Much controversy still exists about maternal-to-infant transmission of human papillomavirus (HPV) infection, specifically about the magnitude of the risk and the route and timing of such vertical transmission. This prospective cohort study examines the risk of vertical transmission of maternal HPV in each trimester of pregnancy.One hundred fifty three healthy pregnant women were followed longitudinally throughout pregnancy and cervical swabs obtained in each trimester and postpartum for HPV detection. Cord blood, neonatal nasopharyngeal aspirates, and placental biopsies were collected at delivery. DNA isolation, polymerase chain reaction, and hybridization were performed using the GG HPV Genotyping Chip Kit . Detection of HPV in neonates was defined as the presence of HPV DNA in either cord blood or neonatal nasopharyngeal aspirate.HPV DNA was detected in 14%(22/153) of healthy women in the first trimester, 18%(22/124) in the second trimester, and 10%(15/153) in the third trimester; 24%(37/153) were positive for HPV DNA on at least one occasion in pregnancy. At birth, 5.2%(8/153) of neonates were HPV DNA positive. Seven of these eight infants were born to HPV-positive mothers. Placental HPV DNA was positive in 3.3%(5/152) of cases, and all five cases were from mothers with at least one HPV-positive test. Detection of HPV DNA in neonates was associated with detection of HPV in mothers during any of the three trimesters of pregnancy.HPV DNA was detected at birth in 5.2%(8/153) of neonates born to healthy women, and was associated with the detection of HPV in mothers during any of the three trimesters of pregnancy. Human papillomavirus (HPV) is a member of the papillomavirus family of viruses that infects stratified epithelium of the skin and mucous membranes. While the majority of the nearly 200 known HPV types cause no symptoms, 30–40 HPV types are capable of infecting the anogenital region and causing disease. HPV infection appears to be the central etiologic agent for the development of most cases of cervical cancer Vertical transmission of HPV from mother to fetus is known to occur. Indeed, up to 80% of neonates born to women with genital HPV have HPV DNA detectable in their nasopharyneal aspirate or oral mucosa There is still much controversy about maternal-to-fetal transmission of HPV, specifically about the magnitude of the risk and the route and timing of such vertical transmission. This prospective cohort study examines the risk of vertical transmission of maternal HPV in each trimester of pregnancy.Healthy women with a singleton pregnancy presenting in the first trimester (<14 weeks of gestation) to a single primary obstetric care clinic  in Incheon, Korea between May 2009 and June 2010 were approached for inclusion in the study. The decision of whether or not to participate was at the patient's discretion, and the patient was not disadvantaged in any other way for declining participation in the study. Subjects were excluded if they had evidence of immunosuppression , a connective tissue disorder, or were receiving medications that may affect immune function. Women were invited to participate whether or not they had a history of genital HPV infection. The study was approved by the local institutional review board , and written informed consent was obtained from all study participants.Each participant was asked to provide a comprehensive medical history and to complete a detailed demographic and medical questionnaire. Thereafter, all subjects underwent a routine obstetric examination. In all cases, cervical swabs were collected for HPV DNA testing  at four time points in pregnancy: at enrollment in the first trimester , at a routine follow-up visit during the second trimester (14–28 weeks), in the third trimester (>28 weeks), and in the postpartum period. The 2001 Bethesda System terminology was used to report the results of the cervical cytology Immediately after delivery, a sample of cord blood was set aside and a neonatal nasopharyngeal aspirate was collected with a bulb syringe using sterile technique so as to avoid possible contamination. Detection of HPV in neonates was defined as the presence of HPV DNA in either cord blood or neonatal nasopharyngeal aspirate. To evaluate placental infection, placental biopsies were routinely obtained from both the central and peripheral portions of the placenta again using sterile technique to avoid contamination. Detection of HPV in placenta was defined as the presence of HPV DNA in at least one placental biopsy.HPV DNA was detected using the GG HPV Genotyping Chip Kit , which identifies 40 HPV types . Genomic DNA extraction, amplification, labeling and hybridization as well as analysis were performed according to the instructions of the manufacturer. In short, extracting genomic DNA was performed with LaboPass™ Tissue mini prep. Kit . The primers chosen were in the L1 gene: primer Ll and L3. PCR with the primer L1 and L3 amplified about 200-base pair DNA fragment of all genotype of HPV. A mixture of 10 µL of HPVDNA-amplified product and 10 µL beta-globin-amplified products were denatured by heating at 95°C for two min, followed by cooling for 3 min on ice. The samples were mixed with 65 µL of hybridization buffer  and put on the HPV DNA chip. The HPV DNA chip was incubated at 50°C for 30 min. And then the HPV DNA chip was washed twice with 3× SSPE for 2 min and 1× SSPE for 2 min. This lead to the formation of visible spots on the array surface, which then were scanned, measured and analyzed using a dedicated reader and software after the chip was dried .Proportions were compared using the Fisher's exact test, and comparisons of continuous variables between groups were performed with a Mann-Whitney U test. Multivariate logistic regression analysis was used to adjust for potential confounding variables. p<0.05 was considered statistically significant. The SPSS software package  was used for all analyses.Of the 384 women originally enrolled in the study, 199 were followed throughout pregnancy. Of these, 46 subjects were excluded from the analysis . The remaining 153 cases were included in the final analysis. Among these, cervical HPV testing was performed in all three trimesters in 124 cases, and only in first and third trimesters in 29 cases.At birth, 5.2% (8/153) of neonates were HPV DNA positive. Among these eight neonates, six were HPV positive in nasopharyngeal aspirate and two in cord blood. HPV DNA was detected in 14% (22/153) of healthy women in the first trimester, 18% (22/124) in the second trimester, and 10% (15/153) in the third trimester. HPV DNA was detected less frequently in third trimester than in first and second trimester (p<0.01 for each). A total of 24% (37/153) of women were positive for HPV DNA on at least one occasion in pregnancy.All 153 women in the study population underwent cervical cytology (Pap smear) screening in the first trimester. Of these, 148 women had normal result, and 5 women had ASCUS. Among the 5 women with ASCUS, 2 were positive for high-risk HPV. One woman with high-risk HPV and ASCUS underwent colposcopy and cervical biopsy, which showed no evidence of cervical cancer. The other woman declined such testing, and a repeat Pap smear was normal.Much controversy still exists about maternal-to-fetal transmission of HPV, specifically about the magnitude of the risk and the route and timing of such vertical transmission. In this cohort of 153 normal pregnant women, HPV DNA was detected in 14% of women in first trimester, 18% in the second trimester, and 10% in the third trimester; 24% of women were positive for HPV DNA in at least one trimester of pregnancy. At birth, 5.2% (8/153) of neonates were positive for HPV DNA, and seven of these eight neonates had mothers who were positive for HPV DNA during pregnancy. Detection of HPV in neonates was associated not only with detection of HPV DNA in mother in the third trimester of pregnancy, but also in the first or second trimester.Several studies have demonstrated an increased risk of HPV vertical transmission in neonates born to mothers at high risk, including women with a history of genital warts, abnormal Pap smears, or cervical dysplasia The strength of the current study is that, in contrast to all prior studies that looked only at HPV status in the third trimester and mother-to-fetal transmission, we followed low-risk pregnant women with HPV testing longitudinally throughout all three trimesters of pregnancy and in the postpartum period. In the current study, 88% (7/8) of newborns who were positive for HPV had mothers with antenatal HPV DNA detection. As shown in Among 8 neonates who were HPV DNA positive, one neonate had different HPV type to its mother and another was born to mother without detectable antenatal HPV DNA. This discrepancy may be explained by contamination of the sampling or PCR technique , or by vertical transmission at other times during pregnancy when maternal sampling for HPV was not done. As we collected maternal samples for HPV DNA detection at only three time points during pregnancy, we may have missed the critical maternal HPV infection responsible for the vertical transmission, which was then cleared from the genital tract by the maternal immune system before the next HPV test.The precise route of vertical transmission remains unclear. Several studies have reported that the risk of vertical transmission is increased with vaginal delivery, suggesting that perinatal transmission occurs as the fetus passes through an infected birth canal In the current study, we measured HPV DNA in the maternal genital tract using a cervical swab. This should be distinguished from serostatus, which refers to evidence of active or past infection We considered neonates positive for HPV if cord blood or nasopharyngeal aspirate tested positive for HPV DNA. This definition may over-estimate the actual burden in neonates, since it does not identify live virus The clinical significance of HPV DNA detection in neonates also needs to be further investigated. The presence of HPV DNA in nasopharyngeal aspirate may represent infection or simply exposure. In the prior studies of Park et al. and Rombaldi et al., all infants who were HPV positive at birth subsequently tested HPV negative at both 6 months and 12 months of life It has recently been suggested that a subset of head and neck squamous cancers, especially oropharyngeal cancer, is HPV-related In conclusion, HPV DNA can be detected in 5% of neonates born to healthy women, and is associated with detection of HPV in mothers during any of the three trimesters of pregnancy. This observation has major implications for our understanding of the route and timing of HPV mother-to-fetal transmission.


