It is well established that cell proliferation is an energetically costly process, which requires a constant supply of energy and nutrients  within the first hours after wound infliction. However, after 6 days proliferation rates did not differ significantly anymore from those in steady-state tissue  (12.8 ± 1.0% in 6 h) (We documented a clear 34% increase in the proliferation rate of choanocytes in elements  during telements . Unfortu in 6 h) .H. caerulea after replacement of the inlet pipe (20.2 ± 3.8% in 6 h) was still substantially lower than found during an earlier study by De Goeij and colleagues (Despite the 34%-increase found in this study, the proliferation rate of choanocytes for lleagues  (46 ± 2.in situ ambient and ex situ aquarium conditions. Our results suggest that this unwillingly compromised the physiology of organisms kept in these aquaria, which can easily lead to the wrong conclusions being drawn from experimental work. This also applies to organisms that may in fact benefit from low bacterial abundances and high inorganic nutrient concentrations, such as algae and corals. Inlet pipes provide an optimal habitat for biofouling organisms due to a lack of predation, reduced competition from a lack of light, and a constant supply of food (in situ conditions as possible.In conclusion, this study has implications for the husbandry of marine organisms in running seawater aquaria systems. We have found that biofouling communities residing in the inlet pipes that feed running seawater aquaria significantly alter the water quality between  of food . They sh10.7717/peerj.1430/supp-1Figure S1Click here for additional data file.10.7717/peerj.1430/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.1430/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.1430/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.1430/supp-5Data S1Click here for additional data file.10.7717/peerj.1430/supp-6Supplemental Information 5Click here for additional data file.


HPV transmission dynamics have rarely been studied in the general population, especially in China. We followed the genital HPV infection status of both partners in 874 couples aged 25-65 years from rural China for up to 7 bi-annual visits during 2009-2013. The positive HPV concordance and transmission rate for partners in a couple were evaluated and relevant risk factors were assessed. The concordance of any, oncogenic, and non-oncogenic HPV was 15.52%, 16.18% and 10.41%, respectively. Male-to-female transmission rate was 7.11, 12.13 and 4.77/1000 person months for any, oncogenic and non-oncogenic HPV respectively. The female-to-male transmission rate was 5.56, 2.37, and 17.01/1000 person months for any, oncogenic and non-oncogenic HPV respectively. The risk of male-to-female transmission was significantly higher than that of female-to-male transmission for oncogenic types. However, for non-oncogenic types, the risk of male-to-female transmission was significantly lower than that of female-to-male transmission. Younger couples, persistent infection with HPV, higher numbers of sexual partners and higher frequency of sexual intercourse were positively associated with HPV transmission in couples. Our results indicate that men in rural China play a more important role than men in western populations as a source of cervical oncogenic HPV infection in women. HPV infection has been demonstrated to be the causative agent of cervical cancer in women, and it also plays an important role in anogenital and oropharyngeal cancers236789676789In this study we followed genital HPV infection status in 874 couples aged 25 to 65 for up to 7 bi-annual visits from 2009 to 2013 by making use of an ongoing population-based cohort study in rural Anyang, Chinand cross-section investigation of the AECCS in 2009-2010. The eligibility criteria for couples included in the current study were as follows: 1) being married and permanent resident in the target villages; 2) age 25-65 years; 3) female partner had an intact uterus and was not pregnant during the study period; 4) no history of cancer, cardiovascular disease or mental disorder; 5) no history of infection with HBV, HCV, or HIV; and 6) willingness to cooperate with the follow up and provide informed consent. All eligible couples were enrolled from 2009 to 2010 and were assessed bi-annually, for up to 7 visits for husbands and up to 6 visits for wives. Centralized examinations were conducted, and all couples were sampled within a period of two weeks at each follow-up visit. Study visits for male and female partners were conducted at the same visit.In 2007–2009 a population-based esophageal cancer cohort study (AECCS) was initiated in rural Anyang, ChinaFor males, exfoliated cells from the penile shaft, glans penis (prepuce was not included), coronal sulcus, and scrotum were collected by an experienced doctor using saline-soaked swabs at each evaluationResearch protocols and materials were approved by the Institutional Review Board of the Peking University School of Oncology, China. All methods were carried out according to the approved guidelines. All participants in this study provided informed written consent.Specimen DNA was extracted using the Biomek 3000 workstation . The β-globin gene was first evaluated by PCR, and only β-globin positive specimens were subjected to HPV evaluation using a pair of SPF1/GP6+ primers131517For concordance analysis, only positive concordance of HPV on the basis of a type-specific principle was evaluated. Any couple meeting the conditions: a) both male and female provided ≥2 valid HPV results within the study period; b) either partner had at least 1 HPV positive visit, was included in the analysis. HPV concordance was defined as the proportion of co-positive couples for a specific HPV type out of all HPV exposed couples. For groups of genotypes , concordance was defined as the proportion of couples co-positive for at least one of any type HPV, 14 oncogenic HPVs and 32 non-oncogenic HPVs. The Chi-square test was performed for specific HPV types to determine whether the observed concordant events significantly exceeded the expected valueAnalysis of male-to-female and female-to-male transmission was conducted separately under a type-specific principle. The infection was treated as the calculating unit and multiple infections in one participant were counted as separate events. In this study, we observed that the transmission event was far less likely to occur after the infection in the partner in whom HPV infection originated had been cleared (data not shown). In view of this fact, we employed an “HPV transmission window” to define the transmission event and calculate the person-time at risk of HPV transmission in couples. The “transmission window” opens at the time of the visit when one partner in a couple is found to be positive for a specific type of HPV for the first time, while his/her partner has been negative for the corresponding HPV type from enrollment to the time of that visit. The window closes at the earliest occurrence of any one of the following: a) the adjacent visit subsequent to the last positive visit of this infection in the HPV index partner, b) a type-specific transmission event occurs, c) the follow-up of the at-risk partner right censored. An HPV transmission event was defined as newly acquired type-specific infection in the at-risk partner within the transmission window. A couple was included in the transmission analysis if the at-risk partner had at least 2 consecutive visits within the “HPV transmission window”. Transmission rates were then calculated by dividing the number of transmission events by the total number of person months of transmission windows, and 95% CIs