Although lower uptake rates of the human papillomavirus (HPV) vaccine among socioeconomically disadvantaged populations have been documented, less is known about the relationships between awareness and acceptability, and other factors affecting HPV vaccine uptake.The current study aimed to estimate the potential effectiveness of increased HPV vaccine awareness on the acceptability of HPV vaccination in a nationally representative sample of women, using a methodology that controlled for potential non-random selection.This study used a population-based sample from the 2007 Health Information National Trends Survey, a cross-sectional study of the US population aged 18 years or older, and focused on the subsample of 742 women who have any female children under the age of 18 years in the household. An instrumental variables bivariate probit model was used to jointly estimate HPV vaccine awareness and acceptability.The proportion of HPV vaccine acceptability among the previously aware and non-aware groups was 58% and 47%, respectively. Results from the instrumental variables bivariate probit model showed that the estimated marginal effect of awareness on acceptability was 46 percentage points, an effect that was even greater than observed.Among populations who are not currently aware of the HPV vaccine, the potential impact of raising awareness on acceptability of HPV vaccination is substantial. This finding provides additional support to strengthening public health programs that increase awareness and policy efforts that address barriers to HPV vaccination. Human papillomavirus (HPV) and related diseases are major public health problems in the United States. Approximately 20 million Americans are currently infected with HPV while another 6 million are newly infected each year . In 2010® in 2006 and Cervarix® in 2009) [The persistent, albeit, diminishing socioeconomic disparities in overall cervical cancer incidence and mortality, can be partly explained by the constantly lower screening rates among disadvantaged populations, despite the widespread acceptance of the Papanicolau (Pap) test -12. The in 2009)  has provin 2009) -5,14 arein 2009) . Althougin 2009) , early ein 2009) -18 indicMajor barriers to HPV vaccine implementation among at-risk populations include high vaccination costs and lack of a usual source of care. Limited community involvement in stimulating awareness of cervical cancer and available screening methods is another important obstacle that needs to be overcome . AwareneLittle attention, however, has been paid to the potential impact of improving HPV vaccine awareness on increasing HPV vaccine acceptability at a population level. Observed differences in HPV vaccine acceptability levels between the previously "aware" and "non-aware" groups, including results from standard regression models, can be misleading because the two groups may differ in many unobserved ways. If the aware group consists of more advantaged individuals who may be more (less) acceptable of vaccinations, a fair comparison in HPV vaccine acceptability between the aware and non-aware groups cannot be achieved because the difference in acceptability may overestimate (underestimate) the marginal effect of improved HPV vaccine awareness.This study aimed to quantify the marginal effectiveness of increasing HPV vaccine awareness on acceptability, using a nationally representative sample of women who reported having any female children under the age of 18 in the household. The hypothesis to be tested is that HPV vaccine awareness increases vaccine acceptability and that this population-level estimate is greater when unobserved heterogeneity between the aware and non-aware groups is taken into account than otherwise. To this end, the current study exploited a unique survey question that captured hypothetical HPV vaccine acceptability, and employed a statistical method that controlled for unobserved differences between the two comparison groups.®) was licensed in the US (June 2006); the bivalent HPV-16/18 vaccine (Cervarix®) was not FDA approved until October 2009 [The present study used data from the Health Information National Trends Survey 2007 (HINTS 2007)  , a studyber 2009 . The sam®. If you had a daughter that age, would you have her get it?" Respondents were given the following options to choose from: "Yes", "No", and "Not sure/It depends". For the statistical analysis, a binary indicator variable was created using "Yes" (= 1) versus "No" and "Not sure/It depends" (= 0), as defined by Fang and colleagues [Acceptability of HPV vaccination was defined using the survey question, "A vaccine to prevent the human papillomavirus or HPV infection is recommended for girls aged 11-12 and is called the cervical cancer vaccine, HPV shot, or GARDASILlleagues . A dichohaving a regular health care provider if she responded positively to the question, "Not including psychiatrists and other mental health professionals, is there a particular doctor, nurse, or other health professional that you see most often?" Similarly, having any insurance referred to an affirmative response to the question, "Do you have any kind of health care coverage, including health insurance, prepaid plans such as HMOs, or government plans such as Medicare?" Health status of respondents were based on their responses to the question, "In general, would you say your health is excellent/very good/good/fair/poor." There were nine census divisions, covering the New England Census Division throughout the Pacific Census Division.The regression analysis included the following covariates: age, race, household income, and education level; having any insurance, any regular health care provider, past medical history of cancer, and family history of cancer; and self-rated health status, and census division. The specific categories used are shown in Table To address the issue of unmeasured differences between the aware and non-aware groups which may influence acceptability of HPV vaccination, the current study employed an instrumental variable (IV) bivariate probit model. This method has been applied in various public health and health economics research to address similar statistical issues -31. In tAccept* is a continuous latent variable for the indicator variable of HPV vaccine acceptability  and Aware* is a continuous latent variable for the indicator variable of whether the respondent has heard of HPV vaccine . X denotes a set of covariates common to Eq. 1 and Eq. 2, and Z denotes a set of IVs included only in Eq. 2. β, 1γ, 2γ, and δ are coefficients for corresponding variable(s) in the models, whereas 1 εand 2 εare error terms of Eq. 1 and Eq. 2, respectively.where 2ε) are not correlated with unmeasured factors in Eq. 1 (1ε) that influence HPV vaccine acceptability. In this case, estimating Eq. 1 alone would already provide a valid estimate of β, which will capture the extent to which being aware of the HPV vaccine leads to HPV vaccine acceptability. If 1 εand 2 εare correlated, however, consistent estimates can be obtained by jointly estimating the two equations in the bivariate probit model. A formal test can be conducted to examine the correlation coefficient (ρ) between 1ε, and 2ε. Estimating the IV bivariate probit model efficiently requires good IVs (Z in Eq. 2) that must satisfy two basic conditions, these IVs: (1) should predict the outcome of Aware and (2) should not directly affect the outcome of Accept. The HINTS 2007 contains such potentially good IVs because access to information related to the various types of cancers is available. For example, a respondent who gave a positive answer to the question, "Have you heard of any tests to find lung cancer before the cancer creates noticeable problems?", would be more likely to have also heard about the HPV vaccine. However, responses to this question would be unlikely to directly influence the extent to which the respondent has greater acceptability of HPV vaccination. In the same vein, a positive response to the question "Have you ever heard of a clinical trial?" would strongly predict a greater probability of HPV vaccine awareness but unlikely affect the level of HPV vaccine acceptability. The current study used these two IVs, after exploring other variables related to the access to different types of information on cancer. Tests for these underlying assumptions suggested that these IVs predict Aware statistically significantly but are not directly associated with Accept other than through Aware. Stata 11.0  was used in all statistical analyses, taking into account a complex survey design.It is possible that unexplained differences in HPV vaccine awareness in Eq. 2  was statistically significant . Through statistical tests, the IVs used were found to be statistically significant determinants of HPV vaccine awareness but were not directly predictive of vaccine acceptability (results not shown).The correlation of the error terms (ρ = –0.689) indicates that unmeasured differences by HPV vaccine awareness (ε2) were correlated with unmeasured factors (ε1) that influenced HPV vaccine acceptability, rendering the estimation of coefficients in the probit model inconsistent. This negative correlation suggests that the unmeasured factors impact HPV vaccine awareness and acceptability in opposite directions. In other words, the marginal effectiveness of increasing awareness of the HPV vaccine on vaccine acceptability at the population level may be much greater than the observed difference between those who are aware of the HPV vaccine and those who are not.This study found that HPV vaccine awareness had a significant positive impact on vaccine acceptability, a result not reported in previous studies which treated awareness as an exogenous variable in the equation for acceptability ,26. The Although caution must be exercised in interpreting the relationship between HPV vaccine awareness and the eventual initiation of the vaccination series in this study, awareness should still be recognized as an important starting point of a continuum that leads to adopting change. The four stages of HPV vaccine adoption described by Allen et al. (2009) include pre-contemplation, contemplation, preparation and action , fairly While low vaccination rates have been previously reported in women from low-income and minority groups who are most at risk of HPV infection and cervical cancer, this study found weak evidence of disparities in HPV vaccine acceptability across ethnicities and socioeconomic status. This suggests that there are presumably other factors that hinder women - who would be, in fact, receptive of the vaccine - from eventual vaccination. These factors present women from socioeconomically disadvantaged groups with a greater obstacle. Perceived barriers to HPV vaccination are essentially multidimensional  and may ®, and levels of vaccine awareness and acceptability may have changed over the past few years.Several limitations should be acknowledged in this study. First, the HINTS 2007 did not offer any insights on how respondents' acceptability of the HPV vaccine may change in view of perceived barriers such as the affordability of the vaccine. Second, while this study quantified the potential effectiveness of improved HPV vaccine awareness on acceptability at the population level, the results of this study cannot directly provide individual-level evidence of the effect of HPV vaccine awareness on acceptability. Third, the key results were sensitive to the definition of HPV vaccine acceptability; when acceptability was defined as including "Not sure/It depends" in addition to "Yes", the main results were not statistically significant. Fourth, the relatively low response rate in the HINTS may not have captured a representative population. Finally, the HINTS 2007 was conducted almost two years after FDA's approval of GardasilDespite these limitations, this study suggests that raising HPV vaccine awareness among those who are not aware of the HPV vaccine in the US - and therefore least likely to uptake HPV vaccination - could substantially increase HPV vaccine acceptability at the population level. This finding provides additional support to strengthening public health programs that increase awareness and policy efforts that address barriers to HPV vaccination.HPV: human papillomavirus; HINTS: Health Information National Trends Survey.No financial disclosures were reported by the authors of this paper. The authors declare that they have no competing interests.YKD conceived the study, analyzed the data, and drafted and completed the manuscript. In addition to conducting the literature review on HPV awareness and acceptability, KYW helped in data analysis and the drafting and completion 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/1471-2458/12/31/prepub


