Studies of human papillomavirus (HPV) awareness and HPV vaccine acceptability have included few non-white participants, making it difficult to explore ethnic differences. This study assessed HPV awareness and HPV vaccine acceptability in a sample of women representing the major UK ethnic minority groups.A cross-sectional study design was used to assess awareness of HPV and acceptability of HPV vaccination. Participants were recruited using quota sampling to ensure adequate representation of ethnic minority women: Indian, Pakistani, Bangladeshi, Caribbean, African and Chinese women (n = 750). A comparison sample of white British women (n = 200) was also recruited.Awareness of HPV was lower among ethnic minority women than among white women (6–18% vs 39% in white women), and this was not explained by generational status or language spoken at home. In a subsample who were mothers (n = 601), ethnicity and religion were strongly associated with acceptability of HPV vaccination. Acceptability was highest among white mothers (63%) and lowest among South Asians (11–25%). Those from non-Christian religions were also less accepting of the vaccine (17–34%). The most common barriers to giving HPV vaccination were a need for more information, sex-related concerns and concern about side-effects. South Asian women were the most likely to cite sex-related concerns, and were also least likely to believe the vaccine would offer their daughters protection.These findings suggest some cultural barriers that could be addressed in tailored information aimed at ethnic minority groups. They also highlight the importance of recording ethnicity as part of HPV vaccine uptake data. The introduction of human papillomavirus (HPV) vaccination aims to reduce the incidence of cervical cancer. The three-dose vaccine is highly effective, providing protection against HPV types 16 and 18, which are responsible for approximately 70% of cervical cancers.In the UK, two large studies have explored attitudes to HPV vaccination.To limit inequalities and support informed choice, it is important to understand levels of HPV awareness and HPV vaccine acceptability across all sectors of the population. UK-based studies have included relatively few non-white participants, making it difficult to explore ethnic differences. Some of the studies were designed to be population representative, and so this was expected from the outset.It is essential that uptake of the HPV vaccine is equitable, or the immunisation programme could widen ethnic disparities in health. A first step is therefore to assess ethnic differences in acceptability of the vaccine, and to try to uncover the reasons for any differences. We carried out a survey recruiting participants from the major ethnic minority groups in the UK, with the aims of considering ethnic differences in awareness of HPV and acceptability of HPV vaccination.http://www.ethnicfocus.com) recruits participants from the largest ethnic minority groups in proportion to their representation in the UK population. Within each ethnic group, quota-based age distributions are fulfilled. The present sample had a total of 750 female participants from six ethnic minority groups  plus 200 white British women.Data were collected in two waves of the monthly omnibus survey conducted by Ethnibus (July and August 2008). Ethnibus . Multilingual interviewers visited households in these areas, and individuals, selected based on their self-reported ethnicity, were invited to participate. Data were collected during face-to-face interviews, and each participant received a £5 incentive. Ethnibus reported that approximately 70% of individuals who met the eligibility criteria agreed to be interviewed (based on figures recorded by the interviewers), and this was similar to response rates in other Ethnibus surveys. The research was exempt from formal ethics committee approval because it ensured complete anonymity.All participants were given HPV information based on the Department of Health’s leaflet designed for girls aged 12–13 years and their mothers. This was translated into the most common languages  by agency-recruited qualified translators, translating into their first language. For all the other languages , the information was verbally translated during the interview. Questions were developed from previous research and qualitative interviews with 20 ethnic minority mothers.After hearing the information, women were read the following: “If you have a daughter age 12–13 years, please think about her when answering the questions; if you don’t have a daughter age 12–13 years, please imagine that you do. Starting in September 2008, girls age 12–13 years (school year 8) will be offered the HPV vaccination in school. If your daughter were invited to have the HPV vaccination at school this autumn, would you agree to her having it.” Responses were made on a five-point scale (definitely not–yes definitely). Women were asked to give a reason for their answer which was recorded verbatim. All participants were asked to indicate the father’s role in deciding about HPV vaccination because qualitative work indicated possible ethnic differences in this.Socioeconomic class (SEC) was based on the occupation of the chief earner in the household and was coded: AB managerial/professional; C1 supervisory; C2 skilled manual; D semi-skilled/unskilled manual; E state pensioners or casual/lowest grade workers. These groupings are often used in market research.Data were analysed using SPSS 15.0. HPV awareness and HPV vaccine acceptability were recoded into binary variables. Women responding to the HPV awareness question were categorised as “aware of HPV” (yes) or “not aware of HPV” (no/don’t know). Mothers responding to the HPV vaccine acceptability question were coded as “acceptors” (yes probably/yes definitely) or “non-acceptors” (probably not/definitely not/unsure). Initially, mothers who were “unsure” were considered as a separate group but, as they were not distinctly different from those who said probably or definitely not, the two groups were combined. Univariate logistic regression analyses were run to identify predictors of HPV awareness and vaccine acceptability. Significant univariate predictors were entered into a multivariate model.2 analyses were used to explore reasons for the vaccine decision (cited vs not cited) and involvement of the father . For each analysis, different ethnic groups were compared with the white group.A coding frame was developed (by LM) and applied (by LM and AF) to the mothers’ verbatim reasons for their decision. Inter-rater reliability was “outstanding” (kappa  = 0.94). χThe sample included 950 women: white British (n = 200), Indian (n = 235), Pakistani (n = 164), Bangladeshi (n = 63), Caribbean (n = 130), African (n = 107) and Chinese (n = 51). Sample characteristics are shown in 2(18) = 123.76, p<0.001; Nagelkerke R2 = 0.21). Marital status, language and generation were no longer significant.Overall, 17% of women reported (at the end of the interview) that they had heard of HPV before taking part in the study. Univariate logistic regression explored predictors of HPV awareness; odds ratios (ORs) and confidence intervals (CIs) are shown in 2(13) = 129.14, p<0.001; Nagelkerke R2 = 0.26).Just under half the sample (n = 440) had a daughter under 16 years old and, of these, 137 had a daughter aged 12–13 years. An additional 17% (n = 161) had a daughter aged 16 years or older. Acceptability did not differ based on the daughter’s age, so all women with a daughter, regardless of her age, were included in the analysis of acceptability (n = 601). Overall, 38% of mothers were “acceptors”  and 62% were “non-acceptors” . Univariate logistic regression explored predictors of HPV vaccine acceptability , sex-related concerns (eg “it encourages promiscuity” or “risk of premature sex”), religious/cultural reasons  and concerns about safety. Other reasons not to give the vaccination included believing it would be unnecessary or inappropriate, age-related reasons (eg 12–13 years is too young), lack of trust in vaccinations and wanting to discuss the decision with other family members.2 analyses had an expected count of less than 5).We explored whether some ethnic groups were more or less likely to cite specific reasons for their decision. Citing the “protection” that the vaccine would offer occurred less among Indian (16%), Pakistani (11%), Bangladeshi (13%) and Caribbean mothers (14%) compared with white mothers (29%), but there were no differences between white and African or Chinese mothers. Needing more information was cited more by Bangladeshi mothers than by white mothers (44% compared with 16%), but there were no differences between white mothers and mothers from any other backgrounds. We were able to look at differences for Indian and Pakistani mothers in citing sex-related reasons, religious reasons, daughter’s health and future benefits. Sex-related reasons were more likely to be cited by both Indian (15%) and Pakistani mothers (20%) than white British mothers (2%). None of the other reasons differed significantly. Limited numbers meant that differences could not be explored for Bangladeshi, Caribbean, African and Chinese women .Acceptability of HPV vaccination for a 12- to 13-year-old daughter was much lower in ethnic minority mothers than in white mothers, with the lowest levels among mothers from South Asian backgrounds. This was the case even after controlling for SEC. This suggests that previous research with predominantly white British parents cannot be extrapolated to ethnic minority parents, emphasising the importance of including minority women in future research. If these findings map on to uptake, it may mean that some areas of the UK achieve particularly low uptake. The non-white population accounts for 8% across Britain, but this rises to 30% in some areas , and is even greater in parts of London .Generation and language spoken at home were not significant predictors of acceptability in multivariate analyses, suggesting that acculturation does not mediate the effect of ethnicity. However, religion remained an important factor, and “religious reasons” were one of the main reasons cited for declining the vaccine. This is consistent with previous work that found parents with “strong religious or cultural views” were less likely to accept HPV vaccination.Across the board, one of the most common barriers to accepting HPV vaccination was concern about side-effects. This is consistent with a recent study of HPV vaccine uptake,We decided to ask about awareness of HPV at the end of the interview, so that women knew what HPV was before saying whether they had heard of it. This makes it unlikely that levels of awareness are underestimated in this study, and means that the findings may not be comparable to other studies which have assessed it at the beginning of the interview. Ethnicity was associated with HPV awareness, with much lower levels in all the ethnic minority women, particularly Indian, African and Bangladeshi women (<10%). Interestingly, language spoken at home did not explain this effect, suggesting that it is not purely the result of a language barrier. One alternative explanation could be variation in exposure to publicity about HPV because of lower coverage in culturally specific media or because sex-related topics are considered “taboo” and as a consequence are discussed less in some cultures. Given that knowledge of HPV is necessary for informed decision-making about vaccination, tailoring information campaigns and channelling these through media outlets aimed at specific ethnic groups may help to raise awareness.One additional finding of interest was that ethnic minority mothers were more likely to believe the father would play an important role in the decision to vaccinate against HPV. To date, there has been little research into fathers’ attitudes in the UK, and the Department of Health leaflet about HPV has been aimed at girls and their mothers. Future research exploring parental decision-making with regard to HPV vaccination among ethnic minorities should include fathers.There are several limitations to this study. The sampling method means the findings may not be generalisable to all ethnic minority mothers in the population, although the quota sampling technique allowed us to focus on and compare ethnic minority groups. The response rate of 70% was good; however, the figure is based on details recorded by the field interviewers. As the interviewers sometimes forget to report these response details, Ethnibus describe the response rate as “approximate” rather than exact. The response rate should therefore be interpreted with caution. Another issue is that, although all mothers in the acceptability analysis had a daughter, not all daughters were aged 12–13 years, and this meant that some mothers were responding hypothetically. Third, we did not assess actual vaccine uptake, and statements about intention can only be indicative of acceptance of HPV vaccination. Expected vaccine acceptance in this sample of white mothers was 63%, which was lower than the 75% and 81% that have been found in other studies.Over the coming years, it will be possible to incorporate behavioural outcomes into work on HPV attitudes, but for now exploring acceptability offers some insight into a decision that is facing parents of adolescent girls right now. It gives a head start in understanding possible inequalities before uptake data from the first cohorts of girls are available. Understanding ethnic differences in acceptability of HPV vaccination will be particularly useful to health promotion specialists working in ethnically dense areas of the country, helping them to limit inequalities and support informed choice by all.HPV vaccination is now offered to all girls aged 12–13 years.In one study, uptake was lower in schools with a higher proportion of ethnic minority pupils.One criticism of the UK-based studies is their limited inclusion of non-white participants.Ethnicity and religion are predictors of HPV awareness and acceptability of HPV vaccination.Sex-related concerns are more likely to be a barrier among ethnic minority parents.The ethnic differences found in this study support the importance of monitoring ethnicity in vaccine uptake data.