were estimated based on Poisson distributionFactors for HPV transmission evaluated in the current study included average age, average lifetime number of sexual partners and sexual frequency per week of study couples, as well as the level of education, type of job and presence of persistent HPV infection in a given couple. Persistent HPV infection was defined as ≥3 consecutive positive visits for the HPV-exposed partner in this study. Factors associated with HPV concordance were assessed using a logistic regression model, in which the Generalized Estimating Equation (GEE) was used to take into consideration of within subject correlationAll statistical analyses were performed using STATA version 11.2 . Tests were two sided, and had a significance level of 0.05.In this study, 93.4% and 98.3% of genital exfoliated cell specimens from men and women were positive for the human beta-globin gene and were thus considered adequate for subsequent HPV testing. A total of 874 couples provided at least one valid HPV positive result for both partners, among whom HPV positivity was found in 749 of 4480 visits (16.7%) in men, and 345 of 4215 visits (8.2%) in women during the study period. The proportion of multiple infection was 8.5% in men (64/749) and 8.7% in women (30/345). Among these 874 couples, 406 (46.5%) HPV exposed couples provided at least 2 valid specimens from both partners and were included in the concordance evaluation. The median follow-up time was 33.6 months for males (IQR 18.8-38.0) and 30.4 months for females (IQR 23.8-31.9). For transmission analysis, 296 HPV exposed couples in whom HPV transmission had potential to occur within the defined transmission window were included with a median follow-up time of 35.7 months for males (IQR 19.4-38.3) and 30.5 months for females (IQR 25.1-32.2).p < 0.001) reported having multiple sexual partner. Around half of these couples  reported having sexual intercourse ≤1 each week. Persistent HPV infections were detected more frequently in males . No appreciable differences were observed in couples included in or not included in the concordance analysis and the transmission analysis, except that greater numbers of lifetime sexual partners were observed in couples included in the concordance analysis as compared with excluded couples (p = 0.013).p = 0.054, for oncogenic vs. non-oncogenic). Specifically, concordance for HPV-16, HPV-18, HPV-11 and HPV-6 was 12.17%, 5.45%, 22.22% and 42.86%, respectively  in male-to-female transmission. The female-to-male transmission rates for any HPV type, oncogenic, non-oncogenic HPV and HPV-16/18/11/6 infection were 5.56, 2.37, 17.01 and 2.73 per 1000 PM, respectively. The transmission rate of oncogenic types was significantly lower than non-oncogenic types (p = 0.012) in female-to-male transmission.Male-to-female transmission rates for any HPV type, oncogenic, non-oncogenic HPV and HPV-16/18/11/6 infection were 7.11, 12.13, 4.77 and 10.48 per 1000 PM respectively as shown in M-to-F vs. F-to-M = 4.89, 95%CI: 1.36-17.55; p = 0.015), but the male-to-female transmission rate was significantly lower than the female-to-male transmission rate in non-oncogenic HPV types . For vaccination types, the risk of male-to-female transmission was higher than that of female-to-male transmission, but this difference did not reach a significant level   .For factor analyses, a significantly lower risk of concordant infection for both any and oncogenic HPV was observed in older couples aged 46-65 years, as compared with couples aged 25-45. Persistent HPV infection was consistently associated with an increased risk of both HPV concordance and transmission. Couples who reported having >1 lifetime sexual partner had a significantly elevated risk of HPV concordance in any and oncogenic HPV, and also had a significantly elevated risk of any type of HPV transmission. Risk of both HPV concordance and transmission in non-oncogenic types was significantly increased in couples who reported having 2 episodes of sexual intercourse per week as compared to couples who reported having sexual intercourse ≤1 per week .In this study, we investigated the positive concordance and transmission rate of genital HPV infection among 874 couples aged 25-65 from northern rural China with population-based sampling. We found that observed concordance in the majority of HPV types was significantly higher than expected. The risk of male-to-female transmission was around 4 times higher than that of female-to-male transmission for oncogenic HPV, and conversely, the male-to-female transmission rate of non-oncogenic HPV was 73% lower than that of female-to-male transmission.et al.et al.et al., the mean number of lifetime sexual partners was 11.0 for men and 5.3 for womenet al. reported that the proportion of >5 lifetime sexual partners was 64.4% for men and 59.4% for womenet al. also reported that the median number of lifetime sexual partners was 12.0 for men and 7.0 for womenet al. found the proportion of ≥3 lifetime sexual partners was 74.7% and 73.7% in men and womenUntil now only 5 prospective studies have investigated HPV transmission in heterosexual couples. All these studies67891926p = 0.003). In the 5 published studies we evaluated for comparison, only Burchell et al. reported the proportion of “persistent” infection after 4 months, and estimates found of 59.5% for men and 60.6% for women were without significant gender difference2930Another possible explanation was the similar prevalence but higher proportion of persistent infection in oncogenic HPVs in males as compared to females observed in our study  younger couples are more sexually active, 2) older couples have longer periods of HPV exposure, and antibodies generated by previous infections may protect against reinfection by identical HPV types192930Two limitations of this study should be noted. First, although we have investigated over 800 couples for as many as 7 visits, statistical power is still limited when analyzing specific types due to the low prevalence and transmission incidence of genital HPV infection in this population. Larger studies with greater numbers of cycles of evaluation are still needed to validate our conclusions. Secondly, as couples in the current study were not asked to avoid sexual intercourse for 48 hours before each visit, superficial virus deposited by sexual partners during recent intercourse may have confounded results. However this would likely not significantly impact our conclusions in view of the fact most couples reported having ≤1 episodes of sexual intercourse per week.The results of our study indicate men in rural China play a much more important role than men in other populations as a source of genital oncogenic HPV infection. While anti-HPV intervention programs are already being planned for women to decrease the burden of HPV-related disease in this population, this further argues for the necessity of such programs in young and sexually active male adults. This is the first population-based prospective study employing a rigorous periodic sampling method to assess genital HPV transmission in a Chinese population. As no national HPV and cervical cancer screening program has been established in rural China, and in view of the fact prophylactic vaccines are still under clinical trial in China pending government approval, our findings may be of significance in targeting a high risk population for development of cost-effective vaccination-based cervical cancer and HPV screening programs.How to cite this article: Liu, M. et al. Transmission of genital human papillomavirus infection in couples: a population-based cohort study in rural China. Sci. Rep.5, 10986; doi: 10.1038/srep10986 (2015).