The control group (n = 102) comprised healthy individuals with normal blood biochemical values. The plasma coenzyme Q10, malondialdehyde (MDA) and antioxidant enzymes activities , superoxide dismutase (SOD), glutathione peroxidase (GPx)) were measured. Subjects with CAD had significant lower plasma coenzyme Q10, CAT and GPx activities and higher MDA and SOD levels compared to those of the control group. The plasma coenzyme Q10 was positively correlated with CAT and GPx activities and negatively correlated with MDA and SOD. However, the correlations were not significant after adjusting for the potential confounders of CAD with the exception of SOD. A higher level of plasma coenzyme Q10 (≥0.52 μmol/L) was significantly associated with reducing the risk of CAD. Our results support the potential cardioprotective impact of coenzyme Q10.A higher oxidative stress may contribute to the pathogenesis of coronary artery disease (CAD). The purpose of this study was to investigate the relationship between coenzyme Q10 concentration and lipid peroxidation, antioxidant enzymes activities and the risk of CAD. Patients who were identified by cardiac catheterization as having at least 50% stenosis of one major coronary artery were assigned to the case group ( Coenzyme Q10  is a lipid-soluble benzoquinone with 10 isoprenyl units in the side chain and is a key component of the mitochondrial respiratory chain for adenosine triphosphate (ATP) synthesis . CoenzymCardiovascular disease (CVD) is the leading cause of death worldwide. The known traditional risk factors for CAD are smoking, obesity, hypertension, a family history of CAD, diabetes mellitus, and hyperlipidemia. In addition to the traditional CAD risk factors, enhanced oxidative stress is a novel risk factor of CAD. Increased oxidative stress is associated with the pathogenesis of CAD –14. Clinn = 51). Case subjects with diabetes, liver, renal diseases, or undergoing statin therapy were excluded. None of our subjects had experienced an acute myocardial infarction within the previous 6 months. Control subjects were recruited from the physical examination unit of Taichung Veterans Hospital. Control subjects did not have any illnesses and a history of gastrointestinal disorder, cardiovascular disease , hypertension, hyperlipidemia, liver and renal disease, diabetes, cancer, alcoholism, or other metabolic disease and exhibited normal blood biochemical values, including fasting blood glucose < 6.11 mmol/L, blood urea nitrogen (BUN) < 7.9 mmol/L, creatinine < 123.8 μmol/L, alkaline phosphates < 190 U/L, glutamic oxaloacetic transaminase (GOT) < 35 U/L, and glutamic pyruvate transaminase (GPT) < 45 U/L. Subjects currently taking antioxidant vitamin supplements were also excluded. Informed consent was obtained from each subject. This study was approved by the Institutional Review Board of Taichung Veterans General Hospital in Taiwan. The age, blood pressures, and smoking habits of all subjects were recorded. Blood pressure was measured in each patient after resting for at least 5 min. Body weight, height, waist, and hip circumferences were measured and the body mass index (kg/m2) and the waist to hip ratio were then calculated.The current study was designed as a case-control study. CAD patients were recruited from the cardiology clinic of Taichung Veterans General Hospital in Taiwan. Patients who were identified by cardiac catheterization as having at least 50% stenosis of one major coronary artery or receiving percutaneous transluminal coronary angioplasty (PTCA) were assigned to the case group (Fasting venous blood samples (15 mL) were obtained to estimate hematological and vitamin status. Blood specimens were collected in Vacutainer tubes  with or without containing EDTA as an anticoagulant as needed. Serum and plasma were prepared and then frozen (−80°C) for storage until analysis. Hematological parameters ) were measured using an automated biochemical analyzer.Plasma coenzyme Q10 was measured using high-performance liquid chromatography (HPLC) according to the method of Chu et al.  and Littt-test or the Mann-Whitney rank sum test. For categorical response variables, differences between two groups were assessed using the Chi-square test or the Fisher's exact test. To examine the relationships of the plasma coenzyme Q10 concentration and the ratio of coenzyme Q10 to lipid profiles with oxidative stress (MDA) and antioxidant enzymes activities , multiple linear regression analyses were used. We adjusted the potential confounders of CAD, including age, gender, systolic blood pressure, waist to hip ratio, creatinine, and smoking. Adjusted odds ratios (ORs) with 95% confidence intervals (CI) for CAD were calculated from the logistic regression models based on the fourth level (75th percentile) of plasma coenzyme Q10 and the fourth level (75th percentile) of the ratio of coenzyme Q10 to lipid profiles. Data were expressed as means ± standard deviations, and results were considered statistically significant at P < 0.05.Data were analyzed using SigmaStat statistical software . The normal distribution of variables was evaluated using the Kolmogorov-Smirnov test. Differences in subjects' demographic data and the hematological measurement data between case and control groups were analyzed using the Student's P < 0.01) and the ratio of coenzyme Q10 to lipid profiles (P < 0.01). The value of MDA was significant higher in the case group (P < 0.01). With regard to the levels of antioxidant enzymes, subjects in the case group had significant lower CAT (P < 0.01) and GPx activities (P < 0.01) but higher SOD activities (P < 0.01) than the control group.  The plasma coenzyme Q10 concentration, lipid peroxidation and antioxidant enzymes activities are shown in Figures β = −0.72, P < 0.05) and the ratio of coenzyme Q10, but the statistical significance were disappeared after adjusting for age and gender or the potential confounders of CAD. The plasma coenzyme Q10 concentration and the ratio of coenzyme Q10 to lipid profiles were significantly positively correlated with CAT and GPx activities but significantly negatively correlated with SOD activities. However, the correlations were not significant after adjusting for the potential confounders of CAD with the exception of SOD. The correlations between coenzyme Q10 concentration, lipid peroxidation, and antioxidant enzymes activities are shown in μmol/L) or with a higher ratio of coenzyme Q10 to lipid profiles  had significant reductions in the risk of CAD. Furthermore, we calculated the ORs of CAD based on the fourth level (75th percentile) of plasma coenzyme Q10 concentration and the fourth level (75th percentile) of the ratio of coenzyme Q10 to lipid profiles . Subjectμmol/L) and a higher ratio of coenzyme Q10 to lipid profiles (coenzyme Q10/TG ≥ 0.52 μmol/mmol and coenzyme Q10/LDL-C ≥ 0.18 μmol/mmol) showed a significantly lower risk of CAD even after adjusting for age, gender, and the potential confounders of CAD. This result is similar to a cohort study conducted by Molyneux et al. [μmol/L) was an optimal cut-off point to predict the mortality of patients with chronic heart failure. Patients with lower coenzyme Q10 concentration might have compromised mitochondrial function and correlating to the severity of disease [μmol/L) in this study is also similar with the CORONA  trial conducted by McMurray et al. [μmol/L) in a univariate analysis but not in a multivariable analysis. The plasma coenzyme Q10 concentration has been shown to be reduced under statin therapy [P = 0.01) (data not shown). The present study showed the plasma coenzyme Q10 concentration had statistically significant reductions in the risk CAD. In x et al. , that fo disease . The cuty et al. , the mor therapy , 22, andμmol/L) [P < 0.01), which is an indicator of free radical-induced damage during myocardial ischemia [Although our CAD subjects were stable and had no experience of acute myocardial infarction within the previous 6 months, their plasma coenzyme Q10 concentration was significantly lower than that of control subjects  and the μmol/L) . Patientμmol/L) –14. Subjischemia , 25. Theischemia , 26. Coeβ = −0.11, P < 0.01) and older patients  had significantly lower levels of plasma coenzyme Q10 in the case group compared to those in the control group. Other CAD risk factors such as blood pressure [β = −0.00, P = 0.01), smoking , and waist to hip ratio . Therefore, we presume that the plasma coenzyme Q10 level was lower in the case group due to oxidative stress and the traditional CAD risk factors. Traditional CAD risk factors such as gender  and age pressure , obesitypressure , 29, andpressure  may alsoCoenzyme Q10 is a lipid-soluble antioxidant, that is, transported by lipids and lipoprotein  in the blood . TherefoFew studies have investigated the relation between plasma coenzyme Q10 and racial difference, especially in Asian population. An observational study was conducted by Hughes et al.  reportedμmol/L, hence the desired power was set at 0.8 to detect a true effect, and α = 0.05 with a minimal simple size of 40 participants in each group). Second, this study was the absence of age and gender matched between case and control groups; as a result, we try to limit these biases by adjusting for the potential confounders of CAD in statistical tests. Lager studies are needed to establish the beneficial effect of coenzyme Q10 in CAD patients. Our study has two limitations. First, the number of participants was small, although we did recruit more subjects than we expected to recruit (sample size calculation: we expected the differences in mean levels of plasma coenzyme Q10 between case and control groups were to be 0.2 ± 0.3 Patients with CAD were exposed to a higher level of oxidative stress and a lower coenzyme Q10 concentration. Our results indicate a strong correlation between the plasma coenzyme Q10 and reductions in the risk of CAD. It might benefit in administration of coenzyme Q10 to CAD patients, especially those with low coenzyme Q10 level.


