Accumulating evidence suggests that cutaneous human papillomavirus (HPV) infection is associated with non-melanoma skin cancer (NMSC). Little is known about the natural history of cutaneous HPV. A sub-cohort of 209 men with no NMSC history, initially enrolled in the HPV infection in men (HIM) study, were followed for a median of 12.6 months. Epidemiological data were collected through self-administered questionnaires. Cutaneous HPV DNA was measured in normal skin swabs (SS) and eyebrow hairs (EB) for 25 and 16 HPV types in genera β and γ, respectively. Any β HPV infection was more prevalent in SS (67.3%) compared to EB . Incidence in SS was higher than 20 per 1,000 person-months for HPV types 4, 5, 23, 38 and 76. Median duration of persistence of β and γ HPV infection was 8.6 and 6.1 months in EB, respectively, and 11.3 months and 6.3 months, in SS, respectively. Older age (>44 years vs. 18-30 years) was significantly associated with prevalent  and persistent β HPV infection . History of blistering sunburn was associated with prevalent  and persistent  β HPV infection in SS. Cutaneous HPV is highly prevalent in men, with age and blistering sunburn being significant risk factors for cutaneous β HPV infection. Human papillomavirus (HPV) is a non-enveloped DNA virus, primarily infecting stratified epithelium The earliest evidence for oncogenic potential of β HPV types came from studies by Jablonska and colleagues, in 1972, which demonstrated the presence of β HPV types in cutaneous lesions of patients suffering from Epidermodysplasia Verruciformis (EV) Few studies have evaluated the prevalence and persistence of cutaneous HPV among healthy individuals. Cutaneous HPV is thought to be ubiquitous in skin, with hair follicles serving as the reservoirs of persistent HPV infection In this study, we describe the natural history of cutaneous HPV infection in a cohort of 209 healthy men in Tampa, Florida. We evaluated a spectrum of epidemiological measures including risk factors associated with prevalence, persistence and incidence across two genera of cutaneous HPV infection. We observed that cutaneous HPV is highly prevalent in men and that, age and blistering sunburn are significant risk factors for cutaneous β HPV infection in men.The study population for the current analysis comprises a sub-cohort of men who participated in the U.S. site of the HPV infection in men (HIM) study, a large, multi-national prospective cohort study of the natural history of HPV infection in men Written informed consent was obtained from all participants in the parent study, and a separate addendum to the consent was completed by all participants in the sub-study. The parent study and sub-study protocols were approved by institutional review boards at each recruiting site, including the Human Subjects Committees of the University of South Florida, the Ludwig Institute for Cancer Research, São Paulo, Brazil, the Centro de Referencia e Tratamento de Doencas Sexualmente Transmissiveis e AIDS, Brazil, and the National Institute of Public Health of Mexico, as described previously At the enrollment visit, HIM study participants completed a comprehensive self-administered questionnaire with information on demographics , socioeconomic status, medical history, smoking status, alcohol consumption, and sexual history. Additional questions on risk factors for skin cancer  were added to the cutaneous HPV sub-study.Although, in the parent HIM study, swabs from both sun exposed and unexposed skin were collected, only swabs from sun-exposed skin were used for HPV DNA measurement in the present sub-study due to limited funding. A 5×5 cm area of normal, sun exposed skin, from the top of forearm, was pre-wetted (0.9% NaCl) and swabbed back and forth five times using a cotton-tipped Dacron swab . All swabs were placed in a separate vial and preserved in 500 µl Digene Standard Transport Medium (STM) for HPV DNA testing. Three to four hairs were plucked from each eyebrow  using disposable tweezers. Hairs with attached follicles were snap frozen in liquid nitrogen. All tissues were stored at −70°C until testing for cutaneous HPV DNA.DNA extraction was performed at the International Agency for Research on Cancer in Lyon, France, using the Qiagen BioRobot EZ1 with the EZ1 DNA tissue kit according to the manufacturer's instructions . Briefly, the Dacron swabs were carefully cut into an Eppendorf tube using scissors and incubated overnight in proteinase K and buffer G2  at 56°C. An EZ1 DNA Forensic protocol was used to extract the DNA from eyebrow hairs according to the manufacturer's instructions. To monitor the possible occurrence of cross-contamination between the different specimens during DNA extraction, tubes containing buffer only were also included (one tube with buffer every ten specimens).The detection of viral DNA was performed by a multiplex PCR/Luminex assay Following PCR amplification, 10 μL of each reaction was analyzed by multiplex genotyping using a Luminex based assay as described Baseline characteristics, including demographics, skin cancer risk factors, lifestyle factors and history of sexually transmitted diseases, were summarized for the cohort using descriptive statistics. HPV prevalence in eyebrow hairs and normal skin swabs was estimated by phylogenetic species along with concurrent infections with other HPV types. Type-specific prevalence of HPV was defined as the proportion of men who were positive for a given HPV out of the baseline cohort with valid samples. Genus- and species-specific prevalence was defined as the proportion of men who were positive for at least one HPV type in the given genus or species. The statistical significance of differences in the prevalence of HPV infection between eyebrow hairs and normal skin swabs was tested using Fisher's exact test. Incidence (in person-months) was determined for any HPV infection, as well as type-specific and species-specific HPV infections in normal skin swabs or eyebrow hairs. For analyses of incidence of any HPV infection, the participant had to be negative for all HPV types (genus β or γ) at baseline, and only the first incidence of any HPV infection was taken into account. Similarly, for species-specific and type-specific HPV infections, the participant had to be negative at baseline for that particular HPV species or type, respectively. Time to incidence of HPV infection was defined as interval, in months, between baseline visit and first visit with a positive HPV sample or time point being censored. Due to fewer events, median time to incidence of γ HPV infection in eyebrow hairs could not be estimated. Time to clearance of HPV infection was defined as interval (in months) between incident HPV infection and clearance of all type-specific HPV infections.Persistence of any HPV (genus β or γ) or type-specific HPV was defined as any or type-specific HPV infection at ≥2 consecutive visits. For a given type of HPV persistence, only participants who were positive for the HPV genus or type and had at least one follow-up visit after that were included. Kappa coefficient (k) was used to determine the concordance of viral infections across eyebrow hairs and skin swabs for each β-HPV type. The kappa coefficients <0 indicates no agreement, 0–0.20 as slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1 as almost perfect agreement Logistic regression models were used to estimate age-adjusted odds ratios (OR) and 95% confidence intervals (CI) for the associations between risk factors and prevalence of overall and species-specific HPV infections (β1 and β2) . Variables that were significantly associated with β HPV prevalence in an age-adjusted model were further evaluated after stratification by β HPV species. Logistic regression was conducted to estimate the association between risk factors and incidence as well as persistence of HPV infection, after adjusting for age. Logistic regression analyses were conducted separately for HPV infection in eyebrow hairs and normal skin swabs. Kaplan-Meier curves were derived for incidence and clearance of any HPV infection. An alpha level of 0.05 was considered statistically significant. Adjustment for multiple comparisons was not conducted. All analyses were conducted using SAS software, version 9.3  and R software, version 2.13.1.The median follow up time was 12.6 months. As seen in As seen in As seen in Incidence and persistence of cutaneous HPV are presented in Men aged >44 years were more than twice and thrice as likely to have prevalent baseline β HPV infection in eyebrow hairs and skin swabs, respectively, compared to men aged 18–30 years . A positEvaluation of species-specific β HPV prevalence indicated that men aged >44 years were twice more likely to harbor β1 and β2 types of HPV in eyebrow hairs and more than threefold likely to have β1 and β2 types of HPV infection in normal skin swabs , compareNo significant associations were seen between baseline factors and incident β HPV infection  and prevFindings on factors associated with persistence of β HPV infection were similar to those of prevalent infections described above. Age>44 years was associated with a six fold  increased persistence of infection with β HPV in eyebrow hairs  but not No significant associations were observed between baseline characteristics and persistence of γ HPV except a significantly increased persistence  in eyebrow hairs among former smokers compared to never smokers .In this HIM study sub-cohort of 209 healthy men, we observed a high prevalence of cutaneous β HPV and γ HPV in normal skin swabs, higher than that in eyebrow hair samples. Although cutaneous HPV seroprevalence rates have been reported previously Results presented here contrast to that of other studies which reported a higher prevalence of cutaneous HPV in eyebrow hairs among individuals without a history of skin cancer Older age and history of blistering sunburns were significantly associated with both prevalence and persistence of HPV but not with incidence of cutaneous HPV infection. Our findings are consistent with previous reports on the association between age and prevalence of HPV The findings of our study should be interpreted with caution. The external validity of these findings is limited to men. Future studies should include women, especially given the previously reported differences in seroprevalence of specific types of cutaneous HPV by gender The study has several strengths. This is the first study to report HPV infection across a continuum of incidence, prevalence, persistence and clearance of cutaneous HPV across different genera, by type and species. We compared HPV infection in normal skin swabs as well as eyebrow hairs, thus providing a comprehensive report on natural history of HPV using biomarkers from two tissues. This is particularly important since concordance between HPV infection in normal skin swabs and eyebrow hairs was found to vary by HPV type. Along with rates of HPV infection, we also evaluated a range of epidemiological factors associated with HPV infection, overall and by species. Analyses were adjusted for potential confounders.In conclusion, we observed high prevalence, incidence and persistence of cutaneous HPV infection among cancer-free men. Age and history of blistering sunburns were significantly associated with prevalence and persistence of β HPV. The association of blistering sunburns with both persistence of HPV infection, but not with incidence, suggests that sunlight exposure does not affect acquisition of new HPV infection but the duration of HPV infection could be affected by sunlight exposure through UV related immune dysregulation or actual promotion of HPV infection. Given the potential role of cutaneous β HPV in NMSC, the findings are valuable in defining a high risk population for development of novel preventive measures.


