Human papillomavirus (HPV) vaccines were designed to prevent cervical cancer in women and their provision remains a major public health need. However, HPV is also a major cause of non-cervical anogenital and oropharyngeal cancers and the potential benefit of vaccination likely extends beyond cervical cancer.A systematic literature search of PubMed (1995–2014) identified publications assessing the incidence, persistence, and clearance of non-cervical anogenital/oral HPV infections. Comparability with cervical HPV was assessed by identifying articles assessing the same or similar populations.Available data suggest high incidence rates of non-cervical HPV infection in men and women, with HPV-16 predominating in all sites. The incidence of high risk HPV per 100 person-years ranged from 11.4 to 72.9 for penile infections, 6.7–47.9 at other male genital sites, and 4.4–36.7 and 5.3–23.4 for anal infections in men and women, respectively. The incidence per 100 person-years of oral infection with any HPV type ranged from 5.7 to 6.7 in men and 6.8–39.6 in women. Within the limitations of the data, there was a general pattern of higher incidence and clearance of non-cervical genital HPV infections, compared to cervical infections. HIV status, circumcision, number of sex partners and partner HPV status significantly influenced high-risk HPV incidence/clearance at male anogenital sites. Few studies assessed risk factors for oral HPV.Parallels appear to exist between the epidemiology of cervical and non-cervical HPV infections in terms of incidence, HPV-type distribution, and risk factors for infection. Available data suggest that non-cervical genital HPV infections may occur more frequently, with higher clearance rates, than cervical infections. More extensive studies could provide useful information for estimating vaccine impact, the wider cost-benefit of HPV vaccination, and guiding vaccination policy.Not applicable, as systematic review of the literature.The online version of this article (doi:10.1186/s12879-016-1633-9) contains supplementary material, which is available to authorized users. Cervical cancer is the fourth most common cancer affecting women worldwide, with an estimated 528,000 new cases and 266,000 related deaths in 2012 , 4. SharAvailable evidence from clinical trials indicates that current HPV vaccines can prevent vulval and vaginal and anal HPV infections, anogenital pre-cancers, and oral HPV infections  in womenThe study objectives were to conduct a systematic review of the literature to describe the incidence, clearance and persistence of non-cervical HPV infections, including comparisons to cervical infections for which data were available for the same/or similar study population. We also examined risk factors for incident and persistent non-cervical HPV infections.A systematic review of the literature in PubMed used three search strings  to identify non-cervical anogenital and oral HPV infections and was limited to studies published in English between 01 January 1995 and 12 July 2014. We used specific searches to obtain articles on cervical HPV infections reporting comparable estimates of incidence, clearance, or persistence in the same or similar populations and using comparable HPV testing methods as in the articles on non-cervical HPV infections. Articles were included if they contained information on incidence, persistence, clearance or duration of non-cervical HPV infections, risk factors for non-cervical HPV infections or risk groups for incidence of non-cervical infections. Articles were excluded if they reported diagnostic test research, if they were case studies, letters to the editor, editorials or comments, literature reviews, or meta-analyses presenting no original data. Articles were also excluded if no information was listed in the inclusion criteria, or if they reported data based on testing of self-collected vaginal samples only. PRISMA guidelines were followed for the report. Independent review in duplicate was undertaken for 30 % of screened titles and abstracts and for 10 % of full text articles. Article selection, data extraction, assessment of risk factors and quality control procedures are provided in Additional file During the search for cervical cohorts, studies were found which used self-collected vaginal samples but in which the samples were referred to as ‘cervical’ swabs. These studies were not identified with the original non-cervical search string that did not include the term ‘cervical’. We did not include studies when vaginal samples had been self-collected, because the original search may have missed studies that assessed self-collected vaginal samples, and because we could not be certain that they represented vaginal-only infections. For similar reasons cervical samples collected using cervical vaginal lavage in D’Souza et al.  were alsFor ease of comparability, incidence, clearance and persistence rates were recalculated, if not provided, per 100 person-years, and all estimates of median (or mean if median not reported) time-to-clearance (or duration of infection) were calculated in terms of months. For example, conversion from 1000 person-months to 100 person-years values was done by multiplying by 12 and dividing by 10. All estimates of median time-to-clearance were calculated in terms of months (dividing days by 30.5). When confidence intervals (CI) were not already provided and there was sufficient data, we used the exact method to calculate 95 % CI for proportions.Ethics approval was not required for this study.The definition of HR-HPV was study specific. The HR-HPV types applicable to individual studies are provided in Additional file Clearance, incidence, persistence, and duration of infection were defined broadly to capture the most studies: For individual HPV types , incidence was defined as at least one positive test for that HPV type following at least one negative test for that type. Clearance of infection was defined as a negative test for the individual HPV type following a positive test for that type.Type-specific incidence of any HPV or HR-HPV was defined as a positive test for at least one HPV type or HR-HPV type not detected at baseline or other previous visit. For non-type-specific incidence, subjects were required to have tested negative for any HPV DNA or any HR-HPV DNA at baseline. We considered this to be non-type-specific incidence even if the HPV type(s) associated with the incident infection was known. In addition, for studies conducting multiple follow-up visits, incidence rates could be calculated by: 1) censoring subjects at the time of the first positive test (first acquired incidence); or 2) considering subjects to be at risk throughout the follow-up period and counting each visit where at least one new type was detected as an event .Type-specific clearance of any HPV or HR-HPV was defined as a negative test for all HPV types or HR-HPV types detected at baseline  or for all newly detected during follow-up (type-specific clearance of incident infection). Non-type-specific clearance was defined as at least one negative test for any HPV DNA or any HR-HPV DNA following a positive test for any HPV DNA or HR-HPV DNA at baseline or during follow-up.Persistence was defined as at least two sequentially positive tests for at least one specific HPV type or HR-HPV type at least three months apart. Definitions for type-specific and non-type specific duration of infection followed those for clearance of infection.For estimates related to any HPV and HR-HPV, calculations were required to be based on the total number of subjects rather than total number of infections as the denominator.We identified 38 articles from 25 unique study cohorts with relevant data on non-cervical anogenital or oral HPV infections Table . A furthMedian follow-up times varied between 6.7 and 84.5 months, with all but 8 studies conducting at least two follow-up visits at intervals ranging between 2 and 12 months. Among the 31 studies reporting data on incidence of any HPV or any HR-HPV, 10 (32 %) used type-specific definitions which counted any newly detected type as an incident infection and 17 (55 %) used non-type-specific definitions that required subjects to be negative for any HPV or any HR-HPV at baseline; an additional 4 studies reported data on incidence of individual HPV types only. A total of 31 studies were known to have multiple follow-up visits. Among the 31, 20 assessed incidence of any HPV or HR-HPV: 13/20 (65 %) estimated incidence based on time to the first acquired infection; 3/20 (15 %) considered each new infection as an event; for 2/20 it was unclear (10 %); 1/20 (5 %) used both methods; and 1/20 (5 %) did not conduct any survival analysis. Among the 29 studies reporting data on HPV clearance, type-specific definitions were used more consistently, with only 5 (17 %) using non-type-specific definitions. Three studies (10 %) required at least two consecutive negative test results to define an infection as having cleared.In studies conducted in Africa, HR-HPV incidence was higher in human immunodeficiency virus positive (HIV + ve) men (42.0–72.9/100 person-years) than HIV negative (HIV-ve) men (19.7–32.9/100 person-years) (Table In three articles that reported age-specific incidence of HPV infection the highest incidence was reported in the youngest age group studied (<30 or 15–24 year olds) , 19, 22.The rate of clearance of incident HR-HPV infection was 123.9/100 person-years  and HIV-ve MSM (range 21.3–46.2/100 person-years), than in HIV + ve MSW (7.9/100 person-years) and MSW . Among MSM, HPV-16 and 18 were among the HPV types with the highest incidence, although depending on the study, the incidences of HPV-51, 52 or 59 were similar or higher –35.In women, anal HPV incidence ranged from 14–56.3/100 person-years. HPV-16 and 52 were the most frequently infecting types . The incA regression analysis conducted in a US study of sexually active women noted a statistically significant inverse relationship between acquisition of a new HR-HPV anal infection and age; with a 57 % (95 % CI 19–77) lower risk of acquisition among older women (≥45 years of age at baseline) than among younger women (<25 years at baseline) .Seven articles reported on the clearance rate of anal infections, of which five were in men who were HIV + ve and/or MSM, and two were in women Table . The cleIn women, the clearance rate of incident anal HPV infections was 89.3/100 person-years, with a median time-to-clearance of 7.8 months  HR-HPV anal infections persisted at 6 months in 51.0 % of MSW and in 24.2 % of MSM who were HR-HPV positive at baseline  Table 33.In Hawaii, anal and cervical samples were collected in sexually active women aged 18–85 years every 4 months for an average of approximately 1.3 years , 39, 41.In two partially overlapping cohorts of sexually active teenager and young adults in the US, clearance by 12 months was also similar for anal versus cervical infections  , 42. In We identified one study of vulvovaginal HPV infections that employed physician-collected swabs . The incEleven articles (considering six cohorts) reported on oral HPV infections: eight in men and five in women Table .Table 6IThe incidence of oral HPV infection was 5.7–6.1/100 person-years in HIV + ve men and 6.7/100 person-years in HIV-ve men , 44. In Two studies reported age-specific incidence rates of oral infections in men. In one study in university students the age-specific incidence was higher in the 21–24 years age-group than in the 18–20 years age-group . In HIV-The incidence of oral HPV infections in HIV-ve women was 6.8–20.4/100 person-years versus 39.6/100 person-years in HIV + ve women , 49. In Clearance rates of oral infections in men were reported in two study cohorts Table . In HIV In US studies, clearance of any oral HPV in women