Previous studies have either investigated the relationship of HPV with oral cancer or the prevalence of HPV on the oral cavity. The purpose of this investigation was to study the prevalence of HPV in oral cavity of women with oral sex practices and cervical lesions.Forty six (46) non-smokers and non-alcoholic patients attended the "Clínica de Displasias" of "Ciudad Juarez" were sampled. This population had a CIN diagnosis sometime between the previous six months. On previous consent they filled out a questionnaire related to their oral sex practices. Afterwards one swab from cheeks and another from palate/gum were taken; PCR was used to determine generic HPV, HPV16 and HPV18.Seventy two percent (72%) of the patients stated to have oral sex practices regularly which all of them were positive to HPV either in oral mucus, palate/gum or both. The total of the given results showed that 35% had HPV16; among those distributed in 26% with regular oral sex practices and 9% stated as never practiced oral sex. An association was found between oral HPV16 positivity and progression to cervical CIN advanced lesions. On the other hand HPV18 was not detected. The frequency of HPV16 was higher in buccal mucosa (23%) versus palate/gum (16%).This study suggests that buccal HPV16 infection is associated with CIN progression. Human papillomaviruses (HPVs) are a family of small 55 nm) icosahedral, non-enveloped virus with a circular double-stranded DNA genome of 7-8 kbp and with a special affinity for epithelial cells ,2. Over 5 nm icosThe potentially oncogenic HPVs has been associated with oral squamous cell carcinoma ,7. Some Despite the recognition of a HPV-associated oral malignancy, it is unclear to what extent cervical HPV infection is translocated to oral HPV infection. Previous studies suggested that oral HPV infection analogously to cervical infection is associated with sexual behavior and immunosuppressant ,13. The This study included 46 voluntary non-smoker and non-alcoholic women that attend to the "Clínica de Displasias del Sector Salud" in the City of Juarez, México. The female subjects attend to this clinic because they presented previous CIN alterations. After they filled out the inform consent and the questionnaire; the patients were instructed by imitation for autosampling the oral cavity. Once in the laboratory and after DNA extraction the human β-globin gene was amplified as an internal control of human DNA as well as a DNA with quality for PCR. Those cases that failed in the β-globin gene amplification were excluded rather than those samples tested positive for β-globin were further used for generic HPV that amplify a wide range of HPV types. After generic HPV positivity the specific PCR for HPV16 and HPV18 were performed. Two regions were sampled in the oral cavity; the first one was the buccal mucosa and the second one was the palate and gum altogether (P/G). All women studied carried generic HPV either in mucosa or P/G, this gives a frequency of 100% for the buccal cavity. However, if the mucosa and P/G are considered separately (Table Sixty-three percent (63%) of the studied female subjects were married, 19% were in common law marriage and 7% were singles. The mean age was 35 years old ranging between 19 to 63 years old (not shown in tables). Table All women were CIN diagnosed along the previous six months (according to Bethesda classification) because of that; it is clearly evident the association between oral HPV16 positivity and CIN progression 51% , followed by 28% with inflammatory alterations; and finally 21% did not present cervical alterations  declared to have only one sex partner, 23% had two partners and 2% had more than two partners (Table The majority (53%) of the women stated not to share spoons, toothbrush or candies Table . But an The prevalence rate of HPV in normal oral mucosa has been reported to vary greatly because of differences in types of samples, collection, detection methods, level of sensitivity, PCR primers used, and PCR inhibitors ,15,22-32In this study, we performed the HPV detection by PCR using the MY09/MY11 primer pair; which, it is widely used on epidemiological studies and showed to be equally sensitive than confirmatory nested PCR with GP5+/GP6+ primers, with a correlation of 94% -35. GeneChlamydia trachomatis [We evaluated HPVs only in buccal samples. Although in cancers of the upper aerodigestive tract HPV has been detected predominantly in the oropharynx and tonsil ,35. A richomatis ,31 or hechomatis , smokingchomatis , age [37chomatis , and thechomatis ,36,39. HIn our study HPV16 in mouth was significantly associated with genital CIN progression , suggesting that women with HPV16 persistent infections and progression to advanced genital lesions have higher risk of HPV16 detection in the oral mucosa. We found HPV16 infection in a media of 37 years. As this type of HPV is related to cancer; it is also considered as high-risk mucosal type virus  and thisThe role of men as possible vectors of HPV has been discussed previously . Our resIn a cross-sectional study of Fahkyr , oral HPOur data hint, it is that oral cavity should be considered as a potential reservoir for HPV and may not be entirely independent of the cervical reservoir as well as the partner genitalia. In this study we did not sample the partner's genitalia but we assume that both genitalia are infected since these are considered as sexually transmissible infections. Thus the presence of the virus in the oral cavity should be explained not only by oral contact with genitalia but also by autoinoculation. We are aware about the different prevalence data in oral HPV DNA detection ought to different populations and methodologies. Consequently comparative studies will be required in parallel studies of the relationship between incident squamous epithelial lesions and persistence oral HPV. This will help to understand the involvement of HPV in the development of oral cancer as well as the role of oral cavity in the cervical infection.In conclusion, this study suggests that women with genital HPV infection have also some kind of HPV infecting their oral cavity and HPV16 detection in the mouth is associated to HPV16 persistence in the genital tract and CIN progression.This study was performed on summer 2008. All women had a previous CIN diagnosis between six months previous to the study. The Inclusion criteria were any women that attend to "Clínica de Displasias" for any cervical associated problem, no matter their origin or place of living (Mexico or USA), sign an informed consent and fill a questionnaire about their sex habits. The exclusion criteria were: smoking and alcoholism habits. All women studied were above eighteen years old. The full protocol was approved by the Ethics Committee of the Universidad Autonoma de Ciudad Juarez and the Clinica de Displasias. Women were asked to autosampling with a cotton swab the two cheeks and with other cotton swab the palate/gum (P/G). In both cases tissues were rubbed for a minute. Cotton swabs were immersed into 15 mL tube containing 1 mL of transporting media  and stored at -20°C in between 24 h after collection.Samples were unfreeze, centrifuged at 3,500 rpm in a clinical centrifuge, cotton swab removed and the transporting media was transferred to a 2 mL microtube that was centrifuged 5 min at 3,500 rpm at 4°C. 300 μL of clear supernatant were taken into a new tube and 25 μL of 5 M sodium acetate and 1 mL of isopropanol were added consecutively and centrifuged at 14,000 rpm at 4°C for 5 min. Pellet were washed with 1 mL of 70% ethanol and dried overnight at room temperature. Pellet was dissolved with 100 μL rehydration solution (Promega A7963) and incubated at 65°C for 20 min.PCRs for generic HPVs were assembled with 5 μL DNA, 12.5 μL 2X GoTaq Green Master Mix (Promega). 5 μL MY11/MY09 primer mix at 2.5 μM each, and 2.5 μL water. PCR conditions were 40 cycles at 94°C for 60 s, 55°C for 60 s, 72°C for 60 s, with an initial denaturation at 94°C for 5 min and a final extension of 72°C for 7 min . PCR proQuestionnaire answers, previous histopathological results and buccal HPVs results were analyzed with SPSS statistical software version 11 and Mann Whitney U Test .There are no conflicts of interest by any of the authors involved neither in this study publication nor for the previous 2 years.LS carried out the molecular genetic studies, participated in the sampling, and questionnaire design, analysis and drafted the manuscript. CD carried out the selection of patients, sampling, questionnaire application, and analysis of data. AM conceived of the study, and participated in its design, statistical analysis, writing and coordination of the study. All authors read and approved the final manuscript.


Trichomonas vaginalis (TV) an inducing factor for the development of (pre-)cancerous lesions of the cervix?Is Cross sectional study.Screening healthy Belgian women with low infection risk.63,251 consecutive liquid based cervical samples.Real time quantitative PCR for presence of TV, 18 HPV types and Pap smear analysis of cytologic abnormalities. Association of TV and HPV with cervix dysplasiaThe overall prevalence of TV DNA was 0.37%, of low risk HPV 2%, of high risk HPV 13.2%, and 8.8 % had cytological abnormalities. Both LR-HPV and HR-HPV were significantly associated with all cytological abnormalities. Presence of TV was associated with LR- and HR-HPV, ASC-US and HSIL, but not with other abnormalities. All women with TV and HSIL also had HR-HPV, while the latter was present in only 59% of women with TV and ASC-US. Amongst HPV negative women, TV was found in 1.3% of women with ASC-US, but only in 0.03% of women with normal cytology . In HR-HPV positive women, presence of TV increased the likelihood of cytological abnormalities somewhat (P=0.05), mainly due to an increase in ASC-US and LSIL, but not HSIL. We conclude that TV infection is associated with both LR and HR-HPV infection of the cervix, as well as with ASC-US and HSIL. TV is a concomitant STI, but is not thought to be a co-factor in the causation of HSIL and cervical cancer. However, TV may cause false positive diagnoses of ASC-US. Trichomonas vaginalis (TV) infection are common sexually transmitted infections (STIs) worldwide [Both human papilloma virus (HPV) and orldwide . The incorldwide . Despiteorldwide -5, probaorldwide ,7. Introorldwide . In someorldwide . We asseth April and 1st November 2008 by the Laboratory for Clinical Pathology (labo RIATOL), Antwerp, Belgium, for cytologic examination and TV testing, were tested for presence of Trichomonas vaginalis and HPV DNA. The medical ethics committee of the University of Antwerp confirmed that ethical oversight is unnecessary for retrospective case studies such as this.  All data were anonymized, no specific tests were performed outside routine practice, and there was no cost or additional risk to the patient.All clinical cervico-vaginal samples, received between 15Trichomonas vaginalis was determined using a multiplex TaqMan-based real-time quantitative PCR. The sequences for the primers and probes used for different type specific HPV qPCRs can be found in the paper by Micalessi and coworkers [Cervico-vaginal cells were collected with the Cervex-Brush Combi . After collection, brush heads were transferred directly into a vial with BD-SurePath preservative fluid . From thoworkers . For detoworkers ,15. HPV oworkers ,18,19. MThe analytic sensitivity of the different PCR assays ranged from 1 to 100 copies and was calculated using standard curves for 16 type-specific PCRs constructed with plasmids containing the entire genome of the different HPV types . Real-tiStatistical analysis was performed with Chi-squared or Fisher exact test when appropriate for absolute numbers, and Student t test for normal distributed continuous variables.From 15th April to 1st November 2008, 64241 consecutive cervical cytology samples were processed in the laboratory. We excluded 966 (1.50%) follow up samples and 24 (0.04%) samples in which no human ß-globin DNA could be amplified.For 63,251 (98.5%) of the remaining samples cytology, HPV and TV results were available 1. The fWhen studying the correlation of LR and HR HPV and cytological abnormalities we found evidence of HPV infection in 7.8% in women without cytological abnormalities, and in 65.2%, 31.8%, 81.6%, 89.3% and 92.6% of women with ASC-US, AGC, LSIL, ASC-H and HSIL respectively. Both LR HPV and HR HPV were significantly associated with all cytologic abnormalities , compared to 0.86% (14/1623) in the HPV positive (LR+HR) ASC-US group .Amongst all HPV negative women, TV was found in 1.3% with ASCUS, and 0.03% with normal cytology . In HR HPV positive patients, the likelihood of having ASC-US+ was slightly higher when TV was present than when TV was absent . However, the majority of lesions in the group with combined HR HPV/TV infections were ASC-US (13/31) or LSIL (11/31) and not HSIL (7/31). In this study, we confirm a correlation of TV with HPV and with cytological abnormalities of the cervix, indicating that STIs are often coexist, even in a setting where the prevalence of TV is very low. This low prevalence makes the interpretation of the role of TV in the pathogenesis of cytologic abnormalities less evident in certain subgroups, despite high numbers of samples examined, a reliable diagnostic technique used  and the completeness of the data obtained, which all add to the strength of the study. T. vaginalis is low in Flanders at 3.7 per 1000. Furthermore, we found that the age-dependent prevalence and epidemiology for HPV and TV were very different in this population, with a peak incidence of HPV infection at the age of 20-25, and of TV infection at 45-55 years [The correlation of LR HPV and HR HPV with different types of cytological abnormalities confirms expected HPV detection rates in cervical dysplastic lesions, as discussed in other studies. The prevalence of 55 years , a findi55 years . Despite55 years ,23. As a55 years . With the present data, in a low prevalence setting, we could demonstrate that in all cases of HSIL where TV was discovered, HR HPV was also present as a causative agent. On the other hand, 36% (8/22) of the women with a combination of TV infection and ASC-US had no HPV. Although the prevalence of TV was higher in the HPV negative ASC-US group (1.3%) compared to the HPV positive (LR+ and HR+) ASC-US group (0.86%), this was not statistically significant. However since cytology reading was performed with knowledge of HPV status, presence of TV could still explain detection of atypical cells in the HPV negative ASC-US group. Also, in the group of HPV negative women, the infection rate of TV was 4 times higher in women with ASC-US than in cytological normal women. Furthermore, in HR HPV negative women, TV was never associated with HSIL or ASC-H, but was present in almost 1% of ASC-US and LSIL cases. These findings strongly suggest that TV can cause ASC-US like findings on cytology, without HR HPV or even any HPV present, and hence could lead to false positive diagnosis of ASC-US, enhancing the likelihood of overtreatment and unnecessary anxiety ,25. OtheAlso, the apparent relation of TV with HSIL (OR 3.2) is almost certainly due to co- infection with HR HPV in all cases, instead of being causative or even a co-factor for the development of HSIL. As only 1% of all HSIL were harboring TV, it seems very unlikely that these sporadic TV would have a significant role in the pathogenesis of HSIL. Still, if present together with HR HPV infection, TV infection was associated with a slightly higher rate of ASC-US+ abnormalities than when only HR HPV were present. As 4 out of 5 of these ASC-US+ lesions were low grade lesions (ASC-US and LSIL), we suppose the small increase in HSIL in combined TV-HR HPV patients may also be due to non-specific cellular aberrations caused by TV, as we found in one third of ASC-US in TV infected women without HPV infection.We conclude that TV infection is associated with both low risk and high risk HPV infection of the cervix, as well as with the cervical cytological abnormalities ASC-US and HSIL. However, TV is unlikely to be involved in the causation or promotion of HSIL and cervical cancer. In women infected with TV, non-specific alterations of the epithelial cells may lead to a number of false positive diagnoses of ASC-US. In order to eradicate TV before making a final cytology diagnosis, we strongly advocate the use of real time PCR or systematic TV detection in liquid media before cytological examination.


Previous analyses from a randomized trial in women aged 24–45 have shown the quadrivalent HPV vaccine to be efficacious in the prevention of infection, cervical intraepithelial neoplasia (CIN) and external genital lesions (EGL) related to HPV 6/11/16/18 through 4 years. In this report we present long term follow-up data on the efficacy, safety and immunogenicity of the quadrivalent HPV vaccine in adult women.Follow-up data are from a study being conducted in 5 sites in Colombia designed to evaluate the long-term immunogenicity, effectiveness, and safety of the qHPV vaccine in women who were vaccinated at 24 to 45 years of age  or 29 to 50 years of age . This analysis summarizes data collected as of the year 6 post-vaccination visit relative to day 1 of the base study (median follow-up of 6.26 years) from both the original base study and the Colombian follow-up.There were no cases of HPV 6/11/16/18-related CIN or EGL during the extended follow-up phase in the per-protocol population. Immunogenicity persists against vaccine-related HPV types, and no evidence of HPV type replacement has been observed. No new serious adverse experiences have been reported.Vaccination with qHPV vaccine provides generally safe and effective protection from HPV 6-, 11-, 16-, and 18-related genital warts and cervical dysplasia through 6 years following administration to 24–45 year-old women.Clinicaltrials.govNCT00090220 Persistent infection of the uterine cervix by 15 to 20 carcinogenic human papillomavirus (HPV) genotypes leads to the vast majority of cervical cancers The quadrivalent HPV (qHPV)  L1 virus-like particle (VLP) vaccine is highly effective in preventing HPV 6, 11, 16, or 18-related high-grade intraepithelial neoplasia and condyloma in men and women aged 16 to 26 naïve to the respective vaccine HPV types at enrollment In this report we elaborate on previous FUTURE III results by presenting the findings of a long-term follow-up of that study evaluating the effectiveness, immunogenicity and safety of the qHPV vaccine in women originally enrolled into the trial from Colombian study sites. Analyses are cumulative incidence since vaccination, and therefore include base study data from all subjects as well as follow-up data from subjects in Colombia only.Between June 18, 2004 and April 30, 2005, 3,819 women between the ages of 24 and 45 years were enrolled at 38 international study sites into a randomized, placebo-controlled, double-blind safety, immunogenicity, and efficacy study (NCT00090220). Subjects were enrolled from community health centers, academic health centers, and primary health care providers in Colombia, France, Germany, Philippines, Spain, Thailand, and the United States. Descriptions of treatments, endpoints, hypotheses and case definitions have been published The protocol for this trial and supporting CONSORT checklist are available as supporting information; see V501-Protocol 019-21 is a study being conducted in 5 sites in Colombia and is designed to evaluate the long-term immunogenicity, effectiveness, and safety of V501, the quadrivalent Human Papillomavirus  recombinant vaccine (qHPV vaccine) in women who were vaccinated at 24 to 45 years of age  or 29 to 50 years of age . This interim analysis report summarizes data collected as of the year 6 post-vaccination visit (relative to day 1 of the base study). Future analyses are planned at year 8 and year 10 . The analyses performed are cumulative incidence since vaccination. Base study data from all subjects contributes for the first 4 years. However, only subjects from Colombia contribute follow-up data after 4 years.This long-term study does not have a placebo group. Subjects who were vaccinated with qHPV vaccine in the base study at 24 to 45 years of age are referred to as the “early vaccination group” (EVG) in this report. Subjects who were vaccinated with placebo in the base study were later vaccinated with a three dose regimen of qHPV vaccine during the first extension of the base study at 29 to 50 years of age (V501 protocol 019-10) and are referred to as the “catch-up vaccination group” (CVG). Because there is no placebo group for the long term follow-up phase, vaccine efficacy cannot be measured. In lieu of efficacy measurements, effectiveness of vaccination with qHPV vaccine is assessed by calculating the incidence of disease endpoints in each of the EVG and CVG and comparing these rates with those observed in groups vaccinated in previous efficacy studies within Merck's qHPV vaccine program. No study vaccinations were provided within the context of this long-term follow-up study.To observationally describe the general safety of a 3-dose regimen of qHPV vaccine [Human Papillomavirus  Recombinant Vaccine] in women 24 to 50 years of age during a period of 5 to 10 years following vaccine dose one by describing vaccine- or study procedure-related serious adverse experiences (SAEs), SAEs resulting in death, prespecified medical conditions, and pregnancy follow-up outcomes.The long-term safety profile will be characterized in terms of incidence of SAEs that a study investigator considers to be possibly, probably, or definitely related to prior administration of the qHPV vaccine or to a study procedure; incidence of death; prespecified medical conditions; and incidence of pregnancy, including pregnancy outcomes and fetal or infant condition.To evaluate the kinetics and age-dependence of anti-HPV 6, 11, 16, and 18 responses following a 3-dose regimen of qHPV vaccine at approximately 6, 8, and 10 years after vaccination dose one among subjects from the qHPV vaccine group of the base study and approximately 1, 3, and 5 years after vaccination dose one among subjects from the placebo group of the base study. The long-term immunogenicity profile will be characterized in terms of geometric means of vaccine-HPV types 6, 11, 16, and 18 titers and seropositivity to the same four vaccine-HPV types. Two different assays will be used; the competitive Luminex immunoassay (cLIA) and the total IgG assay.To observationally describe the incidence of HPV 6/11/16/18 related genital warts or cervical dysplasia, and of HPV 16/18-related CIN2+  up to 6 years following administration of qHPV vaccine in 24 to 50 year-old women. The analyses are cumulative incidence since vaccination. Therefore, the base study data from all subjects contributes for the first 4 years. Only the Colombian subjects have follow-up after 4 years.HPV type replacement will also be observationally described. The incidence of non-HPV 6/11/16/18 related genital warts or cervical dysplasia, and of non-HPV 16/18-related CIN2+  will be assessed in Colombian women. The HPV types that will be studied are those studied in the base study, specifically HPV 31/33/35/39/45/51/52/56/58/59 and all high-grade lesions due to any non-vaccine type.All subjects who received at least one dose of the qHPV vaccine and have follow-up data, starting from when they have signed consent to enter the long-term study will be included in the summaries of safety.The primary effectiveness analysis approach for the EVG will be per-protocol-efficacy (PPE), as defined during the base study. To be eligible for this population, subjects must (i) have received 3 doses of GARDASIL™ within one year, and (ii) have no protocol violations. Subjects will be considered cases related to a given HPV type provided the subject was negative to the respective HPV type by serology and PCR prior to vaccination  and PCR negative through month 7. For purposes of endpoint definition, only the Pathology Panel diagnosis will be considered. Supportive analyses will be done for the group naïve to the relevant HPV type (HNRT) and full analysis set (FAS) populations. The HNRT population consists of all subjects who received at least one vaccination and were seronegative and PCR negative to the relevant type prior to vaccination. The FAS population includes all vaccinated subjects, regardless of HPV status.For the CVG, no serology or swab samples were taken immediately prior to vaccination with qHPV vaccine in the extension. However, any positivity recorded during the base study period (day 1 to month 48) was counted. By comparison, the EVG subjects had a single time-point of assessment for baseline HPV positivity.No new randomization was performed during this long-term follow-up study. The allocation numbers assigned to study subjects from Colombia during the base study were carried over to this study. At visits corresponding to years 6, 8 and 10 after the beginning of the base study, subjects underwent medical and gynecologic histories, pelvic exams, ThinPrep™ Pap tests and a wart/lesion inspection. In addition, serum samples were taken for anti-HPV serology testing. No new vaccinations were provided in this long-term follow-up study.Cytology specimens were evaluated using the Bethesda System—2001. For all cytology diagnoses of Atypical Squamous Cells of Undetermined Significance (ASC-US), a central laboratory automatically performed reflex HPV testing on residual ThinPrep™ material, using the Digene