There is a dramatic rise in the incidence of Human papillomavirus (HPV) – associated head and neck squamous cell carcinoma (HNSCC) in the world, with considerable variation by geography, gender and ethnicity. Little is known about the situation in Bangladesh, where tobacco- and areca nut-related head and neck cancers (HNCs) are the most common cancers in men. We aimed to determine the prevalence of HPV in HNSCC in Bangladesh and to explore the possible value of cell cycle markers in clinical diagnostic settings.One hundred and ninety six archival HNSCC tissue samples were analysed for the presence of HPV DNA. The DNA quality was assured, and then amplified using a nested PCR approach. The typing of HPV was performed by automated DNA sequencing. Cellular markers p53, Cyclin D1 and pRb were tested on all samples by immunohistochemistry (IHC), as well as p16 as a putative surrogate for the detection of HPV.p < 0.001). No statistically significant association was observed between the cellular markers and HPV DNA positive cases. However, p16 positivity had excellent predictive value for the presence of HPV by PCR.HPV DNA was detected in 36/174 (~21%) samples: 36% of cancers from the oropharynx; 31% of oral cancers, and 22% from the larynx. HPV-16 was most common, being present in 33 samples, followed by HPV-33 (2 samples) and HPV-31 (1 sample). Twenty-eight out of 174 samples were positive for p16, predominantly in HPV-positive tissues (There is a significant burden of HPV-associated HNSCC in Bangladesh, particularly in the oropharynx but also in oral and laryngeal cancers. Whilst a combination of PCR-based DNA detection and p16 IHC is useful, the latter has excellent specificity, acceptable sensitivity and good predictive value for carriage of HPV in this population and should be used for prognostic evaluation and treatment planning of all HNSCC patients in South Asia, as in the Western world. Head and neck cancer (HNC) is a major health problem worldwide, with an annual incidence of approximately 600,000 cases and close to 300,000 deaths, mostly in less developed countries GLOBOCAN 2012) [012 [2]. Approximately one-third of the total HNC cases in the world have been shown to be associated with high-risk HPV infection, but wide geographic variation exists , 11.the leading cancer in males and 3rd most common in females in India  and p16 IHC [61/248 (28%)] . A studyThe overall worldwide prevalence of HPV in HNSCC averages approximately 30% with wide variation depending on geographical location and tumour sites . Few stun = 34), included samples only from the oral cavity and found just 3% of HPV positive cases [To our knowledge, there is only one published study from Bangladesh describing the prevalence of HPV in HNSCC, using conventional PCR methods. This had a small sample size (ve cases . Possiblp = 0.025). This accords with the mean age reported from Australia (55.2 years), North America (58 years) and Europe (< 60 years), although the mean age of our Bangladeshi HPV-positive HNSCC patients was slightly higher than reported from other South Asian countries (52.8 years), especially India [HPV16 is the most commonly detected HPV type, being present in 90% of cases of HPV-positive HNSCCs worldwide . Our datly India .In our study, HPV-positive cases tended to have high expression of p16, an association which was statistically significant: however, our HPV-positive cases had low to moderate expression of Cyclin D1, pRb and p53 proteins, none of which were statistically significant. The negative correlation between HPV status and Cyclin D & pRb perhaps indicates that malignancy may not need both aberrant Cyclin D1 and pRb pathways. Our findings in this respect match previously reported studies from China, India and Australia , 53, 54.Several recent studies have demonstrated the prognostic value of cell cycle markers in HPV-positive HNSCC patients. A recent cohort study suggests that high p16 expression is correlated with better survival: p16 positive and HPV-positive cases had a 2 year disease free survival of 86.2%, (95% CI 79–91.1) compared to p16 negative and HPV–negative cases of only 44.2%, (CI 30.2–58.1) . Some stA comprehensive study of 226 patients from Australia suggests a strong association between HPV positivity and downregulation or absent expression of Cyclin D1.In an HPV-positive tumour group, Cyclin D1-positive cancers had 8 fold-increased risk of poor prognosis compared to Cyclin D1-negative cancers with 3.3 years overall survival. However, the effect of Cyclin D1 was small in HPV-negative HNSCC . A stronThere are several limitations to the present study. We have a modest cohort size, with small numbers in some anatomical subsites. Use of FFPE archival samples resulted in poor quality of DNA in some samples, which had to be excluded. Nevertheless, most amplified the L1 target satisfactorily. Expression of E6/E7 mRNA could not be explored in FFPE tissues, as fresh or frozen tissues were not available. We also have limited information on tobacco, areca nut and alcohol habits, and on the sexual lives of our cases. Most importantly no treatment and patient outcome information was available to us.This is the first comprehensive study from Bangladesh and one of the first studies from South Asia to use a combination of detection methods for HPV and their interrelationship with cell cycle markers of putative prognostic value. Nevertheless our cases were derived from a single pathology laboratory, which may limit generalizability. However, this is the largest public laboratory in the nation and receives a wide range of patients and tissue samples from all corners of Bangladesh.Our data show that HPV is associated with, and probably responsible for ~21% of HNSCC in Bangladesh. Because it is now well known that such cases respond comparatively well to treatment, routine assessment of HPV status in HNSCC should be mandated. We strongly recommend the use of ICD-10 for proper site-specific classification of cases. We urge the Bangladesh Government to mandate Tumour Boards and perform longitudinal studies on HNC to confirm the importance of HPV in treatment planning. We recommend the use of both p16 IHC and PCR-based detection of virus, though p16 alone is a useful surrogate marker. Routine use of IHC for the status of p53, pRb and cyclin D1, does not seem to be indicated.


In contrast, parents who had never heard of the vaccine , those who were willing to pay more than HK$2,000 for the vaccine , or had a preference to access it through a private clinic  were significantly less likely to allow their daughter to join the program. Delivery of the HPV vaccine with high uptake rate in a school setting is feasible in Hong Kong. Engaging key stakeholders including school administrators, teachers and community physicians, and providing relevant information on safety and vaccine effectiveness to parents were important to the success of the program.The present study is aimed at assessing the feasibility of delivering the HPV (human papillomavirus) vaccine to girls through a school-based program in Hong Kong, as well as to examine the facilitators and barriers associated with their participation. We approached 1,229 eligible girls aged 9 to 14 at eight schools in Hong Kong to join the program and then delivered the bivalent HPV vaccine at 0 and 6 months over the course of one school year. The students and their parents completed separate questionnaires to indicate their decision on whether or not to participate, and to assess their knowledge of cervical cancer and the HPV vaccine. The overall vaccine uptake was 81.4%  for the first dose and 80.8%  for the second dose. Parents and students were given separate questionnaires and asked whether or not they would like to participate in the vaccination program. 87.1%  of parents and 84.9%  of students indicated that they would join the program. The reasons associated with parents’ decision not to vaccinate their daughters primarily included concerns around side effects and safety. Multivariate regression analysis showed that parents who thought that the vaccine would protect their daughter from getting cervical cancer (OR = 3.16, 95% CI = 1.39–7.15,  Vaccination against human papillomavirus (HPV) has the potential to drastically reduce the incidence of cervical cancer given that approximately 70% of cervical cancers are caused by HPV-16 or HPV-18 NACI, 2015). Vaccination is especially important in regions such as Hong Kong where a comprehensive cervical screening program was conceived in 2004 but never effectively implemented to this date. In the most recent survey conducted by the Department of Health of Hong Kong 015. Vacc. A schooOur methodology relies on the health belief model and theories –8, whichA series of studies conducted among school-aged Chinese girls in Hong Kong showed that the factors associated with the intention to be vaccinated included perceived severity of HPV infection, safety and efficacy of the vaccine, and recommendation by general practitioners or significant others , 10. In The main objective of this study was to determine whether a school-based program for HPV vaccination was feasible in Hong Kong, based on uptake rate among a sample of adolescent, female students. Additionally, we sought to better understand reasons that contribute to both students’ and parents’ decision to participate in a school-based HPV vaccination program. Finally, recommendations were made based on our findings for other schools, administrators, charities and policymakers who may be considering implementation of HPV vaccination programs in Hong Kong.The study was part of a school-based HPV vaccination and intervention program conducted in Hong Kong between May 2015 and July 2016. The participants were female students aged nine to 14 and enrolled in primary grades four to six and secondary grades one to two. Invitations were sent to public and government-funded schools in Tuen Mun and Yuen Long, two districts in New Territory West. These schools previously participated in other health promotion programs organized by the Centre for Health Education and Health Promotion at The Chinese University of Hong Kong (CUHK). The inclusion criteria were: (1) girls between the ages of 9 to 14; and (2) the ability to read and write in Chinese (both students and parents) 3) girls who had not been previously vaccinated for HPV. The program included an education component and surveys that were distributed to both parents and students. The surveys were based on the Health Belief Model  and the  girls whWritten consent from parents was obtained as a pre-requisite to their daughter joining the vaccination program. Ethics approval for this study was obtained from the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (2014-493T).Eight schools (six primary schools and two secondary schools) agreed to participate in the program. A letter