HPV type distribution by cytological status represents useful information to predict the impact of mass vaccination on screening programs.women aged from 25 to 64 who attended cervical cancer screening in five different Italian regions were tested for HPV infection with Hybrid Capture II (HCII) low and high risk probes. Women repeating Pap-test upon unsatisfactory or positive results, or as a post-treatment and post-colposcopy follow-up analysis, were excluded from our study. High risk (HR) HPV positive samples were typed using GP5+/GP6+ primed PCR, followed by Reverse Line Blot for 18 high/intermediate risk HPV types, while low risk (LR) HPV positive samples were tested with type specific primers for HPV6 and HPV11.3410 women had a valid HCII and Pap-test. The prevalence of HR and LR infections was 7.0% and 3.6%, 29.1% and 13.7%, 68.1% and 31.9%, 60.0% and 0.0%, 65.0% and 12.0%, for negative, ASC-US, L-SIL, ASC-H and H-SIL cytology, respectively. The fraction of ASC-US+ cytology due to HPV 16 and 18 ranged from 11.2  to 15.4% (including HPV 16/18 in co-infection with other virus strains), and that due to HPV 6 and 11 ranged from 0.2%  to 0.7% (including HPV 6/11 in co-infection with other LR virus strains).mass vaccination with bivalent or quadrivalent HPV vaccine would modestly impact on prevalence of abnormal Pap-test in screening. In Italy, vaccination against HPV was included in the routine vaccination schedule at the beginning of 2008 and has since been actively offered to all 11-year old girls [Since 2006, both the Ministry of Health and the Centre for Disease Prevention and Control have planned and commissioned a set of studies to assess HPV epidemiology before introducing the vaccine and to predict the impact of a mass vaccination campaign [In particular, the aim of the present study was to measure the prevalence of different HPV types in the general population attending screening programs in the metropolitan area of Rome and southern Italy.Here, we report the proportion of HPV types by cytological status. We calculated the proportion of abnormal cytology attributable to HPV 16-18 and 6-11 infection.The study was conducted within cervical cancer screening programs that actively invite the entire target population (age ranging from 25 to 64) in Rome and southern Italy. A convenience sample of the existing screening programs was selected, because the study needed active participation to the local screening programs and formal approval from the Local Health Unit director. Nevertheless, our final sample included most of the well established programs in the south of Italy. The sample included 7 programs in 5 different regions: Abruzzo, Campania, Lazio, Sardinia, and Sicily. The planned sample size was 4000 women, 800 for each region. The structure and the statistical power of our sample have already been described in detail elsewhere . The recCervical scrape samples were collected in ThinPrep vials containing PreservCyt  transport medium or in Specimens Transport Medium (STM) . Before testing, 400 μl of STM samples, vortexed on a shaking platform at 1000 rpm for at least 10 minutes, were biobanked at -80°C for typing procedures in case of HCII positivity. Only in one centre , the primary screening test was HPV, consequently data from this centre were not informative for this study.in situ). In two centres, pathologists reported also benign cellular changes or other reactive cellular abnormalities that refer to the previous classifications. In any case, these women were all referred to normal screening interval (i.e. three years) and were included in the negative cytology class.The cytology was interpreted locally at each centre according to the Bethesda 2001 system (TBS2001) . We grouAll screening pathology units participate in routine quality control programs based on the circulation of standard slide sets .The molecular biology methods have been described in detail elsewhere (3). Here we briefly report the adopted typing strategies.® (HCII)   using probemix B, specific for 13 HR HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, and probemix A, specific for 5 LR HPV types: 6, 11, 42, 43 and 44 [The presence of high risk (HR) and low risk (LR) HPVs in cervical specimens was evaluated by Hybrid Capture IITyping procedures were centralized in Florence at the ISPO laboratory and all centres involved in the study sent HR or LR HPV positive samples there. The types included in IARC group 1 and 2a were classified as HR strains, the other types as LR ones .® solution and 200 μl of STM samples using QIAamp DNAMini Kit.DNA was extracted from 1.5 ml of samples in PreservCytHCII HR HPV positive specimens were amplified and typed with "consensus High Risk HPV genotyping kit" (Qiagen), a system based on PCR with biotinylated GP5+/GP6+ primers , followeGP5+/6+PCR-negative and reverse Line Blot-negative samples were amplified for the β-globin gene using GH20-PC04 primers (268 bp amplicon lenght) to assess the integrity of DNA .β-globin positive samples were re-typed with "INNO-LiPA HPV genotyping Extra Amp"  following the manufacturer's instructions. This system targets 28 HR or LR HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74 and 82. β-globin negative samples were extracted and typed again.The remaining un-typed samples  were considered untyped HPV (HPV X).HCII LR HPV positive specimens were typed using HPV6 and HPV11 specific primers . No LR tThe prevalences of HR and LR HPV types by cytological class are reported. The proportion of HPV 16-18 and HPV 6-11-16-18 by cytological class are reported, including and excluding cases with co-infection of other HPV types. All analyses were performed separately for women under 35.Co-infections of vaccine types with other types not included in vaccines are classified as follows: for HR vaccine types, HPV 16-18 alone , HPV 16-18 + HR (HPV 16-18 + any non-vaccine high risk type independently from the presence of LR types), HPV 16-18 + LR . Similarly, for LR vaccine types, HPV 6-11 alone , HPV 6-11 + HR , HPV 6-11 + LR .We estimated the age-adjusted Population Attributable Risk of Cytology abnormalities due to HPV vaccine type using a logistic model, according to the algorithm proposed by Fleiss , while 9The analysis was performed taking into account the two stage sample using STATA 8 survey module .The study was approved by the Ethics Committee of the Istituto Superiore di Sanità . Women were informed of an additional HPV test in the screening procedure and asked to sign a consent form.Table Table Prevalence of HR infections is double in women with inadequate cytology as compared to that observed in women with negative cytology.Table In women younger than 35, the HPV16-18 attributable fraction is slightly higher, even if not significantly: 14.6% and 21.2% for ASC-US+ and L-SIL+, respectively including only LR co-infections; 19.7% and 30.3% for ASC-US+ and L-SIL+, respectively, if we also include co-infections with other HR types. The inclusion of HPV 6 and 11 adds 1.9% to the attributable fraction of the ASC-US cytology and has no effect on the L-SIL+ cytology.The overall HPV prevalence found in this study was similar to other Italian population-based studies ,15. A deWhile, as expected, the prevalence of HR infections increases with cytology severity, it is worth underlining that HR HPV prevalence is double in inadequate samples as compared to negative ones. A cost effectiveness analysis of a possible use of HPV DNA test  comparedThe HPV prevalence in general population is quite low in Italy compared to other countries. More surprisingly, the observed low prevalence persists in all cytology classes, compared to other studies -20. TherIn all cytology classes HPV 16 is the most prevalent type and, overall, its prevalence is more than twice the prevalence of the type ranking second, HPV31. Nevertheless, the proportion of cytological abnormalities reasonably due to HPV 16 or 18 is negligible for ASC-US, moderate for L-SIL and relevant only for ASC-H and H-SIL, even if for these two last cytological classes our estimates are based on few observations and cannot be accurate. Given the relative frequency of cytology classes in the screening population, the overall impact on screening program positivity rate of a bivalent vaccine (16-18) would be 12-15% and that of a quadrivalent vaccine (6-11-16-18) would be 13-16%. A modest effect compared to the variability observed among different programs or to what would be obtained with a triage test strategy for ASC-US samples, still scarcely implemented in Italian screening programs.Furthermore, we must take into account that reduction in prevalence will probably increase the proportion of false positives, i.e. a decrease of Pap-test Positive Predictive Value for low grade lesions . ConsequActually, the targets of mass vaccination are pre-adolescent girls and screening programs will see the vaccinated cohorts when these girls are 25. Even if we consider only younger women, the impact of vaccine on cytology positivity would not be substantially different.On the other hand, a more relevant impact on CIN2 and CIN3 reduction is expected: in a large study conducted in the same geographic area  more thaThe shift to HPV DNA as primary screening test may change the scenario ,26, but We tested women who were invited and participated to cervical cancer screening programs in Rome and southern Italy. Response to organised screening is quite low, ranging from 25 to 50% in the selected areas; low participation may introduce a self-selection bias. Nevertheless, active invitation, for the study or for the screening, is the only mean we had of reducing self-selection bias in surveys. Previous studies found few socio-economic differences between participants and not participants ,29.A second limit, due to the nesting of this study into organised screening programs, is that we could not include some regions, such as Calabria and Apulia, because at the time the study started there were no active programs contacting the whole target population.We typed only women positive to HR and LR HCII probes; according to this protocol we may have missed women who are positive to other HPV types not included in the probes or samples with very low number of virus DNA copies. This typing strategy was the same used in all the pre-vaccination studies sponsored by the Ministry of Health making the results comparable ,15 and iIn this report, the vaccine cross-protection against HPV strains not present in the vaccine mix was not taken into consideration. On this issue, only rough estimates can be put forward. Moreover, different definitions of cross-protection values are reported: for quadrivalent vaccines, cross-protection refers to HPV 31, 33, 45, 52, 58 infection and was estimated to be 25% . AccordiThe impact of mass vaccination on cytology screening programs recall rate would be negligible, while probably its impact on CIN2+ detection rate would be much stronger. Our data support the hypothesis that the positive predictive value of Pap test in vaccinated women would be dramatically lower than in non-vaccinated women, as argued by several authors.