was 60.6 % at 3 months , and clePersistence of oral HPV infections was reported for three cohorts Table . In 662 Penile and/or male genital infections with HR-HPV were significantly associated with younger age in only one study , althougAn increased likelihood of clearance of HR-HPV penile infection was associated with an increased number of sex partners and circumcision (in HIV-ve men) , 31. OneRisk factors for anal HR-HPV infection in women were younger age, condom use , higher lifetime number of partners (≥6), and advanced HIV infection and smoking (in HIV + ve women) , 37. TheNo risk factors were significantly associated  with anal HR-HPV infection in men.Risk factors for persistence of oral HR-HPV were only available from one study: a history of smoking was significantly associated with longer persistence whereas a history of genital warts was associated with shorter persistence .The available data suggest a high incidence of non-cervical HPV infections in men and women, with HPV-16 most consistently ranking among the HR-HPV types with the highest incidence rates and the lowest clearance rates in all sites. Despite relatively few studies providing data for cervical and other genital infections in comparable populations, there appears to be a general pattern of both higher incidence and clearance of non-cervical versus cervical genital infections. Around one-half of the identified articles assessed HPV at male penile/other genital sites. There was a wide variation in HPV incidence reported at these sites, although the time-to-clearance was comparable across all studies, with the exception of substantially longer time-to-clearance in HIV + ve men. HIV status, circumcision, number of sex partners and HPV status of the partner significantly influenced HR-HPV incidence, clearance and persistence rates at male anogenital sites in some, but not all studies. Some of these risk factors are the same as those identified for cervical HPV infection, for which the number of sex partners is the most important one .Unlike cervical intraepithelial neoplasia, anal-intraepithelial neoplasia infrequently undergoes malignant transformation in immunocompetent individuals, but the risk of malignant transformation is much greater in HIV + ve individuals . CorrespThe Hawaiian studies by Goodman et al. showed an incidence of anal HPV infection that was three-times higher than cervical HPV infection , 39, 41,The incidence of oral HPV infection was within the same range in men and women, although the prevalence of oral HPV infection has been reported to be higher in men . Our datThe factors that influence oncogenesis at non-cervical sites are not well understood, and appear to differ by site and between populations  , 58. AltA potential limitation of our study is the non-inclusion of articles in which vaginal swabs were self-collected. The single study that we identified using physician-collected vaginal swabs may not be representative for this site as it was limited to university students. However, it was one of the highest quality studies, following more than 400 women aged 18–20 years old every 4 months for 10 years and prospectively collecting sexual behavior data. The studies on cervical infections were selected from specific non-systematic searches aimed to identify comparable populations with which to compare to non-cervical infections. Thus, the results may not be applicable beyond the study setting. Most studies were not properly designed for risk factor analysis and should be interpreted cautiously.The studies we identified were substantially different in terms of their design, including follow-up times and sampling intervals which ranged from several months to 7 years, as well as the number of types and methods used for HPV genotyping. Although it is difficult to apply a standard grading to these studies, the highest quality studies tended to be those that followed cohorts of at least 200 subjects and conducted multiple follow-up visits ≤6 months apart for at least one year. At least one such study provided data for each of the sampling sites: penile , 24, 53,The available published data are limited by the lack of age-specific estimates, and information on clearance/persistence of infection was frequently based on very small sample sizes, resulting in imprecise estimates. Few studies provided incidence or clearance data on oral or anal HPV infections in women versus men, and the relative burden of these infections in each sex is not clear. Specific estimates of incidence and clearance of HPV-16, which is involved in the majority of non-cervical cancers, and HPV-18 which may also be important in some sites, as well as estimation of clearance rates at 6 and 12 months, could inform on potentially important endpoints for clinical trials of vaccine efficacy. Finally, because just a few countries contributed data to this review and given the important differences in study designs, outcomes and populations, it is important to note the limited generalizability of these results to other populations.Cases of cervical cancer are projected to increase, emphasising the importance of ongoing efforts to provide access of effective HPV vaccines to all , 60. As


Prevalence was reduced, and early evidence indicates herd immunity. In 2008, a national human papillomavirus (HPV) immunization program using a bivalent vaccine against HPV types 16 and 18 was implemented in Scotland along with a national surveillance program designed to determine the longitudinal effects of vaccination on HPV infection at the population level. Each year during 2009–2013, the surveillance program conducted HPV testing on a proportion of liquid-based cytology samples from women undergoing their first cervical screening test for precancerous cervical disease. By linking vaccination, cervical screening, and HPV testing data, over the study period we found a decline in HPV types 16 and 18, significant decreases in HPV types 31, 33, and 45 (suggesting cross-protection), and a nonsignificant increase in HPV 51. In addition, among nonvaccinated women, HPV types 16 and 18 infections were significantly lower in 2013 than in 2009. Our results preliminarily indicate herd immunity and sustained effectiveness of the bivalent vaccine on virologic outcomes at the population level. Human papillomavirus (HPV) vaccination programs are established in several countries worldwide  in Scotland through a unique personal identifier, the Community Health Index (CHI), enables us to comprehensively assess the effects of vaccination, including the extent of potential herd immunity. By including samples from women undergoing their first cervical smear testing in 2013, we further assessed the effects of HPV vaccination among young women in Scotland by comparing prevalence of HPV 16 and 18; HPV 31, 33, and 45; and other high-risk HPV types among women who were fully vaccinated as part of the catch-up cohort with prevalence among nonvaccinated women in the same birth cohorts. Additionally, we investigated whether the prevalence of any nonvaccine HPV types was greater among vaccinated women. Using these updated data, we determined whether high uptake of the vaccine protects nonvaccinated women by assessing the trend, over birth cohort, for the proportion of nonvaccinated women with positive results for each HPV outcome.The Scottish Cervical Screening Programme is an organized, national, call–recall program that invites women 20–60 years of age to visit their general practitioner for a cervical smear test . A detailed account of the testing procedures has been described elsewhere  . OverallWe observed a statistically significant decrease in HPV 16 and 18 among vaccinated compared with nonvaccinated women (p<0.0001) . PositivUnadjusted analysis showed a significant linear trend for birth cohort and number of doses a woman received; prevalence of HPV 16 and 18 was lower among women from later birth cohorts and women who had received more doses (both p<0.0001) . The linPrevalence of HPV types 31, 33, and 45 decreased among vaccinated compared with nonvaccinated women  . The posAccording to unadjusted analyses, the odds of being infected with cross-protective types decreased significantly according to birth cohort year (p = 0.0001), but adjusted analyses showed no such significant effect p = 0.2413) because of confounding of the effect with vaccination status , 3. A st because The overall prevalence of nonvaccine, non–cross-protective high-risk HPV types  significantly increased, from 29.1% (95% CI 26.9%–31.3%) in 2009 to 33.9% (95% CI 31.0%-36.8%) in 2013 (p = 0.0128) . PrevaleOdds of nonvaccine or cross-protective high-risk HPV type infection were significantly higher for women in later birth cohorts than for those in earlier birth cohorts (p = 0.0147) . AccordiPrevalence of all 24 HPV types detected by the assay remained unchanged from 2009 to 2012 (58.1% [95% CI 55.7%–60.4%] in 2009 and 58.4% [95% CI 55.3%–61.4%] in 2012) but decreased to 53.8% (95% CI 50.8%–56.9%) in 2013  (5). OveAccording to unadjusted analyses, overall HPV positivity showed a significant linear trend by birth cohort year; HPV infection was more likely among women in later birth cohorts than among than those born in 1988 (p = 0.03171). According to adjusted analyses, however, this trend was not significant p = 0.115) , 5. HPV  , 5. HPVPrevalence of HPV 16 and 18 among nonvaccinated women remained relatively stable at ≈30% during 2009–2012 but decreased to 21.2% in 2013 . During Decline of HPV prevalence in Scotland has been reported (We observed a reduction in the prevalence of HPV types 16 and 18 among nonvaccinated women undergoing their first cervical smear testing in 2013. This finding is encouraging evidence of herd immunity and corroborates the findings of 2 studies from Australia: a sexual health clinic–based national surveillance study that observed a decline in genital warts among heterosexual men and a cross-sectional study that showed a decrease in HPV prevalence among nonvaccinated women (It has been postulated that the reduction of infection with HPV types 16 and 18 and cross-protective types could leave a vacant niche, leading to increased infections with less oncogenic, nonvaccine HPV types among vaccinated women (The main strength of this study is that vaccination and screening data can be linked at the individual level. Coupled with the high levels of vaccine uptake and age of screening in Scotland, this linkage enables us to directly evaluate the effects of HPV vaccination on HPV prevalence. Nevertheless, our study has some limitations. Data from a screened population are not representative of the whole population and possibly underestimate the true prevalence of HPV infection. However, the prevalence of HPV among 20-year-old women who did not undergo a cervical screening test but were invited to send a self-collected urine sample or vaginal swab sample did not differ significantly from prevalence among those who had undergone cervical screening testing in a previous study (Our data preliminarily suggest the presence of herd immunity in the nonvaccinated female population of Scotland. However, we could not assess whether herd immunity is conferring protection to the male population, who are not routinely sampled as part of the surveillance program. We plan to use genital wart consultation data from men to act as a proxy for detecting herd immunity in the male population (


