The design of new healthcare schemes which involve using molecular HPV screening means that both persistence and clearance data regarding the most prevalent types of HR-HPV occurring in cities in Colombia must be ascertained.This study involved 219 HPV positive women in all of whom 6 types of HR-HPV had been molecularly identified and quantified; they were followed-up for 2 years. The Kaplan-Meier survival function was used for calculating the time taken for the clearance of each type of HPV. The role of a group of independent variables concerning the time taken until clearance was evaluated using a Cox proportional-hazards regression model or parametric (log-logistic) methods when necessary. Regarding viral load, the Wilcoxon rank-sum test was used for measuring the difference of medians for viral load for each type, according to the state of infection (cleared or persistent). The Kruskal-Wallis test was used for evaluating the change in the women’s colposcopy findings at the start of follow-up and at the end of it (whether due to clearance or the end of the follow-up period).6 to 9.99 × 109 viral copies had better clearance compared to those having greater viral loads (1.61 time ratio: 1.01-2.57 95% CI). Lower HPV-31 viral load values were associated with this type’s persistence and changes in colposcopy findings for HPV-16 gave the worst prognosis in women having low absolute load values.It was found that HPV-18 and HPV-31 types had the lowest probability of becoming cleared . Women from Colombian cities other than Bogotá had a greater probability of being cleared if they had HPV-16 (HR 2.58: 1.51–4.4 95% CI) or HPV-58 (1.79 time ratio: 1.33-2.39 95% CI) infection. Regarding viral load, HPV-45-infected women having 1 × 10HPV infection clearance in this study was related to factors such as infection type, viral load and the characteristics of the cities from which the women came. Low viral load values would indicate viral persistence and a worse prognosis regarding a change in colposcopy findings. Persistent infection with high-risk human papillomavirus (HR-HPV) types is the main (but not the only) cause of developing pre-cancerous lesions or cervical cancer -3.Several types of HPV considered oncogenic have been found to be responsible for around 90% of cancer cases worldwide , having It has been described that HPV infection represents a transient phenomenon throughout the whole world, leading to high infection prevalence, even though most cases do not produce cervical lesions and those which do, consist of low grade lesions involving spontaneous regression . HoweverIntrinsic viral determinants, such as the infecting viral type or viral load, have been identified as HPV persistence markers ,8,9; howThe present work seeks to describe both viral and host factors which could be intervening in the persistence and clearance of the most common HR-HPV types in Colombia. This study was mainly aimed at providing epidemiological information illustrating how HPV infection can become eliminated in a target population, bearing the state of the infection (single or multiple) and the most prevalent HR-HPV types in mind.Women eligible for the present study were voluntarily attending cervical screening consultations in three Colombian cities  between April 2007 and March 2010.The present study involved 3 cities: Bogotá the country’s capital having an urban population, Chaparral located in the Tolima department is mainly inhabited by mestizos leading a sedentary life style and Girardot in the Cundinamarca department which has become a tourist destination due to its favourable weather and closeness to Bogotá. Data concerning Chaparral and Girardot was combined in a single category called “other city” to ensure a better analysis of women in this study, as both these cities are small, having similar climates and lying at less than 1,000 masl .As inclusion criteria, all women signed a written informed consent form and completed a questionnaire regarding their sociodemographic characteristics, sexual behaviour and risk factor data before undergoing a gynaecological examination and providing a cervical smear sample. The signature of a parent or guardian was required for females younger than 18 years old. Women who stated that they did not intend to move from their home cities for at least 2 years after the study began were included in the follow-up study.HMBS gene were not included. Women who were pregnant at base line and who had less than 3 months or more than 9 months until their next visit were also excluded from the study.Amongst exclusion criteria considered, women who had negative HPV results, those whose samples had little DNA in them  or no amplification for the The Papanicolaou test was used for analysing samples and HPV–DNA detection; real-time PCR was used for selecting just HPV positive women. This study was supervised and approved by the relevant ethics’ committees at Hospital de Engativá Nivel II (in Bogotá), Hospital San Juan Bautista  and Nuevo Hospital San Rafael (in Girardot in the Cundinamarca department).Cervical samples were collected with a cytobrush and kept in 95% ethanol at 4°C . DNA froPolymerase chain reaction (PCR) for human β-globin was carried out on samples to check DNA integrity using GH20/PC04 primers . Three HHomo sapiens hydroxymethylbilane synthase (HMBS) were synthesised according to a study published by Moberg et al., [et al., amplified the same region for HPV-33 and −58; a new set of primers aimed at the E7 region of these viral types was thus designed. Designing probes for each viral type and HMBS was based on four parallel duplex real-time PCRs per patient, taking into account the types included in each reaction , 1× Mango Taq Color reaction Buffer, 2 mM MgCl2, 250 nM dNTPs, 1 mM of each primer and DNase-free water to fulfil the necessary reaction volume. The PCR protocol for each fragment consisted of initial denaturing for 5 min at 95°C, followed by 35 cycles of 30 s at 95°C, 20 s at corresponding melting temperature and 30 s at 72°C. A reaction containing DNA-free water was used as negative control. The amplicons so obtained were purified with a Wizard PCR preps kit (Promega), once their quality has been evaluated on 3.25% agarose gel. A TOPO TA cloning kit was used for ligation, followed by transformation in TOP10 E. coli cells (Invitrogen). Several clones which grew on selective LB plates with 50 μg/mL kanamycin were incubated in LB broth at 37°C with 250 rpm overnight. Recombinant plasmids were purified using an UltraClean mini plasmid prep kit  and sequenced with an automatic ABI PRISM 310 Genetic Analyzer . Each insert’s integrity was checked by aligning the products with the respective theoretical sequenced fragments of each gene using Clustal W software [Processed samples were used as template for PCR , containing 0.5 U/μL Mango software .11-106copies) gave a standard curve for each viral type and HMBS gene . Samples were tested for HPV-16, HPV-18, HPV-31, HPV-33, HPV-45 and HPV-58. The human HMBS (hydroxymethylbilane synthase) gene was amplified in all samples to verify DNA integrity and calculate viral copy number per cell. PCR involved using a CFX96 Touch Real-Time PCR detection system which can detect 6 different fluorescent dyes; four real-time PCR reactions were carried out per sample, one for detecting HPV-16, a second for HPV-18 and −31, a third for HPV-33 and −45 and a fourth one for HPV-58 and HMBS.A NanoDrop 2000 (Thermo Scientific NanoDrop Products) was used for quantifying plasmid DNA and the DNA copy numbers were calculated by using the URI Genomics and Sequencing Center web site  Table 11. Standa2, 250 nM of each dNTP, 250 nM of each primer, 1.5 U/μL MangoTaq Polymerase (Bioline) and 500 nM probe. The second PCR mix for HPV-18 and −31 consisted of 1× reaction buffer, 2 mM MgCl2, 275 nM of each dNTP, 500 nM HPV-18 primers, 250 nM HPV 31 primers, 1.5 U/μL MangoTaq polymerase and 500 nM of each probe. The HPV-33 and −45 PCR mix included 1× reaction buffer, 1.75 mM MgCl2, 275 nM of each dNTP, 250 nM HPV-33 primers, 500 nM HPV-45 primers, 1.5 U/μL MangoTaq polymerase and 500 nM of each probe. The last PCR mix for HMBS and HPV58 contained 1× reaction buffer, 1.87 mM MgCl2, 250 nM of each dNTP, 500 nM of each primer, 1.5 U/μL MangoTaq polymerase, 200 nM HMBS probe and 500 nM HPV 58 probe. All real-time PCR reactions contained 2 μL of the DNA extracted from each cervical sample and DNA-free water to complete 20 μL volume.The HPV-16 PCR mix contained 1× reaction buffer, 1.5 mM MgCl11-106 copy dynamic detection range) and a no template control to rule out DNA contamination.96-well plates were used for each run, including 6 standards for each viral type and HMBS, involving 10-fold plasmid dilutions : number of HPV copies/(number of HMBS copies/2) .Women who had had both a Pap-smear result and HPV-DNA detected by PCR and who fulfilled the follow-up inclusion criteria  were included in the analysis. Women were excluded where the HMBS gene was not amplified by RT-PCR. Analysis was based on type-specific HPV infection rather than on individual women, taking into account that multiple infection is common in the Colombian population .Cox’s multivariate regression model was used when calculating sample size; hazard ratios (HR) of at least 2 were thus considered, whenever they had a 5% significance level, 80% power, 