Participants were selected from one comprehensive university and three to four companies in each city for a total of six comprehensive universities and 16 companies. A 62-item questionnaire on HPV knowledge and HPV vaccine acceptability was administered to participants before and after an educational intervention. The intervention consisted of an informative group lecture.χ2 = 1041.8, p < 0.001 and χ2 = 278.5, p < 0.001, respectively). Post-intervention, vaccine acceptability increased from 881/1146 (77%) to 953/1061 (90%), (p = <0.001) in employed women and 405/557 (73%) in students to 266/325 (82%), (p < 0.001). Women in both groups cited concerns about the HPV vaccine’s safety, efficacy, and limited use to date as reasons for being unwilling to receive vaccination. 502/1146 (44%) of women were willing to vaccinate their children at baseline, which increased to 857/1061 (81%) post-intervention, p < 0.001.A total of 1146 employed women and 557 female undergraduate students were surveyed between August and November 2011. Baseline HPV knowledge was low among both groups— 320/1146 (28%) of employed women and 66/557 (12%) of students had heard of HPV, while only 237/1146 (21%) of employed women and 40/557 (7.2%) of students knew that HPV is related to cervical cancer. After educational instruction, 947/1061 (89%) of employed women and 193/325 (59%) of students knew the relationship between HPV and cervical cancer (Incorporation of our lecture-based education initiative into a government-sponsored or school-based program may improve HPV-related knowledge and HPV vaccine acceptability. Further studies are needed to evaluate and standardize HPV education programs in China. Despite recent strides made in HPV vaccination and education, over half a million women worldwide develop cervical cancer each year. Over 85% of these cases occur in developing countries such as China due to the lack of effective screening and prevention programs ,2. The iBoth the bivalent and quadrivalent HPV vaccines have been demonstrated to effectively prevent the development of high-grade cervical neoplasias, particularly when given to girls before they become sexually active -12. The Previous studies on knowledge and attitudes about HPV among different groups of Chinese women have revealed high vaccine acceptability but low levels of knowledge about HPV and cervical cancer . A pilotIn recent years, there has been a proliferation of HPV education studies internationally. Educational instruction on HPV-related diseases has been demonstrated to effectively raise HPV knowledge and vaccine acceptability in America ,20, SwedThis was a multi-center, cross-sectional study across the five main geographical regions of Mainland China. One large, economically developed city was selected from each of the five geographic regions of China . Participants were then selected from one comprehensive university and three to four companies in each city for a total of six comprehensive universities and 16 companies. Participants were recruited by flyers and poster announcements in lecture halls, student centers, and the employment units at local companies.Prior to enrollment, all participants were fully informed about the objectives of the study, benefits and risks associated with participation, and assured of the confidentiality of the information they provide. After informed consent was obtained, a 62-item, multiple-choice questionnaire was administered to participants. The questionnaire was adapted from those based on expert opinions and used in previous studies among different populations across Mainland China ,18. The The educational intervention consisted of a one-hour group lecture followed by the same questionnaire as the one given after informed consent. Questions were written in Mandarin Chinese in simple terms appropriate for the level of education of the participants as previously validated ,18. PartThe data manager organized the database using EpiData 3.1. To ensure accuracy, two different research members entered data separately. Data analysis was performed using SPSS software version 18.0. Chi-square or F-test analysis was performed on the demographic characteristics of participants such as age, occupation, and geographic region. Multivariate analysis of all descriptive factors was performed to compare pre- and post-interventional HPV and vaccine knowledge. Logistic regression was performed to analyze the association between potential predictor variables and willingness to receive vaccination. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated based on Wald Chi-square statistics. Statistical significance was set at p < 0.05.From August to November of 2011,1146 employed women and 557 female undergraduate students were surveyed. After excluding incomplete questionnaires, 1061 and 325 surveys were included in the post-interventional analysis, respectively.Table At baseline, 1089/1146 (95%) of employed women and 440/557 (79%) of female undergraduate students had heard of cervical cancer. However, HPV knowledge was low in both groups, with only 321/1146 (28%) of employed women and 66/557 (12%) female students having heard of HPV. 237/1146 (21%) of employed women and 40/557 (7.2%) of students knew that HPV infection causes cervical cancer, but only 51/1146 (4.5%) and 18/557 (3.2%) knew the association between HPV and genital warts, respectively. Similarly, only 147/1146 (13%) employed women and 62/557 (11%) students had heard of the HPV vaccine. 212/1146 (18%) and 69/557 (12%) knew that the vaccine could prevent cervical cancer, respectively. 103/1146 (9%) of employed women were not concerned about developing cervical cancer. Knowledge of HPV and cervical cancer did not differ significantly between women from different geographical regions (p > 0.05). Baseline HPV knowledge is summarized in Figure χ2 = 1041.8, p < 0.001). Among undergraduate students, knowledge of this causal relationship increased over seven-fold from 40/557 (7.2%) to 193/325 (59%), .After a brief educational narrative about HPV and cervical cancer, HPV-related knowledge increased significantly. Comparison of pre- and post-interventional HPV knowledge is shown in Figure χ2 = 79.6, p < 0.001) in female students. Employed women also reported increased intention to undergo routine gynecologic exam after the intervention from 601/1146 (52%) to 974/1061 (92%), (p < 0.001).Post-interventional knowledge about HPV-related diseases and risk factors for HPV infection also increased significantly. In particular, correct identification of the ideal time for HPV vaccination as prior to sexual debut increased significantly from 246/1146 (21%) to 725/1061 (68%) in employed women and 205/557 (37%) to 223/325 (69%), (Willingness to receive HPV vaccination increased significantly post-intervention from 881/1146 (77%) to 953/1061 (90%) in employed women and from 405/557 (73%) to 266/325 (82%) in students, (p < 0.01). Female undergraduate students over age 20 were 1.6 times more willing to receive vaccination (0.9-2.9) than those under age 20. Knowledge of cervical cancer, concern about contracting cervical cancer, and approval of widespread usage of the vaccine were factors that influenced willingness to receive vaccination (OR 2.4 (1.3-4.8), 2.8 (1.6-5.0), and 4.3 (2.4-7.6), respectively). 265/1146 (23%) of employed women and 152/557 (27%) of female undergraduate students were unwilling to receive vaccination for themselves. The most common reasons against vaccination given by both groups of participants was concern about vaccine safety (325/1146 (58%) in women and 459/557 (40%) in students), concern about vaccine efficacy (331/1146 (29%) in women and 238/557 (43%) in students), and limited usage of the vaccine to date (258/1146 (23%) in women and 177/557 (32%) in students). Baseline attitudes and vaccine acceptability are shown in Figure χ2 = 261.7, p < 0.001). However, 175/1061 (16%) women were still unwilling to vaccinate their children after educational instruction due to concerns about vaccine safety (249/1061 (24%)), concerns about vaccine efficacy (136/1061 (13%)), and belief that their children are too young to be at risk (233/1061 (22%)). Comparison of pre- and post-educational HPV vaccine attitudes is shown in Figure Post-intervention, willingness of employed women to vaccinate their children increased from 502/1146 (44%) to 857/1061 (81%), (Logistic regression examining the effect of various sociodemographic predictors and cervical cancer risk on maternal willingness to vaccinate their children is presented in Table The brief educational intervention in our study effectively raised HPV and HPV vaccine knowledge in undergraduate students and employed women, and also increased vaccine acceptability. Post-intervention, women were twice as likely to report willingness to vaccinate their children and correctly identified younger ages as ideal for receiving the HPV vaccine. This is of particular significance because a recent study suggests for optimal vaccine compliance, girls in China should be vaccinated between the ages of 13-15, prior to concluding their mandatory schooling and becoming sexually active .While the HPV vaccine may reduce the incidence of cervical cancer in the future, clinical trials across China are ongoing. An effective HPV education program that addresses specific knowledge gaps and common questions among women of different regions, education levels, and cultures has been demonstrated to increase the success of large-scale vaccination programs . StudiesTo our knowledge, the present study represents the first evaluation of a large-scale HPV educational intervention in Mainland China. Kwan et al. ,27 have The results of our present study indicate that baseline HPV knowledge is low among employed women and female undergraduate students in northern, eastern, central, southern, and southwestern regions of China. Through our study, we also gained valuable insight into the preferred methods of obtaining HPV-related information and social beliefs held among Chinese women. Employed women and female college students preferred to receive HPV-related information via school or hospital-based lectures. They also believed that a national organization such as the Center for Disease Control should be responsible for providing the vaccine. In addition, vaccine safety, efficacy, and cost remained concerns and barriers to vaccination. Women had no preference for either the domestic or foreign HPV vaccine, but believed that the government should support and partially subsidize the cost. Future HPV education programs can focus on addressing these aforementioned issues. Furthermore, over 76% of employed females believe that getting vaccinated is a societal responsibility that should be shared by both genders. Thus, a government-sponsored, school-based education program can effectively disperse safe intercourse practices and HPV vaccine information to both male and female students.Strengths of our study include the relatively large population sampled across five main geographical regions of Mainland China. We also targeted employed women and undergraduate female students, two populations who have not been surveyed before. Our findings are consistent with previous studies, which found that HPV knowledge and vaccine acceptability were higher among college-educated women ,25.Limitations of our study include the cross-sectional design. While our educational intervention significantly increased post-test HPV knowledge and vaccine acceptance, the strength of causality is limited by the absence of a control group. Also, although our questionnaire was adapted from previous studies, we did not apply a scale to assess its validity. Future large-scale studies should devise a reliability scale to evaluate the questionnaire. Furthermore, our study was mainly carried out in urban centers among women with higher education levels thus due to the potential sampling bias, our findings may not be generalized to the entire Chinese population. HPV knowledge and vaccine receptivity have been shown to vary widely based on region, sociodemographic factors and education level ,16. In 2Our study demonstrates that incorporation of our educational initiative into a government sponsored or school based program may improve willingness to accept HPV vaccination. The potential implications of our study are far-reaching during this pivotal time of HPV vaccine clinical trials in China. It is important to establish the link between HPV infection and the development of cervical cancer, and to educate women on the ideal time to vaccinate their children. Studies have shown that maternal attitudes towards vaccination greatly impacts vaccination rates among children. It is also imperative to dispel common beliefs that only women with multiple sexual partners need to be vaccinated.Future directions for research include the development of a longitudinal, national HPV education program appropriate for school-based curriculum. Due to our limited participant sampling, our findings may not be generalized to the entire heterogeneous Chinese female population. Widespread implementation among different geographic regions and minority populations across China would shed light on the long-lasting impact of HPV education. Also, it is important develop evaluation and feedback tools to assess the effectiveness of the program. Lastly, follow-up studies must be performed to see if increased HPV and HPV vaccine knowledge translates into higher vaccination rates and vaccine compliance within China.The authors declare that they have no competing interests.IJC participated in data analysis and drafted the manuscript. FHZ and YLQ conceived of the design and coordination of the study. RH, WH and SKZ performed the statistical analysis. SMW and RH participated in the data collection. FZ guided the statistical analysis and revisions of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/916/prepub