®; HPV: human papillomavirus; HR HPV: high risk human papillomavirus types; H-SIL: High Grade Squamous Intraepithelial Lesion; LR HPV: low risk human papillomavirus types; L-SIL: Low Grade Squamous Intraepithelial Lesion; L-SIL+: Low Grade Squamous Intraepithelial Lesion or more severe, it includes L-SIL, ASC-H, H-SIL and cancer; NTCC: New Technologies in Cervical Cancer; PAR: Population Attributable Risk; PCR: polymerase chain reaction; RLU/CO: Relative light units/cut-off; STM: standard transport mediumAGC: Atypical Glandular Cellls; ASC-H: Atypical Squamous Cells non excluding High Grade lesions; ASC-US: Atypical Squamous Cells of Undetermined Significance; ASC-US+: Atypical Squamous Cells of Undetermined Significance or more severe cytology, it includes ASC-US, L-SIL, ASC-H, H-SIL and cancer; HCII: Hybrid Capture IIFinancial: PGR received travel reimbursement for two international conferences by Sanofi Pasteur MSD; FMC is occasional advisor to Gen-Probe, Abbot, Sanofi and GlaxoSmithKline.Non financial: PGR is employed in the HTA Unit of the Agency for Public Health of the Lazio Region, this Agency contributes in establishing public screening programs.PGR designed and conducted the study and drafted the paper; FCa designed the laboratory procedures, coordinated the laboratory quality control and contributed in drafting the paper; SB and EB performed the typing test; FCh designed the data flow, managed the data and performed the statistical analyses; AS, AL, RM, MTP, ADI coordinated the study in the recruiting centres. The Working Group contributed in recruiting the women, performing the HPV tests, collecting the data.All the authors approved the final version of the paper.


This study examined whether there is a positive correlation existed between cervical and oral High Risk-Human Papilloma Viruses (HR-HPV) types 16, 18 infections in patients with clinically confirmed cervical lesions.In this study 50 participants were included (40 were cases and 10 were controls). One hundred DNA materials  were analyzed using HR-HPV subtypes 16 and 18 specific PCR probes.Of the 40 cases, HR-HPV 16, 18 were identified in 16/40 (40%), of the cervical tissues of whom 8/16 (50%) were positive for HPV 16; 6/16 (37.5%) were identified with HR-HPV 18, and 2/16 (12.5%) were detected with both HR-HPV subtypes. All of the clinically healthy cases were found negative. Only one oral tissue sample (case) was 1/40 (2.5%) was found positive for HPV subtype16.The frequency of infection with HR-HPV subtypes 16 and 18 is high among Sudanese women with cervical lesions and suggests a role of HR-HPV in the development of cervical cancer in Sudan. No correlation between cervical and oral HPV infection was noted. Further study with screening of large number of patients with cervical cancer is recommended for further clarification of these findings. Cervical cancer is the second most common cancer found in women with approximately 530,000 new cases each year resulting in an estimated 275,000 deaths, worldwide. In developed countries, cervical cancer incidences have declined, mostly due to cervical cytology screening campaigns, which requires significant medical resources and laboratory infrastructure. Cervical cancer is on the rise in the developing world, with one-seventh of the world’s cervical cancer cases in China, where no nationwide screening program for the disease currently exists . In SudaHPV infection has also been postulated as a potential risk factor for OSCC. Several studies have detected HPV DNA in a considerable proportion of oral cancers, with wide variations from 0% to 100% prevalence in oral tissues, perhaps reflecting the inherent variations in the different populations, .In Sudan, invasive cervical cancer is a leading cause of cancer death among women  . Most cFurthermore, a recent study has reported strong association between HPV types 16 and 18 and OSCC, although, some prior studies have reported a lack .However, due to the lack of published data from Sudan in this context, the aim of this study is to examine whether there is a positive correlation existed between cervical and oral HR-HPV types 16, 18 infections in patients with clinically confirmed cervical lesions.In this descriptive study, 40 patients with clinically confirmed cervical lesions (cases) 10 clinically confirmed with no cervical lesions (controls) were investigated for the presence of HR-HPV subtypes 16 and 18 PCR using molecular techniques. All subjects were randomly selected from those referred to gynecologic clinic at Al Nou hospital. All Participants were with apparently healthy oral cavity.The study was approved by the ethics board of the Faculty of Medical Laboratory Science, Sudan University for Science and Technology. All cervical samples were taken as a part of the specimens required for investigation. All study subjects were consented to participation by completing the self-administered questionnaire.The sample size represents a full coverage of patients that were referred to the gynecologic clinic and agreed to participate within 6 months period. One hundred samples  were collected as a part of the specimen required for screening for cervical cancer. Both cervical and oral cells were obtained from the same person and processed similarly. B scraped cells placed in sterile Caryo-tube containing 5 ml Tris–HCl buffer (PH 8.0) . The components were then centrifugated and the supernatant was discarded, and the deposit was kept immediately in the refrigerator in −200 C.DNA was extracted according to the steps described in DNA extraction kit purchased from Sacace biotechnologies-Casera –Italy. The pellet obtained from previous steps was treated with 300 μl of Reagent 2 (lysis buffer) in addition 100 μl of sample, vortexed, incubated at 65°C for 5 min and centrifuged at (12000–16000 g) for 10 min and transfer the supernatant into new tube (sterile 1.5 ml Eppendorf tube) for DNA extraction . Vortexed vigorously sorbent and added 20 μl to each tube, Vortexed for 5–7 sec and incubated all tubes for 3 min at room temperature, then this step was repeated. Then all tubes were centrifugated for 30 sec at 5000 g and used amicropipette with aplugged aerosol barrier tip, carefully removed and discarded supernatant from each tube without disturbing the pellet. Tips was Changed between the tubes. 500 μl of Washing Solution was added to each tube. Vortexed vigorously and centrifuged for 30 sec at 10000 g. Supernatant was removed and discarded from each tube. This step was repeated and incubated all tubes with open cap for 5–10 min at 65°C. The pellet was resuspended in 100 μl of DNA-eluent. Incubate for 5 min at 65°C and vortex periodically. The tubes were centrifuged for 1 min at 12000x g. The supernatant was containing DNA ready for amplification stored at - 20°C until used.Type specific primers (primer for HPV 16 and HPV18) were used to detect HPV16 and 18 DNA in oral benign and malignant lesion. Amplification was performed according to HPV16/18 kit from Sacace-Biotechnologies S.r.l. Caserta –Italy. The final reaction volume of 40 μl containing 20 μl mix-1 (contained in PCR tubes), 10 μl of mix-2 and 10 μl of extracted DNA (sample). Negative control, positive HPV16 DNA and positive control 18 DNA tubes contained 10 μl of DNA buffer, 10 μl of HPV 16 DNA and10μl of HPV18 DNA respectively. Samples and controls were amplified using Gene Amp PCR system 9700. The PCR program is described in Table The PCR products were visualized in 2% Agarose gel with 0.5 μg/ml Ethidium bromide. Ten micro liters of 100 bp DNA ladder and PCR product was loaded on the gel. Gel-electrophoresis was performed at 120 V and 36 mA for 60 minutes. Pictures were taken by Gel documentation system .According to manufacture HPV16/18 kit  manual, the PCR product length for HPV16 should be 325 bp and 425 bp for HPV18.Data management was done by using the Statistical Package for Social Sciences . SPSS was used for analysis and to perform Fisher exact test for statistical significance . The 95% confidence level and confidence intervals were used.A total of 40 cases  and 10 controls  were included in this study. The age distribution is relatively similar between cases and controls with a range of 18 to 70 years and a mean age of 33 years. Most of cases were middle age 26–35 years representing 21/40 (52.5%) followed by 18–25 group 8/40 (20%), as indicated in Table Table Worldwide, after breast cancer, cervical cancer is the second most common