This study aimed to determine the awareness among fifth-grade girls and boys of sexually transmitted diseases (STDs), cancer, and human papillomavirus (HPV), and to determine the factors associated with intention to obtain the HPV vaccination.n=117). Prior to providing HPV education, the awareness and health beliefs regarding STDs and cancer prevention were assessed according to gender. After 2 hours of HPV education, gender comparisons were made with respect to the awareness and health beliefs, HPV knowledge, and intention to obtain the HPV vaccination, and the factors associated with that intention.A quasi experimental design was employed with Korean fifth-grade students as the subjects for this study  and “cancer is preventable” , and concerns about the pain associated with vaccine injection . After HPV education, there were no significant gender differences in HPV knowledge and intention to obtain the HPV vaccination. Awareness that “HPV vaccine can prevent cervical cancer” was significantly related to intention to obtain the HPV vaccine among both boys and girls.Prior to the 2hours education session, only two boys knew that HPV is a virus. There were significant gender differences with respect to responses to the statements “STD is preventable”  contains supplementary material, which is available to authorized users. The recognition that human papillomavirus (HPV) infection is associated with a high risk of cervical cancer has resulted in a paradigmatic shift in the focus of cervical cancer prevention from female adults to youth, with the introduction of a prophylactic vaccination against HPV . The keyThere are few statistical data pertaining to sexually transmitted diseases (STDs) in Korea, and monitoring of the prevalence of STDs in the past has relied on reports from a sentinel surveillance system. The findings from such reports have led to genital warts (condyloma accuminata), which are caused by HPV infection, being officially designated as one of the seven communicable infections since 2001 . The KorWith respect to Western countries, it has been found that awareness of HPV and the HPV vaccination was relatively low among Hungarian adolescents, and that most of the included students were receptive to receiving further information about sexually transmitted diseases (STDs) . There aOn the other hand, a study conducted in Korea found that adolescents demonstrated an apparent lack of awareness of the HPV vaccine and related health beliefs, and their HPV vaccination rates were reportedly as low as 1.3% among boys and 9.5% among girls . AlthougSince 2009, Korean legislation has stipulated that school health education disseminated by health teachers must be provided to every fifth- and sixth-grade student . The totHowever, it has been shown that while most (89.3%) Korean health teachers were aware of the necessity to provide HPV education to their students, there was no formal HPV education program for them to follow  and littBased on the aforementioned findings, it is necessary to explore the basic understanding of HPV among the Korean youth, and to develop a preliminary HPV education program for Korean health teachers to apply to fifth- and sixth-grade students. The present study applied the Health Belief Model (HBM) to assess the attitudes and intentions among Korean adolescents related to STDs and cancer prevention, which are thought to be associated with intention to obtain the HPV vaccination ,21, and The aim of this study was to determine the awareness and health beliefs of Korean fifth-graders regarding STDs, cancer, and HPV infection both before and after delivery of a 2-h HPV education program. In the first instance, the awareness and health beliefs regarding STDs and cancer among Korean fifth-grade girls and boys were assessed by survey. After delivery of the HPV education program to all of the subjects, gender differences in awareness and health beliefs, HPV knowledge, and intention to obtain the HPV vaccination were examined, as well as the factors associated with that intention.A quasi experimental study was employed to determine the HPV awareness and HPV vaccination intention among fifth-grade girls and boys, and the effect on those parameters of a 2-h HPV education program.n=117), both girls (n=47) and boys (n=70), from an elementary school located in Gangneung city, Korea. Prior to recruitment, permission to access the students was obtained from the school principal, health teacher, and two teachers engaged in fifth-grade teaching. Gangneung city is in the Kang Won Do province, which is one of the eight administrative regions in Korea, and is located on the eastern coast of the country; it is approximately 2.5 hours drive from Korea’s capital, Seoul. In 2013 there were 37 primary schools in Gangneung city, with a total of 11,283 students. The setting for this study was selected by recommendation, mainly because all of the fifth-grade students there were able to participate. The selected school comprised 21 classes, with a total of 506 students overall and 117 fifth-grade students.Convenience sampling was employed to recruit the study participants. This study included all fifth-grade students  HPV as a virus that causes cervical, penile, and oral cancers, and STDs such as warts; (2) low- and high-risk types of HPV infection; (3) the purposes and benefits of, and barriers toward obtaining the HPV vaccination; (4) HPV vaccination of girls and boys for preventing genital warts and genital cancers; and (5) early prevention of STDs and cancers caused by HPV for both genders . The underlying concept of HPV education in this study was that it should be gender based, so that it was fundamentally designed with a view to promoting gender equality regarding HPV prevention. It was intended that both the girls and boys would become aware of their mutually dependent responsibility regarding HPV prevention .The HPV education program comprised two 50-minute sessions that were applied during the period 9.00 a.m. to 1.00 p.m. in the classroom consecutively on the same day. The program comprised a lecture, a PowerPoint presentation, and a discussion. Two coeducational classes comprising 57 and 60 students participated in the HPV education program. The two classes were not allowed to communicate with each other during the program, so that while one class was taking the HPV education session in the classroom, the other class was performing extracurricular activities outside the classroom. A handout for the lecture was given to all students to take home.The data were collected in October 2011 in the classroom before and after HPV education using self-administered questionnaires. The study was conducted in the two classrooms by the research team. There were no dropouts between the pre- and posttests. The research protocol was approved by the Myoungji University Hospital Institutional Review Board. Permissions from the parents and the students were not sought because the school principal and teachers all concluded that it was not necessary for this study. At the first meeting in the classroom, the health teacher and the researchers explained the purposes, content, and schedule for the study, and informed the subjects that the data they provided would be confidential and anonymous. The pre- and posttest were conducted prior to and immediately after delivery of the HPV education, respectively. After the posttest, the students received a small gift, costing approximately US$ 3 , for successfully completing the study.The contents and constructs of the structured questionnaire were validated by three experts in HPV research and sex education, and were tested on ten fifth-grade girls and boys aged 12 years. In the pretest, general information was gathered, including age, sex, feelings regarding the vaccination, awareness of the necessity for the vaccination, whether they had heard of HPV or any other viruses, and general awareness of STDs and cancer. The HBM variables were also assessed. In the posttest, HPV knowledge and intention to obtain the HPV vaccination were added to the survey.The HBM used in this study measures attitudes toward STDs and cancer prevention, and intention to obtain a vaccination to prevent STDs and cancer, and was modified to fit the present study sample based on the findings of previous studies ,14. The Items pertaining to HBM variables consisted of perceived severity (two items), perceived barriers (one item), perceived benefits (two items), and intention to obtain the vaccination (two items); the content of all four variables was validated by HBM researchers. The students recorded their level of agreement on a 5-point scale ranging from 1  to 5 (“very much”). A Cronbach’s α of 0.78 for the HBM scales used in the present study confirmed their reliability.In the posttest, general awareness of STDs and cancer, and HBM variables were evaluated. Moreover, intention to obtain the HPV vaccination was assessed on a 5-point scale ranging from 1  to 5 (“very much”), and HPV knowledge was assessed by eight items that also had been modified to fit the students in this study based on the findings of previous studies ,25. A Crt and χ2 tests. At the posttest, gender differences in measurement variables were analyzed using the χ2 test, the Mann–Whitney U test, and the Wilcoxon signed-rank test. Factors associated with intention to obtain the HPV vaccination were analyzed using the Spearman rho coefficient. IBM SPSS v. 20.0 statistics software was used for all statistical analyses.All variables were analyzed using frequencies, means, proportions, standard deviations, and percentages. At the pretest, the homogeneity test of the general awareness of STDs and cancer, and HBM variables between girls and boys was analyzed using the n=70) and 40.2% were girls (n=47), and all were aged 11–12 years. Before the HPV education session , there were significant differences regarding feelings about the vaccine injection  and awareness of the virus . When asked whether they knew about any viruses, 17.5% of the boys and 34.0% of the girls answered that they did not know, or gave incorrect answers, including referring to bacteria and computer viruses. The most commonly known viruses were flu, influenza, and H1N1 among both boys and girls  and “cancer is preventable”  at pretest, but there were no significant gender differences regarding awareness of STDs and cancer prevention at posttest.The data presented in Table χ2=−2.44, p=0.015) and posttest  and girls . However, there was an increase in the intention to obtain injections to protect against both STDs and cancer among the girls, illustrating that preadolescent girls were motivated by the education strategy to prevent both STDs and cancer . Moreover, the girls were more concerned about the pain at injection than the boys, and their concern about this pain was not reduced by the HPV education. Therefore, future studies should investigate more sensitive approaches for providing information about the subject to girls. Based on the results of the present study, the relationship among rates of HPV infection, HPV vaccination, and cervical cancer should be taught to Korean preadolescent girls and boys. The Korean youth should be supported to recognize and accept not only the concept of HPV vaccination, but also the facts about HPV itself, its potential consequences, and its prevention.Until now it has been considered that health professionals play a critical role in HPV vaccine recommendation to promote HPV vaccine acceptability, particularly among parents with adolescent children ,30-34. OThis study reconfirmed the effectiveness of the HBM framework to guide HPV prevention ,14,40; aThe average age at first sexual intercourse of Korean adolescents appeared to be earlier in 2011 (13.6 years) than in 2009 (14.0 years) , indicatThis study was subject to several limitations with respect to its primitive experimental design, lack of a control group, and the possibility of confounding variables such as the onset of menarche or secondary sexual characteristics, which were not controlled for. Furthermore, the positive perceptual changes in awareness of STDs and cancer prevention, the associated attitudes, and actual vaccine uptake were not used as outcome measures for quantifying the efficacy of the provided HPV education. It should also be noted that the current intentions of Korean students regarding HPV vaccination may also have affected their uptake thereof. Finally, this study did not examine the effectiveness of coeducational classes compared to single-sex classes for HPV education among youth; this needs to be identified in further studies.Unfortunately, no relevant information regarding the rate of HPV infection in Gangneung city was available, and so the data for Kang Won Do were compared with the national average. The results showed that the age-standardized incidence rate of cervical cancer was 6.6 for Kang Won Do in 2010, which is higher than the national average for the same year 5.9 per 100,000 person . RegardlThe findings of this study demonstrate that the implemented 2-h HPV education program was successful for both girls and boys, and could be disseminated to both fifth- and sixth-grade students. Health teachers or school nurses should recognize the concerns and needs that students, parents, and the community have about HPV information, and design an HPV education program that is tailored to these factors.