The molecular epidemiological studies showed that some variants of HPV-16, distributed geographically, would present a higher risk of causing cervical cancer. This study aimed to analyze nucleotide changes of HPV-16 E6 and E7 genomic regions from infected Southwestern Congolese women.DNA of twenty HPV-16 isolates was analyzed by amplifying the E6 and E7 genes using type-specific primers PCR and direct sequencing. The sequences obtained were aligned with the HPV-16 GenBank reference sequences.Thirteen (65.0%) out of 20 DNA-samples were successfully amplified. Genetic analysis revealed 18 and 4 nucleotide changes in E6 and E7 genomic regions respectively. The most frequently observed nucleotide variations were the missense C143G, G145T and C335T in E6 (100%), leading to the non-synonymous amino acid variation Q14D and H78Y. E7 genomic region was found to be highly conserved with two most common T789C and T795G (100%) silent variations. All HPV-16 variants identified belonged to the African lineage: 7 (53.8%) belonged to Af-1 lineage and 6 (46.1%) to Af-2 lineage. The missense mutation G622A (D21N) in the E7 region seems to be described for the first time in this study.This study reported for the first time the distribution of HPV-16 E6 and E7 genetic variants in infected women from southwest Congo. The findings confirmed almost ascendancy of the African lineage in our study population. The Republic of Congo has one of the highest rates of cervical cancer incidence in Sub-Sahara Africa. According to the World Health Organization (WHO), the reported age-standardized incidence rate and age-standardized mortality rate were 25.6 and 13.0 cases per 100 000 women in 2014 [Epidemiological and molecular studies are showed that High-risk human papillomaviruses (HPV) types are the etiological agents of cervical cancer ,3. HPV-1However, most HPV infections resolve spontaneously, suggesting that other co-factors are necessary in the persistence and lesion progression to cancer. These cofactors include some variants of high-risk types such as those of HPV16. Several molecular variants were described and classified according to geographical regions for the HPV-16 genotype ,6. ConveHPV-16 variants studies have shown that certain intratypic-variants would contribute more than others to the persistence of HPV infection and cervical cancer progression ,13. In pIn this perspective and with the advent of prophylactic vaccines against HPV-16/18 and cervical cancer, it becomes necessary to know the HPV genetic variants present in each population.In Congo, our previous study showed that the most common genotype in invasive cervical cancer and its precancerous lesions was HPV-16 . Thus, tOut of 20 HPV-16 single infected cervical cancer samples examined in this study, the E6 and E7 genes were able to be amplified and sequenced in 13  DNA samples. Nucleotide (nt) changes were analyzed between nt 104 to 559 for E6 region and nt 562 to 858 for E7 region.By comparison with the HPV-16 reference sequence , mutation analysis showed that 100% (13/13) of the analyzed sequences contained at least one nucleotide change in the E6 region. Eighteen single nucleotide changes were observed in HPV-16 E6 sequences, of which 13/18 (72.2%) were non-synonymous mutations  and 5/18 (27.8%) were synonymous mutations (silent). The most frequently observed variations were C143G (Q14D), G145T (Q14D), T286A, A289G and C335T (H78Y), which was found in all samples. All nucleotide changes, variants and their prevalence were reported in Table The details of E7 mutations are summarized in Table All HPV-16 positive sequences analyzed belonged to the African lineage, of which 7 (53.8%) out of 13 belonged to Africain-1 lineage (Af-1) and 6 (46.1%) out of 13 to Africain-2 lineage (Af-2). All African lines showed a common pattern of five characteristic mutations in E6, namely, C143G, G145T, T286A, A289G, and C335T, which lead to two non-synonymous amino acid changes Q14D and H78Y. In E7, all HPV-16 isolates showed a common pattern of two silent mutations namely T789C and T795G at codons 76 and 78 respectively. No European (E), Asian (As), Asian-American (AA) or northern American (NA) lineages were detected in this study. Phylogenetic trees were respectively built from eight sequences E6 regions  described and few lead to amino acid changes ,28. Out st cells . In addist cells ,33. The All HPV-16 isolates examined in this study belonged to the African lineage, of which 53.8% belonged to Af-1 lineage and 46.1% to Af-2 lineage. Our results are in agreement with the literature. Indeed, the pioneering studies of Yamada and Wheeler showed that the African lineage was dominated in the continent. More than 90.7% of the variants in Africa region are African lineage, of which 61% belongs to the Af-1 lineage and 29.7% to the Af-2 lineage .Tu et al.  in SouthKnowing that HPV variants data are important in developing HPV diagnostics, vaccines, and other therapeutic approaches to monitoring virus-induced diseases , the majThe present study provides preliminary data on HPV16 E6 and E7 variants in the southwest of Congo. Findings confirmed the predominance of the African lineage in our samples, which could guide control strategies against cervical cancer in Congo. However, future studies with a large sample are needed to better assess the distribution of these variants depending on the grade and the risk of developing cervical cancer.DNA of twenty HPV-16 single infection isolates of Congolese women with cervical cancer from our laboratory DNA bank  constituted from our previous study  was inve2, 0.8 mM of each dNTP, 0.4 μM/μL of sense and antisense primers, 0.025U/μl of Go Taq DNA polymerase (Promega) and 1 μL of template DNA. The thermal program  started with a pre-heat of 94°C for 10 min, followed by 35 cycles according to the protocol: 45 sec denaturation at 94°C, 45 sec annealing at 49.5°C for E6 and 53.5°C for E7 and 45 sec extension at 72°C, with 7 min final elongation at 72°C. For each reaction, DNA from SiHa cell lines as positive controls and Ultra-pure PCR water nuclease-free  as negative PCR control were included.Amplification of HPV-16 E6 and E7 genes was performed using TS primers flanking the encoding regions of HPV16 E6 ORF (nt: 41–576) and HPV16 E7 ORF (nt: 483–911), as previously described . PCR wasPCR products were analyzed based on the expected specific bands in 2% agarose gel. Type-specific PCR products were directly sequenced using the “Big Dye Terminator v3.1 Cycle Sequencing kit” (Applied Biosystems) according to the manufacturer’s instructions in molecular and functional genomics platform .http://www.ncbi.nih.gov/BLAST/) to confirm genotype and amplified region. Multiple sequence alignments of the full-length of E6 and E7 genes were performed using BioEdit Sequence Alignment Software v7.0.4.1 and Clustal W program [The sequences were subsequently analyzed by NCBI using online BLAST 2.0 software server ( program . HPV-16  program .Phylogenetic trees were built by MEGA 5.1 package  using a http://www.ncbi.nlm.nih.gov/GenBank), under the accession numbers KP313765 to KP313772 for E6 and KP313773 to KP313777 for E7 genes.The representative nucleotide sequences from each region sequenced in this study were deposited into GenBank Database .