cancer that affects women. In 99.7% of all cases, cervical cancer results from a history of persistent infection by HR-HPV . The higHPV infection rates vary greatly between geographic regions and population groups ,21. In tIn regard to age, most of positive cases were relatively observed among younger women, and these findings were consistent with global reports . HoweverAccording to the residence, most of cases were from Northern Sudan. However, this might be due to the fact that Northern Sudan consist the most civilized part of the Sudan, this in addition to the fact the study was performed in the Northern Sudan.In this study, oral HPV infection was found to be very low prevalent (2.5%) than cervical HPV infection (40%). Most women with an oral HPV infection also had a cervical infection, likely because the majority of women had a cervical infection. The increased prevalence of oral infections in women with simultaneous cervical infections would suggest that the behaviors that place a woman at risk for oral HPV infection could substantially overlap with cervical infection. Anogenital HPV infections in adults are predominantly sexually transmitted , and indHR-HPV types 16 and 18 are the most commonly detected in Sudan and relatively similar to those described in neighboring African countries, although the relative contribution of HPV16 and HPV18 is substantially lower in cytologically normal women. The oral and cervical infection for HPV infection are likely independent of one another. Further more comprehensive studies are recommended for stronger clarification of association between HPV infection and cervical cancer in Sudan.The authors declare that they have no competing interests.AME: involved in the practical work (PCR). AHSED: involved in the practical work (PCR). RAHA: Patients management. HGA: consultation and Manuscript preparation. All authors read and approved the final manuscript.


To evaluate if human papillomavirus (HPV) self-sampling (Self-HPV) using a dry vaginal swab is a valid alternative for HPV testing.Women attending colposcopy clinic were recruited to collect two consecutive Self-HPV samples: a Self-HPV using a dry swab (S-DRY) and a Self-HPV using a standard wet transport medium (S-WET). These samples were analyzed for HPV using real time PCR (Roche Cobas). Participants were randomized to determine the order of the tests. Questionnaires assessing preferences and acceptability for both tests were conducted. Subsequently, women were invited for colposcopic examination; a physician collected a cervical sample (physician-sampling) with a broom-type device and placed it into a liquid-based cytology medium. Specimens were then processed for the production of cytology slides and a Hybrid Capture HPV DNA test (Qiagen) was performed from the residual liquid. Biopsies were performed if indicated. Unweighted kappa statistics (к) and McNemar tests were used to measure the agreement among the sampling methods.A total of 120 women were randomized. Overall HPV prevalence was 68.7%  59.3–77.2) by S-WET, 54.4% (95% CI 44.8–63.9) by S-DRY and 53.8% (95% CI 43.8–63.7) by HC. Among paired samples (S-WET and S-DRY), the overall agreement was good  and the κ was substantial . The proportion of positive type-specific HPV agreement was also good . No differences in sensitivity for cervical intraepithelial neoplasia grade one (CIN1) or worse between the two Self-HPV tests were observed. Women reported the two Self-HPV tests as highly acceptable.Self-HPV using dry swab transfer does not appear to compromise specimen integrity. Further study in a large screening population is needed.NCT01316120ClinicalTrials.gov:  Cervical cancer incidence and mortality have decreased considerably since the introduction of cervical cancer screening programs in Western countries. However, despite these advances in secondary prevention, there are 500,000 new cases every year worldwide, mostly 85%) in developing countries -3. Cervi5% in devAcceptability studies for Self-HPV indicate that the method is generally well accepted by women, but revealed that some of women have doubts about the validity of the method. One of these was the concern about manipulating the test tube and spilling out the transport medium during the sampling procedure, which some patients interpreted as incorrect and feared that it might affect the test result ,13. ThisThis study was conducted by the Geneva University Hospitals, Switzerland. The Ethics Committee of the Geneva University Hospitals, Switzerland, approved the study . A signed informed consent form (ICF) was required for enrollment of participants in the study. This trial was registered at ClinicalTrials.gov (Identifier: NCT01316120).A total of 120 women were prospectively enrolled from our colposcopy clinic between November 2010 and August 2011. We randomized the sequence of the two HPV tests to avoid any potential biases that may advantage the first test. We included women aged 20 years or older, who understood the study procedures and accepted to participate by signing the ICF. Exclusion criteria were pregnancy and previous conization or hysterectomy.The participants were randomized and received oral instructions by a physician or a research nurse about how to perform the Self-HPV, a self-collected vaginal sample. In brief, they were instructed to wash their hands before performing the procedure, to insert the swab into the vagina and to rotate it three times in both directions. The women were handed two self-sampling kits ) and were directed to a private, well-lit room to perform both samplings. Subsequently the participants were asked to complete a questionnaire on demographic characteristics, knowledge about HPV and preference between the two Self-HPV methods. The questionnaire used a 4-point scale to measure the degree of acceptability, physical discomfort and pain felt using the Self-HPV as well as the preference between the two Self-HPV methods. Participants then underwent a colposcopic examination. During this procedure, a cervical sample for liquid-based cytology and Hybrid Capture (HC) HPV test was obtained using a broom type cervical brush and rinsing it in Preservcyt™ buffer solution . A biopsy for histological analysis was performed if necessary.Self samples were stored at room temperature and the time between sample collection and analysis ranged between 5 to 15 days. HPV test according to the manufacturer’s recommendations. This assay is an automated DNA extraction, PCR amplification and real-time detection of 14 High Risk HPV (HR-HPV) genotypes. It uses the beta-globin gene as an internal extraction and amplification control. PCR amplification and detection are performed in a single tube, where probes with four different reporter dyes track the different targets in the multiplex reaction: (i) HPV 16, and 18 individually, (ii) 12 HR-HPV types  as a group, and (iii) beta-globin.Material from dry swabs (S-Dry samples) was placed into 1 ml of sterile phosphate-buffered saline (PBS)and the tubes were vortexed for 3×15 sec. Then, 0.5 ml of each sample was used for nucleic acid extraction and the rest was frozen at −20°C for storage. Tubes containing S-Wet samples (1 ml) were also vortexed for 3×15 sec and then, 0.5 ml of each sample was used for nucleic acid extraction and the rest of sample was frozen at −20°C for storage. HPV extraction, detection and genotyping were carried out from S-Dry and S-Wet specimens using the Cobas 4800 Postcytology vials processed on the ThinPrep 2000 System  were used. At least 4 mL of remaining PreservCyt™ solution was used for the Hybrid Capture HPV DNA test. Samples were processed in the sample conversion kit and tested with HC2 according to the manufacturer's protocol with probe B for HR genotypes . Sample reactivity was measured in relative light units (RLU). A specimen was considered positive for HR HPV DNA if the ratio of the specimen RLU to the mean RLU of triplicates of a positive control at 1 pg per ml was >3.00. Samples with ratios between 1.00 and 3.00 were retested twice and were considered positive if 2 out of 3 results had a ratio >1.00.Both methods used in this study,  are FDA approved diagnostic methods. Because HC is considered as the reference standard to analyze physician-sampled specimen, as any methods in consideration to replace it should be as accurate as this method of screening.The sample size necessary to validate a difference in test performance of 10% or more was calculated assuming a HPV prevalence of at least 40%. The proportion of positive agreement (PPA) between paired S-WET and S-DRY samples was calculated by dividing the number of samples testing positive for HPV in both tests by the number of samples testing positive in either S-WET or S-DRY. Cohens Kappa was calculated to measure the inter-test agreement between the self-sampling methods in terms of HPV risk categories. Positive agreement between S-WET and S-DRY was calculated as described by Wolfrum et al. .One hundred twenty women were included in the study, of whom we excluded four for not having paired samples, three for inconclusive HPV test results  and one who refused further participation after performing Self-HPV. For the study analysis we included 112 women with 224 paired samples and completed questionnaires. The median age of participants was 31 years (range 21–63 years).The HPV prevalence was 68.7%  59.3–77.2) detected by S-WET, 54.4% (95% CI 44.8–63.9) by S-DRY and 53.8% (95% CI 43.8–63.7) by HC. Mono-infections with HPV 16 or 18 were identified in 19.4% of participants, and combined infections with HPV 16 or 18 and other high-risk types were identified in 36.3%. Infection with one or more HPV types other than HPV 16 or 18 was observed in 44.1% of participants. HPV 16 was detected in 49.3% of cases and HPV 18 in 9%.The