The blue and black dress that “melted the Internet” is thought to have done so because its perceived color depended on people using different prior assumptions about discounting the illuminant. However, this is not the first monochromatic object to have confused the public. For a brief period during WWI, RMS Mauretania was dressed in (dazzle) camouflage shades of blue and black/grey, yet she is sometimes depicted by artists, modelers, and historians in a much showier dress of red, blue, yellow, green, and black. I raise the possibility that this originates from a case of public deception deriving from the momentary misperception of a playful artist who neglected to discount the illuminant, propagating the most (perhaps only) successful application of dazzle camouflage known. Figure 1). Broadly speaking, the generally accepted explanation is as follows: the low-grade photograph was overexposed and its chromatic content became seriously distorted, resulting in a spectrum that can be parsed in at least two different ways: (1) yellowish light from the front that when discounted leaves a dress made from blue and black material; (2) a garment in dim shadowy blue light that when discounted leaves a dress made from white and gold material. A third commonly reported visual interpretation is of a pale blue and brown dress where, presumably, the illuminant is not discounted at all (or is assumed to be made of flat white light), and the pixel wavelengths are seen to be (close to) how they look in isolation.On 26th February 2015, a picture of a dark blue and black dress went viral on social media. The reason? When forced between two alternatives, about half of the population saw it as blue and black, the other half as white and gold  misperception of a rather different carrier of a blue and black (or gray) dress, possibly involving processes of the third kind described above. During the First World War, merchant ships and their escorts were painted in so-called dazzle camouflage consisting of crazy patterns of stripes and geometric shapes. Many schemes used black and white, but blues and pastel colors came to be used in some. It seems that the aim of this striking display was to dazzle the U-boat captains, confusing their assessment of the speed and heading of their targets e.g., 2013. GivFigure 2) shows Mauretania in a scheme of blues and grays. Whether these are the true shades that she carried, or whether the darker blue (such as that to the aft) was closer to black, as shown in Admiralty diagrams , is not clear. However, the camouflage shades of gray in every one of the photographs of the ship I have found on the Internet are consistent with each of these two (rather similar) interpretations. More importantly, I have found no evidence  that Mauritania ever carried different coloration in any of the pale diamonds on the fore of the hull. Builders of scale models have a tenacious interest in the subject of livery/camouflage, and Internet posts from them come to similar conclusions. Nonetheless, an alternative scheme is readily found in reference books  in the exact alternative colors described above; it seems that these have been taken on face value by those who have seen the poster, and the myth has been propagated. But the question arises, why gild the lily? The answer might be that the more colorful scheme was simply thought to be more appealing to potential passengers; but why choose those colors? After all, there is little evidence that bright red was used in British dazzle schemes; available records suggesting that more sombre maritime colors were by far the norm.  2,185 scale models, photographs, paintings, and drawings of dazzle ships, many carrying the curiously named Wilkinson schemes ; in the rare cases where shades of red can be found, they tend toward bauxite or red oxide, not scarlet). We can't know for sure, but it is not so fanciful to suppose that the artist of the publicity poster had seen Mauritania (or other dazzle ships) in port at sunset; reflections of red, and arguably yellow and green, being directed to the artist by some of the paler (more reflective) facets of the ship . With a cognoscente, or even naïve, prior that anything is possible with these dazzle ships, these splashes of narrowband illuminant might well have been perceived as paint, at least for a few moments, and—enjoying the spectacle—the artist chose to share that with the public, in a stylized fashion.The origin of this (assumed) error is almost certainly a post-war publicity poster by the ship's owners, The Cunard Line, which depicts their liner , and it is delightfully ironic that what is probably dazzle's greatest success in deception has nothing to do with the purpose for which it was intended; perhaps also, that a similarly misperceived blue and black dress should be the trigger for Mauritania's public disrobing.Whatever the underlying reason for this misperception of the blue and black ship, it has proved provocative (e.g., the contemporary artwork in


Researchers often group various HPV types into composite measures based on vaccine subtypes, oncogenic potential, or phylogenetic position. Composite prevalence estimates based on PCR genotyping assay results have been calculated to assess HPV infection burden and to monitor HPV vaccine effectiveness. While prevention and intervention strategies can be made based on these prevalence estimates, the discussion on how well these prevalence estimates measure the true underlying infection burdens is limited.A simulation study was conducted to evaluate accuracy of using composite genotyping assay results to monitor HPV infection burden. Data were generated based on mathematical algorithms with prespecified type-specific infection burdens, assay sensitivity, specificity, and correlations between various HPV types. Estimated-to-true prevalence rate ratios and percent reduction of vaccine types were calculated.When “true” underlying type-specific infection burdens were prespecified as the reported prevalence in U.S. and genotyping assay with sensitivity and specificity  was used, estimated-to-true infection prevalence ratios were 2.35, 2.29, 2.18, and 1.46, for the composite measures with 2 high-risk vaccine, 4 vaccine, 14 high-risk and 37 HPV types, respectively. Estimated-to-true prevalence ratios increased when prespecified “true” underlying infection burdens or assay specificity declined. When prespecified “true” type-specific infections of HPV 6, 11, 16 and 18 were reduced by 50%, the composite prevalence estimate of 4 vaccine types only decreased by 17% which is much lower than 48% reduction in the prespecified “true” composite prevalence.Composite prevalence estimates calculated based on panels of genotyping assay results generally over-estimate the “true” underlying infection burdens and could under-estimate vaccine effectiveness. Analytical specificity of genotyping assay is as or more important than analytical sensitivity and should be considered in selecting assay to monitor HPV. Infection with human papillomaviruses (HPVs) can cause warts and various forms of carcinoma in the cervix, anus, vagina, vulva, head and neck in women and men . More thVarious factors could affect accuracy of composite prevalence estimates. Depending on the classification, the number of HPV types included in the composite measures could be different. Since HPV type-specific infections share the same risk factors  and subjects with weaker immune systems are more likely to get infected or stay infected, HPV type-specific infections are likely to be correlated and could result in coinfection with more than one HPV type ,15. In aChallenges to evaluate accuracy of prevalence estimates include not knowing the true values of underlying type-specific infection burdens, low feasibility to recruit subjects from various regions with various levels of infection burdens and to test numerous assays for comparisons. To overcome these challenges, we performed a Monte Carlo simulation study to evaluate composite prevalence estimates. The simulation approach allows us to have data with various levels of known prespecified “true” underlying type-specific infection burdens and genotyping test results based on the prespecified assay performance so the prevalence estimates calculated based on type-specific assay results can be compared to the pre-specified “true” underlying infection burden. Monte Carlo simulation approach has been widely used in statistics, physics, finance, economics and engineering to evaluate the impact of various factors on complex systems/processes and identify an optimal system design/process -25. For A simulation study was conducted to evaluate composite prevalence estimates calculated based on panels of PCR genotyping assay results. Data were generated using mathematical algorithms extended from Lin et al.  with preTwo different levels of infection burdens were considered. For the first setting, the “true” underlying type-specific infection burdens were prespecified as the reported 37 different HPV prevalence of females, aged 14–59, 2003–2006, in the U.S. , having high-risk HPV (14 types in the U.S. and 22 types in Canada), having vaccine type HPV , and having high-risk vaccine type HPV . HPV 16 and HPV 18 are generally considered particularly high-risk genotypes and account for approximately 70% of invasive cervical cancers globally ,28. QuadPrespecified “true” prevalence of each outcome measure is defined as the proportion of subjects with a positive infection status. For each subject, based on the pre-specified “true” type-specific infectious statuses, the “true” composite positive infection status of the four outcome measures is defined as having at least one HPV type-specific infection of the 37 HPV types (45 HPV in Canada), 14 high-risk types (22 high-risk in Canada), 4 vaccine types, and 2 high-risk vaccine types.Prevalence estimate of each outcome measure is defined as the proportion of subjects with positive composite test results. For each subject, a positive test result of the four outcome measure is defined as having at least one positive of the 37 HPV (45 HPV in Canada), 14 high-risk (22 high-risk in Canada), 4 vaccine, and 2 high-risk vaccine type-specific results. To examine how well the composite prevalence estimate measures the “true” underlying composite prevalence, for each outcome measure, estimated-to-true prevalence ratio was calculated. A ratio greater than 1 means the composite prevalence estimate calculated based on a panel of genotyping assay results over-estimates