Risk to harbor cervical lesions was observed in multiple HPV infections with regard to the HPV56  and HPV58  genotypes. In addition, women coinfected with HPV16/31/56 types had 6 and 5-fold increased risk of HSIL  and LSIL , respectively. Multiple HPV infections without HPV16/18 has 2-fold increased risk of HSIL  and LSIL OR=2.03; 95%CI: 1.08 to 3.79; p=0.02). The results of this study suggest that single and multiple vaccine target as well as non-vaccine target HPV types are associated with LSIL and HSIL. These finding should be taken into consideration in the design of HPV vaccination strategies.We performed an association between high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and single or multiple vaccine-target as well as non-vaccine target Human papillomavirus (HPV) types. Using bead-based HPV genotyping, 594 gynecological samples were genotyped. An association between squamous intraepithelial lesion (SIL) and presence of HPV16, 18, 31, 58 and 56 types were calculated. The risk was estimated by using odds ratio (OR) and 95% of confidence intervals (CI). A total of 370 (62.3%) women were HPV positive. Among these, 157 (42.7%) presented a single HPV infection, and 212 (57.3%) were infected by more than one HPV type. HPV31 was the most prevalent genotype, regardless single and multiple HPV infections. Single infection with HPV31 was associated with LSIL (OR=2.32; 95%CI: 1.01 to 5.32;  Clinical and epidemiological studies report that cervical infection with High Risk Human papillomavirus (HR HPV) is necessary but not sufficient to cause the development of cervical cancer ,2. Addithttp://www.hpvcenter.se/html/refclones.html) and approximately 40 of them are known to be able to infect mucosal areas including the epithelium of the ano-genital tract. The classification of HPV types is based on the intrinsic oncogenic potential of these viruses, where 15 oncogenic genotypes are associated with the majority of cervical lesions and cervical cancer [Persistent infection with HR HPV is considered as the main cause of cervical lesions and cervical cancer . To datel cancer . The reml cancer .Multiple HPV infections are commonly found in molecular epidemiologic studies –20. HoweDespite some evidences reported cross-protection of HPV16/18 vaccine among Alpha-9 HPV31, 33, 52) and Alpha-7 (HPV45) species –30, ther, 33, 52 The samples evaluated in this study were obtained by cervical scraping from 594 patients who volunteered to take part in cervical cancer screening at the Gynecological Clinic at the Clinical Hospital (n = 293) and Oswaldo Cruz University Hospital (n = 130) at Recife, Pernambuco State, Northeastern Brazil, and at the Center for Integral Attention to Women’s Health (n = 171) at Aracaju, Sergipe State, Northeastern Brazil. The study included 91 women  with low-grade squamous intraepithelial lesions (LSIL), 113 women  with high-grade squamous intraepithelial lesions (HSIL) and 390 women  with normal cytological results. On the other hand, the exclusion criteria were current pregnancy, undergone hysterectomy or being HIV-positive.Women attended at the three screening centers  were resident in the Metropolitan area of Recife and Aracaju and nobody came from rural areas due to the travel expenses; the costs of patients’ dislocation, is an important social issue to be considered when dealing with individuals enrolled in public health structures; in fact, as it is already known in North East Brazil, people using public health service are from the low income portion of the population with little or no facilities to travel around, being the health service predominant in medium upper social classes. So, patients had very similar socio-economical background, living all in the metropolitan areas of Recife and Aracaju, characterized by low-income economic status, associated with poor scholar training and unemployment .All women participating in this study had not been vaccinated against HPV. The cervical cells collected with cytobrush were placed in phosphate-buffered saline (PBS) pH 7.4 and stored at -80°C prior to DNA extraction.The samples of cervical cells were processed for DNA extraction using the DNeasy Blood and Tissue Kit (Qiagen), in accordance with the following steps: suspension of the cell pellet in PBS (pH 7.4), cell lysis, purification, washing and drying of the material to obtain the DNA elution.All clinical investigation developed in this study was conducted according to the principles expressed in the Declaration of Helsinki. The patients were enrolled in the period between February 2011 and June 2013, and were informed about the objectives of the research. We obtained approval of the Ethical Committee  and all women signed the consent. The inclusion criteria were to agree in participating to the study and be at least 18 years.The multiplex HPV type-specific E7 PCR utilizes HPV type-specific primers targeting the E7 region for the detection of several HR-/pHR-HPV types and 2 LR-HPV types, with detection limits ranging from 10 to 1.000 copies of the viral genome. The amplicon size varies between 210 and 258 bp. Two primers for amplification of the beta-globin gene were also included to provide a positive control for the quality of the template DNA . Each rehttp://www.medcalc.org/index.php. To evaluate the influence of cofactors  in HPV infected patients LSIL and HSIL we used the logistic regression model. The comparison between the groups LSIL, HSIL and Normal cytology was made by the chi-square test using the statistical software R . Significance was defined as P<0.05 for all tests.The most prevalent HPV 16, 18, 31, 56 and 58 types were analyzed separately. The odds ratio of LSIL and HSIL, with single or multiple HPV infections were calculated in comparison with normal group. Odds ratios (OR) and 95% confidence intervals (CI) are reported through the text and in the Tables. Statistical analyses were performed by using MediCal version 14.10.2 A total of 594 cytological samples classified as normal cytology, HSIL and LSIL were genotyped by using BioPlex platform. The prevalence of HPV was 62.3% (370/594), where 42.7% (158/370) of the samples had a single HPV infection, and 57.3% (212/370) were infected with multiple HPV types , HPV16 , and HPV56  . We alsop = 0.0012), although this was not observed in women with LSIL (p = 0.25) (p = 0.0212) (p<0.001) and HSIL  (p = 0.62) and HSIL (p = 0.07), an association was observed between the presence of HPV18 in multiple HPV infections and HSIL  (p = 0.04), but not HSIL (p = 0.32). However, co-infections with HPV31 showed association in both LSIL  and HSIL  (p = 0.0108)  and HPV58  (p = 0.0032)  . Moreove 0.0212) . With re<0.001) . Despite= 0.03) . Single <0.001) . In addi 0.0108) . An assoor HSIL) . Co-infe 0.0032) .p = 0.03) and HSIL  (p = 0.01) and HSIL  (p = 0.1) and HSIL (p = 0.11) (p = 0.03) and 6-fold increased risk to HSIL  (p = 0.01) and HSIL  (p = 0.0007) and HSIL . Multiple HPV infections without HPV16/18 types had a 2-fold increased risk to HSIL  and LSIL   . Similar= 0.01) . In cont = 0.11) . Multipl= 0.002) . In addi= 0.015) . Similar = 0.02) .We performed an association study between cervical lesions and single and multiple VT HPV as well as NV HPV types. Our results showed several meaningful findings, as follow: 1) HPV31 is the most prevalent genotype found in women from Northeastern Brazil; 2) NV HPV56 is the third most frequent genotype; 3) Single infections with either NV HPV51 and HPV56 are frequent in HSIL; 4) Co-infections with HPV31 and NV HPV56 are the most frequent in HSIL; 5) Only single HPV infection with either HPV16 and HPV31 is associated with any SIL; 6) Co-infections with HPV31 had a 3-fold increased risk to any SIL; 7) Co-infections with NV HPV56 have four to five times more risk to any SIL; 8) Co-infections with HPV58 have three times more risk to any SIL; 9) Co-infections HPV31 and NV HPV56 excluding HPV16 have four to five times more risk to any SIL; 10) Multiple HPV infection without HPV16/18 have two times more risk to any SIL. Taken together, these findings suggest an association between the presence of HPV types and the risk to develop any SIL in women from Northeastern Brazil.To date, there is no consensus whether multiple HPV types occur randomly or through competitive or cooperative relationship. Studies have demonstrated that infection with multiple HPV types occurs more frequently than predicted by chance ,13,16. ISeveral authors have shown that HPV16 is the most prevalent in Brazil ,20,37–45For the first time, HPV56 was observed as the third most common HPV (9.24%) found in Northeastern Brazil. The discrepancy between the HPV prevalence studies may be attributed to different HPV DNA detection strategies. In this study, we used a multiplex HPV genotyping, which is a simple bead-based high-throughput hybridization method that allows to detect several HR HPV . In addiSeveral evidences have reported cross-protection of the bivalent vaccine  and the quadrivalent vaccine  against HPV31, 33 and 45 types –30. HoweFinally we have to recognize the limitations of our study including lack of information on cigarette smoking and alcohol habits, age at first intercourse, marital status, due to the possibility not providing these information in the questionnaire administered to the patients; all these factors are known to possibly influence the risk of HPV infection and cervical lesions/cervical cancer development.In conclusion, we have demonstrated that women infected with single and multiple VT HPV as well as NV HPV types have a risk to harbour cervical lesions. Longitudinal studies with these HPV types, could clarify the risk of these HPV types to developing cervical lesions in Northeast Brazilian women.S1 FileTable A, HPV diversity as single and multiple infections found in women from the Northeast Brazil. Table B, Distribution of HPV types among normal cytology, High-grade squamous intraepithelial lesion (HSIL) and Low-grade squamous intraepithelial lesion (LSIL), considering single and multiple infections.(DOCX)Click here for additional data file.