overall test agreement between S-WET and S-DRY was 85.7% (95% CI 77.8–91.6), with a 79.2% positive agreement. Cohen’s kappa for inter-test agreement was 0.70 (95% CI 0.53-0.88). Positive agreement for type specific HPV was 77.3% (95% CI 68.2–84.9). The inter-test agreement was good between S-WET and S-DRY for type-specific detection of HPV 16 and non-16/18 HPV types as well as for all HPV positive cases. The inter-test agreements between HC and S-DRY and between HC and S-WET were inferior to the inter-test agreement of S-WET and S-DRY . No difference in sensitivity or specificity was found between S-WET and S-DRY.In the case of CIN 1 or higher lesions, the stratified overall test agreement was 88.6% (κ=0.70). S-DRY detected HPV in 73.9% of cases of CIN2/3, while S-WET was positive in 91.3% and HC was positive in 69.6%. The agreement between S-WET and S-DRY was highest in CIN1. The performance of both self-tests was comparable to HC samples taken by a physician.We did not observe any statistically significant difference between S-DRY and HC or between S-WET and HC Table .One hundred and twelve questionnaires were completed, and no difference in acceptability between the two Self-HPV tests was observed. Most women (96.4%) were favorable to the idea of performing self-sampling at home, while three women (2.7%) were opposed to this idea and one did not answer the question. Fifty women experienced Self-HPV as slightly to severely painful or embarrassing, but no preference could be highlighted between the two Self-HPV tests. Some of the women  evaluated one of the self-tests as more complex than the other, while the majority did not report any difference in complexity. Twenty-seven women had higher confidence in S-WET, while two had higher confidence in S-DRY, but the large majority (83/112) thought both self-tests were equally reliable.In recent years, we have learned that, with appropriate instruction, self-sampling of HPV specimens yield similar results compared with those collected by health care professionals. The high percentage of agreement for high-risk HPV between these two approaches suggests that Self-HPV is a promising alternative to physician-sampling for HPV testing and cervical cancer screening . BelinsoRecent studies reported that Self-HPV testing is more sensitive than the Pap test in detecting CIN2+ in Chineese and Mexican women of low socioeconomic status ,20. FurtThe possibility of using Self-HPV stored at ambient temperature without transport media would clearly enhance and simplify the utility of Self-HPV. This could reduce the costs of the methods, which might be attractive for low-resource countries. Moreover, it may reduce women’s concerns that the test quality is reduced by accidentally spilling out some of the transport medium ,13. We fIn this study we used different swabs for S-WET and S-DRY. The S-WET swab was a flocked swab that consists of fine and short filaments fixed at the top of the stick while the swab used the S-DRY swab was a standard Dacron swab consisting of a long filament enrolled around a stick. Krech et al. compared flocked and rayon swabs for their sensitivity to detect HPV infection and noticed that the sensitivity of flocked swabs was higher . They exA shortcoming of our study is the use of different HPV DNA detection assays for self-collected vaginal specimens and physician-collected endocervical specimens. The number of variables that differ in addition to wet vs. dry transport weakens our conclusion about the feasibility of dry transportation. Other weaknesses of our study are the small sample size and the fact that our population was recruited in a colposcopic clinic having a high rate of HPV prevalence. Even though the HPV positivity in S-WET compared with S-DRY is not statistically significant, it seems to point in the direction of S-DRY being slightly less sensitive than S-WET. Additional work is needed to conduct a study in a real context of screening with a larger sample size to determine if S-DRY and S-WET have equal sensitivity.Our study shows that Self-HPV swabs can be successfully transported in a dry state at ambient temperature without greatly altering specimen integrity. Self-sampling for HPV testing using S-DRY could be an alternative to other self-sampling methods that require “wet” transport media. Advantages of the dry method include a simplification of the method , as well as its ease of use and lower cost may be of advantage for women with restricted access to health care delivery. However, further research is needed to confirm the equality of both methods in a large screening population.ASC-US: Atypical squamous cells of undetermined significance; AUC: Area under the curve; CIN 1: Cervical intraepithelial neoplasia grade 1; CIN 2/3: Cervical intraepithelial neoplasia grade 2 or 3; HC: Hybrid Capture; HPV: Human Papillomavirus; HSIL: High-grade squamous intraepithelial lesion; ICF: Informed consent form; LSIL: Low-grade squamous intraepithelial lesion; NPV: Negative predictive value; PBS: Phosphate-buffered saline; physician-sampling: Physician collected cervical sample for HPV testing; PPA: Proportion of positive agreement; PPV: Positive predictive value; ROC curve: Receiver Operating Characteristic curves; S-DRY: Self-HPV using a dry swab; S-WET: Self-HPV using a standard wet transport medium; Self-HPV: Self-sampling for HPV testing using a vaginal swab.The authors declare that they have no competing interests.IE conducted the study and drafted the manuscript. VP designed the study. IN participated in coordinating the study. PAM and GA were responsible for the molecular biological analysis. JCP co-designed the study. MB directed the statistical analyses. SU conducted statistical analyses and contributed to the manuscript. PP co-designed the study and led the research project. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/353/prepub


Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. Human papillomavirus (HPV) is the most common sexually transmitted virus in the human population. Although the vast majority of these HPV infections are cleared by the immune system within a year after infection, HPVs may in rare cases persist and cause cancer . HPV is The HPV-16 DNA genome is small, but it contains at least six early genes and two late genes under control of at least two promoters ,8. The ecis-acting RNA elements that interact with cellular splicing factors  were inserted between the unique BamHI site in HPV-16 L1 and an XhoI site located immediately after the L1 stop codon in pBSp and pBSpM . A fragmTo generate pBSpD1MCAT, pBSpD2MCAT and pBSpD3MCAT, site-directed ligase-independent mutagenesis (SLIM)  was perfTo generate pHPV6MCAT, pHPV18MCAT, pHPVMCAT, pHPV5SMCAT, pHPV41MCAT, SLIM mutagenesis  was perfThe complete HPV-16 genome was PCR-amplified and inserted into TOPO followed by regeneration of ends upto the unique SphI site. A 34-nt LoxP site was inserted immediately adjacent to the SphI sites flanking the HPV-16 genome. An SphI fragment was deleted from the TOPO vector to generate a plasmid containing only two SphI sites. A sequence encoding the Rous sarcoma virus long terminal repeat followed by the neomycin resistance gene and the simian virus 40 polyA signal was synthesized by Eurofins MWG GmbH. This sequence was inserted downstream of the LoxP site in the genomic HPV-16 plasmid, resulting in pHPV16AN. Introduction of an XhoI site downstream of the L1 stop codon, and insertion of the poliovirus type 2A internal ribosome entry site (IRES) followed by the gene encoding the secreted luciferase (sLuc)  between The C33A2 cell line is a C33A derived cell line that contains the pBELsluc reporter plasmid stably integrated into the genome, as described previously for pBELCAT in HeLa cells . The pBENeonatal human epidermal keratinocytes were purchased from Gibco Invitrogen Cell Culture and were propagated in EpiLife Medium supplemented with Human keratinocyte Growth Supplement (Gibco Invitrogen Cell Culture) as recommended by the manufacturer. The primary keratinocytes are grown in monolayer cultures and expanded under growth conditions that maintain poorly differentiated cells with properties similar but not identical to the cells in the basal layers of the epithelium. Cells passaged less than three times were used. In all, 150 000 cells were seeded per 60-mm plate for each transfection. Co-transfections of genomic HPV-16 plasmids and plasmid pCAGGS-nlscre  generou,44. Brie4 to obtain the rCAT units displayed in the manuscript figures. Mean values and standard deviations were calculated for all samples.Cell extracts were prepared from transfected HeLa cells at 24 h posttransfection according to the protocol from a CAT enzyme-linked immunosorbent assay (ELISA) kit (Roche). All cell extracts were appropriately diluted to obtain an absorbance reading in the linear range of the CAT ELISA assay. To be able to compare CAT results between experiments, pCMVCAT16 was transfected in triplicate in each transfection experiment. Arbitrary CAT units were calculated by multiplying the absorbance 405 readings with the dilution factor for each sample. CAT units obtained for each sample were divided by CAT units obtained in pCMVCAT16 transfections and thereafter multiplied by a factor of 10Metridia longa sLuc (The nga sLuc  activityCellular DNA was extracted after lysis of cells in lysis buffer . The cell extracts were incubated at 50°C over night and an equal volume of phenol/chloroform/isoamyl alchohol (chisam) was added. The aqueous phase was collected after centrifugation of the samples and subjected to ethanol precipitation. The samples were treated with RNaseA and stored at −20°C until use. To monitor recombination at the loxP sites in pHPV16AN and pHPV16ANsL, PCR was performed with primers 16S (5′-TATGTATGGTATAATAAACACGTGTGTATGTG-3′) and 16A (5′-GCAGTGCAGGTCAGGAAAACAGGGATTTGGC-3′) see A. This P32P-CTP, as described previously  according to manufacturer’s instructions. Briefly, 1 µg of DNA and T7-RNA polymerase (50 U) were incubated at 37°C for 4 h with 50 U Riboblock RNase inhibitor (Fermentas), 10 µl of transcription buffer (Ambion), 40 uCi of 32P-UTP (3000 Ci/mmol) (Perkin Elmer) in a total volume of (100 µl) in the presence of 2 mM each of rATP, rCTP and rGTP. The samples were treated with RNase-free DNase (Fermentas) at 37°C for 30 min, extracted with phenol-chloroform, and ethanol precipitated. The radiolabeled RNAs were pelleted and dissolved in 50 µl of RNase-free distilled water. The unlabeled RNA competitors were transcribed under similar conditions, except that the radiolabeled nucleotide was substituted with 1 mM UTP. The unlabeled transcripts were dissolved in RNase-free distilled water to a concentration of ∼2 µg/µl. The quality of the probes or RNA competitors was examined by electrophoresis on 8% urea polyacrylamide gels. RNAs were visualized by Gel-red staining of gels or by autoradiography.PCR fragments amplified from plasmids pUC19T7-4xWT and pUC19T7-4xMUT with oligonucleotides T7s and 4xWTas or 4xMUTas, respectively, were transcribed 2, 1 mM DTT, 5% glycerol and 5 µg/µl heparin] for 15 min at room temperature followed by UV irradiation in a Bio-link cross-linker (Biometra) for 15 min. For competition experiments, cell extract was pre-incubated with the RNA competitors in the binding buffer for 10 min before addition of the radiolabeled RNA probe. After UV irradiation, RNaseA (20 µg) and RNaseT1 (1000U) were added to the samples that were incubated for 30 min at 37°C. The UV cross-linked RNA-proteins were resolved on 12% SDS–PAGE gels, and the bands were visualized by autoradiography.Ultraviolet (UV) cross-linking was carried out as described previously . Briefly2, 0.5% Triton X-100]. After incubation with rotation for 20 min at room temperature, beads were washed 10 times in binding buffer with 200 mM NaCl. Proteins were eluted by boiling beads in SDS–PAGE loading buffer. After electrophoresis, the gels were either subjected to western blotting as described previously (Sixty micrograms of HeLa nuclear extract was mixed with streptavidin-coated magnetic beads bound to the various biotin-labeled RNA oligonucleotides indicated in the figures in 200 µl of binding buffer  followed by western blotting as previously described  with antHPV-16 splice sites SD3632 and SA5639 are used exclusively to produce the late L1 mRNAs A 15). T. T15). T4 to generate the rCAT values displayed in each figure, as described in ‘Materials and Methods’ section. A comparison between pBSpMCAT and pMT1SDCAT revealed that deletion of 232 nt immediately upstream of SD3632 in pBSpMCAT, as in pMT1SDCAT . Plasmid pBSpOPTCAT produced 21 times more CAT than pBSpCAT . pBSpMCAT, in which also splicing silencers at SA5639 had been inactivated, produced 30-fold more CAT than pBSpCAT . These results confirmed that both SD3632 and SA5639 were subject to negative regulation, and established that SD3632 is suboptimal. Plasmid pBSpOPTMCAT, in which SD3632 had been optimized and splicing silencers at SA5639 had been destroyed, produced 499-fold higher CAT levels than pBSpCAT . However, these CAT levels were >6-fold lower than those produced by pMT1SDCAT , indicating that the splicing inhibitory sequence upstream of SD3632 also inhibited the optimized SD3632, but to a lower extent than wild-type SD3632. The combined effect of optimization of 5′-splice site SD3632 and mutational inactivation of splicing silencers at SA5639 was several fold higher than optimization of each splice site alone, which suggested that splicing silencers at SD3632 or SA5639 acted independently of each other.We optimized HPV-16 SD3632 by changing this 5′ splice site to a sequence with perfect complementary to U1 snRNA  in plasmTo map the location of the inhibitory RNA elements upstream of HPV-16 SD3632, we generated serial deletions in this region in pBSpMCAT, resulting in plasmids pBSp5MCAT, pBSp6MCAT, pBSp7MCAT and pBSp8MCAT F. All plSupplementary Figure S1C). CAT production from pBSpCAT-derived plasmids was overall lower than from pBSpMCAT-derived plasmids . The results confirmed that splicing inhibitory RNA elements at SD3632 are located within the 42 nt immediately upstream of HPV-16 SD3632 , and that additional but weaker splicing regulatory elements are present upstream of nucleotide position 3587. We concluded that the major splicing inhibitory element was located between nucleotide position 3587 and SD3632. These sequences were studied further.We also inserted the same deletions in pBSpCAT, in which splicing silencers at SA5639 were intact, generating pBSp5CAT, pBSp6CAT, pBSp7CAT, pBSp8CAT pBSp9CAT and pBSp10CAT  . Overexpression of the splicing inhibitory factor PTB was performed to show production of the unspliced 13a7 vpr mRNA from pNL13a7.Next we investigated whether the splicing of HPV-16 SD3632 could function in a heterologous context. The sequence immediately upstream of HPV-16 SD3632 (3546–3628 nt) was therefore inserted immediately upstream of HIV-1 5′-splice site SD3 in the HIV-1 vpr cDNA expression plasmid pNL13a7 (ure S2A) . The effTo map the inhibitory RNA elements further, three additional deletions were inserted into pBSp5MCAT . These dTo investigate the sequence specificity of the splicing inhibitory sequence, we replaced this 34-nt sequence with a computer-randomized 34-nt sequence, resulting in pRMCAT . In addiTwo sequence motifs are repeated twice within the 34 nt upstream of SD3632: ACAC and AUAGUA  . Only HPV-16 contained two identical copies of AUAGUA, while other members of species nine alpha viruses contained different, but closely related sequence motifs, i.e. AUAGUG or AUAAUA instead of AUAGUA . A consensus of all species 9 HPVs of the alpha genus revealed that the consensus of the proximal AUAGUA motif was identical to the HPV-16 sequence, whereas the distal motif was not (data not shown). The consensus of the proximal AUAGUA motif in species 7, 9 and 10 of the alpha genus was also AUAGUA, as seen in pHPVMCAT . In contrast, the distal HPV-16 motif was not conserved . We concluded that the presence of two AUAGUA motifs upstream of late 5′-splice site SD3632 appears to be unique for HPV-16. This is of interest in the light of the fact that HPV-16 sticks out as the most common HPV type in the human population and as such must be endowed with unique properties.We compared the 34-nt sequence upstream of HPV-16 late 5′-splice site SD3632 with the corresponding sequence within different HPV genera, including: HPV-6, HPV-18 (Alpha genus), HPV-5 (Beta genus), HPV-4 (Gamma genus), HPV-1 (Mu genus) and HPV-41 (Nu genus) (gure S3) . AnalysiSupplementary Figure S3). The 34-nt sequences of pHPV5SMCAT and pHPV5LMCAT were obtained from two differentially spliced cDNAs . High levels of CAT were produced from all plasmids except pHPV5LMCAT, pHPV1MCAT, pHPVMCAT and HPV-16 plasmid pBSpD1MCAT . Plasmids pHPV6MCAT and pHPV18MCAT encode sequences that are related to the AUAGUA motifs: AUAGUG motif in HPV6 and GUGGUA and AUAAUA motifs in HPV18, but these motifs did not inhibit splice site SD3632 to the same extent as the two AUAGUA motifs in HPV-16 . The pHPVMCAT plasmid that contained an AUAGUA motif at the proximal position and a closely related GUUGUA motif at the distal position produced low levels of CAT , supporting the importance of the AUAGUA sequence. Sequences of the cutaneous types HPV-1 and HPV-5L also inhibited HPV-16 SD3632, but these viral sequences did not encode AUAGUA motif, indicating that other sequences may inhibit splicing. Both HPV-1 and HPV-5L sequences are G-rich, a property of previously described splicing silencers that interact with hnRNP H  overlap the AUAGUA motifs, it is impossible to mutate the AUAGUA motifs without also changing either the E2 or the E4 coding sequence. We therefore constructed two more mutants of the AUAGUA motifs that either affected the E2 ORF or the E4 ORF, resulting in plasmids pME2CAT and pME4CAT, respectively . In pME46 sLuc units at 24 h posttransfection (data not shown). These results demonstrated that splicing silencers at both SD3632 and SA5639 inhibited L1 mRNA splicing in the context of the HPV-16 genome and showed that the splicing silencers were active in human primary keratinocytes that were grown in monolayer cultures.We wished to determine whether the two AUAGUA motifs were active in the context of the HPV-16 genome and in human primary keratinocytes. We therefore generated genomic plasmid pHPV16ANsL, in which a complete HPV-16 genome is flanked by loxP-sites and is released to its episomal form on transfection of pHPV16ANsL with plasmid pCAGGS-nlscre , as prevNext we introduced 2-nt substitutions in each AUAGUA motif upstream of SD3632 in pHPV16MANsL, resulting in pME4 C. These To investigate whether cellular proteins bind to the AUAGUA motifs, we synthesized radiolabeled RNA consisting of four copies of the wild AUAGUA motifs (4xWT RNA) or four copies of the mutant CACUGC motifs (4xMUT RNA) A. The RNSupplementary Figure S4) . Mass spectrometry identified peptides in exon 2, but not in exon 7, suggesting that at least hnRNP D40 was pulled down by the 4xtWT RNA , although it does not exclude binding of hnRNP D37 or hnRNP D42. We concluded that the three members of the hnRNP D family of proteins  as well as hnRNP A2/B1 interacted specifically with the AUAGUA motif found in the HPV-16 splicing silencer located immediately upstream of late 5′-splice site SD3632. We speculate that one or more of these proteins suppress HPV-16 5′-splice site SD3632.Next we performed RNA protein pull-down experiments from cellular extracts followed by mass spectrometry of