the “true” underlying composite infection burden. The number of false positives exceeds the number of false negatives. In contrast, a ratio less than 1 means composite prevalence estimate calculated based on a panel of genotyping assay results underestimates the “true” underlying composite infection burden. The number of false negatives exceeds the number of false positives. The larger the ratio from 1, the less accurate the composite prevalence estimate is.To assess the effects of analytical sensitivity and specificity of genotyping assay on composite prevalence estimates, the initial values of PCR genotyping assay sensitivity and specificity were set to be equal  to reflect the well-performed Linear Array test and then the assay performance was varied. To examine the effect of genotyping assay specificity, assay sensitivity was held unchanged and specificity was reduced from 0.95 to 0.90, 0.85 and 0.80. Similarly, to examine the effect of genotyping assay sensitivity, assay specificity was held unchanged and sensitivity was reduced from 0.95 to 0.90, 0.85 and 0.80.To examine how well the composite prevalence estimates measured intervention effectiveness, for demonstration purposes, assuming the pre-specified “true” type-specific prevalence of the 4 vaccine types  were reduced by 50%. Percent reductions of composite prevalence estimates of 4 vaccine types and 2 high-risk vaccine types  were calculated and compare to the reductions in the prespecified “true” underlying composite prevalence.To examine the effects of levels of underlying type-specific infection burdens and number of HPV types in the composite measure on the composite prevalence estimates, prevalence estimates of the four outcome measures from the U.S. scenarios were compared to those estimates from the NWT, Canada scenarios. The reported type-specific HPV prevalence in the NWT, Canada was generally lower than in the U.S. and the relative rates were also different Figure . In the To examine the effects of correlations between different HPV types on composite prevalence estimates, the correlations between different HPV types were varied from 0, 0.05, 0.1, 0.2, 0.3 and 0.4. The “true” underlying type-specific infections were prespecified as the reported prevalence in the U.S. and type-specific assay sensitivity and specificity were set to be . “True” composite prevalence, estimated composite prevalence and estimated-to-true prevalence ratios of 37 HPV, 14 high-risk HPV, 4 vaccine and 2 high-risk vaccine types were calculated.Table When assay specificity is held unchanged and sensitivity is reduced, the results suggest that composite prevalence estimates are robust to decline of assay sensitivity. As shown in Table Simulation results also suggest that the composite prevalence estimates could under-estimate vaccine effectiveness Table a,b. WhenTable The results of sensitivity analysis for correlations are given in Table Prevalence estimates based on one or panels of PCR genotyping assay results have been used to assess HPV infection burdens and monitor vaccine effectiveness ,29. SincPCR-based DNA genotyping tests can detect the existence of small amount of virus. The PCR process includes denaturation, annealing and extension in each PCR cycle. Each cycle approximately doubles the amount of target viral DNA. Although PCR process is labor-intensive and time consuming, PCR can theoretically produce one million copies from a single double-stranded DNA molecule after 30 cycles. Analysis of the amplification products can be done in different ways including gel electrophoresis, dot blot or line strip hybridization [The initial values of PCR genotyping assay sensitivity and specificity were set to be equal to  in this simulation study to reflect a well-performed Linear Array test. Roche Linear Array genotyping assay is the commercialized version of a PCR test which is designed to standardize the entire PCR process and to detect 37 HPV types. LA is a qualitative test and has been used for research purpose in numerous epidemiological/clinical studies. It is also the most widely-used assay by the labs of the World Health Organization (WHO) HPV labNet to monitor HPV vaccine effectiveness. When specimens are carefully handled and PCR procedures are strictly performed according to protocol, analytical sensitivity and specificity of Linear Array assay can reach . The performance of in-house assays can have greater variation since each of the many steps of the PCR testing procedure can introduce important variability -20. FactType-specific HPV infections are considered to be correlated in this simulation study because the risk factors of getting infected by various HPV types are similar and subjects with weaker immune systems are more likely to get infected and/or stay infected. Without loss of generality, the correlations between HPV types were set to be 0.05. This is the mean of 666 pairwise correlations calculated based on 37 Linear Array genotyping test results collected in the 2003–2006 National Health and Nutrition Examination Survey (NHANES). Although the values of pairwise correlations varied from 0 to 0.3, eighty-five percent of these pairwise correlations were somewhere between 0 and 0.1. In addition, the simulation results  is not discussed in this manuscript. Having a good sample representing the target population is very important. Since PCR genotyping assay results have limited clinical utility, future studies can be conducted to investigate incorporating HPV clinical tests  to monitor HPV infections.Composite prevalence estimates calculated based on panels of genotyping assay results generally overestimate the true infection burden and could underestimate effectiveness. Analytical specificity of genotyping assay is as or more important than sensitivity and should be considered in selecting assay to monitor HPV.


The primo vascular system (PVS), floating in lymph ducts, was too transparent to be observed by using a stereomicroscope. It was only detectable with the aid of staining dyes, for instance, Alcian blue, which was injected into the lymph nodes. Some dyes were absorbed preferentially by the PVS than the lymph wall. It remains a standing problem to know what dyes are absorbed better by the PVS than the lymph walls. Such information would be useful to unravel the biochemical properties of the PVS that are badly in need for obtaining large amount of PVS specimens. In the current work we tried two other familiar dyes which were used in PVS research before. We found that Trypan blue and toluidine blue did not visualize the PVS. Trypan blue was cleared by the natural washing. Toluidine blue did not stain the PVS, but it did leave stained spots in the lymph wall and its surrounding tissues, and it leaked out of the lymph wall to stain surrounding connective tissues. These completely different behaviors of the three dyes were found for the first time in the current work and provide valuable information to elucidate the mechanism through which some special dyes stained the PVS preferentially compared to the lymphatic wall. Long threadlike structures floating in large-caliber lymph ducts were first observed with the aid of staining with the dye Janus green B in the lymph ducts stemming from the lumbar nodes near the caudal vena cava in the abdominal cavity of a rabbit . Since tOne of the important questions concerns the mechanism through which the dye preferentially stains the L-PVS rather than the lymph wall. In answering this question, knowing which dyes do not stain the L-PVS in a similar process would be helpful. If L-PVS affine and nonaffine dyes are compared, valuable information on the mechanism of the staining can be obtained. The current work reports on the findings for two dyes, Trypan blue and toluidine blue, that did not stain the L-PVS when the same protocol as that for the Alcian blue was used. In the first case, we injected Alcian blue into the inguinal node of one side and Trypan blue into the inguinal node of the other side. In the second case, toluidine blue and Alcian blue were compared. Trypan blue was chosen for this study because it is effective for staining the primo vascular system (PVS) on the surfaces of internal organs , in the Effectively visualizing, observing, and isolating the L-PVS is one of the steps necessary to establish the anatomy and the histology of the L-PVS. The current work is one of various efforts with that purpose. The present situation of PVS research is similar to that of the lymph system about a decade ago. Before the first breakthrough with the identification of the vascular endothelial growth factor VEGF-C as a specific lymphangiogenic growth factor, only a rudimentary understanding of the molecular biology of lymphatics existed due to technical difficulties in identifying lymph vessels within tissues and in isolating pure cultures of lymphatic endothelial cells for detailed characterization –22. A moThe possible medical significance of the PVS was suggested by the observation of abundant immune cells in the PVS with the following population ratios: mast cells (20%), eosinophils (16%), neutrophils 15%), lymphocytes (1%), immature cells (3%), and chromatin cells (0.3%) . These d%, lympho ad libitum. The procedures involving the animals and their care were in full compliance with current international laws and policies  and were approved by the Institutional Ethics Committee of the Advanced Institute of Convergence Technology . The rats were anesthetized by intramuscular injection of a regimen consisting of 1.5 g/kg of urethane and 20 mg/kg of xylazine.Rats  were obtained from DooYeol Biotech  and housed in a temperature-controlled environment (23°C). Seventeen rats were injected with Trypan blue and twenty-one rats were injected with toluidine blue in the experiments. All animals were exposed to a 12-hour light-dark cycle and were provided food and waterμm membrane filter  with a syringe . The staining dye Trypan blue solution  0.4% (w/v) in PBS was used. The staining dye with a fiducial volume of 50% toluidine blue working solution was made from the stock solution and 1% sodium chloride (NaCl: 0.05 gm and distilled water: 5 mL). The toluidine blue stock solution was made by melting 0.1 gm of toluidine blue powder  and 10 mL of 70% alcohol.The staining dye 0.2% Alcian blue  solution in boiled phosphate-buffered saline  was prepared and was filtered by using a 0.22-A 2 cm skin midline incision up and down from the navel along the ventral surface of the abdominal cavity was made, and the skin was retracted