Hybrid Capture II™, High Risk/Low Risk Probes. If at least 1 probe was positive or if no result was obtained, the subject was referred for colposcopy. If cervical biopsies and/or endocervical curettage (ECC) specimens were obtained, specimens were sent to the central laboratory for analysis. Endpoint adjudication was performed by an independent pathology panel.The competitive Luminex immunoassay (cLIA) A total IgG Luminex immunoassay (LIA) was developed utilizing yeast-derived L1 VLPs of HPV types 6, 11, 16 and 18 coupled to a set of distinct fluorescent Luminex microspheres In the absence of a control group in the long-term follow-up study, it is not possible to perform analyses of efficacy. Instead, effectiveness will be measured in terms of incidence rates of disease, with analyses performed periodically until the end of the follow-up. Incidence rates based on accrued person-years of follow-up will be estimated together with a corresponding 95% confidence interval, calculated using the exact confidence limits of the binomial distribution, as an approximation to the Poisson distribution. Analyses will combine events and follow-up time from the base study and, for those participating, from the extension study. Exact binomial confidence intervals were calculated for the proportions of subjects seropositiveThis report is comprised of follow-up data from study start through the cutoff date of 02-Nov-2011. The median follow-up time from day 1 of the base study in the EVG was 6.26 years. For the CVG who received qHPV vaccine after the base study, the median follow-up time post-dose 1 of the vaccine was 1.18 years. The rates of positivity prior to vaccination in the CVG are generally higher than those observed in the EVG at the start of the base study, partly as a result of increased ascertainment of infection and partly because of persistent risk of infection in this older age group of women.The cumulative incidence of non-vaccine HPV type (HPV 31/33/35/39/45/51/52/56/58/59)-related genital warts or cervical dysplasia, from the start of the base study through all visits completed before the cutoff date, in the EVG FAS population can be seen in Similar data were seen for vaccine-related CIN or condyloma in the FAS population . For nonThe proportion of subjects who are seropositive to HPV 6, 11, 16 and 18 at month 72 (year 6) from first vaccination, as measured by cLIA and total IgG assays can be seen in Month 72 total IgG seropositivity for HPV 6, 11, 16 and 18 was 87.8%, 84.4%, 98.8% and 81.5%, respectively. It is noticeable that the overall seropositivity for HPV 18 exceeds 80% , which iNo serious adverse experiences have been reported in the context of the long-term extension study period. Previous reports have demonstrated the efficacy, safety and immunogenicity of the qHPV vaccine in women aged 24 to 45 There were no cases of HPV 6/11/16/18-related CIN or condyloma, HPV 16/18-related CIN 2 or worse or HPV 6/11-related condyloma during the follow-up visits in years 4 to 6 in all of the analysis populations. This finding suggests that qHPV vaccine effectiveness continues to be high in this population of women through 6 years following vaccination. The observed person-time of follow-up after the base study was not large enough to demonstrate statistically significant protection against HPV 16/18-related CIN 2 or worse and HPV 6/11-related condyloma. However, the upper 95% CI of the incidence of HPV 6/11/16/18-related CIN or condyloma was below the 4-year incidence of the same endpoint in the placebo group of the base study.There was no evidence of HPV type replacement in those women who were vaccinated with qHPV vaccine. Accordingly, the incidence of non-vaccine HPV types did not increase proportionally over time.The immunogenicity data from the cLIA showed persisting high titers over 72 months and showed no significant reduction in GMT from month 48 to month 72. Similarities in GMTs between the two age strata across all HPV types and time points could be seen when comparing the data over 72 months. While some differences between the two age strata can be seen, these differences are small and the 95% CIs overlap. Additionally, when the entire PPI population and those women who entered the long-term follow-up segment of the study are compared, the GMTs are similar for all HPV types and time points.No significant reduction in seropositivity from month 48 to month 72 was seen. Moreover, seropositivity at month 72 is similar to month 24 for each of the HPV types, including HPV 18. This observation indicates that effectiveness against HPV 18 related disease persisted when the cLIA was positive in approximately 50% of women for the last two years of the base study and for the first two years of the long-term follow-up study.As described A drawback of the current study is its localization to subjects enrolled only from Colombia. While the data are interesting and observational in nature, the generalizability of the findings would be greater if enrolling a larger and/or more geographically diverse cohort.Administration of the qHPV vaccine in the base study was generally well tolerated. Moreover, no serious adverse experiences have been reported in the context of the long-term extension study period. The proportion of subjects who reported serious adverse experiences was comparable between the qHPV vaccine group and the placebo group. Of the four known pregnancy outcomes to date, all have resulted in normal live births.It is clear that HPV vaccination is likely to be beneficial to sexually active adult women as they are at risk of acquiring new HPV infection and related sequelae In summary, we have demonstrated that vaccination with qHPV vaccine provides durable protection from HPV 6-, 11-, 16-, and 18-related genital warts and cervical dysplasia through 6 years following administration to 24–45 year-old women. In addition, the qHPV vaccine shows no tendency to select for disease due to HPV types that are not present in the vaccine through 6 years following vaccination in women 24 to 45 years of age. Lastly, administration of qHPV vaccine to women 24 to 45 years of age is generally safe and well tolerated through year 6 post-vaccination.Table S1Pregnancy outcomes. There were 4 live births in the EVG population and none in the CVG population. No fetal losses have occurred.(DOC)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.


Human papillomavirus (HPV) is the causative agent in cervical cancer and HPV genotypes 16 and 18 cause the majority of these cancers. Natural killer (NK) cells destroy virally infected and tumour cells via killer immunoglobulin-like receptors (KIR) that recognize decreased MHC class I expression. These NK cells may contribute to clearance of HPV infected and/or dysplastic cells, however since KIR controls NK cell activity, KIR gene variation may determine outcome of infection.KIR gene frequencies were compared between 147 patients with a history of high-grade cervical intraepithelial neoplasia (CIN) and a control population of 187, to determine if any KIR genes are associated with high-grade CIN. In addition a comparison was also made between cases of high grade CIN derived from 30 patients infected with HPV 16/18 and 29 patients infected with non-16/18 HPV to determine if KIR variation contributes to the disproportional carcinogenesis derived from HPV 16/18 infection.High-grade CIN was weakly associated with the absence of KIR2DL2 and KIR2DS2  but this association was lost after correction for multi-gene statistical analysis. No difference in KIR gene frequencies was found between high-grade CIN caused by HPV 16/18 and non-16/18.No strong association between KIR genes, high-grade CIN and HPV genotype was found in the Western Australian population. Cervical cancer is the second most prevalent cancer in women worldwide accounting for approximately 230 000 deaths each year and Human Papillomavirus (HPV) is the causative agent. HPV genMany viruses commonly down-regulate major histocompatibility complex (MHC) class I molecule expression to avoid cytotoxic T cell responses and the down-regulation of MHC class I molecules has been a common finding in cervical cancer-12. It hNK cells are cytotoxic lymphocytes which kill target cells with decreased MHC class I molecule expression as a defence mechanism against malignant transformation or viral infection,16. PrevThe current study was undertaken to determine whether there are any associations between KIR genes and high-grade cervical intraepithelial neoplasia (CIN2 & 3), the precursor to cervical cancer, in the Western Australian population. Since 80% of cervical cancers in Western Australia are caused by HPV 16 and 18 genotypes (HPV 16/18), we performed a separate analysis of high-grade CIN samples containing these genotypes on the basis that neoplasia derived from these may be especially resistant to NK cell killing.KIR genotyping was successfully performed on all cervical cytology specimens. Figure All the KIR genes were represented in both the high grade CIN cases and controls. Table Risk factors which determine susceptibility to HPV infection and progression to cervical cancer are likely to be immunogenetic,23 and tAlthough HPV 16 and 18 cause the majority of cervical cancers in Western Australia, this study found no statistical significant difference in any KIR gene frequency between HPV 16/18 and non-16/18 derived high grade CIN. The protective effect of KIR2DL2 and KIR2DS2 was not more pronounced in the cases that were positive for the more oncogenic HPV 16/18 types of virus. However, since HPV has been associated with some head and neck cancers, and theThis study found no strong association between KIR gene frequencies and high-grade CIN in the Western Australian population, although KIR2DL2 and KIR2DS2 may have a very mild protective role against progression to high grade CIN. No association between KIR gene frequencies and HPV 16/18 derived high grade CIN was found.http://bsn.uwa.edu.au/) population which is considered to be reflective of the Western Australian population. This control group had a mean age of 50 with a range of 19 – 88 years. Although CIN status was not obtained for this control group high-grade CIN occurs very rarely[Cervical cytology specimens in ThinPrep  methanol-based fixative, were collected between December 2007 and August 2008 from Western Diagnostics Pathology Laboratory, Perth, Western Australia. The study population consisted of 147 women from Western Australia who had previously been diagnosed with high-grade CIN. This test group had a mean age of 38 with a range of 19 – 71 years. The control frequencies for KIR genes were obtained from 187 women from the Busselton Health Study (BHS)  method in which ThinPrep cervical samples were mixed by shaking and inversion, a 500 μl aliquot was removed and mixed with 1 mL of PCR grade water in a microfuge tube. This was centrifuged at 14,000 RPM for 5 minutes and all traces of the supernatant removed from the pellet. The pellet was then processed according to the manufactures protocol. It is recognized that cells containing integrated HPV would have genomic alterations; however the majority of cells within the ThinPrep sample would not be infected with HPV and would have normal genomes, and so were considered suitable for KIR typing by PCR.KIR genotyping was performed on all samples using multiplexed amplification primers specific for the following KIR genes: 2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3 and 3DS1 as described by Sun et al.. In eachχ2 analysis with Yates’ correction. A p-value less than 0.05 determined significance. Any p values less than 0.05 were corrected for multi-gene statistical analysis using Bonferroni’s correction[KIR gene frequencies in high grade CIN cases and controls, and in HPV 16/18 and non 16/18 derived high grade CIN, were compared by rrection.HPV: Human papillomavirus; CIN: Cervical intra-epithelial neoplasia; KIR: Killer immunoglobulin-like receptor; NK: Natural killer; MHC: Major histocompatibility complex; HLA: Human leukocyte antigen; BHS: Busselton health study; PCR: Polymerase chain reaction; OR: Odds ratio; CI: Confidence interval.The authors declare that they have no competing interests.All authors are from the School of Biomedical Sciences at Curtin University except CW who is a senior research scientist at the Dept of Clinical Immunology and Immunogenetics, Royal Perth Hospital, Western Australia.BB, PC, CM and CW participated in the design of the study and performed analysis of data. MW performed sample extraction, KIR typing for the KIR vs CIN section and contributed to the analysis of that data. RT performed sample extraction, KIR typing for the HPV 16/18 vs non 16/18 section and analysis of that data. All authors read and approved the final manuscript.