individual protein bands detected by the 4xWT RNA and not by the 4xMUT RNA. Multiple silver stained bands were pulled down by the 4xWT RNA, as well as 4xWT ssDNA, but not with the 4xMUT RNA or 4xMUT ssDNA A. The prgure S4) . They eiSupplementary Figure S5). The pBELsLuc plasmid encodes HPV-16 early and late genes but expresses only the early mRNAs as HPV-16 late splice sites are suppressed. The HPV-16 L1 gene has been replaced by the poliovirus IRES followed by the sLuc gene and thus functions as a reporter assay for late gene expression as previously described   following siRNA transfections. As predicted by the sLuc results described earlier in the text, the spliced L1 mRNA levels increased following transfections of the indicator cell line with siRNAs against hnRNP DL, hnRNP AB and hnRNP D compared with cells transfected with scrambled siRNAs  . We reasgure S5) A. The higure S5) A. The pogure S5) . All siRgure S5) B. We werd siRNAs C. We conSupplementary Figure S4). To investigate the role of the individual hnRNP D proteins in HPV-16 late gene expression, we wished to overexpress each hnRNP D isoform and monitor the effect on HPV-16. In contrast to C33A cells, which express approximately equal amounts of all four hnRNP D isoform mRNAs . The hnRNP A1, hnRNP A2/B1 and the hnRNP D proteins are also highly expressed in cervical cancer cells (www.proteinatlas.org). Therefore, there is an inverse correlation between hnRNP A1, hnRNP A2/B1 and hnRNP D protein production in relation to the HPV-16 L1 protein production, which supports an inhibitory role of the proteins in HPV-16 L1 expression.Two HPV-16 splice sites are used exclusively to produce spliced L1 mRNAs: SD3632 and SA5639 ,19. HereThe hnRNP D gene expresses four alternatively spliced mRNAs . AlthougThe hnRNP D proteins act primarily by enhancing decay of labile mRNAs that encode AU-rich RNA elements in their 3′-UTR . hnRNP DDespite the fact that hnRNP D suppresses expression of many cell cycle regulatory factors by promoting degradation of their mRNAs , overexpGenital HPV infections are common in the human population, and in terms of transmission, HPVs are successful viruses. A subset of the genital HPV types is termed high-risk HPV types as a result of its association with cancer, primarily cervical cancer in women. HPV-16 sticks out as the most commonly found HPV type in the human population as well as in cervical cancer, probably due to enhanced capability to establish persistence in addition to its well-defined role in cell transformation (The functional analysis of the splicing silencer elements at HPV-16 SD3632 and SA5639 in the context of the full-length HPV-16 genome, as well as in primary human keratinocytes, established that these RNA elements are important in the HPV-16 gene expression program. The mutations introduced in pME4 affected the E4 amino acid sequence, but not E2  and 5C. Supplementary Data are available at NAR Online.Swedish Research Council-Medicine [2012-1933]; Swedish Cancer Society [CAN 2012/542].Funding for open access charge: Conflict of interest statement. None declared.


Human papillomavirus (HPV) infection is associated with a wide spectrum of disease that ranges from self-limited skin warts to life-threatening cancers. Since HPV plays a necessary etiological role in cervical cancer, it is logical to use HPV as a marker for early detection of cervical cancer and precancer. Recent advances in technology enable the development of high-throughput HPV assays of different formats, including DNA-based, mRNA-based, high-risk group-specific and type-specific methods. The ultimate goal of these assays is to improve the accuracy and cost-effiectiveness of cervical screening programs. HPV testing has several potential advantages compared to cytology-based screening. However, since the cancer to transient infection ratio is always low in the general population, HPV test results are bound to have a low positive predictive value that may subject women to unnecessary follow-up investigations. The wide-spread administration of prophylactic HPV vaccine will substantially decrease the incidence of cancer and precancer. This poses a number of challenges to cytology-based screening, and the role of HPV testing is expected to increase. Finally, apart from technical and cost-effiectiveness considerations, one should also keep in mind the psycho-social impact of using sexually-transmitted agents as a marker for cancer screening. To date, more than 120 types of human papillomavirus (HPV) have been characterizedWith the advances in molecular techniques over the last decade, our understanding of this family of ubiquitous viruses has improved tremendously, a number of previously unrecognized important disease associations have been confirmed, and detection assays for epidemiological study and for confirming diagnosis to assist patient management have been developed. Vaccines targeting two HPV types that show the strongest association with cervical cancer have been developedWith the improvement in the sensitivity of detection assays and coverage for a variety of HPV types, it is now recognized that asymptomatic carriage of HPV on healthy skin is common and can persist over several yearsHPV causes benign warts on the skin, which present as flat or firm non-itchy papulesGenitial tract HPV infection is the most common sexually-transmitted infection. Infections are often subclinical. Clinical lesions (condylomata acuminata) are often multiple and appear as exophytic papillomas, flesh or brown in colour. HPV 6 and 11 are the types most commonly found in visible anogenital warts, but other types, including those typically found in non-genital skin, can also be detected, especially from anogenital areas without visible lesionsThe oral mucosa is also susceptible to HPV infection. Such infection involving the larynx represents a rare but severe diseaseThe hypothesis that HPV plays a role in the development of cervical cancer was proposed in the mid-1970s. Over the last 40 years, a strong body of evidence has accumulated to prove the etiological association between HPV infection and a number of human cancers in addition to cervical cancerEVER1 and EVER2 genesEpidermodysplasia verruciformis (EV), a genetically-inherited chronic skin condition, presents as disseminated flat warts. Recent studies have shown that some EV patients have mutations in the In recent years, some evidence suggests that non-melanoma skin cancers including basal cell and squamous cell carcinoma may be linked to cutaneous HPV infection. The suspicion of a viral cause for cutaneous squamous cell carcinoma is based on the observation that its incidence increases dramatically in solid organ transplant recipients receiving long-term immunosuppressive therapy. While sun-exposure is a recognized risk factor, infection with cutaneous HPV (mainly the beta genus) seems to play a role in the development of non-melanoma skin cancers, especially squamous cell carcinoma. HPV types belonging either to beta species 1 or 2 have been detected from squamous cell carcinoma specimens, but unlike anogenital cancers, no predominant HPV types can be identifiedcarcinoma-in-situ, it can evolve to invasive carcinoma, and it is often associated with HPV 16. A subset of intraepithelial neoplasia and carcinoma of the penis, vulva and vagina is also associated with HPV infection, mainly HPV 16The mucosal group of HPV is associated with several malignant conditions. Bowenoid papulosis presents as multiple small flat pigmented papules on the external genitalia. Histologically, it is a This uncommon cancer in women has an age-standardized rate of 0.3–0.7 per 100,000 worldwideThe age-standardized incidence rates of vulvar cancer lie between 0.5 and 1.5 per 100,000. The geographical pattern of vulvar cancer is different from cervical cancer and high rates are observed in several European populations , whereas the prevalence in sub-Saharan Africa, Southeast Asia, and Latin America is low. Distinct subtypes, such as the warty and basaloid types, have been recognized, but the majority of tumours are squamous cell carcinoma. Etiologically, because vulvar carcinomas are heterogeneous, the prevalence of HPV infection in invasive vulvar cancer cases variesThe vast majority of anal cancers is associated with HPV infection. Cancers arising in the anal canal and tumors of the external skin  are classified as skin cancers. The canal is lined in its upper part by colorectal-type mucosa, and in its lower third by squamous epithelium, with a specialized transitional zone in between. Therefore, cancers are predominantly squamous cell carcinoma, adenocarcinoma, or basaloid and cloacogenic carcinoma. In most populations, squamous cell carcinoma is twice as common in females as males. However the incidence is particularly high among men who have sex with men and the risk is increased further by infection with human immunodeficiency virus, cigarette smoking, anal intercourse, and more lifetime sexual partnersGlobally, this rare cancer accounts for less than 0.5% of all cancers in men. The concordance of cervical and penile cancer in married couples and the geographical distribution of these cancers suggest that they share a common etiologyAmong the cancers for which a confirmed or probable etiological link with HPV infection has been established, cervical cancer has the strongest association and accounts for the largest share of disease burdenAn increase in incidence of oropharyngeal squamous cell carcinoma, specifically those originated from the tonsil and tongue base, has been observed in some parts of the worldin vitro conditions to form virus-like particles which are the main constituent of current prophylactic vaccinesPapillomaviruses have a small double-stranded DNA genome of about 8 kb in length. The