This study was designed to determine the Human papillomavirus (HPV) prevalence and its distribution of genotypes in various regions of Yunnan Province, China.In this study, participants were recruited during routine gynecologic examination between Oct 2013 and Feb 2015. A total of 17,898 women were recruited. Polymerase chain reaction was used for detecting the HPV positive samples and HPV geno-array test was used for genotyping.P = 0.001) than that among the north-western (18.0 %), south-eastern (13.3 %), north-eastern (11.1 %) and central women (12.9 %). The high-risk (HR)  and single genotype  infection rates among the South-western women were also significantly higher than those of among the north-western , south-eastern , north-eastern  and central women , respectively. While, the infections with multiple HPV (4.2 %) genotypes were significantly more common (P = 0.001) among women in north-western Yunnan than women in the south-western , south-eastern , north-eastern  and central Yunnan . A total of 30 HPV genotypes were detected; among them 13 were HR-HPV, 3 were PHR-HPV , 8 were LR-HPV (Low risk) and six were unclassified. The most common HPV genotypes were HPV-52, 16, 58, 53 in control group, HPV-16, 52, 58, 39 and 53 in CINI , HPV-52, 16, 58, 33, 53 and 81 in CINII, HPV16, 58, 18, 52, 81 in CINIII and HPV-16 18, 58, 52 in cervical cancer (CC), respectively. Such variation has also been observed about distribution of HPV genotypes distribution among single and multiple infections.The overall HPV infection rate (19.9 %) among the south-western women was significantly higher (This study gives an epidemiological estimate of HPV prevalence and different genotype distribution in various region of Yunnan province and further explains its prevalence in different neoplastic lesions. Overall HPV-16, 52, 58, and 18 are the leading HR-HPV genotypes. Human papillomavirus (HPV) infections are the most frequently sexually transmitted infections throughout the world. Worldwide, the prevalence of HPV is approximately 11–12 % –5.Papillomaviruses are non-enveloped, circular, double-stranded DNA viruses with a genome length of 8-kb. More than 200 HPV genotypes have been reported, almost 40 of which are frequently transmitted through intercourse and remain in the genital system. Genital HPV infects most sexually active females at some point during their lives and plays a vital role in the development of cervical cancer , 7. HPV The prevalence and genotype distributions of HPV are well documented in developed areas of China. The genotypes HPV-16, 18, 52, 58 and 59 are most commonly detected in Chinese women –14. HoweYunnan Province has a unique geographic location, highly complex topography, and large variations in elevation, and it has a diverse climate because of these distinctive features . Yunnan n = 7065) north-west region (n = 3772), north-east region (n = 3152), south-west region (n = 2164) and south-east region (n = 1745) were recruited. The study designed and method has been described previously [In this study, participants were recruited during routine gynecologic examination between Oct 2013 and Feb 2015. A Women who met the following criteria were recruited: those who (1) was mentally and physically normal, (2) was aged between above 18 years, (3) was a permanent resident of local region; (3) was not a virgin (4) had not undergone a total hysterectomy; (5) had no history of cervical surgery; (6) had never had pelvic radiation therapy. Initially 18,562 participants were recruited for this study. Sixty-three women excluded with due to invalid test result lesions and 45 were not willing for biopsy while 11 were excluded due to HPV-genotype lesions. A total of 17,898 women from the central region (The protocol used in this study was in accordance with the Declaration of Helsinki and was approved by the Ethics Committee at Kunming University of Science and Technology and the Center for Disease Control and Prevention (CDC) in Yunnan Province, China. Written consent was individually obtained from each participant.The women were interviewed at the hospital by trained interviewers using a standardized questionnaire to elicit information on socio-demographic risk factors. After the interview, all women underwent a pelvic examination performed by a senior gynecologist; two cervical scrapings were collected for cytological analysis and detection of HPV DNA. Both conventional smears and liquid-based cytology were performed.All cytological slides were prepared by two qualified technicians individually. First Cytological slides were read locally by a junior cytopathologist from each geographical site according to the Bethesda classification system. All Pap smears with a diagnosis of atypical squamous cells of undetermined significance (ASC-US) or greater and a 10 % random sample of normal Pap smears were sent to the Research Center for Molecular Medicine, Kunming Yunnan for double cytological review by an expert senior cytopathologist who was blinded to the original diagnosis. Additionally, the slides read by the junior cytopathologist were used only for screening. The final diagnosis was the diagnosis of senior cytopathologist.During colposcopy, the cervix was divided into four parts and each part was observed individually. All visually abnormal areas were biopsied. Every part with normal colposcopic impressions had one random biopsy collected at the squamo-columnar junction. Endocervical curettage was then performed. All cervical biopsies were performed with standard 2 mm POI biopsy forceps, which allows rapid healing of the biopsy sites and minimizes patient distress. Histological slides were reviewed by two senior pathologists from Yunnan First Peoples Hospital.(CIN I–III) [The cervical intraepithelial neoplasia grading and cervical cancer was diagnosed according to the World Health Organization classification system N I–III) .Samples that tested positive for β-globin were analysed by PCR amplification of HPV DNA. HPV-positive samples were confirmed by PCR with universal L1 primer MY09/11 and GP5/6 systems . DNA frohttp://blast.ncbi.nlm.nih.gov/Blast.cgi). The sample was identified as a particular HPV genotype if the sequence was 95 % homologous with the reference standard.All the samples that were identified as positive through PCR were genotype with two methods. First we run Geno-Array test and secondly we performed direct sequencing. For the DNA sequencing, new PCR was run with reaction mixture 50 μL containing 6 μL of the DNA extract, 1 μL of 10 μmolar MY09 primer, 1 μL of 10 μmolar MY11 primer and 25 μL of the provided master mixture, and 15 μL dH2O. The consensus primer products were separated by electrophoresis on a 2 % agarose gel and purified with Tiangel PCR purification kit. The DNA was then directly sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction  on an ABI 310 DNA analyzer. The nucleotide sequences were aligned and compared with those of known HPV genotypes available through Genbank by using the BLAST 2.0 software server . All statistical tests were two-sided; All of the participants were recruited from various regions of Yunnan province, China Table .Table 1TP = 0.001) among women in north-western than women in the south-western , south-eastern , north-eastern  and central women . The HPV genotype distribution data by region is shown in Table P = 0.001, P = 0.001) more frequent in the southwest region compared with those in the central, north-east, south-east, and north-west regions. With the exception of the above-mentioned genotypes, no significance differences in the distributions of any of the other HPV genotypes were observed among the five regions of Yunnan province  among the south-western women was significantly higher than that among the north-western (18.0 %), south-eastern (13.3 %), north-eastern (11.1 %) and central women 12.9 %). The high-risk (HR) (18.1 %) and single genotype (16.7 %) infection rates among the South-western women were also significantly higher than those of among the north-western , south-eastern , north-eastern  and central women , respectively. While, the infections with other HPV 4.1 %) and with multiple HPV (4.2 %) genotypes were significantly more common  were also detected, and they were highly prevalent in the CIN1 and CIN2 samples. Only three CC samples were found to have a co-infection with the HPV-66 and HR-HPV genotypes. Interestingly, eight single and multiple LR-HPV infections were detected in both the normal and abnormal cytology groups. Some of the unclassified HPV genotypes, such as HPV-67, 69, 70, 71, 82 and 83, were also found in the control group. Table P = 0.001) in the abnormal histo-pathological group  compared with that in the normal group . The proportion of uncharacterised infection was also higher in the abnormal histo-pathological group  compared with that in the normal group . The distribution of HPV genotypes according to the type of infection is shown in Table P = 0.001), 18 , 33 , and 58  were detected significantly more frequently in the abnormal histo-pathological group compared with the control group. In addition, HPV-31 , 51 , 61 , 66 , and 68  were significantly more common in the normal group compared with the abnormal histo-pathological group  was the most frequently detected genotype in SCC, followed by HPV-58 (18.2 %), 18 (18.2 %), and 52 15.9 %). Interestingly, 8 SCC patients were also found to have a multiple genotype infection . These findings are in complete agreement with previously reported data, indicating that HPV-16, 18, 52, and 58 are most frequently present in cervical cancer  %. Inter. HPV-16,In this study, we also evaluated the HPV genotype distributions among different age groups. HPV-16 was more frequent in women less than 35 years old in both the normal and abnormal cytology groups. However, the distributions of the other genotypes varied among the different age groups with lesions of differing cytology grades. HPV-52 and 18 showed double-peak prevalence in the normal cytology group. The prevalence of HPV-52 in the abnormal cytology group decreased with increasing age; however, that of HPV-18 increased with increasing age in this group. Such variation has also been observed for other genotypes, as shown in Table The results of this study suggest that cervical screening is vital for women, particularly minority ethnic women, to prevent HPV-related cervical cancer . An impoThere are some limitations of this study that must be considered when interpreting our findings. Yunnan includes 26 state-certified ethnic minorities who live in different regions of the province. Most of them do not want to participate in epidemiological studies due to their ethnic beliefs. In this study, we focused only on those women who visited a public hospital with routine gynaecological examination.This study provides epidemiological estimates of HPV genotype distributions in different regions of the province and further reports the prevalence of these genotypes in neoplastic lesions with differing cytological grades. We have found that overall, HPV-16, 52, 58, and 18 are the most common HR-HPV genotypes. However, the prevalence rates of these genotypes significantly differ in different regions of the province. PHR-HPV-53 is also a predominant genotype in some regions. These variations could be due to the large number of ethnic populations residing in the province. A vaccine currently under trial has been formulated for HPV-16, 18, 11, and 6, and it will be introduced nationally in the coming years. However, HPV-52 and 58 are the predominant genotypes in several regions. Thus, based on our observations, a new tetravalent vaccine may be more effective than a bivalent vaccine in China, and particularly in Yunnan province. Further, due to large-scale geographic variation in HPV genotype distribution, a future large-scale, multi-ethnic population-based study must be conducted to obtain comprehensive information on the prevalence and genotype distribution of HPV in various Chinese ethnic populations.HPV, human papillomavirus; HR-HPV, high risk-HPV; PHR-HPV, potential high risk-HPV; LR-HPV, low risk-HPV; CIN, cervical intraepithelial neoplasia; CC, cervical cancer.


Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) −1 and −2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks.Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen:  It is estimated that over 340 million new cases of sexually transmitted diseases (STDs) occur annually throughout the world, with the highest incidence in developing countries. Pelvic inflammatory disease, infertility, ectopic pregnancy, chronic pelvic pain, neonatal morbidity and mortality, and genital cancer all have been associated to some degree to STDs Since 1993, the World Health Organization (WHO) has established a role for genital tract infections in human infertility Semen STDs are prevalent in asymptomatic males over extended periods of time Over the past three decades, diagnostics for STDs have depended nearly exclusively on traditional methods, such as culture, enzyme immunoassay, and fluorescent antibody staining, especially in developing word Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, herpes virus (HSV) types 1 and 2, and Treponema pallidum in semen. Human Papillomavirus (HPV) and genotypes also were detected by single PCR (sPCR) in the same semen samples. We anticipate that M-PCR will potentially impact diagnostics of STD in semen and contribute to diminish male infertility in the near future.Here we report a validated multiplex polymerase chain reaction (M-PCR) diagnostic method to simultaneous screen for The study population was selected in the Sperm Analysis Section (SAS) of Clinical and Research Laboratory of State University of Maringá (UEM)/Brazil between January 2012 and June 2013. Semen analysis was requested as part of a work-up for conjugal infertility investigations after failing to conceive after one year of unprotected intercourse or as a post-vasectomy sterility control. Men who had reproductive system abnormalities , symptoms of genitourinary infections, or those that had received antibiotics within the preceding 3 months or infertility therapy within the preceding year were excluded from the study. Considering this exclusion criteria, the semen samples analyzed between January 2011 and June 2012 (prior to the study period) by SAS/UEM, the sample size calculated for our study was fixed at least 72 semen samples .A total of 76 men were included in the present study with a mean age of 33.4 ± 7.2 years (range 19–51). The men signed a consent form, and this study was approved by the Committee for Ethics in Research Involving Humans at the State University of Maringá (UEM)/Paraná, Brazil (No. 162.209/2012).6/ml, asthenospermia as sperm motility <50% (category [a + b]), necrospermia as sperm vitality <50% and teratospermia as normal morphology <30%.Prior to semen collection and analysis, the men were asked to abstain from sexual intercourse or masturbation for 3–5 days. All samples for analysis were collected on site and into standard containers that had previously been shown not to have any cytotoxic effects on human spermatozoa DNA was extracted using an AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit  according to the manufacturer's instructions. The quality and quantity of purified DNA were measured by spectrophotometry .2, 25 mM of each primer, 5 µL of extracted DNA  and 1 U of Platinum Taq DNA polymerase . The PCR conditions comprised thirty-five amplification cycles of denaturation for 10 minutes at 94°C, annealing for 1 minute at 62°C, extension for 1 minute at 72°C and final extension for 10 minutes at 72°C . The M-PCR products were electrophoresed on a 2.5% agarose gel stained with 1 µg/mL ethidium bromide. In a few cases in which the bands have not been viewed in the 2.5% agarose gel, we analyzed products using an 8% polyacrylamide gel. Positive controls for all studied STDs were derived from positive clinical samples detected by reference methods, including culture and/or single-target PCR (sPCR). For validation, sPCR was also performed for the seven microorganisms in all samples studied and for positive controls using the same primers as for the M-PCR. sPCR (gold standard) is generally more sensitive than M-PCR, and cross-reactivity, which can occur during M-PCR, is avoided We made selected adaptations to previously designed M-PCR assays to achieve simultaneous detection of the seven selected STDs All clinical samples were also tested using human β-globin-specific primers GH20/PC04 as an internal control for amplification and DNA integrity under the same conditions as the M-PCR or sPCR reactions.2, 25 mM of each primer and 50 ng of extracted DNA for a final volume of 15 µL. PCR products were electrophoresed on a 1.0% agarose gel, stained with 1 µg/mL ethidium bromide, and photodocumented under UV light. The samples that gave a positive result by PCR were further analyzed by HPV genotyping. Co-amplification of the human β-globin gene was performed as an internal control using primers GH20 (5′-GAAGAGCCAAGGACAGGTAC-3′) and PC04 (5′-CAACTTCATCCACGTTCACC-3′) under the same conditions as the HPV-PCR. Two types of controls were also included in each reaction series: ‘no-DNA’ (negative control) and ‘HPV-positive DNA’ (positive control).This method has been in use in our laboratory for several years and consists of HPV-PCR amplification carried out using primers MY09 (5'CGTCCMAARGGAWACTGATC-3′) and MY11 (5′-GCMCAGGGWCATAAYAATGG-3′) as described previously HpyCH4 V  according to the manufacturer's instructions. Restriction fragments were resolved on 8% polyacrylamide gels. HPV genotypes were determined by analyzing each band with Labimage 1D software , and comparison of the molecular weights determined the genotypes following carcinogenic potential: HR-HPV (high-risk-HPV), UR-HPV (undetermined-risk-HPV) and LR-HPV (low-risk-HPV). The following types were determined for this genotyping method: HR , undetermined risk (UR- 26) and LR  .HPV-positive samples were genotyped using a PCR-RFLP (Restriction Fragment Length Polymorphism) as described previously p value <0.05 was considered statistically significant.Statistical analysis was performed using EPI INFO 7.0 software . All the variables were expressed as absolute and relative frequencies. The Chi-square test and a crude odds ratio (OR) with 95% confidence interval (CI) were calculated. A We evaluated 52 semen samples from men subjected to semen analysis due conjugal infertility (68.4%) and 24 due post-vasectomy control (31.6%). Overall, the men had a mean of 1.2 ± 1.9 children (range 0–5), and this value was 0.5 ± 0.9 (range 0–4) for those experiencing conjugal infertility.C. trachomatis, M. genitalium, N. gonorrhoeae, T. pallidum, T. vaginalis and HSV-2, the M-PCR showed values of 100% for all parameters. For HSV-1, the M-PCR showed sensitivity and negative predictive values of 100%, specificity of 98.2%, positive predictive value of 75.0%, and accuracy of 98.3%  or in co-infections. Final results were regarded as true positives if the sPCR was positive (gold standard). The overall agreement of M-PCR results with sPCR was 99.2%, and the validation parameters were as follows: sensitivity and negative predictive value 100%, specificity 99.7%, positive predictive value 96.4%, and accuracy 99.8%. When individually analysed the agents of 98.3% . By M-PCn = 29), representing 38.1% of the total samples and 69.0% of the semen positive for STDs. The second most prevalent was T. vaginalis , followed by C. trachomatis , HSV-1 and T. pallidum , M. genitalium and N. gonorrhoeae  and HSV-2  . The most prevalent STD detected was HPV (ith STD) . Co-infeith STD) . Three sn = 20/52; 38.5%), followed by T. vaginalis , C. trachomatis and HSV-1/-2 . In the semen analyzed due to post-vasectomy, the most prevalent STD was also HPV , followed by T. vaginalis  (C. trachomatis or HSV (types 1 and 2) semen infections had a 2.5-fold greater risk of presenting conjugal infertility , although the association between these STDs and conjugal infertility was not statistically significant (p = 0.4 for both).In the semen of men with conjugal infertility, the most prevalent STD was HPV  . Men witn = 29), 17 (58.6%) were infected by 2 or more genotypes simultaneously representing 22.5% of the total samples and 40.5% of the semen with STDs. n = 13/29; 44.8%) and was found in 17.1% of the total samples and 30.9% of the semen with STDs. HPV-82 and HPV-43 (HR) were the second most prevalent , followed by HPV-72 , −58 ; −54 (LR) and −66 (HR) . Other HPV genotypes detected in single or simultaneous infections were HPV-13,-18,-31,-44,-51,-52,-53,-59,-62,-69 and -81 . HpyCH4V) for the initial RFLP and then used a second enzyme ((NlaIII) to confirm the identification. We didn't find any of these genotypes in our analysis and the patter found by the single enzyme was very different and yielded us to well distinguish between all of them, using the 8% polyacrylamide gel as showed in Among HPV positive semen ; semen with simultaneous T. vaginalis and HPV infections had a 10-fold greater risk of presenting teratospermia  (p = 0.03); a tendency toward a greater risk of semen alterations, although not statistically significant, between T. vaginalis and necrospermia  (p = 0.2), simultaneous T. vaginalis and HPV with oligospermia  (p = 0.3) and simultaneous HSV-1/-2 and HPV with teratospermia  (p = 0.2).In regarding to semen quality parameters, the following observations were made: a decrease of seminal volume in samples with 2 or 3 simultaneous STDs or To our knowledge, this is the first study to detect simultaneous bacterial and viral STD pathogens in semen using M-PCR in Brazil and Latin America. The method implemented, which represented an important advance over conventional techniques, allowed the detection of seven STDs in semen, many of which are difficult to identify using standard methods. The overall agreement of the M-PCR with sPCR was elevated (99.2%), and other validation parameters, including sensitivity, specificity, positive and negative predictive value and accuracy, were also excellent (ranging from 99.2% to 100%). Considering the agents individually, the M-PCR also showed excellent values for all the parameters and detected 5 semen samples (6.6%) with co-infections.The M-PCR assay simplifies workflow, allowing for its use in routine diagnostic laboratories with basic molecular facilities n = 29; 38.1% of total semen and 69.0% of semen with STDs). Surprisingly, in 58.6% of semen samples 2 or more genotypes were detected simultaneously  and HR-HPV represented 82.7% of all the HPV genotypes detected. The most prevalent HPV genotype was HPV-16 , followed by HPV-82 and HPV-43, both of which are HR-HPV . The current vaccine protects against HR HPV-16 and -18, but in our study HPV-18 genotype is not the second most frequent HR but HPV-82 and -43, which could have implications for the effectiveness of the vaccine Brazilian men.HPV was the most prevalent STD pathogen in semen , but the population selected for this research included only asymptomatic sexually active young men T. vaginalis . Knowledge of T. vaginalis in men, including in semen, is limited mainly due to difficulties associated with diagnosis including poor sensitivity of available methods T. vaginalis despite the fact that the majority were asymptomatic The second most prevalent STD was C. trachomatis was the third most common STD . The number of men infected by this pathoges was similar to another study that showed that although up to 13.3% of young men carry genital C. trachomatis infection, only half of these will present with any symptom and even fewer are likely to pursue treatment C. trachomatis infection in either partner could potentially contribute to unexplained infertility C. trachomatis infections of male sexual glands may cause severe complications that can threaten male fertility C. trachomatis infection observed in men C. trachomatisT. pallidum , M. genitalium and N. gonorrhoeae  and HSV-2 (n = 2/76). No previous studies were found assessing the prevalence of T. pallidum in semen. Although a direct toxic effect of syphilis on male fertility has not been reported in the literature, it is known that complications of syphilis can affect fertility. Syphilitic epididymitis can cause obstruction of the epididymis. Chronic obliterative endarteritis and interstitial inflammation can occur in congenital or tertiary syphilis and lead to small, fibrotic testes Other STD pathogens were detected in lower abundances as follows: HSV-1 and M. genitaliumAmong men presenting persistent or recurrent urethritis, 19% to 41% are infected with N. gonorrhoeae infection in men can lead to genitourinary tract inflammation , obstruction and infertility N. gonorrhoeae is mostly symptomatic in males, there have been very few studies regarding the relationship between this infection and male infertility. Surprisingly, we detected HSV-2 in semen less frequently than HSV-1. Most commonly, HSV-1 causes oral and genital sores, but HSV-2 is the most common cause of genital herpes. Generally, direct or indirect contact with herpetic lesions is infectious, but HSV-1 and HSV-2 have been detected in semen Other viruses can be highly frequent in semen such as cytomegalovirus (CMV) T. pallidum alone; semen with simultaneous T. vaginalis and HPV infections with a 10-fold greater risk of teratospermia. Correlations were also observed in: T. vaginalis with necrospermia; T. vaginalis and HPV co-infections with oligospermia; HSV-1/-2 and HPV co-infections with teratospermia. These associations confirmed the involvement of STD pathogens with male infertility as described in few previous studies Despite being a secondary goal of our study some associations were observed between STDs and changes in semen parameters such as: decreased seminal volume in samples with 2 or 3 simultaneous STDs or