The low-risk human papillomavirus types 6 and 11 are responsible for approximately 90% of anogenital wart cases, with approximately 190,000 new and recurrent cases reported in the UK in 2010. The UK has recently selected the quadrivalent HPV vaccine, which conveys protection against both HPV6 and HPV 11, as part of its immunisation programme for 2012 and it is expected that this will reduce disease burden in the UK. The aims of the study were to evaluate current strategies used for the monitoring of HPV infection in genital warts and to assess the suitability of laser-capture microdissection (LCM) as a technique to improve the understanding of the natural history of HPV types associated with genital wart lesions.DNA and RNA were extracted from whole wart, surface swabs and LCM sections from 23 patients. HPV types present were determined using the Linear Array HPV Genotyping Test (Roche), with HPV DNA viral load and mRNA expression investigated using qPCR and qRT-PCR, respectively.Results indicated that swabbing the surface of warts does not accurately reflect potential causative HPV types present within a wart lesion, multiple HPV types being present on the surface of the wart that are absent in the lower layers of tissue isolated by LCM. Although it was shown that HPV DNA viral load does not directly correlate with HPV mRNA load, the presence of both DNA and mRNA from a single HPV type suggested a causative role in lesion development in 8/12 (66.6%) of patients analysed, with dual infections seen in 4/12 (33.3%) cases. HPV 6 and HPV 11 were present in more than 90% of the lesions examined.Surface swabbing of warts does not necessarily reflect the causative HPV types. HPV type specific DNA and mRNA loads do not correlate. HPV 6 and 11 were likely to be causally involved in over 90% of the lesions. Dual infections were also found, and further studies are required to determine the biological and clinical nature of dual/multiple infections and to establish the relationship of multiple HPV types within a single lesion. Human papillomavirus (HPV) is the aetiological cause of anogenital warts. Anogenital wart disease is the most common sexually transmitted disease, affecting millions worldwide every year. In 2010, approximately 190,000 new and recurrent cases were reported through GU clinics in the UK . These oMost epidemiological studies on wart disease have involved swabbing of the surface of lesions with subsequent HPV typing being undertaken on the extracted DNA -7. InterThe aim of this study was to determine whether surface swabbing of warts accurately reflects the HPV type or types causing the epidermal proliferation. Surface contamination with other HPV types was determined by comparing the HPV types found in swab samples to those present in whole wart tissue, using the Linear Array HPV genotyping test (Roche). Laser-capture microdissection (LCM) was then used to isolate the surface and the basal areas for further analyses, permitting the identification of potential candidate HPV types responsible for the wart lesion. Furthermore, HPV DNA and mRNA, thought to be the “gold-standard” by which HPV type is linked with cervical lesion progression -14, werestratum corneum and stratum granulosum) and the lower layer includes the uncontaminated stratum spinosum and stratum basale.HPV typing was performed by utilising DNA extracted from swabs and whole wart tissue in 23 samples, and the upper and lower layers of wart tissue dissected out using laser-capture microdissection (LCM) in 16 out of 23 patients. Figure The prevalence of HPV types present in the whole wart, swabs, and the upper and lower microdissected layers, classified into either low-risk or high-risk Table . The fivThere was very good concordance between HPV types detected between swabs and whole tissue, particularly for HPV 6, HPV 11 and HPV 31 . Furthermore, substantial agreement (0.80 ≥ κ > 0.60) (κ = 0.646) occurred for both HPV types 59 and 81. No concordance was observed (κ ≤ 0) for HPV 16, HPV 52, HPV 53 or HPV 54. Remaining HPV types exhibited at least fair agreement (κ > 0.20). Overall concordance for all types found between whole wart tissue and swabs was κ = 0.574 (moderate agreement).The overall concordance for all HPV types detected between upper and lower layers of wart tissue was κ = 0.632 . However, when compared with whole wart tissue, both upper and lower layers only exhibited moderate agreement . Furthermore, concordance estimates for HPV 6 was reduced from κ = 1.000  to substantial (κ = 0.600 for both wart vs upper and wart vs lower). HPV 11 was also reduced from κ = 1.000 to moderate (κ = 0.429 for both). This suggests that the LCM tissue is not representative of its corresponding whole wart tissue in terms of HPV types detected.HPV types were subsequently classified as surface contamination, undetermined or infection candidates, dependent on the presence or absence of HPV in the tissue samples analysed Table . These cQuantitative DNA and mRNA analyses for HPV E6 were performed for HPV 6, HPV 11, HPV 16, HPV 45 and 52 Table . These HPotential infection candidates, identified by subtracting probable and confirmed contaminant types, were then compared to the HPV types with confirmed expression from the mRNA analysis Table . Poor coAnogenital warts is the most common sexually transmitted disease in the world, and is also a marker of transmission of high-risk HPV types, such as HPV 16 and HPV 18, due to similar infection routes. Diagnosis with genital warts is associated with a long-term increased risk of anogenital and head and neck cancers . HoweverIn this study, laser-capture microdissection was used to facilitate the delineation of HPV types likely to be surface contaminants from those likely to be involved in the aetiology of the wart lesion. Subsequent real-time PCR analyses for both E6 DNA and E6 mRNA allowed their suitability for monitoring purposes to be determined. The use of simple equations, based on the premise that HPV types identified from within the deeper epithelium of the wart are more likely to be causal, allowed “infection candidates” to be identified. Messenger RNA detection for five common HPV types found in this cohort was then used to identify the most likely candidate to be involved in actual lesion pathogenesis. HPV 6 was a more frequent cause of lesions than HPV 11. Interestingly, the majority of HPV types identified as surface contaminants were high-risk types (75%), with the remainder being identified as low-risk types (25%). The majority of contaminant HPV types featured only once or twice in the samples (incidence <8%). HPV 16 was the most common surface contaminant (25% of wart lesions). Interestingly, many of the surface contaminants detected via the LCM and typing analyses were high-risk types and we speculate that perhaps high-risk types have an innate survival advantage over many low-risk types  and this aids their transmission rates.The incidence of multiple infections detected in wart studies ranges from 33.8% to 40.1% when using only swabbing techniques ,21, as wModerate concordance exists between whole wart tissue and corresponding swabs in terms of HPV detection κ = 0.574), with high concordance levels for HPV types 6, 11, 31, 59 and 81. This illustrates that anogenital swabs can be used with reasonable reliability to determine HPV types detected in whole wart tissue. However, this study has also shown that HPV DNA viral load estimates from swab samples do not reflect that which is observed in paired wart tissue. Furthermore, the elucidation of surface contamination highlights potential limitations with non-adjusted HPV prevalence data obtained from swab samples 74, with ,18,23 anInterestingly, the lack of correlation between DNA viral loads and mRNA expression suggests that measuring viral DNA levels is not representative of viral activity within the wart. It is possible that the efficiency of the viral transcription machinery differs between individual viruses. Alternatively, it is possible that either the detection sensitivities for the viral DNA/mRNA components using the methods employed vary or there is a differing level of viral E6 gene expression during various stages of the virus replication lifecycle.The importance of HPV 6 and HPV 11 in the pathogenesis of classic condylomata acuminata has been demonstrated here, and has been deemed to be causal in over 90% of this small cohort of 23 patients. These data therefore support published studies in the literature. There has however, been little RNA work performed in wart disease which could serve to either support or refute this finding, only previous publications which quote the 90% level of causation for these two dominant types ,25. It mThe detailed and thorough molecular work involved in this small study has revealed that, although multiple infections are apparently a frequent occurrence when determined by swabbing wart lesions, the actual numbers of types potentially involved in lesion pathogenesis is low. This is an important and potentially confounding factor in all studies that involve only the surface swabbing of wart lesions. We also show for the first time that laser capture microdissection can be utilised successfully to assist in objective analyses of the aetiology of anogenital warts, but there was poor correlation with detected RNA levels. This technique is expensive and requires specialised skills and is likely to remain a research tool only.In this study, samples 4, 9, 11 and 12 demonstrated evidence of dual infection. Interestingly, HPV 16 was shown be elevated for both DNA and mRNA in sample 12, which is evidence of active replication in that lesion. In four cases , HPV 11 was not found by the Linear Array system. The reason for this is not known but may be related to sensitivity levels. The manufacturer has found that their product is able to detect HPV 11 plasmids at a level of 900 copies/ml and it is possible that the virus is present with a DNA quantity lower than this detection threshold. The data shows that HPV 6 or HPV 11 appear to be involved causally in a majority of lesions clinically diagnosed as typical condylomata acuminata. The data also suggests a potential role for HPV 16 or other types in isolated cases of wart disease and this hypothesis needs to be explored in further studies.Clinical samples were obtained from patients attending the Departments of Gynaecological Oncology and Dermatology, Addenbrooke's Hospital, Cambridge, UK. Ethical approval was obtained from the local research ethics committee (Cambridgeshire 3 REC reference no: 07/H0306/127) and all patients gave written informed consent for the use of these samples for this study. Anogenital warts were excised as part of treatment and dissected into five equal segments using sterilised scalpel blades, following which they were frozen in liquid nitrogen until DNA extraction. Prior to excision, a concomitant Dacron swab sampled the anogenital wart surface. The swab was agitated in PBS and discarded. The cells obtained from the swab sample were suspended in PBS and stored at -20°C prior to DNA extraction.One segment of sample was removed from liquid nitrogen and thawed on ice. Fresh unfixed tissue segments were embedded in optimal cutting temperature (OCT) compound. Multiple 7 μm OCT-embedded sections were sliced and mounted to PEN  