This prospective study aimed to examine the combined effect of viral load and alcohol consumption on the risk of persistent high-risk (HR) human papillomavirus (HPV) infection.Among women undergoing health screening between 2002 and 2011 at the National Cancer Center, 284 and 122 women with HR-HPV infection and cytological findings of low-grade squamous intraepithelial or lower-grade lesions were followed up for 1 and 2 years, respectively. Multivariate logistic regression analysis was performed, and the relative excess risk due to interaction (RERI) and synergy index (S) were calculated.Among drinkers, the risks of 1-year  and 2-year persistence  were significantly higher for high HPV loads than for low HPV loads; this association was not seen for non-drinkers. The risks for 1-year  and 2-year persistence  were significantly higher in subjects with a high HPV load who were also drinkers than in those who were non-drinkers. A high HPV load together with a longer drinking duration or higher alcohol consumption was associated with increased risks of 1-year  and 2-year persistence . The synergistic effect of alcohol consumption and HR-HPV load was stronger on the risk of 2-year persistence  than on the risk of 1-year persistence .The synergistic effect of HR-HPV load and alcohol consumption was associated with the risk of HR-HPV persistence and was stronger for longer-term HR-HPV infection. Limiting alcohol consumption might be an important measure to prevent the development of cervical cancer in women with a high HR-HPV load. Persistent high-risk human papillomavirus (HR-HPV) infection is an important cause of cervical intraepithelial neoplasia (CIN) and cervical cancer Among these factors, the use of HR-HPV load as a marker for predictor of persistence remains controversial. Several studies have reported HPV-16 load to be associated with persistent infection in situ (CIS) development and low-grade cytological abnormalities Cigarette smoking and alcohol consumption are critical factors in carcinogenesis and immune suppression. Cigarette smoking is reported to be a critical risk factor for cervical cancer and its high-grade precursors In this prospective study, we assessed the associations of HR-HPV load and alcohol consumption with persistent HR-HPV infection. We used health screening data to investigate the combined effect of HR-HPV load and alcohol consumption, with its different characteristics, on the risk of persistent HR-HPV infection.This cohort study was part of the Korean Prospective Study for the Transition of Human Papillomavirus into Cervical Carcinoma (KOVIC). Among 11,140 Korean women undergoing health screening at the National Cancer Center between 2002 and 2011 who provided written informed consent, 920 were positive for HR-HPV on a DNA test at enrollment and responded to questions about alcohol consumption. Subjects receiving any therapy or surgery or using immunosuppressive agents were excluded at enrollment. Of these, 284 and 122 women with low-grade squamous intraepithelial lesions (LSIL) or lesions of lower cytological grade at enrollment were eligible for HR-HPV persistence follow-up studies 1 and 2 years after enrollment. Four women (3 for the 1-year study and 1 for the 2-year study) were excluded because of lesions with cytology grades greater than LSIL during the follow-up period. Among 284 women in the 1-year follow-up group, 122 with persistent HR-HPV infection (3 consecutive positive results for HPV DNA) at 2 years after enrollment were analyzed in the 2-year follow-up study. Subjects were divided into 2 groups, the clearance and persistence groups, and were analyzed in 1-year and 2-year follow-up studies . ClearanThe Institutional Review Boards and Ethics Committee of the National Cancer Center in Korea (NCCNCS-11-433) approved this study in March 2011 and written informed consent was obtained from all subjects.Detailed self-administered health and lifestyle questionnaires, including questions on behavior related to alcohol consumption, were completed at enrollment. Questions related to alcohol consumption were aimed at determining the alcohol consumption status for the previous 5 years , the frequency of alcohol consumption , the duration of the drinking habit (years), and the typical volume per drink  of beer (200 mL) or soju (50 mL) for the previous 5 years. Daily alcohol consumption was calculated individually for frequency and typical volume per drink HR-HPV DNA was detected using the commercially available Hybrid Capture 2 assay . The chemiluminescent HPV DNA test yielded relative light units (RLU) using a probe designed to detect 13 HR-HPV types. Results were considered HR-HPV positive at concentrations of 1.20 pg/mL or greater than the RLU cut-off ratio (specimen RLU/mean RLU of 2 positive controls [PCs]) and borderline at concentrations of 0.80–1.20 pg/mL. Cervical cytological findings were classified using the Bethesda classification system.t-tests were used to analyze distribution differences in categorical and continuous variables. The Wilcoxon rank sum test was used to analyze differences in raw viral loads. Multivariate logistic regression analysis was performed to evaluate the association between HR-HPV load (low: <100 RLU/PC and high: ≥100 RLU/PC), alcohol consumption (non-drinkers and drinkers), other variables related to alcohol consumption , and HPV persistence (1-year and 2-year persistence). Risk estimates were calculated with low HPV load, non-drinking status, alcohol consumption for <5 years, and alcohol consumption of <15 g alcohol/day as reference categories.Descriptive statistics and logistic regression analyses were performed using SAS 9.1 . Chi-square and The risk of HR-HPV persistence was subsequently analyzed according to HPV load in non-drinkers and drinkers. We divided an ordered distribution of HPV load into tertiles, and risk estimates for 1-year and 2-year persistence were calculated with low viral load as reference. All variables were adjusted for age as a continuous variable and for menopausal status , oral contraceptive use , smoking status , and number of children  as categorical variables.p values were calculated according to previously described methods for measuring biological interaction between risk factors To assess the combined effect of HR-HPV load and alcohol consumption on the risk of persistent HR-HPV infection, interaction tests were performed on additive scale in the model for viral load and drinkers or other variables related to alcohol consumption. The relative excess risk due to interaction (RERI) and synergy index (S) with 95% confidence intervals and p = 0.174), but were significantly different between the 2-year clearance and 2-year persistence groups   . Mean vap<0.001 by the Wilcoxon rank-sum test) and 2-year clearance (p = 0.001) groups, respectively (Median HPV loads were significantly higher in the 1-year and 2-year persistence groups than in the 1-year (ectively . As expep for trend <0.001) and 2-year  persistence. However, among non-drinkers, HPV load was not significantly associated with the risk of 1-year  or 2-year  persistence.We found evidence of a synergistic effect of HR-HPV load and alcohol consumption on the risk of 1-year HPV persistence. The risk was higher for drinkers with a high HPV load  than for non-drinkers with a low HPV load , Table 4p<0.001) and S values .The risk of 2-year persistence was higher for the combination of a high HPV load and alcohol consumption  than for a low HPV load and no alcohol consumption . On straOur findings demonstrate an effect of the combination of HR-HPV load and alcohol consumption on the risk of HPV persistence in women with normal or low-grade cytological abnormalities; thus, a high viral load and alcohol behaviors may synergistically affect the risk of HR-HPV persistence. Furthermore, this synergistic effect is much stronger on longer-term HR-HPV infection.Alcohol consumption is associated with an increased risk of HPV infection or acquisition The usefulness of clinical markers for HPV load has been debated. HPV-16 load has been reported to be associated with persistent infection Several mechanisms could explain the synergistic effects of HR-HPV load and alcohol consumption on viral persistence. Oxidative stress and HR-HPV can act synergistically to initiate and promote carcinogenesis. Viral infection, establishment of persistent infection, and viral integration are promoted by oxidative stress Chlamydia trachomatis or Mycoplasma spp infection and HPV persistence could not be investigated because of the low detection rate of these organisms in the study population C. trachomatis prevalence, with an age-standardized prevalence of 4.3% This study has several limitations. First, data could not be adjusted for sexual behavior or sexually transmitted diseases (STDs) such as chlamydia because the questionnaire did not include questions about these topics. However, the study subjects were mostly middle-aged women (mean age: 48 years), and a decline in sexual interest accompanied by a reduction in the frequency of sexual intercourse is often reported in aging women The HR-HPV load and alcohol consumption had a combined effect on viral persistence; the two factors synergistically increased the risk of persistent HR-HPV infection in women with normal or low-grade cytological abnormalities. Furthermore, this synergistic effect was much stronger on longer-term HR-HPV persistence. Limiting alcohol consumption might be important in preventing the development of cervical cancer in women with a high HR-HPV load.Table S1Odds ratios and 95% confidence intervals of HR-HPV load and alcohol drinking for the risk of persistent high-risk human papillomavirus infection. 1) P value was from Wilcoxon rank sum test after testing for normality. 2) HPV load value was classified as low (<100 relative light units [RLU]/positive control [PC]) or high (≥100 RLU/PC). 3) Multivariate logistic regression analysis was performed after adjusting for age as a continuous variable and for menopausal status, oral contraceptive use, smoking status and the number of children as categorical variables. The risks were estimated with the low HPV load, non-drinkers, <5 years, or <15 g alcohol/day as the reference categories.(DOCX)Click here for additional data file.Table S2Interaction between high HR-HPV load and alcohol consumption on the risk of 1 year-HR-HPV persistence. † N w/wo persistence, the number of subjects with/without persistence; OR, odds ratio. 1) HPV load value was classified as low (<100 relative light units [RLU]/positive control [PC]) or high (≥100 RLU/PC). 2) Logistic regression analysis was performed with adjustment for age as a continuous variable. The risk were estimated with no alcohol consumption, alcohol consumption for <5 years, or alcohol consumption of <15 g/day and a low HPV load as reference categories. 3), 4) The relative excess risk due to interaction (RERI) and synergy index(S) were calculated as described by Rothman et al. RERI>0 and S>1.0 indicate a synergistic effect between HR-HPV load and alcohol consumption behaviors.(DOCX)Click here for additional data file.Table S3Interaction between high HR-HPV load and alcohol consumption on the risk of 2 year-HR-HPV persistence. † N w/wo persistence, the number of subjects with/without persistence; OR, odds ratio. 1) HPV load value was classified as low (<100 relative light units [RLU]/positive control [PC]) or high (≥100 RLU/PC). 2) Multivariate logistic regression analysis was performed with adjustment for age as a continuous variable and for menopausal status and the number of children as categorical variables. The risk were estimated with no alcohol consumption, alcohol consumption for <5 years, or alcohol consumption of <15 g/day and low HPV load combination as reference categories. 3), 4) The relative excess risk due to interaction (RERI) and synergy index(S) were calculated as described by Rothman et al. The RERI>0 and S>1.0 indicate a synergistic effect between HR-HPV load and alcohol consumption behaviors.(DOCX)Click here for additional data file.