In compliance with national and international guidelines, non-pregnant women with cervical intraepithelial neoplasia grade 3 should be treated by cervical conization. According to the definition of the large loop excision of the transformation zone (LLETZ) operation, the lesion needs to be resected, including the transformation zone. It is well known from the literature that the cone size directly correlates with the risk of preterm delivery in the course of a future pregnancy. Thus, it would be highly desirable to keep the cone dimension as small as possible while maintaining the same level of oncological safety.The aim of this study is to analyze whether resection of the lesion only, without additional excision of the transformation zone, is equally as effective as the classical LLETZ operation regarding oncological outcome. We are performing this prospective, patient-blinded multicenter trial by randomly assigning women who need to undergo a LLETZ operation for cervical intraepithelial neoplasia grade 3 to either of the following two groups at a ratio of 1:1: (1) additional resection of the transformation zone or (2) resection of the lesion only. To evaluate equal oncological outcome, we are performing human papillomavirus (HPV) tests 6 and 12 months postoperatively. The study is designed to consider the lesion-only operation as oncologically not inferior if the rate of HPV high-risk test results is not higher than 5 % compared with the HPV high-risk rate of women undergoing the classical LLETZ operation.In case that non-inferiority of the “lesion-only” method can be demonstrated, this operation should eventually become standard treatment for all women at childbearing age due to the reduction in risk of preterm delivery.DRKS00006169. Date of registration: 30 July 2014.German Clinical Trials Register (DRKS) Identifier:  Cervical cancer is the third most common cancer diagnosis and the fourth-leading cause of death in women worldwide, responsible for 9 %  of all new cancer cases and 8 % 275,100) of deaths caused by cancer in women in 2008 [00 of deacervical intraepithelial neoplasia (CIN), which is distinguished in three degrees of severity (CIN 1 to 3) [Precursor lesions of squamous cell carcinoma of the cervix uteri, which represent about 80 % of cervical cancers, are referred to as  1 to 3) . CompareA prerequisite for the development of cervical dysplasia is a persistent infection with high-risk human papillomavirus (HPV) types . AnogeniHPV infections affect undifferentiated basal cells of the cervical epithelium within the transformation zone. The transformation zone is the area of the cervix where the columnar epithelium of the endocervix meets the squamous epithelium of the ectocervix.transformation zone of the cervix. Recently, a hypothesis has been stated that cervical cancer emerges from a newly described embryological cell population. These so-called cuboid cells are located directly at the squamocolumnar junction [Before puberty, the squamocolumnar junction lies within the endocervical canal. Owing to hormonal changes during puberty, the columnar epithelium extends outward and the squamocolumnar junction moves onto the ectocervix. Under exposure to the acidic vaginal environment, columnar epithelium can undergo physiological metaplasia and transform into squamous epithelium. The new squamocolumnar junction is then closer to the endocervix compared with the original squamocolumnar junction, and the zone in between is called the junction .large loop excision of the transformation zone (LLETZ). The rationale for the removal of the transformation zone in addition to the lesion itself is that both the precursor lesion and cervical cancer itself typically arise in the area of the transformation zone.In accordance with national and international guidelines , standarIn addition to general complications such as intraoperative or postoperative bleeding, infection, pain and scarring, cervical conization is associated with a significantly high rate of cervical weakness and a consequent increase in miscarriages and premature birth rates in future pregnancies –12. ThisIn a large retrospective study of over 150,000 women treated for CIN 3 between 1958 and 2008, it has lately been shown that women who received treatment more recently were at greater risk of developing cervical cancer . It is sWith awareness of this development, it is important to respecify the necessary extent of a conization. Primarily, the optimum cone size needs to guarantee the highest oncological safety defined by low recurrence and high R0 resection rates. Simultaneously, the extent of the resection should be as small as possible to reduce the morbidity risk during subsequent pregnancies.The primary objective of the present study is therefore to compare the classical LLETZ operation  with resection of the colposcopically abnormal lesion only in the light of oncological safety.The Evaluation of Clinical Outcome after Reduction of Conization Size (or ECO-ROCS) trial is a multicenter, prospective, parallel-group, patient-blinded, non-inferiority randomized controlled trial in which we are assessing oncological comparability of two conization procedures: (1) classical LLETZ, comprising excision of the dysplastic lesion, including resection of the transformation zone; and (2) excision of the dysplastic lesion only.This study is being conducted in 13 study centers, all of which run a specialized dysplasia clinic (Munich (two), Hannover, Wolfsburg, Berlin, Kiel, Regensburg, Stralsund, Hamburg, Düsseldorf, Aachen, Ulm, Münster). The participating study centers and investigators are fully listed in the German Clinical Trials Register (DRKS00006169).A sample size of 892 patients was calculated with the POWER procedure of SAS for Linux 9.3 software  using the following parameters: non-inferiority margin of 5 %, α = 0.05, 80 % power and one-sided test. A rate of 90 % negative HPV test results 6 months after conization was taken as a reference  for bothThe statistical inference regarding the primary outcome will be based on a one-sided 95 % confidence interval for the risk difference, calculated as the proportion of HPV-positive patients in the control group minus the corresponding proportion in the experimental group. If the upper limit of the confidence interval is less than the non-inferiority margin of minus 5 %, non-inferiority will be regarded as established.Patients are being seen and diagnosed in specialized dysplasia outpatient clinics of the participating study centers. After they provide us their written informed consent, participants fulfilling all required inclusion and exclusion criteria are given a five-digit pseudonym (patient identifier), with the first two numbers indicating the study center and the latter three numbers indicating a sequential patient number. Patients are randomly assigned by their pseudonym with equal probability to either of the two treatment arms, using a 1:1 ratio. Randomization is carried out using the web-based Randoulette randomization service of the Institute for Medical Informatics, Biometry and Epidemiology, University of Munich, Germany. The platform offers a web-based form in which to enter patient identifier, sex, age and study center, as well as the strata of the patient. Access to the randomization service is provided to the two leading investigators at each study center. Patients are stratified by age (>30 or ≤30 years) and study center.After the information is entered, the study arm is selected according to a predefined randomization scheme, in this case an unblended, stratified block randomization. To maintain allocation concealment, the exact length of blocks is kept secret by the Randoulette randomization service. The entered information, as well as the randomization results, are stored in the Randoulette database.Approval of trial protocol and informed consent documents have been obtained primarily from the University of Munich