of invitation was sent to the parents of all eligible students to inform them about the school-based HPV vaccination program and the research nature of the program. Written parental consent was obtained for girls to join the program as well as to complete the questionnaire.After obtaining parental consent, students were asked to watch two educational videos and provided with educational materials to take home to their parents. The educational materials and videos were also posted on a website and made available online for parents to view. Students and their parents were asked to complete a self-administered questionnaire that was developed based on previous studies , 12, 13 A team of research staff, nurses, physicians and volunteers carried out delivery of the school-based vaccination program. A research coordinator and nurse worked with the schools to respond to any questions that parents had about the vaccine. Under the supervision of a primary care doctor, a nurse administered the first dose of the HPV vaccine to all eligible students. Medical staff assessed any students who reported feeling unwell after vaccination and no adverse outcomes were reported. After the first dose, students were given a vaccination card and reminded that they would receive the second dose six months later. Research staff coordinated the delivery of the second vaccination dose with the schools. The second vaccination doses were also provided on-site at the schools and delivered by a nurse under the supervision of a doctor. Students who were unable to obtain their second vaccination dose at school were able to obtain it through a designated family physician in the community. Prior to launch, the program was piloted with four schools in the same district to finalize logistics.Two sets of self-administered questionnaires were developed with reference to previous studies in Hong Kong. These were used in this study to obtain information from parents and students. The questionnaires were designed to measure factors that might be associated with the uptake of HPV vaccine, including awareness and knowledge of cervical cancer, HPV infection and HPV vaccine, perceived impact of the vaccine, attitudes and beliefs about cervical cancer prevention, as well as other socio-demographic information.There were altogether 26 items in the parents’ questionnaire. First, parents were asked to indicate whether their daughter had been given permission to join the school-based HPV vaccination program. Next, it explored reasons for their decisions, such as whether or not they thought the vaccine was safe, whether the vaccine was recommended to them by a doctor, and if they feared possible side effects. Parents were then asked to respond to statements on cervical cancer, such as, “cervical cancer is caused by viral infection” and “females with multiple sex partners have a higher risk of getting cervical cancer.” They had the option to answer “yes”, “no” or “don’t know”. Each correct answer received 1 point while an incorrect answer or “don’t know” received 0 points. Other questions included knowledge of the HPV vaccine such as whether it was able to “prevent all sexually transmitted infections” and if it would “not affect growth”. Parents’ health seeking behaviors and demographic information including age, education level, marital status, and monthly family income were also collected.There were 15 questions in the students’ questionnaire. Similar to the parents’ version, they were first asked to indicate whether they had joined the program and then the reasons impacting their decision. There were questions tapping into their knowledge of cervical cancer, e.g. “cervical cancer is one of the common cancers found in women in Hong Kong” and the HPV vaccine, such as “girls should not receive HPV vaccine before their first menstruation”, as well as their information sources for this knowledge. Both questionnaires were refined by experts in public health and infectious diseases and then tested for face and content validation in the four pilot schools.chi-square test. In addition, responses of parents and students who chose to join or not join the program were compared using chi-square test or Fisher’s exact test. Univariate and multivariate logistic regressions were performed to investigate whether knowledge, attitudes and perception of cervical cancer and HPV vaccine were associated with participation in the program. Variables with significant level less than 0.2 in the univariate regression analyses were included in the multivariate logistic regressions.The denominator for calculating the uptake rate was the number of eligible girls (between the ages of 9 to 14) in participating schools. The difference between the uptake rate among primary and secondary schools was calculated using 2 = 16.93, p>0.001). No adverse events were reported in the program. There were 1,276 girls enrolled at the eight participating schools, of which only 1,229 girls met the inclusion criteria. In total, 1,000 girls received the first dose. Average uptake among the schools was 81.4%. 993 girls received the second dose and average uptake was 80.8%. Of the 1,000 girls who received the first dose, 80.2% came from the six primary schools whereas the remaining 19.8% were from the two secondary schools. The uptake rate was 83.6% among primary school girls and 70.4% among secondary school girls. Chi-square test showed that there was a significant difference in the uptake rate between primary and secondary school girls . The demographics of the parents who completed the questionnaire are summarized in Of the parents who submitted the questionnaire, 1,010 provided consent for their daughters to be vaccinated through the school-based program. Twenty-seven respondents had previously vaccinated their daughters elsewhere. Among those who chose to vaccinate their daughters, over 70% indicated that they believed the vaccine would provide effective prevention of cervical cancer and reported this factor as influential in their decision-making. In addition, over 71% indicated that the safety of the vaccine was an influential or extremely influential factor. Furthermore, not having to pay for the vaccine was also reported as an influential factor with approximately 62% of parents reporting it as being an influential or extremely influential factor in their decision-making .One hundred and twenty-three parents or guardians decided not to vaccinate their daughters for HPV. Among those who declined, 18.1% and 52.0% indicated that fear of side effects was an extremely influential or influential factor in their decision-making, and approximately 46% reported that they did not think the vaccine was effective. Few parents indicated that they were worried about the perceived perception of promiscuity  as a barrier. Over half (64.5%) of all parents indicated that they would be willing to pay less than US$125 for the entire course of the HPV vaccination.The 1,276 girls invited to take part in the program were also asked to complete a survey. A total of 1,147 girls (89.9%) returned the completed questionnaire, with 895 responses from primary school girls and 252 from secondary school girls. There were 974 students who indicated that they would join the program, specific factors that students rated as highly influential in their decision to be vaccinated included the belief that the vaccine could effectively prevent cervical cancer (32.1%), that it was safe (26.1%), and that it was free of cost (23.8%). One hundred and thirty-five girls reported they did not participate in the vaccination program. Of those, 15.9% reported that objection from their parents or a family member was a significant factor that influenced whether or not they would join the program. Fear of possible side effects was also a consideration with 10.5% and 33.9% indicating that it was an extremely influential or influential factor for them respectively. It is also interesting to note that about 33% of girls rated fear of pain to be an influential factor for not participating in the program .The responses from parents who allowed their daughters to join the HPV vaccination program and those who did not allow their daughters to join the program are compared in p < .004), knowledge that the HPV vaccine does not affect growth , perception that the vaccine could protect their daughter , having a doctor recommended the vaccine , and preference for their daughter to receive the vaccine at school  were factors associated with the uptake of HPV vaccine. Conversely, parents who had never heard of the HPV vaccine  and who had a regular family doctor  were associated with a lower uptake of HPV vaccine at school. In multivariate regression analysis, the perception that the HPV vaccine could protect their daughter from getting cervical cancer showed a 3.16 odds  of joining the HPV vaccination program. In addition, having had doctor’s recommendation showed a 4.54 odds for joining the program , whereas parents of girls who had never heard of HPV vaccine , parents who were willing to pay more than HK$2000 for the vaccine  and parents who preferred for their daughter to receive the vaccine at a private clinic  were significantly to have lower uptake.Tables Students’ responses were compared based on whether or not they joined the HPV vaccination program and shown in p = .048), obtaining information about the HPV vaccine from their parents , and having correct knowledge of the HPV vaccine  were associated with the uptake of the HPV vaccine. In particular, students who indicated that the HPV vaccine could prevent cervical cancer had an odds ratio of 4.23  likelihood of participating in the program. Multivariate logistic regression (p = .005) and knowledge that the HPV vaccine could prevent cervical cancer  were independent factors significantly associated with increased participation in the program. However, students who perceived that having multiple sex partners was a risk factor for cervical cancer had a lower likelihood of joining the program .Univariate logistic regressions were performed to explore the association between students’ knowledge and participation in the vaccination program . Resultsgression  revealedThis is one of the first studies to investigate the feasibility and acceptability of a subsidized school-based HPV vaccination program in a region without a universal vaccination program. The findings showed that it is possible to deliver a 2-dose HPV vaccination schedule to girls aged between 9 and 14 years in a school setting with an overall uptake rate as high as 80% across the schools.Prior studies have shown that the uptake rate of the HPV vaccine among females in Hong Kong is low, 7–9% among school-aged girls and 9.7% for university students, as compared to 70% in countries with publicly funded, school-based vaccination programs. The high uptake rates are consistent with results