Usual vulvar intraepithelial neoplasia (uVIN) is the most common VIN type, generally related to a human papillomavirus (HPV) infection, predominantly type 16. The incidence of uVIN has been increasing over the last decades, and a bimodal peak is observed at the age of 40–44 and over 55 years.Almost 40% of patients with uVIN have a past, concomitant or future HPV-associated lesion of the lower genital tract. HPV-related malignancies are associated with a persistent HPV infection. The host immune response is of crucial importance in determining clearance or persistence of both HPV infections and HPV-related VIN. About 60% of the patients present with symptoms. Clinical features of uVIN vary in site, number, size, shape, colour, and thickness of lesions. Multicentric disease is often present.ink4a and p14arf, but negative to p53.Most uVIN lesions are positive at immunohistochemistry to p16Irrespective of surgical treatment used, uVIN recurrence rates are high. Positive margins do not predict the development of invasive disease and the need to re-excide the tissue around the scare remains to be demonstrated. Therefore, considering the low progression rate of uVIN and psycosexual sequelae, treatments should be as conservative as possible.Medical treatments available are mainly based on immunotherapy to induce normalisation of immune cell count in uVIN. None are approved by the food and drug administration (FDA) for the treatment of uVIN. If medical treatment is performed, adequate biopsies are required to reduce the risk of unrecognised invasive disease. Some studies suggest that failure to respond to immunotherapy might be related to a local immunosuppressive microenvironment, but knowledge of the uVIN microenvironment is limited. Moreover, our knowledge of the potential mechanisms involved in the escape of HPV-induced lesions from the immune system has many gaps.HPV vaccines have been demonstrated to be effective in preventing uVIN, with 94.9% efficacy in the HPV-naive population, while studies on therapeutic vaccines are limited. The low incidence of VIN requires large multicentre studies to determine the best way to manage affected patients and to investigate the immunological characteristics of the ‘vulvar microenviroment’ which leads to the persistence of HPV. Until then, these lesions were identified with different terms such as erythroplasia of Queyrat, Bowen’s disease, and hyperplastic dystrophy with atypia. The VIN classification was adopted in 1986 by the International Society for the Study of Vulvar Disease (ISSVD) including intraepithelial neoplasia of the squamous epithelium (divided into three grades based on the thickness of the epithelium affected by cellular atypia) and non-squamous VIN (Paget’s disease and vulvar melanoma in situ) quinolin-4-amine , is a non-nucleoside heterocyclic amine that belongs to immune-response modifiers . In vivoleukin-6 –44. In aleukin-6 .Imiquimod 5% is licensed for the treatment of external genital warts, actinic keratosis, and basal cell carcinoma. Imiquimod 5% acts through a toll-like receptor (TLR7) inducing T cells activation and proinflammatory cytokines release , 46. A tThe largest prospective, randomised, double-blind, placebo-controlled study analysed 52 cases, of which 35% had a complete response and 46% a partial response . This stImiquimod-induced clearance of HPV was associated with the normalisation of immune cell count in uVIN . Many diImiquimod 5% has also been tested after surgery in comparison to surgery alone. The results in 80 patients demonstrated that during 5 years of follow-up, the overall complete response, the recurrence rate, and the disease-free time were similar in the two treatment options .Therapeutic vaccines are designed to reinforce HPV-specific CD4+ and CD8+ T-cell responses in uVIN lesions . Most vaTwo studies investigated the effectiveness of a combination of TA-HPV, a vaccinia virus encoding HPV16/18 E6/E7, and TA-CIN, an HPV 16 L2 E6/E7 fusion protein, in a total of 39 women with HPV 16 high-grade VIN. A reduction in lesion size was seen in 9 patients, while 25 remained stable and 5 progressed. This regimen proved to be immunogenic but no relation between induction of HPV 16-specific immunity and clinical outcome was observed , 54.et al tested a synthetic long peptide (SLP) vaccine in 20 patients with HPV16-positive high-grade VIN. At 12 months of follow-up, 15 patients showed clinical response with a complete response in 9 cases. The complete response was maintained at 24 months of follow-up. In this trial, vaccine-induced T-cell responses were seen in all patients and complete responders had a significantly stronger response of IFNγ-associated proliferative CD4+ T cells and a broad response of CD8+ IFNγ T cells [Kenter  T cells .Small trials showed results with therapeutic vaccines; however, the costs for the development of vaccines are high, and at the moment, the focus is on preventive vaccines rather than therapies.Photodynamic therapy (PDT) uses a tumour-localising photosensitiser, 5-aminolevulinic acid, in combination with non-thermal light to generate tumour-directed cell death and induce local inflammation, which activates antigen-presenting cells (APCs) and induces effector T cells. Several non-randomised and uncontrolled studies were conducted on uVIN with response rates varying from 0% to 71%, with unifocal lesions responding better than multifocal, pigmented, and high-grade lesions –60. RecuIt has long been known that the adaptive immune response, in particular T cells, confer protection against HPV-induced diseases , 62. Oveet al found that initial not-responders to imiquimod seemed to be relatively refractory, probably because of their unfavourable local immune environment, in particular, the increased proportions of T regulatory cells (Tregs) [Winters  (Tregs) . Moreove (Tregs) , 66, 67. (Tregs) .Spontaneous regression and clearance of HPV is associated with systemic HPV-specific CD4+ and CD8+ T-cell responses and normalisation of immune cell infiltration , 68. Theet al showed an alteration in human leukocyte antigen (HLA) molecules not only in HPV-induced cancers but even in the premalignant stage. They detected a partial downregulation of HLA-class I in more than 70% of uVIN lesions and in 80% of HPV-related IVC. However, the downregulation of HLA class I expression in the majority of uVIN was not associated with the loss of heterozigosity (LOH) in contrast with the findings in IVC. This downregulation in uVIN was considered ‘soft wired’ since the expression can be restored applying some changes to the microenvironment, like stimulating with IFNγ. All patients with uVIN evaluated that showed a good clinical response to HPV 16 SLP vaccine had this partial downregulation in uVIN. In fact, the vaccine-induced HPV 16-specific type helper T cells infiltrating the lesion can provide IFNγ in the microenvironment [Moreover, van Esch ironment .Immunotherapy may strengthen the pre-existent activation of the local immune system, and immunologically active primary lesions may be more likely to respond to immunotherapy, also having a lower number of regulatory T cells subsets. Therefore, less immunologically active uVIN lesions, recurrent lesions and IVC may represent the harder challenge for immunotherapies .An increased comprehension of the immunological environment that can be found in uVIN might help to better understand the conditions that favour the persistence of HPV and the development of uVIN and its potential oncogenic transformation to IVC. It might also help to define immunological markers that can predict responsiveness to immunotherapy.HPV prophylactic vaccination has demonstrated to be effective in preventing uVIN. The vaccine efficacy against HPV 16- and/or HPV 18-related VIN is highest in the HPV-naive population, with a 94.9% efficacy . AlthougHPV vaccines have also demonstrated a partial cross-protective efficacy against non-vaccine HPV types, such as HPV 31, 33, and HPV 45 –75. ThisMoreover, studies on previous HPV vaccination in women treated for HPV-related diseases of the cervix, vulva, or vagina showed a reduction in subsequent cervical, vulvar, and vaginal intraepithelial neoplasia and genital warts .An estabilished risk factor for vulvar cancer is smoking. Therefore, smoking should be discouraged .VIN is observed by a wide variety of medical specialists who should be aware of the crucial importance of careful vulvar examination, the malignant potential of the lesion, and the possibilities of conservative treatment depending on VIN localisation, size, focality, associated diseases, age, and psychological conditions of the patient. Long-term surveillance in all treated women is essential as removal of the lesion does not represent a complete prevention of invasive cancer.The low incidence of VIN requires large multicentre studies to determine the best way to manage affected patients and to investigate the immunological characteristics of the ‘vulvar microenvironment’ that leads to the persistence of HPV.The authors have no conflict of interest