Knowledge of the distribution of human papillomavirus (HPV) genotypes among women with cervical lesion and in invasive cervical cancer is crucial to guide the introduction of prophylactic vaccines. There is no published data concerning HPV and cervical abnormalities in Sudan. This study aimed to define the prevalence of HPV and its subtypes in the cervical smears of women presenting with gynecological complains at Omdurman Military Hospital, Sudan.During the period between March 2003 and April 2004, 135 cervical smears collected from these women, were screened using cytological techniques, and analysed by PCR for (beta)-globin and HPV DNA using gel electrophoresis and ELISA.Of these 135 smears, there were 94 (69.3%) negative, 22 (16.3%) positive for inflammation, 12(8.9) mild dyskaryosis, 5 (3.7) moderate dyskaryosis and 2 (1.8) severe dyskaryosis. There were 60.7% ß. globin positive samples for HPV indicating DNA integrity. HPV DNA was identified in three samples (2.2%) by gel electrophoresis and. was positive in four samples (2.9%) as single and multiple infections by PCR-ELISA. The high risk HPV types 16 and 58 were identified in one sample as a mixed infection. The low risk HPV types 40 and 42 were also found as a mixed infection in another patient. HPV types 58 and 42 were identified in the other two patients.HPV type distribution in Sudan appears to differ from that in other countries. The HPV genotypes identified were not associated with cancer. Many secuminata , most HPcuminata . Nearly cuminata . On the cuminata ,6 Women cuminata . Howevercuminata . Thus, tcuminata ,9. HowevDuring the period of February 2003 through May 2004, a cross-sectional study was conducted among, women presenting to the gynecological clinic with symptoms of vaginal discharge, inter-menstrual bleeding, and post-coital bleeding were approached to participate in the study. After informed consent, questionnaires were conducted to gather socio-demographic, medical and gynecological characteristics. Then cervical smears were collected using the standard Szalay cyto-spatula. Cells were initially used to prepare a Pap smear and the remaining cells on the spatula were eluted in Tris HCl buffer PH 8.0. A cell pellet was obtained by centrifugation, re-suspended in 2 ml Tris HCl buffer pH 8.0 and frozen at -20°C until HPV analysis using PCR-EIA in Cardiff, United Kingdom. Cytology and HPV DNA detection Pap smears were prepared with Papanicolaou stain, screened by cyto -screener and positive results revised and confirmed. To target DNA, samples were pre-screened by polymerase chain reaction (PCR) using ß-globin specific primers as described previously . Human pThe mean age of the women was 33.18 years (range 17- 60 years). Cervical smears had normal cytology in 94 (69.6%) out of 135 patients. The rest of the patients had 22(16.3) inflammatory changes 12(8.9) mild dyskaryosis, 5 (3.6) moderate dyskaryosis and 2 (1.5) severe dyskaryosis. Human DNA was detected in 82 (60.7%) out of 135 samples using ß globin PCR. Three samples (2.3%) were positive for HPV DNA as determined by gel electrophoresis; however these samples were not reactive with ELISA for HR and LR groups, suggesting novel HPV subtypes. The cytological findings of these two samples were reported as moderate dyskaryosis and severe dyskaryosis. Nine samples had a reactive result with HPV DNA using ELISA method. Three samples showed reactive result with HPV 58, these were found in samples that were reported as moderate dyskaryosis and one with mild dyskaryosis. Another two samples showed reactive result with HPV types 16 and 42, the cytological result of these were, inflammation and normal cytology respectively. The other four samples were reactive with ELISA and gel electrophoresis for HPV DNA. Those patients showed infection with general high risk (HR) cocktails group and general low risk (LR) cocktails group, table This is the first study we are aware of investigating HPV infection among Sudanese women and one of the few studies that used the PCR-EIA technique  and ELISA) in Sub-Saharan Africa. Previously Ibrahim and his colleagues failed to isolate HPV DNA in a small number of oral cancer tissue samples using classical PCR techniques in Sudanese patients . GP5+/biet al., who described the overall predominant role for HPV 16 and HPV 18. We only identified HPV 16 and could not isolate HPV type 18 often the most common high risk type [HPV 58 was the most common type in the current study. However, HPV 58 is considered a rare type, and not commonly reported in most of Europe, both Americas and Asian countries 14). Yet in Africa, this type was observed e.g. east, central, west and South Africa [. Yet in isk type . All theisk type . SecondlIn summary this preliminary reported concerning HPV types in Sudan will need to be extended to larger studies in the future. Information about HPV types in Sudan will be of great value to help the national health authority and decision makers to introduce HPV vaccine in Sudan. Further studies are also needed to identify the association between HPV infection and cervical cancer.The authors declare that they have no competing interests.MMS and IA designed the study and carried out statistical analyses. MMS and MS carried out the cytological and histopathological investigation participated in the statistical analysis and procedures. KH HPV supervised detection and Identification. KT participated in the clinical work. All the authors read and approved the final version.


Human papilloma virus (HPV) genotypes 6, 11, 16, and 18 impose a substantial burden of direct costs on the Italian National Health Service that has never been quantified fully. The main objective of the present study was to address this gap: (1) by estimating the total direct medical costs associated with nine major HPV-related diseases, namely invasive cervical cancer, cervical dysplasia, cancer of the vulva, vagina, anus, penis, and head and neck, anogenital warts, and recurrent respiratory papillomatosis, and (2) by providing an aggregate measure of the total economic burden attributable to HPV 6, 11, 16, and 18 infection.For each of the nine conditions, we used available Italian secondary data to estimate the lifetime cost per case, the number of incident cases of each disease, the total economic burden, and the relative prevalence of HPV types 6, 11, 16, and 18, in order to estimate the aggregate fraction of the total economic burden attributable to HPV infection.The total direct costs (expressed in 2011 Euro) associated with the annual incident cases of the nine HPV-related conditions included in the analysis were estimated to be €528.6 million, with a plausible range of €480.1–686.2 million. The fraction attributable to HPV 6, 11, 16, and 18 was €291.0 (range €274.5–315.7 million), accounting for approximately 55% of the total annual burden of HPV-related disease in Italy.The results provided a plausible estimate of the significant economic burden imposed by the most prevalent HPV-related diseases on the Italian welfare system. The fraction of the total direct lifetime costs attributable to HPV 6, 11, 16, and 18 infections, and the economic burden of noncervical HPV-related diseases carried by men, were found to be cost drivers relevant to the making of informed decisions about future investments in programmes of HPV prevention. Although infection by the human papillomavirus (HPV) is often asymptomatic and self-limiting, it has been established to be the cause of a number of clinically significant conditions Primary prevention of HPV-related diseases may be achieved by vaccination. Two preventive vaccines against cervical cancer are currently approved in Italy: 1) a quadrivalent vaccine that protects against HPV types 6, 11, 16, and 18 (Gardasil®) and 2) a bivalent vaccine that protects against HPV types 16 and 18 (Cervarix®). In March 2008, a national HPV immunisation programme that uses the quadrivalent vaccine and targets eleven-year-old girls was initiated, This policy decision was informed by several Italian economic studies confirming the cost-effectiveness of the anti-HPV vaccination for the prevention of cervical cancer, cervical dysplasia, and anogenital warts The main objective of the study reported herein was to address the foregoing gap: (1) by estimating the total direct medical costs associated with nine major HPV-related diseases, namely invasive cervical cancer, cervical dysplasia, cancer of the vulva, vagina, anus, penis, and head and neck, anogenital warts, and RRP and (2) by providing an aggregate measure of the total economic burden attributable to infection with HPV 6, 11, 16, or 18. The latter value could be useful to inform further studies of cost-effectiveness and analyses of budget impact, and ultimately future public health policy decisions in Italy.For each of the nine HPV induced diseases we performed an extensive review of the literature to identify the best secondary data available to produce lifetime cost per case estimates, which were expressed in present value. Using an incidence-based approach, we then used the estimated costs per case to produce an aggregate measure of economic burden. The relative prevalence of HPV types 6, 11, 16, and 18 was then applied to the economic burden for each condition to estimate the aggregate fraction of the total burden attributable to HPV infection.As mentioned above, the secondary data used to inform estimates were identified via a literature review. The flow of information through the different phases of the systematic review is shown in In order to collect relevant secondary data, we carried out a systematic search of the following electronic databases: MEDLINE (PubMed), EMBASE (accessed through OVID SP), CDC, CINHAL, and NIHR HTA, covering the period 1990–2011. Details of the search strategy can be found in the Supporting Information files, To ensure the inclusion of all relevant studies, we consulted the websites of official institutions, such as the Cochrane Gynaecological Cancer Group Trials Register, Global Health, and Clinical Trials.Gov. We also searched the grey literature for information from other relevant sources including the Italian Ministry of Health, the Italian National Institute of Health (NIH), the World Health Organization (WHO), the International Agency for Research on Cancer (IARC), the Catalan Institute of Oncology, and various Italian scientific societies , as well as sites dedicated to the rapid dissemination of scientific publications in the socioeconomic and healthcare fields . Generic academic search engines  and meta-engines  were also used to make the search as comprehensive as possible. Peer-reviewed journals, meetings, congresses, and other internet sources were searched up to December 2011 for presentations or communications on any additional data related to our research topic.All the studies used in the qualitative synthesis were required to have met at least one of thefollowing inclusion criteria:they report epidemiological data  derived from population databases, such as national surveys or registries;they report direct cost data accounted from the perspective of the payer, and expressed in monetary values for hospital Disease-Related Groups (DRGs) and outpatient tariffs. The DRG system aggregates all activities, including surgical interventions, drugs administered, materials, and personnel, for each individual diagnosis and stipulates the reimbursement tariff, which corresponds to the sum of all interventions provided, to be paid to the hospital. Outpatient medical treatments and interventions are stipulated in a similar way and reimbursed to the local territorial healthcare service. DRGs and tariffs represent the actual direct cost to the National Health Service and correspond to a codified hospital admission or outpatient intervention;or they report an observational study of epidemiological data on HPV-induced diseases and their associated direct costs, observed simultaneously in the same cohort of patients.Studies used in the quantitative estimates for the burden of diseases were additionally required to have met the following inclusion criteria:they report Italian data relevant to the HPV-related diseases included in the cost analysis;they report estimates of discounted lifetime cost per case.Two independent researchers screened the titles and abstracts of all the identified sources. The full-text versions were then obtained of all articles not excluded on the basis of the foregoing criteria. The best available data to inform the cost analysis were selected on the basis of consensus among the authors. In all phases of the literature review, disagreements were resolved by discussion among the entire team to reach a consensus.For the observational studies, the risk of bias was assessed by two independent researchers through the application of a checklist of essential items, which is reported in We adopted an incidence-based