from a study conducted in Hong Kong  and compFactors influencing familial decision regarding HPV vaccination include attitudes and recommendations of healthcare providers, beliefs and attitudes of parents and adolescents, peer norms, and communication between parents and adolescents on sexual issues . In our On the other hand, the views and emotions of adolescents should also be considered. Some students who did not join the program reported that fear of pain from injection was an extremely influential factor to them, which in turn may affect their parents’ decision. It is possible that by conducting the vaccination in a familiar environment, such as in a school setting with their friends and teachers, could reduce fears around vaccination. During vaccination days, we observed many students supporting one another in spite of the fact that many of them were afraid of pain from injection. In addition, regression analysis also showed that students who received information about the HPV vaccine from their parents and perceived that the vaccine could prevent cervical cancer were more likely to participate in the program. Girls with accurate knowledge about cervical cancer, e.g. starting sex at early age as risk factor were likely to participate in the program but knowledge that having multiple sex partners was a risk factor had lower likelihood to join. It is possible that those students did not believe that the risk factor was relevant to them or HPV vaccine might not be protective for those with multiple risk factors.In places where the HPV vaccination is not subsidized by government, cost has been identified as a crucial factor in many studies , 19. TheEducation and information about cervical cancer and HPV prevention should be tailored around the specific needs of the intended audience, in this case, parents and adolescent female students. For students, having correct knowledge about cervical cancer and HPV prevention did not often translate to participation in the program. Materials should highlight the importance of having the vaccine and encourage students to speak to their parents about it. As an example, students who knew that that having multiple sex partners was a risk factor were less likely to join the program. Educational materials should focus on concerns or information that is more relevant to them.As students require parental consent to receive the vaccine, providing accurate information and knowledge to parents is crucial. Educational materials for parents should contain clear messages around the safety and effectiveness of the vaccine and address concerns around side effects. During the pilot phase of the project, many parents contacted program staff and teachers with questions around safety of the vaccine. To address their concerns, educational materials were revised to specifically address common questions and misconceptions. In addition, a phone line was established so that parents could call and speak to a healthcare provider directly about their questions. Teachers also received education and informational materials in advance so that they could accurately respond to questions from parents.A school-based HPV vaccination delivery strategy could be particularly successful in supporting families with lower socio-economic status and result in higher uptake. In October 2016, the Hong Kong Government launched a 3-year HK$98.75M pilot program offering free HPV vaccines to low income families that is expected to benefit 31,000 girls and be delivered through publicly-funded clinics. Our program cost HK$1.4M and benefited over 1,200 girls in both the pilot and final phases of the program. Thus, it represents a lower cost per client when compared to the government pilot program. It is recommended that government also consider piloting larger scale school-based programs in low income areas to assess the uptake rate and effectiveness among different models of delivery.There are several limitations to this study. First, this is a cross-sectional study which only identifies associating factors rather than causation. Secondly, there may be self-selection bias as the participation of schools, students and parents was voluntary, which could affect the representativeness of our study population. Our study population consisted of schools with higher proportion of families from lower socio-economic income levels and results should be interpreted with caution. Nevertheless, our study removed the financial barriers by providing the vaccine free of cost, which allowed us to develop a clearer understanding of the other facilitators and barriers to vaccination among students and parents.Approximately half of the cervical cancer cases globally are in Asia, but currently only a few large-scale HPV vaccination programs have been implemented in the region, such as Macau and South Korea. In places without such programs like Hong Kong, Singapore, or Taiwan, the uptake rate of conventional vaccine-preventable diseases, such as measles, is high while the uptake of HPV vaccine remains low. The current study provides empirical data that supports the feasibility of a school-based HPV vaccination program as a vaccine delivery model that can significantly improve uptake in Hong Kong.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


The human papillomavirus (HPV) is a major public health concern affectingboth females and males. HPV is associated with cervical, anal, head and neckcancers. About 99% of all cervical cancers are related to HPV. HPVvaccines, Gardasil, Cervarix, and Gardasil 9 are used in the primary preventionof HPV related cancers. Gardasil and Gardasil 9 are available for use in bothfemales and males ages 9 to 26, while Cervarix is available for females ages 9to 25. Gardasil 9 was approved by the FDA for prevention against additional HPVtypes. Despite the availability of this preventative measure against cervicalcancer, the rate of HPV vaccination in the United States remains lower than thatof other industrialized nations. The purpose of this study is to elucidatemechanisms to help increase the HPV vaccination rate by using education as atool; by simplifying the president report so that lay person can understand theinformation presented in the report. Through the quantitative examination of thedata from the states with the lowest and highest vaccination rates, using SPSSstatistical analysis; we analyzed several factors involved with the low uptakeof the vaccines. The results collected show that socioeconomic status,misconceptions about HPV, and misconceptions about the safety of the vaccineswere identified as possible obstacles to the effective uptake of HPVvaccinations. The proposals made by the President’s Cancer Panel toaccelerate the uptake of vaccines include, increasing coverage of the vaccinesthrough government-sponsored programs, and the Affordable Care Act; increasingaccessibility to vaccines through pharmacies, schools, and clinics; anddisseminating more information on HPV to healthcare providers, parents,caregivers, and patients. Allowing greater accessibility to the vaccines for allpopulations regardless of income, education, and eliminating misconceptions ofthe vaccines would play a significant role in eliminating cancer. The Human papillomavirus (HPV) infection is a major public health concern.Approximately 80 million or one in four people are infected with this virus in theUnited States . HPVinfThe Human Papillomavirus (HPV) consists of a group of over 200 types ofdouble-stranded DNA viruses belonging to the Papillomaviridae family. Genitalhuman papillomavirus (HPV) is the most common sexually transmitted infection inthe United States. More than 40 types of HPV are typically transmitted throughsexual contact, and infect the anogential region . “High-rAnnual costs of cervical cancer screening and treatment of HPV-associatedconditions have been estimated at $8 billion U.S. dollars. In 2009,approximately 30,000 HPV-associated cancers were reported in the United States,with 39% prevalence in males ®, a quadrivalent HPV vaccine, was licensed by the Food andDrug Administration (FDA) in 2006 for use in females aged 9–26 years asprevention of cervical cancer and its precursors, vulvar and vaginal cancerprecursors, and anogential warts caused by HPV types 6, 11, 16 and 18[®(GlaxoSmithKline) for use in females aged 9–25 years for prevention ofcervical cancer and its precursors caused by HPV types 16 and 18. The FDAapproved Gardasil 9® , a 9-valent HPVvaccine for prevention against HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58in December 2014 Three HPV vaccines are licensed in the United States for prevention ofspecific HPV types and HPV-associated outcomes. Gardasil6 and 18. Three yHPV vaccines work by stimulating the body to produce antibodies thatrecognize and bind to the HPV virus, preventing it from infecting cells. Thecurrent HPV vaccines are made up of virus-like particles (VLPs) that have beenformed from HPV surface components using recombinant DNA technology. BecauseToday, there are three effective vaccines that prevent infection by thetwo most prevalent cancer-causing HPV types. However, in 2012, 33% ofadolescent females and 7% of adolescent males across the U.S. completedthe three-dose series . These l® or Gardasil®.The vaccination series can be started beginning at age 9 years. Vaccination isrecommended for female’s ages 13–26 who have not been vaccinatedpreviously or who have not completed the three-dose series.In the United States, rates of cervical infection with HPV types coveredby the vaccines fell by more than 50% among girls 14–19 years inthe four years following vaccine introduction. The decline was even moredramatic across the globe, as the prevalence of HPV infections covered by thevaccine among females under the age of 21 plummeting by as much as 90%.Although 56 million vaccine doses have been administered in the United States asof early 2013, The U.S. Advisory Committee on Immunization Practices (ACIP) andthe President’s Cancer Panel called for “urgency foraction” in accelerating the HPV vaccine uptake in the 2012–2013annual report to President Obama . ThepanIn 2007, the first full year after Gardasil was approved in the U.S.,about 25% of 13 to 17 years old girls received at least one HPV vaccinedose, with this rate being similar to the proportion that received otheradolescent vaccines during the first year they were recommended. In 2012,53.8% of 13 to 17-year-old girls had received the first HPV dose, with33.4% completing all three recommended doses.The panel’s report pleads for HPV vaccine uptake to be a publichealth priority and lists a plan of action by recruiting for help from thegovernment, providers, parents and adolescents. The panel recommends thefollowing four goals to help increase uptake of HPV vaccines in the UnitedStates  with hopes of having age-eligible adolescents fullyvaccinated (completion