The prevalence, incidence and persistence of human papillomavirus (HPV) types in sub-Saharan Africa are not well established. The objectives of the current study are to describe (predictors of) the epidemiology of HPV among high-risk women in Kigali, Rwanda.HIV-negative, high-risk women were seen quarterly for one year, and once in Year 2. HIV serostatus, clinical, and behavioral information were assessed at each visit, HPV types at Month 6 and Year 2, and other sexually transmitted infections (STI) at selected visits. HPV prevalence was also assessed in HIV-positive, high-risk women.Chlamydia trachomatis and Neisseria gonorrhoeae infection, concurrent low-risk (LR)-HPV infection and incident HSV-2 were associated with HR-HPV prevalence among HIV-negative women; prior C. trachomatis infection and co-infection with LR-HPV and HPV16-related HPV types with HR-HPV acquisition. HPV16-related types were the most prevalent and persistent.Prevalence of any HPV was 47.0% in HIV-negative women (median age 25 years) compared to 72.2% in HIV-positive women . Among HIV-negative women, cumulative incidence of high-risk (HR)-HPV was 28.0% and persistence 32.0% after a mean period of 16.6 and 16.9 months, respectively. Prior High HPV prevalence, incidence and persistence were found among high-risk women in Kigali. HPV52 had the highest incidence; and, together with HPV33 and HPV58, were strongly associated with acquisition of other HR-HPV types in HIV-negative women. Human papillomavirus (HPV) is the most common sexually transmitted infection (STIs) worldwide . PrevaleAn increasing number of articles report on the type-specific HPV prevalence among women in Sub-Saharan Africa, but incidence and persistence data remain scarce. HPV dynamics, and predictors of incidence and persistence, are best studied in prospectively collected data. This information will aid in the design of primary or secondary prevention programs.As part of secondary data-analysis of an HIV incidence study, we evaluated HPV prevalence, incidence, persistence, and their predictors, among HIV-negative high-risk women in Kigali, Rwanda. In addition, HPV prevalence was assessed among HIV-positive high-risk women cross-sectionally.A cross-sectional HIV prevalence survey was conducted among high-risk women in Kigali, Rwanda. Subsequently, women testing HIV-negative during the survey, and who were 18 years or older, at high risk for HIV (defined as having exchanged sex for money at least once in the last month and/or currently having sex with multiple partners plus having sex at least twice per week) and were not pregnant at enrolment or planning a pregnancy within the next year were invited to participate in a prospective cohort study to assess HIV incidence (2006-2009) [The target sample size for the HIV-negative cohort was 400 and this was based on the primary HIV incidence objective. HPV prevalence, incidence and persistence were among secondary objectives of the study. Enrolled women were followed quarterly for one year, and were invited for one additional visit during year 2 ("Year 2 visit").Women testing HIV-positive during the survey were excluded from cohort participation, but were invited for one additional study visit during year 2 ("Year 2 visit") during which HPV prevalence was assessed.Women were recruited via community meetings in three districts in Kigali. Written informed consent was obtained, including parental or guardian consent for women who were 18-20 years old. The study was conducted at Projet Ubuzima, an international non-governmental organization that operates a research clinic and laboratory, in Kigali, Rwanda.Neisseria gonorrhoeae, C. trachomatis, Treponema pallidum (syphilis), Trichomonas vaginalis, and bacterial vaginosis (BV) at enrolment, Month 6, and Month 12, and for BV also in Year 2. HPV typing and cervical cytology were done at Month 6 and Year 2. HSV-2 serology testing was performed at enrolment, at Month 12 for women testing negative at enrolment, and in Year 2 for women testing negative at Month 12. Standardized questionnaires were administered at each study visit.HIV-negative participants were tested for HIV at every study visit, for HIV-positive women participating in the Year 2 visit followed the same study procedures as described for the Year 2 visit of the HIV-negative cohort participants.Women with abnormal cytology results were referred for visual inspection with acetic acid (VIA) and colposcopy by a gynecologist, and were offered appropriate treatment. Women with curable STIs received treatment and were offered partner notification and treatment. HIV-positive women were referred to HIV care. As part of the study procedures women received HIV prevention, safer sex and family planning counseling at each visit.HPV genotyping was performed at the Institute of Tropical Medicine  using the Linear Array HPV Genotyping Test (LA)  according to manufacturer's instructions. In brief, cervical cells collected in Liquid Based Cytology media PreservCyt were stored at -80°C until testing. An aliquot of 250 μl was used for DNA extraction by AmpliLute Liquid Media Extraction Kit . HPV genotype targets were amplified using standard polymerase chain reaction (PCR). LA applies PGMY09/11 L1 as consensus primer system and co-amplifies human β-globulin as a cellular, extraction and inhibition control. Reverse line blot hybridization and detection identifies 37 high- and low-risk HPV types . An in-house Real-Time PCR (RT-PCR) was established at the ITM to identify DNA of HPV genotype 52 in the mixed probe positive samples. Briefly, DNA was prepared using the NucliSens miniMAG . DNA templates were amplified in a qualitative RT-PCR assay. HPV52-F/R and hydrolysis probe HPV52 labeled at the 5' with FAM and at the 3' with BHQ-1  were used for amplification and detection [HIV testing was done at Projet Ubuzima according to the national testing guidelines, with two positive rapid tests indicating an HIV diagnosis , and a third rapid test  as tiebreaker. Positive results were confirmed with Murex HIV Ag/Ab Combination ELISA . CD4 cytometry was performed at the National Reference Laboratory in Kigali.T. vaginalis diagnosis. Syphilis was diagnosed using RPR  and TPHA . HerpeSelect-2 ELISA  was used for HSV-2 antibody testing. Optical density ≥ 3.5 were considered positive for HSV-2, and OD ≤ 1.1 negative. All tests were performed at Projet Ubuzima. Dry swabs (BD Culturette™ EZ Collection), stored at -80°C were used for Amplicor NG/CT PCR  for N. gonorrhoeae and C. trachomatis testing at ITM. Positive results were confirmed with a strand displacement amplification assay . External quality control for STI testing at Projet Ubuzima was coordinated by ITM. BV was diagnosed by Gram stain Nugent score. Nugent scoring was performed at Projet Ubuzima; at the Academic Medical Center of the University of Amsterdam, Department of Medical Microbiology; and at the Amsterdam Municipal Health Service, Public Health Laboratory. Conventional cytology was performed at Ghent University Hospital, Belgium, using the Bethesda system for classification [InPouch culture  was used for fication .Data were double-entered, and analyzed using STATA 9.2 . Baseline characteristics were estimated as percentages with 95% confidence intervals (CI) for binary and categorical variables; median and interquartile range (IQR) for non-normally distributed continuous variables or mean and standard deviations for normally distributed continuous variables.In these analyses, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 and IS39 were considered HR-HPV types -13. All P-value < 0.10 in univariable models were included in the initial multivariable logistic regression model. Variables were retained in the model based upon Wald statistics (P < 0.05) and/or likelihood ratio tests for the overall fit of the model (P < 0.1). Age was included as a covariate in all models. Pair-wise correlations between the variables in the final models were investigated, and correlated variables were assessed for interaction. The comparison group for HR-HPV analysis included women without HR-HPV, irrespective of LR-HPV detection. Concurrent LR-HPV was included as a covariate. Similarly, the comparison group in LR-HPV analysis included women without LR-HPV, irrespective of HR-HPV, but concurrent HR-HPV was included as a covariate. Age-adjusted odds ratios and 95% confidence intervals (C.I.) were calculated comparing HPV prevalence among HIV-positive (n = 349) and HIV-negative women (n = 126). Women with abnormal cytology results  were excluded.HR-HPV prevalence was estimated from the first HPV results in both HIV-negative (Month 6) and HIV-positive women (Year 2). Multivariable logistic regression models for predictors of HR or LR-HPV detection in HIV-positive and HIV-negative women were fitted using manual backward modeling. Variables associated with the outcome with a P < 0.1). These groups were included in the final incidence model. Sensitivity analyses were performed excluding women with any prevalent HR-HPV and/or LR-HPV at Month 6 from the reference group. These results are not shown because they were similar to those of the incidence model as described above. Of the 349 HIV-negative women with normal cytology results and HPV results at Month 6, 296 women also had HPV results at Year 2 visit and contributed to the incidence analyses. Methods for multivariable logistic regression modeling were identical to those for HPV prevalence.Type-specific HR-HPV incidence was defined as newly detected type-specific HR-HPV at the second HPV measurement (Year 2) among women free of the type of interest at the first HPV measurement (Month 6). The (cumulative) incidence was calculated over the mean interval between Month 6 and Year 2 visit. All incident type-specific HR-HPV cases were grouped together into one binary incident HR-HPV outcome variable, with women without incident HR-HPV as the referent group . The association between prevalent HR-HPV types and incidence of other HR-HPV types was assessed. First, univariable associations between each type-specific HR-HPV at Month 6 and subsequent acquisition of other HR-HPV types were evaluated. To increase statistical power, types were then grouped according to their phylogenetic group and within group according to strength of the association (0.1 >HR-HPV persistence was defined as women who tested positive for the same