Identification of different HPV subtypes in unidentified communities provides sufficient information for screening and monitoring potential impact of a vaccination program. Therefore, the aim of this study was to screen for the presence of HPVs subtypes 31,33,35,39 and 45 among Yemeni women with Cervical Cancer.A total of 200  tissue samples were obtained from Yemeni women with cervical cancer, were investigated for the presence of HPV subtypes 31,33,35,39 and 45 by Polymerase Chain Reaction (PCR).Of the 150 cervical cancer tissue specimens, HPV 31, HPV 33, HPV35, HPV 39 and HPV45 were identified in 10/150 (6.7 %), 6/150 (4 %), 6/150 (4 %), 5/150 (3.3 %) and 10/150 (6.7 %), respectively. The frequency of these HPV subtypes among Yemeni women with cervical cancer was 24 %. Conclusion: HPV 31, HPV 33, HPV35, HPV 39 and HPV45 were prevalent among Yemeni women with cervical cancer. Cervical cancer is the third most common cancer in women Worldwide . OncogenInfection with high-risk human papillomavirus (HR-HPV) is established as the main cause of cervical carcinoma . HPV hasThe L1 and L2 proteins form icosahedral capsids for progeny virion generation. Based on sequence variation of the L1, L2, HPV variants differ in biological and chemical properties, as well as geographical distribution –12. IntrHowever, there are very limited studies about HPVs and their related cancers in the majority of developing countries such as the greater Middle East which including Yemen and its neighboring countries, such as Saudi Arabia, Qatar, United Arab Emirates . HoweverIn this study, 200 formalin fixed paraffin wax processed tissue samples of cervical lesions were obtained from formerly operated patients from National Cancer Centers and different histopathology laboratories in Sana’a, Hadhramout and Aden, Yemen. All specimens and data were obtained from the archive of National Cancer Centers, and different hospitals and different private histopathology laboratories in the three governorates mention above during the period from 2008 to 2012 (full coverage). Cancer specimens included: Squamous Cell Carcinoma (SCC), Adenocarcinoma (Ad), Adenosquamo-Carcinoma (AdSC).DNA was extracted from paraffin-embedded samples, by immersing tissue section in xylene to dissolve the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases were digested by proteinase K. Buffer containing denaturing agent (sodium dodecyl sulfate (SDS)), was added to facilitate digestion . NucleicTo evaluate the DNA quantification after DNA extraction, we had analyzed DNA measurement using a NanoDrop spectrophotometer.Type specific primers  were used to detect HPV31, 33, 35, 39 and 45 DNA in cervical benign and malignant lesions, adopting the classification procedure of the American Joint Committee on Cancer (AJCC) TNM classification and the International Federation of Gynecology and Obstetrics (FIGO) staging system for cervical cancer . AmplifiSamples and controls were amplified using Gene Amp PCR system 9700. The PCR programis described in Table The PCR products were visualized in 2 % Agarose gel with 0.5 μg/ml Ethidium bromide. Ten micro liters of 100 bp DNA ladder and PCR product was loaded on the gel. Gel electrophoresis was performed at 120 V and 36 mA for 60 min. Pictures were taken by Gel documentation system .According to manufacture HPV31, 33, 35, 39 and 45 kit  manual, the PCR product length for HPV 31 should be 520 bp, HPV 33 should be 227 bp, HPV 35 should be 280 bp, HPV 39 should be 340 bp, HPV 45 should be 475 bp.Data management was done by using the Statistical Package for Social Sciences (SPSS version 16). SPSS was used for analysis and to perform Fisher exacttest for statistical significance . The 95 % confidence level and confidence intervals were used.Before the study conducted the proposal of the study were ethically approved by ethical committee of the Sudan University of Science and Technology. Then the inform consent were agreed by the general managers of all national cancer centers, hospitals and private histopathology laboratories in which the study were performed.In this study we investigated 200 formalin fixed paraffin wax tissue blocks obtained from patients previously presented with cervical lesions . The ages of the studied group were ranging from 21 to 75 years with a mean age of 47 years. Of the 150 cervical cancer tissue specimens, HPV 31, HPV 33, HPV35, HPV 39 and HPV45were identified in 10/150 (6.7 %), 6/150 (4 %), 6/150 (4 %), 5/150 (3.3 %) and 10/150 (6.7 %), respectively. Of the 50 benign cervical lesions tissue specimens, HPV 31, HPV 33, HPV35, HPV 39 and HPV45 were identified in 1/50 (2 %), 1/50 (2 %), 1/50 (2 %), 0/50 (0 %) and 1/50 (2 %), respectively, as indicated in Fig. Co-infection was only identified between HPV31 and HPV45, in two cases , which gives a prevalence of 36/150 (24 %) for HPV subtypes .Table The frequencies of infection rates among different age groups were summarized in Table With respect to the residence, most of cases of Cervical Cancer were coming from Al-Janad representing 52/200 (26 %) followed by Aden constituting 45/200 (22.5 %), as shown in Fig. The mortality of cervical cancers is high in Yemen and most cases are diagnosed late stage of the disease. In fact, there is a complete paucity of data in this context from Yemen; due to the absence of sufficient national registries. Although, the prevalence of HPV is unknown in Yemen, but data from the neighboring countries showed increased burden. In present study, the relationship between cervical cancer and the presence of the high risk HPV (HR-HPV) subtypes HPV31, HPV33, HPV35, HPV39, and HPV45 genome was tested. Data on HPV type spreading is essential to assess the possible impact of present and future HPV preventive strategies.The prevalence and distribution of HPV genotypes differs significantly globally, and these variances might be related to the complex geographical and biological interaction between different HPV types and host immunogenetic factors.However, there is a lack of literature regarding the burden of HPV in Yemen. To the best of our knowledge there was only two study investigated the prevalence of HPV in cervical cancer. The first one examined the presence of HPV16 and HPV18 in 84 cervical cancer tissue samples that were previously categorized as harboring HPV by immunohistochemistry using K1H8 (anti-HPV). In the use of PCR testing for HPV subtypes 16 and 18; 62/84 (73.8 %) were identified positive for HPV subtype 16 and the remaining 22/84 (26.2 %) were negative. Then again, on testing the specimens for HPV subtype 18; 23/84 (27.4 %) were found positive and the remaining 61/84 (72.6 %) were revealed negative . AnotherAgain the presence of the high risk HPV (HR-HPV) subtypes HPV31, HPV33, HPV35, HPV39, and HPV45 was previously reported in a number of studies. Prospective follow-up studies have proposed that persistent HR-HPV infections play a crucial role in the progression of CIN lesions and in the development of cervical cancer. The mean clearance time for the distinct HR-HPV type was 16.5 months. HPV16 and HPV31 were the most persistent infections , whereas HPV39 infections cleared quickly. The mean copies per cell in HPV18/45, HPV31, HPV33/52/58, and HPV39 infections were higher in persisting HPV infections than in HPV infections that cleared, but the difference was not significant . In a stRegarding the relationship between age and HPV infection in this study, it was observed that HPV31 and HPV45 were relatively seen in younger ages compared to HPV35 and HPV39; hence, HPV33 was more frequent among elder women. However, the number of infected women was so little to generate specific age range for the studied women.Moreover, there were considerable discrepancies regarding the distribution of the different HPV subtypes among different regions. However, the explanations of these findings require further onsite assessment of different behavioral, social and other demographical measures. A comprehensive screening for HPV subtypes linked to Pap. Test may render more sufficient results for future planning of potential vaccination program in Yemen.However, the limitation of the present study include its investigation included limited HPV subtypes.In conclusion: HR-HPV31, HR-HPV33, HR-HPV35, HR-HPV39, and HR-HPV45 are prevalent among Yemeni women with cervical cancer. Further, measures and strategic plans are urgently required to reduce the burden of cervical cancer in Yemen.


Controversy still exists over whether the benefits of the available HPV vaccinesoutweigh the risks and this has suppressed uptake of the HPV vaccines in comparisonto other vaccines. Concerns about HPV vaccine safety have led some physicians,healthcare officials and parents to withhold the recommended vaccination from thetarget population. The most common reason for not administering the prophylactic HPVvaccines are concerns over adverse effects. The aim of this review is the assessmentof peer-reviewed scientific data related to measurable outcomes from the use of HPVvaccines throughout the world with focused attention on the potential adverseeffects. We found that the majority of studies continue to suggest a positiverisk-benefit from vaccination against HPV, with minimal documented adverse effects,which is consistent with other vaccines. However, much of the published scientificdata regarding the safety of HPV vaccines appears to originate from within thefinancially competitive HPV vaccine market. We advocate a more independent monitoringsystem for vaccine immunogenicity and adverse effects to address potential conflictsof interest with regular systematic literature reviews by qualified individuals tovigilantly assess and communicate adverse effects associated with HPV vaccination.Finally, our evaluation suggests that an expanded use of HPV vaccine into morediverse populations, particularly those living in low-resource settings, wouldprovide numerous health and social benefits. Both HPV vaccines can protect femalesagainst cervical pre-cancers (CIN).Vaccination is the most successful method to control infectious diseases in terms ofboth cost and effectiveness. Human papillomavirus (HPV) belongs to a large family ofmore than 170 double-stranded DNA viruses of which approximately 40 mucosal types arecommonly transmitted mainly via sexual activity. Two prophylactic HPV vaccines have beenapproved by the Food and Drug Administration (FDA) in the USA: the bivalentCervarixSeveral studies have demonstrated that both the bivalent and quadrivalent HPV vaccinesare safe 23. Eac¯ (GlaxoSmithKline) is notlicensed for use in males at this time.It is important to emphasize that the HPV vaccines are not a therapeutic treatment forany HPV-associated disease that might exist at the time of vaccination, nor will itinvariably protect against diseases that are caused by types of HPV not targeted by thevaccines. Furthermore, HPV vaccines are not recommended for females <9 years old orindividuals that are pregnant. Lastly, CervarixDespite the efforts by public health agencies in the United States, the coverage of HPVvaccination remains low. Among adolescent females and males aged 13-16 years, only 33.4and 6.8%, respectively, had received the three recommended doses of the HPV vaccine in2012 . In JuneOne systematic review that involved a total of 29,540 individuals showed that the mostfrequently reported adverse event related to the HPV vaccines was pain and swelling atthe injection site followed by fatigue, fever, gastrointestinal symptoms and headaches.In Japan, HPV vaccination was recommended by the government in April 2013. However,several adverse effects such as complex regional pain syndrome were reported by theJapanese media, which led to a suspension of both the bivalent and quadrivalent HPVvaccines by the Japanese government two months later, in June 2013. Together with thegovernment decision, the media reports also created distrust in the Japanese public thatled to a further decrease in HPV vaccination coverage.Ueda et al.  reportedA group of four clinicians clinically examined 3 young women who were diagnosed withsecondary amenorrhea to analyze a possible association between primary ovarian failureand HPV vaccination. Their serological data showed increased follicular stimulanthormone and luteinizing hormone. In addition, auto-antibodies specific to the ovariesand the thyroid were detected, which the authors suggest might have been triggered bythe HPV vaccine . FurtherIn disagreement with this publication, Pellegrino et al.  wrote a As an opposing response to the previous letter, Colafrancesco et al.  suggeste¯ were capable of crossing theblood-brain barrier, which was suggested to trigger cerebral vasculitis, a severe formof blood vessel inflammation in the brain that can lead to severe autoimmune disordersand even death  has also been investigated . The aut¯ and Cervarix¯ is that placebo groups were oftengiven injections that included the active aluminum adjuvant. Safety concerns existregarding the aluminum, which is widely used as a vaccine adjuvant. Despite its strongneurotoxic potential, the bioaccumulation of aluminum in the brain appears to occur at avery low rate in normal conditions. Recently, mouse experiments designed to assess thebiodistribution of vaccine-derived aluminum have demonstrated that continuouslyincreasing doses of the poorly biodegradable adjuvant may become insidiously unsafe,particularly in cases of repetitive, closely-spaced vaccinations that may alter theblood-brain barrier  vaccine was included in the nationalimmunization program for girls aged 11-13 years old. The vaccine schedule adopted by theBrazilian Ministry of Health involves three doses at 0, 6 and 60 months. The city ofCampos dos Goytacazes in the state of Rio de Janeiro has included the quadrivalentvaccine against HPV in the municipal vaccination program using its own funds sinceSeptember 2010 for residents between the ages of 11 and 15 years. The National System ofNotification (SINAN) of the Brazilian Ministry of Health reported a total of 430 localand systemic events in 36% of the persons who received vaccinations in this program thatwere stratified by dose received in the three dosage administration protocol. No seriousadverse events or hospitalization were reported and there has been a 55% reduction inthe incidence of genital warts observed in women under 21 years old  approved bothprophylactic HPV vaccines for use in Brazil. However, vaccinations were only madeavailable in private clinics since the Brazilian Ministry of Health had not concluded anevaluation for their incorporation into the Public Health system. Starting in March of2014, the HPV quadrivalent  evaluated a school-based HPVvaccination program in the City of Barretos, State of São Paulo, where the vaccinecoverage rates were high and similar between public and private schools. However,according to the authors, the results achieved from this study may not be extrapolatedfor other regions in Brazil, mainly for those with limited access to schools in therural and country areas .Considering that HPV vaccines, like all other vaccines, may not protect all vaccinatedindividuals, regular cancer screening programs should be maintained irrespective towhether or not a person receives HPV vaccination. It remains to be determined if thenewer HPV vaccines against up to seven specific HPV genotypes associated with cancerincrease the efficacy of preventing the onset of CIN and cervical cancer, which isalready high with current vaccine formulations. However, it has been hypothesized thatan increase in the prevalence of other HPV types may occur due to a reduced competitionduring natural infection, although recent studies found no increase of non-vaccine HPVtypes, which would be suggestive of type-replacement ,29.