of the three-dose vaccine).The CDC cites in a recent report that missed clinical opportunities arethe most important reason why vaccine uptake remains low in the United States.Many vaccine-eligible adolescents do not receive HPV vaccines during visits withtheir healthcare providers. The report states that many times, adolescentsreceived other recommended vaccines yet did not receive the HPV vaccine. Factorscontributing to providers’ hesitancy include:Limited understanding of HPV-associated diseases and benefits of HPVvaccination, particularly for malesConcerns about safetyConcerns about inadequate reimbursement for vaccinesPersonal attitudes and beliefsDiscomfort talking to parents and adolescents about a topicrelated to sexual behaviorConcerns about parental resistancevs. youngeradolescentsPreference for vaccinating older Lack of time or incentives to educate parents and patients aboutHPV and HPV vaccinesLack of systems to remind providers to offer vaccines toage-eligible patientsSource: Rimer BK, Harper H, White ON. Accelerating HPV vaccine uptake:Urgency for Action to Prevent Cancer: A report to the President of the UnitedStates from the President’s Cancer Panel.To address provider concerns, the Panel listed strategies to increaseprovider education on the safety and efficacy of the vaccines. Additionally, thePanel recommends the CDC to develop, test, disseminate, and evaluate the impactof integrated, comprehensive communication strategies for physicians and otherhealth care professionals. In order to lead this effort, the Panel also suggeststhat more governmental funding be allocated to this cause. As the panelcampaigns for the increase in demand of the HPV vaccine, they also recommendthat healthcare payers be reimbursed adequately for HPV vaccination storage,administration and services.The panel solicits the help of healthcare organizations and practicesemphasizing the use of electronic health records (EHRs) and immunizationinformation systems (IIS) to avoid missed opportunities for the HPV vaccination.This increase in funding would cover the cost of increasing vaccine uptake inadolescent males and having this information published in the HealthcareEffectiveness Data and Information Sheet (HEDIS) . The HEDHPV has largely been associated with a disease ridden only to women.Recent findings have suggested that HPV may also lead to HPV-related cancers andgenital warts in men . These iIncreasing vaccination uptake in males is a key objective in reducingmissed clinical opportunities. Given that HPV infection can result in genitalwarts, precancerous and cancerous lesions in both males and females, the cancerpanel’s key objective in increasing vaccine uptake is increasing vaccineutilization in adolescent males, where vaccination rates remain the lowest. DespiteParents or other caregivers’ attitudes towards vaccines heavilyinfluences whether their children receive them. One study found that parentswere more likely to refuse HPV vaccines more than other recommended vaccines fora myriad of reasons. These reasons include, but are not limited to, vaccines notbeing needed for males, son or daughter not being sexually active, and safetyconcerns. Surveyed pediatricians and family practice physicians reported thatparents of young adolescents get upset when vaccinations against sexuallytransmitted infections are suggested for their children. Some parents expresstheir concerns that HPV vaccinations promote engaging in sexual activity. Toalleviate parental concerns and ease the conversation between providers andparents about HPV vaccine uptake, the panel suggests that the CDC implementcommunication strategies that focus on messages and modes of delivery that are“sensitive to cultural, literacy/health literacy, and languagedifferences of target populations”. The panel suggests that providers“resonate emotionally” with parents and caregivers, framing HPVvaccines as mechanisms of cancer prevention, while highlighting the safety andefficacy of these vaccines. They also suggest the importance of vaccinating bothmales and females as part of the adolescent vaccine initiative, while stressingthe importance of initiation and completion of the vaccination series. In orderto change the perception of HPV vaccines amongst parents and caregivers, thepanel plans to utilize websites, blogs, social and print media with recruitmentof vaccination proponents who are influential to target audiences to increasevaccine uptake. These efforts are believed to ease physician efforts to conveystrong recommendations regarding HPV vaccination.Although the American Academy of Pediatrics and American Academy ofFamily Physicians prefer that all adolescents receive primary care, includingvaccinations through primary care physicians, the panel recommends increasingthe range of venues to include more providers for HPV vaccination. U.S. Schoolsand pharmacies are among the top two venues suggested in the proposed expansionplan. Survey results indicate that HPV vaccination in alternative settings mayprove effective in increasing vaccine uptake in adolescent males, as many of themales surveyed had not had any recent health care visits.Reports claim that schools can educate adolescents and parents on theimportance of vaccination and can even provide a clinical environment toadminister HPV vaccines. School-based vaccination programs have been reported tobe successful in countries like Australia, the United Kingdom and Canada, whichthe panel suggests for adoption in the U.S. Pharmacies are highly accessible tomost people in the United States, with over 275,000 pharmacists operating out ofover 60,000 pharmacies nationwide. During the 2012–2013 flu season,pharmacists were influential in administering the flu vaccination as theyaccounted for 20 percent of vaccine received. The panel recommends thatpharmacists be allowed to administer vaccines to adolescents, as many of themare prohibited from administering HPV vaccines, especially to youngadolescents.vs. theU.S. may be attributed to the lack of cervical cancer education and screeningprograms. With data consistently showing the relationship between HPV types 16and 18 and the progression of cervical cancer, the panel strongly recommendsthat vaccine uptake increase globally. In a global perspective, high-resourcedareas such as Australia, the United Kingdom and Canada have astonishinglydifferent health care systems than that of the United States and have done wellin implementing HPV vaccination programs with rates as high as50–85%.HPV-associated cancers affect various populations worldwide, withcervical cancer being the most prevalent. The disparities in cervical cancerrates in countries such as Africa (15 times higher) Efforts to expand HPV vaccination to low- and middle-income countries(LMICs) are recognized through the efforts of the GAVI Alliance  and the Pan AmericanHealth Organization (PAHO) Revolving Fund. In 2013, GAVI launched a program thatprovides HPV vaccines to more than 180,000 girls in eight countries. GAVI isalso helping countries like Rwanda establish a HPV vaccination program . They havealso partnered with the Islamic Development Bank (IDB) to catalyze theimplementation of many vaccines (including HPV vaccinations) in IDB membercountries. The panel suggests that the U.S. continue to support GAVI and otherworld health organizations with a heavy emphasis on increasing vaccineintroduction and uptake in low-income countries.In addition to the four goals outlined in the Cancer Panel2012–2013 report, the panel also suggested funding“high-priority” research to advance prevention of HPV-associatedcancers. The panel hopes that the government would continue to fund HPV vaccineresearch that:Investigates more convenient dosing schedule for currentvaccines. With hopes of producing evidence that HPV can be treated withfewer vaccine doses.Develops next-generation vaccines that provide better protectionand/or are easier to store and administer. This would allow for changesin vaccine formulation that would increase coverage of HPV types andwould allow for easier storage and management.Explain the natural history of or pharyngeal HPV infections.Knowledge of the pathology of or pharyngeal HPV remains to beconclusive. Research would allow for the investigation and introductionof biomarkers that aid in the recognition and diagnosis of thismalignancy, which can possibly be eradicated through vaccination.Develop more effective ways to communicate about HPV-associateddiseases and HPV vaccines through mobile health interventions. Specificstrategies should be tested among different populations in accordancewith sociocultural factors in order to determine the best way to conveymessages regarding the HPV vaccination series.Determine how best to integrate HPV vaccination with cervicalcancer screening. This is of particular importance because studies showthat African-American females are not being screened for cervicalcancer, which may account for the high incidence and mortality rates inthis population.The Human Papillomavirus vaccination is a novel approach to combat theincidence of HPV and its associated health outcomes in both male and femalepopulations. The panel’s report investigates and addresses causes of lowvaccine uptake not only in the U.S., but globally . The lowvs. 2.2 per 100,000 (CW)). Previous researchers demonstratedthat Black women experience inadequate access to preventive services or do not usethese services for a variety of reasons, including but not limited to transportationissues, availability of insurance, level of income or education, fear and mistrustof the health care system [In the United States, the current cervical cancer mortality rate for Blackwomen (BW) is almost twice that of Caucasian Women (CW) e system . Inadeque system . An intee system ,26. Itie system ,28.Accelerating HPV vaccination uptake will require the help of parents, thenation’s public health providers and other public health professionals inorder to make this a high priority issue. There are 600,000 cases worldwide eachyear with an HPV infection that could have been prevented with the completion of theHPV vaccination series, therefore reflecting the great efforts to eradicate thisdisease in the future. The current vaccines; Cervarix, Gardasil, and Gardasil 9 havebeen scientifically proven to be effective in decreasing the amount of disparitiesfound in underrepresented areas. With other countries achieving success in HPVvaccinations, it is proven that it can be done. Incorporating the FOUR main goalsfrom the presidents cancer panel; reducing missed clinical opportunities torecommend and administer vaccines, increase parents, caregivers, and adolescentsacceptance of HPV vaccines, maximize access to HPV vaccination services, andpromoting global HPV vaccine uptake we are a step closer to eliminating Humanpapillomavirus which in turn will aid in eliminating cancer disparities.