0.55 standard deviation of tested covariates and 0.1 correlations between tested covariates. The probability of clearance was set at 0.7, according to previous reports ,19. SuchClearance was defined as at least two consecutive type-specific HPV DNA samples proving negative, such samples taken at 6-month intervals following a positive sample . PersistThe first step was evaluating each variable independently to assess their importance regarding clearance time Table ; those vThe independent variables included in the multivariable model were city, ethnicity, age of first sexual relationship, number of lifetime sexual partners, family planning method, coinfection and viral load  concerning time taken to clearance using a Cox’s proportional hazard (PH) regression model or parametric methods (log-logistic) when the PH assumption was violated. The PH assumption was graphically evaluated using log-log plots and a PH test based on weighted residuals using Grambsch and Therneau tests . The choThree categories were assigned to the variable “changes in colposcopy”  for evaluating changes in colposcopy findings between the results of colposcopy at the start of follow-up and the end of it (whether due to clearance of the virus or not). The Kruskall-Wallis test was used for evaluating the difference in viral load for each viral type and change in colposcopy findings since the sample did not have a normal distribution. The Mann–Whitney test was also used for evaluating viral load according to the state of infection (persistent or cleared).Categorical variable distribution amongst groups was assessed by Chi-squared test or Fisher’s test, as appropriate. Median and interquartile ranges were used for quantitative variables, according to the data distribution. Incidence ratios were estimated using months of follow-up as denominator. A ≤ 0.05 p value was considered statistically significant; STATA 12 was used for all statistical analysis.The present work has consolidated data concerning 219 women infected by several HR-HPV types; they became voluntarily incorporated into our follow-up study. All the women included guaranteed to attend a base-line visit and at least 3 follow-up visits with around 6 month difference (±3 months); 23.3% of the population being sampled managed to attend follow-up 4 (i.e. data became available from 5 visits).Regarding baseline information, 23.7% (n = 52: 18.3-29.9 95% CI) of the women in the study were infected by a single type of HR-HPV (single infection); the rest of the population, 76.3% (n = 167) had multiple infections, distributed as follows: 26% (n = 57: 20.3-32.4 95% CI) had infections having simultaneous detection for 2 types of HR-HPV, 29.7% (n = 65: 23.7-36.2 95% CI) infection by 3 types of HR-HPV, 13.2% (n = 29: 9.1-18.4 95% CI) had positive identification for 4 viral types, 5% (n = 11: 2.5-8.8 95% CI) were infected by 5 high risk types and 2.3% (n = 5: 1–5.2 95% CI) had viral DNA identification for 6 types of HR-HPV.Participants’ age ranged from 17 to 71 years-old . Most of the participating population was mestizo , and from the city of Girardot . The population’s socio-demographic and sexual behaviour data was described regarding follow-up (base-line), according to the state of the infection  of the 4 most prevalent viral types in molecular determination , bearing in mind the most important variables in univariate models, for each type (data not shown) .6 to 9.99 × 109 compared to loads equal to or greater than 1 × 1010.Regarding the types belonging to species A7, HPV-18 infection did not have a statistically significant association with any variable evaluated here. However, it was observed that the probability of clearance regarding HPV-45 became significantly increased when the absolute viral load for this type ranged from 1 × 10It should be highlighted that single or multiple HR-HPV infection was not associated with time to clearance in the present sample, since coinfection values for any HPV type were not statistically significant in this model Table .When evaluating the medians for normalised viral load (per cell) for each type, according to the state of infection at the end of follow-up (cleared or persistent), it was observed that the median for HPV-31 type in the group of women where clearance was found  was greater than the median for those where this virus was not cleared . There were no differences in any of the groups regarding the medians for the other HR-HPV types.6; IQR = 1.1 × 107; alike: median = 2.9 × 106; IQR = 9.9 × 106) (p = 0.046). There were no differences in the rest of the types evaluated here regarding viral load according to change in colposcopy findings.The change in colposcopy findings was also evaluated concerning the result at the start of follow-up and the result at the moment of clearance, or at the end of follow-up Figure . There wWhen evaluating normalised viral load according to the degree of HPV infection and colposcopy findings at the start and end of follow-up, it was found that median viral load for HPV-58 was greater for women who had a better prognosis  compared to those whose prognosis remained the same  , only in the group of women having persistence for this virus (p = 0.012).The number of reinfections for each viral type was determined Table ; HPV-58 This study has provided detailed epidemiological data for six HR-HPV types present in a Colombian cohort. This has been the first study in Colombia (to the best of our knowledge) aimed at using real time quantification of DNA from the 6 most prevalent types of HR-HPV, giving absolute and normalised load values.Findings concerning the virus’ persistence for the 6 types included here have demonstrated that the risk of acquiring a later HPV infection becomes increased in women already infected by any type of HPV  and its relationship to clearance time, this work did not reveal an important relationship between such aspects. Various studies have shown that type specific HPV clearance seems to occur regardless of coinfection in an immunocompetent population ,27.The highest clearance rates in our cohort were observed for HPV-33 and HPV-16. Previous reports have shown that HPV-16 clears out after other HR-HPV types ,28,29, bThe reduced clearance rates observed for infections with HPV-18 and −31 types is particularly important bearing in mind that HPV-31 was found in high prevalence in our country  and thatDespite HPV-31 was not evaluated in the multivariable model due to the low sample size, it is worth noting that it displayed the highest viral load values and one of the lowest clearance rates. Future studies analysing more women infected with this viral type might help to a better understanding about the influence of viral loads in the type-specific clearance process.HPV-16, −18 and −58 viral load values did not have a clear relationship with clearance time. This has already been shown for a population from Bogotá in a study involving semi-quantitative identification of viral DNA . The preAnother factor associated with clearance time was city of origin, showing that women infected by types from the A9 species became cleared more rapidly if they came from Girardot/Chaparral. It is supposed that these cities have factors related to sexual behaviour or cultural characteristics which were not measured in this study and which would have modulated such findings. When analysing the control arm of the large randomised PATRICIA study, it was found that region of origin was one of the behavioural determinants of clearance time, as north-American women took less time to clearance than their European counterparts .Regarding ethnicity, fine control was not used for obtaining it; thus, other ethnic characteristics which were not controlled in this study may have intervened in such marked association between city and clearance time. Another aspect concerned the women’s nutritional state; most women were from low socio-economic strata. However, nutritional and/or feeding data was not controlled and may have provided more detailed characteristics concerning the population’s idiosyncrasies. Previous studies have shown that women who consumed one or more servings of vegetables per day cleared their HPV infections more quickly than women who did not consume vegetables daily . The intOther factor that was not measured in this study but that could influence HPV clearance is hygienic practices. In a cohort of university students, it has been shown that the use of tampons was associated with a reduced rate of HR-HPV clearance .Very interesting data for three A9 species types  was revealed when determining viral load according to the state of infection and colposcopy findings since these had low viral load values (absolute or per cell) associated with greater lesion severity at the end of follow-up or when infection did not become eliminated. Besides intermediate viral load values for HPV-45 (A7 species) were associated with faster time to clearance, this may be a factor related to transient infection. Such results could have been due to immune system evasion mechanisms since it has been reported that low HPV viral load values have been related to persistent infection  and it cThis is contradictory with studies proposing that high viral loads facilitate persistence, specifically, it has been shown that HPV-16 viral loads in LSIL and HSIL were higher compared