approach, wherein a lifetime cost per case associated with each condition was applied to the estimated number of incident cases attributable to HPV 6, 11, 16, and 18 that occurred in men and women over a one-year period. We sought to develop cost-per-case estimates that represented the present value of the total direct medical costs that accrued from the time of diagnosis to follow-up. Whenever possible, we sought published estimates of discounted lifetime cost per case which were available for cervical cancer, cervical dysplasia, and anogenital warts. However, for other conditions, due to the scarcity of available Italian studies due to the paucity of available Italian studies, especially with respect to noncervical cancers and RRP, we estimated the discounted lifetime cost per case by modelling the costs related to diagnosis, treatment, and follow-up. Treatment paradigms for noncervical cancers  were based on the recommendations of generally accepted Italian Medical guidelines Only direct medical costs from the perspective of the Italian National Health Service were included in the analysis. Costs were calculated on a DRG basis at 2006 tariffs Incidence was estimated on the basis of the population resident in Italy in 2011, i.e., 31,213,168 women and 29,413,274 men, giving a total of 60,626,442. Census data on the Italian resident population were obtained from the Italian National Institute of Statistics Costs were adjusted for inflation at 2011 values using compound annual Italian National Consumer Price Indexes (NIC) Future costs (more than 12 months after diagnosis) that were associated with the follow-up of HPV-related diseases were not adjusted for inflation to match the nominal discount rate used (3%) to obtain their net present value. The chosen discount rate reflects the opportunity cost of financing from the perspective of a public payer  and it does not include the expected rate of inflation.Similarly to a recently published estimation of the burden of HPV-related disease in Europe Crude incidence rates were calculated directly by source The annual numbers of cases of abnormal Papanicolau (Pap) smears and colposcopies To estimate the annual number of incident cases attributable to HPV 6, 11, 16, and 18, prevalence rates for HPV were identified from the literature review. For all cancer diagnoses , the rate chosen was the pooled prevalence for HPV 6, 11, 16, and 18 To develop an aggregate monetary measure of the burden of HPV-related diseases, the fraction of incident cases attributable to HPV 6, 11, 16, and 18 was applied to the total lifetime costs per year for each disease . The estFurther details of the estimation approach are provided in the disease-specific sections of the Results.Incidence rates for HPV-related cancers included were obtained from 22 Italian registries α/2  =  t-value with an area of α/2 to its right, s  =  sample standard deviation, n  =  sample size.where: No upper and lower bounds were available for some cervical abnormalities  or for anogenital warts. Due to the lack of Italian data, upper and lower bounds for annual incident cases of RRP were estimated from epidemiological data from the US For most of the malignancies  and cervical neoplasia (CIN1-3), the upper and lower bounds of the incidence of HPV 6, 11, 16, and 18 were based on 95% confidence intervals obtained directly from the literature A sensitivity analysis of the impact of input uncertainty on the economic burden of HPV 6, 11, 16 and 18-related disease was performed using the SensIt’s Many Inputs, One Output option.Cost estimates for cervical cancer were obtained from published Italian data from a retrospective observational study conducted for 351 patients Estimates of the annual incidence of cervical cancer in Italy were obtained from the literature The fraction of the cost of cervical cancer in Italy that was attributable to HPV 6, 11, 16, and 18 was estimated to be €53.0 million . When the upper and lower bounds for the prevalence of HPV 6, 11, 16, and 18 were considered, this cost ranged from €47.7 to €61.8 million.Although some regions in Italy started to implement screening programmes for cervical cancer in the 1970 s, an organised nationwide programme was only recommended in 1996, following the European Commission Guidelines on Quality Assurance in Cervical Cancer Screening. It is estimated that each year in Italy, approximately 5.2 million women aged 25–69 undergo opportunistic screening In our analysis, we estimated the total direct lifetime costs associated with the clinical and surgical management of cervical dysplasia from published Italian data Consistent with our primary methodological constraint, the estimated annual number of incident patients and cost data were derived from the same Italian source mentioned in the previous paragraph The cost of diagnosis of cervical dysplasia attributable to HPV 6, 11, 16, and 18 was derived from published data The total cost associated with the management of cervical lesions, including the diagnosis and treatment of CIN1/2/3 stages was €34.4 million per year (range €34.4–€52.6 million). The fraction attributable to HPV 6, 11, 16, and 18 was €15.2 million (range €13.4–€17.5 million). A previous Italian cost analysis We evaluated the direct costs associated with treatment and follow-up for HPV-related cases of five noncervical malignancies: cancer of the anus, head and neck, vulva, vagina, and penis. The diagnosis of head and neck cancer included oral, naso-pharyngeal, pharyngeal, and laryngeal cancers.The lifetime cost per patient was modelled for each diagnosis on the basis of national treatment guidelines Crude rates of the annual incidence of noncervical cancers in Italy were obtained from the literature The crude rates that we used to estimate the number of annual incident cases for head and neck cancers were confirmed by analogous rates collected from 1988 to 1992 by the Italian Istituto Superiore di Sanità (ISS) For all cancer diagnoses, most of the fraction that was attributable to HPV was constituted by types 16 and 18 (defined as high-risk or oncogenic), although a minor contribution of types 6 and 11 (defined as low-risk or nononcogenic) was observed in cervical and vaginal cancer. A small but significant prevalence (range 2 to 5%) of HPV 6 and 11 has been associated with noncervical cancers by an increasing number of authors The estimated fractions of the total annual direct costs attributable to HPV 6, 11, 16, and 18-induced malignancies are reported in Head and neck cancers were responsible for the highest annual burden of direct costs among the HPV 6, 11, 16, and 18-induced malignancies , followed by anal cancers , vulvar cancers , vaginal cancers , and penile cancers .Lifetime direct costs per patient, stratified by gender and by diagnosis , were obtained from a published study The estimated lifetime cost per incident case was calculated as the sum of the mean, inflation-adjusted direct costs incurred by new patients (€416 for women and €311 for men) and the probabilised, discounted, inflation-adjusted mean direct costs of a recurrent (€199 for women and €66 for men) and a resistant (€48 for women and €92 for men) episode observed one year after the initial diagnosis. Probability rates were assigned on the basis of the number of recurrent (50.6% for women and 34.8% for men) and resistant (28.2% for women and 38.5% for men) cases as a percentage of the annual number of newly diagnosed cases, assuming a steady state in the total number of genital warts treated in Italy Consistent with the difference in the mean number of examinations observed between women and men in the present study, the estimated lifetime cost for the management of anogenital warts was significantly higher in women than in men .The total annual economic burden of anogenital warts in Italy that was attributable to HPV 6 and 11 amounted to €70.9 million, and was significantly higher for women . Data on upper and lower bounds were not available. Anogenital warts were the second highest contributor to the economic burden of HPV 6, 11, 16, and 18-related diseases in Italy, preceded by total cervical diseases (€104.6 million) and followed by head and neck cancers (€59.1).No Italian data were available on the lifetime cost per patient, the annual number of incident cases, and HPV prevalence for this HPV 6 and 11-induced disease. Consequently, we proceeded to evaluate the economic burden of RRP using the relatively few data available in the international literature. Because of the lack of benchmarks for calibrating the inputs to the cost model, we decided to use a conservative approach to the valuation of the annual direct costs that were attributable to RRP.To estimate the lifetime cost per patient, elective surgical procedures and medical treatments were selected on the basis of published reports from the US To estimate the number of incident cases of RRP per year, we applied a crude incidence rate  The total direct costs (expressed in 2011 Euro) associated with the annual incident cases of the nine HPV-related conditions included in the analysis were estimated to be €528.6 million, with a plausible range of €480.1– 686.2 million. The fraction attributable to HPV 6, 11, 16, and 18 was €291.0 (range €274.5–315.7 million), accounting for approximately 55% of the total annual burden of HPV-related disease in Italy.Under the base case assumptions, the fraction of the annual direct costs of noncervical HPV-related diseases that could be attributed mainly to HPV 6 and 11  and 3.5%; in this case, we obtain values for the economic burden of HPV 6,11,16 and 18-related diseases of €291.5, €291 (base case) and €290.4 million.To our knowledge, the present study is the first attempt to evaluate the annual economic burden of diseases associated with HPV 6, 11, 16, and 18 infections in Italy. From the aggregate measure of the cost estimates, we make three basic observations that could inform future public health choices in terms of programmes of immunisation and secondary prevention of HPV infections.First, the dominant prevalence of HPV 6 and 11 in cases of anogenital warts and RRP meant that the total costs for noncervical diseases that were attributable to the low-risk HPV types 6 and 11 were at least as high as the direct costs attributable to the high-risk HPV types 16 and 18. This occurred because anogenital warts and RRP were associated cumulatively with 58.8% of the total costs of noncervical HPV 6, 11, 16, and 18-related diseases. This observation was consistent with data already in the literature from the US Second, Third, RRP was the fourth largest driver of the economic burden of HPV 6, 11, 16, and 18-related diseases in Italy . However, epidemiological and cost data are still not available for Italy. To reduce the uncertainty that is presently associated with the estimates of the economic burden of RRP, the incidence, prevalence, aetiopathogenesis, and management of RRP should be observed in Italy over a sufficient period of time.The present study has several limitations that merit acknowledgement. Italian sources of cost data were limited and the quality of available information was variable. Published estimates of lifetime cost per case were only available for cervical cancer The data used to derive the lifetime cost per patient for noncervical cancers were not comprehensive, but were based on the valuation of the interventions recommended by the generally accepted Italian Medical guidelines The attribution of direct costs to HPV and HPV 6, 11, 16 and 18 was derived mostly from prevalence rates obtained from Italian regional registries. The use of population-based cancer registry data to assess the proportion of diseases attributable to HPV might have resulted in an overestimation of the contribution of HPV, due to its high prevalence in healthy subjects Cost data based on DRG tariffs did not capture the actual direct costs incurred by hospitals and outpatient practices. Direct or indirect nonmedical costs were also not included, due to the lack of availability of published Italian data. One aim of any future research should be to fill some of the gaps in the Italian epidemiological and cost data to reduce the uncertainty associated with the present estimates.In summary, our results have provided an approximate but plausible estimate of the significant economic burden imposed by the most prevalent HPV-related diseases on the Italian welfare system. The fraction of total direct lifetime costs attributable to HPV 6, 11, 16, and 18 infections and the economic burden of noncervical, HPV-related diseases carried by men were found to be cost drivers that are relevant to the making of informed decisions on future investments in programmes to prevent HPV infection.Appendix S1Details about the search strategy and the terms used to query the main following electronic databases: MEDLINE (PubMed), EMBASE (accessed through OVID SP), CDC, CINHAL, and NIHR HTA, covering the period 1990–2011.(DOCX)Click here for additional data file.Table S1Checklist of essential items used by two independent researchers to assess the susceptibility to bias of observational studies.(DOCX)Click here for additional data file.Table S2Risk assessment for major systematic biases of the studies identified by the systematic literature review which reported epidemiological or economic data used to inform the burden of disease.(DOCX)Click here for additional data file.


