COMP mutation  in a Chinese PSACH family. We detected the plasma levels of COMP and type II collagen (CTX-II) in the four affected individuals. The results showed the levels of plasma COMP significantly decreased and plasma CTX-II significantly increased in the three PSACH patients with COMP mutation. However, both plasma levels of COMP and CTX-II were not to have found significant difference between the presymptomatic carrier and the age-matched subjects. In vitro analysis and immunofluorescence displayed wild type COMP homogenously expressed in cytoplasm, but mutant proteins were irregularly accumulated inside the HEK-293 cells. Western blot revealed that the quantity of the mutant COMP was more compared to wild type COMP in cells after transfection for 12 hours and 24 hours. Subsequently, 3D structural analysis showed three changes have taken place in secondary structure of the mutant COMP. In conclusion, the novel mutation of COMP may result in intracellular accumulation of the mutant protein. Decreased plasma COMP and increased plasma CTX-II may potentially serve as diagnostic markers of PSACH but may not be applicable in the presymptomatic carrier. Pseudoachondroplasia (PSACH) is an autosomal dominant osteochondrodysplasia caused by mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Accurate clinical diagnosis of PSACH is sometimes difficult. Here, we identified a novel Pseudoachondroplasia  is an autosomal dominant osteochondrodysplasia characterized by disproportionate short stature, brachydactyly, joint laxity, scoliosis, early onset osteoarthritis, and epiphyseal and metaphyseal abnormalities. The affected individual is normal at birth and usually recognized at 2-3 years of age on the basis of a waddling gait and decreased linear growth [ COMP also results in multiple epiphyseal dysplasia , a relatively milder skeletal dysplasia and genetically heterogeneous disorder. To date, more than 100 mutations in COMP have been reported in about 300 patients with either PSACH or MED, but without any mutations in exons 15, 17, and 19 of COMP having been identified [PSACH is believed to be caused exclusively by mutations in the gene encoding cartilage oligomeric matrix protein (COMP), which located on chromosome 19p13.1 –6. COMP,entified , 12.Diagnosis of PSACH is usually based primarily on family history, clinical manifestation, physical examination, radiographic evaluation, and genetic diagnosis. However, accurate diagnosis of PSACH sometimes may be difficult, especially for nonexpert doctors, and because clinical presentation varies substantially between PSACH individuals, most radiographic changes are not disease-specific and DNA testing is expensive and not used widely . There i COMP in a Chinese PSACH family with four affected members in three generations. The plasma levels of COMP and CTX-II in the affected individuals were determined. In vitro expressions of normal and mutant COMP protein were performed to investigate the supposed pathological mechanism. Additionally, to explore the influence of the variant reported in this study on the protein structure and function, three-dimensional homology models for the normal and mutant proteins were built.In this study, we identified a novel variant  in exon 15 ofThe subjects are the three dwarf members  and the child  of a Han nationality family in Southwest China  by polymerase chain reaction (PCR) and subjected to DNA sequencing. A heterozygous nucleotide change c.1675G>A (p.Glu559Lys) was identified in exon 15 (nucleotide numbering is according to cDNA sequence with GenBank accession number XM_009336). Secondly, we amplified and sequenced the COMP exon 15 in the two affected individuals: III-3 and III-5, as well as the daughter of proband (V-1). Thirdly, PCR and sequencing of the COMP exon 15 in three unaffected individuals: IV-2, IV-5, and IV-6 were performed. Lastly, PCR and sequencing of the candidate variant were performed on gDNA from 100 unaffected individuals of the same ethnic background as the patients. Two different software programs including PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2) and SIFT (http://sift.jcvi.org/) were used to predict whether the amino acid substitution affects protein function.Firstly, genomic DNA (gDNA) was extracted from blood samples of the proband using standard method . Exons 8–19 and their flanking intronic sequence of the COMP were amplified using eight pairs of primers  of the family participated this study, and they were individuals III-3, III-5, IV-3, and V-1. The control group were selected at random  from the West China Hospital, Sichuan University, and participants selected were, respectively, matched to the age of the four affected individuals. Three milliliters of peripheral blood was collected into an EDTA-containing tube for plasma separation. The blood samples were centrifuged and the plasma was aspirated and stored at −80°C until further processing. Plasma COMP and CTX-II concentrations of the four affected individuals and twelve controls were analyzed using enzyme linked immunosorbent assay kit  according to the manufactures' protocol. Each measurement was performed in triplicate.Four individuals with the COMP was constructed following the standard procedure of the Ligation-Free-Cloning Kit . Thereinto, DNA insert fragments were amplified using special primer (Supplement Table  2) including the homologous sequence of vector and desired gene-specific region. In the meanwhile, the linearized pcDNA3.1 vector carrying Myc tag was prepared using restriction enzyme digest method. Afterwards, cloning procedure was set up as standard protocol, and selected colonies were confirmed through PCR amplification following gel electrophoresis and plasmid extraction following sequencing.Total RNA was extracted from human leukocytes of peripheral blood using TRIzol reagent  and RNA was reverse transcribed into cDNA . PcDNA3.1 expression vectors of human wild type and mutant type (c.1675G>A)5 HEK-293 cells were plated 24 hours before transfection. Cells were transiently transfected with 2 ug of each pcDNA3.1-wtCOMP or pcDNA3.1-mtCOMP or pcDNA3.1-myc empty plasmid using jetPRIME® transfection  according to the manufacturer's protocol. HEK-293 cells were incubated with the transfection reagents for 4 h at 37°C with 5% CO2 in DMEM supplemented with 1% antibiotics and 10% fetal calf serum, followed by further 12 hours or 24 hours of incubation in fresh full DMEM. After incubation, cells were lysed in lysis buffer followed by determination of protein concentration with BCA Protein Assay Kit .Approximately 1 × 10Prepared proteins were subjected to Western blot for myc, and GADPH was used as an internal control. In brief, proteins were subjected to SDS-PAGE, transferred to PVDF membrane, and detected with specific appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized using an enhanced chemiluminescence (ECL) Western blot detection system. Semiquantification of mean band intensities by Quantity One was shown.The immunofluorescence was performed using a standard protocol. The anti-Myc mouse antibody  was used as primary antibody in combination with a rabbit anti-Grp78 antibody , a representative marker of endoplasmic reticuluma, each in a 1 : 100 dilution. As secondary antibodies the anti-mouse-DyLight 594-labeled antibody and the anti-rabbit-DyLight 488-labeled antibody  were used in 1 : 100 dilutions, respectively. The cells were mounted with DAPI  for nuclear staining and the images were acquired with laser scanning confocal microscope .http://www.rcsb.org/pdb/explore/explore.do?structureId=3fby); ID: 3FBY is the crystal structure of the signature domain of COMP including amino acid sequence of 225–757 with resolution of 3.15 Å [https://www.swissmodel.expasy.org/) based on the 3FBY for template [To explore the effect of the novel variant on the protein structure and function, 3D models for the normal and mutant proteins were analyzed. Wild type COMP was downloaded from PDB . All data were presented as the mean ± standard deviation using GraphPad Prism 5.0 software. Differences between groups were determined using the nonparametric Mann–WhitneyThree PSACH patients in this family  were heterozygous for the nucleotide change c.1675G>A  in exon P < 0.001). But, in the proband's daughter (presymptomatic carrier), the mean plasma COMP level had no difference with equal-aged controls (P = 0.864). Besides, the mean plasma COMP level was significantly higher in the adult controls than in the controls less than three years old (P < 0.001) (P < 0.001) (P = 0.497). Also, there was no discrepancy of CTX-II levels in normal adults and children (P = 0.887) (As shown in < 0.001) . As for < 0.001) . But, in= 0.887) .The results of Western blot detecting the transfected cells using anti-myc showed mutant and wild type proteins were expressed in HEK-293 cells. And, also, quantity of the mutant COMP in cell was significantly more than wild type COMP after transfection for 12 hours and this trend was more pronounced after 24 hours . Meanwhiβ-sandwich that is composed of 15 antiparallel β-strands, and the novel mutation is located on β3 strand  in exons 15 of COMP in the three PSACH patients and an asymptomatic carrier. The variant was not detected in the three unaffected individuals in this family and 100 individuals of the same ethnic background, which demonstrate this variant is pathogenic mutation and not a single nucleotide polymorphism. This is the first report of the variant in exons 15 (aa 557–572) of COMP in PSACH and/or MED patients.In this study, we report a PSACH family with six affected members in Southwest China. The five PSACH patients were all in dwarf with disproportionately short limbs and short fingers and severe osteoarthrosis and scoliosis. Through COMP mutation carrier showing the clinical symptom of PSACH. The mechanism for the lower circulating COMP in children remains unclear, probably because the children under three years of age do not begin rapidly epiphyseal development, and the chondrocytes express relatively fewer COMP. This also interprets why affected individuals do not exhibit the symptom of PSACH before 2-3 years of age.There is evidence that peripheral blood COMP levels decrease in PSACH patients with COMP mutations, but circulating COMP levels in asymptomatic carrier have not been reported , 15. OurAs a crucial ECM protein, COMP binds to type II and type I collagens in cartilage and tendons, respectively, for providing the tissues with resistance to tensile forces. And COMP interacts mainly with free type I and type II collagen molecules for promoting further assembly . Since pSubsequently, we performed in vitro expression of normal and mutant COMP protein to investigate the supposed mechanism. Early electron microscopy studies of PSACH iliac crest biopsies showed dilated rough endoplasmic reticulum (rER) in chondrocyte . SubsequApproximately 90% of the reported COMP mutations were identified in the type 3 repeat but only 9% in CTD . CTD of In summary, our findings expand the spectrum of mutations in COMP leading to PSACH. We illustrate that the novel mutation (Glu559Lys) changes the secondary structure of COMP, which possibly leads to misfolding and intracellular retention of mutant COMP proteins. Importantly, we suggest decreased plasma COMP and increased plasma CTX-II may serve as a pair of diagnostic markers of PSACH patient but may not be applicable in the presymptomatic carrier. However, there are still some limitations in our paper. Except for intracellular pathogenic mechanisms, it has been reported that mutations in CTD of COMP could allow secretion causing PSACH by interference of the collagen fiber assembly and disruption of ECM structure in cartilage and tendons , 28. Fur COMP gene.Supplement table 1: List of primers used for amplification of exons 8–19 of theSupplement table 2: List of primers used for amplification of cloning insert fragment.