Poor attendance to cervical cancer (CC) screening is a major risk factor for CC. Efforts to capture underscreened women are considerable and once women agree to participate, the provision of longitudinal validity of the screening test is of paramount relevance. We evaluate the addition of high risk HPV test (HPV) to cervical cytology as a primary screening test among underscreened women in the longitudinal prediction of intraepithelial lesions grade 2 or worse (CIN2+).Women were included in the study if they were older than 39 years and with no evidence of cervical cytology in the previous five years within the Public Primary Health Care System in Catalonia (Spain). 1,832 underscreened women from eight public primary health areas were identified during 2007–2008 and followed-up for over three years to estimate longitudinal detection of CIN2+. Accuracy of each screening test and the combination of both to detect CIN2+ was estimated. The risk of developing CIN2+ lesions according to histology data by cytology and HPV test results at baseline was estimated using the Kaplan–Meier method.At baseline, 6.7% of participants were HPV positive, 2.2% had an abnormal cytology and 1.3% had both tests positive. At the end of follow-up, 18 out of 767 (2.3%) underscreened women had a CIN2+, two of which were invasive CC. The three-year longitudinal sensitivity and specificity estimates to detect CIN2+ were 90.5% and 93.0% for HPV test and 38.2% and 97.8% for cytology. The negative predictive value was >99.0% for each test. No additional gains in validity parameters of HPV test were observed when adding cytology as co-test. The referral to colposcopy was higher for HPV but generated 53% higher detection of CIN2+ compared to cytology.Underscreened women had high burden of cervical disease. Primary HPV screening followed by cytology triage could be the optimal strategy to identify CIN2+ leading to longer and safe screen intervals. Infection with high-risk human papillomavirus types (HPV) is the necessary cause for the development of cervical cancer (CC) . HistoriPrimary CC screening with HPV detection has been shown in randomized controlled trials (RCTs) to have higher longitudinal sensitivity to detect cervical intraepithelial neoplasia grade 2 or worse (CIN2+) than conventional cytology, maintaining a high negative predictive value (NPV) –14.In the Autonomic region of Catalonia (Spain), routine screening with cervical cytology is recommended to women aged 25–65 with a 3-year interval. Although screening is opportunistic, within the Public Health System efforts to increase CC screening coverage in underscreened women have been established . These aThe aim of this study was to evaluate the addition of HPV test to cervical cytology as a primary screening test among the underscreened population in the longitudinal prediction of CIN2+.1,832 women older than 39 years old were included. Women were selected if they had no evidence of cervical cytology in the public primary health registries in the previous five years. Women identified in eight public primary health areas of Catalonia during 2007 and 2008 were included in this study and followed-up until June 2012  provided information on age, results and date of cytologies, histologies and HPV tests during the study period for each woman. The overall project was approved by the ethical committee of the Catalan Institute of Oncology. Any information regarding the identification of patients was anonymized before analysis.−1, which corresponds to 1.0 relative light unit (RLU/CO). All HPV reference laboratories participated in an inter-laboratory quality control with kappa values over 90% [HPV detection was performed with the FDA-approved Hybrid Capture 2 test  which detects 13 high-risk HPV types . An HPV sample was considered positive if attained or exceeded the FDA-approved threshold of 1.0 pg HPV DNA mlCytologies were performed largely with conventional Pap smears. Few centres used liquid based cervical cytology and in such cases the HPV and the cytology was performed in the same sample. All the cytological results were classified according to the 2001 Bethesda system . AbnormaThe CIN classification was used to categorize histological results .The end-point of follow-up was established at the moment of the most severe diagnosis or June 2012. For women with a positive test during the study period, a final diagnosis of “normal” was assigned if at least two negative tests were registered in subsequent visits. For women with both tests negative at baseline a subsequent negative test was requested to be categorised as being negative for CIN2+.When concomitant cytological and histological results were available, the highest histological grade of abnormality was used for the final diagnosis. Women not having any additional test to those obtained at baseline were considered as lost to follow-up.Whenever appropriate, estimates are presented by different combinations of screening tests results. Three year longitudinal sensitivity, specificity, positive predictive value (PPV) and NPV for CIN2+ detection and their 95% CI were calculated for cytology, HPV test and the combination of both. Estimates were corrected taking into account the proportion of women who returned to the next screening round in each screening strata of results as reported elsewhere . We estiAt baseline, the average age among the 1,832 included women was 54.1 years (range 40–88 years). Most of them (92.4%) had both tests negative. 338 women were relieved from further screening because of being 65 years old or older and had both tests negatives, leaving 1,494 undescreened women to be followed-up. Of them, 767 women (51.3%) completed follow-up. Lost to follow-up was higher in women with both tests negative when compared to those with at least one positive test (p < 0.05)   had to be screened again within a year in order to correct for poor cytology sensitivity. This was not implemented in our population because of the co-testing recommendation.Positive cytology was detected in 2.2% of the women, percentage that was overall similar to that observed in the general population of the same age group (2.1%), although lesions in our study were more severe than expected . HoweverThe cumulative detection of CIN2+ among women with normal cytology at baseline was high for HPV positive women at baseline compared to those HPV negative and comparable to that seen in other screening cohorts , 24. OurEuropean RCTs and American screening cohorts have shown that among HPV negative women, the risk for CIN2+ was very low  suggesting that safe intervals can go beyond five years if a validated HPV tests is being used , 31, 32 In this study we have explored the strategy to protect poorly screened women by introducing a more complete screening approach. However, screen negative women were prone to a poor follow-up at 3 years as almost half of them did not return during the follow-up period consistently with that observed in other studies . A behavDue to the differential attendance to control visits according to the screening baseline results we corrected the accuracy parameters  by follow-up estimates to minimize any bias . We coulWe were concerned about the low longitudinal sensitivity of the cytology test but it is well accepted that quality of cytology depends on many factors as is extremely amenable to poor reproducibility. Although a relevant proportion of the women were menopausal, we did not find differences by age strata or by pathology laboratory but our sample size was relatively small and estimates by strata were unstable.Strengths of this study are the performance of screening and follow-up processes in many centres across Catalonia as part of the routine CC screening. Co-testing with cytology and HPV allowed us not only the comparison between tests but also to speculate about different screening scenarios as testing was done blind to the other test result. Finally, the HPV testing used complied with its recognized clinical validity and reproducibility .In a group of underscreened women participating in opportunistic screening, HPV test, as primary screening tool, was superior to cytology for CIN2+ detection with a higher longitudinal sensitivity over 3-year follow-up. Both tests had a very high NPV. Primary HPV screening followed by cytological triage could be the optimal strategy to identify CIN2+ in poor screening attenders in developed countries leading to longer and safe screen intervals.