membrane coated slides , being stored at -70°C prior to LCM. An additional section of tissue was sliced onto Biobond-coated Superfrost slides  and stained with haematoxylin and eosin (H&E) in order to allow tissue strata to be identified.2O) and stained with 0.4% cresyl violet solution for 10 seconds and immediately washed with distilled water to remove any residual staining medium. Sections were allowed to dry prior to transportation on ice.Prior to LCM, slide-bound tissue sections were stained with cresyl violet. Tissue dehydration was performed by a series of alcohol baths for 1 minute . Lipids were removed from the tissue by bathing in 1:1 chloroform:ethanol for 3 minutes. The tissue was then rehydrated . The slide was inverted and fixed under the microscope where a 0.5 μm laser beam was used to dissect selected areas of tissue. Dissected tissue was allowed to fall into a collecting tube, containing 40 μl PBS, placed underneath the inverted slide as summarised in video format, tp?ID=309. The colSamples of whole wart tissue were inserted into a FastPrep lysing matrix tube  and disrupted in a Bullet Blender™  prior to genomic DNA (gDNA) extraction . gDNA waThe Roche Linear Array HPV Genotyping Test , allowing detection of 37 high- and low-risk HPV types, was carried out according to the manufacturer’s instructions .2O. After DNAse I digestion, RNA was recovered by column purification using PureLink™ RNA extraction kit (Invitrogen) following manufacturer’s instructions and a maximum of 5 μg reverse transcribed using Bioscript™  after preincubation with random hexamer primers for 5 min at 65°C. Reactions were performed as follows: 25°C for 10 min, 42°C for 60 min and 70°C for 15 min. No-RT controls were performed without the addition of enzyme.RNA was extracted from whole wart tissue, as previously described . Briefly2 , 375 μM dNTPs , 100 nM primer pairs and probe , 0.5 units Hotstart Platinum Taq  and template.The primers and probes targeting the HPV E6 gene for types 6, 11, 16, 18, 45 and 52, together with human GAPDH  and β-globin are described elsewhere (20–23). All qPCR reactions were performed in 20 μl containing 1x PCR buffer , 4.5 mM MgClRelative cellular mRNA for HPV E6 was determined by normalizing HPV values, obtained following qPCR of wart mRNA, with TBP (TATA-Box binding protein), YWHAZ  and HMBS (hydroxymethylbilane synthase) using primers and probes that have been previously described (24–26).qPCR amplification was performed using Rotor-Gene 3000 (Corbett Life Sciences). Cycling conditions were as follows: an initial activation/denaturation step at 95°C for 15 min, then 40 cycles of 95°C for 15 sec and 60°C for 60 sec, with acquisition of fluorescent signal at 60 sec. Data was analysed using the Rotor-Gene analysis software (version 6.1). The quantification of HPV DNA viral load and mRNA expression in samples was accomplished through the use of a standard curve incorporated into each experiment.*The choice of E6 DNA and mRNA as a target for our qPCRs for DNA and RNA viral loads was taken because E6 and E7 are crucial early proteins active in all alpha-group papillomavirus replication, DNA synthesis, abortive infections and productive infections but not in latent infections -32.The concordance and agreement between samples for each HPV type detected was calculated. Kappa values of 0–0.2 (slight), 0.21-0.4 (fair), 0.41-0.6 (moderate), 0.61-0.8  and 0.81-1.0  indicated the level of agreement between the methods used . StatistLCM: Laser-capture microdissection; qPCR: Quantitative PCR; qRT-PCR: Quantitative reverse-transcribed PCR.MAS is consultant to Sanofi Pasteur-MSD, Lyon, France and PKCG & MAS were in receipt of an unrestricted educational grant. PKCG was previously consultant to SP-MSD two years ago. MAS is also consultant to Merck Research Laboratories, Westpoint, USA, and GSK Biologicals, Rixensart, Belgium. CS has been in receipt of travel funds from SP-MSD and GSK.MH carried out the molecular genetic studies, LCM, performed the statistical analysis and helped draft the manuscript. DW, SB, KV helped perform the molecular genetic studies. CS, MAS participated in the drafting of manuscript. JCS participated in the design of the study and helped draft the manuscript. PG conceived of the study, and participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript.


As human papillomavirus (HPV) vaccines become available in developing countries, acceptability studies can help to better understand potential barriers and facilitators of HPV vaccination and guide immunisation programs.n = 31) and group discussions (n = 12) were conducted with a total of 169 respondents .Prior to a cluster-randomised phase IV trial of HPV vaccination delivery strategies in Mwanza Region, Tanzania, qualitative research was conducted to assess attitudes and knowledge about cervical cancer and HPV, and acceptability of and potential barriers to HPV vaccination of Tanzanian primary schoolgirls. Semi-structured interviews (While participants had heard of cancer in general, most respondents had no knowledge of cervical cancer, HPV, or HPV vaccines. Only health workers had heard of cervical cancer but very few knew its cause or had any awareness about HPV vaccines. After participants were provided with information about cervical cancer and HPV vaccination, the majority stated that they would support HPV vaccination of their daughter to protect them against cervical cancer. Opt-out consent for vaccination was considered acceptable. Most preferred age-based vaccination, saying this would target more girls before sexual debut than class-based vaccination. Potential side effects and infertility concerns were raised by 5/14 of participating male teachers.Reported acceptability of HPV vaccination amongst parents, teachers and other community members was high in this population. Respondents stressed the need to provide adequate information about the vaccine to parents, that also addresses side effects and infertility concerns. We conducted 31 in-depth interviews (IDI) and 12 group discussions (GD) with a total of 169 respondents, using a purposive sampling strategy with the purpose of recruiting particular kinds of respondents, in our case specific groups or types of people who would be involved in the different sites of school-based vaccination. Participants included parents/caregivers, female students, teachers, religious leaders (seven Christian and two Muslim), and health workers. Aside from parents in two group discussions (discussed below), these participants had not received any project-related sensitisation. A small monetary incentive  was provided to adult participants to compensate them for the time spent during the interview or group discussion. For interviews with teachers, parents, and pupils, different school strata were selected: government urban, government rural, and private schools. When possible, individuals were recruited from the three strata .Head teachers assisted in recruiting parents, female students, and teachers; selection criteria were that these persons would be involved in the actual vaccination program, either as a parent, a student, or a teacher of Year 6 or 12-year-old girls. The girls selected were asked for written assent after their parents/caregivers gave their permission. Two group discussions were held with parents after a cultural dance and drama troupe performed a show on cervical cancer and HPV. We chose nine health facilities at random, representing rural and urban sites and interviewed one health worker in each, exploring the following themes: knowledge of cervical cancer and HPV, HPV vaccine acceptability, views on delivery strategies, decision-making, and other experiences with vaccines or school-based health services. When respondents demonstrated no knowledge of cervical cancer, HPV, and/or the HPV vaccine, the interviewer gave a brief, standard explanation about the planned HPV vaccination project, and then continued with questions. IDIs and GDs were recorded, transcribed and translated into English; the source and/or location of IDI and GD are given after quotations in the main results. Initial coding, which used a list of pre-set codes based on the research themes with further codes added that emerged during repeated readings, was reviewed by a second researcher who conducted the final analysis.33.1The age range of teachers and health workers interviewed was between 19–51 years and 33–55 years respectively. The 54 student respondents had a median age of 12 years and were aged between 11 and 17 years whilst parents were aged between 18 and 59 years. The majority of parents worked as farmers, fisherman or operate small businesses . Most had completed primary school; a minority (12/60) had completed secondary school. The religious leaders were aged between 35 and 50 years and came from the following denominations: Roman Catholic, Anglican, Lutheran, Tanzania Assemblies of God, African Inland Church, Seventh-Day Adventist, Glory City Church, and Muslim.Large scale qualitative research suggests that the median age of sexual debut is approximately 14 although self-reporting in surveys suggests 16–17 years 3.2Nine female health workers were interviewed; all but one had two years of nursing education. All had heard of cervical cancer but their knowledge was limited and often inaccurate. When asked about cervical cancer symptoms, they mentioned vaginal bleeding, smelly vaginal discharge, or pain during sexual intercourse. Only two nurses identified HPV as the cause of cervical cancer. Both had heard about HPV through preparatory work for an immunogenicity and safety trial of the bivalent HPV vaccine in Mwanza (2009–2010). Another nurse had heard of HPV vaccines on the radio but could not remember any details. All nurses mentioned a wide range of, sometimes incorrect, causes of cervical cancer such as poor genital hygiene, early age at childbirth, frequent childbirth, abortion, wearing nylon undershorts and insertion of traditional medicines.Most parents recognized cancer as a serious, potentially deadly illness, but knew little about cervical cancer. Two parents (participating in an GD) had heard about it on the radio but did not remember any details. One 53-year-old father (participating in an IDI) heard information on the radio but incorrectly thought that cervical cancer affected women during pregnancy or menstruation, when poor vaginal cleansing caused women to contract germs and then cancer. Four parents (GD) and two mothers (IDI) had heard of uterine, but not cervical cancer. No parent had heard about HPV or the HPV vaccine. The female pupils had heard of cancer in general, but none of the 49 girls in GDs had heard about cervical cancer, HPV or the HPV vaccine. Similarly, teachers had heard of cancer but only 1 of 37 knew about cervical cancer, and no teacher had heard of HPV or the HPV vaccine. One 48-year-old female teacher (IDI) talked about a family member who had “died of cervical cancer” but recalled little about the disease. Religious leaders interviewed knew about cancer in general but nothing of cervical cancer, HPV, or the HPV vaccine.3.3Most respondents welcomed a vaccine that prevents cervical cancer. Almost all the adults said they would allow their daughter to be vaccinated since “prevention is better than cure” . All the girls interviewed said they would