A divergent human papillomavirus (HPV), isolated from a lung lesion of a patient with recurrent respiratory papillomatosis, was fully cloned, sequenced, and genetically characterized. DNA analysis revealed that the HPV contained a 10.4-kb genome, with a duplication of 2,493 bp that includes partial L1-long control region (LCR)-E6-E7-partial E1 sequences. Recurrent respiratory papillomatosis (RRP) is the most common benign neoplasm of the larynx . Most caA male patient with a 19-year history of recurrent respiratory papillomatosis developed progressive, bilateral tumor invasion of the lung parenchyma . DNA anaOur data suggest a link between the duplication of the HPV-11 promoter and E6/E7 oncogenes and the clinical aggressiveness of the tumor in RRP. The present report describes the first sequence-confirmed duplication of the LCR-E6-E7 region in HPV. An earlier study had noted duplications of HPV-11 genomes in RRP that had progressed to squamous cell carcinoma and metastasized (JN644141.The complete genome sequence of HPV-11 associated with lung progression is available in GenBank under accession number


Glossina fuscipes. Tsetse use a range of olfactory and visual stimuli to locate their hosts and this response can be exploited to lure tsetse to insecticide-treated targets thereby reducing transmission. To provide a rational basis for cost-effective designs of target, we undertook studies to identify the optimal target colour.Most cases of human African trypanosomiasis (HAT) start with a bite from one of the subspecies of G. fuscipes fuscipes attracted to targets consisting of a panel (25 cm square) of various coloured fabrics flanked by a panel  of fine black netting. Both panels were covered with an electrocuting grid to catch tsetse as they contacted the target. The reflectances of the 37 different-coloured cloth panels utilised in the study were measured spectrophotometrically. Catch was positively correlated with percentage reflectance at the blue (460 nm) wavelength and negatively correlated with reflectance at UV (360 nm) and green (520 nm) wavelengths. The best target was subjectively blue, with percentage reflectances of 3%, 29%, and 20% at 360 nm, 460 nm and 520 nm respectively. The worst target was also, subjectively, blue, but with high reflectances at UV (35% reflectance at 360 nm) wavelengths as well as blue (36% reflectance at 460 nm); the best low UV-reflecting blue caught 3× more tsetse than the high UV-reflecting blue.On the Chamaunga islands of Lake Victoria , Kenya, studies were made of the numbers of G. f. fuscipes should be blue with low reflectance in both the UV and green bands of the spectrum. Targets that are subjectively blue will perform poorly if they also reflect UV strongly. The selection of fabrics for targets should be guided by spectral analysis of the cloth across both the spectrum visible to humans and the UV region.Insecticide-treated targets to control  G. f. fuscipes attracted to different coloured targets. Our results show that the attraction of tsetse is correlated positively with reflectance in the blue region of the spectrum but negatively with the UV- and green regions. The best blue targets attract and kill three times more tsetse than the worst because of different UV reflectance levels in the different blue cloths. Hence selecting fabrics for use in targets must be based on spectral analysis of the fabrics' reflectance across the spectrum visible to tsetse, which includes UV, and not simply on the ‘rule of thumb’ that targets to control tsetse should be blue.Efforts to control human African trypanosomiasis (HAT) would be strengthened by the development and application of more cost-effective methods of controlling the various species of tsetse fly vector. Among the most promising approaches is the use of insecticide-treated targets which use various olfactory and visual stimuli to attract and kill tsetse. Following on from previous studies of the responses of tsetse to odours and target size and shape, we compared the numbers of  Glossina spp.) are restricted to sub-Saharan Africa where they transmit the trypanosomes causing the diseases of nagana in livestock and sleeping sickness, also known as human African trypanosomiasis (HAT), in humans. Tsetse are commonly divided into three groups: i) the Morsitans group (savannah species) which are the main vectors of the trypanosomes causing nagana; ii) the Palpalis group (riverine species) which are largely responsible for transmission of Trypanosoma brucei spp, the causative agents of HAT, and iii) the Fusca group (forest species) which currently are usually only minor vectors. There is no vaccine against trypanosomiasis, and the use of drugs is limited by problems of toxicity and resistance Glossina fuscipes .Tsetse flies  that are treated with insecticide G. f. fuscipes, one of the two most important vectors of sleeping sickness.The present paper reports screening tests for the attractiveness of various colours and types of fabric in small cloth-and-net targets for T, Colgate-Palmolive, active ingredient: sodium hypochlorite) to create a material with high reflectance in the UV-region (supplementary −2) supplied by VF.Targets consisted of a 25×25 cm panel of cloth flanked by the same-sized panel of fine black polyester netting . Various materials, obtained from different sources, were used for the cloth panel. Phthalogen blue cotton  (used as the standard) and black cotton (Black 1) were already available at the research station and were from the same stocks used in previous studies of target design http://www.dhigroup.com) on a Shimadzu dual beam photometer, from 190–900 nm at 10 nm intervals  , Lake Victoria, Kenya, using targets in which the cloth and netting panels were each covered on both sides with an electrocuting grid of fine black wires Studies were performed from February to December 2009 on Chamaunga Island , 410–500 nm (‘blue’), 510–600 nm (‘green’) and 610–700 nm (‘red’) G. f. fuscipes to baits of different colour. The colours can be divided into two groups: i) “cut-off” colours, i.e., colours with a steeply sloped spectrum  was low for all colours , averagiThe second set of experiments  focused Of all the fabrics tested the most promising alternative to the Phthalogen blue C standard was purple polyester . Furthermore, the purple polyester bait performed well in relation to the blue polyester material (Blue 7). Two experiments compared different shades of purple polyester cloth (Exp. 14 and 15). For males, Purples 4, 5 and 8 had high catch indices , while PAs with the first set of experiments , the secDetails of the regression models are shown in G. f. fuscipes to colour are broadly similar to those of other tsetse: blues, and phthalogen blue sensu stricto in particular, are more attractive than other colours whereas reds and blacks are intermediate and green-yellow is least attractive G. pallidipes in Zimbabwe, G. palpalis palpalis in Côte d'Ivoire using three colour bands: 300–380 nm (ultraviolet), 380–480 nm (ultraviolet-blue) and 480–620 nm (blue-green-yellow- red) MuscaG. pallidipesG. palpalis palpalisThe results show that the responses of G. f. fusipes and other tsetse species to visual cues, the data also confirms the observed differences between Palpalis and Morsitans group tsetse flies. In the present study there is a 2–3× difference in catch between the best  and worst (yellow and targets with high UV reflectance) targets. This range is similar to that reported for other Palpalis-group tsetse G. p. palpalis and G. tachinoides) phthalogen blue is more attractive than black, and black does not seem to elicit a marked landing responses G. f. fuscipes  and red range  was much more effective for males and females than the yellow-green range . Furthermore, light blue fabrics  were in general of poor effectiveness. Our results underline the important negative effects of UV reflectivity on the attraction of tsetse to targets. Hence the selection of fabrics must be guided by spectral analysis and not just visual inspection of the cloth to identify fabrics that reflect strongly in the blue- but weakly in the UV-region of the spectrum. Second, if the only highly effective and colour-fast dye that is available can be used only on cotton, it might be acceptable to employ targets where the cloth panel is not treated with insecticide. Previous work has shown that treating a net with 0.8% deltamethrin results in >70% mortality for at least 9 months The data presented support the view that phthalogen blue cotton is at least as effective as any other material tested, and probably more effective than most or all of them. This is unfortunate given the declining availability of phthalogen blue dye, the technical problems with cotton, and the difficulties of dyeing artificial fibres with phthalogen blue. There seem to be two main options. First, it might be useful to look for other alternatives to phthalogen blue dyes – ones that can be used with polyester. Scientists have searched for such options previously In any event, allowing that it might be useful to screen cloth materials for use with those types of target in which only the netting is impregnated with insecticide, it would be useful if future screening tests employed not only the present fully-electrified targets but also targets in which the grid is restricted only to the net.Appendix S1Dying procedure of polyester materials Blue 9–13, Purple 2, White 2. Detailed dying procedure for materials Blue 9–13, Purple 2, White 2.(DOCX)Click here for additional data file.Figure S1Reflectance spectra data. Data for reflectance spectra of the 37 different materials used measured at Danish technological service institute (http://www.dhigroup.com) on a Shimadzu dual beam photometer, from 190–900 nm at 10 nm intervals.(XLSX)Click here for additional data file.Figure S2Reflectance spectra graphs for each experiment. Graphs of spectral reflectivities of materials used in each experiment, on the y-axis is percent reflectivity and on the x-axis wavelength in nm.(XLSX)Click here for additional data file.