OPSCC) should be divided into subsites when evaluating the presence of human papillomavirus (HPV) and prognosis. More specifically, tonsillar squamous cell carcinoma (TSCC) and base of tongue squamous cell carcinoma (BOTSCC) have much higher HPV prevalence compared to other OPSCC. Moreover, patients with HPV positive (HPV+) TSCC and BOTSCC have a better prognosis as compared to patients with HPV negative (HPV−) corresponding tumors, while the prognostic role of HPV in other OPSCC is unclear. Furthermore, in a recent report from Denmark, TSCC was further subclassified into specified TSCC (STSCC) and nonspecified TSCC (NSTSCC), with HPV significantly more prevalent in STSCC. In this study, the histopathological influence of HPV prevalence and survival in TSCC was analyzed in a TSCC cohort with known HPV status, of patients diagnosed 1970–2002 in Stockholm. In total, 139 TSCC biopsies with both tumor and adjacent normal tissue were separated into STSCC and NSTSCC. HPV was significantly more commonly found in STSCC than in NSTSCC. Patients with HPV+ STSCC had a better disease‐specific and overall survival as compared to patients with HPV+ NSTSCC, but no survival differences were observed in patients with HPV− STSCC and NSTCC. These findings confirm previous reports and suggest that TSCC subsite may also be of relevance for clinical outcome and should be further followed up in future studies.Current data advocate that oropharyngeal squamous cell carcinoma ( It is only a decade ago since the International Agency of Research on Cancer (IARC) declared a casual role of human papillomavirus (HPV) in the etiology of cancer of the oropharynx and the tonsil n = 139) were separated into STSCC  and NSTSCC  based on the histomorphological context according to Garnaes et al., with the exception of including only tumor specimens containing both tumor tissue and normal tissue In total, 203 patients diagnosed with TSCC ICD‐7 145.0) between 1970 and 2002 together with clinical data and HPV status (presence of HPV DNA tested with PCR) were obtained from previous publications  145.0 beStatistical analyses were performed in STATA  and SPSS . The Chi‐square test was used to compare categorical data. Survival was measured in months from the date of diagnosis until a defined event or until 5 years from diagnosis. An event was defined as death of any cause  or death with disease . Patients who died without a documented TSCC were censored at the death date in DSS. All patients were censored after 5 years (60 months). Only patients treated with the intent to cure were included in the survival analysis, as previously described P < 0.0001) . In a multivariate analysis including tumor stage, tumor differentiation and patient age upon diagnosis, histomorphological subclassification was still correlated with survival in patients with HPV+ TSCC. The unadjusted and adjusted HR for DSS were 4.4  and 7.3 , respectively. Corresponding HR for OS were 2.3  and 1.9 , respectively.Moreover, patients with HPV+ STCC had a significantly better 5‐year DSS and OS as compared to patients with HPV+ NSTSCC Fig. A and B. No significant differences in survival between STCC and NSTSCC were observed in patients with HPV− TSCC Fig. C and D. In this brief report, HPV+ status was significantly more frequently associated with STSCC as compared to NSTSCC, and moreover, patients with HPV+ STSCC had a significantly better prognosis compared to those with HPV+ NSTSCC. In contrast, HPV− status was significantly more often found in NSTSCC, but the histomorphological subtype of HPV− TSCC (NSTSCC or STSCC) did not affect patients’ prognosis.The relation of HPV status and TSCC subsite was clearly in agreement with the study by Garnaes et al. where a similar relationship was described in a Danish cohort Interestingly, patients with HPV+ STSCC had a better DSS and OS as compared to HPV+ NSTSCC. These data are also in line with previous data, in which HPV only had a prognostic impact in tumors arising in tissues harboring crypts There are recognizable limitations in this study. A major limitation was that for this cohort we only had access to hematoxylin‐eosin stained slides; moreover, no tumor material was available for p16 IHC staining. However, in cohorts partly overlapping with the present one, we have reported that p16 overexpression was highly concordant and significantly associated with HPV+ DNA status Taking these limitations into account, our data still support the data of Garnaes et al. In conclusion, the present study shows that HPV+ status is mainly observed in STSCC as compared to NSTSCC and vice versa. Moreover, HPV+ status only had a positive survival value in the context of STSCC and not in NSTSCC. Together these data suggest that the context of the tumor is essential for evaluating HPV prevalence and tumor prognostication and that the concept of OPSCC is too broad when separating patients with HPV+ and HPV− tumours.No conflict of interest to declare.Table S1. Total patient cohort with included and excluded cases and their characteristics, obtained from previous publications Click here for additional data file.


Among the nearly 21 million military veterans living in the United States, 64.0% of women and 76.1% of men are overweight or obese, higher rates than in the civilian population (56.9% of women and 69.9% of men). Attributes of the residential environment are linked to obesity. The objective of this study was to characterize the residential environments of the US veteran population with respect to availability of food and recreational venues.We used American Community Survey data to determine the concentration of veterans (the percentage of veterans among the adult population) in all continental US census tracts in 2013, and we used proprietary data to construct measures of availability of food and recreational venues per census tract. Using descriptive statistics and ordinary least-squares regression, we examined associations between the concentration of veterans per census tract and those residential environmental features.In census tracts with high concentrations of veterans, residents had, on average, 0.5  supermarkets within a 1-mile radius, while residents in census tracts with low concentrations of veterans had 3.2  supermarkets. Patterns were similar for grocery and convenience stores, fast food restaurants, parks, and commercial fitness facilities. In adjusted analyses controlling for census-tract–level covariates, veteran concentration remained strongly negatively associated with availability of those food and recreational venues. In nonmetropolitan tracts, adjusted associations were greatly attenuated and even positive.Where veterans live is strongly associated with availability of food outlets providing healthy  foods and with recreational venues, raising questions about the contributions of veterans’ residential environments to their high obesity rates. Additional research is needed to address those questions. Among the nearly 21 million military veterans living in the United States, approximately 64.0% of women and 76.1% of men are overweight or obese  and as barriers (convenience stores and fast food restaurants) to the adoption and maintenance of healthy behaviors. The sample comprised all 2,013 census tracts in the continental United States with an adult population greater than zero.Concentration of veterans. We used 5-year estimates from the American Community Survey 2009–2013, including data on self-reported veteran status, to determine the percentage of veterans among the adult population in each census tract  and Dun & Bradstreet (fast food restaurants) databases. Data on the locations of recreational venues were obtained from InfoUSA  and Navteq and TeleAtlas (parks) databases. Details on data collection are described elsewhere , economic characteristics (median household income), and geographic characteristics (census division) (ivision) . As a meivision)  and clasivision)  to calcu2 tests for categorical variables. We also examined location quotients graphically. Finally, to gain further insight into relationships between veterans’ residential locations and their potential access to dietary and physical activity–related resources and barriers, we fit ordinary least-squares regression models of availability measures as a function of veteran concentration while controlling for census-tract–level covariates. We estimated 2 models for 2 groups of tracts: all census tracts and census tracts located in nonmetropolitan areas. Model 1 included only the sociodemographic, economic, and geographic characteristics of census tracts. Model 2 added concentration of veterans and a measure of distance to the nearest military installation. Stata version 13.1 (StataCorp LLC) was used for all analyses.We compared the availability of food and recreational venues in census tracts grouped according to quartiles of concentration of veterans, using means, standard deviations (SDs), and interquartile ranges (IQRs); we used 1-way analysis of variance for continuous variables and χThe mean concentration of veterans, by census tract, ranged from 0% to 100%; the mean concentration of veterans among all tracts was 9.1%  . In censP for all <.001).In census tracts with high concentrations of veterans, residents had 0.5  supermarkets on average within a 1-mile radius of their residence, while in census tracts with low concentrations of veterans, residents had 3.2 supermarkets  . When viAmong all census tracts, location quotients in census tracts with high concentrations of veterans were less than 1.0 for all food outlets: for example, 0.12 for grocery stores, 0.50 for chain supermarkets, and 0.51 for chain fast food restaurants . LocatioP < .001; mid-high, β = −0.205, P < .001; high, β = −0.226, P < .001). Furthermore, associations of sociodemographic, economic, and geographic characteristics with supermarket availability changed little when variables indicating veteran concentration were added to the model.In regression analyses of the number of all supermarkets (chain and nonchain combined) within 1 mile of residents , Model 1P < .001; mid-high, β = 0.048, P < .001; high, β = 0.028, P = .04), but coefficients were smaller than those from metropolitan and nonmetropolitan areas combined. Results for all food and recreational variables were similar in direction and significance to those for supermarkets in both metropolitan and nonmetropolitan areas (Appendix Table).In nonmetropolitan tracts , adjusteThe objective of our study was to characterize the residential environments of the US veteran population with respect to availability of food and recreational venues. We found that areas with high concentrations of veterans were different from areas with low concentrations of veterans. The greater the percentage veterans in an area, the fewer the supermarkets, grocery stores, convenience stores, fast food restaurants, fitness facilities, and parks nearby. We found this relationship in both metropolitan and nonmetropolitan areas. Likewise, the higher the concentration of veterans in a census tract, the fewer the food and recreational venues when compared with the average tract in the continental United States. The associations between the concentration of veterans and the availability of food and recreational venues remained even after we adjusted for a broad set of sociodemographic, economic, and geographic factors with which veteran concentration is correlated and that are themselves known to be associated with residential environmental attributes.This pattern means that a large percentage (89%) of the veteran population (about 19 million veterans) lives in areas where food outlets that provide variety in healthy food options at the most affordable prices  and places to engage in recreational physical activity are less available than they are for the average adult living in the continental United States. Areas of high concentrations of veterans also have few outlets that predominately offer energy-dense, nutrient-poor foods . Although the dearth of convenience stores and fast food restaurants in areas of high concentration of veterans reduces residents’ access to unhealthy foods, supermarkets and grocery stores are still important to facilitate access to healthy foods.Our findings have implications for veterans’ health. A large body of evidence documents the vulnerability of many veterans to poor health outcomes after military service. After discharge, weight increases, and physical activity declines –22. SomeConsidering how the environment may contribute to health declines is important. A better understanding of veterans’ residential environments can inform efforts to protect and improve their health. The US Department of Veterans Affairs (VA) has invested heavily in finding ways to help veterans manage their weight. It operates a large-scale nationwide behavioral weight-management program designed to help reduce obesity rates among veterans (25). Information on veterans’ geographic access to food and recreational venues could be incorporated into individual self-management plans and educational content. Successful partnerships between the VA, other federal agencies, and communities such as those targeting homelessness among veterans have demonstrated policy makers’ acknowledgment of and commitment to addressing a broader set of influences on veterans’ health and well-being than is within the reach of individually focused health care services and other benefits provided through the VA. Access to healthy foods at affordable prices and to places for physical activity may be among those influences.This study has several limitations. Commercial data sources for retail food outlets are prone to error. We carefully cleaned and processed the data to maximize its quality . It is rOur study suggests that where veterans live is strongly associated with availability of food outlets that provide healthy  foods and recreational venues close to home. Given recent recognition of high rates of obesity among veterans and evidence of a link between availability of healthy foods and fast foods and diet and a link between the availability of recreational venues and physical activity, these environmental variations raise questions about their potential effect on veterans’ health. Additional research is needed to address those questions. Furthermore, the findings highlight the potential need for programs aimed at promoting veterans’ health to address or take into account their residential environments.