Australia was one of the first countries to introduce a publicly funded national human papillomavirus (HPV) vaccination program that commenced in April 2007, using the quadrivalent HPV vaccine targeting 12- to 13-year-old girls on an ongoing basis. Two-year catch-up programs were offered to 14- to 17- year-old girls in schools and 18- to 26-year-old women in community-based settings. We present data from the school-based program on population-level vaccine effectiveness against cervical abnormalities in Victoria, Australia.Data for women age-eligible for the HPV vaccination program were linked between the Victorian Cervical Cytology Registry and the National HPV Vaccination Program Register to create a cohort of screening women who were either vaccinated or unvaccinated. Entry into the cohort was 1 April 2007 or at first Pap test for women not already screening. Vaccine effectiveness (VE) and hazard ratios (HR) for cervical abnormalities by vaccination status between 1 April 2007 and 31 December 2011 were calculated using proportional hazards regression.a priori for age at first screening, socioeconomic status and remoteness index, for women who were completely vaccinated, was greatest for CIN3+/AIS at 47.5% (95% CI 22.7 to 64.4) and 36.4% (95% CI 9.8 to 55.1) for women who received any dose of vaccine, and was negatively associated with age. For women who received only one or two doses of vaccine, HRs for HG histology were not significantly different from 1.0, although the number of outcomes was small.The study included 14,085 unvaccinated and 24,871 vaccinated women attending screening who were eligible for vaccination at school, 85.0% of whom had received three doses. Detection rates of histologically confirmed high-grade (HG) cervical abnormalities and high-grade cytology (HGC) were significantly lower for vaccinated women (any dose)  compared with unvaccinated women  HR 0.72 (95% CI 0.58 to 0.91) and HR 0.75 (95% CI 0.65 to 0.87), respectively. The HR for low-grade (LG) cytological abnormalities was 0.76 (95% CI 0.72 to 0.80). VE adjusted A population-based HPV vaccination program in schools significantly reduced cervical abnormalities for vaccinated women within five years of implementation, with the greatest vaccine effectiveness observed for the youngest women. Randomized clinical trials have shown that prophylactic human papillomavirus (HPV) vaccines are highly effective at preventing infection with HPV types 16 and 18, which cause about 70 to 80% of cervical cancers worldwide  and in p©. Commencing in April 2007, the Australian vaccination program targeted 12- and 13-year-old girls in schools and offered catch-up vaccination for 14- to 17-year-old girls in schools and 18- to 26-year-old women in community based settings.These vaccines have been widely adopted in high income countries since 2007 with most countries targeting girls prior to commencement of sexual activity, between 9 and 13 years of age, with varying catch-up programs for older adolescent girls [Australia is in a unique position to evaluate the impact of the HPV vaccine at the population level. The recommended starting age for cervical screening is 18 years or 2 years after the onset of sexual activity, whichever is later, and thus screening already overlaps with the vaccination program. Screening is recommended every two years using conventional cytology until the age of 69 years. Australia’s State based Pap test registers systematically record cervical cytology and histology results and send reminder and follow-up letters to women and their practitioners according to national guidelines. Participation in both screening and vaccination programs is high: catch-up HPV vaccine coverage in Victorian females 12 to 17-years old was 86/82/75% for doses 1/2/3  and 77% of 20 to 24-year-olds attended at least one screening between 2007 and 2011 .In this retrospective cohort study, we linked data from the Victorian Cervical Cytology Registry (VCCR) and the National HPV Vaccination Program Register (NHVPR) and evaluated the effectiveness of the HPV vaccine against cervical abnormalities in a screening population of women eligible for vaccination in the school-based cohorts (aged 17 or younger in 2007). We focus on this cohort for two reasons: (1) effectiveness in this age group will most closely approximate the eventual impact of vaccination in the ongoing target group of girls prior to sexual activity and (2) vaccination status in the school cohorts is more completely notified than in the adult women catch-up program, where notification was not compulsory and under-notification has been estimated at 10 to 15% .The VCCR captures cervical screening results for all women resident in Victoria, Australia, which has a population of more than 2.7 million women, and receives data on cervical cytology and histopathology directly from laboratories in a timely manner. Fewer than 1% of women request to opt-off the VCCR . DetailsThe NHVPR was established in June 2008 and records HPV vaccination doses administered nationwide. Details on the establishment of the NHVPR, and initial coverage in both the schools program and the catch-up program have been published elsewhere ,13,14. ICross-referencing of data against cervical cytology registers for monitoring of the effectiveness of HPV vaccine is allowed for by legislation enabling operation of the NHVPR. However, because the VCCR was established far earlier, there is no similar provision for the VCCR and thus individual identifiers could not be used for the data linkage in this study.A deterministic linkage procedure was undertaken to link records from the VCCR, which included Victorian women who are age-eligible for the HPV vaccine as per product indication (date of birth on or after 1 July 1962), to the NHVPR which included all Australian women who received the HPV vaccine prior to 1 January 2012  to 31 December 2011. Women were counted as at risk of a diagnosis of a cervical abnormality from the time they commenced cervical screening, and were entered into the cohort at their first Pap test (or on 1 April 2007 if their first Pap test was prior to that time). We found that our analyses were robust when the entry criteria to the cohort for those screening prior to April 2007 were altered to include person-time from their first Pap test after 1 April 2007. Women were followed until the outcome of interest, date of death, hysterectomy or the end of the study period.Unvaccinated women were those who had no doses of HPV vaccine recorded on the NHVPR; vaccinated women were those who received any doses of HPV vaccine. Completely vaccinated women were those vaccinated with three doses compliant with the vaccine schedule  . Person-We chose the dose assignment method, with no time-lag until abnormalities are ‘counted’ after vaccination, because we wanted to reflect apparent incident abnormalities as they occurred in the real world . However, we recognize that abnormalities diagnosed during the vaccination course are predominantly caused by HPV infection acquired prior to the onset of vaccination. Therefore, we also evaluated the cohort using a measure closer to vaccine efficacy assessment, excluding time for vaccinated women where they were in the process of being vaccinated, that is, censoring time occurring between dose 1 and dose 3. This second method  cervical disease (CIN2+/AIS), defined as CIN2, CIN3, adenocarcinoma the AIHW  and Austthe AIHW . For allU test for ordinal variables and the Pearson chi-square test for nominal variables. Detection rates were calculated as the number of events per 1,000 person-years at risk and vaccine effectiveness was (1-rate ratio) × 100.Analyses of demographic and exposure characteristics of women in the cohort by vaccination status used the Mann-Whitney a priori the other predictors: age at first screening , socioeconomic status and remoteness. Remoteness areas were assigned based on postcode of residence and as defined according to the Australian Standard Geographic Classification. Socioeconomic status was assigned by postcode of residence according to the Australian Bureau of Statistic’s Socio-Economic Indexes for Areas (SEIFA) Index of Relative Socio-Economic Disadvantage [Cox proportional hazards regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI), and included dvantage . Lower sdvantage . Individdvantage .Ethics approval was obtained from the Department of Health and Ageing and the Australian Institute of Health and Welfare’s Human Research and Ethics Committees. Approval for use of NHVPR data was given by the Department of Health and Ageing, the data custodian and for the VCCR data by the Victorian Department of Health.P <0.05).Between 1 April 2007 and 31 December 2011, 24,871 women aged between 12 and 17 years who were vaccinated against HPV had commenced cervical screening. Of these women, 21,151 (85.0%) were completely vaccinated and 3,690 women had received one or two doses of vaccine. There were 14,085 unvaccinated women of the same age who had commenced cervical screening. The follow-up period was a maximum of 4.8 years with an average of 1.5 years for both vaccinated women and unvaccinated women. There was no difference in mean age at entry into the cohort or mean age at first screen between vaccinated and unvaccinated women, with almost all first Pap tests occurring after April 2007, Table The number and rate of cervical abnormalities detected by vaccination status as per our primary analysis (dose assignment method for assessing vaccine effectiveness) is shown in Table Figure Vaccine effectiveness (VE), adjusted for remoteness, SES and age at first Pap test, was highest for CIN3/AIS at 47.5% (95% CI 22.7 to 64.4) for women who were completely vaccinated (compared to no doses),  analyses of the clinical trials of the quadrivalent HPV vaccine, which vaccinated women between the ages of 16 and 25, and was highest in younger women, who as a group are least likely to have been sexually active prior to vaccination. Vaccine effectiveness was greatest for outcomes most strongly associated with vaccine related HPV types 16 and 18, in particular CIN3/AIS, and lowest for low-grade cytological