Infections with high-risk human papillomaviruses (HPV) account for approximately 5% of all human cancers. Cervical cancer, in particular, is almost universally caused by “high-risk” HPV infections and these HPVs also cause a large fraction of other anogenital tract cancers as well as oral carcinomas. The viral E6 and E7 proteins are the universal oncogenic drivers of HPV-associated cancers, and persistent E6/E7 expression is necessary for the maintenance of the transformed state. Hence the HPV E6 and E7 proteins should represent excellent therapeutic targets. The HPV E6 and E7 proteins lack intrinsic enzymatic activities and attempts to discover small molecule inhibitors of E6 or E7 containing cellular protein complexes have been futile. HPV E6 and E7 function by associating with host cellular proteins, thereby functionally re-programming cellular signal transduction pathways. The resulting rewiring of cellular circuitry causes “addictions” to certain signaling pathways that are non-essential and/or redundant in normal cells. This in turn generates specific cellular vulnerabilities that may be more readily druggable.INK4A (p16) tumor suppressor and executed by the retinoblastoma tumor suppressor protein, Rb. In normal cells, p16 expression is epigenetically silenced by polycomb repressive complexes. p16 inhibits cyclin dependent kinase 4 and 6 (CDK4/6). This causes accumulation of hypophosphorylated Rb, which in turn signals cell cycle arrest  identifies a surprising and potentially druggable vulnerability in the retinoblastoma tumor suppressor pathway caused by the high-risk HPV E7 protein. High-risk HPV E7 protein expression triggers a cellular defense response referred to as oncogene induced senescence (OIS), which is mediated by the p16It has been noted early on that p16 expression is confined to those tumors that contain pRB mutations, and a number of non HPV-associated cancer types, including some breast, prostate, lung and high-grade serous ovarian carcinomas express p16. It will be important to determine whether some of these tumors are similarly addicted to p16 expression and whether they are vulnerable to inhibition of KDM6B and/or other epigenetic enzymes that are necessary for p16 expression.


Information on human papillomavirus (HPV) type distribution is necessary to evaluate the potential impact of current and future HPV vaccines. We estimated the relative contribution (RC) to invasive cervical cancer (ICC) and precancerous cervical lesions of the nine HPV types (HPV 6/11/16/18/31/33/45/52/58) included in an HPV vaccine currently under development.Estimations on ICC were based on an international study of 8,977 HPV positive cases and estimations on precancerous cervical lesions were extracted from a published meta-analysis including 115,789 HPV positive women. Globocan 2008 and 2010 World Population Prospects were used to estimate current and future projections of new ICC cases.RC of the 9 HPV types in ICC was 89.4%, with 18.5% of cases positive for HPV 31/33/45/52/58. Regional variations were observed. RCs varied by histology, ranging between 89.1% in squamous cell carcinomas (SCC) and 95.5% in adenocarcinomas (ADC). HPV 16/18/45 were detected in 94.2% of ADC. RC of the 9 types altogether decreased with age (trend test p < 0.0001), driven by the decrease in older ages of HPV 16/18/45. In contrast, the RC of HPV 31/33/52/58 increased with age. Due to population growth alone, projected estimates of ICC cases attributable to the 9 types are expected to rise from 493,770 new cases in 2012 to 560,887 new cases in 2025.The RCs of individual high risk HPV types varied by cytological and histological grades of HPV-positive precancerous cervical lesions, and there was an under representation of HPV 18 and 45 compared to ICC.The addition of HPV 31/33/45/52/58 to HPV types included in current vaccines could prevent almost 90% of ICC cases worldwide. If the nine-valent vaccine achieves the same degree of efficacy than previous vaccines, world incidence rates could be substantially reduced. Cervical cancer and other HPV related cancers represent an important global public health problem, both in more and less developed countries. Cervical cancer is the third most common cancer among women worldwide, with an estimated 530,000 new cases and 275,000 new deaths in 2008. More than 85% of the global cervical cancer burden occurs in developing countries and in 12 of the 22 regions of the world, cervical cancer remains the first or second most common female cancer [HPV carcinogenicity has been convincingly established for cervical cancer and it is generally accepted that HPV infection is necessary for the development of invasive cervical cancer (ICC) . The IntHPV prophylactic vaccines using virus like particles (VLP) have been recognized as a major advance and the most effective intervention to control for HPV and cervical cancer . CurrentFuture impact of HPV vaccination with current commercialized vaccines has been largely modelled ,14. ModeAfter HPV 16/18, data confirm HPV 31/33/35/45/52/58 as the most frequently detected types in ICC worldwide . Merck iThe aim of the present article is to estimate the potential impact of a new nine-valent vaccine in HPV-related cervical disease by summarizing the evidence on specific relative contribution (RC) of the 9 HPV types (HPV 6/11/16/18/31/33/45/52/58) in ICC and precancerous cervical lesions, in the pre-vaccination era.To estimate the RC of the 9 HPV types included in the V503 Merck's investigational HPV vaccine, we used data from an international study on HPV in ICC  and the The estimations on RC in ICC were based on data from a retrospective cross-sectional study carried out at Catalan Institute of Oncology, Spain (ICO) in collaboration with DDL laboratory, The Netherlands, to estimate the distribution of HPV types in women with ICC between 1949 and 2009 . BrieflyRC of the 9 HPV types included in the nine-valent vaccine (HPV 6/11/16/18/31/33/45/52/58) in ICC was expressed as the proportion of women positive for a given type among all the HPV-positive samples. The availability of HPV-disaggregated data made possible the calculation of RCs for HPV types alone or for the groups of HPV types. Multiple infections were added to single types in accordance with a proportional weighting attribution . For exaSubjects were classified into 10 geographical regions defined by the United Nations Population Divisions . RC was provided for the following histological categories: Squamous Cell Carcinoma (SCC), Adenocarcinoma (ADC), Adenosquamous Cell Carcinoma (ADSCC) and Other histology that included undifferentiated, neuroendocrine, not otherwise specified, basal adenoid and cystic adenoid carcinomas. Estimations of HPV RCs by age groups were also provided. Evaluations of the trends by age were determined by trend test analysis. Statistically significant p-value for this analysis was set at 0.05 level.Projections of new ICC cases attributed to HPV 16/18 and HPV 31/33/45/52/58 were calculated for year 2012 and projected to year 2025, worldwide and by region. Estimations were based on the following assumptions and data sources: a) Oncogenic HPV types may be detected in virtually all cases of cervical cancer, being generally accepted that the virus is necessary for the development of cancer, so the RC of all HPV types altogether is assumed to be 100%. Therefore, we assumed that the attributional fraction (AF) for each HPV type in ICC corresponded to the RC in ICC. The overall RC was used as the world AF, without weighting regional estimations; b) Globocan 2008 incidence rates were used with the assumption that these rates will apply in the future [The estimations on RCs in precancerous cervical lesions were based on data from a recent IARC meta-analysis . BrieflyThe RCs of the 7 individual HR HPV types (16/18/31/33/45/52/58) were expressed as the proportion of samples positive for a given type among all the HPV-positive samples tested for the mentioned type, therefore denominators vary by type. Individual-level data on HPV types in multiple infections were not available, so RC includes single and multiple infections and the total sum of RCs of the 7 HR HPV can exceed 100%. Subjects were classified into seven geographical regions defined by the United Nations Population Divisions  .In an attempt to estimate a combined RC estimation of the 7 HR HPV types altogether for CIN2/3 or HSIL lesions similar to that in ICC, we used Smith et al. meta-analysis   worldwide was 89.4% (95%CI: 88.8-90.1), with some regional variations, from 84.6% (95%CI: 81.9-87.1) in Central America to 95.5% (95%CI: 91.2-98.2) in North America. HPV 16/18 were the most common types accounting for almost 70% worldwide, except for Central America and North America where a RC of 64.4% (95%CI: 60.9-67.8) and 78.8% (95%CI: 71.6-84.8) were observed respectively. The additional RC of the other 5 high risk HPV types (HPV 31/33/45/52/58) was 18.5% (95%CI: 17.7-19.3); ranging from 8.8% (95%CI: 5.0-14.1) in Oceania to 22.3% (95%CI: 20.4-24.3) in Eastern Asia. HPV 45 was the most frequently detected type after HPV 16 and 18 worldwide except in Eastern Asia where RCs of HPV 52/58 were higher, and in Europe where RC of HPV 33 was higher. RC of HPV 45 was especially prominent in Africa , Western Asia  and Latin-America . HPV 31 was relatively more prevalent in Latin-America  and HPV 33 was more prevalent in Europe , as previously remarked. Infections by HPV 52/58 were more usually diagnosed in Eastern Asia . Addition of HPV 6/11 did not modify the cancer burden estimates (RC: 0.1%)  in SCC to 95.5% (95%CI: 93.3-97.2) in ADC. HPV 16/18/45 were consistently the three most common types in all histological categories accounting for 94.2% (95%CI: 91.7-96.2) of ADC cases compared to the 75.4% (95%CI: 74.5-76.3) contribution in SCC. A greater contribution of HPV 18/45 was observed in ADC, ADSCC and other histologies compared to that in SCC and conversely, HPV 16 RC was higher in SCC. Substantial differences in the RCs by histological category were observed for the other HPV types, with much higher contributions of HPV 31/33/52/58 in SCC (RC: 13.6%) and ADSCC (RC: 7.2%) compared to the RC of less than 1% and 3% observed in ADC cases and other histologies, respectively ; however, this was mainly explained by the decrease in older ages of HPV 16/18/45. In contrast, ICC cases in women positive for HPV 31/33/52/58 increased with age (trend test p < 0.0001), although HPV 16/18/45 were also the most common types in older ages. Remarkable differences were found in ICC cases positive for HPV types other than those included in the nine-valent vaccine, with RCs ranging from 4.2% (95%CI: 0.5-14.3) in women aged less than 25 years to 19.4% (95%CI: 13.7-26.0) in women older than 80 years  in HPV-positive HSIL and 58.2% (95%CI: 57.3-59.1) in HPV-positive CIN3 diagnoses. HPV 45 was consistently detected less often than other HR types included in the nine-valent vaccine in all cytological and histological categories . Regarding HPV-positive high-grade lesions, HPV 31 was relatively more prevalent in Western and Central Asia, and similarly to that found in ICC; HPV 52/58 showed higher RCs in Eastern Asia . Similar to ICC lesions, multiple infections were added to single types in accordance with a proportional weighting attribution. Multiple infections were higher in included studies compared to that in not included studies . Despite the consistency on the overall RC of the 9 HPV types observed in ICC, large variations between articles were observed for CIN2/3 or HSIL lesions, with RCs of the 7 HR HPV types ranging from 58.8% to 93.6%. Individual information on LR HPV 6/11 was available in 10 studies. Addition of HPV 6/11 did not modify the CIN2/3 or HSIL burden estimates (less than 1%).We provided information on HPV distribution of 9 types included in a broad spectrum vaccine currently under development for 10 regions of the world and across different grades of cervical lesions. Although existing vaccines cover HPV 16/18 that are responsible of 70% of ICC worldwide, the inclusion of HR HPV 31/33/45/52/58 would increase the protection to almost 90% of the infections responsible for ICC.As previously reported, HPV 16 is uniformly the most common HPV type in all histological types of cervical cancer and all the regions of the world ,17. Our RC estimations in Africa from ICC study reflect that 52% of ICC cases were infected with an HPV type different from HPV 16. The different RCs on HPV genotypes in Africa are not fully understood. Different RCs may be related to host genetic determinants or could be the result of a different distribution on HPV types in HIV-infected women compared to that of the general population ,25. In AOur data confirm that HPV 16 is the most common type in all cervical lesions. By contrast, HPV 18/45 are underrepresented in HPV-positive women with precancerous cervical lesions compared to the high contribution in ICC, where these types are consistently the second and third most common types in most of the world regions ,17. ReceIt should be remarked that HPV 16/18/45 were detected at younger ages in ICC compared to other HPV types. Particularly for HPV 18/45, the detection at younger ages together with the underrepresentation in precancerous cervical lesions suggest a rapid progression to ICC of both HPV types, with or without transition through preinvasive stages ,28.Due to the restricted genotype contribution in the pathogenesis of ADC subtype, higher rates of protection with the vaccine could be expected, compared to SCC . Similar to previous findings a greater contribution of HPV 18/45 in ADC, ADSCC, and other histologies (such as neuroendocrine tumours) was observed compared to that in SCC and conversely, HPV 16 RC was higher in SCC -31. Our The existence of multiple infections complicates the estimation of the attribution of cases to HPV types. Assessment of RC of multiple infections was possible for