with no intraepithelial lesion or malignancy .This work has several strengths, such as having compiled data from two important focuses of HPV infections (i.e. Girardot and Bogotá), determined viral load using the most sensitive technique for doing so and the percentage of multiple infections revealing an important Colombian populational characteristic. However, the study had difficulties in terms of follow-up times, since infection transience meant that shorter follow-up times than the ones established here may probably have led to obtaining more precise clearance and incidence values. Our information was limited to using prevalent high risk infections for analysing persistence and clearance of infection; the foregoing means that follow-up studies are needed to facilitate understanding the most prevalent epidemiological HPV patterns for Colombia.Diagnosing HPV infection in clinical specimens has been widely accepted to date in Colombia; viral DNA identification in this type of sample has been included in the Obligatory Healthcare Plan, 2012. The following step must thus be to incorporate monitoring from the identification of HPV infection in cervical cancer control schemes. Such work thus contributes towards the search for a correct algorithm for defining HPV DNA screening since the time taken for most women to clear the virus must be determined for calculating the determinants of such scheme and be referred to regular monitoring .Time to clearance in Colombian females infected by the most frequently occurring HR-HPV types in the sample population was not modulated by infection status (single or multiple). However, viral load played a role in terms of infection regarding HPV-45 and the origin of HPV-16 and −58 infection. Viral persistence and worsening of cytological findings were related to lower HPV-16, −31 and −58 viral loads. All women in our sample who eliminated HPV-58 were not infected again by this viral type. Given that time to clearance was related to lesion development, such information should prove significant when designing HPV DNA primary screening in Colombian healthcare systems, as well as in developing countries.HPV: Human papillomavirus; HR-HPV: High risk human papillomavirus; L-SIL: Low squamous intraepithelial lesion; H-SIL: High squamous intraepithelial lesion; ASCUS: Atypical squamous cells of undetermined significance; HMBS: Hydroxymethylbilane synthase; PCR: Polymerase chain reaction; RT-PCR: Real-time PCR; DNA: Deoxyribonucleic acid; STD: Sexually-transmitted diseases; SD: Standard deviation; CI: Confidence interval; HR: Hazard ratio; Tm R: Time ratio; IQR: Interquartile range; AIC: Akaike information criterion; BIC: Bayesian information criterion.The authors declare that they have no competing interests.All authors contributed ideas to this paper and reviewed the manuscript for important intellectual content. SCSDL and MC provided the concept, acquired, analysed and interpreted data, designed the study and wrote the manuscript. LDRO and DAMP developed the methodology and were involved in drafting the manuscript. RS provided statistical analysis, interpreted data and helped in writing the manuscript. The study was supervised by APP, MEP and MAP who provided expertise regarding the discussion of results. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/395/prepub


To understand knowledge about, and acceptability of, cervical cancer screening and HPV vaccines among medical students; and to explore potential factors that influence their acceptability in China.We conducted a survey among medical students at six universities across southwest China using a 58-item questionnaire regarding knowledge and perceptions of HPV, cervical cancer, and HPV vaccines.P<0.001) or screening (P<0.001). Students who showed negative or uncertain attitudes towards premarital sex were less likely to accept either HPV vaccines , or screening . Non-clinical students showed lower acceptability of cervical screening compared to students in clinical medicine .We surveyed 1878 medical students with a mean age of 20.8 years (standard deviation: 1.3 years). Of these, 48.8% and 80.1% believed cervical cancer can be prevented by HPV vaccines and screening respectively, while 60.2% and 71.2% would like to receive or recommend HPV vaccines and screening. 35.4% thought HPV vaccines ought to be given to adolescents aged 13–18 years. 32% stated that women should start to undergo screening from the age of 25. 49.2% felt that women should receive screening every year. Concern about side effects (38.3% and 39.8%), and inadequate information (42.4% and 35.0%) were the most cited barriers to receiving or recommending HPV vaccination and cervical cancer screening. Females were more likely to accept HPV vaccines  or cervical cancer screening . Students with a higher level of related knowledge were much more willing to receive or recommend vaccines (The acceptability of HPV vaccines and cervical cancer screening is relatively low among medical students in southwest China. Measures should be taken to improve knowledge about cervical cancer and awareness of HPV vaccines and screening among medical students at university. Cervical cancer is the third most common malignancy among women, with an estimated 528,000 new cases and 266,000 deaths worldwide in 2012 Cervical cancer can be effectively controlled through primary and secondary prevention such as cervical screening and prophylactic HPV vaccination. Since the Papanicolaou (Pap) smear test was introduced for routine screening, a substantial decline has been witnessed in cervical cancer deaths in developed countries in the last four decades However, health care providers have lagged behind in their efforts to improve screening for cervical cancer in China. There are no national guidelines for cervical cancer screening, though non-binding recommendations were made by the China Foundation of Cancer in collaboration with the Ministry of Health in 2009, that proposed cervical screening every 3 years for women between 25 and 65 years of age in urban areas, and between 35 and 65 years of age in rural areas To date, the two prophylactic HPV vaccines available internationally are still to be approved by the China Food and Drug Administration, and are not commercially available in mainland China With the rapid scale-up of cervical cancer screening and anticipation of licensure for prophylactic HPV vaccines in China, it is important to understand acceptability of, and possible barriers to, screening and vaccination among the population. To date, previous studies have explored these issues among women, parents of adolescents, female university students, health care providers, and health management staff for HPV vaccines This cross-sectional study was conducted between May and September 2013 at six universities across southwest China, including Sichuan University and Chengdu Medical College in Chengdu, Luzhou Medical University in Luzhou, North Sichuan Medical College in Nanchong, Chongqing Medical University in Chongqing, and Kunming Medical University in Kunming. These universities are the principle institutions of medical education in three major provinces in southwest China. We planned to use 200–400 medical students from different years of study from each university as sample to ensure diversity. In addition, 5–6 whole classes of medical undergraduates were selected from clinical medicine and other non-clinical major subjects such as nursing, dentistry, public health, pre-clinical science, and medical technological science. Clinical medicine and non-clinical majors have different foci in China: clinical medicine and dentistry involve more clinical work, while other major subjects may focus on medical lab science, health management, public health, and other health related work. Due to financial and logistic constraints, the classes from each university were surveyed based on convenient contact points instead of a random sampling process. The study was approved by the Ethics Committee of Sichuan University Fourth Hospital/West China School of Public Health.We used a 58-item questionnaire comprised of three sections of closed- and open-ended questions relating to basic information concerning participants, knowledge and perceptions of HPV, cervical cancer, HPV vaccines, cervical cancer screening, and sexual attitudes and behavior . The proCollaborating staff and medical students were trained on administering the questionnaire, and conducted the survey before or after class at each university between May and September 2013. Survey objectives were explained to potential participants before each survey. The medical students were free to participate, or not, at their discretion. The informed consent form was bound together with the questionnaire when it was handed out to them. Since the survey was anonymous to protect privacy and ensure data integrity, there was no request for a signature on the consent form if they agreed on the survey; they could choose to answer the questionnaire if they consented verbally or just leave it blank if they did not after reading the consent form. In accordance with the ethics committee document, no consent, verbal or written, was requested from parents or guardians of any participants. Our surveyors were responsive to queries about the questionnaire itself during the survey.Completed questionnaires from each university were delivered to the Data Management Team in the West China School of Public Health. Data were entered and managed by the team in a database that had been developed by EpidData v3.1 .IBM SPSS 19.0  was used to analyze the data. Demographic information, selected questions regarding HPV and cervical cancer related knowledge, and perceptions of cervical cancer and screening were presented as frequencies and percentages. Perceptions and concerns of primary and secondary prevention of cervical cancer were compared between males and females using the Chi-square test. Univariable logistic regression analysis was conducted to predict factors influencing the acceptability of cervical cancer and HPV vaccination, and the overall predictive model was established using multivariable logistic regression analysis based on factors of statistical significance. All tests were two-tailed with a significance level of 0.05.A total of 2150 medical students were approached, and 2000 successfully completed the questionnaires. However, another 122 questionnaires were discarded because they contained answers to only a few questions, or contained inconsistencies in the answers. Demographic characteristics of the final 1878 medical students are summarized in Responses to selected knowledge questions from the questionnaire are presented in P<0.001). 