A novel human papillomavirus (HPV ICB1) was fully characterized from a skin swab by using a sensitive degenerate PCR protocol combined with next-generation sequencing. The L1 open reading frame of HPV ICB1 shares 70.54% nucleotide homology with its closest relative, HPV164, and thus constitutes a novel human gammapapillomavirus. Human papillomaviruses (HPVs) are nonenveloped double-stranded DNA viruses approximately 8 kb in size with an epithelial tropism. HPVs colonize normal skin and mucosa and can induce cutaneous and mucosal lesions –4. The L–Degenerate PCR primers  were useThe complete viral genome of a new HPV type , with a G+C content of 38.09%, was obtained by DNA amplification using multiply primed rolling circle amplification (RCA) according to the manufacturer’s instructions . RCA was combined with long-range PCR  performed with outward-directed primers specific for the putative new HPV . An amplicon of approximately 8 kb in size was cloned in the pCR-XL-TOPO vector using the TOPO-XL PCR cloning kit  and sequenced by a primer walking strategy . The sequence was validated using a proofreading polymerase, followed by Sanger sequencing.6-GGT). Like all HPV types, E6 and E7 have zinc-binding domains [CxxC(x)29CxxC] containing two and one zinc-binding domains, respectively. In addition, E7 contains an LxSxE retinoblastoma (RB)-binding motif (HPV L1 sequences that share less than 90% sequence similarity to the closest papillomavirus type are traditionally considered to be distinct HPV types , 8. The ng motif . Analysing motif . Moreoveng motif , 11 haveMF356498.The complete genome sequence of HPV ICB1 is available in GenBank under the accession number


HPV is the major causative agent for cervical cancer. Study on the risk of cervical cancer associated with different hr-HPV genotypes would be useful for disease management and new vaccine strategy. With limited reports available, the present study aimed to investigate the pattern of HPV genotypes coinfections and risk of cervical carcinoma associated with them in Indian population. 15 HPV genotypes were detected by E6/E7 multiplex nested type-specific PCR in the HPV-positive cervical samples of 172 cervical cancer cases and 174 subjects with normal cytology. Association between the genotypes and cervical cancer was estimated by calculating the Odds ratio and 95% confidence interval. Risk of cervical carcinoma was associated with multiple genotypes excluding HPV16 , multiple genotypes excluding HPV18 , multiple genotypes of α9 species, and multiple genotypes of α7 species . Genotypes not targeted by quadrivalent vaccine types  conferred 2.94 fold higher risk of cervical carcinoma. Cases those coinfected with phylogenetically related genotypes  p = .17) were at 2.9 fold higher risk of invasive cervical carcinoma than those infected with other genotypes although it is not statistically significant. Whereas phylogenetically unrelated genotypes coinfection is negatively associated with cervical carcinoma (OR = .44 95% CI (.244-.8) p = .007) and it is statistically significant.Genotypes not targeted by 9-valent vaccines  associated with lesser risk of cervical carcinoma as compared to other genotypes. Subjects infected with any HPV genotype/genotypes excluding HPV16 in association with HPV 18  were at 4.1 fold higher risk of developing invasive cervical carcinoma.In conclusion, the risk of development of cervical cancer is genotype specific and might be associated with type-specific interactions between the genotypes in multiple infections. HPV infection is the major cause of cervical cancer. HPV 16 and 18 are the most prevalent carcinogenic hr-HPV types associated with 70% of the high-grade cervical lesion and targeted by the current vaccines such as Gardasil and Cervarix .The important genital HPV genotypes belong to the genera of alpha(α) papillomaviruses. The members of α -9 species show distinct genomic sequences and similar pathological properties resembling with HPV16 genotype due to their phylogenetically relatedness. α-9 includes HPV types 16,31, 33, 35, 52, 58 and 67. Viruses of α- 7 species resembles with HPV 18 which includes HPV types 39, 45, 59, 68, 70 and 85 along with HPV 18 [Cross-protection of the bivalent vaccine (Cervarix) and the quadrivalent vaccine against HPV 31, 33 and 45 types have been reported . TheoretCervical cancer is often associated with multiple HPV genotype infection. Thus it is considered that co-infection may be a risk factor that drives carcinogenesis , 16.But Suspected cases of cervical cancer attending OPD of Acharya Hari Hara Regional Cancer Center and SCB medical college, Cuttack, Odisha between January 2014 and December 2015 have been enrolled in the study. Married women, above 18 years showing any of the symptoms like abnormal vaginal bleeding/discharge, pain during coitus, lower abdominal pain and clinician suspicion of cervical malignancy were included in the study after clinical examination by a gynecologist. Unmarried women, pregnant cases and patients undergoing treatment were excluded. Cases and control were identified on the basis of cytology.Informed written consent to participate in the study was obtained from all the enrolled cases. This study is approved by institutional ethics committee of Regional Medical Research Center (ICMR), Bhubaneswar, Odisha, India.Clinical data including signs and symptoms and socio-demographic data such as age, education, economic status, age at marriage, the age of menopause etc were collected by interviewing the patients with a predesigned questionnaire.Cervical swab specimen was collected using cytobrush and stored inside the viral transport media (Hi-Media) and transported to Virology laboratory, Regional medical research center, Bhubaneswar, Odisha for further analysis. Pap smears were prepared from the collected cervical sample for cytological analysis. Cytological classification was done according to Bethesda system .50-GCMCAGGGWCATAAYAATGG-30; MY09: 50-CGTCCMARRGGAWACTGATC-30–) targeting a 450-bp region of the HPV L1 gene. E6/E7 Type specific primers were used for the detection of 15 HPV genotypes. Type-specific primers were chosen according to the previously published study [DNA was extracted from a 200μl aliquot of exfoliated suspended cell samples using the QIA amp DNA Blood Mini Kit (Qiagen) as per the manufacturer’s instructions. Amplification of the human β-globin gene was performed to test sample sufficiency. To detect HPV, PCR was performed by using PGMY09/PGMY11 primers , 250mM dNTP , 5U Taq DNA polymerase , primers and 50ng of DNA was processed for the first PCR cycle. The temperature conditions were: 4 min of an initial denaturation step at 94°C, followed by 35 amplification cycle. Each cycle includes the 30s of denaturation at 94°C, 30s of annealing at 56°C, elongation at 72°C for 45s and a final extension of 4 min at 72°C. First PCR was followed by nested type-specific PCR. 2μl of the PCR product was used for the nested PCRs. PCR product was analyzed by electrophoresis on 2% agarose gel stained with ethidium bromide followed by visualizing under UV light.This is a case control study. Infection with more than 1genotypes was considered as multiple infections. In triple/quadruple genotype combinations only those having all the three genotypes of different species were considered as phylogenetically unrelated clad. Odds ratio were calculated for estimating the association of genotypes with cervical carcinoma. Statistical analyses were performed by using MediCal version 14.10.2 . A p-valSubject enrollment and outcomes are presented in 346 HPV positive samples were genotyped for 15 genotypes of HPV. Out of 346 samples 172 cases  were invasive cancer and 174 cases  without abnormal cytology. The genotypes detected were HPV16,18,51,39,66,68,45,35,6/11,58,43 and 42.Prevalence of multiple genotypes was 23.41% with double, triple and quadruple genotypes while the prevalence of single genotype infection was 76.59%.The study population was positive for 6 species  of HPV genotypes . Among bHPV genotypes belong to α5, α6, α8 and α10 species were always present as co-infection. Genotypes of α7 and α9 species were present both as single and co-infection where co-infection percentages were 66% and 31.56% respectively .Cases and controls were compared by demographic and other factors.It was fThe risk of cervical cancer associated with single and multiple infections have been evaluated . There wGenotypes not targeted by quadrivalent vaccine types  conferred 2.94 fold higher risk of cervical carcinoma. Cases those coinfected with phylogenetically related genotypes  were at 2.9 fold higher risk of invasive cervical carcinoma than those infected with other genotypes although it is not statistically significant. Whereas phylogenetically unrelated genotypes coinfection is negatively associated with cervical carcinoma (OR = .44 95% CI (.244-.8) p = .007)and it is statistically significant. Genotypes not targeted by 9-valent vaccines  associated with lesser risk of cervical carcinoma as compared to other genotypes. Subjects infected with any HPV genotype/genotypes excluding HPV16 in association with HPV 18  were at 4.1 fold higher risk of developing invasive cervical carcinoma.To the best of our knowledge, this is the first study to address the association of risk of cervical cancer with specific HPV genotypes coinfection among the Indian population.However the study population was drawn from tertiary care hospital and not from community screening. The present study has demonstrated many meaningful facts that could have profound implications for disease management and devising new vaccine strategy.The present study reported thathigher age at diagnosis, multiple parity, early age of marriage and low education found to be significantly associated with cervical carcinoma. This is in consistence with other previously reported studies ,25.The relationship between multiple HPV type infections and the progression of cervical cancer is debatable. However, competitive or cooperative interactions between HPV types could increase the risk of progression to cervical cancer. The present study reveals that women infected with multiple genotypes with phylogenetically related clad had the higher risk of cervical carcinoma as compared to the population infected with phylogenetically unrelated clad. Multiple infections with alpha-9 genotypes conferred asignificantly 5.3 fold higher risk while coinfection with alpha-7 genotypes conferred 2.5 fold risk of cervical cancer. It might explain a synergistic action of the same species of HPV genotypes when coinfected together for the induction of cervical cancer. Prevalence of Alpha 9 species is shown to be higher than alpha-7 is in agreement with other studies . Our stuIn vitro study reveals that co-infection of a single cell with both HPV16 and HPV18 results in replication interference between them [Interactions between different HPV types during their life cycle may affect the propagation, pathogenesis, and evolution of HPVs .The preseen them . Severaleen them , 29. AntHPV vaccination may not have a profound impact on non-vaccine targeted genotypes . Hence tThe present study reported that most of the non-vaccine targeted genotypes were co-infected with HPV16/ 18 except HPV45 and HPV58 . It was Present data shows that genotypes from certain species  appear as co-infecting more frequently than genotypes from other species (α9 and 7). These findings may be indicative of differential dependency for each genotype or species to colonize cervical epithelium. However, the strength of the association and whether this probable dependency is intra or interspecies need to be addressed with large sample size. These results may support the theory of elimination of certain genotypes by vaccination may affect the distribution of other genotypes or the theory of type replacement. In conclusion, the risk of development of cervical cancer is not common for all the genotypes. There might be type specific interactions (synergistic/antagonistic) between the genotypes in multiple infections that increases or decreases the risk of development of cervical cancer. The preliminary findings of the present study suggest further longitudinal study with large population size to shed more light on the carcinogenic potential of the HPV genotypes.