Institutional Review Board (project number 275-14), followed by the responsible ethics committees and/or state chambers of physicians of the 12 other study centers .http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6/E6_R1_Guideline.pdf) and the European Medicines Agency’s CPMP/ICH/135/95 guideline . Important protocol modifications will be reported to all relevant parties. All participants complete informed consent forms prior to participation in the trial.All study staff commit themselves to the Declaration of Helsinki (2013 version), as well as to all pertinent national laws and the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) harmonized tripartite guidelines for Good Clinical Practice issued in June 1996  who havPatients are excluded at any time upon their request or if any of the following applies: pregnancy at time of inclusion and during the first 6 postoperative months, intake of immunosuppressants, known HIV infection, malignant disease requiring treatment or inadequate colposcopy.To exclude bias by unconsciously different behavior, the study is patient-blinded for the first 6 postoperative months. In case of complications, however, access to the surgical report is possible at any time.loop electrosurgical excision procedure electrode. Procedures are performed by experienced surgeons at 13 German dysplasia centers under colposcopic control and are carried out as shown in Figs. Both surgical techniques are performed using a thin, low-voltage, electrified wire loop known as a In the lesion-only resection, the dysplastic area is removed with a safety margin of 2 mm. In the classical LLETZ, the lesion, including the transformation zone, is resected. Because the outer border of the transformation zone cannot be determined colposcopically, the following definition for resection of the transformation zone was stated after expert consensus. After identification of the squamocolumnar junction, an area of 4 mm surrounding the junction is defined as the transformation zone. In the event that the iodine-negative zone is greater than the defined area, the iodine-negative area is considered the transformation zone and should be removed as well.Postoperatively, cone size is measured before fixation by submergence in a fluid-filled cylindrical vial using Archimedes’ principle. Afterward the specimen is evaluated histologically for the grade of dysplasia and achievement of resection margins.According to German guidelines, the first follow-up examination is performed after an interval of 6 months. A Pap smear, HPV test and colposcopy are performed. In cases of colposcopically suspect lesions, a biopsy is taken. A second postoperative control is carried out 12 months after conization.The primary endpoint is the rate of negative high-risk HPV tests after 6 months, because a negative HPV test is generally regarded as evidence of successful treatment. The negative predictive value for a negative HPV test after conization is between 92 %  and 100 Because it is possible, from a clinical point of view, that patients who have received the lesion-only operation simply need more time for the viral infection to be eliminated, HPV is tested again in the postoperative control after 12 months. Further secondary endpoints are Pap smear results in both postoperative controls, biopsy results (if applicable) and resection margins.It should be noted that, even with classical conization, resection margins are positive or only questionably negative in up to 25 % of cases.If there is CIN 1 detectable at the cutting margins, recurrence or persistence is expected in only 0 % to 5 % of participants. In the case of CIN 3 at the surgical margins, recurrence can be expected in 20 % to 25 %. If non in sano resection happens at the endocervical margin, risk increases to 30 %. If both, endo- and ectocervical resection margins are affected with CIN 3, risk is highest with 50 % . ImportaFor this reason, an immediate reconization usually is not indicated, but should be assessed according to individual risk and discussed with the patient. Only after further histological confirmation of CIN during the first postoperative control after 6 months will reconization be recommended.Thus, the initially conservative approach in terms of a checkup after 6 months is also justified in the case of an increased incidence of positive surgical margins. This can be seen especially in light of the expected reduction of complications associated with surgery.An interim analysis will be performed at the postoperative 6-month control of the first 250 patients included. The primary outcome is the result of HPV tests. If the rate of positive HPV tests after 6 months in patients who have undergone lesion-only surgery is higher by more than 15 % than among patients who have had classical LLETZ, the study will be terminated. Because the interim analysis was included exclusively for safety reasons and thus does not involve a test of the non-inferiority study hypothesis, the probability of a type I error  should not be affected.Because HPV infection is the cause of dysplasia, further analyses to investigate HPV infection are performed in a subset of 250 patients. A particular focus is on the genotyping of HPV infection because often multiple infections with different HPV types are present simultaneously, especially in young patients. Thus, there is the risk that, despite eradication of the lesion-causing HPV type, the conventional HPV test result may continue to be positive because infection with another HPV type is present without clinical consequence.To make a statement with respect to a more valid clinical consequence of HPV infection, detection of transcriptional activity of E6 and E7 is analyzed. Expression of viral oncoproteins E6 and E7 plays a crucial role in HPV-mediated carcinogenesis, as it interferes with the cell cycle, causes cell cycle progression (E7) and at the same time prevents apoptosis (E6). This occurs through the inactivation of oncosuppressor proteins p53 and retinoblastoma protein (pRb). pRb in particular prevents cells from entering S-phase and thus has antiproliferative effects –37. As oThe described additional analyses are performed at five study centers .With knowledge about the obstetric complications of cervical conization, surgical radicalism has increasingly declined in recent years. To date, it has not yet been systematically analyzed whether consistent oncological safety can be guaranteed by removing less tissue. In discussions about the optimal extent of conization with leading colposcopists in Germany, it furthermore turns out that the size of the cone varies significantly, depending on the individual surgeon.The aim of this study is thus to define a universal standard for cervical conization that combines the highest level of oncological safety with minimal obstetric risk. To that end, we compare two surgical methods with each other: (1) classical LLETZ, in which the lesion is removed, including the transformation zone; and (2) excision of the lesion only.There are certain limitations to our study. In patients in whom the entire transformation zone is affected by the lesion but who will be randomized into the lesion-only group, the extent of the operation would in fact be the same as in patients randomized to undergo the classical LLETZ operation. This could ultimately lead to limited discrimination between the two study groups. To evaluate this influence on the results, every surgeon must indicate the extent of the dysplastic area (CIN/ectocervix in percent) at the time of the operation. However, including only patients with small lesions would lead to a severe selection bias by recruiting patients according to more subjective criteria. Furthermore, generalization of the study results could no longer be guaranteed, because only a subgroup of patients with high-grade dysplastic lesions would be included.As of May 2015, we are still recruiting participants.