towards the rat's spinal column. The inguinal nodes (INs) are bilateral and are situated close to the bifurcation of the superficial epigastric veins. After the left-hand side IN had been exposed, the prepared 0.2% Alcian blue dye was injected into the node at a slow rate. Then, Trypan blue was injected into the right-hand side IN. The order of injections was varied and the left and the right sides were alternated from one experiment to another. The other experiments with Alcian blue and toluidine blue were done using similar procedures.The main lymph duct to observe was located along the superior epigastric vein and connected the IN to the axillary node (AN); most of the time, it had several branches. Natural circulation of the lymph fluid had to be promoted in order to ensure that the dyeing agent had been thoroughly washed out. Therefore, each rat was covered with paper tissue and kept in the bedding to maintain its body temperature.Two hours after dye injection, the IN and the lymph duct were exposed to observe the L-PVS. All procedures were performed under a stereomicroscope . After the stained L-PVS had been observed, the rats were sacrificed by using an intracardiac injection of urethane (1 mL). The skin, including the lymph ducts, was fixed with a 10% neutral buffered formalin (NBF) solution at 4°C for 24 hours. The lymph ducts, including the L-PVS, were collected from the fixed skin.For the staining of nuclei with 4′, 6-diamidino-2-phenylindole (DAPI), the specimen was stained with 300-nM DAPI  solution for 20 minutes. After the specimen had been washed, it was covered with a mounting solution. The stained specimen was investigated under a phase contrast microscope , a microscope with a black and white charge-coupled device , a contrast-enhanced microscope , and a confocal laser scanning microscope .The position of the relevant lymph duct in the skin from the inguinal node to the axillary node along the prominent epigastric blood vessel is illustrated in Compared with Alcian blue and Trypan blue, the dye toluidine blue showed a completely different behavior, as shown in The primo node is an oval-shaped tissue packed with various cells .  FigureBecause the distribution of nuclei and their shapes are critical, we present one more specimen with DAPI staining. A phase contrast microscopic image of the extracted primo vessel is shown in To the present time, several dyes have been used for the visualization of the L-PVS in lymph ducts: Janus green B , fluoresAlcian blue, Trypan blue, and toluidine blue showed different behaviors in this experiment. Alcian blue is used for the staining of acidic polysaccharides in histology . It was  in vivo and in situ, not as a staining dye of tissue specimens for histological purposes. By using it, we were able to make the following significant contributions: (1) a web-like network of PVS was observed on the omentum and the visceral peritoneum [The dye Trypan blue is used in histology for staining connective tissues, such as collagen, muscle, and cornified epithelium . Anotherritoneum ; (2) theritoneum –18; and ritoneum . In the The behavior of the toluidine blue was beyond our expectation in the sense that it leaked out through the lymphatic wall and left randomly-distributed dots in the lymph wall and its surrounding tissues. In addition, the dye flowed in some venules around the lymph duct and strongly stained the vascular wall, but it was not absorbed by the L-PVS. These results were newly found in this experiment. Toluidine blue has often been used in the staining of PVS tissues for light microscopic imaging in transmission electron microscopic studies of PVS tissues .The possible medical significance of the PVS is suggested by the observation of abundant immune cells in the PVS . The datThe present work was limited in that it only compared the efficacies of the three dyes in visualizing the L-PVS and did not uncover the biochemical mechanism for their different behaviors. The understanding of this mechanism is left for a future study. For this purpose it is valuable to know the dyes that were used to stain lymph vessels. For example, Evans blue, and guanylate cyclase that was used to stain dermal lymphatic capillary .In conclusion, Trypan blue and toluidine blue were quite different from Alcian blue in their abilities to visualize the L-PVS. Trypan blue was cleared completely by a two-hour natural washing and did not stain either the lymph wall or the L-PVS. To the contrary, toluidine blue left scattered strongly stained dots in the lymph wall and surrounding tissues after leaking out from the lymph duct. It did not, however, stain the L-PVS at all. Sometimes, it entered some small veins and stained the vessel strongly. These were first observed in this experiment, but this phenomenon, in terms of biochemical processes, is still not understood.


Additional issues include the unmet need for a reliable screening strategy for HNSCC, as well as the ongoing assessment of the efficacy of prophylactic vaccines for the prevention of HPV infections in the head and neck regions. This review also outlines a number of emerging prospects for therapeutic vaccines, as well as for targeted, molecular-based therapies for HPV-associated head and neck cancers. Overall, the future for developing novel and effective therapeutic agents for HPV-associated head and neck tumors is promising; continued progress is critical in order to meet the challenges posed by the growing epidemic.The rise in human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) has elicited significant interest in the role of high-risk HPV in tumorigenesis. Because patients with HPV-positive HNSCC have better prognoses than do their HPV-negative counterparts, current therapeutic strategies for HPV Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, with an incidence of over half a million new cases annually ,2,3,4,5.The link between HPV and oropharyngeal carcinoma was initially suggested four decades ago, when it was still considered a risk factor ,15. Howeet al., surmised that within the US, a sexually active individual is likely to encounter an HPV infection at one or more points during their lifetime [et al. [HPV infection has been extensively studied in the context of its association with cervical cancer ,9,23, thlifetime ,41. Howelifetime ,23,41,42lifetime ,28,29,38 [et al. ,43,44. I [et al. ,29 as ce [et al. ,34,45. I [et al. ,32,46, a [et al. ,48. Ther [et al. ,34.vs. 34% in their Caucasian counterparts) [Since HPV-positive oropharyngeal cancers display a different etiology than do HPV-negative cancers ,21,49, Herparts) ,58,59. Ierparts) . Therefoerparts) ,32.Current therapeutic interventions for HNSCC patients include surgery, chemotherapy, and radiotherapy ,15,52,60Surgery is one of the standard treatments for early stage I/II HNSCC. In the past, surgical procedures sometimes consisted of extensive open transmandibular, and open pharyngotomy procedures ,62,64,752 laser [via a direct transoral approach using transtumor cuts to assess tumor depth and microscopic magnification to aid in margin control [TLM is one of the procedures available for early head and neck cancer . This pr2 laser ,77. TLM  control ,54,77; a control ,53.en bloc in the oral cavity [In 2009, TORS, an alternative method for transoral surgery, became approved for small primary tumors of the head and neck region ,54,62,75l cavity ,62,77. Il cavity ,52,54,62l cavity ,54,62. Hl cavity . There il cavity ,75. To hl cavity ,62,76. Tl cavity .Cisplatin is the most widely used chemotherapeutic agent with the best prognostic outcome, achieving about a 90% 3-year survival rate ,67. CispHistorically, radiotherapy has been thought of as a conventional treatment for HNSCC and is usually a component of a multi-modal therapy plan ,55. RadiResearch has shown that disease control is attainable in both HPV-related and HPV-unrelated subsets when TORS is employed as an initial surgical approach followed by chemoradiation ,35. UnfoClinicians are becoming increasingly aware of the need for differential therapeutic regimens between HPV-positive and HPV-negative patients  due to tThe reason(s) HPV-related HNSCC are associated with an improved survival outcome as compared to HPV-unrelated cancers remains speculative ,14,60, bTP53 is mutated and inactive, while the TP53 gene in HPV-infected tumors is wild-type and functionally intact, with the protein being degraded by the HPV oncoprotein E6 [E6 oncogenes, allowing the TP53 gene to carry out its normal function [TP53 gene and the lower mutation rate [An alternate or possibly complementary explanation focuses on differences in biological mechanisms. That is, even though the biologic mechanisms leading to divergent prognoses in HPV-dependent and independent oropharyngeal cancer have been elusive ,57, the otein E6 ,5,30,35.function . Therefoion rate  observedion rate ,20,53.in vitro demonstrated increased survival [in vivo, where the cells are surrounded by an immunologic microenvironment. Likewise, an apparent higher response in immunocompetent vs. immunodeficient mice further supports this finding [+ T cells that are specific to HPV [Another possibility is that HPV-positive cancer cells express viral proteins that induce and enhance the immune response, which becomes involved in clearing cancer cells during treatment ,8,74,96.survival  and resisurvival ,28 as co finding . In addi finding  and a hic to HPV ,3,37 comLastly, the difference in the degree of intratumor heterogeneity between HPV-dependent and HPV-independent OPSCC could contribute to their divergent prognoses. Intratumor heterogeneity refers to a tumor population comprised of subpopulations that display differing genetic makeups . AssuminTo date, an effective mono-dimensional therapy approach suitable for head and neck carcinoma is not available . MoreoveSome researchers contend that concurrent radiochemotherapy may confer excess treatment . MoreoveClinical trials testing various de-intensification strategies for HPV-positive head and neck carcinoma patients are under examination ,28. The Another strategy is to employ the new minimally invasive TOS technique as a primary surgical therapy ,52. A raAnother possibility is the administration of a less toxic alternate agent, such as cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody . The RadEver since the presence of HPV was demonstrated in tissues of HNSCC patients in 1983, the study of molecular mechanisms in HPV-associated HNSCC has garnered significant attention ,20,98. IApproaches already