HR-HPV type at both visits during which HPV typing was performed (Month 6 and Year 2). Persistence was calculated over the mean interval between these two visits. All persistent type-specific HR-HPV cases were grouped into one binary persistent HR-HPV outcome variable, irrespective of LR-HPV. The reference group included women who cleared all HR-HPV infections at the second visit that were present at the first visit, but who may however tested positive for new HPV types at the second HPV measurement. Of the 111 HR-HPV positive women with normal cytology at Month 6, 90 women also had HPV results at Year 2 visit and contributed to the persistence analyses. Multivariable logistic regression modeling was conducted as described for HPV prevalence above.Ethical approvals were obtained from the National Ethics Committee and the National AIDS Control Commission (CNLS) in Rwanda; the Institutional Review Board of Columbia University Medical Center, New York, USA; and the Medical Ethics Committee of the University of Antwerp, Belgium.N. gonorrhoeae was 10% (CI 7.3-13.6), C. trachomatis 5% (CI 2.7-7.2), T. vaginalis 17% (CI 13.4-21.2), syphilis 7% (CI 4.7-10.1), and BV 45% (CI 39.4-49.9).Eight hundred women participated in the HIV prevalence survey between October 2006 and August 2007 and 192 women tested HIV-positive. Four hundred three (403) HIV-negative, non-pregnant women were enrolled in the cohort. The flow-diagram of the study is given in Figure Among 349 HIV-negative women with no cervical abnormalities, the HPV prevalence at the first HPV measurement visit (Month 6) was 47.0% for any HPV, 31.8% for HR-HPV and 32.4% for LR-HPV. The five most prevalent HR-HPV types among HIV-negative women were HPV16 (5.4%), HPV45 (4.9%), and HPV52 (4.3%) HPV58 (4.0%) and HPV68 (3.7%)  more likely to have any HPV detected; 2.2 times (CI 1.5-3.4) HR-HPV and 2.5 times (CI 1.7-3.9) LR-HPV. The five most prevalent HR-HPV types among HIV-positive women were HPV52 (13.9%), HPV58 (11.1%), HPV51 (11.1%), HPV16, HPV 45 and HPV35 (each 7.9%). Multiple HR-HPV types were detected in 29.4% of HR-HPV positive/HIV-positive women.N. gonorrhoeae or C. trachomatis infection at baseline or among women with concurrent BV or LR-HPV infection. Women experiencing incident HSV-2 infection at Month 6 and women using hormonal contraceptices for longer than 6 months were also more likely to have HR-HPV detected at Month 6 (P < 0.05). Sexual behavior indicators  were not associated with HR-HPV prevalence among this highly sexually active population. In multivariable analysis, being single, having N. gonorrhoeae or C. trachomatis infection at baseline, concurrent LR-HPV infection, and incident HSV-2 infection remained independent predictors of HR-HPV prevalence among HIV-negative women  women Table . The odden Table .P < 0.05) (data not shown). Hormonal contraceptive use for longer than 6 months and co-infection with HR-HPV were positively associated with LR-HPV (P < 0.05). Age, and concurrent HR-HPV remained independently associated with LR-HPV in multivariable analysis, and there was a trend in that direction for hormonal contraceptive use for longer than 6 months .In univariable analysis, LR-HPV was negatively associated with older age , and the interaction between CD4 count and cART were not associated with HR-HPV detection.P = 0.087). Cumulative HR-HPV incidence was 39.2% among LR-HPV positive women at Month 6 compared to 22.1% among LR-HPV negative women (P = 0.002).Of the 296 women who contributed to the HPV incidence analyses, incident HR-HPV types were detected in 28.0% (95% CI 23.0-33.1) after a mean interval of 16.6 months (SD 1.8). The mean duration between the two HPV measurements was not significantly associated with acquisition of type-specific HR-HPV (data not shown). Among HR-HPV positive women at Month 6, 34.4% acquired a new HR-HPV type by Year 2, compared to 24.8% among women who were HR-HPV negative at Month 6 . Mean duration between the two HPV measurements was not associated with clearance or persistence. Persistence was most common for HPV31 and 68 (data not shown). In univariable analyses, only BV at Month 12 was associated with a decreased likelihood of persistent HR-HPV infection  and LR-HPV (54.8% and 32.4%) in HIV-positive and HIV-negative high-risk Rwandan women, respectively. This is considerably higher than the prevalence rates that we found in women from the general population in Kigali, Rwanda . Our reVery few prospective studies on HPV epidemiology have been conducted in Africa. We found a cumulative HR-HPV incidence of 28% and persistence of 32% over a mean interval of 17 months, in HIV-negative high-risk women. In comparison, HR-HPV incidence among a rural Ugandan population was estimated at 7.0 per 100 PY among HIV-negative and 17.3 per 100 PY among HIV-positive women; 66% of these infections were cleared by HIV-negative women compared to 40% by HIV-positive women . HR-HPV HPV52 had the highest incidence rate, the presence of infection by HPV52, 58, and 33 combined at Month 6 was strongly associated with increased acquisition of other HR-HPV types in Year 2. In a previous analysis of the same cohort, the presence of HR-HPV at Month 6 was strongly associated with HIV acquisition, and HPV52 was the most frequent HR-HPV type detected in HIV seroconverters . InfectiC. trachomatis and N. gonorrhoeae infection were associated with HR-HPV prevalence in HIV-negative women, and prior C. trachomatis infection with HR-HPV incidence. The literature is mixed, with some studies finding associations between C. trachomatis and HR-HPV [C. trachomatis infection and HPV infection are not yet fully understood [C. trachomatis infection by the next visit. Active epithelial regeneration following infection might increase the susceptibility of target cells to HPV. Conversely, insufficient Th1 cells responses (which are required for resolving C. trachomatis infection and clearing HPV) may promote persistent C. trachomatis and persistent HPV infection [C. trachomatis infections in our population were treated or cleared, we were not able to establish an association between persistent C. trachomatis infection and HPV persistence. We also did not find an association between BV and any or HR-HPV, as has been found in other studies [In our study, prior d HR-HPV ,27, and d HR-HPV . The bioderstood ,30. Inflnfection -33. As t studies ,35. SexuThere is no consistent definition of HPV persistence in the scientific literature, with some published studies using a duration of 6-12 months, and others using a duration of longer than 12 months . The meaC. trachomatis was associated with HR-HPV incidence.In conclusion, high HPV prevalence, incidence and persistence rates were found in this study among high-risk women in Kigali. HPV52 had the highest incidence; and, together with HPV33 and HPV58, was strongly associated with acquisition of other HR-HPV types in HIV-negative women. HPV52 and 58 were also the most prevalent in HIV-positive women and, as previously shown, in HIV seroconverters. In addition, prior Women at high-risk for sexually transmitted infections (including HIV) experience a higher incidence and prevalence of HPV infections, including infections with high-risk HPV types. Therefore these women are at greater risk for developing cervical cancer. Cervical cancer prevention programs should incorporate strategies to ensure a high coverage among these high-risk women.The authors declare that they have no competing interests.JvdW, JV and SB have been involved in the conception and design; CI, JN, EK, CT, AU, MU, CT, JV, SB and NV in the acquisition of data; and NV, FW and JvdW in data analysis and interpretation. NV took the lead in drafting the manuscript, all authors critically reviewed manuscripts drafts and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/333/prepub


This study explores whether gender, age and race differences in oral sexual behavior account for the demographic distribution of oral human papillomavirus infection (HPV) and HPV-positive oropharyngeal cancer (HPV-OSCC)This analysis included 2,116 men and 2,140 women from NHANES (2009–10) who answered a behavioral questionnaire and provided an oral-rinse sample for HPV detection. Weighted prevalence estimates and prevalence ratios (PR) were calculated for sexual behaviors and oral HPV infection by gender, age-cohort , and race, and contrasted with incidence rate ratios (IRR) of OSCC from SEER 2009. Multivariate logistic regression was used to evaluate predictors of oral sexual behavior and oral HPV16 infection.Differences in oral sexual behavior were observed by gender, age-cohort and race. Most men (85.4%) and women (83.2%) had ever performed oral sex, but men had more lifetime oral and vaginal sexual partners and higher oral HPV16 prevalence than women (each p<0.001). 60–69 year olds (yo) were less likely than 45–59 or 30–44 (yo) to have performed oral sex , although oral HPV16 prevalence was similar. Prevalence ratios (PR) of ever oral sex in men vs. women (PR = 1.03), and 45–59 vs. 30–44 year-old men (PR = 0.96) were modest relative to ratios for oral HPV16 infection (PRs = 1.3–6.8) and OSCC (IRR = 4.7–8.1). In multivariate analysis, gender, age-cohort, and race were significant predictors of oral sexual behavior. Oral sexual behavior was the primary predictor of oral HPV16 infection; once this behavior was adjusted for, age-cohort and race were no longer associated with oral HPV16.There are differences in oral sexual behaviors when considering gender, age-cohort and race which explain observed epidemiologic differences in oral HPV16 infection across these groups. Human papillomavirus (HPV), a sexually transmitted infection, causes a subset of oropharyngeal squamous cell cancers (OSCCs). In recent decades sexual behaviors have changed; the age of sexual initiation has decreased, and the lifetime number of sexual partners has increased. Similar to the differences seen in HPV-OSCC rates, oral HPV prevalence in the general population varies by gender, age, and race. Data from the 2009–10 National Health and Nutrition Examination Survey (NHANES) were used in this analysis. NHANES is a stratified multistage probability sample of the U.S. population, that oversamples blacks and Hispanics, and people ≥60 yo Demographic data was collected using an interviewer administered survey. Race and ethnicity were self-identified by participants and reported by NHANES as: Mexican-American, other Hispanic, non-Hispanic white, non-Hispanic black, and other race including multi-racial Sexual behavioral data were collected by audio computer-assisted interview (ACASI). Variables included ever having had any sex, ever performing oral sex , age at first sex, and age first performed oral sex. Age at first oral sex was compared to age at first sexual act ; we considered first oral sex to be “at” sexual debut if age at first oral sex was the same as age at first sex. Number of lifetime partners for any kind of sex, vaginal sex, and performing oral sex, were each collected separately. Number of lifetime partners reported in this paper is a sum of both male and female partners. Participants ≥60 yo were not asked about lifetime number of oral sex partners therefore analysis for this variable was restricted to those between 20–59 yo. Women ≥60 yo were not asked about number of female (i.e. lesbian) sexual partners. Subjects <20 years of age were excluded because of more limited sexual data in this group and because their current sexual experience does not reflect their expected cumulative exposure.Oral rinse sample collection and HPV detection (Roche Linear Array HPV Genotyping Test) are described on the NHANES website and by Gillison et al. Analysis for this study was restricted to individuals 20–69 yo, as the ACASI was not administered to older individuals. 