60.8% of female students though HPV vaccines could be given to boys, as compared to 50.0% of males students (P<0.001). 36.8% of male, and 34.6% of female, students preferred HPV vaccination for adolescents aged 13–18 years, and 63.5% and 66.0% of them thought that the best time for HPV vaccination would be before becoming sexually active. Most students stated that the local Center for Disease Prevention and Control (CDC) is the most appropriate venue for HPV vaccination, and the least selected venue was school (24.2% versus 22.2%). Over 70% of medical students were willing to pay a price lower than 500 RMB (82 USD) for either imported or domestic vaccines. In addition, 45.8% medical students expressed preference for imported vaccines, while 36.5% would like to make their choice according to the price.Perceptions of HPV vaccination among medical students are shown in P<0.001). However, 45.2% and 39.6% of males and females were not aware of any existing screening techniques. 41.9% and 32% stated women ought to start being screened from 20 or 25 years, and 49.2% and 42.1% were of the opinion women should receive screening every year or every 2–4 years. 82.7% medical students thought a price below 100 RMB (16.5 USD) was reasonable for cervical cancer screening per occasion.Perceptions of cervical cancer screening among medical students are presented in The concern about side effects (38.3% and 39.8%), and inadequate information (42.4% and 35.0%), were the obstacles most cited against receiving or recommending HPV vaccination and cervical cancer screening . 81.5% oP<0.001) or screening (P<0.001). In addition, non-clinical students showed lower acceptability of cervical screening compared to students in clinical medicine . There were no statistically significant differences in acceptability of HPV vaccination or cervical cancer screening between students at different year groups (P = 0.218 and 0.091), and between students with or without clinical internship (P = 0.854 and 0.180). Moreover, non-clinical students did not express lower degree of likeliness to accept HPV vaccination (P = 0.376).Associations of acceptability of HPV vaccination and cervical cancer screening, with other factors, are presented in To our knowledge, this is the largest multicenter study that has explored the acceptability of HPV vaccines and cervical cancer screening among medical students in China. One major finding is that 60.2% and 71.2% of medical students were willing to accept or recommend HPV vaccines and cervical cancer screening, respectively. Female students and students with improved knowledge were positive predictors for both HPV vaccines and screening, while a negative attitude towards premarital sex was a negative correlate for both. In addition, non-clinical students were less likely to accept cervical cancer screening. We also noticed certain concerns and perceptions of these students regarding future HPV vaccines and screening programs. In particular, the concern about side-effects and inadequate information were the two most cited concerns connected with HPV vaccines and cervical cancer screening among medical students.The acceptance of both primary and secondary prevention of cervical cancer was relatively low in our study. The acceptability of HPV vaccines was similar to another study (67.8%) among medical students in India Cervical cancer education is relevant also in regards to the poor knowledge of cervical cancer and its prevention. Fewer than 85% of medical students knew the right answer to all of the seven selected knowledge questions in our study. Surprisingly, only 14.4% and 29.0%, respectively, knew that persistent HPV infection was the necessary cause of cervical cancer and that there was a prophylactic HPV vaccine available for cervical cancer in the world. Fewer than 50.0% agreed that cervical cancer could be prevented by HPV vaccines, while 80.0% thought cervical cancer could be prevented by screening. All these daunting statistics point to the lack of necessary education about cervical cancer prevention for medical students in school. In addition, we found that a higher level of knowledge was positively associated with acceptance of HPV vaccines and cervical cancer screening, which is consistent with findings in other studies in mainland China In addition to the knowledge level, factors such as gender, attitude towards premarital sex, and choice of major subject were important predictors of the acceptability of HPV vaccines and cervical cancer screening. Female medical students were more likely to accept vaccines and screening in our study, which is consistent with the findings for HPV vaccination among health care providers Perceptions of the timing, target population, and venue for immunization were explored in our study. 35.4% and 46.4%, respectively, thought that HPV vaccines ought to be provided at age 13–18 years, junior or senior middle school or equivalent, which is fairly consistent with the findings in another study among parents of adolescents Perceptions of techniques, onset, interval and pricing of cervical cancer screening were assessed in this study. To our surprise, 41.4% of medical students did not know any screening techniques; only 31.6% and 41.1%, respectively, knew the two common techniques, Pap smear and HPV DNA testing. The fact that these two techniques are widely used in clinical practice reflects problems with emphasis on cancer treatment instead of screening and prevention There are certain limitations in the study. Firstly, the medical students surveyed were selected from medical universities or colleges in southwest China. The results may not be readily generalizable to the medical students in other areas, since cities in this part of China are generally less developed economically. However, since students from both cosmopolitan areas such as Chengdu and small cities were sampled, the conclusion may reflect part of the actual medical education scenario. Secondly, since we did not use a standard validation procedure for the questionnaire design, information collected might not be completely representative of actual conditions due to lack of validity and reliability measures. However, we did carefully incorporate input and comments from former staff in the field and participants in pilot surveys in designing the questionnaire, and this transparent procedure can ensure a relatively reliable and valid questionnaire. Thirdly, caution should be taken when interpreting the acceptability of HPV vaccines and cervical cancer screening among medical students. Acceptability may not necessarily mean that they will advise use of vaccines in practice. Knowing something good does not translate into practicing something good. Some factors that might influence their acceptability such as religion and concern for promiscuity are not analyzable in current study. Fourthly, our results only reflect the status quo of current cervical cancer education for medical students. Most of them may increase their knowledge rapidly in health care practice. Finally, constrained by the design, the study might be subject to selection and information bias due to some participants' refusal to participate and data missing in completing the questionnaires by some medical students.In conclusion, the study indicates low acceptability of HPV vaccines and cervical cancer screening among medical students. The acceptability is associated with relevant knowledge, gender, attitude towards premarital sexual behavior, and the area of major in a course of medical studies. It is important to improve the knowledge of HPV and cervical cancer among medical students in order to supply well-informed health care providers for prevention and control of cervical cancer.Figure S1Supplementary questionnaire: the questionnaire was used to survey the knowledge and awareness of HPV, cervical cancer, HPV Vaccines, and cervical cancer screening among medical students in Southwest China.(DOC)Click here for additional data file.