developed for the treatment and prevention of cervical cancer may be of great help in combating HPV-derived HNSCC . Nonethei.e., HPV-16, 18) categories, based on their ability to develop precancerous lesions and their potential to cause malignant transformation [HPV is transmitted through the mucosal and non-mucosal skin epithelia ,37. Abouormation ,109. Theormation ,110 of cormation ,33, 99% ormation , 88% of ormation , and 70%ormation ,14,33 leormation , HPV-16 ormation ,39, accoormation ,16, follstratum, and as they move up the suprabasal layer [stratum of the epithelium, HPV will proceed with protein coat formation, assembly of the new viral components, and eventual viral release [The HPV is a non-enveloped, double-stranded DNA virus that displays a predilection for squamous cell epithelium ,37,111. al layer ,37, theyal layer ,74. HPV al layer ,20,37,74al layer ,15,42. Hal layer . When th release .E1, E2, E4, E5, E6, and E7 are involved in viral replication, and the structural or late genes L1 and L2 control the viral packaging [The HPV genome is composed of approximately 8,000 base pairs  with duaackaging ,107,111.ackaging , and is ackaging . E2 can ackaging ,111. Theackaging ,114.The immortalizing qualities of the virus are attributable primarily to the oncoproteins E6 and E7 ,2,15,112The oncoproteins E6 and E7 are constitutively expressed throughout the progression of the carcinoma , making The central role of the oncogenic protein E6 is to inhibit apoptosis of the infected cells by accelerating the degradation of apoptotic mediators, including the well-known tumor suppressor protein p53 ,121,122,E7, enhances cellular proliferation by inactivating the retinoblastoma protein (pRb) and other proteins involved in the control of cell division [Another oncogene, division ,130,131.division ,123,132.The majority of head and neck carcinomas are discovered at late stages of tumor progression, arguing for the need of a reliable detection tool that is clinically relevant to facilitate early detection of HNSCC ,27. ConsIn contrast to the case with cervical cancer, there are no reliable screening methods or routine check-ups equivalent to the Pap smear to detect early HPV neoplasia in the oral cavity ,15,29,35E6 or E7 sequences [L1 region of the HPV genome [L1 region is that this region can be compromised or deleted following integration into the host genome [At this point, the technique(s) to be employed for determining the HPV status of head and neck cancers is controversial, due to variations in available methods in terms of cost, sensitivity, technicality, specificity, and reliability ,135,136.equences ,138 throequences ,139,140.equences ,141. It V genome . A disadt genome , therebyt genome . Furthert genome ,138,139.t genome ,144. On t genome ,139. It t genome . Nevertht genome ,135.The detection of HPV E6/E7 mRNA is the “gold standard” validation of active HPV oncoprotein transcription, and is considered clinically applicable in the evaluation of carcinogenesis ,139,146.CDKN2A gene [+ head and neck carcinomas [Another major alternative for detecting the virus is the IHC of the CDK inhibitor p16, a transcript encoded by the N2A gene ,54,138. N2A gene , technicN2A gene ,139,144,N2A gene ,139,144,N2A gene . p16 is N2A gene , and is N2A gene ,138. p16N2A gene ,20,37,96rcinomas ,139.et al. reported that a HPV DNA test such as PCR in addition to an evaluation of p16 overexpression through IHC is regarded as “specific and sensitive as utilizing a gold standard” [Many investigators propose that using RT-PCR to detect the presence of E6/E7 mRNA may be suitable as a gold standard for fresh samples, since the expression of these two oncogenes is characteristic of a functional HPV infection and cell transformation ,17,19,27tandard” ,148. Yettandard” ,139. If tandard” . This setandard” ,139. Othtandard” ,139. In tandard” .A steep upward shift in the incidence of HPV-derived HNSCC demands a search for a vaccine that can avert the infection of oral HPV before an opportunity to develop a malignant lesion arises, especially considering the lack of a reliable routine screening tool for those at risk of oropharyngeal SCC ,21. PastPreventive vaccines against HPV in the cervix have been developed and have become available to the public within the past decade . The firIn contrast to the large diversity of high-risk HPV types observed in cervical carcinoma , HPV typTherapeutic vaccines for HPV-driven malignancies are currently undergoing clinical investigations ,23. UnliIn the design and development of therapeutic vaccines, HPV-16 E6 and E7 oncoproteins have become popular viral targets since they are consistently expressed in HPV malignancies and are critical for transformation ,118,119.Due to their safety, ease of production, purity and stability, DNA vaccines have become attractive therapeutic candidates for HPV-associated HNSCC ,118,119.In contrast, peptide vaccines are taken up by antigen presenting cells (APC) directly without the need for encoding and are loaded onto MHC molecules for antigenic presentation ,107. ThiProtein vaccines are similar to peptide vaccines in many ways, but they can bypass MHC restriction since the protein contains a variety of antigenic epitopes ,118. Addex vivo before injecting them back into the patient [The cell-based vaccine technique entails the pulsing of dendritic cells (DC) with an antigen ,119, all patient ,118.Listeria monocytogenes fused to E7 has shown the ability to cause regression of solid tumors and has progressed to phase I clinical studies in oropharyngeal cancer patients [A live vector, consisting of either a bacteria or a virus, can be employed to deliver antigens such as those found in the E6 and E7 oncoproteins to the host APCs in order to enhance antigen presentation and the induction of a cell-mediated response ,111,118.patients . Anotherpatients ,111,117.patients ,117. Thepatients ,117.Current treatment for HNSCC patients is confined to standard therapies, such as irradiation, surgery, and chemotherapy ,67; and We have come a long way in understanding the molecular biology of head and neck cancer over the past few decades . InteresTP53 and amplification of EGFR [The role of EGFR is to transmit signals to intracellular pathways that regulate a host of cellular activities including proliferation, cell cycle progression, apoptosis, migration, metastasis, differentiation and angiogenesis ,80,151.  of EGFR . Thus, t of EGFR . Several of EGFR ,150. The of EGFR ,57,68,80vs. 36.4% for radiotherapy alone) [Cetuximab is a recombinant chimeric immunoglobulin (Ig)G mAb, specifically targeting the extracellular domain of EGFR ,80,150. y alone) ,80,152. y alone) ,80,150. y alone) ,80,93. Ty alone) ,80.Other fully humanized IgG anti-EGFR antibodies under consideration include zalutumumab  and panitumumab , and these are being investigated in phase II and III studies ,37,67,80EGFR TKIs have also demonstrated some clinical activity in HNSCC but without as much success as seen with the mAbs ,80. The EGFR is involved in downstream intracellular pathways such as the PI3K/Akt/mTOR pathway. Alterations in the phosphoinositide 3-kinase (PI3K) pathway have been found in patients with head and neck cancers, and appear even more predominately in patients with HPV-derived tumors ,57,153. Vascular endothelial growth factor (VEGF) is another type of growth factor and is considered one of the most critical angiogenic cytokines in tumor vasculogenesis ,150. TarDetermining the molecular differences between HPV-dependent and HPV-independent head and neck cancers will be crucial in the discovery of therapeutic targets specific for HPV-dependent malignancies . VariousThe interaction between E6 and E6AP represents an attractive antiviral target, as agents that target this interaction may be able to inhibit the degradation of p53 and sensitize cells to agents that induce apoptosis ,90,112. A small molecule that has been widely studied in multiple types of cancer is the p53 protector, RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis) ,163. Thiin vitro as well as in vivo [A similar approach is taken by the non-peptide small molecule compound Nutlin-3A, an imidazoline analog and potent MDM2 antagonist. Nutlin causes substantial cell death in a variety of wild-type p53 expressing cell lines ; however in vivo . CH1iB i in vivo . This ac in vivo . A precl in vivo ), indica in vivo .Another attractive target is the interaction of E6 and caspase 8, a protein involved in the extrinsic apoptotic pathway . The extAnother strategy is to inhibit the interaction between E7 and pRb, thereby preventing E7 from inhibiting pRb’s ability to inhibit cell division. The small compound thiadiazolidinedione inhibits HPV-E7 from disrupting the pRb-E2F complex by blocking the E7-pRb interaction . Lastly,Research into small molecules useful for the treatment of HPV-dependent cancers is ongoing and encouraging. However, concepts developed during studies conducted on cervical cancer will have to be assimilated and translated to oropharyngeal carcinoma. Further developments in our understanding of the molecular biology underlying the development of HNSCC will be necessary to refine the efficacy of these early phase agents.The current epidemic of HNSCC has sparked significant interest in the role of HPV in oncogenesis, and the emergence of HPV-positive head and neck cancer has shifted the demographic of HNSCC from an older population to a younger generation. Current treatments, which consist of transoral surgery, platinum-based chemotherapy, and intensity-modulated radiotherapy, are increasingly recognized as requiring improvements. While advances in standard therapies have improved outcomes, the new group of younger patients is at high risk of morbidity and consequently a compromised quality of life. Therefore, the demand for major progress in the therapy and diagnosis of HPV-associated carcinoma remains current and compelling . The betSeveral advances in biotherapy have led to the identification of a number of small molecular compounds with the potential for contributing to the development of less toxic treatments. The field of small molecular targeted therapy is in its infancy, but current findings are encouraging, advocating for the rapid progression of the field. The studies presented above reveal the urgency of the burden and the impetus to identify better targets and antiviral therapies effective in attenuating the incidence of HPV infection and counteracting the growing epidemic of HPV-associated head and neck cancers .