5,001 individuals of this age completed any part of the 2009–10 ACASI, however, analysis was restricted to the 4,256 (85%) who answered the sexual behavior questions.Weighted prevalence estimates were reported. The primary outcomes of interest were history of ever performing oral sex and number of lifetime oral sex partners. These outcomes were stratified by gender, age-cohort, and race/ethnicity. Age categories were chosen a-priori to represent generational cohorts who had expected peak sexual exposure around the 1960s, 1970–85, 1986–99 & 2000–10; subjects were divided by age into these corresponding strata of 60–69 yo , 45–59 yo (“middle-aged”), 30–44 yo (“adults”), and 20–29 yo (“young adults”). To account for the complex NHANES survey design, all analyses used NHANES 2009–2010 MEC sample weights, and thus provided unbiased estimates of behavior and oral HPV prevalence for the civilian, non-institutionalized U.S. population. Variances were computed with SUDAAN software version 10.0.1  using the Taylor series linearization method. Wald F p-values were used to compare weighted prevalence between groups. For continuous variables, weighted means were compared using analysis of variance with Wald F p-values.The association of gender, age-cohort and race with odds of ever having performed oral sex was explored using multivariate logistic regression. Multivariate risk factors for any prevalent oral HPV and with oral HPV16 infection were each similarly modeled. These models were adjusted for potential confounders including: any college education, ever married, ever smoked cigarettes (>100 cigarettes in lifetime), and ever drank alcohol regularly (≥1 drink/month), each yes/no; models for HPV also adjusted for either ever oral sex , or for number of oral sex partners (among 20–59 yo). With the exception of age-cohort, gender, and race, only covariates that were statistically significant were retained in the final multivariate models. Multivariate associations with number of lifetime oral sex partners were similarly explored using linear regression, restricting to individuals <60 years olds since this data was not collected among older individuals. Prevalence ratios (PR) were calculated for oral sexual behaviors of interest and oral HPV16 infection.Age-adjusted incidence rate ratios (IRR) of OSCC were similarly compared by gender, age-cohort, and race, using data from the 2009–10 Surveillance, Epidemiology and End Results (SEER) Program Data from 2,116 men and 2,140 women between the ages of 20 and 69 were included in this analysis  had the oldest age at sexual debut (mean = 18.8 years) and were less likely to have ever performed oral sex (72.7%), or to have had >5 sexual partners (39.4%) , and the youngest age of first performing oral sex (mean = 19.0 yo) compared to all other racial/ethnic groups , p<0.001Reporting fewer lifetime oral than vaginal sexual partners was significantly more common among blacks than whites . Similar differences were observed when comparing whites with other racial/ethnic groups .The behavioral differences observed by gender, age-cohort, and race are consistent with changing HPV-OSCC trends; i.e. males, younger age-cohorts, and whites were significantly more likely to report oral sexual behaviors of interest. To understand whether these behavioral differences could explain the observed demographic differences in oral HPV16 infection and HPV-OSCC incidence, we compared the prevalence ratios of oral sexual behaviors and oral HPV16 infection, with SEER incidence rate ratios of OSCC across these groups of interest .While prevalence of ever oral sex was similar in men and women (PR = 1.03), men were ∼seven times more likely than women to have oral HPV16 infection and ∼five times as likely as women to be diagnosed with OSCC . Number When examining prevalence ratios by age-cohort, older men were moderately less likely to report oral sexual behaviors (PRs 0.77–0.96), but had non-significantly higher oral HPV16 prevalence (PR = 1.3). As expected, OSCC rates increased dramatically with older age  and OSCC (IRR = 1.2) among white compared to black men .To further understand the contribution of these differences in oral sexual behaviors to the demographic differences in oral HPV16 prevalence, multivariate analyses were performed. We first evaluated the association between demographic characteristics of interest and oral sexual behaviors . While mBy contrast, when evaluating the association between these demographic characteristics and prevalent oral HPV, after accounting for oral sexual behavior, age-cohort and white race were not associated with increased odds of oral HPV16 or any oral HPV . Indeed,This study describes gender, age-cohort, and racial/ethnic differences in oral sexual practices in a representative sample of the U.S population. These data reveal that there are differences in oral sexual behaviors when considering gender, age-cohort and race. Men, younger age-cohorts, and whites have higher exposures to the oral sexual behaviors associated with oral HPV16 infection and OSCC. The relative differences in prevalence of oral sexual behaviors, oral HPV16 infection, and OSCC incidence are dramatic when considering gender . Prevaleindependently influence the presence of oral HPV16 infection . This finding may be due to the age-categorization used, as the presence of a bimodal relationship of age and oral HPV prevalence has been suggested when this NHANES data was modeled. Notable age-cohort differences in oral sexual behaviors were observed. Our finding that oral sex was significantly more common among young adults than older adults is consistent with prior data. In recent decades, the age of sexual debut has decreased and the number of lifetime sexual partners has increased. less common in blacks than whites, prevalence of any oral HPV was significantly more common in blacks than whites; it is unclear why this difference was observed. Given that tumor HPV status for SEER OSCCs is unknown, the ratio of HPV-OSCC comparing whites to blacks may be higher than our estimated incidence rate ratio for OSCC . Interestingly, while oral HPV16 infection was for OSCC . HoweverIn our analysis, we show that oral sexual behaviors vary by gender, age-cohort and race. We also describe similar relative differences in prevalence of oral HPV16 infection and incidence of HPV-OSCC. However, after accounting for oral sexual behavior, we learn that the age and race do not independently affect the odds of oral HPV16 infection. Instead, sexual behavior appears to be driving the prevalence of oral HPV16 infection and therefore, is likely to have a similar effect on HPV-OSCC . After controlling for number of oral sexual partners, gender remained associated with oral HPV prevalence, and was elevated but not statistically significantly associated with oral HPV16 prevalence; this suggests that while number of oral sexual partners may be the primary risk factor for oral HPV infection there may be other sexual factors , hormonal factors (sex steroids), or other unknown factors which also influence the observed gender difference in oral HPV prevalence. While this study suggests that changes in oral sexual behavior likely contribute to the observed increases in HPV-OSCC, there may be other unknown factors that may also contribute to these epidemiologic differences.This study has several limitations and strengths. Study limitations include the lack of some sexual data in 60–69 yo, the lack of temporal data, and the limited number of individuals with oral HPV16 infection. The NHANES and SEER data are not linked, and HPV tumor status of OSCC in SEER is unknown. However, since ∼70% of current OSCCs are HPV-positive, This data provide an important context regarding changing sexual behaviors and the role these behaviors may play in the increasing incidence of HPV-OSCC. We learn from this data that oral sexual behaviors are common and that males, younger age groups, and whites report increased oral sexual behaviors, and thus likely have a higher exposure to oral HPV infections. However, age-cohort and race are not independent predictors of oral HPV infections, but rather affect the odds of oral HPV infection through differences in oral sexual behaviors. Similar to U.S. epidemiologic trends, OSCC appears to be rising among younger men in some economically developed countries, while incidence is decreasing or stable OSCC in other regions. Recent research suggests HPV vaccination is likely effective in preventing oral HPV infection among women, with efficacy expected among men as well. File S1Table S1. Characteristics of the 4256 individuals in NHANES study population contributing data to this analysis. Table S2. Sexual behavior and oral HPV prevalence by age-cohorts. Table S3. Multivariate risk factors associated with ever oral sex, oral HPV infection, when controlling for ever performing oral sex, among 20–69 year olds, and stratified by gender.(DOCX)Click here for additional data file.