¯ could extend theduration of efficacy, but would lead to a significant escalation in costs. It would alsobe a challenge to identify those women in need of revaccination (¯ vaccine that includes direct protection against HPVtypes 31, 33, 45, 52 and 58 in addition to the HPV types 6, 11, 16 and 18 of thequadrivalent vaccine.Duration of efficacy is a key question when discussing the HPV vaccines. According toDr. Harper, if the duration is at least 15 years, then vaccinating 11-year-old girlswill protect them until they are 26 years old, which would prevent some pre-cancers, butprimarily postpone most cancers. If duration of efficacy is less than 15 years, thenmost cancers are not prevented, only postponed. Administration of boosters after thediminishing antibody production elicited by Gardasilcination . Since tAlthough vaccines undoubtedly reduce the incidence of several infectious diseases, whichstrongly supports that the benefits outweigh the risks, side effects still need to beclosely monitored and reported without bias. For HPV vaccines, the body of evidencesuggests that their potential to elicit life-threatening side effects is very low withautoimmune responses being the greatest concern. While the reduction in the occurrenceof genital warts conferred by the HPV types present in the vaccines strongly suggests anultimately lower incidence of HPV-positive cancers, the time period since the initialvaccinations has not been sufficient to determine the absolute reduction in cervicalcancer, the major benefit expected. For this reason, it is important to stress the needto maintain routine surveillance for lower genital tract diseases especially in women,even if there is widespread use of the HPV vaccine.We also strongly believe that a regular systematic review of the literature by qualifiedindividuals with no financial interests should be conducted. In many instances,financial resources originate from parties within the competitive HPV vaccine market,which certainly have economic interests, causing major complications in interpreting theresults and conclusions from the various studies. Funding also comes from foundationsand organizations that are strongly against vaccination programs.


Human papillomavirus (HPV) are firmly established as the principal causative agent for cervical carcinoma. Current vaccines may provide some protection for women from cervical carcinoma linked to HPV genotype 16 and 18. This may be the best vaccine for Western women, but the geographical variation in HPV distributions may not make it the most appropriate vaccine for China or Asia. This study provided an observational, retrospective, hospital-based cross-sectional study on the distribution of HPV genotypes among 5410 women with invasive cervical cancer (ICC) or cervical intraepithelial neoplasia (CIN). Overall, the positive rates of the four HPV types included in current prophylactic vaccines were counted, the two high-risk types (HPV-16 and -18) covered by current vaccines represented 66.9% of women with squamous cancer, 55.0% with adenocarcinoma, 64.9% with adenosquamous carcinoma and 77.4% of other type ICC, as well as 59.5% of CIN III, 45.0% of CIN II and 38.1% of CIN I cases. As expected, two low-risk types (HPV-6 and -11) included in the quadrivalent vaccine did not show good coverage data. Particularly worth mentioning is the fact that the addition of HPV-52 and -58 to the vaccine cocktail would increase cancer protection in our population, potentially preventing up to beyond 16% of squamous/adenosquamous carcinoma and other type of cervical cancers, and 7.75% of adenocarcinomas. It might also potentially reduce the rate of CIN III by a further 28.6% and CIN II and I by a third. This study established the baseline for surveillance in Zhejiang Province, and provides data for further vaccine designs: a quadrivalent HPV vaccine covering HPV-16/-58/-18/-52, would be more welcome in our region in the forthcoming year compared to the currently available vaccine. Invasive cervical cancer (ICC) ranks as the third most common malignancy and the fourth cause of cancer-related death among women worldwide ,2, with HPV is firmly established as the principal causative agent for cervical carcinoma. To date, more than 200 HPV genotypes have been identified, 40 of which are sexually transmitted and 15 of these are carcinogenic and classified as high-risk human papillomaviruses (hr-HPVs) . Infecti®  targets HPV-16/-18 and the quadrivalent vaccine Gardasil®  targets HPV-6/-11/-16/-18. The vaccines may offer some crossover and protection from other HPV types, but vaccinated women will not gain significant protection against oncoviruses other than 16 and 18. HPV vaccines have been commercially available since 2007. The bivalent vaccine CervarixTherefore, the knowledge of the regional HPV genotype is crucial to guiding estimates of the effectiveness of vaccination programs, health economic calculations and the development of improved vaccines. HPV-16 and -18 are the two most common hr-HPVs types but there are geographic variations. A survey of healthy women by the International Agency for Research on Cancer established that some genotypes were particularly prevalent in different continents  squamous cell carcinoma (SCC), 258 adenocarcinoma (ADC), 91 adenosquamous carcinoma (ASC) and 31 other type ICC , 1160 were CIN . The Local Institutional Ethics Committee approved the study.HPV genotyping by HybriMax used an HPV GenoArray Test Kit  according to the manufacturer’s instructions . This as2 test was used to compare HPV prevalence across groups. Statistical significance was determined at p < 0.05 level.The data were key-entered twice using SPSS 16.0 software package . The χp < 0.001). The 4250 samples of ICC, included SCC, ADC, ASC and other carcinomas, the rate of hr-HPV genotype was higher with SCC than ADC (p < 0.001), but no difference was observed between SCC and ASC (p = 0.08). Of the 1160 samples from women with CIN, the HPV positive rate was higher with CIN III than CIN I (p < 0.001) and CIN II (p < 0.001), but no difference was seen between CIN I and CIN II (p = 0.97) The proportions of single- or multiple-type  infection are shown in 4405 (81.4%) of the 5410 cervical samples from women with known pathology had detectable HPV: 80.3% with ICC, compared to 85.5% with CIN (The mean age of the positively detected women was 49.27 (range 16–93 years). HPV-16, -58, -18 and -52 were the most prevalent types found in Zhejiang Province. Invasive cervical cancer. The relative distribution of HPV types among the 4250 cases of cervical carcinoma, regardless of the infection status , HPV-51 (23.8%), HPV-52 (19.1%) and HPV-58 (14.3%); in CIN II they were HPV-16 (40.4%), HPV-58 (23.8%), HPV-52 (9.6%) and HPV-33 (9.2%); in CIN III they were HPV-16 (55.5%), HPV-58 (20.4%), HPV-33 (8.5%) and HPV-52 (8.2%). As shown in The cumulative contribution of six hr-HPV and two lr-HPV types for each stage of cancer shows a generally lower detection with advancing stage . HPV-16, -58 and -18 were the most prevalent types found in ICC, HPV-16 attributed to the majority of ICC staging , with the subsequent types showing lower attribution rates . HPV-16 plus -58 accounted for the majority of ICC stage with attribution rate of 68.0%, 69.3%, 62.3% and 59%, respectively . StratifThe positive rates of the four HPV types included in current available prophylactic vaccines were counted. The two high-risk types (HPV-16 and -18) covered by current available vaccines represented 66.9% of SCC, 55.0% of ADC, 64.9% of ASC, 77.4% of other type ICC, 59.5% of CIN III, 45.0% of CIN II and 38.1% of CIN I cases. The two low-risk types (HPV-6 and -11) included in the quadrivalent vaccine only represented a small proportion of ICC (1.2% of SCC and 1.3% of ADC) and CIN . Particularly worth mentioning is that addition of HPV-52 and -58 to the vaccine cocktail would result in the biggest marginal increase in coverage, with 16.6% for SCC, 7.8% for ADC, 16.5% for ASC, 16.1% for other type ICC. It might also potentially reduce the rate of CIN III by a further 28.6% and CIN II and I by a third. This study adds to the growing literature on the distribution of HPV genotypes among cervical cancer and its precursors. Controlling HPV infection is thought to be the key to minimizing the impact of cervical cancer. The knowledge of HPV prevalence, its subtype and age distribution might guide local public health policies and prevention measures, including vaccination . Hence, There are several limitations of the data in this report. We do not know how many women had cancer from our region but never had a diagnosis, and this could introduce as certain bias. Another source of potential bias is false negative laboratory reporting. In order to provide internationally comparable data, High-Risk Hybrid Capture 2 (HR HC2) was selected. In our study, the HPV positive rate was only 80.3% of women with ICC, yet others have found values of up to 99.7% . Part ofet al. concluded that the infection with multiple HPV types is a significant risk factor for high-grade CIN [Multiple HPV infections occurred in 24.3% of women with CIN  and 16.5% of ICC . So far, neither the role played by each HPV type found in multiple-type infections in a lesion (in a random fashion or a competitive or cooperative relationship exists), nor the type-specific contribution of HPV to CIN and ICC progression is clear. Interestingly, Jaisamrarn found a paradoxical result that co-infection could increase the risk of progression to a CIN but also increase the chance of clearance . Pista, rade CIN . The clarade CIN . In addiThe five most common hr-HPV types in the general worldwide female population are HPV-16, -18, -31, -58 and -52, while the rank varies by geographical regions. HPV-16/-58/-18/-52 were the most commonly identified types in our population. The attribution of HPV types in southeast China seems different from elsewhere. As HPV immunity is type-specific, this should be considered in prioritizing HPV types for vaccine development. Since 2007, specific vaccination programs have been implemented into clinical practice . The twoOur data showed a low prevalence of HPV vaccine types (HPV-6 and -11) and relatively high prevalence of HPV-58 and -52, which could serve as a baseline for future monitoring. Hence, we suggested that in Asia, a quadrivalent HPV vaccine covering HPV-16/-58/-18/-52, would be more useful in the forthcoming years.