Viruses that were detected include phages, adenoviruses, herpesviridae and HPV. HPV, which causes virtually all cervical cancers, was identified in 95% of the carcinomas, 100% of the CIN2/3 samples, both CIN1 samples and in 55% of the normal samples. Comparing the amount of mapped fragments on HPV for each HPV-infected sample yielded a significant difference between normal samples and carcinomas or CIN2/3 samples , reflecting different viral loads and/or methylation degrees in non-normal samples. Fragments originating from different HPV types could be distinguished and were independently validated by PCR-based assays in 71% of the detections. In conclusion, although limited by the a priori knowledge of viral reference genome sequences, the proposed methodology can provide a first confined but substantial insight into the presence, concentration and types of methylated viral sequences in MBD-seq data at low additional cost.Most next generation sequencing experiments generate more data than is usable for the experimental set up. For example, methyl-CpG binding domain (MBD) affinity purification based sequencing is often used for DNA-methylation profiling, but up to 30% of the sequenced fragments cannot be mapped uniquely to the reference genome. Here we present and evaluate a methodology for the identification of viruses in these otherwise unused paired-end MBD-seq data. Viral detection is accomplished by mapping non-reference alignable reads to a comprehensive set of viral genomes. As viruses play an important role in epigenetics and cancer development, 92 (pre)malignant and benign samples, originating from two different collections of cervical samples and related cell lines, were used in this study. These samples include primary carcinomas ( The advent of next generation sequencing (NGS) has initiated a revolution in molecular biology. Due to massively parallel sequencing, new insights could be revealed in genetics, transcriptomics and more recently epigenomics. However, the processing of the sheer amount of data produced by these methods proved to be a challenge. Identification of nucleotide sequences is often the first step in many NGS analyses, yet a substantial fraction cannot be properly identified. These unidentified fragments might arise from low-complexity regions , bacteria, viruses, other organisms or artificial noise , Epstein-Barr virus (EBV), Kaposi's sarcoma associated herpesvirus (KSHV), Human cytomegalovirus (CMV) and Merkel cell polyomavirus (MCP). It is estimated that viruses have a causal role in about 16% of all human cancers , which are putative cervical cancer precursors  in Amsterdam, further referred to as Set 1. Of this set, 10 samples were obtained from carcinoma, 12 are high-grade cervical intraepithelial neoplasia (CIN2/3), 3 are low-grade cervical intraepithelial neoplasia (CIN1) and 15 originate from cell cultures .To obtain the DNA methylation profiles, the MethylCap kit from Diagenode was combined with Illumina Genome Analyzer IIx paired-end sequencing as described in   specific counts (Nvs) for each sample (s). Whenever we observed an Nvs > 0, we reported the virus to be present for that sample. Scripts for the execution of the pipeline can be found here: https://github.com/klamens/viral-pipeline.We aimed to identify fragments of viral origin. This was achieved by searching for sequence similarity between the non-canonical reads and a set of viral reference genomes. For this purpose, we used FR-HIT  and the presence of HPV in a sample was performed by Pearson's Chi-squared test with 2000 simulated permutations. Association of high-risk HPV type occurrence and histological groups was tested as well. The most abundant HPV type per sample was used for the assessment of high/low risk HPV type occurrence. When abundances of the most and second most abundant type were equal and their risk was different, the sample was rejected for testing. For a comparison of the fraction of viral fragments, Wickham, .Samples of Set 1 were assessed for HPV (type) presence using the GP5+/6+ PCR followed by enzyme immunoassay (EIA) read-out system using a probe cocktail of 14 high-risk HPV types   of all fragments in each sample could not be aligned to the human reference genome. Of these reads, only 0.31% (SD = 0.17%) could be mapped to the viral reference genomes. In total, we tried to map reads of 4.3×108 non-canonical fragments to 6433 different viral genomes, obtained after removal of very similar genomes . More details about the mapping statistics can be found in the Supplementary Material. As MBD-seq enriches for methylated CpGs, a high-quality dataset should include only a limited amount of fragments without any CpG, and most fragments should have multiple CpGs , but post-hoc tests revealed only significantly higher HERV-K113-like fractions in the cell cultures compared to normal tissue, CIN2/3 and carcinomas .In a first phase, the presence of specific viruses in the different sample sets was assessed .Human adenoviruses were discovered in multiple samples. More fragments were observed in samples originating from Set 1 compared to those of Set 2. The most occurring types were human adenovirus C and human adenovirus B. Two CIN2/3 samples contained a single fragment of the Merkel cell polyomavirus. Multiple, particularly carcinoma and CIN2/3, samples were found to contain one to 25 fragments of the Epstein-Barr virus. Human cytomegalovirus was only detected in cell culture samples. However, these fragments most likely originate from the CMV promoter which is included in the pcDNAI neo plasmid. Human herpes virus 1, 6, and 7 were also identified, each in just a single sample.p < 0.001).HPV was detected in all but one sample in the carcinoma group and the cell culture group. It was discovered in all samples originating from the CIN2/3 group and in 11 of the 20 normal samples. Also in both samples of CIN1 and in one of the two leukocyte samples HPV was detected. Association between the presence of HPV and cervical groups (excluding cell culture samples and leukocytes) was assessed for by Pearson's Chi-squared test with simulated . Post-hoc analyses reveal significant differences between the normal group and cell carcinoma and CIN2/3 samples (see Table n = 2).Next to assessing the  presence of specific viruses, also a quantitative analysis can be performed. To illustrate the feasibility, HPV N = 30), followed by HPV31 (N = 12), HPV39 (N = 9), HPV18 (N = 6), and HPV36 (N = 6). Other HPV types could not be observed in more than 4 different samples , which we consider here as gold standard. The independent validation of the HPV types yielded a positive verification in 71% of the detections. For HPV types indicated to be present by these methods, results were 66% concordant with the MBD-seq approach. Vice versa, verified absence of viruses was 98% concordant with the proposed methodology. The more fragments that were detected for an identified HPV type, the more likely it was to be validated as can be seen in Table In this study, we demonstrated that the non-canonical fraction of MBD-seq fragments can be used to identify viruses. Considering the increasing importance of sequencing methods, this strategy can provide key evidence regarding the involvement of specific viruses in pathologies at minimal additional cost. Given the roles of DNA methylation in virus biology, the outlined pipeline is capable to generate valuable hypotheses from otherwise unused data. As the outlined application has also several disadvantages (see below), the generated hypotheses should of course be additionally validated by state-of-the art methods. The observed CpG content in many cervical samples, in comparison with De Meyer et al. , suggestRecently, some studies already achieved viral identification in RNA-seq experiments by comparable methods  is very limited compared to the amount of mammalian viruses estimated at 320.000  association of HPV presence and histological group (ii) differential quantities of HPV fragments in HPV positive samples between normal samples and carinoma or CIN2/3 samples (iii) type detection with good concordance as verified by independent methods. In other words, if the impact of HPV in cervical cancer would have been unknown, it might have been picked up by the outlined approach, though additional validation would of course have been absolutely necessary. It is therefore clear that the methodology can generate novel knowledge regarding the presence of viruses in disease, and that the inherent disadvantages are by far outweighed by the major benefit of obtaining information regarding the presence of any sequenced virus in otherwise typically discarded data.Ed Schuuring, Renske D. M. Steenbergen and G. Bea A. Wisman provided the data regarding the cervical samples and performed the HPV type verifications for these samples. Tim De Meyer and Klaas Mensaert conceived the general idea and approach. Klaas Mensaert and Tim De Meyer have designed the pipeline, analyzed the data and wrote the manuscript. Wim Van Criekinge and Olivier Thas have contributed to the conceptual development and provided critical advice. All authors have reviewed the article and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cervical cancer is the most common female cancer in Bhutan, the first low/middle-income country to implement a national human papillomavirus (HPV) vaccination programme.To provide a robust baseline for future evaluations of vaccine effectiveness, cervical cell specimens were obtained from 2,505 women aged 18–69 years from the general population, and biopsies from 211 cervical intraepithelial neoplasia grade 3 (CIN3) and 112 invasive cervical cancer (ICC) cases. Samples were tested for HPV using GP5+/6+ PCR.Among the general population, HPV prevalence was 26%, being highest (33%) in women ≤24 years, but remaining above 15% in all age-groups. Determinants of HPV included age, marital status, and number of sexual partners. Among the eight percent with cytological abnormalities, 24 CIN3 and 4 ICC were histologically confirmed. Even after additional testing with a sensitive E7 PCR, no infections with vaccine-targeted HPV types were detected in the few vaccinated women (n = 34) compared to 6% prevalence in unvaccinated women of similar age (p = 0 · 215).Based upon type-specific prevalence among biopsies, at least 70% of