The Ministry of Health in Uganda in collaboration with the Program for Appropriate Technology for Health (PATH) supported by Bill and Melinda Gates Foundation in 2008–2009 vaccinated approximately 10,000 girls with the bivalent humanpapilloma virus (HPV) vaccine. We assessed parent's knowledge, risk perception and willingness to allow son(s) to receive HPV vaccines in future through a cross-sectional survey of secondary school boys aged 10–23 years in 4 districts. 377 questionnaires were distributed per district and 870 were used in analysis. Parents that had ever heard about cervical cancer and HPV vaccines; those who would allow daughter(s) to be given the vaccine and those who thought that HPV infection was associated with genital warts were more willing to allow son(s) to receive the HPV vaccine. Unwilling parents considered HPV vaccination of boys unimportant (p = 0.003), believed that only females should receive the vaccine (p = 0.006), thought their son(s) couldn't contract HPV (p = 0.010), didn't know about HPV sexual transmissibility (p = 0.002), knew that males could not acquire HPV (p = 0.000) and never believed that the HPV vaccines could protect against HPV (p = 0.000). Acceptance of HPV vaccination of daughters and likelihood of recommending HPV vaccines to son(s) of friends and relatives predicted parental willingness to allow sons to receive HPV vaccines. Probable HPV vaccination of boys is a viable complement to that of girls. Successfulness of HPV vaccination relies on parental acceptability and sustained sensitization about usefulness of HPV vaccines even for boys is vital. Humanpapilloma Virus (HPV) infections are among the most common sexually transmitted infections (STIs) worldwide In Uganda, the Ministry of Health (MoH) in collaboration with PATH and supported by the Bill and Melinda Gates Foundation vaccinated approximately 10,000 girls in two districts with the bivalent HPV vaccine in 2008 and 2009 to demonstrate a viable strategy of giving the vaccine using existing public health delivery systems HPV types 6, 11, 16, and 18 are known to be prevalent in both cervical and non-cervical diseases Information regarding HPV infection and HPV-associated diseases is often directed at young women and parents of girls. Consequently, many men tend to be ill-informed about HPV and are unaware of its consequences for male health Like elsewhere, there are still many questions about HPV vaccines including; acceptability of vaccinating boys and girls, alternative delivery models of HPV vaccines, influence of HPV vaccination on sexual behaviors and the impact of sensitizing adolescents about HPV vaccination in Uganda. The study assessed parents' knowledge and perception of their son(s) risk to HPV infection and willingness to allow their son(s) to receive probable HPV vaccines in future as a complement to HPV vaccination of females.Ethical approval clearance to carry out the study was obtained from Makerere University School of Medicine Research and Ethics Committee and Uganda National Council for Science and Technology Committee on study of Human Subjects; District Directors of Health Services, District Education Officers and authorities in charge of immunization in the concerned districts. Data reported does not arise from a clinical study and is not from patients. Parents that participated in the study gave written informed consent. Parental refusal to participate was respected. The overall conduct of the research adhered to Helsinki Declaration In the study, a cross-sectional research design was used. The study was done in 4 districts, 2 (Ibanda and Nakasongola) where HPV vaccination of girls took place in 2008 and 2009 and 2 (Mbarara and Luwero) where it did not. Parents from districts where HPV vaccination of girls never took place were included for comparison purposes.Participants were recruited during the process of recruiting a corresponding sample of 10–23 year old 1,600 in-school boys into a related study. Data analysis for a separate manuscript from the 1,600 boys is on-going. A consent letter requesting for participation of parents and their sons into the two related studies was delivered to 1,600 parents. Parents of secondary school boys that consented were recruited. A parent was taken to be either biological or a guardian who was living with and taking care of the boy(s). In selecting parents, authors worked with secondary schools which had students that were trekking to and from home every school-day. Secondary schools in study districts fulfilling this description were listed and probability random sampling was applied to select those to participate. Parents were accessed through their sons in the age range of interest.Raosoft sample size calculator Since variability in the proportion of parents willing to allow son(s) to receive HPV vaccines if availed in future was unknown, we adopted the most conservative proportion of 50% which represents maximum variability in any population. We set the desired confidence level at 95% and the level of precision at ±5%. Using the online Basing on study objectives and literature review, a pre-coded self-administered questionnaire was developed and used to collect data from parents on their socio-demographic characteristics, knowledge about HPV and HPV vaccine, perception about their son(s)' risk of HPV infection, and willingness to allow probable HPV vaccination of boys in future. Socio-demographic data included: gender, domicile, educational attainment, religious affiliation, and occupation.‘HPV is usually sexually transmitted’, ‘Males cannot get HPV’, ‘Condom use can prevent HPV’, ‘Sexual abstinence prevents HPV’, ‘HPV Vaccine effectively protects against HPV’, ‘HPV Vaccine can cure HPV’, ‘HPV Vaccine can cause infertility’, ‘HPV vaccine protects against HIV’, ‘HPV vaccine protects against STDs’, ‘HPV infected young girls can develop cervical cancer’ and ‘A healthy looking person can have HPV’. Parents were also asked about what they perceived to cause cervical cancer and genital warts. At analysis, the response of ‘I don’t know’ and ‘No’ were merged to form ‘No’.Knowledge was assessed by asking parents to choose one response from: 1 =  Yes, 2 =  No and 3 =  I don't know (if they were not sure) to each of these statements: ‘I feel that chances are high that my son(s) can get the HPV’, ‘I am afraid that my son(s) might contract the HPV’, ‘I believe that my son(s) can be exposed to HPV infection if he engages in sexual intercourse’, ‘I believe my son(s) can get HPV even if he/they are only having sex with one partner’ and ‘My son(s) would rather have HIV than HPV’. Parents were also asked about what they perceived to be the current likelihood of their son(s) to acquire HPV and other STDS.Perceived son(s)' risk of infection with HPV was assessed by a set of 5 statements requiring responses on a 5-point Likert scale of: 1 =  strongly disagree, 2 =  disagree, 3 =  neither agree nor disagree, 4 =  agree and 5 =  strongly agree. The statements were framed in such a way that parental response referred to a son(s). Sample statements included: ‘In the future, how likely are you to . . . ‘to ask a health worker to give your son(s) the HPV vaccine?’, ‘do additional research to understand the HPV vaccine?’, ‘discuss the usefulness of the HPV vaccine to your son(s) with health workers?’, ‘make an appointment to have your son(s) get the HPV Vaccine?’ and, ‘do nothing to have your son(s) get the HPV vaccine?’. Parents were also asked whether they would recommend the HPV vaccine to sons of their friends or relatives, whether they would allow their own sons to be given the HPV vaccine, and who should receive the HPV vaccine. They were further asked to give reasons for their views. The dependent variable in the study was parental willingness to allow son(s) to be given current HPV vaccines in future.Parents’ willingness to have their sons to be vaccinated against HPV was assessed using a set of statements that required responding to a 5-point Likert scale rating of: 1 =  not at all likely, 2 =  not likely, 3 =  somewhat likely, 4 =  very likely, 5 =  extremely likely. The statements were stated as follow; Luganda translation  or a Runyankore translation (for western Uganda districts) to help parents who did not understand English. Luganda and Runyankore are commonly spoken Bantu language dialects in the study areas. To attain face validity, feasibility visits to assess the appropriateness of the proposed study sites were done. This was followed by a pre-test of the research tool on a sample of articulate respondents (not part of this study), that had similar demographic characteristics as the targeted study participants. The pre-test phase also enabled assessment of the adequacy of the research tool, likely data analysis techniques and identification of appropriate strategies to recruit study participants. Appropriate changes were made in the research tool after the pre-test.Each participating parent gave oral and written consent. School boys delivered the questionnaires and consent forms to parents and returned them filled to research assistants. Data from a few parents who were semi-literate or illiterate was collected with the guidance of a research assistant who oversaw filling of the questionnaire. Each question item in the questionnaire had either a Data was collected in November, 2012. Four research