Lactobacillus depletion and increased microbial diversity facilitate human papillomavirus (HPV) infection and might be involved in viral persistence and cancer development. To define the microbial Community State Types (CSTs) associated with high-risk HPV−persistence, we analysed 55 cervico-vaginal samples from HPV positive (HPV+) women out of 1029 screened women and performed pyrosequencing of 16S rDNA. A total of 17 samples from age-matched HPV negative (HPV−) women were used as control. Clearance or Persistence groups were defined by recalling women after one year for HPV screening and genotyping. A CST IV subgroup, with bacterial genera such as Gardnerella, Prevotella, Megasphoera, Atopobium, frequently associated with anaerobic consortium in bacterial vaginosis (BV), was present at baseline sampling in 43% of women in Persistence group, and only in 7.4% of women in Clearance group. Atopobium genus was significantly enriched in Persistence group compared to the other groups. Sialidase-encoding gene from Gardnerella vaginalis, involved in biofilm formation, was significantly more represented in Persistence group compared to the other groups. Based on these data, we consider the CST IV-BV as a risk factor for HPV persistence and we propose Atopobium spp and sialidase gene from G. vaginalis as microbial markers of HPV−persistence.Changes in cervico-vaginal microbiota with  Persistence of oncogenic human papillomavirus (HPV) infection contribute to the development of CC.Cervical cancer (CC) is one of the most common cancer in women, with an estimated incidence of 485 000 new cases and 236 000 deaths in 20134, viral persistence occurs in almost 10% of infected women. The factors responsible of persistence, as well those that promote and initiate the carcinogenesis process, need to be fully elucidated. Many other factors such as immunodeficiency, age, smoking, oral contraceptives and Chlamydia trachomatis infection are related with higher persistence rates6. Recently, several scientific reports indicated the role of vaginal microbiota in the acquisition and persistence of HPV and risk of CC development7.While the virus is cleared in more than 90% of infections within 6–18 months9. However, in the vaginal environment, health is commonly associated with low microbial diversity and prevalence of only a few species of Lactobacillus13. Lactobacillus spp. prevent colonization of exogenous pathogens by producing lactic acid, bacteriocins and reactive oxygen species (ROS), and compete with them for adherence sites to mucous layer15. Five major community-state types (CSTs) discriminate vaginal microbiota in healthy women11. Lactobacillus crispatus, L. gasseri, L. iners and L. jensenii dominate CST I, II, III and V respectively, while depletion of Lactobacilli identifies CST IV11. In previous studies differences in vaginal microbiota by ethnicity was observed16. African American women may have increased L. iners and decreased L. crispatus compared with Caucasian women17.In the majority of human body sites, highly diverse microbial communities are generally considered a signature of healthGardnerella, Prevotella, Peptostreptococcus genera and/or aerobic bacteria of Enterobacteriacee family usually populate vaginal environment depleted of Lactobacillus species20.Anaerobic bacterial species of 7. In this disorder Atopobium vaginae, Clostridiales and selected Gardnerella vaginalis strains usually form biofilm on the vaginal epithelium, resistant to antibiotic therapies21.CST IV is frequently associated with bacterial vaginosis (BV), the most common vaginal infection in women of reproductive age26. CST IV is also frequent in aerobic vaginitis (AV). In this disorder, Lactobacillus spp. are predominantly replaced with enteric commensals or pathogens. Group B Streptococci (GBS), Escherichia coli, and Staphylococcus aureus are microorganisms most frequently associated with AV30.BV emerged as a public health problem due to its association with sexual transmitted infections including human immunodeficiency virus (HIV) and HPVIn this paper, to define the Community State Types (CSTs) associated with HPV-persistence, we used cervico-vaginal samples collected in a study for HPV screening program. Cervico-vaginal microbiota was characterized by metagenomic analysis in samples of HPV positive women and in HPV negative, selected as control. Microbial profiles were associated with viral clearance or persistence.Cervico-vaginal samples were collected in the contest of a pilot study aimed to evaluate the efficacy of HPV test in a primary screening program involving 1029 women aged between 26–64 years old (see Methods).A total of 55/1029 samples were positive for high risk HPV (HR-HPV+) at baseline screening. We were able to determine the HPV genotype in 50/55 samples collected at baseline screening and no significant prevalence of HPV genotype was recorded. Multiple HPV genotype were present in 13/50 samples (Supplementary Table All HR-HPV+ women attended the one-year follow up visit and were subjected to a second HR-HPV assay. The results of the second assay were used to stratify the baseline sampling in (i) Clearance Group (n = 27), HR-HPV+ women in which infection clears and had no evidence of HR-HPV DNA after one year; (ii) Persistence group (n = 28), HR-HPV+ women who developed persistent infection and maintained the expression of at least one of HPV-DNA genotypes revealed at baseline sampling. HR-HPV+ women were triaged to cytological testing. When Atypical Squamous Cells of Undetermined Significance (ASC-US) or a more severe lesion was ascertained, women were referred for colposcopy to define the grade (low or high) of lesions (see Methods). We reported Cervical Intraepithelial Neoplasia grade 1 (CIN1) and/or condiloma in 6 out of 28 (21.4%) and CIN grade 2 (CIN2) in 2 out of 28 (7.14%), as shown in Supplementary Table A total of 17 samples from HPV negative (HPV−) women matched for age were selected as control group. All the available epidemiologic and clinical information about the enrolled women were reported in Table via pyrosequencing of the V3-V5 hypervariable region of 16S rDNA gene, amplified in DNA from samples of HPV+ and HPV−women, as controls. In Supplementary Fig. Meta-taxonomic investigation of cervico-vaginal microbiota was performed Lactobacillus represented the most abundant genus of cervico-vaginal microbiota in the three groups  was observed, enrichment in aerobic and anaerobic bacteria, likely derived from gut environment or faecal contamination, was found in the three groups. In particular, bacterial genera such as Gardnerella, Prevotella, Atopobium, Escherichia/Shigella and Streptococcus,associated with bacterial vaginosis31, were frequently found in our cohorts , Chao1 and Shannon indices. We observed that biodiversity was higher in the group of HPV+ women and, in particular, in samples from Persistence group compared to Control group  and pairwise Spearman’s correlation was performed with two-tailed probability of t for each correlation. This analysis indicated that few genera of anaerobic bacteria  significantly correlated with depletion in Lactobacillus spp. in the Persistence group. In contrast, Streptococcus, Streptophita, Aerococcus, and numerous genera from Enterobacteriacee family significantly correlated with Lactobacillus depletion in the Clearance and Control groups  which were designated as CST I, II, III, IV, according to Ravel and Mitra32. Performing hierarchical clustering analysis of the samples based on taxonomic abundances in each enrolled women we found association with HPV status, age range and CSTs  and by a wide array of strict or facultative anaerobes. Most of CST IV samples were dominated by anaerobic bacteria belonging to genera Gardnerella, Prevotella, Atopobium, Sneathia, which are frequently found in women with bacterial vaginosis (BV). We named this subgroup of CST IV as CST IV-BV.Based on taxonomic distribution and accordingly with previous studiesLactobacillus depletion (<60%) and a mixture of aerobic and anaerobic bacteria of the genera Pseudomonas, Brevibacterium, Peptostreptococcus, Enterococcus, Streptococcus, Propionibacterium, Bifidobacterium, Shigella. Differently than CST IV-BV, strictly anaerobic bacteria of the Gardnerella, Prevotella, Atopobium, Megasphoera genera were absent or poorly represented. This subgroup of CST IV was named CST IV-AV.The remaining samples ascribed to CST IV showed p = 0.026; Fig. We observed an increased frequency of CST IV-BV (42.9%) in Persistence group compared either with Clearance group (7.4%) or with Control group (11.7%). These data Fig.  stronglyTo rule out that these data are mainly determined by the high frequency of CST-IV in post-menopausal women, we repeated the analysis considering only women in reproductive age exposed to HPV infection. The results obtained confirmed that CST IV-BV is significantly correlated to viral persistence in this group of women .Lactobacillus species, only selected microbial communities (CST IV-BV) play a role in the persistence of viral infection.Alpha diversity analysis of the CST-IV subtypes showed lower species richness in CST IV-BV compared to CST IV-AV Fig. , especiap = 0.001), age range of women (p = 0.009) or for both CST and age range (p = 0.002). In contrast, microbial diversity was not dependent on reproductive age  and Non-metric Multi Dimensional Scaling (NMDS), based on Bray-Curtis dissimilarities Fig. . By PERMSneathia (Leptotrichiaceae family)33, Megasphaera (Veillonellaceae family) and Pseudomonas (Pseudomonaceae family)34 and also in Pediococcus (Lactobacillaceae family) and Brevibacterium (Brevibacteriaceae family) in the group of HR-HPV+ women compared to HPV− controls  Effect Size (LEfSe) analysis (see Methods). In agreement with previous reports we found a significant enrichment in ols Fig. . Furtheroup Fig. .Figure 6Gardnerella is one of the dominant genera in our survey. It is known that selected strains of G. vaginalis adopt the biofilm mode of growth as a survival strategy36 and through symbiotic relationships with normally dormant vaginal anaerobes  may lead to increase of the latter. The production of sialidase is an important step in the biofilm formation37. To investigate whether sialidase-producing Gardnerella spp. were differently represented in microbial communities of the four CSTs, we studied the presence and the relative amount of sialidase-encoding gene (GVSI) by quantitative Real Time PCR. The amount of sialidase-encoding gene of G. vaginalis was significantly higher in the Persistence group compared with Clearance group , indicating a strong association among species of G. vaginalis producing sialidase, relevant for biofilm formation, CST IV-BV and HPV persistence.Lactobacillus spp. and higher diversity in HPV-infected women compared to HPV-negative38.Metagenomics has been previously used to assess the impact of vaginal microbiota composition in HPV infection. Most of these studies showed lower proportion of 39 performed a large longitudinal study in a cohort of 32 premenopausal women using self-sampling at twice-weekly intervals over the course of 16 weeks. The results of this study showed that CST III (dominance of L. iners) and IV (depletion in Lactobacilli) were most likely associated with HPV infection40.Brotman and co-workersIn our survey, the use of cervico-vaginal samples collected in the course of a primary screening and stored in a biological bank, gave us the opportunity to discriminate samples from women in which viral infection was cleared at one-year follow up, from those who developed persistent infection and to evaluate cervico-vaginal microbial profiles as a predictive risk factor of HPV persistence.Microbiota characterization was performed at baseline sampling in all of HPV+ women (55/1029) involved in the study and HPV negative as control, with age between 26–64 years, in order to evaluate microbial profiles independently on the