abnormalities for which the majority of disease is accounted for by non-vaccine HPV types. The reduction in risk of cervical abnormalities was restricted to women who received three doses of vaccine.The follow-up time in our study was five years and further follow-up will likely impact upon the effectiveness of the vaccine. Our data confirm that VE diminishes with age of administration in catch-up programs, which is a proxy for the onset of sexual activity. The ongoing vaccination programs in Australia and elsewhere target girls prior to onset of sexual debut and at the population level higher reductions will be observed as this large cohort reaches screening age, as a greater proportion of vaccinated women will have been unexposed to HPV at the time of vaccination. The VE was higher in the youngest women in our study than observed in the ITT analyses of either the quadrivalent or bivalent vaccine trials -21. The Studies have shown that a greater proportion of CIN3+ lesions in younger women (under 30 years of age) is due to HPV type 16, with other less oncogenic types more prevalent in women over 30 years of age -7. HPV16We observed a significant impact of vaccination on LG cytological abnormalities that was similar in magnitude to that observed for HG cytological lesions. In addition, the effect of partial vaccination was significant for LG cytology, but not for HG cytology, although this may be due to the larger number of outcomes in this group. Because a greater proportion of HG cytology is due to vaccine-related HPV types than LG cytology, we would have expected to see a greater impact on HG as compared with LG cytological lesions. However, this result was sensitive to whether we counted person-time in between dose one to three for those women who ended up being fully vaccinated  is minimized.If fewer than three doses could provide sufficient protection against disease, vaccine programs could achieve higher coverage at a lower cost -27. TherOur results contribute to the growing body of evidence in Australia of a reduced burden of HPV-related disease since implementation of the vaccination program in 2007. Data from HPV prevalence studies show a significant reduction in the prevalence of vaccine HPV genotypes  in a post-vaccination sample compared with a pre-vaccination sample and lower prevalence in both vaccinated and unvaccinated women compared with the pre-vaccine reference population . A substIn conclusion, we have demonstrated the first population level impact of a school-based HPV vaccination program on cervical abnormalities. The program included a two-year catch-up for girls aged 14- to 17-years and achieved high vaccination coverage rates. The impact of the vaccine was greatest for women completely vaccinated at younger ages and for the more serious cervical abnormalities CIN3/AIS, which are more likely to be due to the vaccine HPV types. The reduction in risk of cervical abnormalities was restricted to women who received three doses of vaccine; however, relatively few women received fewer than three doses and these women were older at vaccination than the other women. Over the next five years, the effect of the vaccine is expected to increase as cohorts of women vaccinated prior to sexual debut move through the screening program.HG: High-grade; HPV: Human papillomavirus; HR: Hazard ratio; HSIL: High-grade squamous intraepithelial lesion; LG: Low-grade; LSIL: Low-grade squamous intraepithelial lesions; NHVPR: National HPV Vaccination Program Register; SEIFA: Socio-Economic Indexes for Areas; SES: Socioeconomic status; TVC: Total Vaccine Cohort; VCCR: Victorian Cervical Cytology Registry; VE: Vaccine effectiveness.DMG, JMLB and MS were investigators on an Australian Research Council Linkage grant, for which CSL biotherapies was a partner organization. JMLB has been an investigator on investigator designed unrestricted epidemiological research grants partially funded through bioCSL but received no personal financial benefits.MS, DMG and JMLB were principal investigators of the study, with assistance from KD and GC. JMLB, DMG and MS designed the study. AB conducted the statistical analysis and interpretation of results was led by DMG, JMLB and MS. DMG led the writing of the manuscript, together with AB, JMLB and MS, and all authors contributed to the final report. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/227/prepubRate of histological and cytological cervical abnormalities for completely vaccinated, partially vaccinated and unvaccinated women using final dose status method .Click here for file


The pEGFP-N1-KAI1 plasmid was transfected into human gastric carcinoma SGC7901 cells using liposomes. The effect of transfection with the KAI1 gene was measured using a reverse transcription-semi-quantitative polymerase chain reaction (RT-sqPCR) assay. The Transwell chamber assay was used to study the metastatic and invasive ability of SGC7901 cells. Gastric cancer metastasis-associated genes, including hypoxia-inducible factor (HIF)-1α, matrix metalloproteinase (MMP)-2, MMP-9, basic fibroblast growth factor (bFGF), and urease plasminogen activator (uPA) were measured by RT-sqPCR prior to and following transfection with the KAI1 gene. The expression of KAI1 protein and mRNA was associated with the differentiation degree of gastric cancer, presence of lymph node metastasis, tumor-node-metastasis stage, depth of invasion and the survival time of patients. The migratory and invasive abilities of SGC7901 cells were significantly decreased subsequent to transfection with the KAI1 gene, and the expression of bFGF and uPA was downregulated. It was concluded that the tumor suppressor gene KAI1 inhibits the migration and invasion of gastric carcinoma cells, possibly by suppressing the expression of uPA. Patients that expressed KAI1 may demonstrate an improved prognosis.The present study aimed to explore the effect and mechanism of the Kangai 1 (KAI1) gene in regulating the migration and invasion of gastric carcinoma cells, and the prognostic significance of this gene in gastric cancer patients. Immunohistochemistry and  Gastric cancer is the second highest cause of cancer-associated mortality worldwide. Despite the advances in diagnosis and treatment, the prognosis of patients with gastric cancer remains poor. The median survival time of patients with advanced gastric cancer is ≤10 months, and only 10–20% of patients survive >5 years.in situ hybridization were used in the present study to evaluate KAI1 expression in various stages of gastric cancer.Kangai 1 (KAI1), also termed cluster of differentiation (CD)82, is a tumor metastasis suppressor that was first identified as a metastasis suppressor for prostate cancer. KAI1 is located on human chromosome 11p11.2 . It has At present, no specific studies have been conducted to investigate the effects and mechanisms of the KAI1 gene on the migration and invasion of gastric carcinoma cells. To detect these aspects, the pEGFP-N1-KAI1 plasmid was transfected into the gastric carcinoma SGC7901 cells through liposomes in the present study.Tissue specimens obtained from 128 patients with gastric adenocarcinoma that underwent resection at the Shandong Cancer Hospital  between January 2007 and April 2009 were used in the present study. The patients consisted of 81 males and 47 females, aged between 30 and 74 years . The inclusion criteria for the present study were as follows: Complete surgical R0 resection of the primary tumor; pathologically confirmed diagnosis of gastric adenocarcinoma; no chemotherapy or radiotherapy administered; and the absence of secondary malignancies. All patient records contained complete clinical, pathological and follow-up data. Normal gastric mucosa tissue (≤5 cm) adjacent to the tumor was excised and confirmed to be tumor-free by pathological analysis.Tumor histology was determined according to the criteria provided by the World Health Organization . The patThe tissue sections were conventionally dewaxed, hydrated and subjected to antigen repair with EDTA. The monoclonal mouse anti-human KAI1/CD82 antibody  was diluted at 1:200. The immunohistochemical staining was performed using the of streptavidin-peroxidase two-stage method, according to the instructions of the kits . Negative controls were stained following the same procedure, with the exception that the primary antibody was replaced with PBS. The KAI1-positive tissue provided by Fuzhou Maixin Biotech Co., Ltd. was used as a positive control.The staining intensity and percentage of cells stained for KAI1 expression were evaluated in a blind manner by three pathologists simultaneously, and a consensus was reached for each score. Cells positive for the expression of KAI1 were considered to be cells with brown plasma membranes and cytoplasm. The presence of KAI1 expression was assessed through the ratio of stained to non-stained cells. At least nine visual fields were observed for each section under a high power lens . The staining intensity was judged based on the ratio of KAI1-positive to total cell numbers observed in the visual field. Sections with ≤10% KAI1-positive cells were considered to not express KAI1 and sections with >10% KAI1-positive cells were considered to express KAI1.2O2 at room temperature for 10 min, and were then digested using Proteinase K, diluted in 3% saline sodium citrate, at 37°C for 20 min. The in situ hybridization was performed according to the instructions for the kits . Blank controls were operated following the same procedure, but without the probe. The expression of KAI1 was indicated by in situ hybridization as clear yellow brown granular material, which was located on the cell membrane. The KAI1 staining intensity was scored as follows: 0, absent; 1, weak; 2, moderate; and 3, strong. The percentage of KAI1-positive cells was scored into four categories, as follows: 1, 0–10%; 2, 11–30%; 3, 31–60%; and 4, 61–100%. The sum of these two scores was classified as follows: 1–3, absent; 4–5, positive; and 6–7, strongly positive.The mRNA sequence of the KAI1 gene was retrieved from the National Center for Biotechnology Information database . The oligonucleotide probe sequences were designed