ICC lesions, where multiple infections were added to single types in accordance with a proportional weighting attribution. We also calculated a more conservative RC by combining single type infections and multiple type infections restricted to the exact combination of the 9 mentioned types obtaining a RC close to 86% worldwide, meaning that the HPV types included in the nine-valent vaccine could cover the majority of infections responsible of ICC.No similar attribution of multiple infections could be performed for precancerous cervical lesions from Guan et al. meta-analysis, meaning that HPV positive women with multiple infections could be counted more than once. Hence RCs for precancerous lesions was shown only for individual, and not combinations of HPV types. Although this approach does not allow to estimate type specific risk, it can be useful to identify HPV variations by region or lesion severity, as it has been confirmed that HPV type is the strongest factor that affects the risks of progression of precancerous cervical lesions given viral persistence, with HPV 16 and 18 less likely to regress than other HPV types .CIN2/3 lesions rarely occur in absence of HR HPV infections ,34,35, bFor estimations of HPV attributable cases, we assumed that current incidence rates will apply in the future, and calculated expected numbers using the appropriate population forecast. We did not take into account the impact of screening programs and current vaccines that will influence future rates since this type of analysis is more suitable for more complex analysis such as simulation models. Particularly for HPV vaccines it has to be taken into consideration that vaccination programs are not implemented worldwide, the coverage is still very low  and the impact on the reduction of invasive lesions will need more than ten years to be observable. The overall RC was used as the world AF. Even weighting regional estimations by world populations, the world attributable number of cases did not differ. Irrespective of changing risk, demographic changes will have a major impact in the future burden of ICC and this will be especially remarkable in developing countries where, in general incidence and mortality rates have been relatively stable over decades, reflecting the lack of screening implementation . These eThe good concordance of HPV estimations with previous reports suggests the representativeness of the study data, and especially in those regions with large sample sizes. Eighty five percent of all ICC from the ICC study were positive for DNA, compared to the ninety percent estimated in the most recent IARC meta-analysis on ICC available in the literature . Given tThe low number of cases in the ICC study from Oceania and North America could be a potential limitation. Although RCs for Oceania are similar to that observed in previous reports, estimations of RCs for North America based on this study differ to that observed in the IARC meta-analysis and the largest study performed in this region by Wheeler et al. ,36, withIrrespective of histological type and similar to previous reports , HPV 16/The inclusion of additional HPV types in nine-valent vaccine currently under development could have direct implications for cervical cancer incidence and prevention in all regions of the world. If the nine-valent vaccine achieves the same degree of efficacy as has been shown for HPV 16 and 18, and vaccination programs are effectively implemented, almost the 90% of ICC cases worldwide could be prevented. This means that Globocan 2008 world incidence rates could be substantially reduced. If all our estimations are true, and assuming high vaccination coverage and no HPV type replacement, this intervention would provide a higher impact than any screening programme has ever produced in the very best scenarios.GMC has no potential conflict of interest. BS and ST: Institutional support: HPV vaccine trials and epidemiological studies sponsored by GlaxoSmithKline, Merck and Sanofi Pasteur MSD. LA: Institutional support: HPV vaccine trials and epidemiological studies sponsored by GlaxoSmithKline, Merck and Sanofi Pasteur MSD. Personal support: Travel grants to conferences occasionally granted by Merck and Sanofi Pasteur MSD. LB: Institutional support: HPV vaccine trials and epidemiological studies sponsored by GlaxoSmithKline, Merck and Sanofi Pasteur MSD. Travel grants to conferences occasionally granted by Sanofi Pasteur MSD. TW: Is currently working at Merck. FXB: Institutional support: HPV vaccine trials and epidemiological studies sponsored by GlaxoSmithKline, Merck and Sanofi Pasteur MSD. Personal support: Travel grants to conferences / symposia / meetings and honorarium are occasionally granted by either GlaxoSmithKline, Merck, Sanofi Pasteur MSD, Roche or Qiagen. SS: Institutional support: HPV vaccine trials and epidemiological studies sponsored by GlaxoSmithKline, Merck and Sanofi Pasteur MSD. Personal support: Travel grants to conferences / symposia / meetings are occasionally granted by either GlaxoSmithKline, Sanofi Pasteur MSD or Qiagen.BS, LA and ST were responsible for the data analysis. All authors contributed to the writing of the manuscript and BS was responsible for the preparation of the manuscript for submission. All authors read and approved the final manuscript.


The purpose of this study was to identify attitudes toward HPV vaccination among US women 27 to 45 years of age. A survey was mailed to 2,750 insured US women to assess perceptions of relevance or irrelevance of the HPV vaccine, the underlying reasons, and, for those reporting relevance, the likelihood of vaccination if it became available. Among the 451 eligible respondents, 304 (67.4%) reported that the HPV vaccine was relevant to them, whereas 143 (31.7%) stated that it was not at all relevant. The most common reasons for relevance were protection from cervical cancer (62.8%), vaginal cancer (58.2%), precancerous cells (55.9%), HPV (55.6%), and genital warts (46.4%). Reasons for irrelevance were most commonly being married (54.0%) or in a monogamous relationship (39.6%). Most respondents reporting relevance of the HPV vaccine were likely (33.4%) or extremely likely (37.7%) to receive the vaccine if approved for their age group. Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States . Low-risTwo HPV vaccines are licensed in the United States. A quadrivalent vaccine targeting HPV types 6, 11, 16, and 18 was approved in 2006 for females aged 9–26 , and a bHPV vaccine acceptability is generally high in women over 26 , 12 but This is the third of several reports on a survey of adult women's attitudes toward the HPV vaccine , 20. SubWomen were identified in the health plan database if they had an outpatient medical claim on an index date occurring between January 1 and April 30, 2007 (the identification period), were 27–45 years of age on the index date, and were continuously enrolled in the health plan during an observation period extending from 6 months before to 12 months following the index date. Women were excluded if they had a medical claim related to cervical cancer during the 18-month observation period or a claim for HPV vaccination during the identification period. A total of 141,130 women met the above criteria. Samples of 1,375 women were randomly selected from among those aged 27–34 and 35–45, for a total of 2,750 study subjects.Survey packets were mailed beginning in April 2008. Packets contained an invitation letter, an informed consent form, a 7-page survey, and a payment of $10 as compensation for participation. the reminder postcards were sent one week following the initial mailing, and a second survey packet was sent to nonrespondents after three weeks. The total survey collection period was 8 weeks.The survey collected information on demographic characteristics , overall health status, and general attitudes about reproductive health. A series of questions then assessed the respondents' attitudes toward HPV vaccination. After being asked to read a short paragraph about HPV, the HPV vaccine, and the types of diseases the vaccine prevents, respondents were asked “How relevant is this vaccine to you?” Possible answers were “extremely relevant,” “very relevant,” “relevant,” “slightly relevant,” and “not at all relevant.” If the respondent indicated that the vaccine was relevant (defined as answers ranging from extremely to slightly relevant), she was asked about her reasons for believing so. Possible reasons included the desire for protection from HPV and its various sequelae and beliefs about the safety and efficacy of the vaccine. Respondents who thought the vaccine was relevant to them were asked with which type of health care provider they would specifically discuss the vaccine. Possible answers were “gynecologist (OB/GYN),” “general/family practitioner (GP/FP),” “internist or internal medicine doctor (IM),” and “nurse practitioner/physician's assistant.” They were also asked how likely they would be to get the vaccine if it were offered in the future: “extremely likely,” “likely,” “unsure,” “unlikely,” or “extremely unlikely.” Respondents who indicated that the vaccine was not relevant to them were asked why they believed it was not relevant. Possible answers included not being at risk for HPV and its sequelae, needing more information about the vaccine, and being married or in a monogamous relationship.A descriptive analysis was conducted on the entire study sample. The results are presented as the number and percentage of respondents who gave a particular answer to each survey question. Demographic characteristics are given for all respondents, but the frequencies of reasons given for relevance or irrelevance of the vaccine are presented as a proportion of respondents in those subgroups. A stratified analysis of age groups 27–34 and 35–45 was carried out to determine whether there were any attitudinal differences between them.Of 2,750 surveys sent out, 457 were completed and returned, for an overall response rate of 16.6%. Six respondents were subsequently found to be ineligible due to HPV vaccination or disenrollment from the health plan, leaving 451 surveys available for analysis. Respondents  were preOf the 451 respondents, 304 (67.4%) reported that the HPV vaccine was relevant to them. Among these women, the most frequent reasons given for believing the vaccine to be relevant  were proAlmost all (92.8%) women who thought the HPV vaccine relevant would plan to discuss the vaccine with a gynecologist . Among wThere were 143 (31.7%) respondents who felt the HPV vaccine was not relevant to them. Among this group, the most frequently reported reasons for HPV vaccination not being relevant  were beiIn this insured population of women aged 27 to 45, about two-thirds felt the HPV vaccine was relevant to them, and, among these, nearly three in four were likely or extremely likely to get vaccinated if HPV vaccination were offered in the future to women of their age. One-third saw the vaccine as not relevant, most commonly because they were married or in a monogamous relationship or because they believed that they were at low risk for getting HPV infection and HPV-related disease.Only one other survey has specifically elicited the reasons older women have for wanting or not wanting the HPV vaccine , 21. In Younger women have similar reasons for wanting or not wanting the HPV vaccine. Belief in the severity of cervical cancer was associated with higher intent to be vaccinated among 13–26-year olds , and knoIn previous studies of nonvaccinees, 7 to 14.6% of women aged 18–26 cited cost as a reason for not being vaccinated , 25. CosAwareness of HPV and the HPV vaccine is generally high in women over 26. The National Immunization Survey of 2007 found that 82.9% of US women aged 27–48 had heard of HPV and 76.5% had heard of the HPV vaccine  . In CanaHealth care providers are women's most trusted source of information about HPV and the HPV vaccine , 29. In Physician recommendation may play an especially important role for married women, who in both younger and older adult age groups feel less at risk for HPV infection. Their perception is supported by prevalence data showing that HPV infection is twice as likely in never married women and three times as likely in widowed/divorced/separated or cohabiting women compared to married women . NeverthBecause the prevalence of HPV peaks among women aged 20–24 . PrelimiP < .001) and less likely to reside in the South . It is possible that this geographic imbalance may have produced bias in the estimation of reports of HPV vaccine relevance. In addition, respondents were given limited information on the efficacy of the vaccine, which may have affected their perceptions of relevance. Because of the study population chosen, the results may not be generalizable to women living outside the United States or women without health insurance. Finally, intention to get vaccinated may not translate into actual uptake [One limitation of our study was the low survey response rate. We were able to compare the age and geographic locations of respondents and nonrespondents using claims data. There were no statistically significant differences between these two groups in age, whether analyzed as a continuous variable or as a categorical variable (age categories 27 to 35 and 36 to 45 years). Similar proportions of respondents and nonrespondents resided in the Northeast and West, but respondents were more likely than nonrespondents to be located in the Midwest (32.6% versus 24.8%, In this sample of women aged 27 to 45, most felt the HPV vaccine was relevant to them because of its ability to protect them from HPV infection and HPV-related diseases. The majority of women reporting relevance were likely to consider vaccination if the vaccine became available to their age group. Women who did not feel the vaccine was relevant did not perceive themselves at risk for HPV infection or HPV-related diseases.