reproductive age.Lactobacillus-dominated microbiota) could become a negative indicator in the more simplified vaginal communities. When compared with HPV− women, we found selected genera  significantly enriched in the HPV+ women. These results are in agreement with those reporting the prevalence of Sneathia in HPV+ patients with Squamous Intraepithelial Lesion (SIL)34.In accordance with previous studies, the higher alpha diversity observed in the group of HPV+ women and especially in the Persistence group suggests that increase in species richness  as the most represented bacterial community in HR-HPV infected women compared to negative controls. However, two subgroups of CST IV could be identified in our records. Few strictly anaerobic bacteria of the Gardnerella, Prevotella, Atopobium, Megasphera, Dialister genera dominated CST IV in women developing persistent infection. We referred this type of CST as CST IV-BV, since the same bacterial communities are frequently found in women with bacterial vaginosis (BV). A mixture of aerobic and anaerobic bacteria including Pseudomonas, Brevibacterium, Peptostreptococcus, Enterococcus, Streptococcus, Propionibacterium, Bifidobacterium and Shigella, namely CST IV-AV subtype, was prevalent in the Clearance group. The differences among the groups in bacterial communities populating vaginal environment, following depletion in Lactobacillus species, were evident through the Spearman's correlation analysis. Beta diversity analysis showed that, microbial variability among samples is correlated with CSTs and age range of women. However, the adoption of the CST classification proposed by Ravel11 allowed us to perform a risk association analysis, and despite the limited sample size, we found a significant association between CST IV-BV and the persistence of HR-HPV infection, which was not dependent on the reproductive age. Unfortunately, the lack of additional clinical and behavioural information of the enrolled women did not allow the revealing of further correlations considering CST subgroups and in particular with CST IV-BV.The classification of cervico-vaginal microbial communities based on the CSTs reported by Ravel Atopobium as key phylotype in the Persistence group compared to the Clearance group.However, we observed a very low alpha diversity in the CST IV-BV in women of the Persistence group, indicating that only a limited number of bacterial genera are likely involved in viral persistence. Based on our results, we suggest that CST IV-BV subtype play a role in viral persistence. To define potential metagenomic biomarkers of persistent infection, we identified Gardnerella vaginalis species in the three groups of women. As known, selected strains of G. vaginalis are able to form biofilm and the production of sialidase is an important step in the biofilm formation37. This enzyme facilitates the destruction of the protective mucus layer on the vaginal epithelium allowing resistant adhesion of bacteria, which can start to form biofilm36. The observed higher amount of sialidase-encoding gene in the Persistence group compared to other groups and in particular the prevalence in CST IV-BV suggest that sialidase-producing G. vaginalis strains are likely involved in formation of biofilm entrapping anaerobic pathobionts, such as Prevotella and Atopobium, favouring their overgrowth41, and that these biofilms may contribute to viral persistence.Finally, to define molecular markers associated with viral persistence, we evaluated the relative amount of sialidase-encoding gene from Atopobium vaginae, which is significantly more represented in microbiota of women in Persistence group compared to Clearance group, may play a role in the disruption of epithelial barriers42, favouring either HPV infections or viral dissemination. A recent study reports that anaerobes as Prevotella, Gardnerella, Atopobium, Sneathia, induce a strong inflammation in vaginal environment with high recruitment of T-helper 17 lymphocytes, which favours HIV infection26. Although no differences in lymphoid population were associated with clearance or persistence of HPV infection43, a high concentration of inflammation markers (IP-10 and MIG chemokines) was found in the vaginal environment of women who cleared the virus. The differential inflammatory potential of bacterial taxa populating vaginal environment in the Clearance and Persistence groups need to be elucidated. While a high inflammatory environment could facilitate viral clearance, immune-modulating bacterial genera, such as Faecalibacterium45, could play a role in the progression of disease in later stages of infection. Further investigations are needed to establish the role of bacterial biofilms and/or the induction of immunomodulation in HPV disease.Finally, although we could not ascertain microbiota stability in our patients during one-year intercourse, we can conclude that the early cervico-vaginal microbiota characterization may help to discriminate women at risk to develop persistent HPV infection and provide useful insights to develop new therapeutic strategies, modifying the vaginal microbiota ecosystem.A total of 1029 women, resident in Florence district  in both reproductive and post-menopausal age, who never received HPV vaccine, were enrolled in a HPV DNA based cervical cancer screening program in the contest of pilot study aimed to evaluate efficacy of HPV testing for the detection of invasive cervical cancer and cervical intrahepithelial neoplasia. The study was approved by the Ethics Committee of Azienda USL 10, Florence, Italy (Protocol ref. 263/2009). The protocol and the methods were performed in accordance with relevant guidelines and regulations. Informed consent for using biological samples for scientific investigations, including microbiota characterization, was obtained from all women at the time of the first screening.All enrolled women were of Caucasian ethnicity. Group Age ranged between 26–64 years. Exclusion criteria were: (i) pregnancy (ii) HIV infection (ii) immunodeficiency-related diseases, including AIDS. Abstinence from sex and from using vaginal antibiotics and/or vaginal washes was required until three days before collection.Cervico-vaginal samples were collected in Specimen Transport Medium  by trained midwives at Istituto per lo Studio e la Prevenzione Oncologica .HR-HPV status was determined through HR-HC2 assay (see paragraph below) and the remaining samples were stored at −80 °C for bacterial genomic DNA extraction.Samples from all of HPV+ women 55/1029) and from 17 selected age-matched HPV− women (who gave consensus for scientific investigations) were used for microbiota characterization. All information obtained by consensus are summarized in Table 029 and f46. When Atypical Squamous Cells of Undetermined Significance (ASC-US) or a more severe lesion was ascertained, women were referred for colposcopy to define the grade (low or high) of lesions, according to the Italian cervical cancer screening protocol . Furthermore, HPV genotyping was used to confirm the viral persistence.HR-HPV+ women were not directly referred to colposcopy but were triaged according to cytological testing. If the result of this test was abnormal  the women were immediately referred to colposcopy. If cytology was normal, the women were invited to repeat a new HPV test after one year, following the HTA protocolSupplementary Table 38. Quality control was carried out by gel electrophoresis and measuring ng/µl of DNA and 260/280 OD at Nanodrop 1000 (Thermo Scientific).Bacterial genomic DNA was extracted from the cervico-vaginal samples stored at −80 °C, by using a QiAmp Mini DNA kit  as previously describedHPV DNA test was performed according to manufacturer’s guidelines using the Hybrid Capture 2 assay .The HC2 assay is a nucleic acid hybridization assay with signal amplification using microplate chemiluminescence for the qualitative detection of thirteen high-risk types  of HPV DNA in cervical specimens.47.Genotyping was performed on HR-HC2 positive samples. Specific genotyping, which identifies thirty-one HPV types, was performed by amplifying the target DNA with PGMY09/11 and HLA biotinylated primers, using the validated method of the WHO HPV LabNet groupet al.48. The 454 pyrosequencing was carried out on the GS FLX+ system using the XL+ chemistry following the manufacturer’s recommendations.We amplified the 16S rRNA gene by using primer set specific for V3-V5 hypervariable regions, and the FastStart High Fidelity PCR system  as described in Strati http://www.ebi.ac.uk/ena/data/view/PRJEB18720) under the accession study PRJEB18720. Pre-processing of the reads was performed using the MICCA pipeline . Trimming of the primers and quality filtering were performed using micca-preproc, truncating reads shorter than 280 nt . Operational Taxonomic Units (OTUs) were assigned by clustering the sequences with a threshold of 97% pair-wise identity. The representative sequences were classified using the RDP classifier version 2.7 against RDP 11 database (update 5) of 16S rRNA. Template-guided multiple sequence alignment was performed using PyNAST (version 0.1) against the multiple alignment of the Greengenes 16S rRNA gene database (release 13_05) andfiltering at 97% similarity. A phylogenetic tree was inferred using FastTree and micca-phylogeny (parameters: -a template-template-min-perc 50). Rarefaction was performed in order to reduce the sampling heterogeneity. A total of 15,400 sequences per sample was obtained.Pyrosequencing resulted in a total of 2,112,426 16S rDNA reads with a mean of 26,405 sequences per sample. Average sequence lengths were 589 nt (±SD 28.4), 591 nt (±SD 25.7) and 594 nt (±SD 24.39) for the first, second and third run, respectively. Raw 454 files were demultiplexed using Roche’s.sff file software, and made available at the European Nucleotide Archive  and Bray-Curtis dissimilarities (indicators of beta diversity) were calculated using the phyloseq package of the R software suite.Lactobacillus spp. as >60%, and aerobic and anaerobic bacteria ranging from 14 to 40%.Community State Type (CST) of each cervico-vaginal microbiota was defined considering the relative abundance of To evaluate the differences in overall bacterial community composition, Principal Coordinates Analysis (PCoA) and Non-metric Multidimensional Scaling (NMDS), based on Bray-Curtis dissimilarities were performed. The significance of between-groups differentiation  was assessed by PERMANOVA, Adonis function, using the R package vegan with 999 permutations.49, heatmap of the relative abundances of bacterial taxa was generated. Variables such as HPV status, CST, age range of the women were associated with the respective microbiota sample. Heatmap was supported by a dendrogram obtained with hierarchical clustering of the samples and based on average distances among samples calculated by Pearson correlation.By Clustvis toolGVSI) was performed by using Platinum TaqUniversal SYBR® green supermix (InvitrogenBiorad) and primers specific for G. vaginalis sialidase-encoding gene (Fw-5′GGGTTTATGCACACGCTT TT-3′ and Rv-5′GAAAATGCAGACAACGC AGA-3′)50 and universal bacterial 16 S rRNA gene  as reference of the total bacterial community51. RT q-PCR was performed in Applied Biosystem 7900 instrument following these conditions: 50 °C for 2′; 95 °C for 10′, 40 cycles of denaturation at 95 °C for 15″, annealing at 55 °C for 30″, and extension at 68 °C for 30″ followed by a dissociation stage :95° for 15″; 60° for 15″ and 95° for 15″ with a rampe rate of 2%. Samples with a melting temperature (Tm) value at 84° for sialidase-encoding gene (n = 26) and between 87 and 91 °C for the 16S r RNA gene were considered positive (n = 55).Quantitative Real Time PCR (RT q-PCR) of sialidase-encoding gene  Effect Size (LEfSe) methodLactobacillus abundance data and all the other taxa were calculated using the pairwise Spearman’s correlation and two-tailed probability of t for each correlation.For correlation analysis, microbiota abundance data were divided into independent data matrices . The correlation coefficients and significant negative correlations (p < 0.05) between Odd ratio with 95% confidence interval was calculated to correlate CSTs with HPV clearance and persistence in all women population and considering also the only women in reproductive age. P < 0.05 was considered statistically significant.Statistical analysis of RTq-PCR of sialidase-encoding gene was performed with non parametric Mood’s Median test (MDTest) and Wilcoxon Signed Rank test using the Origin software. P < 0.05 was considered statistically significant.Supplementary InformationSupplementary Dataset 2Supplementary Dataset 4