The expected reduction in cervical cancer incidence as a result of increased access to antiretroviral therapy is yet to be seen. In this study we investigated the effect of HIV infection and treatment on high-risk (hr) human papilloma virus (HPV) prevalence and distribution.Cervical cells from 515 (220 HIV positive and 295 HIV negative) women, recruited during community cervical cancer screening programme in states of Ogun and Lagos and at the cervical cancer screen clinic, Nigerian Institute of Medical Research Lagos were evaluated for the presence of 13 hr HPV genotypes by polymerase chain reaction based assay.The prevalence of high-risk HPV was 19.6% in the studied population. HPV 16 (3.9%), 35 (3.5%), 58 (3.3%) and 31 (3.3%) were the most common hr HPV infections detected. We observed that the prevalence of hr HPV was higher in HIV positives (24.5%) than 15.9% in HIV negative women . A multivariate logistic regression analysis showed a lower hr HPV prevalence in HIV positive women on antiretroviral drugs  and with CD4 count of 500 and above . A higher prevalence of hr HPV was also noted in HIV positive women with CD4 count < 200 cells/mm3 .HPV 16, 35, 58 and 31 genotypes were the most common hr HPV infection in our study group, which could be regarded as high risk general population sample; with higher prevalence of HPV 16 and 35 in HIV positive women than in HIV negative women. The use of antiretroviral drugs was found to be associated with a lower prevalence of hr HPV infection, compared to those not on treatment. This study raises important issues that should be further investigated to enable the development of robust cervical cancer prevention and control strategies for women in our setting. In sub-Saharan Africa, the cervical cancer rates are on the rise, paralleling the HIV epidemic ,2. The oWhile studies from southern and eastern Africa have noted a strong association between HIV, HPV co-infection and the development of genital cancers ,3,9,10, The introduction of antiretroviral therapy in the last few years and the increased access to HIV care in low-income countries have resulted in the improvement of clinical outcomes and life expectancy in HIV infected persons ,13,14. CEpidemiological studies, including meta-analyses mostly from high-income countries, have shown that HIV infected women are at increased risk of infection with hr HPV, yet studies from sub-Saharan Africa that accounts for over 70% of global cervical cancer burden contribute less than 1% of the evidence ,9,19-21.The two currently available vaccines that protect against the acquisition of hr HPV are based on HPV 16 and 18, the reported most common HPV genotypes globally ,22-24. HUnfortunately, several low-income countries, including Nigeria, have approved and deployed the HPV vaccine without such a detailed characterization of the HPV genotypes in their settings . ConsideIncreasingly in Nigeria and other West African countries, a large number of women have been offered an opportunity to HIV treatment ,31. It iSince the introduction of HIV treatment in Nigeria in 2002, over 350,000 HIV infected adults have been initiated on antiretroviral therapy  and 60% This study therefore aims to determine the hr HPV prevalence and distribution by HIV status among women residing in two southwestern Nigerian states of Lagos and Ogun. The effect of HIV infection, immunosuppression and antiretroviral drug use on the hr HPV distribution was also assessed.A total of 536 (231 HIV positive and 305 HIV negative) women were enrolled into the study over a period of 12 months. The HPV results of 21 (3.9%) women were invalid and thus excluded from the analysis. There were no differences in demographic profiles of these 21 women and the remaining 515 (96.1%) with valid HPV results (p = 0.8). Table The socio-demographic characteristics of 515 women with valid HPV results by HIV status is shown in Table The HIV related characteristics of HIV positive women enrolled in the study is presented in Table Table The prevalence of hr HPV was higher in HIV positive (24.5%) women than in HIV negative women . Multiple infection rates were also higher in the HIV positive women (8.2%) compared to 3.9% in HIV negative women . A similar trend was observed in HPV 16  and HPV 35  infections. No difference was observed in other 11 hr HPV genotypes studied.Among the HIV positive women, HPV 16 and 18 constituted 32.7%  among HITo determine the association between HIV infection, treatment and hr HPV infection, we compared the sociodemographic and HIV related characteristics with hr HPV status among the women in the study Tables , 3 and 5At univariate analysis Tables  and 3, aTable Among women enrolled in this study, the overall prevalence of hr HPV infection was 19.6% and the prevalence of multiple hr HPV infection was 5.4%. The prevalence of hr HPV infection in HIV positive women (24.5%) was significantly higher than the prevalence in HIV negative women . The most common hr HPV strains were 16 (3.9%), 35 (3.5%), 58 (3.3%) and 31(3.3%), however it was only HPV 16  and 35  genotypes that were found to have significant association with HIV infection. HIV positive women with a CD4 count of < 200 were found to be at higher risk of hr HPV infection than their counterpart with higher CD4 cell count . The use of antiretroviral drugs  and CD4 cell count above 500 cells  were found to be associated with a lower HPV prevalence than HIV positive women not on therapy or with CD4 count <500 cells respectively.The overall hr HPV prevalence of 19.6% though similar to 18.3% reported from Ibadan, Nigeria , is highHPV 16, 35, 58 and 31 were the most common hr HPV genotype detected in this study, which is similar to findings reported by previous studies in our sub region ,24,34. HIn the studied sample, the prevalence of hr HPV and multiple hr HPV infection were found to be significantly higher in HIV positive women (24.6% and 8.2%) than in HIV negative women (15.9% and 3.9%) respectively . However statistically significant association with HIV infection was noted only for hr genotypes HPV 35 and 16 after controlling for confounders  as HIV positive women on antiretroviral therapy were found to be at lower risk of acquiring hr HPV infection than those not on antiretroviral drugs . This finding supports previous studies and also the hypothesis that ART has the potential to restore the immune response against HPV -44. The The combined relatively low prevalence of HPV vaccine related HPV 16 and 18 reported in this study were comparable both in HIV positive and negative women. Also the combined prevalence in this study corroborate other findings reported from our sub-region ,24,34, sOur results need to be interpreted with caution, as it was conducted in 2 states out of 36 states in Nigeria. However, Lagos as the commercial capital of Nigeria and former administrative capital is the sociocultural, behavioural and diverse ethnic melting point of the country. As such information obtained may be generalizable to the entire country. Secondly, being a cross-sectional study, it may have missed possible variations of specific HPV types overtime. However, as the study was conducted over a one year period this concern may have been taken care of. In addition, the recruitment of about 90% HIV positive group from the clinic and majority of HIV negative women from the community may have introduced some selection bias. This method of recruitment was used because of low HIV prevalence in Lagos and Ogun states. The HIV positive women in the HIV treatment Centre were used in this study because over 65% of them reside in the same setting as the women recruited in the community. Secondly if this strategy was not adopted, sufficient number of HIV positive women and those on antiretroviral therapy will not be recruited. In addition, there were no statistically significant differences in the sociodemographic characteristics between the two groups except in their HIV status. Mindful of the possible effect of this on the validity of our results, we controlled for the possible confounders in multivariate logistic regression using three models.The major strength of this study is the recruitment of women of diverse characteristics including HIV positive and negative women from rural as well as urban areas, those on treatment and not on treatment, thus increasing the feasibility of generalizing the findings. It is also the first study, to the best of our knowledge, in our sub-region that evaluated the effect of HIV infection and treatment on hr HPV prevalence and distribution. In addition two sample size estimates were calculated . This assured us that the study had sufficient statistical power to detect the expected effects.HPV 16, 35, 58 and 31 were the most common hr HPV infections in the population studied and HIV positive women are at higher risk of acquiring HPV infection. The current HPV vaccine prevents genotypes 16 and 18, which accounted for only a minority of hr HPV infection (21.7%) with no significant difference between HIV negative and positive women. The use of antiretroviral drugs was found to be protective against HPV infection. Although further studies with a larger number of participants and wider geographic spread are required in order to comprehensively characterize the HPV distribution in Nigerian women, this study raises important issues such as;1) The effectiveness of the currently available HPV vaccine in our sub region.2) The rate of HPV 35, 58 and 31 in invasive cancer cells in our setting.3) The role of long term antiretroviral drugs on cervical cancer incidence, that should be further investigated to enable the development of a robust cervical cancer prevention and control strategy for all women in our sub region.A cross sectional study among women of known HIV status enrolled into a study primarily to evaluate the utility of direct visual inspection of the cervix in diagnosing premalignant lesions of the cervix in HIV positive women.The study was conducted at Nigerian Institute of Medical Research (NIMR) Lagos, cervical cancer screening Clinic and during community cervical cancer screening outreach programs in Lagos and Ogun States of Nigeria. These are two contiguous states in southwestern Nigeria, which together are inhabited by approximately 13 million people of whom 48.8% are women . The cerNIMR is the apex medical research institution in Nigeria charged with the responsibility to conduct research into diseases of public health importance. The Institute currently provides comprehensive HIV care, treatment and support for over 20,000 patients of whom 62.9% are women. Over seventy-five percent of the patients come from Lagos and Ogun state (where majority of the HIV negative women in this study were recruited during community outreach programmes); with the remaining from the other neigbouring states and west African countries. All services at the Centre are provided free of charge.The study population was adult females aged 18 years and above seen at the cervical cancer screening clinic, Nigerian Institute of Medical Research Lagos and during community cervical cancer screening outreach programmes. While 80% of the HIV positive women were recruited from the cervical cancer screening clinic; the remaining were recruited during the community outreach. The HIV negatives were mainly recruited from the women who attended the various community outreaches cervical screening programme. However it is important to note that over 50% of the patient in our HIV treatment centre are from these same communities. The women were recruited after obtaining a written informed consent in both settings.2P (1-P)/d2, where Zα is the Z statistic for a 95% confidence level, N is the sample size, p is the prevalence of hr HPV, and d is the precision [The sample size for this study was based on a reference prevalence value of hr HPV among women in a community based study conducted in Ibadan, Nigeria of 18.3% . The sturecision . Based ohttp://epitools.ausvet.com.au.http://epitools.ausvet.com.au/content.php?page=2Proportions&P1=0.15&P2=0.4&conf=0.95&Power=0.8&Ratio=1&Tails=2α + Zβ) × ((p1)(1-p1) + (p2)(1-p2))/(p1-p2)2, where we used confidence interval of 95% for computation (Zα = 1.96). Assuming sample sizes of 57 for each group and using an estimate of 40% in ARV drug naive group and 15% in ARV treatment group (difference of 25%), we arrived at 80% power to detect reduction in the rate of hr HPV in the antiretroviral drug group using a 2-tailed test.These site calculators use standard assumptions based on normal approximation to the binomial distribution. The basic formula for comparing two proportions is: N = . The laboratory information  was extracted from the participant’s laboratory results and entered into the relevant portion of the study record form. The entered information was cross checked with the laboratory results by the PI to avoid entry error.Trained Midwives and Doctors conducted the pelvic examination. Samples of cervical exfoliated cells from the ectocervix and endocervix were obtained using a cytobrush. The tip of the cytobrush, which contains cellular material was then placed into a transport medium and stored at 2-8°C immediately. All specimens were coded with the participants study number.HIV test was conducted according to Nigerian National HIV testing and counseling guidelines in women with unconfirmed HIV status before enrolment into the study. Diagnosis was based on positive test on double ELISA based algorithm. However before initiation of antiretroviral therapy the initial test was reconfirmed using Western Blot.The tests were conducted at the Human Virology Laboratory, NIMR. Whole blood of the HIV positive women were used to perform CD4 assay using the Cyflow Counter and Kits  according to the Manufacturer’s instructions. The viral load assay was performed using Roche Amplicor HIV-1 monitor test (version 1.5) according to manufacturer’s instruction.All samples were subjected to centrifugation at 200 rpm for 10 minutes. Precipitated cells were digested using digestion buffer containing protein kinase. HPV DNA was extracted with phenol- chloroform, re-suspended in 100 μl elution buffer and stored at −20°C prior to molecular analysis. Quality and integrity of DNA sample for PCR was verified by amplification of a 268 base pair region of human B-globin gene. Specimens with negative internal control amplification were excluded. All the B-globin positive samples were amplified with HPV4A ACE PC . To detect the genotype of the 13 high risk HPV strains, samples were amplified using the specific primers HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68.Information entered into the study record forms were coded and analyzed using the SPSS version 19.0  statistical packages. The main outcome variable was hr HPV positivity, defined as presence of any of the 13 virus strains mentioned above. First level analysis was to determine the overall prevalence of hr HPV (defined as presence of any of the 13 hr HPV strains) among the women and the prevalence of specific hr HPV strains by HIV status and adjusted for age, treatment history and life time sexual partnership. Since the hr HPV prevalence was not very high, we used multivariate model to estimate the odds ratio for independent variables associated with hr HPV infection. Bivariate analysis was first performed to assess the association of variables with hr HPV infection. All factors independently associated with hr HPV infection in the bivariate analysis were considered as confounders and introduced in a step wise manner in the models. In the first step, we started with the crude association between the variables and main outcome adjusted by age , then we adjusted for HIV status and treatment (Model 2), and finally in a step wise manner introduced the variables that were shown to be statistically associated with hr HPV infection in the bivariate analysis . The results were reported in percentages, Odds ratios (OR) or adjusted ORs and their 95% confidence intervals (CI).Approval for the study was obtained from the Institutional Review Board, Nigerian Institute of Medical Research, Lagos Nigeria. A written informed consent was obtained from the women invited to be part of the study after detailed information about the study. Impartial witnesses who were not members of NIMR staff assisted the consenting process for the low literates.•HIV treatment status: The use of antiretroviral therapy among the HIV positive women for more than 3 months.•Viral load: The level of HIV RNA copies/mL of plasma measured by Roche Ampliclor HIV-1 monitor test (version 1.5).•CD4 Cell count: The level of body’s immunity measured as the number of CD4 T lymphocytes per mm3 of blood by Cyflow Counter and Kits.•High risk Human Papilloma Virus (hr HPV): The 13 oncogenic HPV strains of 16, 18, 31, 33, 35, 39, 45, 51, 56, 58, 59 and 68.The authors declare that they have no competing interests.All the authors participated in the planning and design of the study, and all read and approved the final manuscript. EO conceived the study, recruited the women, performed the statistical analysis and produced the first draft. In collaboration with NF and IAOU, OE defined the protocol for molecular analysis, while NF performed the molecular laboratory studies. PO and KOP reviewed the data analytic plan, the statistical output for accuracy and appropriateness and reviewed all the draft manuscripts for important intellectual content. All authors read and approved the final manuscript.