Oral squamous cell carcinoma is the sixth most common malignancy worldwide. In Bangladesh, it comprises 20% of the whole body malignancies. Several studies found that 15% to 25% of oropharyngeal cancer cases are associated with human papilloma virus (HPV). This study is done to find the association of human papilloma virus subtypes, particularly HPV type 16 and HPV type 18, with the oral squamous cell carcinoma in Bangladeshi patients. In total, 34 diagnosed patients of oral squamous cell carcinoma were included in the study. Extracted DNA from the cancerous tissues was checked for PCR reaction to detect the subtypes of human papilloma virus. Data of the present study suggest that oral squamous cell carcinoma are almost absent in Bangladeshi patients with human papilloma virus, particularly HPV 16 and 18. Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy worldwide, with major incidence in western countries as well as India and Southeast Asia ,2. Risk Detection of HPV may be dependent on several factors, e.g. molecular technique employed to detect the DNA materials, treatment of the sample material, ethnic and geographical differences, and the anatomic site of the lesion . Since 0In western countries, researchers have used retrospective case studies to investigate the possible link between the high-risk HPV strains and oral squamous cell carcinoma (OSCC). A recent study  showed tIn Bangladesh, no studies on the association of HPV subtypes with oral squamous cell carcinoma were published. This study is expected to assess whether Bangladeshi people possess the risk of having oral squamous cell carcinoma of HPV origin as reported in the literature of the western countries.It is a case series study done during the period of January 2009 to December 2009 in the Department of Oral and Maxillofacial Surgery, Bangabandhu Sheikh Mujib Medical University (BSMMU) and in the laboratory of the Department of Physiology and Molecular Biology of BIRDEM.Clinically-diagnosed 45 subjects were enrolled in the study, irrespective of race, religion, socioeconomic status, and geographic distribution. Among them, 34 cases were diagnosed by biopsy as oral squamous cell carcinoma.After infiltration with local anaesthesia, tissue was incised, with all aseptic precaution, in such a manner that both normal and pathological tissues are included.Extraction of integrated DNA from the sample tissue was done, using GenElute DNA extraction kit . Tissue sample was minced and placed in a micro-centrifuge tube to which protienase K was added. Contents of the tube with tissue buffer solution were incubated. Cell lysis buffer solution was readded to the tube and again incubated. After cooling the tube contents to room temperature, absolute ethanol was added and centrifuged. The column formed due to centrifuge was washed with buffer 1 solution, re-centrfuged, and again washed with the buffer solution 2. Then the resultant contents were taken in a DNA-collection tube to which DNA AE buffer was added and incubated, and the centrifuging was repeated. GenElute DNA kit was used for checking the yield and stored for future use.All samples were subjected to PCR, using primers specific for consensus sequence spanning the E6 open reading frame of high-risk HPV type 16, 18, 31, and 33.PCR was carried out in 15 μL reaction volume which contained :5 μL DNA solution21.5 mM MgCl200 μM dNTPs500 μM of each primer1 unit of HotStart Taq DNA polymerase The PCR conditions were as follows: initial incubation at 94 °C for 15 min followed by 35 cycles of reaction with step of denaturation at 95 °C for 45 seconds, annealing at 57 °C for 45 seconds and elongation at 72 °C for 45 seconds, and the 35th cycle was followed by a step of final elongation at 72 °C for 10 min.PCR product was checked for amplification in a 3% agarose gel stained with ethidium bromide. The optimum size of the product was ascertained comparing it with 100 bp DNA ladder. Positive samples were subjected to PCR, using HPV 16 and 18 type-specific primers. PCR product-size for HPV 16 is 238 bp and for HPV 18 is 268 bp.For HPV 16, PCR was carried out using the primer set:Forward: 5’-ATTAGTGAGTATAGACATTA-3’Reverse: 5’-GGCTTTTGACAGTTAATACA-3’.Reaction was carried out in 15 μL reaction volume, and the conditions were :Step of denaturation at 95 °C for 30 sec, annealing at 52 °C for 45 sec. and elongation at 72 °C for 45 sec for 35 cycles with an initial incubation at 95 °C for 15 min, and a step of final elongation at 72 °C for 10 min.For HPV 18, PCR was carried out using the primer set:Forward: 5’-ACTATGGCGCGCTTTGAGGA-3’Reverse: 5’-GGTTTCTGGCACCGCAGGCA-3’.Reaction was carried out in 15 μL reaction volume, and the conditions were as follows:Step of denaturation at 95 °C for 30 sec, annealing at 64 °C for 45 sec, and elongation at 72 °C for 45 sec for 35 cycles with an initial incubation at 95 °C for 15 min, and a final elongation at 72 °C for 10 min.For both reactions, PCR product was checked for amplification in 3% agarose gel stained with ethidium bromide.Among the 34 respondents, those who were diagnosed as quamous cell carcinoma patients, only 1 (2.9%) was in histological Grade 1, and the majority 23 (67.6%) were in Grade 2, and rest 10 were in Grade 3 .Only one specimen was found to have HPV, out of 34 oral squamous cell carcinoma patients .In DNA sequencing is a standard procedure to confirm DNA product. The specific primer set did not produce any product of optimum size; so, absence of HPV DNA was inferred.The human papilloma viruses are a family of icosahedral, non-enveloped viruses with a circular, double-stranded DNA genome, which has special affinity for epithelial cells. So far, more than 120 different types of HPV have been isolated , althougHigh-risk human papilloma viruses type 16 and 18, as aetiological agents of anogenital carcinomas, have been firmly established in the literature . BecauseA predilection of HPV for certain, especially non-keratinized anatomical sites in the oropharynx, was confirmed when HPV 16-related DNA sequences were detected in 16%, 51%, 60%, and 13% of SCC of the tongue, tonsil, Waldeyer's ring, and pharynx respectively ,16,17.Estimates of the prevalence of oral HPV in normal mucosa, pre-malignant lesions, and OSCC are highly variable, and comprehensive reviews have reported results that varied from 0% to 100%. These results seemed to depend on the sampling methods, patient's profile ,19, deteThe major aim of this study was to see the possibility of having any evidence for HPV association as a risk factor of OSCC in our community, and if so, to find out the presence of type 16 and type 18 particularly.et al. . Shroyer et al.  reported . This study provides some evidence as mentioned.It has been noted that there is decrease in the HPV PCR positivity with increasing distance from the site of the tumour. Part of the tumour where the DNA copy number is mostly available is designated as focal point of HPV infection . So, sitDespite the mismatches at the base nucleotide inserted in the outer reverse primer region, the primers are able to amplify HPV. This is probably due to the low annealing temperature which allows for maximum amplification efficiency and for mismatches between the primers and template DNA. The primers have also shown that these are able to detect a broad range of mucosal HPV types and are, therefore, likely to be able to amplify novel mucosal HPV types . Also, tInfection of the mucosa with HPV may increase the susceptibility of the epithelium to subsequent chemical carcinogenesis, and the nature of these co-carcinogens may vary from region to region and with the social and dietary habits of various people. This variation in both incidence of HPV infection of the oral mucosa and the nature of the chemicals that act as promoters, initiators, or as simple co-carcinogens of the SCC, may be important in explaining the wide variability of HPV detection in various head and neck carcinomas around the world . This vaPolymerase chain reaction (PCR) is the sensitive method available for detecting HPV in the tissues. However, a problem with the PCR-based detection is the possibility of false positivity, due to carry-over of the product and contamination of paraffin-embedded tissue during routine processing . Also, dIn this study, we tried to find any association of HPV with oral squamous cell carcinoma among Bangladeshi population. The association between HPV and head/neck cancers are well-established, although the aetiological factors are still unknown. It is seen that HPV has predilection for primary tonsillar carcinomas but is rare for the pharynx or the hypopharyx . As this