High-risk HPV DNA testing has been proposed as a primary tool for cervical cancer screening (HPV-CCS) as an alternative to the Papanicolaou cytology- method. This study describes factors associated with women’s intentions to attend cervical cancer screening if high-risk HPV DNA testing (HPV-CCS) was implemented as a primary screening tool, and if screening were conducted every 4 years starting after age 25.This online survey was designed using the Theory of Planned Behaviour to assess factors that impact women’s intentions to attend HPV-CCS among women aged 25–69 upon exit of the HPV FOCAL trial. Univariate and regression analyses were performed to compare the demographic, sexual history, and smoking characteristics between women willing and unwilling to screen, and scales for intention to attend HPV-CCS. A qualitative analysis was performed by compiling and coding the comments section of the survey.Of the 981 women who completed the survey in full, only 51.4 % responded that they intended to attend HPV-CCS with a delayed start age and extended screening interval. Women who intended to screen were more likely to have higher education , while both positive attitudes  and perceived behavior control  were significant predictors of intention to screen. Among women who provided comments in the survey, a large number of women expressed fears about not being checked more than every 4 years, but 12 % stated that these fears may be alleviated by having more information.Acceptability of increased screening intervals and starting age could be improved through enhanced education of benefits. Program planners should consider measures to assess and improve women’s knowledge, attitudes and beliefs prior to the implementation of new screening programs to avoid unintended consequences. Globally, cervical cancer is the fourth most common type of cancer in women . In highClinical trials have confirmed increased detection of pre-cancerous lesions with the use of HR-HPV DNA testing compared to cytology –20. A reCurrently in British Columbia (BC), Canada, screening begins at age 21, or approximately 3 years after sexual debut, and screening is recommended every 2 years. In our previous study, women’s intention to be screened with HPV decreased from 84.0 to 51.4 % when they were advised about the delayed start for screening and the extended interval . In thisThe goal of this study was to determine the factors associated with women’s intentions to be screened for cervical cancer if HR-HPV DNA testing was used instead of Pap smears when screening was conducted every 4 years and screening started at 25 years of age.Survey participants were recruited at study exit from the HPV FOCAL trial (ISRCTN79347302). HPV FOCAL is a clinical trial determining the efficacy of HPV based primary screening to liquid based cytology (LBC) for the detection of cervical intraepithelial neoplasia grade 2 or greater (CIN2+). Trial methods are described in detail elsewhere , 27 but Participants were invited to complete the survey at exit if they had completed the study per protocol . At the Theory of Planned Behaviour (TPB) [p < 0.5 then items within the scale are dropped until acceptable agreement is achieved. The details of how scale consistency was achieved for the questions used in this survey, and scale characteristics are detailed elsewhere [At study exit, women were invited via email to complete an electronic survey. Women were provided with background information on HPV, transmission, and a rationale for the use of primary HR-HPV DNA testing including an extended screening interval Appendix . The surur (TPB) , which hur (TPB) . It examlsewhere . Particip ≤ 0.2 were considered for stepwise logistic regression modeling [Univariate and regression analyses were performed using SAS (v. 9.3.1) to compare the characteristics of survey responders to non-responders, in addition to presenting demographic, sexual history, smoking data, and scales for intention to attend HPV-CCS. All variables that reached significance of modeling . A logisIn total, 2016 email invites were sent to women who had exited the HPV FOCAL trial. 1538 received the survey, 1466 unique women clicked onto the survey, and 981 completed the entire survey (Table p = 0.007). No other demographic characteristics  were statistically significant. All scale items for attitudes, subjective norms (direct & indirect), and perceived behavior control were significantly associated with intention to screen with HPV with an extended interval and delayed start date (p < 0.001), while items assessing the impact of partners were not significant (p = 0.33).The results of the univariate analysis are captured in Table In the final logistic model, women with the intention to screen at age 25 and every 4 years were more likely to be educated beyond high school   participants provided comments: 14 from the control arm, 284 from the safety arm and 18 from the intervention arm. The mean age for these women is 46. They were predominantly married (67 %), half of whom were university graduates or had a university advanced degree (52.2 %). Twenty-two percent of all comments were related to the extended screening interval. While some women stated that the 4-year interval would be an improvement over screening every year, a large number of women expressed fears about not being checked more often than every 4 years. This was especially true of older women who have historically been told to have a Pap yearly for most of their lives. As one participant stated, “I worry that only being tested every 4 years gives plenty of time for issues to arise and go untreated”. Seven percent of women who commented expressed concerns over the delayed screening age, for example one participant stated “Age (25) is too late. I had a 19 year old staff member with cervical cancer. Girls have sex as early as 14…11 years is too long to wait”. Another stated “How did you come up with the every 4 years and starting at 25 years old? Should it not be more frequent at a younger age? It just seems to be an arbitrary number”. Twelve percent of the comments suggested that their fears about the new HPV testing technology and the extended interval may be alleviated by having more information.Many countries are considering, or have confirmed implementation of HPV testing as the primary method for cervical cancer screening including the United States (U.S.), Australia, and Canada –36. The The lower acceptability of the extended screening interval could lead to unintended consequences and impacts on screening programs. Women may choose to return for screening every 2 to 3 years instead of the recommended 4 to 5 years. This could lead to over-screening and potentially over-treatment for cervical lesions that could resolve without treatment. In studies conducted in the U.S., the majority of women surveyed preferred to screen annually despite knowledge of changes to guidelines recommending an extended screening interval , 38. In It has been reported elsewhere that the adverse emotional responses experienced by women are directed at the HPV infection itself rather than HPV testing . FeelingNot all email addresses to which the survey was sent were functional, and the overall response rate was 63 %, therefore, selection bias is possible. However, our previous study  demonstrAs programs begin planning for implementation of HR-HPV DNA testing as the primary tool for cervical cancer screening, it becomes important to assess the acceptability and willingness of this screening technology among women currently engaged in screening programs. This study assessed women’s intention to attend HPV-CCS, using scales for attitudes, direct and indirect subjective norms, and perceived behaviour control based on the Theory of Planned Behaviour. All women were provided information about HPV and HPV testing at when they consented to participate in HPV FOCAL. Our results demonstrated that women’s intention to attend HPV-CCS decreased significantly with the introduction of the longer screening interval and the delayed starting age for screening. Attitudes toward HPV-CCS, and perceived behavior control were particularly important factors in women’s intention to attend screening. This has important implications for program planners who are considering changes to screening programs and want to avoid unintended consequences such as reducing the uptake of screening, or over-screening. Our findings also indicate a need for robust and targeted messaging and education on HPV and HPV based CCS for women and healthcare providers.Data requests can be directed to the corresponding author.


The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening.The study, conducted by the “HEllenic Real life Multicentric cErvical Screening” (HERMES) study group, involved the recruitment of 4,009 women, aged 25–55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein.Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25–29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+  as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology).HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18 genotyping could represent a more accurate methodology for primary cervical cancer screening in comparison to liquid-based cytology, especially in older women. Low coverage and poor patient compliance, subjective interpretation of results, in conjunction with factors concerning public health and legal issues, are some of the possible reasons for the substandard results that have been noted in cytology-based screening  was significantly higher than the specificity of HPV testing [90.3% (95% CI 89.3%–91.2%)] (P<0.05). The specificity difference (ASCUS+ cytology—14HR HPV testing) was 0.07 (95% CI: 0.06–0.08). Testing for types 16&18 only had similar diagnostic accuracy indices to cytology. Positivity for the combination of the two tests (co-testing), that is positivity for either ASCUS+ or 14HR HPV testing, resulted in reduced specificity , which was higher than the sensitivity of cytology [ASCUS+: 53.7% (95% CI 37.4%–69.3%)] to a statistically significant degree (cificity .P<0.05). The specificity difference (ASCUS+ cytology—14HR HPV testing) was 0.07 (95% CI: 0.06–0.08). Testing for type 16 only had similar diagnostic accuracy indices to cytology. Testing for types 16&18 only had at least equal accuracy compared to cytology for CIN3+ but not in a statistically significant degree. The combination of the two tests (co-testing) resulted again in reduced specificity (At the threshold of CIN3+ HPV testing had a sensitivity of 100% (95% CI 76.8%–100.0%), which was higher than the sensitivity of cytology [ASCUS+: 64.3% (95% CI 35.1%–87.2%)] but the difference did not reach statistical significance. The sensitivity difference (ASCUS+ cytology—14HR HPV testing) was -0.36 (95% CI: -0.61–-0.07). In contrast, the specificity of cytology  was significantly higher than the specificity of HPV testing [89.7% (95% CI 88.7%–90.6%)] (cificity .P<0.0001).With the exception of 14HR HPV testing, which has a sensitivity of 100% in both cases, changing the threshold from CIN2+ to CIN3+ has led to an increase in sensitivity and NPV for all tests examined. The largest increase in sensitivity, from 58.5% (95% CI 42.1%–73.7%) to 78.6% (95% CI 49.2%–95.3%), was noted for HPV 16 or 18 positivity. PLR was slightly increased if the threshold was CIN3+ for all tests apart from positivity for the 14HR HPV types, and NLR as well as PPV markedly decreased. Comparison of the diagnostic accuracy of the cobas HPV test to cytology with the McNemar’s test revealed a statistically significant difference , but also in 86% more cases of CIN2+ detected (22 cases with ASCUS+ vs 41 cases with ASCUS+ and/or 14HR HPV positive). The number of colposcopies needed to detect a CIN2+ after a positive cytology or positive 14HR HPV testing or both (co-testing) was 11.1 [41 CIN2+ detected after performance of 454 colposcopies (454/41)], whereas the respective number for 14HR HPV testing alone was 10.0 (409/41) and for cytology (ASCUS+) alone was 6.4 (141/22).HPV infection in general, and especially asymptomatic or