The standard of care for oropharyngeal squamous cell carcinoma (OPSCC) has consisted of definitive radiation and chemotherapy for many decades. While early-stage tumors can be managed with surgery or radiation alone, locally advanced, stage III-IV OPSCC is typically treated with definitive radiation to the standard definitive radiation dose of 70 Gy, with treatment delivered daily over 7 weeks . The ben2 weekly) to reduced-dose radiation alone using an accelerated regimen (6 treatments per week). The Eastern Cooperative Oncology Group (ECOG) is investigating whether radiation dose can be reduced in the post-operative setting after trans-oral robotic surgery (TORS) for HPV-positive OPSCC. It must be recognized, however, that without any prospective evidence to support de-escalating treatment for HPV-positive OPSCC patients, published data to this date has been largely limited to retrospective series. However, these studies for this population have demonstrated excellent outcomes, and it is now evident that HPV-positive OPSCC represents a unique entity with distinct clinical and molecular characteristics.There have been a number of recent efforts to de-escalate treatment for patients with HPV-positive OPSCC under the hypothesis that they can be cured equally as well with lower doses of radiation and/or systemic therapy. The RTOG developed a phase III trial (RTOG 1016) investigating de-intensification with the recombinant human/mouse chimeric epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab instead of cisplatin for HPV-positive OSPCC patients; results are currently pending. The NRG Oncology Group is currently accruing HPV-positive patients for a phase III trial (NRG HN002) comparing reduced-dose radiation with low-dose cisplatin (40mg/mWe recently presented the results of a phase II trial for patients with HPV-positive OPSCC using a strategy of induction chemotherapy with paclitaxel and carboplatin followed by de-intensified radiation, with the reduced dose of 54 Gy or 60 Gy determined by the radiographic response to initial chemotherapy. This study was designed based off of promising results of an ECOG phase II trial with the theory being that because local control rates are so high in HPV-positive cancers, patients may benefit from upfront chemotherapy to control any distant micro-metastatic disease [The literature on de-escalated treatment for HPV-positive OPSCC is rapidly expanding. Despite the attractiveness of this approach, the data focusing on possible de-intensification remains preliminary, and treatment paradigms will likely continue to evolve as findings from innovative clinical trials yield answers to many provocative questions. While the optimal strategy of de-escalation for patients with HPV-positive OPSCC remains to be determined, promising data continues to emerge validating the notion that less may indeed be more for the treatment of this disease.


A major challenge for developing countries like Ghana has been the unavailability and costs of HPV DNA-based testing. This study compares the performance of careHPV and Anyplex-II HPV28 genotyping assay, and by conventional cytology.A study comparing between frequency matched HIV-1 seropositive and HIV-seronegative women was conducted in the Cape Coast Teaching Hospital, Ghana. A systematic sampling method was used to select women attending clinics in the hospital. Cervical samples were tested for HPV by careHPV assay. The inter-assay concordance was 94.3% , similar by HIV serostatus. The careHPV assay was equally sensitive among HIV-1 seropositive and seronegative women  and slightly more specific among HIV-seronegative women . careHPV had good sensitivity (87.5%) but low specificity (52.1%) for the detection of low SIL or greater lesions, but its performance was superior to genotyping . Reproducibility of careHPV, tested on 97 samples by the same individual was 82.5% (95%CI: 73.4–89.4%).A total of 175 paired results (94 from HIV-1 seropositive and 81 from HIV-seronegative women) were analyzed based on the ability of both tests to detect the 14 hr-HPV types included in the careHPV compared to genotyping suggest that this simpler and cheaper HPV detection assay could offer a suitable alternative for HPV screening in Ghana.The performance characteristics of  Modend China –7. A stuTM II HPV28 . The assay detects 28 HPV genotypes including 19 hr-HPV types of which 13 are considered carcinogenic , six possible carcinogenic , and nine low-risk HPV types , according to the Interagency for Research on Cancer (IARC) classification [careHPV would considerably enhance access to HPV-based cervical cancer screening in the country. In particular, since HPV molecular assays have very high negative predictive value for the detection of cervical cancer lesions, they could become very useful as a primary screening test or as triage in combination with cytology. However, because they cannot distinguish between transient and persistent infections, their specificity is low. They are thus recommended for use among women aged 30 years and older when most HPV infections should have cleared. Molecular tests can also indicate complete viral eradication if the result is negative 12 months following cervical lesions treatment, hence they can be useful for patients’ follow-up. Studies have compared various strategies including those which combine HPV genotyping with concurrent cytology and those which offer HPV screening without concurrent cytology. The results of the ATHENA trial conducted among 42,209 women in the United States of America comparing various single or combination screening strategies suggest that both strategies are feasible and have equivalent performance depending on factors like age of the woman [In Ghana, HPV testing has remained confined to research laboratories where genotyping is used. One such genotyping assay is the recently developed Anyplexhe woman .careHPV with HPV genotyping for the molecular diagnosis of HPV and at evaluating the performance of both assays against cytology among HIV-1 seropositive and HIV-seronegative women in Ghana. This is an essential step as the country is looking to inform the development of its cervical cancer screening algorithms.The present study aimed at comparing careHPV evaluation study.Participants were recruited as part of a larger HPV/cervical cancer epidemiological study (comparing HIV-1 seropositive to HIV-seronegative women) conducted in the Cape Coast Teaching Hospital (CCTH) in Ghana. Briefly, a comparative frequency-matched study was conducted in a systematic (1 in 5) sample of women attending the HIV and the general outpatient clinics at CCTH. Every fifth woman aged ≥18 years was systematically selected from the list of attendants, starting by a randomly selected attendance number for the first woman. If a woman was deemed not eligible , the next available patient was offered her place, and every fifth woman whence, to a maximum of 10 women per clinic day. Participants who met the inclusion criteria (i.e. aged ≥18 years and willing to be tested for HIV) were given an explanation of the protocol after which written informed consent was obtained. This method was used to recruit the women for the parent study and then a sub set target of about 50% of participants (every other recruited woman) were asked to be part of the TM Cervical SamplerTM and transported in Swab Specimen Collection Kit  for genotyping by Anyplex II HPV 28. For careHPV testing, the careHPV specific brush and transport medium were used . Cervical smears were taken for cytology with a cervical brush and immediately alcohol-fixed at the clinic.Gynaecological examination with speculum was performed, during which cervical swabs were collected from the ecto and endocervix targeting the squamo-columnar junction using a DNA PAPcareHPV test was performed on the samples collected into the careHPV transport medium. This test is a semi-rapid test designed based on simplification of the Digene HC2 test technology to be used for the detection of the DNA for 14 hr-HPV types . This test takes 2.5 to 3 h to perform for a 96 well and involves 6 easy-to-follow steps of denaturation, hybridization and capturing, conjugation, washing, additions of substrate and detection with the illuminometer. The results obtained are qualitative for hr-HPV without indicating the specific genotype [The Anyplex II HPV 28 test was performed from its specific transport medium as per manufacturer’s protocol previously described . The isogenotype , 12, 13.Cervical smears were prepared in the laboratory following a standardized protocol for Papanicolaou (Pap) staining. Slides were read by a consultant cytopathologist at CCTH using the Bethesda 2001 guidelines for SIL classification .careHPV performance compared with genotyping was done for 14 hr-HPV genotypes detectable by both tests . Sensitivity, specificity, positive and negative predictive values (PPV and NPV), and Cohen’s Kappa values for agreement between the two tests were calculated with their 95%CI. These were calculated for the total results and then also done separately for HIV-1 seropositive and HIV-seronegative women separately. Data analyses were performed using Stata version 13 software .Analysis of Ethical approval for this study was obtained from the Committee on Human Research Publications and Ethics (CHRPE) of the School of Medical Sciences (SMS), Kwame Nkrumah University of Science and Technology (KNUST) before the study commenced. Study participants were recruited only after obtaining signed written informed consent.careHPV and genotyping samples from the subsample of women  and 97 HIV-seronegative  randomly selected into the careHPV validation study were tested, and 175 results (89%) were available for analysis based on the ability of both tests to detect the 14 hr-HPV types. For 21 careHPV results (6 from HIV-1 seropositive and 15 from HIV-seronegative women), Anyplex II HPV 28 detected genotypes which are undetectable by careHPV . In addition, one careHPV sample (from an HIV-seronegative woman) gave an invalid result despite repeat testing. These 22 samples (11.1%) were not included in the analysis of careHPV performance.Overall, 333 eligible women were included in the parent study, 163 HIV-1 seropositive women  and 170 HIV-seronegative women . A total of 197 paired careHPV was 55% (95%CI: 48.0–62.9) overall, 79% (95%CI: 69.0–86.5) among HIV-1 seropositive women and 28.0% (95%CI: 19.0–39.5) among HIV-seronegative women (p ≤ 0.0001) , and the agreement was similar among HIV-1 seropositive  and seronegative  women   and slightly more specific among HIV-seronegative women , but these differences were not statistically significant  between careHPV and Anyplex II HPV 28 was 100%. For 4 genotypes careHPV missed one positive sample, and for HPV58 it missed 2 samples , the concordance between careHPV prevalence increased according to severity of cytological lesions, from 47.8% among women with normal cytology to 100% among women with high-grade lesions (HSIL/ASC-H) (p-trend = 0.08). Similarly, hr-HPV prevalence by genotyping increased by cytological grade severity (p-trend = 0.07)  but careHPV had statistically significantly higher specificity , PPV and NPV than genotyping . In 5/97 initially negative careHPV results the second result was positive, 2/97 became invalid and in 8/97 positive careHPV result, the second result was negative, and 2 became invalid.A random sample of approximately 50% of all Given the high cost and resource intensive nature of genotyping for HPV screening, both in terms of skills and materials, it is essential that developing countries find acceptable alternatives to move into the modern cervical cancer screening era. The advantage of full genotyping is its higher analytical sensitivity and ability to specifically identify the genotypes present in a population. While this is essential for research or epidemiological monitoring purposes, it is not absolutely necessary for clinical care. The role of HPV screening for clinical practice is to help establish a protocol of screening which is cost effective and helps identify women having hr-HPV infection so they can have further evaluation , whilst careHPV represents an alternative HPV screening assay that has been specifically developed for resource-constrained settings. This assay requires just bench top and 3 portable equipment which has a backup battery to store power enough to run a full set of 96 samples without the need for an external supply of electricity. The samples for careHPV can also be stored at room temperature for up to 4 weeks, do not require DNA extraction and require very limited technical knowledge to be performed. While careHPV has been evaluated in some settings, this research presents the first such evaluation done among women including HIV seropositive and seronegative women in Ghana. The simplicity of the assay and its relative robustness in the context of a resource-constrained laboratory setting was confirmed in this study. Various studies have been conducted using this assay including in Uganda [n Uganda , rural Cn Uganda , rural Tn Uganda  with gook = 0.88) between careHPV and full HPV genotyping for the detection of 14 hr-HPV genotypes, and the result was similar among HIV-1 seropositive and HIV-seronegative women. The careHPV assay was slightly more sensitive among HIV-1 seropositive women but more specific among HIV-seronegative women. Investigators in Burkina Faso and South Africa found very similar excellent agreement (94.6%) for careHPV compared to HC2 [careHPV detected 83.3% of all cases with LSIL and all cases (100%) of HSIL/ASC-H. Other studies have demonstrated good clinical performance of careHPV in HIV-seronegative African women [careHPV for the diagnosis of HSIL among 929 HIV-1 seropositive women in Burkina Faso and South Africa to be 88.8 and 61.8% respectively. The negative predictive value of careHPV for detection of cytological lesions was 99.0% in this study. This is very important because it implies that it could serve as an essential screening tool. Given its good sensitivity but low specificity, careHPV testing might be best performed as triage test with cytology or visual inspection (VIA) to reduce unnecessary referrals for colposcopy. It will be useful to study the cost effectiveness of such strategies in the Ghanaian socio-economic context.This study found an excellent agreement  and inter-rater  agreements. Intra-rater agreement was 98.8% (k = 0.97) and 98.9% (k = 0.97) for Technicians 1 and 2, respectively, and the inter-rater agreement was 96.3% (k = 0.90), suggesting that careHPV results were reliable [To check reproducibility and hence reliability of results, 97 samples were tested in duplicate in this study by the same individual. Reproducibility was found to be 82.5%. This is good but still implies that all of the 6 processing steps be completed without fault by a meticulous lab technician to reduce the risk of having variable results, and that quality control should be routinely implemented. The positive and negative controls included for each plate serve to ensure that only valid results are read, and only a small proportion of retests we found invalid (4%). To assess the reliability of results in a study evaluating careHPV was relatively low, owing to financial constraints and dependence on donated careHPV kits; and for the same reasons a more extensive repeat testing could not be organized. Furthermore, a full economic evaluation of cervical cancer screening was beyond the scope of this study. However, based on market prices and personal communication with relevant people, the cost of genotyping (including reagents and technician time) would come to approximately US$ 100.00 per sample, whereas careHPV cost could be about US$ 15–20.00 per sample. Other forms of cervical screening include cytology (Pap smears) being offered at a minimum of US$ 15.00 in Ghana , whereas visual inspection using VIA could cost as little as US$ 5.00 (up to US$ 15) [The present study had some limitations. The number of samples tested by  US$ 15) . As indi US$ 15) . The fac US$ 15) , 24 and  US$ 15)  are impocareHPV has very good concordance with, and good performance characteristics compared to, HPV genotyping for the detection of cervical squamous intraepithelial lesions, and whilst reproducibility could be improved, the findings support the possibility of setting up HPV screening without the need for resource-intensive genotyping as a suitable alternative for cervical cancer screening in countries like Ghana.This study has demonstrated that


AboutFace, a digital storytelling (DST) resource designed to help veterans recognize PTSD and motivate them to seek evidence-based treatment. The Ralph H. Johnson Veterans Affairs Medical Center (VAMC) and the National Center for PTSD have partnered to conduct pilot work to evaluate veterans’ reactions to AboutFace to set the stage for a large-scale study to examine whether AboutFace effectively reduces stigma and improves attitudes toward treatment-seeking among veterans. If effective, this DST approach may serve as a valuable national model for a variety of treatment-seeking populations.Research suggests that at least 10% of veterans returning from Iraq and Afghanistan meet criteria for posttraumatic stress disorder (PTSD) related to their military experiences. National dissemination initiatives have increased veterans’ access to best-practice interventions. However, treatment-seeking remains low among veterans with PTSD, often due to perceived stigma and other associated barriers. The National Center for PTSD recently developed and launched AboutFace will be conducted via semi-structured interviews with 20 veterans. Audio recordings of interviews will undergo transcription and coding. A report of the results of qualitative analyses of these interviews will be provided to the National Center for PTSD and will inform revisions to the site. In the second phase of the pilot, 60 veterans referred to a specialized PTSD clinic will be recruited to demonstrate and refine the methodology that we propose to use in a larger randomized controlled trial evaluation of AboutFace. Veterans will be randomly assigned to receive AboutFace plus standard education vs. standard education alone. Baseline and 2-week telephone assessments will be conducted with participating veterans to measure stigma, attitudes toward seeking mental health services, and treatment access/engagement.During the first phase of the pilot, in-person usability assessments of AboutFace is found to increase access to care, this finding would have broad and significant implications for overcoming barriers to care for veterans and other populations with stigmatized conditions.The feedback we receive in this pilot will be used to strengthen the quality of the DST website in preparation for a large-scale evaluation. Future work will involve evaluation of reach and impact of the site relative to stigma, attitudes toward seeking mental health service, and utilization of care. If NCT02486692Clinicaltrials.gov,  Military operations in Iraq and Afghanistan frequently involved life-threatening experiences, such as dangerous patrols and direct fire, witnessing violence and human suffering, and open hostility from civilians , 2. ManyEvidence-based treatments are available for veterans , and natThere is a critical need for innovative strategies to overcome barriers to care  and assist veterans with mental health problems who do not seek services from traditional sources . Widely The role of peer education has been examined with individuals at risk for breast cancer , 27, humDST involves the use of recorded audio, video, graphics, and text that are disseminated electronically  to a target population for a particular purpose . This is accomplished through several storytelling modalities, including re-telling of historical events, informational or instructional videos, and personal narratives. DST has been used in a number of contexts and has traditionally been used in educational settings. For example, instruction-based and student-driven digital storytelling methods have led to positive learning effects in educational settings , and, whDespite positive findings for the role of DST as an educational and therapeutic tool, little is known about its value in decreasing mental health stigma and increasing treatment-seeking behavior, particularly in veterans. A quasi-experimental study to evaluate a peer education program for active-duty service members of the UK armed forces found that this program improved attitudes about mental health and help-seeking . HoweverAboutFace is a web-based video gallery that introduces viewers to a community of 77 veterans, diverse with regard to military experience, age, gender, and race/ethnicity. The veterans have experienced PTSD and received treatment through the Veterans Health Administration (VHA). The site also contains testimonials from 23 family members and 22 clinicians. AboutFace aims to use the shared bonds of military service to educate veterans and their families and help normalize common reactions that veterans may experience due to their military service or deployment experiences. Consistent with this, visitors to the site can “meet” veterans and hear how PTSD has affected them through unscripted, authentic personal stories. Veterans are filmed in natural settings looking directly at the camera. For the viewer, the eye contact is intimate, as if the veterans have invited you into their homes and are sharing personal details of their lives.Developed by the National Center for PTSD, AboutFace can learn about other veterans’ military histories, the common symptoms of PTSD, treatment options, and the struggles of other veterans regarding decisions to seek care. They can get detailed descriptions about what treatment was like for other Veterans. There is even a section where veterans give advice on what they think fellow veterans with PTSD should know about seeking help. Thus, through honest and open testimonials, the veterans of AboutFace serve as encouraging peer supports to other veterans as they consider their own need for mental healthcare, what the process of PTSD treatment will look like, and how they can obtain and initiate needed services through VHA. This use of a peer-to-peer approach is innovative, relevant to a wide range of healthcare conditions, and has the potential to increase access to care through trusted narratives that promote hope in recovery.Veterans who access AboutFace has an open format that allows visitors to navigate the site freely based on their own user preferences. Visitors can learn about one veteran’s experience at a time by selecting that veteran's image and then choosing from a series of statements that represent topics addressed by the veteran. Alternatively, the visitor can choose a specific topic and review video clips of all veterans, who have addressed that topic. Veterans also can receive advice from expert clinicians and hear how PTSD can affect family members, via a separate component of the AboutFace site. Topics addressed in the brief (~1 min) video clips are included in Table e brief ~ min videFew studies have examined the impact of peer education approaches in the healthcare field. Most evaluations of peer education interventions have relied upon in-person, live interactions. Costs associated with training peers and supporting their interactions with the target population limit the scalability of these approaches. Peer education approaches have not been examined extensively in veteran populations, and there has been no rigorous evaluation of veterans’ receptivity to a digital storytelling approach. This low-cost, highly sustainable and scalable approach to peer education may have particular value for veterans with stigmatized conditions. Although the current study will focus on veterans with PTSD, data derived from this study may have broad implications for the value of implementing and disseminating similar programs for other mental health and chronic disease populations.AboutFace as a public awareness campaign designed to help veterans recognize the symptoms of PTSD and to motivate them to seek evidence-based assessment and treatment. Although it is believed that AboutFace has tremendous potential to reduce stigma and improve attitudes toward seeking mental health services among veterans, it has not yet been formally evaluated. The current study is designed to use veteran feedback to strengthen the quality and applicability of the AboutFace site. This will be accomplished by conducting in-person usability assessments of AboutFace via semi-structured interviews with 20 veterans. A report of the results of qualitative analyses of these interviews will be provided to the National Center for PTSD and will inform revisions to the site. These revisions, in turn, may improve the reach and impact of the site with veterans who have PTSD and other mental health conditions. We will conduct a feasibility trial to prepare for a large-scale examination of this aim by randomly assigning 60 veterans, referred to a specialized PTSD, to receive AboutFace plus standard education vs. standard education alone. Baseline and 2-week telephone assessments will be conducted with participating veterans to measure stigma, attitudes toward seeking mental health services, and treatment access/engagement.A number of veterans with PTSD do not seek mental health services due to perceived stigma and other barriers , 22. TheThe current investigation was informed by the framework for program evaluation in public health, which was developed by the Centers for Disease Control and Prevention, to provide a practical and systematic method for effective program evaluation . Under tUsability testing explores a user’s experience with a product by having consumers from the target population use the product while being observed by an evaluator. Observations are systematically recorded and, later, analyzed and interpreted to gain a unique depth of understanding around user experiences with the product. One of the main purposes of this type of testing is to obtain objective usability metrics and to identify opportunities to strengthen the quality of a product. Common usability issues include anything that prevents task completion, takes the user off course from the task, creates confusion, or decreases satisfaction with the product; more specific examples include performing the wrong action, misinterpreting something, and not understanding the navigation . Many usAboutFace is highly relevant to veterans, who have recently attended a PTSD evaluation session. This approach will ensure that feedback is received from those who will be most likely to use and benefit from the site: veterans who are in the process of considering whether or not to seek mental healthcare. Second, this recruitment approach was chosen to ensure the feasibility of meeting sample recruitment goals and other milestones and study aims. Third, the sample of veterans recruited from this clinic will be heterogeneous, with about 30% who do not follow-up with treatment. Thus, the goal is to obtain feedback from veterans at different levels of treatment readiness.Usability testing will include individually-administered, in-person, semi-structured interviews with 20 veterans who are recommended for PTSD treatment following the completion of a diagnostic evaluation by PTSD clinical team (PCT) and Telehealth Program clinicians at the Ralph H. Johnson Veterans Affairs Medical Center (VAMC) in Charleston, SC. The use of semi-structured interviews for this stakeholder group was chosen for several reasons. First, AboutFace, while an interviewer observes their use of the site. This stage of testing will involve an introductory observation phase during which the veteran will be encouraged to freely navigate the site, followed by an evaluation phase using a cognitive interviewing approach ).Triangulation is a method used by qualitative researchers to verify and minimize bias in their findings , 60. We AboutFace  will converge with quantitative data that will be gathered from the Website Analysis and Measurement Inventory (WAMMI). However, investigators will explore areas of divergence across these data sources, should they arise, in order to better understand how to address barriers to care in the refinement of the intervention.With regard to data and method triangulation, a best-case scenario is that the results will prove to be mutually reinforcing. In the case of divergence, every effort will be made to look for patterns of inconsistencies and understand these differences in a manner that provides a more comprehensive understanding of the data. For example, it is anticipated that qualitative data regarding veterans’ reaction to Generalized linear models (GLM) will be used to examine the associations of age, gender, race/ethnicity, urban/rural residence, and comorbidities with the WAMMI and questions around attitudes toward mental health treatment. We will examine the distribution of the measures to employ the appropriate link function for the model. GLM has been shown to be good for estimating models of mental health outcomes .The feasibility trial will provide qualitative and descriptive data that we will use to assess the feasibility of our study methodology and to plan for a large-scale RCT. Experts strongly recommend that pilot grant mechanisms support evaluation of the feasibility and implementation of novel methods . ResearcAboutFace condition who use the site; (c) retention of veterans in the study protocol; (d) variability in data relating to stigma and attitudes toward seeking mental health services; (e) veterans’ reactions to the AboutFace site; and (f) preliminary data on treatment attendance across conditions.For veterans randomized to both conditions, we will assess and describe several variables. Recruitment will be assessed by the proportion of patients who agree to participate as compared to the total number solicited to enroll. Study retention refers to the proportion of veterans enrolled in the study that complete the follow-up interview. This pilot will provide an opportunity to examine the feasibility of the study design and will yield pilot data such as (a) rate of recruitment into the study, including recruitment of rural, minority, and female veterans; (b) number and percentage of veterans in the p value. Logit models are often used to assess binary mental health outcome treatment outcomes [p < 0.05. As this will be an underpowered study, we will provide 95% confidence intervals for all analysis estimates and all results will be treated with caution.We will estimate a logit model to examine the association of veteran age, gender, race/ethnicity, rural/urban residence, and comorbidities with the likelihood of dropping out of the study, measured as a binary variable, to understand where outreach efforts may need to be reinforced. The logit model will provide an odds ratio on each veteran factor along with a 95% confidence interval and outcomes . We alsoAboutFace , attitudes toward seeking mental health services (quantitative), and sociodemographic variables. Again, these data sources will be used to better understand reactions to AboutFace and attitudes toward mental health treatment-seeking that can be used to refine the intervention for use across a diverse range of veterans. Similar to the usability component, convergence and divergence between data sources will be explored and reconciled to provide a more comprehensive picture of feasibility related to uptake and dissemination of AboutFace.Coding and analysis of qualitative data will follow a similar procedure as that outlined above, with particular emphasis on data gathered from the experimental group regarding reactions to PTSD is a prevalent, but undertreated, mental health problem among our nation’s veterans. Many veterans in need of mental health services are reluctant to seek care because PTSD is a stigmatized condition. Education may assist in addressing stigma, , 67 as wAboutFace is an ideal DST resource with which to initiate this line of research for several reasons. First, PTSD is a prevalent and stigmatized condition. Thus, a peer-to-peer approach may be particularly valuable for conditions where stigma may play a role in help seeking. Survey research has found that US adults with stigmatized illnesses  were more likely than those with at least one other chronic illness  to have used the Internet for health information or to have communicated with providers via the Web [AboutFace is a widely accessible resource that has already been developed and launched. Third, the clinician component of the resource is unique and will allow us to examine veterans’ use of, and reactions to, this educational content in addition to the peer education content. Data on veterans’ reactions may provide insight into the incremental value of expert content integrated into peer education resources. the Web . Thus, w. More immediately, data will guide improvements to AboutFace that will be led by the National Center for PTSD. Usability testing data will inform an improved site design, such as changes to navigation, layout, and length of videos. Information on impact will guide next steps in the development process. In addition, the feasibility trial will allow for preliminary data on stigma, attitudes toward seeking help, and access to treatment. On each of these outcomes, we expect significant benefit ultimately at the population level. If not observed, additional videos could be added that more directly address these topics or, in some cases, new site features or components might be required. Upon completion of these improvements, the Center is prepared to immediately increase the marketing of the site through new social media avenues, as well as notices in the PTSD Monthly Updates. Resources directed to dissemination will be increased still further if data from the future planned RCT show clear benefits of AboutFace relative to stigma and access to treatment.Data obtained in this pilot will be used to guide planning in preparation for a large-scale RCT evaluation of AboutFaceIt is important to note that this study can serve as a model that can be implemented and have great potential with a wide range of treatment-seeking populations. This study’s methodology is easily translatable for researchers seeking to develop and/or improve other web/smartphone-based DST resources to improve relevance, interest, and effectiveness in decreasing stigma. Promisingly, this can be accomplished on both the disease  and demographic  level. Technology-based DST resources hold great promise for engaging various treatment-seeking populations, and through the rigorous methodology found in this investigation, such resources have potential to reach these populations on an even greater level.The study is registered on clinicaltrials.gov . Usability testing was undertaken between August 2015 and January 2016. Participant recruitment for the feasibility trial began in March 2016 and is due to finish in September 2016.