RPS19 and rs2342700 on TYMS, and prevalent hrHPV infection. Multivariate analysis of hrHPV risk adjusted for age, body mass index, smoking, age of menarche, age at sexual debut, lifetime total number of sexual partners and the total number of pregnancies as covariates, yielded a p-value of 0.071 and 0.010 for rs2305809 and rs2342700, respectively. Our findings in this unique population suggest that a number of genetic risk variants for hrHPV are shared with other population groups. Definitive studies with larger sample sizes and using genome wide approaches are needed to understand the genetic architecture of hrHPV risk in multiple populations.High risk HPV (hrHPV) infection is a necessary cause of cervical cancer but the host genetic determinants of infection are poorly understood. We enrolled 267 women who presented to our cervical cancer screening program in Abuja, Nigeria between April 2012 and August 2012. We collected information on demographic characteristics, risk factors of cervical cancer and obtained samples of blood and cervical exfoliated cells from all participants. We used Roche Linear Array HPV Genotyping Test® to characterize the prevalent HPV according to manufacturer's instruction; Sequenom Mass Array to test 21 SNPs in genes/regions previously associated with hrHPV and regression models to examine independent factors associated with HPV infection. We considered a p<0.05 as significant because this is a replication study. There were 65 women with and 202 women without hrHPV infection. Under the allelic model, we found significant association between two SNPs, rs2305809 on  With 75,000 new cases and 50,000 deaths in 2008, cervical cancer is the commonest cancer in men and women combined and among women in Sub-Saharan Africa et al in a study of Swedish cancer registry data demonstrated an association between heritability and cervical cancer in situin situ estimated the heritability to be between 11 and 15% Persistent high risk HPV infection (hrHPV) is recognized as a necessary but not sufficient cause for Cervical Intraepithelial Neoplasm (CIN) grades 2/3 and cervical cancer (CIN2+) OAS3), sulfatase 1 (SULF1), epidermal dysplasia verruciformis (EV)-associated EVER1 and EVER2 genes, transmembrane channel-like 6 and 8 , peroxiredoxin 3 (PRDX3), ribosomal protein S19 (RPS19), interleukin 2 receptorα (IL2RA), telomere maintenance 2 (TELO2), thymidylate synthetase (TYMS) and complement component 1, r subcomponent-like (C1RL) were associated with components of the cervical carcinogenesis pathway Improved understanding of the etiology, development of new methods of risk stratification including identification of genetic markers of hrHPV infection and CIN2+ will benefit citizens in developed and developing countries. In previous studies, SNPs in 305 genes based on a priori hypotheses of association with HPV infection and cervical cancer were evaluated and several loci of interest identified In this study, we sought to replicate the association between these genes/regions and risk of prevalent hrHPV infection in an African population.The study was approved by the National Health Research Ethics Committee of Nigeria.Women were recruited from 2 cervical cancer screening clinics within the National Hospital, Abuja and University of Abuja Teaching Hospital, Abuja, Nigeria during the period April 2012 to August 2012. To be eligible to enroll in the study, the participants had to be above the age of 18 and provide written informed consent for the study. We excluded women who had had a total hysterectomy, were pregnant or could not provide an informed consent. A total number of 278 women were screened, of which 267 (96%) had complete phenotype data and were successfully genotyped (as described below). These 267 women comprised 65 women with prevalent hrHPV (cases) and 202 controls.Using interviewer administered questionnaires, participants provided information on demographics, socio-economic status, physical activity, smoking, alcohol use, sexual and reproductive history. A gynecologic examination was carried out on each participant. During the gynecologic examination, exfoliated cervical cells were collected from the cervical os. A cervical brush was inserted into the cervical os and rotated 3 full turns to collect exfoliated cells from the cervical os. The head of the brush was subsequently snapped off and placed at the bottom of a specimen transport tube containing 95% ethanol. In addition to the gynecologic specimens collected, blood samples were also collected from the antecubital veins. All samples were transported to the IHVN laboratory. The exfoliated cervical cells were stored at −80°C until the time of analysis. Buffy coat was separated from the whole blood samples and stored till the time of analysis. Data were collected and managed using REDCap electronic data capture tools hosted at the Institute of Human Virology, Nigeria Frozen buffy coat was thawed quickly in a 37°C water bath with mild agitation and stored on ice before beginning the DNA extraction procedure. Approximately 500ul leukocyte-containing fraction was used in the extraction of germline DNA using Gentra Puregene DNA kit® according to manufacturer's instructions. Some 250 µl of DNA hydration solution was added and incubated at 65°C for 1 hour to dissolve the DNA which was then stored. DNA samples were shipped to the Center for Research on Genomics and Global Health, National Human Genome Research Institute, National Institutes of Health, Bethesda for analysis.DNA samples were quantified using Quant-iT™ PicoGreen® dsDNA reagents and kit . After quantification, DNA samples were normalized to 100 ηg/µL. To obtain the quantity of DNA required for the genotyping protocol, samples were further diluted to 3.3 ηg/µL and 2 µl of the later concentration were used to stamp the 384-well plates used for genotyping.The SNPs were genotyped at the National Human Genome Institute  using iPLEX Gold assay on the MassArray platform  as previously described The PCR reaction was at a final volume of 5 µl per well. The PCR mixture contained 10× PCR buffer, MgCl2 (25 mM), dNTPs (25 mM each), Primer mix (500 nM each) and HotStar Taq (5 U/uL). PCR conditions were as follows: denaturation at 95°C for 15 minutes, followed by 45 cycles of 20 seconds at 95°C, 30 seconds at 56°C, 1 minute at 72°C, and a final extension of 3 minutes at 72°C. Then all wells are treated with Shrimp Alkaline Phosphatase (SAP) to remove un-incorporated dNTPs. After optimization of the extension primers and adjustment of the input concentrations, the iPlex extension primer reaction was carried out to identify genotypes following the manufacturer's recommendations. Each sample was run in duplicate. A total of 29 SNPs split into 2 plexes of 13 SNPS and 16 SNPs respectively, were genotyped. The Typer 4.0 software (Sequenom) was used to process and analyze genotype calls.The genotyping assay succeeded for 26 SNPs of the 29 SNPs tried. Four samples were excluded for having failed genotyping at 4 or more loci (i.e. sample success rate <80%). Applying further quality control filters resulted in dropping one SNP that had <90% success rate, 1 SNP for being inconsistent with Hardy-Weinberg equilibrium (HWE) expectations (P (HWE) <0.05), two SNPs for being monomorphic or invariant and one SNP with a Minor Allele Frequency (MAF) <0.05. The final association dataset comprised 21 SNPs on 267 subjects. The overall genotyping rate for this dataset was 98.7%. Concordance of duplicate genotypes was 99.9%.HPV DNA extraction and genotyping were done from ectocervical cell samples. The HPV DNA genotyping was done using LINEAR ARRAY HPV Genotyping Test  based on the principle of DNA amplification by Polymerase Chain Reaction (PCR) and nucleic acid hybridization. This genotyping test qualitatively detects 37 high- and low-risk human papillomavirus genotypes  following a four stage process of sample preparation, DNA amplification through PCR protocol using biotinylated PGMY 09/11 L1 region consensus primer, hybridization and immobilization of the amplified product to HPV genotype-related oligonucleotide probes and the capturing of detectable colored bands of amplified products on a LINEAR ARRAY HPV genotyping strip.The ectocervical samples were into two separate Sarstead tubes. These were centrifuged at 20,000 rpm for 5 minutes to pellet the cells. The liquid supernatant was removed and the tubes were allowed to dry at either 50°C in hot oven or overnight at room temperature in a level 2 safety working cabinet. The dry cell pellets were underwent enzymatic cell lysis, denaturing by detergent and elution at high temperatures. The process isolates the human β-globin gene. The digestion solution is a mixture of 1x Proteinase K-Laureth Digestion Buffer containing proteinase K/ml, 2% (vol/vol) Laureth-12 (50 mM), Tris-HCl (pH 8.5) –1 mM EDTA. A total of 100 µL of the buffer is added to the tubes containing the dry sample. The tubes were vortexed vigorously and digested at 65°C with shaking at 1050 rpm for 30 minutes. The vortexing and digestion condition were repeated once followed by inactivation of the proteinase K lysing enzyme at 95°C for 15 minutes without shaking. Gloves were changed intermittently to avoid cross contamination of sample.Amplification involved a pool of biotinylated primers in a PCR mastermix that specifically binds to designated sequences of nucleotide on the L1 region of the HPV genome. The primer pool which includes additional primer pair for the human β-globin allows for the amplification of HPV DNA from 37 high and low-risk human papillomavirus genotypes and β-globin gene. The amplification of the β-globin gene serves as an internal control for cervical cell adequacy, DNA extraction and amplification. A total of 5 µL of the digested DNA sample was added to 96 well PCR plate with 50 µL of HPV LINEAR ARRAY mastermix solution. The amplification was run on the conventional principle of polymerase activation and denaturing of the viral and human genomic DNA to expose the template DNA for primer to anneal to the target DNA sequence upon cooling. The polymerase extends the primers to produce complementary bases of double stranded HPV or the human β-globin DNA molecules. As the cycle continues, the target nucleic acid sequence on HPV or the human β-globin DNA is amplified. The target nucleic acid amplification in the Linear array genotyping test was enhanced by the inclusion of AmpErase enzyme and deoxyuridine triphosphate (dUTP) in the master mix. The addition of denaturing solution and high temperature above 55°C during the amplification inactivates the AmpErase and prevents the degradation of the target amplicon by the enzyme. The amplification was performed on Applied Biosystems Gold-plated 96-well GeneAmp PCR System 9700 as specified by the manufacturer. Appropriate volumes for controls of contamination and assay sensitivity were added in the 96-well assay.The hybridization assay was performed on Tecan ProfiBlot-48 robots  using the Roche HPV Linear Array detection solutions. The samples were the denatured HPV or the human β-globin amplification products. Each Linear Array