transient HPV infections are more common in younger age groups compared to older, whereas the opposite is the case for the prevalence of high-grade cervical lesions (CIN2 or worse) ,40. TherFinally, By combining genotyping with cytology, testing positive for HPV16 or ASCUS+ cytology, as well as testing positive for HPV16 or HPV18 or ASCUS+ cytology had a markedly higher sensitivity of 75.6% (95% CI 65.4%–88.8%) and 92.6% (95% CI 91.8%–93.5%) respectively than ASCUS+ alone, the former being marginally significant and the latter being clearly significant and the highest among all test combinations. By replacing ASCUS+ by LSIL+ or HSIL+, combined with HPV16 testing alone or HPV 16 or HPV 18 or both, a gradual decrease in sensitivity was noted, although all rates were not statistically significantly different than ASCUS or worse cytology alone. Testing positive for HPV 16 or HPV 18 or both, and additionally ASCUS+ cytology would significantly increase PPV and PLR for the histological identification of CIN2+, in comparison to ASCUS+ alone [32.4% (95% CI 17.3%–47.5%) and 46.4 (95% CI 25.1–85.9) vs 10.4% (95% CI 6.3%–14.6%) and 11.2 (95% CI 8.2–15.4) respectively]. Similar significant increase in PPV and PLR in HPV 16/18 cases is noted when ASCUS+ is replaced by LSIL+ compared to ASCUS+ cytology alone [39.1% (95% CI 19.2%–59.1%) and 62.3 (95% CI 28.6–135.7) vs 10.4% (95% CI 6.3%–14.6%) and 11.2 (95% CI 8.2–15.4) respectively].Detection of HSIL or worse cytology or HPV 16/18 genotyping for the triage of HPV positive women has a slight, not statistically significant increase of sensitivity and PPV than ASCUS or worse cytology alone [68.3% (95% CI 54.0%–82.5%) vs 53.7% (95% CI 37.4%–69.3%) and 17.4% (95% CI 11.5%–23.2%) vs 10.4% (95% CI 6.3%–14.6%)]. However, considering that the PPV of HSIL alone is 46.7% (95% CI 21.4%–71.9%), testing positive also for HPV 16 or 18 or both would result in an almost similar PPV of 50.0% (95% CI 10.0%–90.0%), whereas the sensitivity would drop from 17.1% (95% CI 5.6%–25.6%) to 7.3% (95% CI 0.0%–15.3%)]. According to that, the addition of HPV 16/18 genotyping to HSIL cytology would not improve diagnostic accuracy concerning triaging HPV positive women.Finally, no statistically significant changes in relative risk between the different combinations of triage tests used to detect CIN2 or worse cases among HPV-positive women were noted.Many things have changed since 1943 when Papanicolaou and Traut published their paper on the role of the vaginal smear in cervical cancer diagnosis , leadinget al. taking into account the follow-up data of 176,464 women from 4 European studies have shown that HPV DNA-based screening alone provides a 60–70% better protection against cervical cancer compared to cytology . Sensitivity rates follow similar trend when ASCUS or worse is replaced by LSIL or worse cytology. Testing positive for HPV 16 or HPV 18 or both and additionally ASCUS+ cytology would also significantly increase PPV and PLR for the identification of CIN2+, in comparison to ASCUS+ alone [32.4% and 46.4 vs 10.4% and 11.2 respectively]. Regarding cytology of HSIL or worse, the addition of HPV 16/18 genotyping would result in reduced sensitivity and similar PPV, a sign for the relatively low additive value of 16/18 genotyping in HPV-positive women if a HSIL+ cytology result is present.As a conclusion, triaging of HR HPV positive women with HPV 16/18 genotyping alone has a similar or slightly better performance than using LBC alone, whereas combining cytological indices with HPV 16/18 genotyping results in an even more sensitive method to triage HPV-positive women than either genotyping or cytology alone. The latter, however, can only be achieved at the cost of performing two different diagnostic procedures, (i.e.) HPV 16/18 genotyping and LBC, additionally to the initial HPV testing. On the contrary, genotyping for HPV 16/18 using the cobas HPV test as alternative to cytology for triaging HPV-positive women, can be performed by one sole diagnostic procedure offering concurrently HPV testing and HPV 16/18 genotyping results.et al. using cobas was 10% [The superior sensitivity of HPV testing is an established finding in the literature, but it has also been shown that there are variations in performance depending on the HPV testing method used and the study population especially between continents . Therefo was 10% . It is oThe design of our study, in some aspects and in a much smaller scale, is similar to that of the larger ATHENA trial and, based on the respective results, we drew similar conclusions. As weaknesses of our study one can notice the relatively small sample of women tested, as well as the fact that it is more subject to verification bias since double screen negatives were not offered colposcopy, apart from a small fraction of the NILM and HPV-negative women used for verification bias adjustment. Taking in mind that this percentage of double negative women tested was relatively small, there were no cases of CIN2+ found in the random sample of double negative women that had colposcopy, making actual adjustments of the results not necessary. This could cause an overestimation of the HPV test sensitivity but should not affect the relative difference between HPV testing and cytology. Apart from that, the lack of histological verification in all HPV-positive or/and cytology-positive women by using the presence of abnormal colposcopic impression as a prerequisite for the performance of biopsies, as well as the relatively high number of HPV-positive or/and cytology-positive women lost to colposcopy, represent additional limitations in our study, which could cause a diminished identification of women with high-grade disease. This fact could probably affect to some extent the final accuracy of the two screening tests.As strengths of our study one could note the use of LBC, the fact that all women enrolled had both, HPV DNA testing and cytology performed, that cytology examinations were performed at the corresponding cytology laboratories of the hospitals cooperating in the study reflecting real-life routine laboratory conditions, that cytologists weren’t aware of the HPV DNA test results, nor were molecular biologists of the cytology results, that colposcopy and histology were conducted in each of the cooperating hospitals by experienced specialists and pathologists were not aware of the HPV DNA test result. As an additional strength of the present study one could consider the sub-analysis performed for women aged below and above 30, in order to identify the best option for starting age of cervical cancer screening, by assessing the performance of HPV DNA testing with genotyping as well as of LBC for the two age groups.In summary, our study is the first multicenter trial in Greece evaluating the performance of HR HPV DNA testing with concurrent HPV 16/18 genotyping in comparison to LBC for primary cervical cancer screening. According to our results from the first screening round, HPV DNA testing has much better sensitivity than cytology to identify high-grade cervical lesions (CIN2+ or CIN3+), with slightly lower specificity. Changing the starting age of screening from 25 to 30 years, leads to significant increase on both, specificity and PLR of HPV DNA testing, advocating for its optimal use in women over 30 years of age. Additionally, genotyping for HPV 16 or HPV 18 or both in order to detect high-grade lesions has similar sensitivity and specificity to ASCUS or worse cytology in young (25–29 years) as well as in older women (30–55 years). In conclusion, HPV DNA testing with concurrent individual genotyping for HPV 16, HPV 18 or both, could represent a more accurate methodology for primary cervical cancer screening in comparison to liquid-based cytology.


Recently, the studies on the prevention and treatment of human papillomavirus (HPV) which is closely related to the cervical cancer and other genital diseases are attracting more and more attention all over the world. Marine-derived polysaccharides and other bioactive compounds have been shown to possess a variety of anti-HPV and related cancer activities. This paper will review the recent progress in research on the potential anti-HPV and related cancer agents from marine resources. In particular, it will provide an update on the anti-HPV actions of heparinoid polysaccharides and bioactive compounds present in marine organisms, as well as the therapeutic vaccines relating to marine organisms. In addition, the possible mechanisms of anti-HPV actions of marine bioactive compounds and their potential for therapeutic application will also be summarized in detail. Papillomavirus is a diverse group of non enveloped DNA viruses that infect the skin and mucosal tissues of a range of vertebrate species, including humans ,2,3. In Although HPV infection is considered a sexually transmitted infection, HPVs can also be transmitted by on-sexual routes including casual physical contact and erinatal vertical transmission ,10. SomeThis review presents an overview of recent progress in research on the prophylaxis and therapy for HPV and related cancer diseases. Moreover, this review will mainly focus on the heparinoid polysaccharides and bioactive compounds present in marine organisms. Recent developments in the possible mechanisms of anti-HPV actions of marine bioactive compounds and their potential for therapeutic application will also be discussed in detail.E1, E2, E4, E5, E6 and E7) and two late genes (L1 and L2) . It. It65]. 0 µg/mL) .In summary, the sulfate polysaccharides such as heparin and heparinoid polysaccharides can inhibit the entry process of HPV by interfering with the initial attachment of the viral particle to the host cell, which suggests these polysaccharides can be developed into novel anti-HPV drugs in the future.d-galactose and 3,6-anhydro-d-galactose extracted from red algae  proliferation [It was reported that some sulfated polysaccharides from brown algae possess good antiviral and anti-tumor activities and some of them have been developed into novel antiviral agents ,79,80,81 seaweed . Some al seaweed , anti-in seaweed , and ant seaweed . It was feration . In addiet al. [Laminaria brasiliensis (molar ratio M/G 1.2) and its M and G blocks (DP = 20) all could promote atypical mitoses in HeLa cells, besides the presence of acidophilic material in the cellular cytoplasm and occurrence of multinuclear cells present in the monolayer. In addition, the polysaccharide fraction SF isolated from the brown seaweed Sargassum stenophyllum containing mainly fucose could promote much accentuated morphologic modifications in HeLa cells at low concentrations (2.5 μg/mL) [Moreover, Stevan et al. , reporte5 μg/mL) . SF coulin vitro with the IC50 value of 1.1 µg/mL [50 < 0.1 µg/mL), it also merits further investigation as novel anti-HPV or anti-cervical cancer candidate in the future. Taken together, brown algae-derived bioactive compounds, in particular the alginate polysaccharides and fucans, have good antiviral or anti-tumor activities, thus the sulfated polysaccharides from brown algae have the potential to become new resources for the development of anti-HPV and related cancer agents.Furthermore, it was reported that fucoidan produced from brown algae could also inhibit HPV pesudovirus infection .1 µg/mL . AlthougAspergillus species are filamentous saprophytic fungi that can be found in almost all aerobic environments, which possessed antitumor, anti-inflammatory, antiviral and antibacterial activity [Aspergillus, Gliocladium and Penicillium species, is a tricyclic alkaloid [Aspergillus sp. may induce apoptosis in HPV related cancer cells via the mitochondrial pathway followed by downstream events leading to apoptotic mode of cell death [Marine-derived fungi have proven to be a promising source of bioactive metabolites and a growing number of marine fungi have been reported to produce bioactive secondary metabolites ,89. Aspeactivity . Gliotoxalkaloid ,92,93. Galkaloid . In a woll death .Microsporum sp. [Marine microbes are valuable sources of structurally diverse bioactive compounds with anticancer