key functions and proteins encoded by different regions of the HPV genome that are relevant for designing detection assays are shown in Papillomaviridae which comprises members infecting humans, non-human primates, birds, and reptilesHPV are classified under the family Because the replication cycle of papillomaviruses can be completed only in differentiated epithelial cells, isolation of viruses from clinical samples is difficultHPV is equipped with several immune evasion mechanisms. It multiplies in keratinocytes that have a short-life span, and thus the progeny viruses can be released in a natural way without inducing cell lysis that is seen with other non-enveloped viruses. Tis avoids triggering of infammation and immune responses associated with cellular damage. The lack of a viremic phase also minimizes stimulation to the systemic immune system. Furthermore, the virus actively downregulates the synthesis of interferon. As a result, although the immune system plays an important role in clearing infection and it is associated with a strong localized cellular immune response, natural infection with papillomaviruses does not result in a robust antibody responsein situ hybridization). It is necessary to distinguish analytical sensitivity (minimum number of HPV genomes to be present in a sample to generate a positive test result) from clinical sensitivity (proportion of women with disease who test positive)  target-amplification methods , and 2) signal amplification methods (liquid-phase or ositive) .The choice of the HPV test depends on the application . Assays On the other hand, when applied in the clinical situation for cervical cancer screening and post-treatment follow-up, assays with lower analytical sensitivity may produce a better positive predictive valueHybrid capture (HC2) is based on liquid phase hybridization using long synthetic RNA probes complementary to the genomic sequences of 13 high-risk types  and five low-risk types ; these are used to prepare high-risk (B) and low-risk (A) probe cocktails that are used in two separate reactionsHC2 does not require special facilities to avoid cross-contamination because it does not rely on target amplification to achieve high sensitivity, as do PCR protocols. The recommended cut-of value for test-positive results is 1.0 relative light unit . Several studies have noted that the high-risk probe cocktail in HC2 cross-reacts with HPV types that are not represented in the probe mix. It has been reported that HC2 using the high-risk probe at a 1.0-pg/mL cut-off detected HPV types 53, 66, 67, 73, as well as other undefined types, and raising the cut-of to 10.0 pg/mL did not eliminate the cross reactivity to types 53 and 67Currently, the most important clinical application of HPV detection is to identify women with a higher risk of developing cervical intraepithelial neoplasia or invasive cervical cancer . HPV DNAL1: The L1 region is the most frequently-used target for amplifying HPV genome from clinical samples . LabNet, with the collaboration of the National Institute of Biological Standards and Control (UK), is establishing international standards (IS) for HPV types, to harmonize HPV nucleic acid amplification technology-based assays. These efforts will be immensely helpful in monitoring the impact of HPV vaccination programs worldwide and in evaluating data on uniform platforms. One of the main aims is to harmonize various HPV detection assays and to minimize inter-laboratory variation by collectively establishing strong and effective quality control and quality assurance programs.The need to monitor vaccine effectiveness by effective surveillance programs and the increasing use of HPV assays in the field have stimuated the World Health Organization (WHO) to develop a structured Global Laboratory Network (WHO HPV LabNet). To date, this LabNet includes two Global Reference Laboratories (Sweden and USA) and eight Regional Reference Laboratories . Work is currently being conducted in the areas of scientific and technical advice, quality assurance, training, and communication . It is only the latter that carries an increased risk of cancer developmentE6 and E7. Among women with normal cytology, atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL), E6/E7 mRNA was detected in 30% of the HPV 16, 56% of the HPV 18 and 75% of the HPV 31 DNA-positive women, respectively. The mRNA test therefore potentially has a higher specificity compared to the HPV DNA test, and thus fewer patients would be referred for further testing or close follow-upINK4a, Ki-67, topoisomerase IIA (TOP2), and minichromosome maintenance proteins (MCMP). Among these, p16INK4a is the most promisingINK4a is inactivated by genetic deletion or hypermethylation, which leads to an increase in cyclin-dependent kinase activity and inactivation of RbINK4a as a result of the lost of negative feedback regulation that depends on Rb activityAnother approach to improve the positive predictive value of HPV testing as a primary screening tool is to carry out a “reflex” follow-up test for HPV-positive samples. The “reflex” approach can prevent an extra visit and unnecessary anxiety while waiting for the follow-up test results. Biomarkers indicating the transforming activity of HPV can potentially serve this purposeThe patterns of viral integration at different stages of neoplastic progression have been investigated. Diverse, even conflicting, results have been reported. Some studies observed viral integration mainly from specimens of high-grade lesionsDNA methylation is an epigenetic event that is linked to cancer development. A number of studies have been conducted to examine the association between the viral DNA methylation pattern and lesion severity. However, as with viral integration, most of the available data suggests that the patterns observed from low-grade and high-grade lesions are too diverse to achieve a clinically-useful predictive valueCo-test refers to the use of both HPV and cytology tests in parallel as first-line screening. The main advantage of co-test is the improvement in sensitivity for CIN II+ lesions; women who are double negative will have an extremely low-risk for CIN II+. The potential gain in cost-effectiveness from the expense of the extra test is the longer interval of safety supported by a double negative result. Data have suggested that the safety interval following an HPV-negative result could be as long as 5–7 yearsThe first clinical application of HPV testing was on the triage of patients presented with ASCUS on Pap smear. Patients who were HPV-positive would be referred for colposcopy whilst those who were HPV-negative could be followed by repeating the Pap smear 12 months laterThe cost-effectiveness of using HPV testing to triage women with LSIL depends mainly on the context. For instance, it has been shown that about 80% of patients presenting with LSIL were HPV-positive; thus triage of LSIL by HPV testing was not recommendedThe underlying risk of having CIN II+ lesions among women with high-grade squamous intraepithelial lesions (HSIL) or atypical squamous cells is high, but HSIL (ASC-H) cannot be excluded, and colposcopy may be indicated for CIN II+ even if the HPV test result is negativeCIN II is often regarded as a threshold for treatment . The preThe currently available prophylactic HPV vaccines are highly effective for the prevention of HPV16/18-related cervical neoplasia and offer some degree of cross-protection for lesions caused by related HPV types. A number of countries have implemented vaccination programs for adolescents and the coverage is expected to increase in the coming yearsIn the coming decades, several issues regarding cervical screening will need to be addressedSince HPV is a sexually-transmitted infection, using HPV testing for cervical screening may lead to anxiety and concerns about sexual relationships, and may have an emotional impact on the quality of life and a negative impact on mental healthOther practical challenges are the diversion of resources towards vaccination, and the resulting pressure for less frequent screening. At present, the recommended frequency in most countries is not less than once every 2–3 years. To lengthen the screening interval means getting closer to the interval required for advancing from low-grade to high-grade intraepithelial lesions or even to the development of invasive cancer. In other words, there may be just one chance to pick up at-risk women before the full development of invasive cancer. This will require a test with extreme sensitivity that is subject to a low positive predictive value, especially in the vaccinated population with a low incidence of disease. Lengthening of the interval for cytological examination is not advisable because the sensitivity of the test is low and repeated negative cytological tests are required to ensure no underlying precancerous or cancerous lesions. The high sensitivity of HPV testing, on the other hand, is more reassuring and allows lengthening of the screening interval. To overcome the low positive predictive value of highly-sensitive HPV testing in the vaccinated population with a low incidence of disease, a supplementary test for other biomarkers or co-testing with cytology is the way to move forward.Technically speaking, HPV testing has several advantages over cytology-based screening, particularly in the situation where the incidence of cervical cancer and precancer decreases substantially following the widespread use of vaccination. However, although it is beyond the scope of this review, the psycho-social stigma associated with testing for a sexually-transmitted infection needs to be addressed. It has been shown that, even among well-educated women, most have not heard of HPV and do not know its association with cervical cancer