using Primer3 software  as follows: 5′-CAGCCTTTCTGTGAGGAAGG-3′ (800–819 bp); 5′-GATGGTCCTGTCCATCTGCT-3′ ; and 5′-GCAGTCACTATGCTCAT-3′ (438–454 bp) . The primers were marked by digoxin. The tissue sections were conventionally dewaxed and underwent gradient alcoholic dehydration. The slides were incubated in 3% HpEGFP-N1 is a eukaryotic expression plasmid without the objective gene, and this plasmid possesses a selectable marker gene for G418 resistance. The eukaryotic expression plasmid vector was supplied by Proteintech Group Inc. .2 atmosphere.The SGC7901 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences . The cells were cultured in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  at 37°C in an incubator with a humidified 5% COOnce the cells had reached 70–90% confluency, the pEGFP-N1-KAI1 plasmid was transfected into the SGC7901 cells using Lipofectamine 2000 , in accordance with the manufacturer's instructions. The vector control plate was transfected with the pEGFP-N1 plasmid, and cells without transfection acted as a blank control.2O, and 0.5 µl cDNA template. The PCR conditions were as follows: 95°C for 3 min; 30 cycles at 94°C for 20 sec, 55°C for 20 sec and 72°C for 20 sec; and 72°C for 5 min.The effect of KAI1 gene transfection was measured using an RT-sqPCR assay. Total RNA with isolated using TRIzol  using an RNeasy Mini kit  and cDNA was generated by reverse transcription, according to the instructions of the Reverse Transcription System kit . The primers used for KAI1 PCR were as follows: Forward, 5′-CCCCAAGTACTGAGGCAGC-3′, and reverse, 5′-AACCACAGAACAGCCAGGG-3′. This generated a 217-bp product (1040–1256 bp) . The PCR6/ml. The three groups of cells were seeded in the upper chamber of the Transwell insert, and 600 µl DMEM containing 10% FBS was added to the lower chamber. The cells were cultured at 37°C in a humidified 5% CO2 incubator for 24 h, the inserts were washed with PBS. A cotton swab was used to remove adherent cells on the inner side of the upper chamber membrane. The upper chamber was then dried naturally and stained with 0.5% hematoxylin. Six visual fields of each insert were randomly counted under an upright light microscope , and the average cell number was calculated.A Transwell chamber assay was used to perform the cell migration analysis. The cells were fasted for 24 h with serum-free medium DMEM containing 0.1% bovine serum albumin (BSA), and then trypsinized and resuspended with medium to a density of 1×102). This process was repeated 3 times. The plate was then incubated at 4°C overnight. The subsequent experimental steps were in accordance with the cell migration assay.The Transwell chamber invasion assay was performed to investigate the role of KAI1 in gastric cancer cells. Laminins  were diluted in serum- and BSA-free DMEM and were then added to a 24-well plate at 0.95 ml/well  and reverse transcribed into complementary DNA using the kit (Takara RT-PCR), according to the manufacturer's instructions. RT-sqPCR was performed by using the Roche Cobas 4800 System (Roche). The sequences of the HIF-1α2 test. The Kaplan-Meier survival curve and log-rank test were used to analyze the association between KAI1 expression and patient survival. The correlation analysis was performed using Spearman's rank correlation coefficient test, and the inspection level was α=0.05. P<0.05 was considered to indicate a statistically significant difference.The PEMS 3.1 software  was used for the statistical analysis. All data were expressed as the mean ± standard deviation from at least three independent experiments. The KAI1 staining level in tissues from various stages of gastric cancer was compared using the χ2=24.382; P=0.000). The expression of KAI1 was associated with the differentiation degree of gastric cancer tumors . There was no significant difference (P>0.05) between the presence of KAI1 mRNA expression and the tumor location, or between the age and gender of the patients. The rate of KAI1 mRNA expression in the superior differentiation group, which consisted of well- and moderately-differentiated adenocarcinoma, was increased compared with the expression in the inferior differentiation group, which consisted of poorly-differentiated and mucinous adenocarcinoma and signet-ring cell carcinoma (P<0.05). Correlation analysis was used to analyze the association between the depth of invasion and KAI1 mRNA expression, and there was a significant negative correlation between these variables . Similarly, there was a significant negative correlation between the TNM stage and KAI1 mRNA expression . The rate of KAI1 mRNA expression in gastric cancer patients with lymph node metastasis was markedly decreased compared with the rate in gastric cancer patients without lymph node metastasis, and the difference was statistically significant P<0.05; .2=11.523; v=1; P<0.05; The result of Kaplan-Meier analysis indicated that the survival time of the group that expressed KAI1 was significantly longer compared with the group that did not express KAI1. The log-rank test indicated that the difference between the two groups was statistically significant  to examine 16 cases of well-differentiated adenocarcinoma, 28 cases of moderately-differentiated adenocarcinoma, 66 cases of poorly-differentiated adenocarcinoma, 10 cases of mucinous adenocarcinoma and 8 cases of signet-ring cell carcinoma. In addition, the effects of KAI1 expression on the migration and invasion of gastric cancer cells were investigated, as migration and invasion are the critical steps in cancer progression. The results of the present study demonstrated that KAI1 staining occurs mainly in the cytoplasm (KAI1 was first identified as a metastasis suppressor in prostate cancer ,10. The ytoplasm , which iytoplasm ,10,11.Statistical analysis of the positive rate of KAI1 revealed a significant reduction in cytoplasmic KAI1 expression in internal gastric cancer compared with superficial gastric cancer, indicating that reduced KAI1 expression may be critical for the progression of gastric cancer. The present data revealed that decreased KAI1 expression was significantly associated with the presence of lymphatic metastasis, TNM stage, patient survival time, depth of invasion and the degree of gastric cancer differentiation. The results also indicated that reduced KAI1 expression was not involved in distant metastasis, but significantly stimulated gastric cancer cell migration  and invaThe KAI1 protein plays an important role in signaling pathways, as it mediates the signal transduction between cells and the surrounding environment, which affects the movement and differentiation of cells and ultimately inhibits the invasion and metastasis of tumor cells. KAI1 may regulate the infiltration and metastasis of tumors through the following four mechanisms. First, KAI1 binds with integrin to form a complex and affects cell adhesion by regulating the functions of integrin. Second, the protein promotes adhesion between tumor cells by acting on the signal pathway of Src kinase . Third, In addition, KAI1 is a highly glycosylated protein, and a deficiency in the glycosylation process leads to a lack of KAI1 function, without affecting the expression of the protein . The sigIn the present study, the expression of HIF-1α, MMP-2, MMP-9, bFGF and uPA was detected in order to further reveal the underlying mechanism of the function of the KAI1 gene in gastric cancer. The current study identified that the inhibitory effect of KAI1 expression on the cell migration and invasion of gastric cancer was not mediated by HIF-1α, MMP-2 and MMP-9, but may be associated with the reduction in uPA and bFGF expression.bFGF may promote angiogenesis and degrade the extracellular matrix by upregulating the expression of MMPs . bFGF maet al (The present study demonstrated that KAI1 may be used as a prognostic marker in human gastric cancer, which is not consistent with the findings of a previous study by Knoener et al . This isFurthermore, the experimental data revealed that the expression of MMP-2 significantly increased following transfection with the KAI1 gene, which was not consistent with the anti-metastatic role. This discrepancy may be due to the initiation of malignant tumor development being an extremely complicated process that involves multiple anomalies of signaling pathways, including the induction of apoptosis, regulation of the cell cycle and inhibition of tumor cell adhesion. Proto-oncogenes, anti-oncogenes and multiple other factors participate in this process. The association between these factors is complex, and the effect may be negative, positive or modulatory, and in various forms. In addition, the association may change with the progression of tumors. Anti-oncogenes may not inhibit all the cocarcinogens. Therefore, it is not notable that the expression of MMP-2 significantly increased subsequent to transfection with the KAI1 gene. This result also indicated that the inhibitory effect of KAI1 may not be mediated by MMP-2.The present results demonstrated clearly that the invasive ability of gastric cancer SGC7901 cells was significantly decreased subsequent to transfection with the KAI1 gene. In addition, the KAI1 gene may suppress the migration and invasion of SGC7901 cells by reducing the expression of uPA and bFGF. Therefore, the present data indicated that the expression level of KAI1 protein may affect the migration and invasion ability of gastric cancer cells. Upregulation of KAI1 gene expression in gastric cancer cells may represent an important direction to inhibit the invasion and metastasis of gastric cancer, which requires additional investigation.In summary, the present study confirmed that KAI1 plays an important role in the inhibition of metastasis and invasion of gastric carcinoma, and downregulated KAI1 expression was significantly associated with advanced gastric cancer and a poor five-year survival rate in patients. The present study provides a theoretical and experimental basis for the investigation and clinical application of KAI1. It was concluded that KAI1 may be used as a potential therapeutic target and is a promising prognostic marker for gastric cancer.