activity. It was reported that a prenylated indole alkaloid, neoechinulin A, could be isolated from the culture broth extract of a marine-derived fungus, orum sp. . Neoechiorum sp. . In summStichopus Japonicus is a class of natural polysaccharides, which is rich in marine coelenterata. Some studies reported that marine japonicus polysaccharide SJAMP could significantly inhibit the proliferation of human cervical carcinoma Hela cell in vitro [in vitro through interfering with the cell cycle of cervical cancer cell [Marine life is a treasure trove of natural medicine, and the polysaccharides isolated from a variety of marine animals have been shown to possess good pharmacological activities such as antitumor, anti-inflammatory, and antiviral activity. Marine japonicus polysaccharide isolated from in vitro . SJAMP ccer cell .Meretrix meretrix Linnaeus) is one of the main beach cultured shellfishes in China. Clams possess many biological activities, such as anti-tumor, antiviral, and antioxidant effects [in vitro and in vivo. Zhang et al. [Moreover, Clam ( effects . There ag et al.  reportedg et al. .N-acetylglucosamine, is produced by deacetylation of chitin derived from the shells of crabs and shrimps, and has been reported to have good pharmacological properties such as antiviral activities [Furthermore, chitosan, a partially deacetylated polymer of tivities . Recentltivities ,102. Mortivities . In summin vitro, which suggests use of such carrageenan based gels may be able to block the sexual transmission of HPV [HPV-caused cancer is a major health problem worldwide, especially in developing countries. The discovery of medicinal agents specifically capable of inhibiting HPV and related cancer is urgently required to prevent HPV infection. Marine derived bioactive compounds especially the heparinoid polysaccharides have similar pharmacological activities to the natural heparin, which can effectively block the entry process of HPV. Carrageenan-containing sexual lubricant gels were reported to be able to inhibit the infectivity of HPV16 pseudovirus n of HPV . Moreoven of HPV . So natuin vitro or in mouse model systems until now. Therefore, further studies are needed to explore their activities in clinic in the future.However, the possibility that some genital HPVs might exhibit natural resistance to inhibition by carrageenan or other marine derived natural compounds would be an important factor to consider in the design of clinical efficacy trials . In addiIn summary, the anti-HPV and related cancer agents derived from marine organisms have the potential to treat both unapparent HPV infection as well as visible clinical diseases. Besides that, marine natural products especially polysaccharides and polypeptides have many advantages, such as relatively low cytotoxicity, low production costs, and wide acceptability, which suggest these compounds merit further investigation as novel drug candidates to reduce or regulate HPV infection related cancer ,107. Fur


Whole grain rye products have been shown to increase satiety and elicit lower postprandial insulin response without a corresponding change in glucose response compared with soft refined wheat bread. The underlying mechanisms for these effects have not been fully determined The primary aim of the study was to investigate if whole grain rye crisp bread compared to refined wheat crisp bread, elected beneficial effects on appetite and postprandial insulin response, similarly as for other rye products.2, were served a standardized breakfast with unfermented whole grain rye crisp bread (uRCB), fermented whole grain rye crisp bread (RCB) or refined wheat crisp bread (WCB), Appetite was measured using a visual analogue scale (VAS) until 4 h after breakfast. Postprandial glucose and insulin were measured at 0-230 min. Breads were chemically characterized including macronutrients, energy, dietary fiber components, and amino acid composition, and microstructure was characterized with light microscopy.In a randomized cross-over trial, 23 healthy volunteers, aged 27-70 years, BMI 18-31.4 kg/mReported fullness was 16% higher (P<0.001), and hunger 11% and 12% lower (P<0.05) after ingestion of uRCB and RCB, respectively, compared with WCB. Postprandial glucose response did not differ significantly between treatments. Postprandial insulin was 10% lower (P<0.007) between 0-120 min but not significantly lower between 0-230 min for RCB compared with WCB. uRCB induced 13% (P<0.002) and 17% (P<0.001) lower postprandial insulin response between 0-230 min compared with RCB and WCB respectively.Whole grain rye crisp bread induces higher satiety and lower insulin response compared with refined wheat crisp bread. Microstructural characteristics, dietary fiber content and composition are probable contributors to the increased satiety after ingestion of rye crisp breads. Higher insulin secretion after ingestion of RCB and WCB compared with uRCB may be due to differences in fiber content and composition, and higher availability of insulinogenic branched chain amino acids.NCT02011217ClinicalTrials.gov  Obesity and overweight are becoming more prevalent and causing increasing concern in large parts of the world . LifestyProducts based on whole grain rye, mainly porridge and soft bread, have repeatedly been shown to induce higher subjective satiety –13 and lIn most studies to date on whole grain rye products such as porridge and soft bread, refined wheat bread has been used as the control product. Whole grain rye porridge and soft bread typically contain more water than soft, refined wheat bread, which gives lower energy density and may influence postprandial responses ,25. HoweThe aim of the present study was to evaluate whether whole grain rye crisp bread, compared with refined wheat crisp bread, exerted beneficial effects on appetite and postprandial insulin response, as reported for other rye products. A further aim was to evaluate the effect of yeast fermentation by including one yeast fermented and one unfermented rye crisp bread as part of a breakfast.The study was carried out in agreement with the Helsinki Declaration and was approved by the regional ethics board in Uppsala on August 14, 2013 (dnr 2013/201). Recruitment started immediately after approval by the ethics board and the last participant was enrolled on August 27. The experimental work was completed by September 20, 2013. Individuals interested in participating were given written and oral information about the study and made aware of their option to withdraw from the study at any time they desired. Prior to inclusion in the study, written consent was obtained from all participants.The study was registered at ClinicalTrials.gov (NCT02011217) after completion. The delay was due to unawareness by investigators regarding time frame for trial registration. The authors confirm that all ongoing and related trials for this drug/intervention are registered. The protocol for this trial and completed CONSORT checklist are available as supporting information, see 2, physical activity level <2, fasting plasma glucose <6.0 mmol/L, fasting serum insulin <11 mE/L, serum TSH <2.5 mIE/L, plasma LDL <5.3 mmol/L, fasting plasma triglycerides (p-TG) <1.8 mmol/L, non-smokers, no medications likely to affect appetite, no medical conditions involving the gastrointestinal tract, no gluten intolerance or food allergies, no physical problems with eating, habitual consumption of breakfast, lunch and dinner, no dieting or self-reported fluctuations in body weight of more than 10% three months prior to screening, no recent participation in a dietary study, and no pregnancy, lactation or intention to become pregnant during the study period.Participants for the study were recruited through an advertisement in a local newspaper, through a website for clinical trials advertisements, on the Ultuna campus of the Swedish University of Agricultural Sciences (SLU), and through an internal e-mail to employees at SLU. After initial screening, men and women who met the eligibility criteria were invited to take part in the study. These criteria to were: age 18–70 years, BMI 18.5–30 kg/mThe study was conducted as a randomized, crossover trial with three different isocaloric breakfast treatments consisting of crisp breads served as part of a complete breakfast . Test meCrisp breads were milled with a cyclone sample mill . Extractable and unextractable DF content and composition were analyzed according to the Uppsala method . β-glucaSubjective appetite ratings were recorded 30 min before breakfast, at breakfast and at 30, 60, 90, 120, 150, 180, 210, and 240 min after breakfast, using an electronic visual analogue scale (VAS). On arriving at the study center, each participant was provided with a Palm computer model z22  which prompted the participants to answer three questions: “How hungry do you feel right now?”, “How full do you feel right now?” and “How strong is your desire to eat right now?”. The participants marked their answer on a line anchored at each end with an extreme answer to each question; “Not at all hungry”/“Extremely hungry”, “Not at all full”/“Extremely full” and “Extremely strong”/“Not strong at all”. The computer then translated the mark to a number between 0–100. This method has been shown to give results which are in agreement with the conventional 100 mm VAS –34.Venous blood was collected by trained nurses 15 minutes before breakfast and at 15, 35, 65, 95, 125, 185 and 230 minutes after breakfast. Blood samples were collected into ice cold tubes prepared with either potassium EDTA or lithium heparin . Immediately after blood sampling 160 μl protease inhibitor cocktail (1 SigmaFast tablet (Sigma S8820) in 2.2 ml H2O + 5.5 μl 10 mM KR-62436(DMSO)) were added to the test tube to prevent breakdown of appetite related gut peptides and incretines, which may be analyzed later for other purposes. The samples were kept on ice and processed immediately. Blood plasma was separated from buffy coat and erythrocytes by centrifugation at 2500 g for 10 min (4°C), aliquoted into 2.0 mL screw cap micro tubes  and immediately placed in a freezer at -20°C. Plasma samples were transferred and stored in a freezer maintaining a temperature of -80°C. Analyses of serum insulin and plasma glucose concentrations were performed by established routine methods at the certified laboratory of the Department of Clinical Chemistry at Uppsala University Hospital. Serum insulin was analyzed on a Roche cobas 8000 e602 module  and plasma glucose on an Architect c8000 and c16000 .Samples were fixed with glutaraldehyde vapor for 12 h followed by 2 h fixation with OsO4 vapor. Samples were then washed twice in distilled water, dehydrated in a series of aqueous ethanol of increasing concentration and finally infiltrated and polymerized with Technovit 7100.The embedded samples were cut into 1.5 μm thick sections with an ultra-microtome . The sections were stained with Lugol’s solution which stains proteins yellow, and starch dark blue/violet. It also distinguishes between amylopectin rich areas (beige/brown), and amylose (blue). The stained sections were examined using a Nikon Eclipse Ni-U microscope and the images were captured with a Nikon Digital Sight DS-Fi2 camera and processed with the software NIS-Elements BR .t = 0–230min) as well as for the first 120 min (AUCt = 0-120min). Response variables were log-transformed in order to approximate a normal distribution in cases where data were skewed as judged by a Shapiro-Wilks test with P<0.05. Reported values in curves are unadjusted mean unless otherwise stated. Treatment effects are reported as model-adjusted least square mean (LSM) ± standard error (SE). P-values for all main effects reported are based on models in which non-significant (P>0.10) interaction terms were removed. When a significant interaction term between treatment and time was found, t-tests were performed at each time point. P<0.05 was considered statistically significant for all tests, except for removal of interaction terms (P>0.10).Differences between treatments were evaluated with models appropriate for cross- over