teams were simultaneously deployed, one per district. The research assistants were professional health workers with prior experience in HPV-related data collection. They were given more training about the study, field surveys, dynamics of fieldwork, content of research instruments, and their ethical obligations as data collectors. They were asked to inform participating parents not to ask or allow anyone to help them respond to questions. Parents were requested to put a tick or circle on the response option that represented their thoughts. Parents were also promised and accorded confidentiality by delinking any identifiers from the data.t-test were evaluated. Binary logistic regression (backward stepwise) to adjust for possible interaction and confounding of different independent categorical variables was used in evaluation of their association with the dependent variable. Parental willingness (dependent variable) was entered as: ‘willing to allow a son(s) to be given the HPV vaccine’  = 1 and ‘not willing to allow a son(s) to be given the HPV vaccine’  = 0. Level of significance was set at p≤0.05 and corresponding Odds Ratios  were generated.Data in questionnaires were checked for completeness and consistency and entered into a computer using EpiData software. Double data entry was done by two independent data entry clerks and later, compared to clean out inconsistencies. Data was then exported to the Statistical Package for the Social Scientists (SPSS) version 11 for analysis. Frequencies and percentages of parents' socio-demographics, knowledge, risk perception and willingness to accept probable vaccination of young males with Human Papillomavirus (HPV) vaccines in future were produced. At a descriptive level, parents' socio-demographics, knowledge about HPV and HPV vaccine, and perception of son(s)' risk for HPV infection were compared using a 2-way contingency table analysis (Pearson's chi-square statistic) on the basis of willingness to allow sons(s) to receive HPV vaccines in future. Differences in age through means and standard deviations using a Student's Parents from districts where HPV vaccination of girls had taken place were 456 (52.4%) with a mean age of 40.8 (SD = 15). Those from districts where HPV vaccination of girls had not happened were 414 (47.6%) with a mean age of 40.6 (SD = 12.7). Parents that were willing to allow a son(s) to receive the HPV vaccine in future were 681 (78.3%) with a mean age of 41 (SD = 14). Those not willing to allow a son(s) to receive the HPV vaccine were 85 (9.8%) with a mean age of 39.5 (SD = 12.3). Parents that never responded to the question on willingness to allow a son(s) to receive the HPV vaccine were 104 (12%). Parental age did not significantly differ between districts where HPV vaccination of girls had occurred and districts where vaccination had not occurred.see p = 0.003).There was no significant statistical difference between parents willing and parents unwilling to allow their son(s) to receive HPV vaccines in future on all the demographic variables considered. However, parents that had ever heard about cervical cancer, parents that had ever heard about a vaccine to prevent cervical cancer or HPV, those who thought that HPV infection was the major cause of genital warts, those who would allow their daughter(s) to be given the HPV vaccine, and those who would recommend the HPV vaccine to sons of friends/family members were significantly more likely to be willing to allow their son(s) to receive the HPV vaccine in future see Table 1Parents were asked about the importance of the HPV vaccine for son(s), what they thought cervical cancer meant, how they thought HPV was transmitted, reasons they would consider to allow their sons to be given the HPV vaccine in future, reasons they would consider to recommend the HPV vaccine to sons of friends and family members, who they believed should be given the HPV vaccine, their perception of a possibility that their son(s) could get an HPV infection and what they thought were the chances that their son(s) could get an STD as displayed in p = 0.003). Parents who believed that all males should receive HPV vaccines before sexual debut were more likely to show willingness to allow son(s) to receive HPV vaccination . Also, parents who were unsure about recommending the HPV vaccine to sons of friends and relatives were significantly less likely to be willing to allow their own son(s) to receive the HPV vaccine in future  to get the HPV vaccine were significantly less likely to be willing to allow the same son(s) to receive the HPV vaccine in future  to receive HPV vaccines on most responses. However, parents who considered HPV vaccines to be very important for boys were more likely to show willingness to allow son(s) to receive HPV vaccination . Parents who were afraid that their son(s) might contract HPV were significantly more likely to show willingness to allow their son(s) to receive HPV vaccination in future . Parents who believed that their sons could be exposed to HPV infection if they had sexual intercourse were significantly more likely to be willing to allow their son(s) to receive HPV vaccination in future . Parents who were neutral with the possibility that their son(s) could contract HPV even if they had sexual intercourse with one partner were significantly more likely to be willing to allow their son(s) to receive HPV vaccination in future . Also, parents who strongly agreed that their son(s) could contract HPV even if they had sexual intercourse with one partner were significantly more likely to be willing to allow their son(s) to receive HPV vaccination in future .On the basis of parental attitudes and beliefs towards cervical cancer, HPV, HPV Vaccine and HPV Vaccination of boys, there were not many significant differences between parents willing and parents unwilling to allow their son(s) to receive HPV vaccines in future . Howeverp = 0.001) and those who were extremely likely  to ask heath workers to give their son(s) the HPV vaccine in future exhibited more willingness to allow their son(s) to receive HPV vaccination in future.Parents responded to statements about their likelihood to accept HPV vaccination of their boys with HPV vaccines in future. As shown in p = 0.002), those who knew that males could acquire HPV , and those who knew that the HPV vaccine effectively protects against HPV  had significantly more willingness to allow their son(s) to receive HPV vaccines in future. Generally, considerable proportions of parents lacked factual knowledge on each of the statements used to assess knowledge in this study.Parents responded to 12 knowledge statements about cervical cancer, HPV, HPV vaccine and probable HPV vaccination as presented in In a multivariate analysis, we assessed the association between different covariates and willingness to allow sons to receive the HPV vaccines in future. All the significant factors  associated with willingness to allow son(s) to receive the HPV vaccines in future were entered into a binary logistic regression. The final step in the model showed few significant differences between willing and unwilling parents. Acceptance of HPV vaccination of daughters  being likely to recommend the HPV vaccine to son(s) of friends and relatives  predicted parental willingness to allow son(s) to be given the HPV vaccine in future. Conversely, being unsure about whether to recommend the HPV vaccine to sons of friends and relatives  and disagreeing with the possibility that their sons could be exposed to HPV infection if they had sexual intercourse  predicted parental unwillingness to allow son(s) to be given the HPV vaccine in future.Similar to what has happened in many countries, the introduction of HPV vaccination in Uganda targeted only girls. Concerted efforts were put in the preparation of the population before the HPV vaccination of girls High proportions of parents were willing to allow sons (78.3%) and daughters (90.6%) to receive HPV vaccines if availed. This is consistent with studies that have established high HPV vaccine uptake among comparable adolescent populations The higher proportion of parents willing to allow daughters to receive current HPV vaccines compared to those willing to allow sons in this study may reflect differences in knowledge and risk-perception of HPV infections for males and females. The lower proportion of parental willingness to allow sons to receive HPV vaccines could have been related to the focus of information about HPV infection and HPV-associated diseases on young women and parents of girls during sensitization that was a precursor to the introduction of HPV vaccination in Uganda Parents that had ever heard about cervical cancer were 83% while those that had ever heard about the HPV vaccine were 60.9%. Those who believed that HPV infection was a major cause of genital warts were 78.4%. The proportion of parents that would recommend HPV vaccines to sons of their friends and relatives was as high 91.6%. More parents from districts where HPV vaccination of girls had happened accepted the assertion that HPV infection is a major cause of genital warts, possibly indicating the role of sensitization before introduction of the vaccine Parents who thought that vaccination of boys against HPV was very important were more likely to show willingness to allow son(s) to receive HPV vaccines in