ICC in Bhutan are theoretically preventable by HPV16/18 vaccination, but screening programmes should be expanded among older women, who have an important underlying burden of CIN3 and ICC. In the Himalayan kingdom of Bhutan, cervical cancer represents the most common cancer among females, with an age-standardised incidence rate of approximately 13 cases per 100,000 person years . Since 2The impact of the vaccine programme on cervical cancer will not be seen for at least 20 years and, therefore, short-term evidence of vaccine programme effectiveness is crucial to encourage national planners to sustain HPV vaccination services. Hence, the Bhutan Ministry of Health, in collaboration with the International Agency for Research on Cancer (IARC), initiated a series of studies to evaluate the short term impact of HPV vaccine on HPV prevalence.As no data existed on the burden of HPV among women in Bhutan, a baseline study of the prevalence of, and risk factors for, HPV infection prior to vaccination was initiated in 2011 according to the standardised protocol of the IARC HPV prevalence surveys . CervicoBetween December 2011 and October 2012, a survey was conducted by the Ministry of Health of Bhutan in collaboration with the IARC, Lyon, France. The study aim was to enrol 2,500 women from the general population using an age-stratified approach, namely 1,000 women aged below 25 years old, 500 women aged 25 to 29 years old, 200 women in each five-year age group between 30–34 and 45–49 years, and 200 women aged ≥50 years. Over-representation of young women was done in order to allow robust assessment of vaccine-induced changes in HPV prevalence in a repeat survey after 5 years. All mentally and physically competent women were eligible for the study, regardless of their marital status.Study procedures were performed in the Reproductive Health Departments of Jigme Dorji Wangchuck National Referral Hospital (JDWNRH) in Thimphu, and in the hospital of Lungtenphu, a satellite town of Thimphu. To reach a broad and representative sample of the population living around the two hospitals, women were recruited in two ways: 1) Women residing in the pre-defined areas surrounding the two hospitals were visited at home by social workers and invited to join the study. Participation among the 1217 and 673 women invited from Thimphu and Lungtenphu was 25.7% and 54.2%, respectively. 2) In addition, women consulting outpatient clinics in the two hospitals , were also invited to join. Participation among women invited through the hospital was close to 100%.Some limited sociodemographic information was obtained from all women invited from home in Thimphu, among whom non-participants tended to be younger and more likely to be unmarried in comparison to participants. Following signature of an informed consent form, a structured questionnaire including information on socio-demographic characteristics, sexual behaviour of the women and of their partners, reproductive factors, use of contraceptive methods and smoking habits was administered to all study participants. History of HPV vaccination was asked to women aged ≤20 years.A cytobrush  was used for the collection of exfoliated cervical cells from the endocervix and ectocervix. After preparation of a conventional Pap smear, the brush containing cellular material was placed in a vial containing PreservCyt medium  and stored at +4C until shipment.Conventional Pap smears were read in the Department of Cytology, JDWNRH, and reported according to the 2001 Bethesda System . All womFormalin-fixed paraffin-embedded biopsies were retrieved from all cases of ICC and CIN3 diagnosed between 2010 and 2012 at JDWNRH, Thimphu or the Eastern Regional Referral Hospital, Mongar. Biopsies were sent to the VU University Medical Center, Amsterdam, for HPV DNA testing and review of histology. After exclusion of 11 biopsies negative for beta-globin PCR and 177 biopsies without any histological evidence of CIN3 or cancer, 211 CIN3 and 112 ICC cases were included in following analyses .2 of tissue were predigested with proteinase K after which DNA was extracted using magnetic beads (Macherey-Nagel).HPV testing was performed on all exfoliated cervical cells and biopsies in the Department of Pathology at the VU University Medical Center, Amsterdam. DNA was extracted from the PreservCyt sample using magnetic beads (Macherey-Nagel) on a robotic system (Hamilton Robotics) according to the manufacturer’s instructions. Biopsies were sectioned using a ‘sandwich’ approach, whereby inner tumour sections were destined for HPV testing and outer sections for histological confirmation of tumour tissue (see above). One or more sections representing approximately 1 cmBeta-Globin PCR analysis was conducted first to confirm the presence of human DNA in all specimens . The preDNA extracted from cervical cells of women aged ≤ 24 years, was additionally tested for the 13 HR types plus HPV6 and 11 using an alternative PCR-based assay based upon multiplex E7 gene PCR and DNA microarray  at IARC.In addition to crude prevalence, age-standardised HPV prevalence was computed using the world standardised population to allow comparisons of HPV prevalence with other IARC surveys . PrevaleThe present study had the approval of both the Research Ethical Board of the Bhutan Ministry of Health and the IARC Ethics Committee.Of the 2,525 participants who provided cervical cell samples, 2,505 had valid HPV results and were included in the following analyses, including 1,638 and 867 recruited at JDWNRH and Lungthenphu hospitals, respectively. Among the 2,468 women with a valid PAP smear result, 196 (8%) had an abnormal cytological diagnosis, including 123 (58%) with atypical squamous cells of undetermined significance, atypical squamous cells cannot exclude high-grade lesion or atypical glandular cells of undetermined significance (ASCUS/ASC-H/AGUS), 59 (2%) low-grade squamous intraepithelial lesions (LSIL), and 14 (1%) high-grade squamous intraepithelial lesions or worse (HSIL+). Histological confirmation was obtained for 135 women with abnormal cytology, among whom 23 CIN2, 24 CIN3 and 4 ICC were histologically confirmed.Overall HPV prevalence was 26% (95% CIs: 24–28), being 23% (95% CIs: 21–25) among women with normal cytology , source of recruitment  , particularly among women younger than 25 years (22%). Findings can be confidently compared with other IARC HPV surveys that were conducted according to a similar sampling and HPV testing protocol. In this respect, the age-standardised HR HPV prevalence in Bhutan (18%) ranks below that of Guinea (31%) , but is HPV16 was by far the most frequently detected type in Bhutan, increasing from 3% of normal cytology up to 63% of HPV-positive ICC. Such a high proportion of HPV16 in cervical cancer agrees with findings of a meta-analysis in which the proportion of HPV16-positive ICC in Western/Central Asia (66%) was the highest among all world regions . InfectiRisk factors associated with cervical HPV infection were largely consistent with previous IARC HPV Surveys. Lifetime number of sexual partners was an important determinant of HPV positivity, although the mean reported number of women’s lifetime sexual partners (1.1) was relatively low. Being single, separated or divorced, as well as a history of receiving cash for sex , were also positively associated with HPV prevalence. Nulliparous women had a significantly higher risk of being HPV-positive, even after adjustment, in agreement with a pooled analysis of previous IARC HPV Prevalence Surveys , but theForty-five percent of study participants reported to having a prior PAP smear, which gives an estimation of the coverage of the cytology-based programme that has been targeting women living in Thimphu since 1999. Cervical screening is unavailable, however, in most rural and higher-altitude areas of Bhutan. One possibility to improve nationwide coverage is the application of an HPV testing approach, e.g. careHPV in women aged 30 or older. Our data suggest that approximately 15% of screened women aged 30 or older might test positive for HR HPV and require diagnostic follow-up and treatment.This study has weaknesses and strengths. In particular, the extent to which our study population is representative of the general female population in Thimphu, and Bhutan as a whole, is difficult to evaluate. However, HPV infection is asymptomatic and self-selection of women by level of HPV risk is unlikely to have occurred. Furthermore, there was no significant difference in HPV prevalence by whether women were recruited through hospital consultations or from their homes, neither was there any substantial difference in their distribution of any of the study variables in Table Strengths include the relatively large number of women, especially those younger than 25 years, and the high rate of colposcopical follow-up and histological confirmation for women with abnormal cytology, allowing a strong gold standard of disease ascertainment. Furthermore, we used two high-quality PCR tests of which one, GP5+/6+, is clinically validated and conducted in a central laboratory that allows comparability with other studies, notably previous IARC HPV Surveys.We made a particular effort to sample a large number of women aged ≤24 years, and to test with a second highly-sensitive E7 multiplex PCR assay . As expeThis baseline survey represents the first part of a long-term protocol to monitor HPV vaccine impact in Bhutan, which will include a repeat HPV prevalence survey in cervicovaginal samples in 2016/17 among women aged ≤24 years to demonstrate the 5-year impact of the HPV vaccination programme, as well as repeated HPV prevalence surveys in urine samples of adolescents aged 17–19 years at high-schools in Thimphu, Bhutan.These data will serve as a robust baseline for future evaluations of HPV vaccine programme effectiveness on HPV prevalence as cohorts of vaccinated girls grow up. Nevertheless, our survey did also capture a few young vaccinated women in the baseline survey, among whom there was a very reassuring, albeit statistically weak, evidence of 100% protection against vaccine-targeted HPV type (HPV6/11/16/18) infection, irrespective of the HPV test. Whilst awaiting more statistically robust evaluations in the future, this ad hoc finding represents the first ever real-life evidence of HPV vaccination programme impact in a low- or middle-income country.ASCUS/ASC-H/AGUS: Atypical squamous cells of undetermined significance, atypical squamous cells cannot exclude high-grade lesion or atypical glandular cells of undetermined significance; CI: Confidence interval; CIN: Cervical intraepithelial neoplasia; HR: High-risk; HPV: Human papillomavirus; HSIL: High-grade squamous intraepithelial lesions; IARC: International Agency for Research on Cancer; ICC: Invasive cervical cancer; JDWNRH: Jigme Dorji Wangchuck National Referral Hospital; LMIC: Low- and middle-income country; LR: Low-risk; LSIL: Low-grade squamous intraepithelial lesions; OR: Odds ratio; PR: Prevalence ratio.P.J.F. Snijders has Honoraria from Speakers Bureau from Roche and is a consultant/advisory board member for Roche and Gen-Probe. C.J.L.M. Meijer has Honoraria of Speakers Bureau from GlaxoSmithKline and is a consultant/advisory board member for Qiagen. No competing interests were disclosed by the other authors.GC and SF conceived and designed the study. GC, SF, IB, UT and DD were involved in the development of the methodology. DD (cytology), PJFS (HPV testing), CJLMM and MCGB , TG and MT (HPV testing) collected data. GC, IB, SF, and VT performed data analysis and interpretation. All authors read and approved the final manuscript. DD was responsible for administrative, technical, or material support. GC supervised the study.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/408/prepub