An increasing incidence of anal cancer among men, especially men who have sex with men (MSM) suggests a need to better understand anal human papillomavirus (HPV) infection among this group.A cross-sectional study was conducted among MSM in Shenzhen, China. Blood was collected for HIV serological testing and syphilis serological screening, and anal swabs were collected for HPV genotyping. Difference of HPV prevalence between HIV seropositive and HIV seronegative MSM was assessed by chi-square test. Factors associated with anal canal HPV infection were assessed by univariate and multivariate logistic regression.A total of 408 MSM were recruited. HIV and HPV prevalence were 6.9% and 36.4%, respectively. HPV was detected in the anal canal in 71.4% of the HIV-positive MSM and in 33.8% of the HIV-negative MSM (P<0.001). Oncogenic types were seen more often in anal specimens of HIV-positive MSM than in specimens of HIV-negative MSM (P = 0.001). The HPV genotypes detected most frequently were HPV06 (8.2%), HPV16 (7.2%), HPV11 (6.4%), HPV18 (4.7%), HPV58 (4.7%), and HPV52 (4.2%).In this study, HIV positive MSM had a higher burden of HPV infection, especially oncogenic HPV infection. HPV types 52 and 58 were as popular as those types designed for the currently available vaccine . Infection with Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide Approximately 85% of anal cancers worldwide are attributed to the oncogenic HPV In China, most studies on prevalence of anal HPV infection among MSM were in northern part of China, while the data on molecular epidemiological studies of anal HPV infection in southern China is limited The study protocol was reviewed and approved by the Medical Ethics Committee of the Chinese Academy of Medical Sciences Institute of Dermatology in Nanjing. Written informed consent was obtained from each study participant before the interview and testing. Publication of the study data is in accordance with the community standards and approved by the ethics committee.The study was carried out between July and September of 2009 in a sexually transmitted diseases (STD) clinic specially providing health care for MSM in Shenzhen, China. Those eligible to participate were males, at least 18 years old, ever had sex with another man, willing to provide anal swabs and blood for the tests, physically able and willing to provide written informed consent. After an informed consent was obtained, socio-demographic and sexual behavior data were collected through a face-to-face interview by the trained clinical staff in a separate room using a standardized questionnaire. The questions included age, ethnicity, education, marriage status, place of domicile, dwelling status, self-reported sexual orientation, ever had skin lesions in genitalia , number of male sex partners in the previous 6 months, lifetime number of male anal sex partners, et al. Blood was collected for serological tests of HIV and syphilis infections, anal swabs were collected for detection of HPV, and urine was collected for detection of Chlamydia and gonococcus infections.Blood samples were collected for the HIV and syphilis serological assessment. The HIV serologic status was determined by enzyme linked immunoassay . And positive tests were confirmed by HIV-1/2 Western blot assay . The syphilis serologic status was determined by Treponema pallidum-enzyme linked immunoassay . And positive tests were confirmed by tolulized red unheated serum test kit Anal swabs were collected by trained personnel by rotating a dry nylon flocked swab in the anal canal for about 3 to 4 rotations. All specimens were coded and stored at −70°C before processed further. HPV DNA was extracted by QIAamp DNA Mini Kit  according to the manufacturer's instructions. Then HPV DNA was amplified and viral types were determined using the HPV Geno Array test kit  according to the manufacturer's instructions. The GenoArray test is a L1 consensus primer-based PCR assay and is capable of amplifying 21 HPV genotypes including 15 oncogenic HPV types  and 6 non-oncogenic HPV types . We considered a specimen to be positive for HPV if it was positive for any of 21 genotypes. We labeled specimen as oncogenic if any of the 15 oncogenic types were detected , regardless of the presence of other genotypes. In contrast, specimens with single or multiple non-oncogenic HPV genotypes  and not co-infection with oncogenic types were classified as non-oncogenic. If a specimen was positive for single or multiple types of HPV6, 11, 16 and 18, it was classified as HPV6/11/16/18 positive.The operating protocol has been described in detail by previous study All data from the questionnaires and laboratory tests were double entered and compared using EpiData software  to ensure the quality of data entry.Outcome variables include prevalence rates of HPV infections and their 95% confidence intervals (CI). Univariate analysis was used to determine association between socio-demographic, behavioral and biomedical characteristics and the infections, and crude odds ratio (COR) and its 95% CI were calculated. Factors with significance level of p≤0.05 in univariate logistic regression analyses were included in multivariate logistic regression model to explore the association of indicators with acquisition of each of the four HPV outcome variables . Adjusted odds ratio (AOR) and its 95% CI were calculated. Values of p<0.05 were considered statistically significant. Data were analyzed using SPSS .The characteristics of the study population (N = 408) were outlined in Prevalence of HPV infection among HIV positive MSM (71.4%) was significantly higher than those MSM negative by HIV . Specifically, the difference in prevalence between HIV-positive and HIV-negative MSM was only statistically significant for any types, oncogenic types, multiple types, and single types HPV; however, we did not found that difference for non-oncogenic HPV. HPV6 (8.2%), HPV16 (7.2%), HPV11 (6.4%), HPV18 (4.7%), HPV58 (4.7%), HPV52 (4.2%) were found to be the most frequently identified types. Among oncogenic types, HPV16 and HPV 18 accounted for 40.2% and HPV 58 and HPV 52 are also predominant types of oncogenic HPV.p = 0.028). Ever had skin lesion in genital increased the possibility of any HPV infection , oncogenic HPV infection  and multiple HPV infection . Having more than 10 male sexual partners in the recent 6 months facilitated any HPV infection compared to 0–2 male sexual partners . HIV and syphilis seropositivity were both associated with significantly increased infections by any HPV, oncogenic HPV and multiple HPV, while MSM infected with chlamydia had an increased infection of any HPV, non-oncogenic HPV and multiple HPV. Ethnicity, education, marital status, place of domicile, dwelling status, self-reported sexual orientation, lifetime number of male anal sex partners and co-infection with gonorrhea were not significantly associated with the four HPV infection outcomes.The association between potential risk factors and anal HPV infection was assessed by univariate logistic analyses . No signp = 0.006) and oncogenic HPV infection  after adjusted by confounders and variables remaining in the model. Age was the only factor associated with non-oncogenic HPV infection. Compared with the age group of ≥40 years, those aged 30–39 years had a decreased infection of non-oncogenic HPV . No factor was found to be associated with multiple HPV infection in multivariate logistic analysis.In multivariate logistic analysis , ever haThe prevalence and genotype distribution of HPV varied widely in different places Our data was similar with a clinic-based study in Rotterdam of the Netherland The overall prevalence of HIV and anal canal HPV infection in our study was lower than that from Beijing and Tianjin  The higher prevalence of any type HPV, oncogenic HPV and multiple HIV in HIV positive MSM than HIV negative MSM was in accordance with several previous studies Previously reported risk factors for anal canal HPV infection in MSM included younger age, minority, larger number of sexual partners a men has currently or has had in his lifetime, having ever had oral sex, and having ever found sex partners in gay venues, recent history of anal warts, and recent receptive anal intercourse In the current study, the prevalence of any HPV, oncogenic HPV and multiple HPV were all similar across four age groups, just as several previous studies In summary, our study confirms a high burden of anal HPV infection among MSM, particularly those MSM positive for HIV in Shenzhen, China. HPV types 52 and 58 are as popular as those types designed for the currently available quadrivalent vaccine (HPV6/11/16/18). The high prevalence of oncogenic HPV infection among MSM calls further for the importance to consider HPV vaccination for protection against anal cancer in this population. Innovative formats of vaccines to cover more oncogenic types of HPV may be needed to designed and developed for the population in China.Table S1Factors associated with anal HPV infection among MSM: univariate logistic analyses.(DOC)Click here for additional data file.


Implementation of Human Papillomavirus (HPV) vaccination should be considered a key cervical cancer prevention strategy in Tunisia, where Pap smear screening is not efficient. This study aims to estimate the prevalence and to identify risk factors associated with HPV infection among women from Grand Tunis, Tunisia. We conducted a cross-sectional study, between December 2012 and May 2013. Eligible women for this study were those aged 18–65 years, sexually active, who sought medical attention at their primary health care centre or clinic in Grand Tunis, Tunisia and who gave written consent. A liquid-based Pap smear sample was obtained from all women using a cervical brush. Only women with betaglobin positive test were further analysed for HPV detection and typing. A nested-PCR of the L1 region was performed followed by reverse line blot hybridization to facilitate the specific detection of 31 HPV genotypes. Multiple logistic regression modeling was used for the analysis of associations between variables with some considered possible confounders after checking for interactions. A total of 391 women were enrolled in this study and 325 out of the 391 cervical samples were positive for the betaglobin test. Overall HPV prevalence was 13.2% [9.8%−17.5%], with the following most prevalent HPV genotypes: HPV6 (40%), HPV40 (14%), HPV16 (12%), HPV52 (9%), HPV31 and HPV59 (7%), followed by HPV68 (4%). Mean age of HPV positive women was 40.7±0.92 years. Independently associated risk factors of HPV infection were smoking (OR:2.8 [0.8–9.6]), low income (OR:9.6 ), partner with multiple sexual relationship (OR:4.5 [0.9–22.9]) and single women  (OR:6.9 [1.1–42.2]). This study provides the first national-based estimate of HPV prevalence in Tunisia. Our findings contribute to the evidence on the current burden of HPV infection, the critical role of sexual behaviour and socioeconomic status and call for increased support for the screening program