Cis-acting HPV RNA elements and cellular RNA-binding proteins control HPV mRNA splicing and polyadenylation. These interactions are believed to play a particularly important role in the switch from early to late gene expression, thereby contributing to the pathogenesis of HPV. Indeed, it has been shown that the levels of various RNA binding proteins change in response to differentiation and in response to HPV induced cervical lesions and cancer. Here we have compiled published data on RNA binding proteins involved in the regulation of HPV gene expression.The human papillomavirus (HPV) life cycle is strictly linked to the differentiation program of the infected mucosal epithelial cell. In the basal and lower levels of the epithelium, early genes coding for pro-mitotic proteins and viral replication factors are expressed, while terminal cell differentiation is required for activation of late gene expression and production of viral particles at the very top of the epithelium. Such productive infections are normally cleared within 18–24 months. In rare cases, the HPV infection is stuck in the early stage of the infection. Such infections may give rise to cervical lesions that can progress to cancer, primarily cancer of the uterine cervix. Since cancer progression is strictly linked to HPV gene expression, it is of interest to understand how HPV gene expression is regulated.  Human papillomaviruses (HPV) are small DNA viruses with a strict tropism for human epithelial cells ,2. They The HPV virion is approximately 50 nm in diameter and consists of eight kilobases, double stranded, circular DNA genome embedded in an icosahedral capsid formed by the HPV L1 and L2 capsid proteins . The virin vitro . T. T63]. TIn addition to the U-rich region that binds CUG-BP, the bipartite negative RNA element in the HPV16 late UTR element encodes 5'-splice site-like sequences ,39,57. TThe splicing factor U2AF65 has been reported to bind GU-rich sequences immediately downstream of the U1snRNP binding sites in the HPV16 late UTR ,62, but in vitro of SR proteins and the HPV E2 protein by SRPK1 [HPV E4 proteins colocalize with the cellular SR-proteins kinase 1 (SRPK1) in human cells . Since Sby SRPK1 . HoweverIn this regard, it is of interest to compare with the adenovirus E4orf4 protein binds to SR proteins and to the major cellular phosphatase PP2A, which results in dephosphorylation of SR proteins . As a coin vitro and inhibits in vitro splicing [in vitro, suggesting that E2 interferes with cellular polyadenylation factors at the HPV16 early polyA signal pAE, thereby activating HPV late gene expression [The HPV E2 proteins is a DNA binding protein that binds to HPV LCR DNA to control transcription, and to recruit the cellular DNA polymerase together with HPV E1 to replicate the HPV genomic DNA . It alsosplicing . A recensplicing , but it splicing . In addisplicing . E2 affepression . The exppression . The recpression  is consiEpigenetic changes of the HPV16 genome have been described and include: CpG methylation of HPV DNA, and posttranslational modifications of HPV16-associated histones. More than hundred CpG motifs that could potentially be methylated are present in non-coding as well as protein-coding regions of the HPV16 genome, and they are unevenly distributed over the HPV16 genome . AlthougCellular RNA-binding proteins control HPV16 gene regulation at the level of RNA processing by binding to the HPV16 mRNAs. In this capacity, cellular RNA binding proteins play an important role in the natural course of the HPV16 infection, as well as during progression to cancer. Indeed, expression levels of many RNA binding proteins are altered in both premalignant lesions caused by HPV16 as well as in cervical cancer. A better understanding of the control of HPV16 gene expression by RNA binding proteins is necessary in order to understand how HPV16 causes disease. This research may uncover novel targets for antiviral drugs to HPV16 as well as predictive biomarkers for women at risk of developing cervical cancer.


PCR,products were purified and sequenced on the genetic analyzer ABI Prism 3100 Avant. Software programs Sequencing Analysis 5.1.1., SeqScape v2.6, BioEdit were used. Phylogenetic analysis was performed using the MEGA4.1 software with the Neighbour-Joining and Kimura 2-parameter method.Of 764 PCR positive samples there were sequenced 77 and 10 DNAs on genes L1 (296 bp) and E6/E7 (620 bp) HPV-16 regions, respectively. The phylogenetic analysis of gene L1 region established that generally all samples from Belarus clustered round reference-sequences from South East Asia and Europe, only 15 and 9 samples formed independent groups with each other. p-distances between the Belarusian samples fluctuated from 0.000 to 0.024 that points as to new virus penetration to the country, and on circulation of our "old" HPV-16 types which formed independent groups of samples on a phylogenetic tree. Separately the sample 63 from Gomel settled down, it didn't enter one of groups. Most likely, it is our "domestic" HPV-16 variant. Three viruses, two from Minsk and one from the Grodno region settled down in one group "Africa" with African reference - sequences, but separately from each other that points to a different origin of a virus at these patients. Sequencing on gene of E6 and E6/E7 region also divided viruses into groups. Also as well as on L1 gene on gene E6/E7 region virus formed, generally the general groups with Asian HPV-16 variants. In too time part of isolates settled down separately and formed independent groups.Thus, carried-out sequencing and phylogenetic analysis have showed that on the territory of Belarus circulation as "old" HPV-16 variants takes place, and there is a penetration of new types HPV because of borders of Belarus, generally from the countries of South East Asia.