future. This suggests that parental acceptability of HPV vaccination for boys would be high if targeted sensitization focused on benefits their sons would receive if they were to receive HPV vaccines in future and the protection this would give to their spouses Parents who agreed that chances were high for their son(s) to contract HPV, those who were afraid their son(s) might contract the HPV and those who were either neutral or strongly agreed with the possibility that their son(s) could contract HPV even if they had sexual intercourse with one partner were more likely to show willingness to allow their son(s) to receive HPV vaccines in future. Parental acceptance of HPV vaccination of boys against HPV seemed to be associated with son(s) risk to HPV infection.Up to 36% of parents thought that their sons were at risk of contracting HPV. These parents either agreed that their sons had high chances of acquiring HPV or were apprehensive of the likelihood that their son(s) could contract HPV if they had sexual intercourse. Up to 27% agreed with the assertion that their son(s) would rather have HPV than HIV which is indicative of a low perceived severity associated with HPV infection. Being an STI, HPV could be associated with stigma and some parents could have perceived that vaccinating their son(s) against an STI would tantamount to condoning high risk sexual behavior, similar to what has been noted elsewhere If HPV vaccination of males was to be complement that of females in a socio-economic setting like Uganda, it will be necessary to emphasize the protective effects of HPV vaccines against genital warts other than mainstreaming prevention of cervical cancer only. The view of parents preferring their children to receive HPV vaccines which protects against both cervical cancer and genital warts has been articulated before Parents who knew that HPV is usually sexually transmitted, that males can acquire HPV, and that HPV vaccines effectively protects against HPV were more likely to allow their son(s) to receive HPV vaccines in future. This underscores the positive influence that provision of accurate information about the sexual transmission of HPV, HPV risk for boys and girls once they get exposed through sexual intercourse and efficacy of the HPV vaccine has on parental acceptance of HPV vaccination of boys. However, considerable proportions of parents lacked factual knowledge in this study, which is indicative of a huge information gap. This study showed that there were concerns about the safety, likely effects and cost of the HPV vaccines for males. Therefore, widespread parental sensitization about risks and consequences of HPV infections and benefits of HPV vaccination not only to females but for males as well if HPV vaccines are to be well accepted is necessary Inherent methodological weaknesses that characterize often cross-sectional studies were in this study as well and should be borne in mind when interpreting these findings. We could only infer association and not causality in this study. We admit that there are no definitive inferences about the direction of observed relationships between covariates and parental willingness to accept HPV vaccination of their son(s) in future. There could be threats to validity and reliability of findings since questions to investigate all the differences between willing and unwilling parents, in districts where HPV vaccination of girls had or had not happened may not have been included in the questionnaire we used. Parents' responses were hypothetical since HPV vaccines for males were yet to be approved in the country.‘a Luganda’ or ‘a Runyankore’ dialect. The process of translation into local dialects was carefully done to avoid alterations in meaning and conceptualization. An already described process that ensures conceptual equivalence, cultural sensitivity and validity was followed By allowing some parents with modest schooling to respond to questions on their own, complexities related to distortions in response interpretation might have occurred. We tried to solve this by providing each parent a cover letter and assigning research assistants to guide data collection for parents that felt uncomfortable to do it alone. Each question in the questionnaire was in English with a translation into either The strength of this study was the relatively big sample size which implies that findings could be generalized with relative comfort to the parent population. Data that was collected and the subsequent interpretations were reasonably valid and reliable and should be taken as a starting point for further research in this area. Future studies, perhaps interventional in nature should adopt a longitudinal design to the assessment of parental willingness to allow sons to receive availed HPV vaccines in order to identify potentially modifiable risk factors.Successfulness of HPV vaccination in developing countries like Uganda heavily rely on parental acceptability of the exercise. As a complement to HPV vaccination of females, HPV vaccination of males could be an important public health strategy since HPV vaccines are known to be efficacious in men as well. Men are not only an important vector in the transmission of the virus but they too can develop genital warts and anogenital cancers as a result of HPV infection.Contrary to expectation, parents' knowledge of HPV and HPV vaccines in general was not a strong predictor of parental acceptance of vaccination of sons against HPV in future. If HPV vaccination of males is to be adopted as a complement to that of females, sustained sensitization about usefulness of HPV vaccines is necessary to enhance their acceptability not only for girls but for boys too. This sensitization should emphasize the high vulnerability of boys and girls to HPV infection if they are exposed. Future attempts to vaccinate males against HPV in Uganda and other comparable countries should be anchored on the linkage between HPV infection, men's health in general and sexual health in particular. Besides, parents willing to let sons get HPV vaccines in future agreed that this should happen before sons' sexual debut.On comparing HIV and HPV, parents seemed not to perceive the likelihood of HPV infection of their sons to be too severe. This could be indicative of lack of awareness about consequences of HPV infection. It is vital to raise HPV risk and severity perception among parents if acceptability of HPV vaccination for males is to complement that of girls. This is because parents who believed that their sons could be exposed to HPV infection if they had sexual intercourse and those who were apprehensive about the likelihood that their son(s) could contract HPV showed more willingness to allow sons to receive HPV vaccination in future.Lastly, it is necessary to elevate parent's confidence in health workers if they are to accept current HPV vaccines for their male children. Apparently, parents who were likely to ask heath workers to give their son(s) HPV vaccines in future showed more acceptability. Secondly, parents that generally oppose vaccination seemed to be the ones likely to refuse letting their sons to receive HPV vaccines in future. Most parents that accepted HPV vaccination of daughters were also likely to recommend HPV vaccines to son(s) of friends and relatives as well as to allow their own son(s) to receive the HPV vaccine in future.


Intratypic molecular variants of human papillomavirus (HPV) type-16 and -18 exist. In the Netherlands, a bivalent vaccine, composed of recombinant L1 proteins from HPV-16 and -18, is used to prevent cervical cancer since 2009. Long-term vaccination could lead to changes in HPV-16 and -18 virus population, thereby hampering vaccination strategies. We determined the genetic diversity of the L1 gene in HPV-16 and -18 viral strains circulating in the Netherlands at the start of vaccination in order to understand the baseline genetic diversity in the Dutch population.DNA sequences of the L1 gene were determined in HPV-16 (n = 241) and HPV-18 (n = 108) positive anogenital samples collected in 2009 and 2011 among Dutch 16- to 24-year old female and male attendees of the sexually transmitted infection (STI) clinics. Phylogenetic analysis was performed and sequences were compared to reference sequences HPV-16 (AF536179) and HPV-18 (X05015) using BioNumerics 7.1.For HPV-16, ninety-five single nucleotide polymorphism (SNPs) were identified, twenty–seven (28%) were non-synonymous variations. For HPV-18, seventy-one SNPs were identified, twenty-nine (41%) were non-synonymous. The majority of the non-silent variations were located in sequences encoding alpha helix, beta sheet or surface loops, in particular in the immunodominant FG loop, and may influence the protein secondary structure and immune recognition.This study provides unique pre-vaccination/baseline data on the genetic L1 diversity of HPV-16 and -18 viruses circulating in the Netherlands among adolescents and young adults. Human papillomaviruses (HPVs) are small double stranded DNA viruses that have the potential to infect mucosal and cutaneous human epithelial cells. Over 160 HPV types have been identified based on the DNA sequence of the L1 open reading frame of which about 60 types infect the human anogenital tract . Of thesNaturally occurring intratypic molecular variants of HPV-16 and -18 are known to occur