Cervical cancer is the major cause of morbidity and mortality in Thai women. Nevertheless, the preventive strategy such as HPV vaccination program has not been implemented at the national level. This study explored the HPV prevalence and genotypic distribution in a large cohort of Thai women.A hospital-based cervical cancer screening program at Chulabhorn Hospital, Bangkok and a population-based screening program at a rural Pathum Thani Province were conducted using liquid-based cytology and HPV genotyping.Of 5906 women aged 20–70 years, Pap smear was abnormal in 4.9% and the overall HPV prevalence was 15.1%, with 6.4% high-risk (HR), 3.5% probable high-risk (PR), and 8.4% low-risk (LR) HPV. The prevalence and genotypic distribution were not significantly different between the two cohorts. Among HR-HPV genotypes, HPV52 was the most frequent (1.6%), followed by HPV16 (1.4%), HPV51 (0.9%), HPV58 (0.8%), HPV18 (0.6%), and HPV39 (0.6%). Among LR-HPV genotypes, HPV72 and HPV62 were the most frequent while HPV6 and HPV11 were rare. HPV infection was found to be proportionately high in young women, aged 20–30 years (25%) and decreasing with age (11% in women aged >50). The more severe abnormal cytology results, the higher positivity of HR-HPV infection was observed.In conclusion, HPV52, HPV16, and HPV51 were identified as the most common HR-HPV genotypes in Thai women. This study contributes genotypic evidence that should be essential for the development of appropriate HPV vaccination program as part of Thailand’s cervical cancer prevention strategies. Cervical cancer is the third most common female cancer worldwide after breast cancer and colon cancer with age standardized incidence rate (ASR) of 14.0/100000 person-year -6. As a Since HPV genotypic distribution can be area-specific, it is necessary to determine its genotypic distribution before establishing health care policies and vaccination programs in each area ,9. This After approval by the Ethical Committee for Human Research of Chulabhorn Hospital, the first hospital-based study was performed among 4550 Thai females, aged 20–70 years, who were voluntarily registered into the screening program at Chulabhorn Hospital, Bangkok, Thailand during July 19, 2011 - November 5, 2012. Exclusion criteria included absence of cervix, previous HPV vaccine vaccination, history of abnormal cytology or cervical intraepithelial neoplasia or cervical carcinoma, prior HPV infection, active disease for any types of cancer during the last 5 years, or unable to receive follow-ups throughout the program. Sixty-three women were excluded. All participants received detailed information regarding the study objectives and consented to the study. Demographics, obstetric and gynecologic history, and cervical cancer screening data were collected. The population-based study was subsequently undertaken in the Bangkhayaeng District, Pathum Thani Province using permanent living (named in the census registration) or current living status in Bangkhayaeng area at the day of pelvic examination to recruit a total of 1668 Thai females, aged 20–70 years who then underwent cervical cancer screening during February 4-June 16, 2013. Exclusion criteria were the same as our previous study.Samples were obtained using a cytobrush by gynecologic oncologists or well-trained general practitioners for pelvic examination of Chulabhorn Hospital. The brush was then placed in the preservative fluid in the BD SurePath Pap test kit  for both liquid-based cytology and HPV DNA testing. The investigators performing HPV typing were blinded to cytology results. All cervical cytology slides were interpreted per normal routine by qualified pathologists at Chulabhorn Hospital, using the Bethesda 2001 report system .For the identification of HPV genotypes, we used the Linear array HPV testing . This kit was capable of identifying 37 HPV types including 12 high-risk (HR), 8 probable high-risk (PR), and 17 low-risk (LR) types those classified by oncogenic potentiality -16. In bEach 100 μL reaction consisted of 50 μL working master mix and 50 μL of DNA sample. Amplification was performed in an Applied Biosystems GeneAmp PCR System 9700 using the recommended parameters: 50°C for 2 min, 95°C for 9 min and 40 cycles of 95°C for 30 s, 55°C for 1 min, 72°C for 1 min and 72°C for 5 min before holding indefinitely at 72°C.Following PCR amplification, the HPV and the ß-globin amplicon were chemically denatured to form single stranded DNA by addition of 100 μL Denaturation Solution. Aliquots 100 μL of denatured amplicon were then transferred to the appropriate well of typing tray containing hybridization buffer and single LINEAR ARRAY HPV Genotyping Strip coated with HPV and ß-globin probe lines. The biotin-labeled amplicon was hybridized to the oligonucleotide probes only if the amplicon contained the matching sequence of complementary probe.A blue colored complex precipitated at the probe positions where hybridization occurred. Then, the linear array HPV genotyping strip was read visually by comparing the pattern of blue lines to the linear array HPV genotyping test reference guide.Descriptive statistics were used to determine the distribution types and frequencies of HPV positivity, age, and cytology results. Frequency tables were done for qualitative variables. Independent samples t-test was used compare the means of age and Pearson’s chi-square test were performed to verify the association of demographic data and HPV prevalence between the two cohorts. p <0.05 was considered statistically significant. Licensed Stata program version 12 was used for analysis.Demographic characteristics of 2 cervical cancer screening populations were shown in Table Table Figure Pap smear was abnormal in 4.9% of the population. The abnormal Pap smear rate was highest in those young women aged 20–30 years (6.6%) and decreased by advancing age  n = 189, 3.2%), atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) , low grade squamous intraepithelial lesion (LSIL) , and high grade squamous intraepithelial lesion (HSIL)  was 31.2%, 45.5%, 86.4% and 90.0%, respectively. The more severe cytological result, the higher HPV-positive proportion was found.HPV, HR-HPV positive samples with normal cytology in 5614 women amounted to 13.3%, 5.1%, respectively. The most common HR-HPV type was HPV52 (1.4%), HPV16 (0.9%), HPV51 (0.6%), HPV58 (0.6%), HPV18 (0.5%), and HPV39 (0.5%), respectively. The overall HPV prevalence in 292 women with abnormal cytology was 48.6% (HR-HPV 31.2%).The most common HR-HPV types in this group included HPV16 (9.6%), HPV51 (6.9%), HPV52 (5.8%), HPV58 (3.8%), HPV59 (3.4%), and HPV18 (2.4%) as shown in Figure .2%, atypWe investigated the prevalence and distribution of HPV genotypes among 5906 Thai women enrolled for cervical cancer screening at Chulabhorn Hospital, Bangkok, or Bangkhayaeng District, Pathum Thani and revealed the 15% HPV infection rate among these women. The overall prevalence in both hospital-based and population-based cohorts was not statistically different despite the significant differences in the cohorts’ demographic characteristics as shown in Table Major studies from the Western countries reported the prevalence of HR-HPV infections around 11.3%-18.3% which were higher than our results -21. DispWith respect to the HR-HPV genotypes that are the major risk factors for cervical cancer, it was of interest to find that in our Chulabhorn Hospital Cohort, the most common genotype was HPV52 followed by HPV16 and HPV51. This finding was in contrast to most studies in the Western countries and two smaller studies in Thailand in which HPV16 was identified as the most frequent ,24. NeveWhen considering the significance of HPV52 in Thai women compared to worldwide invasive cancer cases in the meta-analysis of 30848 cases from 243 studies, the prevalence of global HR-HPV was 89.9%, with HPV16 and HPV18 as the most common types, followed by HPV types 58, 33, 45, 31, 52, 35, 59, 36 and 51, respectively . In our HPV prevalence is strongly associated with age worldwide ,9. We noFuture cervical cancer screening is moving towards HPV-based screening because of its very high sensitivity -38. A raIn conclusion, this study represents the largest report from Thailand with respect to detailed and comprehensive genotypic analysis of HPV subtypes. The prevalence and genotypic distribution of HPV did not significantly differ between hospital-based and population-based cohorts. HPV52 was the most frequently identified high-risk genotype in Thai women followed by HPV16 and HPV51. The majority of Thai women undergoing cervical cancer screening programs were not infected by readily available vaccine-preventable HPV genotypes . This study thus provides scientific evidence essential for the development of appropriate HPV vaccination programs targeted for certain HR-HPV genotypes prevalent in the Thai women.