Viral infections contribute to approximately 12% of cancers worldwide, with the vast majority occurring in developing countries and areas. Two DNA viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), are associated with 38% of all virus-associated cancers. The probability of one patient infected with these two distinct types of viruses is increasing. Here, we summarize the co-infection of EBV and HPV in human malignancies and address the possible mechanisms for the co-infection of EBV and HPV during tumorigenesis. Oncogenic viruses include various classes of DNA and RNA viruses that are necessary but not sufficient for the development of all types of malignancies. A unifying theme is that cancer develops in a minority of persistently infected people and only after many years of chronic infection . The majThe probability of a single patient who is infected with two or more distinct types of viruses is increasing , 12. VirThe implication that HPV has a role in the carcinogenesis and prognosis of cervical cancer is well established. However, many studies have found that HPV infection is linked to NPC as an etiologic factor –17 and tNPC is a common tumor in South China and Southeast Asia, but it is a rare disease among Caucasians , 25. ForThe co-infection of EBV and HPV is usually found in NPC patients from endemic regions. In one study, NPCs in Iranian patients were detected by in situ hybridization, and a low percentage (15%) of EBV-positive NPC patients had HPV sequences (HPV type 6/11 or HPV type 16/18) . ApproxiHowever, co-infection of EBV and HPV is less common in NPC patients from some regions in the United States compared with that from the South China. HPV might be the etiologic factor in some EBV-negative, non-keratinizing NPC patients, including Caucasian North American patients . A recenUsing established EBV-negative NPC cell lines and advanced molecular biology techniques, our understanding of molecular alternations in these cells has been tremendously improved –43. UntiCo-infection of EBV and HPV is frequently found in endemic NPC, but it is less common in Caucasians than in Asian populations; HPV is an important etiologic factor in EBV-positive NPC among Caucasians . FurtherCervical cancer is the third most common malignancy in women and the fourth most common cause of death from cancer . It is wMany studies have found that there is a co-infection of high-risk HPV and EBV in cervical tissues , 59, 60,EBV can transform cells bearing the EBV/C3d  receptor, making them receptive to other oncogenic stimuli , 64. TheHPV types 16 and 18 are the two most common high-risk HPV types and were involved in the development of cervical cancer , 70. HPVBreast cancer is the second leading cause of cancer-related death in women around the world . The etiInterest in EBV as a possible breast agent arose from the presence of EBV in breast tissue . Some reEBV is a major contributor to Hodgkin lymphoma, and significant associations exist between the incidence of Hodgkin lymphoma and breast cancer, suggesting that EBV may act as an etiologic factor for some breast cancers . Some EBCo-infection of EBV and HPV appears to be present in a significantly higher proportion in breast cancers than in normal breast epithelial cells , 83. TheAs many studies provide insufficient evidence, testing in larger sample groups is needed to demonstrate that EBV and HPV are associated with breast cancer . To clarAmong men, prostate cancer is the most common cancer and the second leading cause of cancer-related death . BecauseAlthough EBV is a ubiquitous virus with known oncogenic potential, few investigations have demonstrated its association with prostate cancer. Several reports showed that EBV was present in prostate tissues. In Sweden, EBV was present in 8.8% (31 of 352) of benign and malignant prostate tissues , and in Both HPV type 18 and EBV gene (EBNA1) sequences were identified in a high and approximately equal proportion of normal, benign, and prostate cancer specimens . The seqWe summarized the ratio of co-infection of EBV and HPV in several malignancies Table . It is pLung cancer is the leading cause of cancer-related death in the world, and it is unlikely that HPV or EBV are important etiologic agents in lung adenocarcinoma, even among people who have never smoked ; in factEsophageal squamous cell carcinoma (ESCC) is prevalent worldwide and is particularly common in certain regions in Asia, including Southeast China. One group of researchers showed that EBV infection contributed to the molecular pathogenesis of ESCC , whereasAltogether, it is unclear whether the co-existence of EBV and HPV acts as a promoter or bystander in the development of these carcinomas aforementioned. To improve the treatment and prognosis, it is important, from the standpoint of genetic etiology, to clarify the connection.Oncogenic viruses induce cancer by a variety of mechanisms that do not include the viral episome or lytic replication; for example, the immune system can have a deleterious or protective role, with increasing immunosuppression in human virus-associated cancers and with activating DNA damage response , 101. ReStudies on the roles of chromatin in the regulation of viral infections have revealed the significance of dynamic viral-host chromatin interactions in determining viral infections as well as the interplay between viral-encoded proteins in modulating chromatin. These interactions are especially significant for multiple epigenetic events, such as chromatin assembly, histone and DNA modifications, and higher-order chromosome structures that control viral episome generation and maintenance after viral infection –113. TwoThe effect of such reprogramming on the viral life cycle also remains unknown; it will be interesting to evaluate the host-virus interactions through combined genome-wide analyses. Recently, a large-scale functional genomic analysis of EBV, together with over 700 publicly available high-throughput sequencing data sets for human lymphoblastoid cell lines, were mapped to the EBV genome, and viral lytic genes were found co-expressed with cellular cancer-associated pathways, indicating that the lytic cycle may play an unexpected role in virus-mediated oncogenesis , 115. MoOncogenic virus-induced cancers are an important global concern. Remarkable progress continues to be made in the global fight against cancer by preventing the spread of these viruses, by developing and distributing safe and effective vaccines against them, and by identifying the oncogenic mechanisms used by these viruses, enabling the development of new treatment options for these virally associated human malignancies. Although there is a vaccine for HPV , there i