HPV genotyping strip coated with HPV, human β-globin and cross-reactive oligonucleotide probes  was placed on wells of the hybridization tray. Denatured biotin labelled amplicons and hybridization buffer were also transferred to the wells. As the reaction progresses with continuous but slow agitation rate, the amplicons sequence hybridizes and matches complementary oligonucleotide probes on the strip.The strips were automatically washed to remove any unbound material followed by binding of Streptavidin-Horseradish Peroxidase conjugate (SA-HRP) to the strips. A second wash removes unbound SA-HRP. The SA-HRPcatalyzes 3,3′,5,5′ -tetramethylbenzidine (TMB) in the presence of Hydrogen peroxide to form detectable blue colour complex that emerges at the point of hybridization. Each HPV type was detected visually by a type-specific probe masked on the genotyping strip except for HPV 52. The detection of the latter depends on a probe that cross hybridizes with HPV 33, 35, 52 and 58. HPV 52 can only be inferred if the cross-reactive probe is hybridized in the absence of any of the HPV 33, 35 and 58 type-specific probes. The HPV genotypes strip result was interpreted by two independent readers across a reference template supplied by the manufacturer along with the detection kit. The consensus result was compared by a third reviewer with custom computer application result reader.p-values <0.05 were considered significant.Association analysis was done using PLINK version 1.07 There were 267 women, 65 with hrHPV and 202 controls enrolled in this study. The characteristics of the participants are shown in RPS19) and rs2342700 (TYMS), were significant under the allelic model (−3 (data not shown). Further adjustment of the model for the age of menarche, age at sexual debut, lifetime total number of sexual partners and the total number of pregnancies yielded a p value of 0.071 and 0.010 for rs2305809 and rs2342700, respectively  0.258 YRI versus 0.642 CEU). This suggests that these two SNPS are significantly associated with hrHPV across multiple ethnic groups despite allele frequency differences. The association findings for all 21 SNPs tested are presented in Two SNPs, rs2305809 (ic model . Adjustiectively . NotablyRPS19) and Thymidylate Synthase gene (TYMS), in the allelic model. This risk remained significant, after adjusting for age, body mass index, smoking, age at menarche, age at sexual debut, lifetime total number of sexual partners and the total number of pregnancies.In this study of genetic risk of prevalent hrHPV infections in Nigerian women, we found significant associations with SNPs on ribosomal protein gene S19  RPS19 has also been shown to be associated with the risk of cervical cancer and persistence of HPV TYMS gene, catalyzes the synthesis of thymidylate, or deoxythymidine monophosphate (dTMP), from deoxyuridine monophosphate (dUMP) with 5,10-methylenetetrahydrofolate as the methyl donor et al and together with the RPS19 finding raises innovative questions about biological pathways that may be associated with hrHPV and possibly other viral infections TYMS, the protein product of the This is the first study investigating genetic determinants of hrHPV infections in Africans and while we confirm 2 of the previously reported genetic risk factors, we did not find any association with the other SNPs that had been previously reported. Our results supports a hitherto unsuspected role for ribosomal and mitochondrial processes in hrHPV infection. It is possible that such processes play a role in other viral oncogenes or are uniquely related to hrHPV infection.RPS19 and TYMS with specific types of hrHPV because of small sample size. Since our study is continuing, we should be able to do this in future. We focused on hrHPV infection in this paper and did not consider cervical cancer or precancer. While these genetic factors may be associated with hrHPV infection, they may not be or have very weak association with cervical cancer. Nevertheless, given the strong association between hrHPV infection and cervical carcinogenesis, they may contribute to understanding the biological mechanisms and risk stratification.Our study has several limitations. We evaluated association between these SNPs and prevalent hrHPV infection which has a weaker association with cervical cancer and precancer. However, we continue to follow up these participants and will be able to evaluate whether these associations change with persistence of hrHPV infection in future. We were not able to evaluate the association of In conclusion, we present novel findings of the genetic risk factors for hrHPV infection among African women. Although several risk factors for hrHPV infection have been established, there has been little work on the genetic risks. Our findings support the use of hypothesis free methods to evaluate these risk factors particular in the African population which is characterized by marked genetic heterogeneity.


Maize x Teosinte Hybrid Cobs Do Not Prevent Crop Gene Introgression. Whether introgression from crops to wild relatives can occur is an important component of transgene risk assessment. In the case of maize, which co-occurs with its wild relative teosinte in Mexico, the possibility of introgression has been controversial. Maize is cross-compatible with teosinte, and spontaneous hybridization is known to occur. Some scientists have hypothesized that the maize x teosinte cob infructescence will prevent progeny dispersal, thus preventing introgression. Motivated by a prior study where we found maize x teosinte hybrid fruits naturally dispersed under field conditions, we tested whether hybrid cobs hold their fruits as tightly as maize cobs. We found the force required to detach hybrid fruits was substantially and significantly less than that for maize. Consequently, we expect that introgression of transgenes from maize into teosinte in Mexico should occur largely unimpeded by the hybrid cob.La mazorca o elote híbrido de maíz x teocintle no impide la introgresión de genes transgénicos provenientes del cultivo. La introgresión entre el maíz cultivado y el maíz silvestre, o teocintle, es un componente importante en la evaluación ambiental relacionada con los riesgos de la introducción de genes transgénicos. La posibilidad de introgresión entre el maíz domesticado y el teocintle ha sido un tema controversial, en particular en México, donde maíz y teocintle coexisten. El maíz es compatible con el teocintle y la hibridización espontánea ocurre entre ellos. Algunos científicos han planteado como hipótesis que al cruzar el maíz con teocintle, la estructura interna de la infrutescencia que sujeta los frutos conocida como la mazorca de maíz o el elote, impide la dispersión de la progenie evitando que la introgresión ocurra. Los resultados de un estudio previo evidencian la dispersión de los frutos híbridos del maíz x teocintle en condiciones naturales. Motivados por estos resultados, hemos decidido investigar si la mazorca o el elote de las infrutescencias del híbrido sujetan los frutos con una fuerza comparable o mayor a la del maíz. Nuestras mediciones implican que la fuerza necesaria para liberar los frutos híbridos son substancial y significativamente menores que aquellas necesarias para desprender los frutos del maíz. Como conclusión sugerimos que en México, la mazorca o el elote no representan una barrera que impida la introgresión de los genes transgénicos del maíz al teocintle. When closely related domesticated and wild plants grow within cross-pollination distance, it is not uncommon for hybridization to occur, resulting in movement of crop genes into wild populations  and what is known as mexicana teosinte (Z. m. ssp. mexicana) have accumulated over the last century. Wilkes  hybrids might be evolutionary dead ends because those hybrids create an infructescence that is a cob, like its domesticated parent  maize, Zea mays ssp. mays, and (3) their F1 hybrids. As part of a prior study at the University of California Riverside Agricultural Experiment Station in Riverside, California  teosinte, We measured the force needed to free the fruits using a Chatillon® DEM 50 force gauge. Modified fish hooks were attached to the device for the purpose of pulling individual fruits from the cobs. The device was set to peak tension; the force was measured in Newtons (N).Fruits were pulled from the distal, middle, and proximal portions of each infructescence; 18 teosinte individuals, 54 hybrid individuals, and 24 maize individuals were used. Each maize and hybrid infructescence was fastened horizontally by a table clamp to hold it firmly. In the case of teosinte, fruits are attached by such a fragile rachis that a horizontal position caused multiple fruits to be dislodged spontaneously. Thus, teosinte infructescences were clamped in place vertically, and grains were dislodged using horizontal force. Because the fruits of hybrids and maize are densely attached, fruits adjacent to and above the selected grain were carefully removed to make room for the hook.Fishhooks of varying sizes were used to best accommodate varying fruit sizes. The fishhook was affixed to the individual fruit and pulled with a forward motion until the fruit detached. The reading for the peak tension was then recorded. All trials were performed by the same person to minimize variation.Given that our data could not be normalized, we used a non-parametric Kruskal-Wallis test with two degrees of freedom to test for differences between the forces necessary to dislodge fruits from our three different samples. This test was followed by a pairwise comparison between the data of the maize, teosinte, and hybrids using a Wilcoxon rank sum test with a Bonferonni adjustment for multiple comparisons so that specific significant differences could be elucidated. The same tests were done to detect differences in dispensability of different portions of the infructescence. Calculations were done using R statistical package . Maize fruits required the most force . Hybrid fruits required intermediate force . Pairwise comparisons using Wilcoxon Rank Sum test with a Bonferroni correction for multiple comparisons demonstrated highly significant differences between the average shattering forces for each pair of comparisons at each of the positions .For all three positions measured on the infructescence, we found substantial differences in the force necessary to detach the fruits of teosinte, maize, and their hybrid Fig. . A Krusk-4. Pairwise comparisons using Wilcoxon Rank Sum test with a Bonferroni correction for multiple comparisons showed no significant difference between the proximal and middle positions, but found the average shattering force for the distal position was significantly lower than both the proximal and middle positions .We also found overall systematic and significant differences in shattering forces between the different infructescence positions. A Kruskal-Wallis test comparing the detachment force for distal, mid, and proximal positions showed significant heterogeneity with a P = 1.40 x 101 hybrid cobs should have the same dispersability as maize cobs (Martinez-Soriano and