Clinical management of HPV positive women is difficult since many of the infections, including high-risk oncogene genotypes (hr-HPV), are transient. Therefore only a limited number of patients have a high-grade lesion and sending all HPV positive women for colposcopy would only increase costs and unnecessary treatment, with serious psychological consequences for patients. The need has emerged to identify other HPV related markers able to correctly detect women with a high-risk of developing high-grade lesions. Genotyping and the search for E6/E7 mRNA are among the possible candidates.The study was carried out by means of an observational analysis of the data relative to 674 HR-HPV positive women who we had observed from January 2013 to June 2015; the data had been gathered in a database at the HPV Center of the University Hospital of Catania, Italy.Women were considered eligible for this study if the following data was present in the database: Pap TEST, histologic evaluation, HPV TEST and E6/E7 mRNA detection.We calculated the Odds Ratio (OR) of woman who were mRNA positive, with CIN2+ lesions, and Odds Ratio of HPV16 positive women.Transcripts were detected in 23.6% (69/292) of the women with CIN1 and in 97.2% (210/220) of those with CIN2 + .Regarding genotyping, the 81,8% (180/220) of the women with CIN2+ had genotype 16, while only 18.1% (40/220) had genotype 18, 31, 33, 45.We calculated the OR in the group of HPV16 women with CIN2+ , this value increased  in women with CIN2+ and positive mRNA.The presence of the HPV16 genotype in our study was associated with a risk 5 times greater of developing a high-grade lesion (CIN2+) (OR = 4.62 95% CI:3.13–6.82); this supports the hypothesis that it would be opportune to have targeted protocols for the management of HPV 16 positive women. The results showed that there was an association between E6/E7 mRNA expression and histology . The E6/E7 mRNA test showed a higher prevalence of E6 and E7 transcripts in patients with higher-grade lesions.The results of this study suggest that the HPV genotype determination and E6/E7 mRNA detection would find an important application for management of HPV positive women. Numerous studies carried out in various countries on a large number of women have confirmed the greater sensitivity of the HPV test to detect CIN2+ lesions with respect to traditional cytology –3 increaThe problem arises with HPV positive woman; how best to manage them if only a limited number have a high-grade lesion and sending all HPV positive women for a colposcopy would only increase costs and unnecessary treatment, with severe psychological consequences for the patients. Over the last 10 years many researchers have tried to identify, among the patients positive at the HPV test, those at high-risk of neoplastic progression (CIN2+ lesions). In fact, with the simple negative/positive result to the HR-HPV virus it is possible to evaluate the prognostic role of various markers; this knowledge can help clinicians manage HPV positive cases better.In the light of the numerous studies, it appears that genotype determination and the search for E6/E7 mRNA are still two markers that help clinicians in the management of woman with an anomalous Pap test.Scientific evidence has shown that the evolution to CIN2+ lesions is greater with persistent HR-HPV infections , 5, therKnowing the incidence of the genotypes in the population can be fundamentally important as regards the preparation of prophylactic vaccines, which could vary their multivalency based on the most frequent genotypes in that given area .Many studies associate E6/E7 mRNA positivity with an integration of the progression of carcinogenesis, revealing an association between degree of integration, and histological, and cytological lesion severity –10. HoweIn our retrospective, observational study we wanted to investigate the positive HPV population that came to our center for secondary screening. We evaluated the incidence, the frequency of the various genotypes, their relationship with the histological lesions, and the capacity of the mRNA test to identify women at risk of CIN2+ lesions.The study was carried out by means of an observational analysis of the data relative to 674 HR-HPV positive women who we had observed from January 2013 to June 2015; the data had been gathered in a database at the HPV Center of the University Hospital of Catania, Italy.□ Positive for HPV genotype 16, 18, 31, 33, 45;□ not pregnant;□ no evidence of any immunodeficiency;□ no history of therapy for neoplasms.Women were considered eligible for this study if they satisfied the following criteria:The average age of the women was 36.6 ± 9.5 years (range 22–62). To include a patient in the retrospective study, the following data had to be present in the database: pap smears, histologic evaluation, HPV TEST and mRNA test.The smears, by conventional cytology, were classified according to the 2001 Bethesda System Criteria : negativIf patients were HPV DNA positive for at least one of the 5 HR HPV types  they were analyzed for the expression of viral oncogenes E6 and E7, looking for the mRNA by the NucliSens EasyQ HPV assay method (bioMerieux).Analyses of the samples were performed by the Virology Laboratory at the University Hospital of Catania, Italy.Histologic evaluation was performed with specimens collected by a colposcopy-directed biopsy and/or cone specimens collected by the loop excision procedure. The histological slides were diagnosed according to the WHO classification as CIN2+ for all the cases of CIN2, CIN3, and SCC lesions.The women who showed progression to CIN2+ underwent large loop excision of the transformation zone (LLETZ).Exo-endocervical cytology samples were collected in ThinPrep solution for total nucleic acid extraction (DNA and RNA) for detection and genotyping of viral DNA by means of gene amplification. This was followed by hybridization with genotype-specific probes able to identify most of the genital region HPV types.The automated DNA extraction was carried out with 1 ml sample on the NucliSenseasyMAG system  following the manufacturer’s HPV 1.1 protocol, with a 55 μl final elution volume.A total of 5 ml of Preserv Cytsolution was used for the detection of E6/E7 mRNA of HPV types 16, 18, 31, 33, and 45 by means of the PreTect HPV-Proofer Kit (referred to as mRNA test)  within 14 days of sample collection, according to the manufacturer’s instructions. mRNA was extracted from cervical samples using NucliSENS mini MAGMagnetic Extraction . The PreTect HPV-Proofer technology uses an isothermal nucleic acid sequence–based amplification (NASBA) that amplifies mRNA in a DNA background, detecting and genotyping HPV transcripts in the same reaction.The mRNA testing was performed according to the manufacturer’s instructions NucliSENS Easy Q HPV  and in accordance with national guidelines for HPV testing.Statistical analysis was carried out using SPSS for Windows . Descriptive statistics were expressed by frequency, arithmetic mean, standard deviation (SD) and percentages. We calculated the Odds Ratio (OR) of women with mRNA positive, with CIN2+ lesions, the Odds Ratio (OR) of women with HPV16.The 674 HR-HPV positive women underwent biopsy; Table The results of HPV test are shown in Table The results of E6/E7 mRNA expression are shown in Table Regarding genotyping, the 81.8% (180/220) of the women with CIN2+ had genotype 16, while only 18.1% 40/220) had genotype 18, 31, 33 and 45 , this value increased  in women with CIN2+ and positive mRNA.The onset of a carcinoma is not due to the presence of the virus but its integration . On thisThe method is a qualitative assay based on the NASBA method that detects the mRNA of 5 HR-HPV ; it has a cellularity check that targets the human U1A gene. Studies reported in the literature show its greater specificity with respect to the Aptima test and HC2, but it shows a lower sensitivity for the detection of CIN2+ lesions .The rationale behind the mRNA test is convincing, with a good sensitivity and greater specificity. Among the various problems to be overcome there are: sample storage (mRNA is very unstable and the possible use of this method in screening protocols requires efficient collecting systems to avoid degradation); the management of the woman with a negative mRNA test but a positive HPV DNA test.The growing interest in molecular studies has led various authors to compare the performance of the HPV DNA test with HPV mRNA test, evaluating the diagnostic accuracy in identifying high-grade lesions –20.From a recent systematic review , it was However, very few studies have verified the role of the mRNA test in predicting the development of a high-grade lesion during follow-up. The results showed that there was an association between E6/E7 mRNA expression and histology evaluation.. However, it cannot be verified whether an mRNA test negative in CIN2+ cases could be associated with a higher probability of regression, since CIN2+ women, according to local on-going protocols, usually undergo excisional treatment.In our study, the mRNA test was negative in 4.5% 10/220) of CIN2+ lesions. Taking into account that some CIN2+ lesions may spontaneously regress, mainly when they are CIN2 0 of CIN2 (2011) fCattani P. et al. , (2009) Genotype determination for woman with alterations on the Pap test is fundamental for the following reasons as it: detects the HPV genotype responsible for infection; detects multiple infections, which can be a marker for lesion progression; and discriminates between new infections and pre-existent infections. The presence of HPV16 genotype in our study was associated with a risk 5 times greater of developing a high-grade lesion (CIN2+) (OR = 4.62 95% CI:3.13–6.82), supporting the hypothesis that it would be opportune to have targeted protocols for the management of HPV16 positive woman; in fact, according to the latest up-date of the guidelines of the American Society for Colposcopy and Cervical Pathology (ASCCP), since the HPV16 virus is much less common than CIN1 lesions, with respect to CIN3 lesions, women positive for HPV 16, even with negative cytology, (women 30 and older who have discordant results) should be sent for colposcopy . From a