in Tunisia to prevent cervical cancer. These results allow us to evaluate the cost-effectiveness of vaccine program implementation in Tunisia in future. Human Papillomavirus (HPV) is the most common cause of sexually transmitted diseases and causes a wide range of pathologies , 2. AlthIn the 1980s, the link between cervical cancer (CC) and HPV was established . During In Tunisia CC is the third cause of cancer in women resulting in an estimated 1,000 deaths per year often in young women . It reprThe present study is a part of a national pilot study. It aims to estimate the prevalence and distribution of HPV genotypes and identify related risk factors among women in the Grand-Tunis region .A cross-sectional descriptive study was conducted between December 2012 and May 2013. Eligible women were those aged 18 to 65 years old, sexually active resident in the Grand Tunis region, seeking medical attention at their local healthcare centre (CSB) or at a regional reproductive health centre (CRSR) and who gave written consent. The two health centres are considered as a first line healthcare in Tunisia. Selection of CSBs and CRSRs was made proportionally to the size of the governorates between December 2012 and May 2013. As shown in The sample size (n) was calculated for each governorate  using th studies ,18, an αProperly trained medical doctors and midwives conducted face-to-face interviews using a standardized questionnaire, collecting information on socio-demographic status, sexual behaviour of the woman and her partner, reproductive and socio-economic status and medical history. For all women who provided written informed consent, a liquid-based cervical sample was obtained using a cervix brush . Samples were kept at 4°C and sent to the Laboratory of Human and Experimental Pathology at the Pasteur Institute of Tunis (which is the EMRO WHO HPV Reference LabnetLaboratory) in less than one week. Part of the sample was used for a Pap smear and the remaining content stored at -20 C for further use in genomic DNA extraction.Cytological diagnosis was performed blindly by two pathologists of the laboratory of Human and Experimental Pathology at the Pasteur Institute and the final conclusion was taken by consensus according to the 2001 Bethesda system .DNA was extracted from a 200μl aliquot of the suspended cell samples using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. The quality of extracted DNA was evaluated by betaglobin-specific primers PC04/GH20. Only samples positive for betaglobin were further analysed for HPV detection and genotyping.HPV was detected using nested PCR with biotinylated PGMY09/11 primers for the first PCR and GP5+/GP6+ primers for the second PCR. Briefly, 50μl mixture containing 3mM MgCl2, 10 μmol of each primer, 1.5 mM of dNTP , 5μl of the Taq DNA polymerase buffer, 1U of Taq DNA Polymerase, and 10 μl of DNA preparation was aliquoted. The PCR cycling parameters were composed of a 10 minutes initial denaturation at 94°C, followed by 30 amplification cycles of 30 s at 94°C, 1 min at 50°C and 1 min at 72°C, and a final extension step for 7 minutes at 72°C. This reaction was followed by a nested PCR using 10μl of the PGMY PCR product in a reaction mixture containing 50 μmol of GP5+/GP6+ primers, 3mM MgCl2, 1.5 mM each of the dNTP, 1U of Taq DNA Polymerase and 5μl of the Taq DNA polymerase buffer. The PCR cycling parameters comprised of a 10 minutes initial denaturation at 94°C, followed by 40 amplification cycles of 1 minutes at 94°C, 2 minutes at 40°C and 1.5 minute at 72°C, and a final extension step for 7 minutes at 72°C.Genotyping of positive samples was performed by Reverse Line Hybridization as described in the Human Papillomavirus Laboratory Manual published by the World Health Organization . BrieflyQualitative variables were described by the simple counts and percentages; quantitative variables by the mean ± SEM. The distribution of HPV genotypes was summarized using frequency distributions. The relation of HPV positivity with demographic, epidemiological and clinical differences was first examined by univariate analysis using the two-sample t test for normally distributed continuous data, the Mann-Whitney test for non-parametric data, the chi-squared test for frequencies and the Fisher’s test and the chi-square test for trend. Odds Ratios (ORs) and 95% confidence intervals (CI) were used to quantify the association between risk factors and positivity for HPV. Multivariable analysis was done using multiple logistic regression to study the relationship between HPV infection (HPV-positive vs. HPV-negative) and the explanatory variables, while adjusting for confounding factors and effect modification if needed. Model building was done using backward procedures. Only variables that retained statistically significant associations with the outcome variables were left in multivariate analyses. A p-value <0.05 was considered statistically significant. Data analysis was performed using SPSS version 22 software.The study was approved by the Ethics Committee of Institut Pasteur de Tunis and conducted in accordance with Good Clinical Practice, ensuring confidentiality and anonymity. Written informed consent was obtained prior to enrolment of study participants.Three hundred ninty-one women were enrolled in this study. Sixty-six out of 391 samples collected were negative for betaglobin and were excluded. HPV detection and typing were performed on the remaining 325 samples. Of note, no significant differences were observed between excluded women and included women in this study .Socio-demographic characteristics of the participants are presented in HPV prevalence in 325 cases was 13.2% . Prevalence of HPV infection according to socio-demographic, clinical and behaviour characteristics is summarised in The prevalence of HR-HPV and low-risk HPV (LR-HPV) types were 3.1%  and 6.5% , respectively. Multiple infections were detected in only 18% of positive samples (8/43) and single infections in 55.8% (24/43). Of the 8 multiple HPV infected samples, 5 were infected with 2 HPV types, 2 were infected with 3 HPV types, and 1 was infected with 4 HPV types. Multiple infections with both HR-HPV and LR-HPV types were the most common (62.5%). mixed infections with HR-HPVs were observed in 25% of cases while mixed infections with LR-HPVs were seen in 12.5% of cases. The most common HR-HPVs were HPV16 , HPV31 and HPV52 . HPV6  and HPV40  were the most common in LR-HPV. The prevalence of detected HPV types is shown in Prevalence of HPV infection was highest among women aged less than 30 years , followed by a non-significant decline in HPV prevalence in women aged 30 to 50 years and a new increase after 50 years of age (p = 0.45). There was a statistically significant difference between low- and high-risk HPV prevalence trends by age . PrevaleThe distribution of detected genotypes according to age groups showed that HPV 16 was the most common type in women aged 31–40 and over 50 years-old. HPV31 was present in all age groups except in women aged 41–50 year. Here HPV6, HPV40, and HPV35 were the major genotypes .A total of 27 (8.3%) cervical cytology samples were inadequate. For those with adequate results, Pap smear was normal for 115 women . It was atrophic or inflammatory for 141 women , Low-squamous intraepithelial lesions (LSIL) were seen for 38 women  and High-squamous intraepithelial lesion (HSIL) and Atypical glandular undetermined significance (AGUS) accounted for two cases . HPV prevalence was 16.5%  in normal smear, 10.6%  in atrophic or inflammatory smears and 18.4%  in LSIL. HPV infection in HSIL accounted for 50% of cases and all were single HPV31 infections. The 2 cases of AGUS were HPV negative.Associated factors with HPV infection in univariate analysis were marital status, tobacco use, sexual behaviour and partner sexual behaviour .Multivariate analysis suggests that marital status, partner with multiple sexual encounters outside the stable relationship, housing type and income index were independently associated with HPV infection . HoweverBaseline information on HPV prevalence and genotype distribution is crucial to evaluate the impact of HPV vaccines and inform the best approach for cervical cancer prevention. Our cross-sectional study estimated HPV prevalence in women residing in the Grand Tunis region and is a part of the first large-scale national-based epidemiological study on the prevalence of HPV infection and genotype distribution.In this study, the prevalence of HPV infection in the Grand Tunis region was estimated as 13.2%. The few previous studies in Tunisia that focused on a small series of participants in cervical screening programs have shown divergent results, that is 43.8% among Tunisian female prostitutes , 7.8% inIn this study, HR-HPV accounted for about half of all HPV infections, with the most prevalent HR-HPV being HPV-16, 31, 52 and 59. Comparing with reported series, HPV16 was the most common genotype , 25,26 aTwo peaks of infection by age appear clearly in our results. One peak in ages less than 30 years, which could represent the beginning of sexual activity, and another peak in ages greater than 50 years. This second peak may partly be explained by a relative lack of viral clearance and insufficient adaptive immune responses at this age caused by hormonal changes, contributing to HPV persistence or reactivation of latent HPV infections –31. Our In this study HPV infection in normal Pap smears accounted for 16.5% . This prevalence is similar to reported series in Africa (7.3%-37.1%), America (4.6%-42.2%) and Europe 8.5%-22.7%) [.7% [22].In our study, risk factors were shown to be specific: the better the income, the greater the risk of HPV infection. Several risk factors related to HPV infection and persistence, such as age, smoking, age at first sexual relation and multiple sexual partners, are reported in the world with differences due to the specificities of each studied population , 25, 29.It is established that smoking increases the risk of HPV infection and persistence through a vulnerability of immune system which facilitates infection and persistence of HPV and an increasing risk of turning into cancer . In our Various aspects of sexual behaviour were reported to be related to the acquisition of HPV infection , 29, 40.HPV infection prevalence in the Grand Tunis region was 13.2% and is larger than previous estimates in Tunisia. HR-HPV types accounted for half of all infections and were mainly HPV-16, 31, 52 and 59. Smoking, sexual behaviour of partner and high level income were the main risk factors. This study is the pilot phase of a national survey which aims to assess the national HPV prevalence in the whole of Tunisia and provide baseline data to enhance the understanding of HPV infection. Those findings will inform recommendations to optimize HPV screening efforts for high-risk genotypes, the most affected age groups and to evaluate the cost-effectiveness of vaccine program implementation in Tunisia in future.S1 Table(DOC)Click here for additional data file.