Leal-Klevezas We found significant differences in the force necessary to detach fruits from teosinte, maize, and their hybrids. Not surprisingly, we found teosinte fruits are easily dispersed. In contrast, depending on the position on the cob, the force necessary to detach maize fruits was more than 50-fold greater than that of teosinte. We found that the hybrids require an intermediate force for detachment, considerably more than teosinte and considerably less than maize. These results contrast with prior claims that maize x teosinte F1 generation hybrid cob is an absolute barrier to subsequent introgression. After all, hybrid cobs would be expected to occur in agro-ecosystems where cobs could be broken into individual fruits or small fragments by a plow, foot, or hoof; units would be small enough to be easily and passively dispersed. Given the finding of significant variation in force required for dispersal between different portions of the infructescence, we would expect that fruits from the distal portion would be more easily dispersed.Our results suggest that the potential for dispersal, and consequently introgression, exists. They are congruent with our prior discovery of dispersed hybrid fruits that we mentioned in the Introduction. They are compatible with our observation that stepping on a hybrid cob typically results in the release of fruits, while stepping on a maize cob typically has no effect. There is no question that hybrids disseminate more efficiently than maize, but less efficiently than teosinte. It is clearly wrong to assume that the FAnother avenue of introgression has been overlooked. Even if the hybrid plants were 100 % seed-sterile, under field conditions they can serve as male parents and backcross to nearby pure teosinte plants because their flowering time overlaps substantially (Guadagnuolo et al. The foregoing data, our experimental results, and our prior observations of dispersed hybrid seed, as well as prior genetic data of others supporting maize-to-teosinte introgression (Blancas et al. 2 recombinants or by fathering seeds on nearby teosinte plants to create BC1s. Transgene flow in itself does not necessarily represent a hazard (NRC Such introgression has immediate consequences for transgenic maize biosafety. It should be assumed that if transgenic maize is grown spatially close enough to teosinte to hybridize, then some level of hybridization is apt to occur if its flowering time overlaps with that of teosinte. The resulting hybrid progeny will have opportunities to continue to the next steps to introgression either by mating among themselves and creating Fzard NRC . But if zard NRC .Teosinte populations could serve as a genetic bridge for transgenes to move from one maize population to the next. Once a transgene has made its way into a teosinte population, unless it confers a fitness disadvantage, it is likely to persist in the wild population, outcrossing to all maize populations within cross-pollination distance (Ellstrand That bridge could occur over time as well as over space. If an introgressed teosinte population persists over generations, the potential exists for that population to outcross with a future nearby maize plantation, reintroducing the transgene locally. In the regions of Mexico where teosinte is a part of the native flora, maize is often grown as landraces, where seed is saved every year and replanted (Ellstrand


Male circumcision protects against cancer of the penis, the invasive form of which is a devastating disease confined almost exclusively to uncircumcised men. Major etiological factors are phimosis, balanitis, and high-risk types of human papillomavirus (HPV), which are more prevalent in the glans penis and coronal sulcus covered by the foreskin, as well as on the penile shaft, of uncircumcised men. Circumcised men clear HPV infections more quickly. Phimosis (a constricted foreskin opening impeding the passage of urine) is confined to uncircumcised men, in whom balanitis (affecting 10%) is more common than in circumcised men. Each is strongly associated with risk of penile cancer. These findings have led to calls for promotion of male circumcision, especially in infancy, to help reduce the global burden of penile cancer. Even more relevant globally is protection from cervical cancer, which is 10-times more common, being much higher in women with uncircumcised male partners. Male circumcision also provides indirect protection against various other infections in women, along with direct protection for men from a number of genital tract infections, including HIV. Given that adverse consequences of medical male circumcision, especially when performed in infancy, are rare, this simple prophylactic procedure should be promoted. Penile cancer is a devastating disease, although uncommon in developed countries. It accounts for less than 1% of all malignancies in men in the USA and 0.1% of cancer deaths. The 5-year survival rate is approximately 50% , 3. The In the USA, Hispanic men have the highest incidence , then Black men , White (0.39), American Indians (0.28), and Asian-Pacific Islanders (0.24) . These fAn extensive review in 2006 concluded that penile cancer is an “emerging problem”, noting that “public health measures, such as prophylactic use of circumcision, have proven successful” . NeonataThe penile cancer incidence data for the USA have to be viewed in the context of the high proportion of circumcised men, especially in older age groups, and the age group affected, where mean age at presentation is 60 years . Thus anIn five major series in the USA, starting in 1932 , not onein situ —only 16 had been circumcised as newborns, that is, incidence was 7.3-fold higher in the uncircumcised [In circumcised men, the very low lifetime risk has been estimated as 1 in 50,000 to 1 in 12,000,000 , 19. Forcumcised , 21. A scumcised . In Denmark, where circumcision prevalence is 2%, penile cancer has nevertheless been decreasing steadily  in paralAs a historical point of interest, Diego Rivera, the famous Mexican muralist, who was renowned for having multiple sexual partners in a country where most men are uncircumcised, developed penile cancer . He refuIn Australia, cases averaged 66 per year over the decade to 2003 . TypicalIn Israel, where almost all males are circumcised, the rate of penile cancer is extremely low: 0.1 per 100,000, that is, is 1/10th that of Denmark , 29.Low- and middle-income countries have a much higher incidence of penile cancer: approximately 3–10 cases per 100,000 per year . In counA statement made in 1973 that “despite overwhelming evidence from urological surgeons that neoplasm of the penis is a lethal disease that can be prevented by removal of the foreskin, some physicians continue to argue against routine newborn circumcision in a highly emotional fashion”  is just in situ or invasive penile cancer. In the USA the proportion of each of these is similar, 45% and 55%, respectively. Invasive penile cancer is lethal, whereas carcinoma in situ is comparatively benign. The former is not necessarily a continuum of the latter [Cancer of the penis can present as carcinoma e latter . HPV is present in most basaloid and warty carcinomas which comprise 50% of cases . SimilarIn a review of 31 studies, representing 1466 penile carcinomas, overall prevalence was 46.9% [There is good reason to suspect that the high-risk HPV types  found in a large proportion of cases, are involved in the causation of many penile cancers , since tHigh-risk HPV types produce flat warts that are normally only visible by application of dilute acetic acid  to the penis. The majority of HPV infections are subclinical; moreover, HPV infection is more prevalent in uncircumcised men having balanoposthitis . High-riThere have been numerous studies comparing HPV prevalence in circumcised and uncircumcised men in different countries, racial groups, and ages , 43–58 .New England Journal of Medicine in 2002 detected HPV in 19.6% of 847 uncircumcised men, compared to only 5.5% of 292 circumcised men  after adjusting for potential confounding factors = 0.37; 95% confidence intervals (CI) = 0.16–0.85; P < .001) [P = .05 level unless otherwise indicated.) In this study, samples were collected from the urethra and glans penis/coronal sulcus. A study at an STI clinic in Copenhagen, Denmark, found that being uncircumcised was associated with a 5-fold higher likelihood of being infected with HPV [A large multinational study published in the  < .001)   . The metA subsequent meta-analysis, published in 2009, examined high-risk HPV types in 14 studies . It assessed data for 5,880 circumcised men and 4,257 uncircumcised men, finding circumcision to be protective, the OR for HPV infection being 0.52 (95% CI = 0.33–0.82)  Figure . This meP < .0001) [High-risk HPV types exhibit a much higher prevalence with proximity to the tip of the penis . In an e< .0001) . In AmstNew England Journal of Medicine, found that 24 months after circumcision the prevalence of high-risk HPV in swabs from the coronal sulcus of the penis was 18% compared to 28% in the uncircumcised men in the control arm of the trial, giving an adjusted prevalence ratio of 0.65, indicative of a 35% protective effect of circumcision [β-globin (meaning cellular DNA was present), HPV was found in 14.9% of the circumcised group compared with 26.5% of the uncircumcised group, pointing to a 44% protective effect [β-globin DNA. At 12 months, high-risk HPV was present in 15.5% of shaft samples from 121 circumcised men and 23.8% of 171 uncircumcised men  = 0.65), indicating 35% protection [The protection afforded by circumcision against HPV prevalence on the penis is supported by RCTs in two localities. One of these, conducted in Uganda and published in the umcision  (Table 1e effect . Multiple effect . The pree effect . These rotection  (Table 1P = .01) [In another report from the Rakai trials, among 230 circumcised men, 14% acquired new HPV infections over 24 months, compared to 25% of 267 uncircumcised men, giving an adjusted incidence rate ratio of 0.58, meaning a 42% protective effect . The acqP = .01) . The incP = .01) . StatistP = .01) . The absIn another RCT, in South Africa, high-risk HPV in urethral swabs was 34% lower in the circumcised group at 21 months after surgery  Table 1. The autP < .001), an association that suggested that high-risk HPV could facilitate HIV acquisition [P = .03) [The South African RCT found that HIV infection was higher in men positive for high-risk HPV (adjusted incidence rate ratio (IRR) = 3.8; uisition . Howeveruisition . In the P = .03) .In uncircumcised men, the moist subpreputial space likely provides a more hospitable environment for infection by viruses than the drier environment of the penis lacking a foreskin –79. In wP = .04). There was no difference for the shaft or scrotum. In the RCT in Rakai, Uganda, clearance of pre-existing HPV was higher in circumcised men at 216 cases per 100 person years in the intervention arm compared to 159 cases per 100 person-years in the control arm—adjusted RR = 1.39 [Although HPV seroprevalence was found to be similar in circumcised and uncircumcised