No HPV16-specific CMI response or antibodies were detected in two children with persistent oral HPV16. The profiles of induced cytokines indicated higher levels of IL-5, IL-10, and IL-17A in children with HPV DNA in placenta and/or cord blood than in other children. HPV16-specific CMI is common in HPV DNA-negative children. The cytokine profile in children infected with HPV16 during early life suggests that the viral dose and/or specific environment created by the placenta may have significant impact on the type of HPV-specific immunity.The purpose of this study was to evaluate if an early exposure to human papillomavirus (HPV) during the prenatal period or infancy could result in HPV16-specific T helper (Th) responses resembling those of adults with HPV-induced lesions. We tested HPV16-specific cell-mediated immunity (CMI) in children born with HPV-positive umbilical cord blood and/or placenta or having persistent oral HPV infection and in constantly oral HPV-negative controls. Peripheral blood mononuclear cells from 33 children from the Finnish HPV Family Study cohort (mean age 14.7 years) were stimulated with peptide pools covering the amino acid sequence of the HPV16 E2, E6, and E7 proteins. Lymphocyte proliferation, secretion of cytokines , and the frequency of Foxp3+ regulatory T-cells were determined in relation to the HPV DNA status during a 14-year follow-up. 73.6% of cases and 85.7% of controls responded against HPV16 E2, while reactivity against E6 was found in 10.5 and 35.7%, respectively. The proliferative response against E6 and E7 was more frequent in controls than in cases ( Human papillomavirus (HPV) infection is generally regarded as a sexually transmitted disease. However, several studies, including those of our own , 22, 31,We have shown a strong concordance between the HPV genotypes detected in the mother’s genital tract and her offspring’s oral samples , implyinp = 0.0001 and OR = 4.7; 95% CI, 1.4–15.9; p = 0.015, respectively) -thymidine incorporation assay. The cut-off value for counts per minute (CPM) values was determined by the average plus 3 × standard deviation (SD) of the eight medium-only control wells. The stimulation index (SI) was calculated as the average of the eight tested wells divided by the average of the medium-only control wells. The proliferative response was defined as positive if the CPM values of at least six of the eight wells were above the cut-off value and if the SI was ≥3.LST was performed as described previously , 39. BriThe supernatants from PBMC cultures were analyzed using the Cytometric Bead Array (CBA) human enhanced sensitivity flex set system , according to the manufacturer’s instructions. In this array, the levels of IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-10, and IL-17A were determined, as previously described , 39. TheRegulatory T-cell identification was performed as previously described , 15, 39.p-value ≤0.05. All statistical analyses were run using the IBM SPSS®  software package .The means of secreted cytokine concentrations and proliferative responses of all groups were analyzed using one-way analysis of variance (ANOVA). The Bonferroni correction was used to control for multiple comparisons between the groups. All statistical tests were two-sided and declared significant at a All 33 children were actively followed up from birth until the age of 6 years and passively thereafter, until the collection of the present samples taken now from the children with a mean age of 14.7 years . The HPV DNA status of the oral mucosa was determined at birth, at the age of 3 days, 1, 2, 6, 12, 24, 36, and 72 months, and, finally, at 14.7 years. HPV serology to genotypes 6, 11, 16, 18, and 45 was determined at the same time points from the age of 1 to 36 months. Fourteen children stayed constantly HPV-negative during follow-up and HPV16-negative at final time point, and were considered as controls, while 19 children, who had either HPV DNA detected in umbilical cord blood and/or placenta and/or HPV DNA detected in oral mucosa either continuously (> 24 months) or HPV16 only at the final time point, were defined as cases. Tables Among cases, 16 of 19 children were tested for HPV at the last follow-up visit, and six of them had oral HPV: HPV11 was detected in one, HPV16 in four, and HPV6/16 coinfection in one child. Overall, oral HPV of any genotype was detected in all case children during the follow-up of 14.7 years, except one (ID201), who was HPV cord blood positive but remained oral HPV DNA-negative during the entire follow-up. In the control group, 9 of 14 children were tested at the last follow-up visit, one having oral HPV11. None of these 14 control children had any HPV DNA detectable in their oral mucosa at any study point during the original 72 months of follow-up. The bivalent HPV vaccination showed no significant effect on HPV detection at the last visit.Altogether, HPV antibodies for genotypes 6, 11, and 16 were found in 13 of 19 case children, nine of them having antibodies during the first 2 months of life, i.e., prior to the emergence of their own antibody production, indicating the maternal origin of these HPV antibodies. Among the controls, HPV antibodies were detected in 11 of 14 children, and six of them had antibodies during the first 2 months of life. Interestingly, of the 11 children with persistent HPV infection, ten had the same HPV type detected already at delivery or during the first 2 months, implicating that HPV acquired at very early age may predispose to HPV persistence.The HPV16-specific proliferative responses and frequencies of CD4+CD25+Foxp3+ regulatory T-cells (Tregs) after 7-day stimulation of PBMCs in both groups are shown in Fig. In the control group, the PBMCs of 13 of 14 children responded by proliferation to MRM. The child without an MRM response (ID006), however, showed reactivity to the HPV16 peptides. Altogether, 12 of 14 control children displayed reactivity to E2, of whom eight had a proliferative response to both HPV16 E2 peptide pools. Five children responded to the E6 peptides and two to the E7.2 peptide pools. Two control children (ID001 and ID010) were negative in the proliferation test for HPV16. As controls, they did not show any oral HPV positivity during the follow-up, but were HPV-seropositive . Increased percentages of Tregs specifically responding to HPV16 peptides were observed in four control children. In three children , these Treg frequencies were relatively high and were induced by several peptide pools.p = 0.047). The responses to the early antigen E2 were not significantly different between the groups. Similarly, no differences were found when the responses were stratified by HPV seropositivity (children with HPV antibodies detectable at 1 or 2 months and children with HPV antibodies after 2 months of age).On the whole, the proliferative response induced to the HPV16 E6 and E7 oncoproteins was significantly different between the cases and controls, with a higher frequency in the control group than in the case groups , but the range was wide . The cytokine secretion was higher in the cultures displaying cell proliferation after stimulation than in the other cultures. IFN-γ, IL-10, and IL-2 were the most frequently detected cytokines in all children, followed by IL-17A, IL-5, and TNF-α. The concentrations of IFN-γ and IL-5 were slightly higher than that of other cytokines. Similar to our previous study, IL-4 was not detectable except in one case child (ID303) as a response to the E6.2 peptide pool.p = 0.043), IL-10 (p = 0.013), and IL-17A (p = 0.043) in groups I–III (children with HPV DNA in placenta and/or cord blood) than in other case subgroups. Furthermore, when the children who tested HPV-positive in the placenta and/or cord blood (groups I–III) were stratified by their oral HPV persistence, those with no persistence showed higher TNF-α (p = 0.013), IL-2 (p = 0.009), and IL-5 (p = 0.006) secretion after E2.1 stimulation (pooled data). Figure Similar to the proliferation data, the cytokine concentrations were compared between the five case subgroups and the control group. No differences between the groups were found when the cytokine secretion by the proliferative PBMC cultures was analyzed. However, when we considered the data of all peptide-stimulated PBMC cultures, there was a significantly higher HPV16 E2-induced production of IL-5  also without any HPV16 antibodies and no response against HPV16 peptides. No HPV DNA was detected in the oral sample at the final visit, which, however, does not entirely exclude the presence of oral HPV. The mother of this child had genital and oral HPV16 detected once (at 24 months) and persistent genital HPV45, but no HPV antibodies at any time point. Both children were born by vaginal delivery and the breast milk tested HPV DNA-negative after delivery. Clinical examination revealed normal oral mucosa at the 72 months visit.In this study, we examined the HPV16 E2-, E6-, and E7-specific immune responses in children having distinct HPV profiles during the first 6 years of their lives. We found CMI responsiveness for E2 peptides in the majority of all tested children (78.8% of 33 children). This responsiveness was similar regardless of the child’s HPV status during the follow-up. The CMI responses against HPV16 E6 and E7 peptides were detected in 24.2% of children and were significantly more frequent in children who stayed constantly negative for oral HPV than in children with oral, placental, or cord blood HPV DNA. Interestingly, the analysis of the secretion patterns of Th1 and Th2 cytokines as a response to HPV16 peptides indicated more pronounced Th2 response in children born with HPV DNA-positive placenta and/or HPV-positive cord blood.Our previous studies , 15 wereThe cytokine polarization of HPV16 E2-, E6-, and E7-stimulated PBMCs was determined to canvass the possible alterations between types 1 and 2 cytokines. As the defects in the immune response against HPV have been linked with an increase in type 2 cytokines , it was Earlier studies in mice have shown that inoculation of newborn mice with high doses of murine retrovirus induces type 2 cytokine responses, while low viral doses induce type 1 responses. Further, the initial viral dose with relation to the number of potentially responsive T-cells is critical for the induction of Th1 and Th2 responses and development of protective immunity in newborn mice. Thus, the lower T-cell number in neonates compared to adults explains the reduced immune responses to same antigen , 21, 26.However, normal pregnancy in humans is known to be associated with Th2 skewing and the Th2 cytokine-dominated milieu at the placenta was suggested to establish a kind of protective homeostasis . This ThAs in our previous observations, HPV16 E2 peptides were, once again, the most antigenic epitopes, inducing proliferative response similarly among the cases and the controls. The proliferative response to HPV16 E6 peptides was also found frequently. The responsiveness of control children to E2 and E6 peptides is in line with the previous observations in healthy adults , 11, 38,Because HPV E2 is the main factor in the transcription initiation and regulation of viral genes and, therefore, abundant in all HPV-infected cells at the stage of viral amplification, the individual’s responsiveness to HPV16 E2 peptides alludes to the existence of circulating HPV16 E2-specific memory T-cells, originating from prior or present replicative HPV16 infection . FurtherInterestingly, we could identify two case children (ID305 and ID306) with persistent oral HPV16 (HPV DNA detected in repeated samples over 24 months) who showed no response to any HPV16 peptides. In general, the measured cytokine production was below the cut-off values as expected, since no HPV16-specific T-cell proliferation was detected in these children. Both of them had demonstrated oral HPV16 persistence during their active follow-up without HPV16 antibodies, and ID305 had oral HPV16 DNA detected also at the last (15 years) visit. This child had also received three doses of bivalent HPV vaccination, while child ID306 was not vaccinated. At the moment, we cannot ascertain whether the repeatedly detected DNA of a given HPV type in the oral mucosa is a sign of persistent HPV infection or if it is a new encounter with the same HPV type. However, our recent (unpublished) data on persistent oral HPV16 infections in adults have shown that HPV16 is either in an integrated or mixed physical state, confirming the concept of HPV persistence. In the same study, all subjects who cleared their oral infection had HPV16 in an episomal state . It is tempting to consider that children who received prophylactic HPV vaccination and who show persistent oral HPV16 infection might have an integrated HPV16, undetectable by the antibodies elicited by the HPV vaccination. In addition, it might well be that these two children, especially ID305, carrying persistent oral HPV16 without any detectable immunological responses, can have a specific defect in their immune system or even conceivable features of HPV tolerance. We also noticed that, in children with persistent HPV during the follow-up, the same HPV type was detected already at birth or during the first 2 months of life, implying that these very early infections could be more prone to remaining persistent. In addition, another child (ID011) in the case group showed no HPV16-specific response, but still had HPV16 DNA detectable in the oral mucosa. In this case, one possible explanation for the lack of immune response could be a very recent infection, not yet recognized by the adaptive immune system . This paTo conclude, these results support our earlier observations on the existence of HPV16-specific CMI in children , 15. It Altogether, the present findings of HPV16-specific CMI, together with our earlier data on the natural history of HPV infections during infancy and childhood, attest the view that an early exposure to HPV might be highly relevant for the subsequent HPV infections in adulthood, and also have some influence on the effectiveness of prophylactic HPV vaccines.