We conducted a community-based study to evaluate genotype-specific prevalence of high-risk HPV (HR-HPV) and potential predictors of its presence in young, asymptomatic, female college students. Self-administered surveys and vaginal swabs for self collection were distributed to students of participating schools. A sufficient cellular component in cervical samples was verified by examining for the presence of human β-globin DNA by PCR. A total of 1,118 valid cervical samples were subjected to screening for HR-HPV infection with the Digene Hybrid Capture 2 assay, followed by identification of HPV genotypes with GENOSEARCH HPV31 kit. Logistic regression was used to adjust for confounding factors associated with HR-HPV positivity and the adjusted odds ratio (AOR) was calculated.p < 0.01), total number of partners , number of partners in the past 6 months , improper use of condoms , and chlamydia infection . The most common HR-HPV genotype was type 52 (6.4%), followed by 16 (3.1%), 56 (3.0%), and 58 (2.6%).The median age of recruited students was 20 years. Of the 1,118 women who provided valid cervical samples for testing, 770 had sexual intercourse in the past, of which 125 (16.2%) were positive for HR-HPV. Logistic regression analysis revealed that HR-HPV infection was associated with smoking history (AOR 2.13; 95% confidence interval [CI] 1.98 to 5.05; Compared with previous reports in East Asian coutries, the prevalence of HR-HPV infection among young, asymptomatic, female students before the nationwide use of vaccination in Japan was in the intermediate range. The most common HR-HPV genotypes were HPV 52, 16, 56, and 58. Cervical cancer ranks as one of the most common cancers affecting women in most countries. A total of 250,000 women die of cervical cancer each year worldwide . In JapaData accumulated by long-term follow-up studies indicate that infection of 12 HPV types  classified as class I (established) carcinogens were more common in women with cervical cancers than those without cervical lesion. Several other rare HPV types  were also found more commonly in women with cervical cancers [HPV infection is acquired through sexual intercourse and therefore, the sexual behavior of young people has a significant impact on HPV infection. Therefore, community-based, baseline epidemiological data, including HPV genotypes, in young women who are at a particularly high risk of HR-HPV infection are indispensable for monitoring the impact of vaccination. However, previous epidemiological studies were hospital-based and no community-based epidemiological information is available at present. The purpose of our study was therefore to elucidate the prevalence, potential predictors, and genotype-specific prevalence of HR-HPV among female students at colleges and vocational schools in a prefecture in southern Japan.This was a community-based, cross-sectional study investigating asymptomatic HR-HPV infection in young women. There are 6 universities and 18 vocational schools in the city of Miyazaki, Japan. Initial inquiries were sent to the administrative offices of these schools to find out their willingness to participate in the study. Among those, 3 universities and 6 vocational schools agreed to participate in the study, and female students attending those schools who were 18 years or older were potential candidates. An investigator visited the participating schools to present the purpose and design of the study to students. Student health personnel of the schools also solicited participation and campus posters were used to promote the study. Written consent was obtained from each voluntary participant prior to distribution of study material. The study was conducted from October 2011 to February 2012 and was approved by Tokyo Healthcare University Institutional Review Board.The aim of the study was to assess the prevalence of asymptomatic HPV infection. Therefore, candidates who reported that they were aware of urogenital symptoms suggestive of or were being treated for sexually transmitted disease were excluded from the study. We distributed survey sets consisting of an envelope containing a self-administered blank questionnaire on sexual behavior, a HPV test kit, a chlamydia test kit, and seals stamped with an ID no. (set of four with the same number) to candidate students. The questionnaire on sexual behavior asked about the history of past sexually transmitted diseases, age at first sexual intercourse, total number of sexual partners, number of sexual partners in the past half year, and use of condoms. The other questions included in the questionnaire were smoking and drinking habit. Candidates were instructed to apply seals to the two test kits and the questionnaire, and to keep the one remaining seal as their personal ID no. To access the test results, participants were instructed to log in on the study website by cell phone or computer approximately 3 months after submitting the HPV and chlamydia test kits, and to retrieve the test results using their personal ID no. To completely to protect the privacy of the study participants, they were not contacted about treatment, but general information on HR-HPV infection and cervical cancer, and prevention, diagnosis, and treatment were posted on the website where the test results were provided.Study participants were instructed to obtain the cervical specimen for the HPV test by rubbing the cervix with a cotton swab included in the Cervical Sampler . The cotton swab was then placed in a tube filled with Specimen Transport Medium. Participants were instructed to avoid handling the capped tube roughly or placing it in a hot environment prior to sample submission. Subjects were also told to collect the urine sample on the first urination in the morning for chlamydia testing. The cervical specimens and urine samples were to be taken to school by midmorning and placed in a refrigerated box and kept refrigerated at 2 to 8°C. The collected samples were carefully packaged and shipped refrigerated to a central laboratory in Tokyo. The specimens and samples were processed for testing within 24 hours after being submitted. HR-HPV infection in the samples was evaluated in two steps. First, the presence of HR-HPV was tested by the Hybrid Capture 2 (HC2) method (Digene). The denatured samples were mixed with a RNA probe mixture that contains probes for 13 HPV types known to be high-risk types, and the DNA/RNA complex was detected by immunoassay using an antibody detecting the DNA:RNA hybrids. Specific HPV genotypes were then identified with the samples that were positive for HR-HPV by HC2, using the GENOSEARCH HPV31 kit . The kit can identify the following 31 HPV genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 66, 68, 70, 71, 73, 82, 84, 90, and CP6108, utilizing reverse sequence-specific oligonucleotide PCR (PCR rSSO). A probe detecting a portion of genomic DNA that encodes human β-globin was also included as an internal control to verify presence of sufficient cellular component in the samples. Briefly, DNA extracted using QIAsymphony DSP viral/pathogen kit (Qiagen) from 400 μl of the sample that had been alkaline-denatured for the HC2 assay was amplified by multiplex PCR for 31 HPV DNA using biotin-labeled specific probes in 96-well Gene Amp PCR system 9700  with the following program: 35 cycles of denaturing at 94°C (30 sec), annealing at 61°C (1 min) and extension at 72°C (30 sec). The resulting amplicon mixtures were hybridized with fluorophore-loaded beads, followed by detection with phycoerythrin-bound streptavidin by the Luminex System .Chlamydia DNA in urine samples was simultaneously assayed by SDA .Associations between HR-HPV positivity and its potential predictors were evaluated by univariate analysis using Pearson’s chi-square tests or Wilcoxon rank-sum tests. Logistic regression analysis was used to adjust for potential confounding factors associated with HR-HPV positivity. All covariates were entered into the model, and analyses were executed using stepwise variable selection. Odds ratios were estimated for each independent potential predictor and 95 percent confidence intervals were calculated after adjusting for all other variables retained in the model. A two-sided significance level of 0.05 was used. All statistical analyses were performed using Statistical Package for Social Sciences .The sample collection kits and questionnaire sets were distributed to 1,627 candidates. Among them, 1,183 subjects whose cervical sample contained human β-globulin DNA and who answered the questionnaire properly were analyzed. A total of 770 of these women had experience of sexual intercourse  and theyA total of 125 women  had HR-HPV infection . The mosThe following factors were found to be associated with HR-HPV infection by univariate analysis: older age, smoking history, number of sexual partners , inappropriate use of condoms, and chlamydia positivity. Age at first intercourse was not significantly associated with HR-HPV infection.p < 0.01), total number of partners , number of partners in the past 6 months , inappropriate use of condoms , and chlamydia infection   .Most studies identifying the prevalence of HR-HPV infection have thus far been conducted in visitors to medical institutions or health centers and may have been biased. Accurate epidemiological information on HPV infections, including genotype-specific prevalence, is essential for achieving further progress in prevention, early detection and appropriate treatment of cervical cancer, and therefore, population- or community-based studies are desired. In this study, the subjects were sexually active young women attending educational institutions in a relatively closed community. Women who had urogenital symptoms at the time of the questionnaire survey were excluded (no cytology was performed). The prevalence of HR-HPV infection (16.2%) in this study was close to the HR-HPV prevalence of 18.5% reported in a recent UK community-based study of young female students  and the The AOR for smoking history was 2.13. While several previous studies reported that smoking history is a potential predictor –14, otheHPV 52 genotype was the most common , followed by HPV 16 (3.1%), HPV 56 (3.0%), and HPV 58 (2.6%). The prevalence of HPV 18 was only 0.9%. The combined prevalence of HPV 16 and 18 in this study was 4.0%, but appears to vary between regions, being 1.5% and 0.8% in the US , 4.2% anThe present study has several limitations. First, the effective sample size of 1,183 subjects was not large. The number of potential candidates was limited by study design as we aimed to conduct a community-based study. Also, our approach was to recruit students in the area, excluding women who were not attending schools. This may have caused selection bias. Our findings should therefore be interpreted with caution. Second, the accuracy of self-reporting is limited. Four of the subjects who reported that they have never had sexual intercourse also reported a history of sexually transmitted disease, implying a possible error in interpreting the question or false entry in the questionnaire. It is impossible to fully eliminate possible bias due to misunderstanding of questions and/or false answers in the self-administered questionnaire, especially when questions are related to sexual behavior. For example, in a previous study that involved more than 10,000 subjects, 1.4% (13 of 914) of participants who reported having had no vaginal sex were chlamydia-positive . Memory This study provided community-based, basic epidemiological information on HPV infection and genotype-specific prevalence in young women. These findings will hopefully be used to track changes in HPV prevalence and HPV genotype-specific prevalence, as well as changes in the prevalence of HPV-induced cervical cancer with the introduction of HPV vaccines in Japan.