P<0.01), mother’s education level (P<0.01), mother’s occupation (P<0.01), household income (P<0.01) and residence type (P<0.01). Knowledge about HPV/HPV vaccine was poor with a mean total score of 3.56 (SD = 2.40) out of a possible score of 13. Mothers with a higher education level reported higher levels of knowledge (P = 0.02). Slightly more than one-fourth (26.49%) of mothers expressed their potential acceptability of HPV vaccine for their daughters. Acceptability increased along with increased daughters’ age (P<0.01), household income (P<0.01) and knowledge level (P<0.01). House wives and unemployed mothers had the highest acceptability (P<0.01). The most common reasons for not accepting HPV vaccination were “My daughter is too young to have risk of cervical cancer (30.95%)”, “The vaccine has not been widely used, and the decision will be made after it is widely used (24.91%)”, “Worry about the safety of the vaccine (22.85%)”. Awareness and knowledge of HPV/HPV vaccines are poor and HPV vaccine acceptability is low among these Chinese mothers. These results may help inform appropriate health education programs in this population.In preparation for the introduction of human papillomavirus (HPV) vaccine, we investigated awareness and knowledge of HPV/HPV vaccine and potential acceptability to HPV vaccine among mothers with a teenage daughter in Weihai, Shandong, China. A cross-sectional survey was conducted in 2013 with a sample of 1850 mothers who had a daughter (aged 9–17 years) attending primary, junior and senior high schools. In the final sample , awareness of HPV was reported by 305 (19.32%) mothers. Awareness varied significantly by daughter’s age ( More than 270 000 women die of cervical cancer worldwide every year and over 85% of these deaths occur in low and middle income countries . CervicaThe first HPV vaccine was licensed by the US Food and Drug Administration (FDA) in 2006 . CurrentHPV vaccination rates in many countries have been suboptimal since HPV vaccines were licensed –18. The Currently, a number of phase III clinical trials with two international and one domestic HPV vaccines are ongoing in China ,19,20. TAccording to WHO  and lessBefore HPV vaccines are licensed, it is important to understand HPV-related awareness, knowledge and potential acceptability of HPV vaccine among both adolescent girls and significant others. The aim of this study was to explore these constructs in a highly influential population—mothers of teenage daughters to inform the forthcoming introduction of HPV vaccines in China.From November to December, 2013, we conducted two cross-sectional surveys both using self-administered anonymous questionnaires among a sample of mothers and a sample of adolescent girls, respectively. These surveys were conducted in Weihai, a city in eastern Shandong Province, PR China, with a total population of about 2.8 million. This paper reports results from the mother survey only.The study was approved by the Ethics Review Board of the Center for Disease Control and Prevention, Shandong province. A written consent was obtained from each mother prior to the survey.This survey targeted mothers of daughters aged 9–17 years old. According to Chinese education system, the great majority of adolescents within this age range are students attending the last two grades (4–5) of primary schools (9–10 years old), and the 4 grades (1–4) of junior high schools (11–14 years old), and the 3 grades (1–3) of senior high school (15–17 years old). We used a multi-stage cluster sampling approach to determine our sample of mothers of these female students.Firstly, all public schools with at least 2 classes in each of the aforementioned grades in the study region were identified and were grouped by school level  and location type . For each level and location type, 2 schools were approached by the research team in a convenient manner. A total of 12 schools, including 4 primary schools, 4 junior high schools and 4 senior high schools balanced by school location, agreed to participate. For each grade in these schools, 2 classes were randomly chosen and all female students’ mothers in selected classes were invited to participate in the survey.The questionnaire was developed by the research team based on literature review and a pilot study. The questionnaire includes three parts: (1) demographic characteristics, such as age, education level, occupation and monthly family income et al; (2) awareness and knowledge of HPV and HPV vaccine; (3) acceptability and reasons for not accepting vaccination.Awareness of HPV was assessed using the question “Before today, had you heard of HPV?”. Mothers who answered “yes” to this question were considered to be aware of HPV. This question has been previously used in other studies in China  and otheKnowledge of HPV and HPV vaccine was collectively assessed using 13 multiple choice questions with single correct answers. The first 8 were used to assess knowledge on HPV and the rest were used for knowledge on HPV vaccine . Only moThe question “Are you willing to vaccinate your daughter with HPV vaccine once it is available?” was used to assess the potential acceptability to HPV vaccine. This method was also used in previous research ,7, 12. FA pilot survey with a small sample (n = 71) of mothers was conducted to test the acceptance and feasibility of the questionnaire. Based on the results from the pilot, minor wording changes were made in the final version which was then used in the main survey.The questionnaire was taken home by female students along with a written instruction. Mothers of female students were asked to fill out the questionnaire independently without consulting with any other persons or resources. Filled questionnaires were then brought back to school to the head teachers the second day.The head teachers for selected classes agreed to collect and scan the returned questionnaire to examine 1) if there are obvious errors or unexpected answers in terms of mothers’ and daughters’ ages; and 2) if there are any missing items for other demographic questions excluding household income due to privacy concerns. In total, 17 questionnaires  were identified by the head teachers and were returned to mothers to complete.Key variables included awareness and knowledge of HPV/HPV vaccine, potential acceptability for daughters and reasons for not accepting HPV vaccination.Correct answer to each item of HPV/HPV vaccine knowledge was assigned with 1 point; 0 point was given if the question was incorrectly answered or not answered. The possible range of the total knowledge score was 0 to 13. In regression analysis, those who did not hear of HPV and skipped these items were given a total knowledge score of zero. Finally, all respondents were divided into three groups based on their total knowledge scores: no knowledge (score = 0), low knowledge (score = 1–4) and high knowledge (score = 5–13).α level of 0.05.The data were entered into a database developed using EpiData  independently by two persons. The SPSS 13.0  was used to analyze the data. The Chi-square test was used for analyzing categorical data. Logistic regression analysis was used to analyze the associations between potential predictors and vaccine acceptability. Statistical significance was assessed by two-tailed tests with A total of 1850 mothers were invited to participate in this survey. With 258 mothers refusing to participate, a total of 1592 questionnaires were collected. Among these questionnaires, 14 questionnaires being excluded due to logic errors in question response (such as expressing willingness to vaccination while choosing reasons for not accepting vaccination for daughter). 