men in a longitudinal study in New Zealand , indicatR = 1.39 . The ability of circumcised men to clear high-risk HPV faster would further explain their lower risk of penile cancer, and of cervical cancer in their female partner(s). Moreover, as mentioned above, in healthy Mexican military men, OR for persistent HPV infection was 10-times higher in those who were not circumcised . InteresFemale-to-male transmission of HPV involves cervix to penis transmission most frequently, with the glans being most vulnerable . The risHigh transmission potential with a low impact on herd immunity means extensive vaccination would be required to substantially reduce the incidence of cancer of the cervix and penis caused by high-risk HPV types . FurtherUrology, the 2009 International Consultation on Urologic Disease Consensus Publishing Group pointed to the well-established role of HPV subtypes in the etiology of cancer of the penis and suggested circumcision and early treatment of phimosis, together with significant changes in global health policy, in addressing this problem [The International Consultation on Penile Cancer determined in November 2008 that the factors associated with invasive penile cancers were high-risk HPV infection (level of evidence 3a–4), phimosis (level of evidence 3a), and balanitis (3a) . In the  problem .Interestingly, 93% of men whose female partner was positive for early signs of cervical cancer by having CIN had the male equivalent, PIN . This unPhimosis is strongly associated with invasive penile carcinoma, the adjusted OR for this being 16 in one study . In factSmegma is a whitish film found under the foreskin of uncircumcised males. It contains bacteria, other microorganisms, dead skin cells, mucous, and other components. Evidence for a role of smegma in the etiology of penile cancer was obtained in an early study . The carin situ [These conditions are all more prevalent in uncircumcised men. Chronic relapsing balanitis of bacterial, mycotic, or viral origin increases the risk of invasive penile cancer , 108. A in situ . Other sin situ , 44% [11in situ , and 50%in situ  of casesin situ . Lichen in situ . Althougin situ . A meta-A cocarcinogenic role of recurrent HSV-2 in penile cancer has also been suggested , 117.The widely used vaginal spermicide, nonoxynol-9, which is abrasive, greatly increases susceptibility of the genital epithelium to HPV16 infection . The vegIn addition, other factors, such as smoking (4.5-fold increase in risk ), poor hIt therefore seems there may be two etiologic routes to penile cancer: one via sexual transmission of oncogenic HPV in younger men and the other, unrelated to HPV, that mostly affects older men (reviewed in ). In eacP < .005) [Risk of prostate cancer correlates with a history of STIs, most consistently syphilis, gonorrhoea, Chlamydia, and HPV –129. In  < .005) . For HPV < .005) . In cont < .005) . A study < .005) . Consist < .005) . The moloney murine leukemia virus homologue known as xenotropic murine leukemia virus (XMRV) (gene: HPC1) was implicated in prostate cancer, initially in patients homozygous for a genetic variant of HPC1 that encodes RNase L, an important component of antiviral defence mechanisms . In a USThe polyomavirus BKV has been found in 19% of cases of prostate cancer in Crete, leading to a suggestion that it could play a role in some of these .Trichomonas vaginalis, the most common bacterial STI, was positively correlated with risk of prostate cancer later in life in the US Physicians Health Study [T. vaginalis in samples collected a decade before prostate cancer was diagnosed. Seropositivity was associated with a 2-fold increased risk for advanced prostate cancer and a 3-fold higher risk for prostate cancer leading to death. Most men who have T. vaginalis infection do not have symptoms. An RCT has found that circumcision can protect against T. vaginalis infection, this organism being 46% lower in the men who had been circumcised [T. vaginalis to be even lower, 51%, the adjusted OR being 0.41 (P = .030).th Study . This stcumcised . An as-tSuch infections may establish a state chronic active inflammation in the prostate, which is associated with a variety of cancers . The ratP = .022), supporting the possibility of circumcision having a protective effect against this cancer .Uncircumcised men have a 1.6- to 2.0-fold higher incidence of prostate cancer compared with circumcised men –146, andAscending passage of a particular STI to the prostate could be a causative factor in prostate cancer. An extended clinical trial of the role played by circumcision in the prevention of prostate cancer is needed , but thiIn the USA, 1 in 6 men develop/get prostate cancer during their lifetime  with an Treatment by radical prostatectomy leads to shortening in length of the penis by an average of 1.3 cm for the flaccid penis and 2.3 cm (one inch) for the stretched penis, although this generally resolves about a year after surgery .A simple cost-benefit analysis for the USA  considerAny discussion of penile cancer in men cannot fail to mention cervical cancer in women. Sexual transmission of high-risk HPV infection is responsible for virtually all cervical cancer. The incidence of cervical cancer is 10 times higher than that of penile cancer, with 12,000 new cases and 4,000 deaths from cervical cancer each year in the USA . AustralThe study in Denmark referred to earlier that found 5-fold lower HPV in circumcised men concluded that “the female partners of circumcised men are less exposed to cervical cancer because these men are less likely to be infected with HPV” .High-risk HPV types 16, 18 and over a dozen other less common types are responsible for virtually every case of cervical cancer –161 and New England Journal of Medicine mentioned earlier irrefutably implicated lack of male circumcision in cervical cancer [P < .001). Monogamous women whose male partner had had 6 or more sexual partners were over 5.6 times more likely to have cervical cancer if their partner was uncircumcised . Male circumcision was also protective in women whose partner had an intermediate sexual behavior risk index . In this study, penile HPV infection was associated with a 4-fold increase in the risk of cervical HPV infection in the female partner, and cervical HPV infection was associated with a 77-fold increase in the risk of cervical cancer. In an accompanying editorial it was suggested that “reduction in risk among female partners of circumcised as compared with uncircumcised men may well be more substantial than reported" [The large, well-designed, multinational study by the International Agency for Research on Cancer published in the l cancer . It invoeported" . NEJM study condom use provided only a slight protective effect—the odds ratio between condom users and nonusers (0.83) was not statistically significant [Genital HPV types are highly infectious and can infect skin throughout the genital region. Skin-to-skin contact that does not extend to actual sexual penetration by an uncircumcised penis could infect women. In the nificant . A studynificant . SquamouP < .001) [An ecological analysis of data from 117 developing countries revealed a cervical cancer incidence of 35 per 100,000 women per year in 51 countries with a low (<20%) circumcision prevalence compared with 20 per 100,000 in 52 countries with a high (>80%) circumcision prevalence ( < .001) . Of all A meta-analysis of 14 studies up until September 2007  found an OR of 0.75 (95% CI 0.49–1.14) for the association between male circumcision and cervical cancer in monogamous women .P = .006) in the women whose male partner had been circumcised, but did not change significantly (+1.6%) in the women whose male partner had remained uncircumcised. The study also found that by 2 years women with circumcised partners had cleared 82% of high-risk HPV acquired during the first year of the trial, compared with 70% for women with uncircumcised partners (P = .14). The authors pointed out that the estimated efficacy of male circumcision in prevention of high-risk HPV (28%) could, for a number of reasons, have been an underestimate.A RCT in Rakai, Uganda, studied the female partners of men who underwent circumcision and those of men who remained uncircumcised . Of thesThus the epidemic of cervical cancer worldwide in women would appear to be facilitated, at least in part, by the lack of circumcision in men. We speculate that in countries that have experienced a downturn in the uptake of neonatal circumcision, as occurred in the USA and to a greater extent in Australia in the late 1970s and 1980s, the incidence of cervical cancer can be expected to increase. This is because these males would now have reached sexual maturity. The higher proportion of uncircumcised men in the male population increases the overall risk to women today, more than would otherwise have been the case if male circumcision prevalence had remained high.Prophylactic vaccines against HPV 16 and 18 became available for administration to girls prior to sexual activity in 2007. These two HPVs represent 70% of the HPV types found in cervical cancers. They were also the two genotypes that had the highest population prevalence in the past. In 2007, however, the CDC reported that HPV 16 and 18 are now less prevalent, type 16 becoming only the 6th most common and HPV18 now being even less prevalent . MoreoveVarious studies have demonstrated increasing infection with genital HPV types at a younger age. In the UK, 5% of girls under 14 had HPV antibodies, indicating current or prior infection . By age HPV can be transmitted to the mouth during oral sex and is an independent risk factor for some oropharyngeal cancers . It should be noted that there might be as many as 200 types of HPV, up to 50 of which have been described in the anogenital region. Most of these range from uncommon to extremely rare. The number of HPV types relevant to screening for cervical cancer risk in the population is approximately 20. Ideally, many expect that vaccination against the most common types (HPV 16 and 18) could prevent two-thirds of cervical cancers. A randomized, placebo-controlled, double-blind trial involving 5,455 women aged 16–24 years found that vaccination reduced the rate of cervical lesions by only 20% . The stuComplete elimination of HPV 16 and 18 from the population by vaccination might, under optimal conditions of uptake and efficacy, take 20–30 years or more. In the meantime at the population level, other oncogenic HPV types not included in vaccines might take over and replace these two types of HPV . Partici Chlamydia trachomatis [Trichomonas vaginalis, bacterial vaginosis, genital ulceration [Given the high cost of vaccinating all girls compared with the lesser cost and proven protective effect of universal male circumcision against a raft of other conditions and diseases in men , 179, thchomatis , 190, thceration –193, andceration .There is now overwhelming evidence that male circumcision affords very strong protection against penile cancer. Unlike the many other conditions that affect up to half of uncircumcised males over their lifetime , penile