To determine the prevalence of sexually transmitted diseases in female athletes.Chlamydia trachomatis, human papillomavirus and Neisseria gonorrhoeae were performed. Blood samples were collected to test for the human immunodeficiency virus, syphilis, hepatitis B and C. The athletes presenting clinical diseases or conditions identifiable by laboratory tests were treated and followed up in the unit.An observational, cross-sectional study was conducted including 50 female athletes with mean age of 20±3 years. Colposcopy, pap smear, and polymerase chain reaction for C. trachomatis. There were no cases diagnosed of infection by N. gonorrhoeae, syphilis, hepatitis B, hepatitis C and human immunodeficiency virus. However, only 26 athletes had been vaccinated for hepatitis B.Forty-six percent of the participants were unaware of sexually transmitted diseases. The prevalence of sexually transmitted diseases among athletes was 48% (24 cases). Human papillomavirus was the most frequent agent (44%). Considering the human papillomavirus genotypes, subtype 16 was the most prevalent (53%), followed by 11-6 (22%) and 18 (13%). Two athletes tested positive for The prevalence of sexually transmitted diseases in female athletes was high. Primary prevention measures (hepatitis B and human papillomavirus vaccination) and secondary  must be offered to this specific group of women. The matter should be further approached in sports. The truth is that up to this event, athletes were seen as super-heroes and little was known on the risk of acquiring sexually transmitted diseases (STD) while practicing sports and what was the prevalence of these conditions in athletes. The risk of contamination is currently known to be very low and the possibility lies on the presence of bleeding wounds.,3In 1991, basketball player Magic Johnson announced he had acquired immunodeficiency syndrome, commonly known as AIDS. The news had a major impact on the sports world, and many of his teammates began to get tested, because they were afraid of having acquired the disease.taekwondo, box and American football.  However, the risk is extremely low, estimated to be around 1/85 million games.,4 Conversely, transmission through contaminated syringes in athletes who use illicit drugs (doping), has been associated to several sports modalities.The risk of transmission of the human immunodeficiency virus (HIV) may occur in some contact sports, such as wrestling, The risk of transmission of the hepatitis B virus is 50-fold greater than of the HIV virus. This occurs because the virus is resistant to drying, room temperature, to detergents and to alcohol, in addition to being stable on surfaces, during at least 7 days. Therefore, the transmission of hepatitis B in sports is estimated at about 1/10 thousand to 1/50 thousand games.-8Regarding hepatitis C, there have been reports on former football players that used injections of vitamin complexes.,10 Although most conditions are curable, many lesions increase the risk of HIV infection three-fold and others are precursors of neoplasms – such as human papillomavirus (HPV), Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections .STD are among the five major causes for seeking medical care and the second major cause of not having a healthy lifestyle among women.,12 Young athletes begin contact with the opposite sex early, on competition trips, and the lack of knowledge on transmission of STD makes many of them not practice safe sex. Moreover, this population has a greater consumption of anabolic substances and a higher number of sexual partners.-16Some studies appointed a positive association between being an athlete and having risky sexual behavior. In soccer, over 80% of players declare acknowledging HIV but few (20%) know about other STD. They also have questions on how the virus is transmitted, given many believe in contamination by exchanging shirts and kissing.High performance athletes and, especially those in team sports, have little perception of risks, and also engage in non-programmed sexual activity. It would be a timely moment for detecting these diseases, treating them whenever necessary, and counseling on means of prevention.In recent years, professionals who work with athletes have proposed STD screening at the beginning of every season, using a pre-participation sports assessment (PPSA).To determine the prevalence of sexually transmitted diseases among women athletesEscola Paulista de Medicina da Universidade Federal de São Paulo (UNIFESP), from January to July 2011. The Project was approved by the Ethics Committee of the institution, under number 1,269/08. All participants read and signed the consent form.An observational, cross-sectional study was performed at the Sports Gynecology Sector of the Investigators went to the athletes' training site and explained the objective of the study. Those who agreed to participate answered a questionnaire that emphasized gynecological data . Athletes, family members and coaches were offered talks on STD/AIDS on the training site and, after answering questions, athletes were invited to come to the outpatient clinic, where they would be seen by a gynecologist.Women who practiced performance sports in Olympic modalities of the International Olympic Committee (IOC) were considered athletes. All of them had been federation affiliates for at least three years and had a mean training load of 20 hours/week.At the outpatient clinic, blood samples  were drawn, and cervical and vaginal secretion were collected during colposcopy . A biopsy of the lower genital tract was done whenever necessary.The flocculation method was used (VDRL), along with the titer for syphilis; whenever necessary the result was confirmed by passive hemagglutination methods (HTPA) and EIE (ELISA).HIV was diagnosed by two ELISA tests. If any of the two were positive, immunofluorescence tests were performed. If a question remained a Western blot test was performed.ELISA was used for hepatitis B serology, using the following the algorithm: determination of titers of HBs antigen and total anti-HBc antibodies. If both were negative, the result was considered negative. If the HBsAg was negative and total Anti-HBc positive, an anti-HBs antibody detection test was performed, and the positive result indicated immunity or previous hepatitis B infection.® qualitative immunoenzyme test and MONOLISA™ anti-HCV plus, an indirect immunoenzyme test.Serology for hepatitis C used two techniques: EIA-HepanostikaRegarding cervical and vaginal samples, results with superficial, intermediate squamous cells and/or endocervical cells without abnormalities were considered normal cytology, as were those with squamous metaplasia and/or with inflammatory changes. A diagnosis of low grade lesion, high grade lesion, squamous carcinoma, squamous cell atypia of undetermined significance and glandular atypia of undetermined significance were considered abnormal cytology.Real time polymerase chain reaction (PCR) was used to test for CT, NG and HPV. All athletes with clinical or identifiable laboratory diseases were treated and followed up at the service.The population was analyzed as to age, race and sports modality. The following gynecological data were analyzed: age at menarche, menstrual cycles, contraceptive method, and number of pregnancies and of deliveries. Analysis of variables was descriptive. Minimum and maximum values, medians, means and standard deviations were used for quantitative variables.Fifty athletes with a mean age of 20±3 years were assessed. Of the total, 66% declared themselves as white and 34% as non-white. As to socioeconomic status, 35% of participants had completed College, 11% High School and 4% Elementary School. Monthly family income ranged between R$ 370.00 and R$ 2,100.00.When each athlete was asked on knowledge of STD, 46% declared not having knowledge on such conditions, and five athletes reported a previous HPV infection.The prevalence of STD among athletes analyzed was 48% (24 cases). HPV was the most frequent agent (44%) and, considering mixed infections, there was a case of CT com HPV. No cases of infection by NG, or syphilis, hepatitis B, hepatitis C and HIV were diagnosed .Regarding the type of HPV genotype, that was detected in 23 of 50 women athletes, subtype 16 was the most prevalent (53%), followed by 6-11 (22%) and by 18 (13%) .Although no cases of hepatitis B were detected, 24 (48%) athletes were not immune to hepatitis B virus (negative anti-HBs).Three athletes presented low grade cervical lesions and one athlete had a high grade lesion detected by the cervical-vaginal cytology. The results of the present study alert on the importance of measures to explain and orient about STD in sports, given half of athletes had no knowledge on these conditions.Although muscle-skeletal lesions are strongly associated with exercising, infectious diseases cause significant morbidity among athletes. Moreover, premature initiation of sex life and of non-programmed intercourse contribute to the growth in susceptibility to STD infections and to unwanted pregnancy.,22The sports environment has a distorted image of being healthy and with no risk behaviors. Several authors appointed toward an excessive use of alcohol, tobacco, illicit drugs and unprotected sex among young athletes.,18The general prevalence of STD among athletes was high, and the isolated or associated occurrence with HPV should be underscored. The prevalence of the HPV genotype with high risk for cervical cancer is also worth mentioning. A possible explanation would be the complex interaction between the immune system and exercise. In general, moderate intensity exercise promotes protection against infections, because it drives cell immune response (mainly of type 1 T-helper – Th1 cells). However, high intensity activities generate increase in the concentrations of anti-inflammatory cytokines (type 2 T-helper – Th2), increasing susceptibility to infections.The prevalence of HPV among asymptomatic women seen at public gynecological clinics, using the PCR method, is about 16% – much lower than the values found among athletes.The higher prevalence of oncogenic HPVs among athletes was a concerning finding. Epidemiological studies showed that HPV 16 is responsible for most cervical neoplasms, followed by HPV 18. HPVs 6 and 11 account for 90% of genital warts. Although infection by oncogenic HPV is a necessary condition to develop cervical cancer, it is insufficient. In two years, the infection reverts spontaneously in almost 90% of cases. Moreover, the progression of infection to cervical cancer is very slow and controllable. Thus, cervical cancer screening, using pap smears, should be part of the routine for young athletes, along with other routine tests requested regularly by sports physicians.HPV vaccination is part of the recommendations proposed by the Brazilian Federation of Gynecology and Obstetrics Societies (FEBRASGO) and by the Brazilian Association of Immunizations (SBIm) and should be better promoted among athletes. A timely moment to offer vaccination would be PPSA. In one of the studies with 9 to 12-year-old boys and girls, the prevalence of CT in girls was 6.5% and of gonorrhea 2.0%. In another study, the general prevalence of CT among athletes was 2.7% (3.2% men and 2.2% women). In our study, the prevalence of CT was in agreement with the literature, highlighting that one of the athletes had a co-infection with the HPV virus.The United States and Australia have put in place routine tests for CT and gonorrhea during the PPSA.Chlamydia is its cervical carcinogenesis capacity, which occurs through proteins synthetized by bacteria. These proteins have an anti-apoptotic action during persistent infection, which eases the action of oncoproteins on cells simultaneously infected by high risk HPV (HPV types 16 and 18). Another severe sequela is pelvic inflammatory disease which can lead to ectopic pregnancy and to infertility.CT infections have a high morbidity and also have a preference for youth between 15 and 29 years. Considered as having a silent epidemiology, its diagnosis is critical, given the infection is asymptomatic in almost 80% of cases. One of the problems of persistent infection by  Therefore, the prevention of these infections is still one of the most efficacious and less costly measures that exist.There is a consensus that the major means of transmission of STD among athletes is not while practicing sports, but similar to the general population – by unprotected sex and exchange of contaminated syringes.The major international organizations for athletes have stimulated hepatitis B vaccination, contact sports judges wearing gloves, the interruption of a game when there is a bleeding lesion and the education of coaches, fitness coaches and athletes on STD/AIDS.Athletes should be asked about their knowledge and difficulties regarding sexuality and prevention of STD, which would also mean helping them to deal with their own vulnerability.As the sports world is a strong information media, capable of spreading several topics to the general population, athletes with appropriate knowledge of STD may boost the importance of using preventive measures, such as safe sex and vaccination for some diseases.The prevalence of sexually transmitted diseases in female athletes analyzed was high and the human papillomavirus was the most frequent agent. Results point toward the vulnerability of athletes to sexually transmitted diseases and toward the need for primary (hepatitis B and human papillomavirus vaccination) and secondary  prevention measures in this specific group of women.