like be vaccinated to avoid a dangerous disease like cervical cancer.Five (35%) of the 14 male teachers participating in this study  dissented and said they would not allow their own daughters to be vaccinated. They feared side effects; especially whether the vaccine would have a potential effect on future reproduction: “vaccinations in this country that are linked to issues of reproduction have had very bad results later on,” or the vaccine could “disorder and destroy the eggs that a girl has, and reproducing would be a problem.” The aunt of one student was suspicious of the vaccine and had told her: “they are coming to implant cancer in people… they are coming to reduce reproduction” (GD Nyakato).Most participants trusted the safety of the vaccine, since it had been explained that the Tanzanian government had approved the vaccine: “I know the government cannot do something malicious to children” .All parents stated they would agree to have their daughters vaccinated, but some hesitated when confronted with an unknown infection (HPV), disease , and vaccine: “That disease you are talking about, we are completely in the dark about it” , and “The vaccine will have a benefit if it does not have harmful side-effects” .The five male teachers  who opposed vaccination also commented that the vaccine might give girls a license to start sexual activity: “if this is introduced, a person would have the freedom to do anything.” A few religious representatives also echoed this concern but most found the vaccine a ‘good thing’ because it would protect adolescent girls. No parents thought that the vaccine would encourage sexual activity among the targeted girls.Generally, teachers, parents, students, and health workers preferred age-based vaccination as they believed that this would target more students who had not yet started sexual activity; choosing students in School Year 6 [where the mean age is 13.9 years (range 11–22 years)] would include a greater age-range and older girls who might have started sex. Participants suggested vaccinating much younger girls: “a ten-year-old child has already started with sex, the ones who have not started are those aged seven” . A few suggested testing girls’ HPV status before vaccination. If class-based delivery was to be used, participants preferred classes lower than Year 6. A few parents preferred class-based delivery because of simpler logistics, since each girl's age would not need to be checked. Other interviewees focused more on student understanding and preferred 12-year-olds: these would be “mature enough” to understand the vaccination information and could help to “educate parents” ; those in Year 6 would “value” the vaccine more .A few girls and a few parents said that boys might also want the vaccine, and could feel discriminated against or jealous of the girls, but the general consensus was that, because cervical cancer only affects girls, boys would understand if only girls were vaccinated. Health workers anticipated that questions from boys could be resolved by explaining the underlying reasons: “when we educated [the boys], they understood” .A few respondents asked how out-of-school girls could get the HPV vaccine. Some parents suggested organising door-to-door visits to identify and vaccinate all girls of a certain age, regardless of education status. Some religious representatives asked what could be offered to their wives and adult sisters.3.4The majority of participants were positive about other vaccinations, such as for measles, tetanus or polio. They saw that “when children are vaccinated, they grow up healthy and do not get that disease” . Health workers confirmed that there was “much awareness” about infant vaccinations; mothers knew that minor side-effects  might occur post-vaccination (IDI Igoma).Reactions to a new HPV vaccine being delivered through primary schools were influenced by past experiences with vaccinations and/or school-based health programs. Many participants remembered rumours undermining previous vaccination or de-worming campaigns 3.5When asked about adding HPV vaccination to their workload, health workers all mentioned familiar concerns about public health services: insufficient staff serving a large population and lack of transport. One nurse said, “some places are far away and some of us have become old” and, with not enough staff, “you might find yourself alone at work for the whole month” (IDI Nyegezi). Health workers encountered various shortages; of drugs, vaccines, or consumables: “we might lack drugs for two weeks… sometimes we have the drugs but would not have the syringes” (IDI Makongoro).One nurse summed up ways to alleviate these issues: the necessary “facilities” for storing vaccine, “enough medicines,” “motivation [i.e. salary supplements] for those who go to do the work,” and training “so that she can administer the vaccine correctly” (IDI Igoma).3.6All respondents emphasised that parents need appropriate information and intensive sensitisation about HPV infection and the new vaccine. Without this, parents would quickly oppose a new vaccine: “we’d charge you [in court]” . All viewed school-based meetings as an essential sensitisation strategy: “[parents] should get educated like how you [the interviewer] have come here” . Teachers said inviting parents to school meetings was not always successful. Not all parents may attend and, even when they did, “you might educate the wife, but when she gets home to her husband, he refuses” . Some suggested adding radio and television announcements, so that even those who do not attend meetings are informed.Students thought sending letters to parents via students would work, provided they themselves also received sufficient information: “It won’t be difficult [to deliver letters] because many children will agree to be vaccinated and very few won’t want to get the vaccine.” (IDI Buhongwa). Most respondents liked the letter strategy but some teachers cautioned about relying on written information: not all parents know how to read.Most teachers, parents, and students said it was necessary to get parental permission, but not necessary to ask each parent for individual written consent. Most interpreted consent as a process whereby parents would be informed about the school-based vaccination programme, either by letters, meetings, by the targeted child, or other types of announcements (like radio or television); parents could refuse to allow their child to be vaccinated by making this known to the school or by keeping the child home on vaccination day. A few teachers (GD Ng’ombe) suggested that active consent should be required from parents, or that parents should accompany their daughter on the day of vaccination to ensure that parental wishes are respected. Teachers feared parents might threaten them at school, as happened during past health programmes, or take them to court. Some health workers suggested that teachers might have coerced their students during prior vaccination campaigns: “when we go to administer a vaccine, we find the teachers have gathered the girls, and they are standing by the door with a stick, …” .Some parents, teachers and students said that if a student has sufficient understanding and wants to be vaccinated, she should get the HPV vaccine even if her parent(s) refused. “The child ought to be given the vaccine because it's for her benefit, provided she's willing and has got sufficient education. If the parent isn’t willing, it's the right of the child to get it” (teachers GD Serengeti); “I should be vaccinated because I’m the one who’ll contract the disease” .Health workers were accustomed to giving infant and child vaccinations without parental consent. With nationally-mandated vaccinations, health workers go to schools, inform the teachers, and on vaccination day, inform and vaccinate the children. These are vaccines that “the community knows and understands [to not be] harmful” . Most health workers felt that, if the government mandates HPV vaccine as part of the school vaccination program and the community has been ‘educated’, this should be sufficient. Two (of nine) health workers said children should not be vaccinated if their parents refuse, but health workers should try to convince these parents of the vaccine's benefit. Most health workers said that if the child understands and wants the vaccine, she should be vaccinated: “what I aim at is to save the life of the child, not the parent” (IDI Nyegezi). Another said: “A child is yours when she's in the womb. Once she's born, she belongs to the government … it can protect her” (IDI Butimba).4We found that teachers, parents, pupils and health workers interviewed in our qualitative sub-study had limited or no knowledge about cervical cancer, HPV, and the HPV vaccine. Generally, most welcomed a vaccine to prevent cervical cancer and most parents said they would agree to have their daughter vaccinated although some adopted a “wait and see” approach. Most had a strong belief that vaccines prevent diseases. Our findings are similar to formative research results by PATH in Uganda, Peru, Vietnam and India prior to HPV vaccination Anti-fertility rumours, raised as a potential issue for the vaccine in our study and the study in Uganda, are widespread in Africa in relation to vaccines and health-related products and reflect underlying suspicions about public health interventions Issues of power and control over health emerged in the discussions about opt-out consent. Health workers saw public health actions as mandatory and considered that individual parent consent was not a necessary part of national immunisation policy, although provision of information to parents and communities was important. This was also stressed by other respondents. In Mwanza, parents wanted to be involved in the decision-making process but the consensus was that opt-out consent was acceptable, and there was considerable support for a girl's right to be vaccinated, even if parents refused their consent. Uganda's pilot HPV vaccination program also used a similar opt-out approach Many respondents among the different groups  stated that parents should be given adequate information about the vaccine and cervical cancer, and that a school meeting would be an essential strategy to reach the parents. People were eager to learn about the HPV vaccine. Religious leaders reported that this was the first time that staff from a health programme had come to discuss a health intervention with them, and that they would discuss cervical cancer and HPV vaccination with their congregations.Limitations of the qualitative sub-study included the fairly small purposive samples and the fact that, in schools, a teacher selected the parent, student and teacher participants for GDs who might have been the most accepting of new health interventions. However, the interviewer then selected IDI participants from the groups. These included several teachers who opposed vaccination, parents who asked critical questions, and female students who stated they would defy parental wishes in terms of accepting vaccine.In USA, beliefs about the safety of vaccines, likelihood of HPV infection, as well as doctor's recommendations, have been associated with increased HPV vaccine acceptability ® vaccine for low-income countries In light of the recent price reduction of the Gardasil