designs using PROC mixed in SAS version 9.3 . Effects of treatments were evaluated by two separate models . In modeThe primary outcome metric of the study was AUC for appetite ratings. With a power of 0.9 and a level of α 0.05 for a two sided test, 12 participants are sufficient to detect a 10% difference in mean appetite ratings over 4.5 hours in a comparison between two foods . Seconda2 . Hunger was 11% (P<0.05) lower for uRCB and 12% (P<0.05) lower for RCB compared with WCB . For fult = 0–230min (P<0.0001) and insulin secretion was 13% and 17% lower for uRCB compared with RCB (P<0.002) and WCB (P<0.001) respectively. There was no significant difference in AUCt = 0–230min between RCB and WCB, but the insulin response for uRCB was 12% and 21% lower compared to RCB (P<0.02) and WCB (P<0.001), respectively. Treatments also differed significantly when comparing AUCt = 0-120min (P<0.0001) and the insulin response was 10% lower for RCB compared with WCB (P<0.007).There was no significant difference in postprandial plasma glucose concentrations between any of the treatments . For insThe microstructure of the rye crisp breads differed from that of the wheat crisp bread in several respects. In uRCB and RCB  was charThis study evaluated effects on appetite and postprandial glucose and insulin responses after consumption of unfermented whole grain rye crisp bread, fermented whole grain rye crisp bread and fermented refined wheat crisp bread as part of a complete breakfast. The rye crisp breads induced higher fullness and less hunger as well as lower insulin response postprandially than refined wheat crisp bread. Furthermore, the postprandial insulin response appeared to be different between fermented and unfermented crisp bread.Intake of whole grain rye products in comparison with refined wheat bread has been shown to have an appetite-reducing effect in most studies where rye products such as porridge –8,11 andCertain DF have been shown to have an appetite-reducing effect ,38 and tThe microstructural features of crisp bread products may also affect the absorption rate of nutrients. The rye crisp breads contained intact cells and fragments of aleurone layers, which were both absent in the refined wheat bread. These cells and fragments could lead to decreased digestion rate and lower digestibility of nutrients due to encapsulation ,44, LoweThe more swollen starch granules observed in the rye crisp breads would be expected to make the starch more susceptible to enzymatic degradation . HoweverWhen combined, the composition and content of DF and the microstructural properties of the rye crisp breads may also have contributed to a prolonged effect on gastric distension and release of satiety hormones , resultiDecreased rate of release and absorption of nutrients, e.g. glucose, would be expected to result in a corresponding decrease in concentration in the blood. However, all treatments in our study elicited similar glucose response but the rye crisp breads gave slightly lower insulin concentrations, which is in line with results from earlier studies –16,21. TA recent study by Eelderink et al  showed tThe higher content of viscous DF in the rye crisp breads could be expected to result in lower postprandial glucose responses compared with WCB. Decreased postprandial blood glucose concentrations have been shown for foods supplemented with different DF, and have been attributed to increased digesta viscosity –56. In aFermentation of RCB most likely induced changes in the composition and content of DF compared with uRCB, which may also have influenced digesta viscosity and glucose absorption. Fermentation has been shown to reduce the content of fructans in RCB ,62, and The differences in postprandial insulin response could also be due to different amounts of insulinogenic amino acids such as the BCAA valine, isoleucine and leucine as well as the amino acids lysine and threonine. These have been suggested to mediate increased secretion of insulin and to be correlated with higher concentrations of the incretins gastric inhibitory peptide (GIP) and glucagon-like peptide-1 (GLP-1) ,65. HighUnfermented (uRCB) and fermented (RCB) whole grain rye crisp bread as part of a complete breakfast induced higher satiety up to 4h after intake compared with refined wheat crisp bread (WCB). This difference was most likely due to higher content of dietary fiber, mainly viscous arabinoxylans and β-glucans, and microstructural features of the rye crisp breads.Lower postprandial insulin concentrations, but no difference in glucose concentrations, were observed for both rye crisp breads compared with WCB and for uRCB compared with RCB. These differences in insulin responses may be due to altered glucose absorption kinetics caused by content- and process-induced changes in composition of dietary fiber in RCB and WCB, and higher content and availability of insulinogenic branched chain amino acids in WCB. Additionally, we cannot rule out effects of slight differences in particle size distribution.S1 CONSORT Checklist(DOC)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.


Cervical screening by using cytology was proven efficient in reducing the mortality secondary to cervical cancer, but this method has limitations. High risk HPV infection is essential for cervical cancer development so HPV testing is a new tool used for screening patients for cervical neoplasia. HPV testing was proven most useful for women over 30 years old, in cases in which cytology identified ASC-US and after treatment for CIN. This article outlines the clinical significance of HPV-DNA testing for precancerous cervical lesions and the evidence that stands behind these recommendations. Abbreviations: HPV = Human Papilloma Virus, ASC-US = Atypical Squamous Cells of Undetermined Significance, CIN = Cervical Intraepithelial Neoplasia, LSIL = Low Grade Squamous Intraepithelial Lesion, DNA = Deoxyribonucleic Acid  In spite of the results obtained by using cervical cytology, this screening method has multiple limitations that led to the research for the other screening tests. Taking into consideration the fact that the high oncogenic risk HPV infection is essential for the development of precancerous and cancerous cervical lesions, recent studies have focused on the detection of these types of infections [2]. As a screening test for cervical cancer, HPV-DNA testing has a high sensitivity but a low specificity compared to cervical cytology [3] due to the high prevalence of limited HPV infections in younger women. The cervical cancer screening by using cervical cytology has significantly reduced the mortality secondary to squamous cell cervical cancer [HPV-DNA testing for women over 30 years old3-5]. Combined screening is recommended at 5 years interval because the rate of cervical cancers associated with this method is lower than the one associated with the 3 yearly cervical cytology [6]. The 5 years cervical cancer incidence is 3,2 per 100000 women per year for women who have negative HPV and cytology, 3,8 per 100000 women per year for women with negative high risk HPV test only, and 7,5 per 100000 women per year for women with negative cytology only [6]. Abnormal cytology for women with positive high risk HPV test increases the 5 year risk of CIN3 and cervical cancer from 5,9% to 12,1% compared to women with a normal cytology and a positive HPV test. 3 yearly combined testing is associated with a risk for cervical cancer similar to 5 yearly combined testing  but is associated with a significantly higher number of negative colposcopies [7]. Studies have demonstrated that HPV-DNA testing, adjacent to cervical cytology increases CIN3 detection with 7-31% for the first testing, and reduces CIN3 and cancer detection rate for subsequent tests (absolute reduction in cancer rate at second test 0.03-0.05%) . A positive high risk HPV test is highly associated with cervical adenocarcinoma  [9]. A combined screening technique, by using HPV-DNA testing and cervical cytology, increases the detection rates for cervical adenocarcinoma, a very important benefit taking into consideration that unlike squamous cell cervical carcinoma, the invasive cervical adenocarcinoma incidence has not reduced substantially after the initiation of cervical screening by using cytology [HPV-DNA testing in patients with ASC-US10]. For patients with this result, other tests are necessary in order to determine the risk for precancerous cervical lesions. For women with positive high-risk HPV test and ASC-US cytology, colposcopy is recommended because these women have a 2 years risk of CIN3 and invasive cervical cancer of 10% [11]. Studies have demonstrated that for women with ASC-US and negative high-risk HPV test, the risk for precancerous cervical lesions is very low, similar to women with negative cytology and HPV test. The absolute risk of CIN3 for women with ASC-US and negative HPV is 0,28% at that moment and 0,54% at 5 years . Because this risk is less than 1% for these women, it is recommended to return to routine cervical screening [10]. The cytological interpretation of ASC-US represents a category of morphologic uncertainty. The definition of ASC-US is “some, but not all” the features of a LSIL and as such, includes both poorly sampled and poorly represented LSIL and the many morphologic mimics of LSIL [HPV-DNA testing after CIN treatment13]. These patients need postop follow up in order to reduce the risk of invasive lesions. Old follow up guidelines recommended yearly cytology after CIN treatment. This type of follow up is excessive for approximately 90% of the patients and patients compliance over such a long period is low [14]. Also cervical changes after CIN treatment can make cytology evaluation difficult especially in case of cervical stenosis. High risk HPV testing for these patients increases the efficiency of the screening programme.CIN treatment reduces the risk of invasive cervical cancer with over 90%, but these women still have a 5 times higher risk of developing invasive cervical lesions compared to women with normal cytology because treatment does not always manage to eliminate the HPV infection [15]. A British study has demonstrated that only 0,2% of women with negative HPV and cytology post treatment had CIN2 at 5 years and only 2,4% of women with negative HPV test and inadequate cytology or ASC-US post treatment had CIN2 at 5 years. In conclusion, 97,6% of the women with ASC-US and negative high risk HPV have not developed CIN2 at five years, so colposcopy can be deferred for these patients [14]. Kocken et al. evaluated the efficiency of cytology and HPV testing follow up for women with cone biopsy for CIN2. 6 months post treatment high risk HPV testing had a higher sensibility than cytology testing  without a reduction in specificity  [16]. Combined high-risk HPV testing and cervical cytology had a higher sensitivity for the detection of recurrent high-grade cervical lesions than cytology or HPV testing alone. In order to increase the screening programme sensitivity it is recommended to repeat the testing at 18-24 months [17]. The persistence of HPV 16 post treatment should lead to a more intense monitoring because this HPV type has lower disappearance rate compared with the other high risk HPV types [18]. Genotype specific testing is important because the 6 months post cone biopsy persistence of the same type HPV is an independent risk factor for CIN recurrence at 24 months. Unlike the general high risk HPV testing, the type of specific testing predicts residual disease with similar sensitivity but higher specificity  [The use of high risk HPV testing as a screening tool increases the detection rates for all the precancerous and cancerous cervical lesions and decreases the screening interval in a safe manner. Also, HPV testing for women with ASC-US cytology reduces the number of unnecessary colposcopies by underlying the women who are at high risk of cervical precancerous lesions. Post CIN treatment, high-risk HPV testing points out the women who are at risk of recurrence, reducing the number of women who require frequent and long-term follow up.