The diagnostic performance of p16 in HIV infection is unclear.Between June-December 2009, HIV-infected women underwent Papanicolaou (Pap) smear, human papillomavirus (HPV) testing, visual inspection with acetic acid (VIA), and colposcopy-directed biopsy as the disease gold standard at a HIV clinic in Kenya. Pap smears were evaluated for p16 expression. Sensitivity, specificity, positive predictive value (PPV), and area under the receiver operating characteristic curve (AUC) of p16 to detect CIN2/3 on histology and the impact of immunosuppression and ART was assessed.Of 331 cervical samples with p16 expression, p16 sensitivity and specificity to detect CIN2/3 was 54.1% and 72.4% respectively, which was lower than Pap and HPV in sensitivity, but higher in specificity than Pap, HPV, and VIA. Combining tests and p16 reduced sensitivity and increased specificity of Pap from 90.5% to 48.7% and 51.4% to 81.7%; of VIA from 59.5% to 37.8% and 67.6% to 89.9%; and of HPV from 82.4% to 50.0% and 55.3% to 84.8%. Combination p16 increased the PPV of Pap from 34.9% to 43.4%; of HPV from 34.7% to 48.7%; and VIA from 34.9% to 51.9%. Adjunctive p16 did not change AUC (P>0.05). P16 performance was not altered by immunosuppression or ART use. Combining p16 with HPV and VIA reduced the variation in HPV and VIA performance associated with CD4 and ART.As an adjunctive test in HIV-infected women, p16 immunohistochemistry increased specificity and PPV of HPV and VIA for CIN2/3, and was not altered in performance by immunosuppression, ART, or age. Cervical cancer is one of the most prevalent cancers worldwide with the greatest burden among women in resource-limited settings . CervicaHIV-infected women are disproportionately affected by HPV and at increased risk of developing HPV-associated cervical neoplasm and invasive cervical carcinoma , 8. WhilIn HIV-uninfected women, p16 protein expression has been shown to distinguish mild cervical intraepithelial neoplasm (CIN1) from moderate to severe intraepithelial neoplasm (CIN2/3) with similar sensitivity but higher specificity for CIN2/3 than HPV testing , 10. MorThe objective of this study was to determine the performance of p16 immunohistochemistry to detect CIN2/3 among HIV-infected women while assessing the effect of immune status, ART use, and age, and to evaluate the utility of p16 staining alone and in combination with Pap smear, HPV, and VIA.A total of 500 HIV-infected women were recruited from the Coptic Hope Center for Infectious Diseases in Nairobi, Kenya between June-December 2009. HIV-infected women were eligible for cervical screening if they were between ages 18 and 55 years, had an intact cervix, and never received treatment for cancerous or pre-cancerous cervical lesions. Methodology has been described elsewhere , 16. ColWritten informed consent was obtained from all study participants. Ethical approval was received by the University of Washington, Kenyatta National Hospital, and International Agency for Research on Cancer.After Pap smears were read for cytology, slides were stored until shipment to Seattle, Washington to undergo p16 immunohistochemistry. After removing coverslips, the CINtec® histology kit  was used to detect and stain for qualitative expression of p16 protein per manufacturer’s instructions . PositivDisease was defined as the detection of CIN2/3 by colposcopy-directed biopsy on all women. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the receiver operating characteristic curve (AUC) of abnormal Pap smear, defined as atypical squamous cells of undetermined significance or greater (≥ASCUS), HPV, and positive VIA were compared to the positive detection of p16 protein alone and in combination with other screening tests. Combinations included Pap with p16, HPV with p16, and VIA with p16. In these combinations, a positive screening result was defined as both tests being positive.Comparisons of sensitivity and specificity were stratified by CD4 count (≤350 and >350 cells/μl), ART , and age (<40 and ≥40 years) and compared using chi-square tests. Statistical analyses were performed using STATA 13 .Of 500 women screened, 471 had histology results, of which 331 (70.3%) cytology samples had adequate p16 results, 117 (24.8%) had indeterminate p16 results, and 23 (6.9%) were missing. Of the 331 women with p16 results, median age was 38.0 years and 80.7% were between ages 30–49 years. Forty percent were married and 34.7% reported one lifetime sexual partner. At screening, the median CD4 count was 371 cells/μl , 70% had CD4 <500 cells/μl and 52.4% had been on ART ≥2 years. There was no difference in age, CD4 count, or ART use between subjects with known results and those with indeterminate p16 results.On histology, 125 (37.8%) were normal, 132 (39.9%) CIN1, 43 (13.0%) CIN2, and 31 (9.4%) CIN3. On Pap, 128 (38.7%) were normal, 56 (16.9%) ASCUS, 79 (23.9%) LSIL, 57 (17.2%) HSIL, and 11 (3.3%) indeterminate. On p16, 111 (33.5%) were positive; on Pap, 192 (58.0%) were positive; on HPV, 176 (53.2%) were positive; and on VIA, 126 (38.5%) were positive. There was no difference in HPV, Pap, and histology between subjects with known results and subjects with indeterminate p16 results.P16 sensitivity, specificity, PPV, NPV, and AUC were: 54.1%, 72.4%, 36.0%, 84.6%, and 0.63% , Fig 1. P16 performance was not altered by immune status, ART duration, or age at screening . SensitiHPV specificity was higher at CD4 >350 than ≤350 cells/μl , however, addition of p16 to HPV reduced this difference . Combining p16 with HPV reduced the difference in HPV specificity associated with ART ≥2 years versus no ART and ART <2 years , and younger vs. older age . Adjunct p16 testing reduced the variability in VIA sensitivity associated with age  and the differences in VIA specificity associated with ≥2 years ART vs. no ART and <2 years ART .Among HIV-infected women, the use of p16 for the detection of histologically confirmed CIN2/3 had higher specificity (72.4%) and lower sensitivity (54.1%) compared to Pap, VIA, and HPV. While the performance of p16 alone was comparable to VIA, p16 sensitivity was markedly lower than HPV and Pap (≥ASCUS) and AUC was comparable between methods. As an adjunctive screening test for cervical neoplasia among HIV-infected women, p16 decreased sensitivity but increased specificity and PPV and its performance was not altered by immune status, ART duration, and age at screening.P16 protein expression as a screening method and adjunct test among HIV-infected women is not well documented. Studies of p16 in HIV-negative women have shown sensitivity from 79–97%, specificity from 71–85%, and PPV from 41–91% , 19–21. The effect of immunosuppression and ART on the performance of cervical neoplasia tests in HIV-infected women has been documented , 6. UnliA major strength of this study is the use of colposcopy-directed biopsy on all women. As a result, our findings may better reflect p16 sensitivity in combination with other cervical neoplasia screening methods. In addition, this study included detailed data on CD4 count and ART exposure. However, this study has several limitations. P16 staining was performed on archived samples, of which 24.8% had indeterminate results. Sensitivity analysis showed that there was no difference between subjects with indeterminate and determinate p16 results by sociodemographic characteristics, clinical factors, or CIN2/3. The use of archived samples may have reduced quality due to long-term storage and the de-staining and re-staining procedures. Cell loss may have occurred during the removal of plastic coverslips, reducing p16 interpretability. Additional studies are needed to investigate p16 staining on cytology samples without prior staining. This study did not use positive p16 control slides to ensure internal quality assurance and this may limit interpretability of our results. Finally, while we had detailed data on immune status and ART duration, we did not have longitudinal data related to the duration of immunosuppression, HIV diagnosis date, and progression or persistence of cervical lesions/neoplasm.As an adjunctive test to HPV and VIA in HIV-infected women, p16 increased specificity and PPV and reduced the variation in HPV and VIA performance associated with immunosuppression and ART duration. As WHO and U.S. guidelines recommend HPV genotyping as the primary screening for referral and treatment, adjunctive p16 testing may reduce unnecessary colposcopy and subsequent overtreatment among HIV-infected women.S1 Filep16_PlosOne.xls.(XLS)Click here for additional data file.