and have been shown to be specific or more prevalent in certain parts of the world . IntratyHPV vaccination of sexually naïve girls has been introduced in many countries in order to prevent cervical cancers caused by persistent infection with HPV-16 or -18. To what extent HPV vaccination will affect the distribution of HPV types and variants in a  vaccinated population is not yet known. Therefore, monitoring of type-specific HPV prevalence before and after the start of HPV vaccination is of great importance. Previous HPV monitoring studies have shown that the three most prevalent high-risk HPV types (among women) at the introduction of HPV vaccination in the Netherlands were HPV-16, HPV-51 and HPV-52 –13.Since 2009, a bivalent vaccine, containing virus-like particles (VLPs) composed of pentamers of recombinant major capsid protein L1 from HPV-16 and -18, is used for vaccination of young girls in the Netherlands. This HPV vaccine is known to elicit high-titre neutralizing antibodies directed against the L1 protein and confer type-specific and long-lasting protection against a persistent infection and cervical abnormalities caused by HPV-16 and -18 . An addiIn order to detect a long-term effect of L1 vaccination on the HPV-16 and/or -18 virus population, knowledge of the genetic diversity of L1 gene in HPV-16 and -18 viruses circulating in the Netherlands at the start of vaccination is required. The aim of this study was to determine the genetic diversity in the L1 protein and HPV variant distribution of circulating HPV-16 and -18 viral isolates found in young adolescents before the start of HPV-16/-18 vaccination, at baseline.Between February and April 2009, a cross-sectional study was started in 12 sexually transmitted infection (STI) clinics spread throughout the Netherlands prior to the introduction of vaccination. Follow-up surveys in this setting have been conducted every two years since 2009. For the results presented here only samples obtained in 2009 and 2011 were analyzed. The study population and methods have been described in detail previously . The stuSamples were obtained from anogenital  swabs collected in 2009 and the follow up year 2011 within the PASSYON (PApillomavirus Surveillance among Sti clinic YOungsters Netherlands) study among Dutch 16- to 24-year old male and female attendees of STI clinics. Details of the PASSYON study were described elsewhere , 13. All2, 0.2 mM dNTPs (Roche) and 10 pmol of each primer. The cycling conditions were as follows: 15 min 95°C denaturation; 15 sec 95°C, 30 sec 55°C, 90 sec 72°C for 35 cycles; 10 min 72°C final extension. Amplicons were visualized on 2.2% agarose gels. PCR products were treated with ExoSAP-IT® and sequenced by automated DNA sequencing using Big Dye Terminator 3.1.HPV-16 (n = 241) and HPV-18 (n = 108) positive samples were selected for further molecular characterization of the L1 gene (1596 bp for HPV-16 and 1707 bp for HPV-18) by sequence analysis. The entire L1 gene of HPV-16 and HPV-18 was amplified in three overlapping fragments using the primers listed in All sequences from a given sample were combined and used to construct a contig. Contig sequences were aligned using Bionumerics version 7.1 and compared with HPV-16 European German reference sequence AF536179 (sublineage A2), or HPV-18 reference sequence X05015 (lineage A/European). For clustering of HPV-16 the L1 gene, GenBank reference sequences from K02718 = NC001526 (sublineage A1/European), HQ644236 (sublineage A3/ European), AF534061 (sublineage A4/European-Asian), AF536180 (sublineage B1/African-1a), HQ644298 (sublineage B2/African-1b) and AF472509 , HQ644257 (sublineage D1-North American), AY686579-, AF402678  and AF043286 (vaccine variant) were included in the analyses. For clustering of HPV-18 the L1 gene, GenBank reference sequences from X05015 (lineage A/European), AY262282 , EF202146 , EF202151 , GQ180787 (sublineage A5/ European) were used. HPV-16 and -18 L1 sequences sequenced here were deposited into the NCBI GenBank database (accession numbers KU70477-KU707717 for HPV-16 L1 sequences and KU707718-KU707825 for HPV-18 L1 sequences). The presence of the variations found infrequently, in one, two or three strains were confirmed by resequencing at least 2 additional times.http://www.hiv.lanl.gov) [To estimate selection pressure acting on HPV-16 and HPV-18 L1 sequences, codon-specific, non-synonymous (dN) and synomymous (dS) substitutions rates were inferred by using Nei- Gojobori method  and Jukeanl.gov) . Non-synL1 HPV-16 sequences were determined and analyzed by aligning the entire L1 gene from 241 HPV-16 positive samples collected in the PASSYON study rounds 1 (2009) and 2 (2011) . Forty-sIn total we identified ninety-five single nucleotide polymorphisms (SNPs) among the observed Dutch HPV-16 strains compared to the reference strain AF536179. Of these SNPs, 68/95 (72%) were synonymous variations and 27/95 (28%) were non-synonymous variations. The majority of the non-synonymous variations 21/27 (78%) was located in the L1 region encoding alpha helix, beta sheets, surface loops or connecting loops, in particular in the immunodominant FG loop, and therefore may influence the protein secondary structure. The position of the non-synonymous variations is shown in We calculated the non-synonymous/synonymous substitution rates for HPV-16 L1 amino acid residues. Based on the HPV-16 sequences analyzed here the dN/dS ratio was 0.176 suggesting that for the entire L1 protein no evidence for positive selection was found (P<0.01).L1 HPV-18 sequences were determined and analyzed by aligning the L1 contigs for in total 108 HPV-18 positive samples from the PASSYON study, which is 35% of the HPV-18 positive samples . Table 1Seventy-one (71) single nucleotide polymorphism (SNPs) were identified among the 108 Dutch HPV-18 strains related to the reference strain X05015. Forty-two of seventy-one (42/71) (59%) were synonymous variations and 29/71 (41%) were non-synonymous variations. For HPV-18 fourteen of the non-synonymous variations 14/29 (48%) were located in sequences encoding the alpha helix, beta sheet, surface loops or connecting loops. The position of the non-synonymous variations is shown in Non-synonymous and synonymous substitution rates for HPV-18 L1 amino acid residues were calculated based on the HPV-18 L1 gene sequences analyzed here and shown be significantly different (P<001). No evidence for positive selection was found for the entire L1 gene since the dN/dS ratio was 0.172 suggesting that it was subject to purifying selection.Human papillomavirus (HPV) vaccination using VLPs composed of the L1 protein has been widely implemented in many countries, with vaccines targeting high-risk HPV-16 and -18 alone, or additional HPV types. While type-specific protection is expected, genotypic and phenotypic variants within HPV types have been recognized, with some evidence for geographic association of this diversity and differences in progression to cervical intraepithelial neoplasia grade 3 (CIN3). At present it is not known if immunity to viruses within an HPV-type is equally efficient for all variants, with some evidence that this may not be the case: although studies have suggested that immunization with L1 VLPs of an European variant induces antibodies able to neutralize different HPV-16 variants . Other sIn the past, several studies have addressed the intratypic variation of the major capsid protein from HPV-16 and/or HPV-18 –26. MostAs HPV variants have been shown to differ in geographic origins  it was tOur study identified known and new variants of HPV-16 and -18, with amino-acid variations in or close to epitopes. The new variants of HPV-16 and -18 were found both in European and non-European group of viral strains. Whether these changed L1 proteins are less susceptible to the vaccine induced immunity is unclear at this time. In general, human papillomaviruses mutate slowly  because Some study limitations should be noted. First, we have sequenced a relative small number of HPV strains, especially strains sequenced from males and persons from the non-Dutch population group were low in number. Specific variants are not expected in males but have been seen in persons from non-Dutch population group. A second limitation is that we have selected swabs collected in 2009 and 2011 for studying the pre-vaccination genetic diversity. However, vaccination was introduced in the Netherlands in 2009 in a catch-up campaign in girls aged 14- to 16-years old before introduction in 12-year old girls in 2010. Therefore it is possible that samples collected in 2011 from 16–18 year old girls were from vaccinated girls. Since the number of strains collected in 2011 from 16 to 18 years girls is very low and the vaccination uptake at that time was about 50% we believe that no (or very little) dilution of vaccination effects have been introduced the pre-vaccination genetic diversity.Whether in the future we will observe the emergence or expansion of (new) HPV-16- and or HPV-18 variants caused by the selective pressure induced by mass vaccination will remain unclear until investigated. Knowledge of the genetic diversity of HPV-16 and -18 at the start of vaccination (baseline), as is presented in this study is essential in order to understand the genetic variability of these proteins over time.S1 Table(XLSX)Click here for additional data file.