Methods for surveillance of cervical precancers and associated types were developed to monitor effect of HPV vaccination. >18 years of age residing in defined catchment areas; collecting relevant clinical information and detailed HPV vaccination histories for women 18–39 years of age; and estimating the annual rate of cervical cancer screening among the catchment area population. The first few years of the project provided key information, including data on HPV type distribution, before expected effect of vaccine introduction. The project’s success exemplifies the flexibility of EIP’s network to expand core activities to include emerging surveillance needs beyond acute infectious diseases. Project results contribute key information regarding the impact of HPV vaccination in the United States.In 2007, five Emerging Infections Program (EIP) sites were funded to determine the feasibility of establishing a population-based surveillance system for monitoring the effect of human papillomavirus (HPV) vaccine on pre-invasive cervical lesions. The project involved active population-based surveillance of cervical intraepithelial neoplasia grades 2 and 3 and adenocarcinoma in situ as well as associated HPV types in women  Human papillomavirus (HPV) vaccines are primarily designed to prevent HPV-associated cancers that typically occur years to decades after exposure to HPV-16 and -18. Three prophylactic HPV vaccines are available in the United States: bivalent, quadrivalent, and 9-valent vaccines. The bivalent vaccine protects against HPV-16 and -18, the most common oncogenic HPV types, which are responsible for ≈70% of HPV-associated cervical cancers and a large proportion of other HPV-related cancers  has recommended routine HPV vaccination of girls 11–26 or 12–26 years of age who have not previously been administered the quadrivalent vaccine , which are detected through routine screening, take less time to develop and were used as a surrogate for cervical cancer in vaccine trials. Real-world reductions in CIN2+ have been shown in countries with high vaccination coverage and catch-up programs for older persons and where it is possible to link data across population-based disease, screening, and vaccination registries  recommended screening begin at age 18 or the age of onset of sexual intercourse, whichever was first  received funding to determine the feasibility of establishing a population-based surveillance system that could, in addition to monitoring overall CIN2+ trends, enable monitoring of trends in HPV type distribution in CIN2+ lesions among vaccinated and unvaccinated women with a diagnosis of CIN2+. Although the EIP network has traditionally focused on acute infectious diseases with typically short incubation periods, their extensive expertise and proficiency for enhanced surveillance and demonstrated ability to develop local infrastructure to support complex surveillance activities made the EIP network an ideal candidate for collaboration on developing this new system.>18 years of age) ranged from ≈260,000 to 350,000 for the 5 catchment areas. The size of each catchment area was selected to maximize successful implementation of all elements of the system while allowing adequate precision for measuring trends over time. The objectives of the HPV-IMPACT project were to 1) conduct active population- and laboratory-based surveillance of CIN2+ diagnoses in women >18 years of age residing in defined catchment areas, 2) determine HPV types in CIN2+ lesions among women 18–39 years of age, 3) collect relevant clinical information and detailed HPV vaccination history for women 18–39 years of age, and 4) estimate annual rates of cervical cancer screening among the catchment area population.The 5 EIP US sites selected to participate in the pilot HPV-IMPACT project were in California, Connecticut, New York, Oregon, and Tennessee. Catchment areas comprised 8 contiguous cities in northwest Alameda County, California; New Haven County, Connecticut; Monroe County, New York; a contiguous region including Portland and most of Washington and Multnomah Counties, Oregon; and Davidson County, Tennessee. On the basis of the 2010 US census, the population of women , along with histopathologic findings. Reports were deduplicated, anonymized, and entered into the project database.Additional information was obtained for women 18–39 years of age who received a diagnosis of CIN2+. Reporting laboratories were requested to provide samples of archived specimens. At most sites, 1 laboratory agreed to process all specimens according to standard procedures, and prepared specimens were sent to CDC  for HPV DNA typing. Laboratory methods have been previously described (http://www.census.gov/acs/www/) and the Behavioral Risk Factor Surveillance Survey (http://www.cdc.gov/brfss/data_documentation/index.htm) to estimate the relative proportion and the difference in screening rates between the groups. The insured and uninsured groups were then combined to estimate overall annual screening rates by age group  adjusted for insurance status. New York obtained deidentified cervical cancer screening reports from cytopathology laboratories serving the catchment area. The reports, which were deduplicated within the laboratory, included patient age and results of the first screening in a given calendar year and combined and categorized into specified age groups. Screening rates were estimated by using data from the 2010 US census.Site-appropriate methods for obtaining screening rates in the catchment areas’ populations were investigated. Self-reported cervical cancer screening is subject to misclassification ; thus, novel methods to determine screening rates were explored at each EIP site, and methods using existing resources were developed to obtain screening estimates in 3 sites: California, New York, and Oregon. In California and Oregon, weighted estimates of screening were calculated by using available data from national and state-based surveys and administrative databases. National survey data indicated differences in screening rates between insured and uninsured women, so women in the catchment areas were divided into 2 groups on the basis of insurance status (insured or uninsured), and annual screening rates were obtained for each group by using data from the American Community Survey  were in women 21–29 years of age. Women in the youngest (18–20 years of age) and oldest (>50 years of age) age groups represented only 3.9% and 7.2%, respectively, of all cases  of 18- to 39-year-old women with a diagnosis of CIN2+ during 2008–2012. Of these specimens, 6,745 (87.7%) were histologically adequate and underwent DNA testing; HPV DNA was detected in 6,721 (99.6%) of the specimens. The prevalence of HPV types in CIN2+ lesions is shown in the Vaccination and screening history data were used to demonstrate that proportions of HPV-16– and HPV-18–attributable CIN2+ in women who initiated vaccination at least 24 months before receiving an abnormal screening test result were statistically significantly lower than proportions in unvaccinated women (The HPV-IMPACT project has provided valuable information for monitoring the effect of HPV vaccine among the US population, including data on the type distribution of HPV before widespread introduction of the vaccine. A variety of challenges to developing a sustainable population-based system for monitoring CIN2+ and associated HPV types were identified and addressed during the project’s pilot phase. Since 2011, active surveillance has been ongoing at all 5 EIP sites, and the systems have been periodically evaluated. Laboratories serving the catchment areas are monitored through routine contact with health care providers, regional and local surveys, and other means to ensure completeness of reporting is maintained over time for all women in the catchment areas. Case ascertainment methods are continually updated and refined as the recommended diagnostic terminology evolves. New mechanisms for obtaining complete vaccination histories, such as interviewing case-patients and contacting vaccine providers to verify self-reported vaccination, are being explored. Efforts are ongoing to improve methods for measuring cervical cancer screening rates.The success of the HPV-IMPACT project exemplifies the flexibility of the EIP network to expand core activities to include emerging surveillance needs beyond acute infectious diseases. Results from this project contribute key information on the effect of HPV vaccination among the US population.