1578 mothers (85.30%) were finally included in the analysis ..The mean age of these mothers and daughter were 40.25±4.20 years (range: 30–56 years) and 13.03±2.44 years (range: 8–19 years), respectively. Most commonly reported education level, occupation and annual household income were junior high school, farming, and 50–100 thousand CNY . Urban and rural mothers made up 38.09% and 45.94% of the sample, respectively. All demographic characteristics are presented in CI: 17.38–21.28) mothers had heard of HPV before investigation. There was no statistically significant difference in the proportion of HPV awareness by mothers' age group . Awareness was significantly more common among mothers of daughters older than 16 years , having an education level of senior middle school or above , having a professional occupation , with an annual household income above 100 thousand CNY , and residing in urban areas   .SD = 2.40) out of a possible score of 13. A total of 45 (14.75%) mothers were classified as having no knowledge related to HPV/HPV vaccine, 179 (58.69%) had low knowledge, and 81 (26.56%) had high knowledge. There were no statistically significant differences in the knowledge level by mothers’ age , daughters’ age , mother occupation , household income , and residence type . Mothers with a higher level of education were more likely to be classified as having high level of HPV-related knowledge  (Among mothers who were aware of HPV (n = 305), the mean total score was 3.56 ( = 0.02) .Among those who responded any of the knowledge questions (n = 305), nearly half or more correctly answered “What’s the main risk factor of cervical cancer?” (Item 7) (58.36%), “What is HPV?” (Item 1) (57.05%), “What’s the main hazard of HPV infection for female” (Item5) (48.85%), and “Is persistent HPV infection the cause of cervical cancer?” (Item 8) (47.54%). Poorer responses were found for the items “Can wives be infected with HPV in normal sexual behavior?” (Item 4) (17.38%), “What’s the main hazard of HPV infection for male?” (Item 6) (15.08%), “Can HPV vaccine effectively prevent genital warts for males?” (Item 10) (13.77%), “How many times does HPV vaccines need to be injected” (Item 11) (6.89%), “Are HPV vaccines effective on HPV infected person?” (Item 13) (5.25%), and “What is the best timing of HPV vaccination?” (Item 12) (4.92%) .CI: 24.31–28.67) mothers expressed willingness to vaccinate their daughters. Five variables were significantly associated with acceptability in Chi-square tests, including daughters’ age , education , occupation , household income , knowledge level . Four variables were significantly associated with acceptability in multivariate regression analysis. Increased acceptability of HPV vaccination was associated with higher daughters’ age, household income and knowledge score. The acceptability of professional and unemployed women is relatively higher , followed by “The vaccine has not been widely used, the decision will be made after it is widely used” (24.91%), and “Worry about the safety of the vaccine”(22.84%) .According to the recommendation by WHO , one tarIn this study, only 19.33% of all mothers were aware of HPV before investigation which is slightly lower than the results from another study in China 25.1%) [5.1% [2] We found although a number of factors are associated with the awareness of HPV, but only higher education among mothers was significantly associated with a higher level of knowledge, which is similar to another study . This fiA large variation in the awareness was found among different knowledge items, with the correction answers ranging from 4.92% to 57.05% among mothers who heard of HPV (n = 305). In general, knowledge on HPV vaccines is poorer than that on HPV. Knowledge on HPV vaccines may be of particular importance for the introduction and implementation of vaccination programs. Such knowledge may help resist misinformation about HPV vaccines and to boost public confidence in vaccination. Future education programs should have a focus on HPV vaccine-related knowledge.Different knowledge items may have different implications for HPV vaccination. For example, women who lack of knowledge of “Wives can be infected with HPV in normal sexual behavior” may underestimate the risk of HPV infection and refuse vaccination. In fact, the HPV infection is very common that 50% of women contract a genital HPV infection within 2 years after onset of sexual activity . The infThe potential acceptability 26.5%) for HPV vaccine of this study is lower than the results from another study in China (36.20%) 6.5% for ,25. TherSecondly, Lack of confidence in the efficacy and safety of the vaccine. The against reason of“the vaccine has not been widely using, the decision will be made after it is widely used (24.91%)” and “Worry about the safety of the vaccine (22.85%)” ranked the second and the third respectively, account for about half of the total reasons. It reflected the doubts of vaccine efficacy and safety essentially. In today’s China, more and more adverse events after vaccination were reported incorrectly, which triggers an irrational panic for vaccination safety .When a nThirdly, the cost of HPV vaccine is another obstacle for acceptance of HPV vaccine. Despite only 3.79% mothers against HPV vaccination due to the cost of HPV vaccine in this study, but the result of this study shows the higher household income, the higher acceptability of HPV vaccination. A study  in AustrThe essence of acceptability analysis is “vaccine hesitancy”. This issue is complex and context specific, varying across time, place and vaccines . In thisThere is no “magic bullet” or single intervention strategy that works for all instances of vaccine hesitancy . The taiThis study is strengthened by analyzing awareness, knowledge and acceptability of HPV/HPV vaccine in a large sample of Chinese mothers comprehensively. For the first time we examined some core knowledge items of HPV/HPV vaccines that were developed based on previous international and national studies and perceived importance in the Chinese social context. This will help identify specific factors that affect acceptance at a micro level, and to develop tailored vaccination strategies.There are a number of limitations in our study that must be addressed. Firstly, our study was conducted only in an economically developed city in China and was not a multi-center study. Therefore our results may not be generalized to other areas, especially underdeveloped regions of China. Secondly, there may be a potential response bias in the study, since the questionnaire was filled by mothers themselves who may tend to answer socially desirable responses. Thirdly, because only a few (n = 9) mothers reported a total knowledge score of 9 or higher, we used a wide range to define the “high-level knowledge (scored 5–13) which may have resulted in some misclassification. However, one of our key results, that is, more knowledge was significantly associated with potential acceptance, remained unchanged when we removed the 7 respondents from the analysis. Finally, mothers with a daughter attending primary and senior high schools were slightly over sampled because the classes in these schools are smaller than those in junior high schools. In this study, we found positive relationships between daughters’ age and better awareness and higher acceptability. Therefore, the overall awareness and acceptability among all mothers of girls of study ages may have been overestimated.The awareness and knowledge of HPV/HPV vaccine and acceptability of HPV vaccine are poor in Shandong province of China, which may be a major obstacle for the potential introduction of HPV vaccination. The acceptability of HPV vaccination is associated with knowledge. Therefore it is critical to conduct effective education programs to raise awareness and knowledge of HPV/HPV vaccine, and in turn to boost public confidence regarding vaccine safety and effectiveness to improve the HPV vaccine acceptability.S1 Dataset(XLS)Click here for additional data file.