Public health educational program focusing and tailored on benefits perception of HPV vaccine and HPV disease severity, carried out at school or during medical visits, can be useful to improve HPV vaccination uptake.In Italy, the Human Papillomavirus (HPV) vaccination was implemented for twelve years old girls in 2007, but its coverage was lower than the recommended level. Sicily is one of the Italian administrative regions with lower vaccination coverage, with a value of 59% for those born in 1996 increasing to 62% coverage for those born in 1999. The aim of the study was to investigate factors associated with the refusal of HPV vaccination among young adult women of Palermo, Italy. The study was approved by the Ethics Committee of the Policlinico “Paolo Giaccone” Hospital  and the questionnaire was validated in a convenience sample representing 10% of the young women. A cross-sectional study was conducted through the administration of a telephone questionnaire, consisting of 23 items on HPV infection and vaccination knowledge based on the Health Belief Model framework. The eligible population were young women (18–21 years old) who had at least a vaccination among all included in the Sicilian vaccination schedule, without starting or completing HPV vaccination. Overall, 141 young women were enrolled (response rate 22%). Among them, 84.4% were unvaccinated and 15.6% had at least one dose of the HPV vaccine. In multivariate analysis, the factors associated with the refusal of the HPV vaccination were a bachelor’s as the education level (OR = 10.2,  Human Papillomavirus (HPV) vaccination represents the best strategy for primary prevention of cervical cancer. HPV vaccines have high efficacy against cervical pre-cancerous lesions, if given to females before they are exposed to the virus; therefore, the World Health Organization  recommends offering HPV vaccination to pre-adolescent girls .As of January 2018, 30 of 31 European (EU) countries had implemented HPV vaccination. Target age, financing and vaccine delivery differed among countries [In Italy, HPV vaccination is free and has been actively offered to all girls during their 12th year of life since 2007, and the National Health Department established a target vaccination coverage of 95% within five years of the start of the campaign ,5. HowevPrevious studies conducted in Italy and in Europe among adolescent and young women have indicated that common reasons for not receiving the HPV vaccine were the perception of low risk or not needing the vaccine, lack of vaccine awareness, doubt about the safety and efficacy of the vaccine, fear of side effects, inadequate testing of novel vaccines that may be harmful and weaken the immune system, lack of physician recommendations and cost of the vaccine ,8,9.The Health Belief Model (HBM) attempts to explain and predict health behaviours, and is used in assessing health-behaviour interventions by focusing on the attitudes and beliefs of individuals. The HBM has been used extensively to study vaccination beliefs and behaviours, and has also been used in vaccination research to identify people’s perceptions of disease and vaccination ,10,11.The objective of the study was to investigate factors associated with refusal of HPV vaccination using HBM among young adult women of the City of Palermo, Italy, after ten years of vaccine implementation.n = 26,153). The sample was recruited through the vaccination registries of two public vaccination services of Palermo LHU. These services were selected to be as representative as possible of the Palermo geographical area, relative to knowledge, attitudes and behaviours towards HPV vaccination and reliable sources of information. The vaccination registry was filled in for each girl residing in the area of public vaccination services, and who had performed at least one immunization recommended by the Sicilian vaccination schedule. A structured questionnaire was administered through the telephone by a trained healthcare professional from May to September 2017. The eligible population was represented by young adult women who refused or did not expect complete HPV vaccination schedule. The interviewer invited eligible young women to participate in the study, ringing up them to explain objectives of the study and data treatment. At a later time, the interviewer rang young women to administer the questionnaire. The exclusion criteria from the study were: erroneous telephone numbers, not responding after at least six attempts, already vaccinated, and refusal to participate in the study. At the beginning of the interview, informed consent was obtained and survey aims were explained as well as methods used to ensure confidentiality of data. The study was approved by the Ethics Committee of the Policlinico “Paolo Giaccone” Hospital  on 5 April 2017 (protocol number 04/2017). The questionnaire consists of 23 items based on previous studies conducted about HPV infection knowledge in Sicily and HBM in Europe, and it was divided into two sections (8–9). The first concerned demographic characteristic, HPV infection knowledge and use of gynaecologist services. The second section consisted of HPV vaccination knowledge and health belief investigating: perceived susceptibility of risk of developing cervical cancer (one item), perceived severity of the disease and its consequences (one item), perceived benefits related to vaccination (four items), perceived benefits related to HPV vaccination (three items), perceived vaccination barriers (two items) and perceived HPV vaccination barriers (three items) using HBM as the theoretical framework. The available response options, using a five point Likert scale, were: 1 = absolutely disagree; 2 = disagree; 3 = neither disagree nor agree; 4 = agree; and 5 = absolutely agree. In order to make the results easier to understand, all questions were scored towards beliefs that would result in adherence to vaccination. Therefore, lower scores reflect stronger beliefs about vaccination refusal. This questionnaire was validated in a convenience sample representing approximately 10% of the young women.An observational study was conducted among young adult women of the local health unit (LHU) of Palermo , including girls born from 1996 to 1999 . Normal distribution and homogeneity of variables were tested with Kolmogorov–Smirnov and Levene’s test, respectively. Mean values with standard deviation (SD) or median with interquartile range(IQR) were calculated for quantitative variables, while frequencies were counted for qualitative variables. Mean data were compared by a one-way analysis of variance (ANOVA) with Scheffe’s post hoc test, while comparisons of differences in the medians were analysed with the Mann–Whitney test. A univariate logistic regression analysis was performed in order to evaluate the factors associated with refusal of HPV vaccination. Study covariates, which were found to be significantly associated with the study outcome after the univariate analysis (p < 0.1), were evaluated into the multiple logistic regression models. Multivariate analysis was performed to investigate the independent effect of a risk or protective factor after adjustment for one or several other factors or to adjust for confounding variables. Only age was considered an a priori confounder.Statistical analyses were performed using STATA v14.2 software . For all analyses, Furthermore, a subset was selected to conduct an exploratory factor analysis on reasons to refuse HPV vaccination. Principal factor extraction was used followed by orthogonal rotation, which allows correlation among the factors. A three factor model was fitted, examining factor loadings for each model.n = 311) or not being available after at least six attempts . Only 5% (n = 34) of women had already performed HPV vaccination outside Sicily.Overall, 638 young women were eligible, although 72% of them did not have a useful phone number (erroneous phone number or not responding). n = 119) were unvaccinated and 15.6% (n = 22) received at least one dose of the HPV vaccine. Demographic characteristics, HPV infection knowledge and use of gynaecologist services of young adult women were described in n = 44) of young women participated in school informative meetings about HPV, 58.2% (n = 82) had a complete sexual intercourse, 11.3% (n = 16) a sexually transmitted disease (STD) and 50.3% (n = 71) a gynaecologist visit. Young women refusing vaccination more frequently had a school diploma (odds ratio (OR) = 4.56, p = 0.034) than women with an incomplete HPV vaccine schedule. On the other hand, young women refusing vaccination had less commonly taken part in a school informative meeting about HPV , and they had a lower amount of gynaecologist visits .A total of 141 young women were enrolled in the study. Of these, 84.4% (n = 60), followed by gynaecologists  and parents . The HBM answers on benefits of all vaccines had a mean score of 2.5 (SD = 0.1), benefits of HPV vaccine 2.5 (SD = 0.1), barriers of all vaccines 2.6 (SD = 0.1), barriers of HPV vaccine 2.0 (SD = 0.1), susceptibility of disease 4.3 (SD = 0.1) and disease severity 3.2 (SD = 0.1). Young women refusing vaccination had lower scores on HBM questions of perceived HPV vaccination benefits , perceived HPV vaccination barriers  and perceived severity , compared to women with at least one vaccination dose received.As showed in p = 0.028), the lower participation in school seminars on HPV  and the lower perception of anti-HPV vaccine benefits .In multivariate analysis , factorsn = 47), followed by fear of vaccine adverse events (33.6% n = 40), vaccine was not efficacy (15.1% n = 18) and logistic reasons (11.8% n = 14).Main reasons for vaccination refusal of HPV vaccine were lack of information . The UK HPV vaccination programme had several variations: some schools hold assemblies to promote and discuss the programme in advance of vaccination days, and other schools offer science lessons associated with HPV vaccination . In SwedIn addition, participants with a lower score for perceptions about HPV vaccinations’ benefits were more likely to be unvaccinated. This finding is in agreement with Marlow et al. where participants with a low score for HPV vaccination benefits (safety and efficacy of HPV vaccination) were more likely to be unvaccinated . A possiA consistent lack of information is mainly reported among young women who never performed vaccination. This data was similar to what was reported in a study conducted in the USA and in a survey conducted in Italy ,23. In aYoung women who reported fear of vaccine adverse events as the reason to refuse HPV vaccination were suffering from comorbidities and were critical of the roles of the pharmaceutical industry in HPV vaccination. Furthermore, their inclination to refuse HPV vaccination was dominated by fear of potential long-term side effects, in accordance with other findings ,24. ThosFinally, another reason for refusing HPV vaccination was revealed to be logistical reasons. The most frequently reported components of this category were the distance from the vaccination center and the lack of time to administer HPV vaccines. In particular, the latter was reported above all in a study as a reason for not completing the vaccination schedule . AccordiThere are two main limitations of this study. Firstly, the study was based on self-reported information, so personal perceptions may have been overestimated, although self-reporting was recognized as a cost-effective and feasible method for gathering data from large population samples. Secondly, the study had a low participation rate; therefore, the possibility of non-response bias should be taken into consideration. The low participation rate was mainly due to invalid phone numbers in the vaccination registry, suggesting a reorganization of the telephone catch-up procedure of Palermo vaccination services.These findings suggest that educational interventions focused on sexual transmitted diseases and conducted in a school setting may be necessary to enhance HPV vaccination rates among Sicilian girls. Therefore, supplying correct and unambiguous information to young women about vaccine efficacy and safety, and the value of vaccinations in preventing cervical cancer may be needed in order to increase HPV vaccination coverage in the future.