High-risk strains of human papillomavirus (HPV) cause cervical cancer. American Indian (AI) women in the Northern Plains of the U.S. have significantly higher incidence and mortality rates for cervical cancer than White women in the same geographical area. We compared HPV prevalence, patterns of HPV types, and infection with multiple HPV types in AI and White women living in South Dakota, U.S.n = 235) or an urban clinic in the same area serving mostly White women (n = 246). Data collection occurred before HPV vaccination was available to study participants. HPV DNA was amplified by using the L1 consensus primer system and an HPV Linear Array detection assay to identify HPV types. We used chi-square tests to compare HPV variables, with percentages standardized by age and lifetime number of sexual partners.We analyzed the HPV status of cervical samples collected in 2006-2008 from women aged 18-65 years who attended two rural AI reservation clinics (p = 0.01) and reported more sexual partners (p < 0.001). A lower percentage of AI women tested negative for HPV infection compared to Whites , and a higher percentage of AI women were infected by oncogenic types . Infections among AI women showed a wider variety and very different pattern of HPV types, including a higher prevalence of mixed HPV infections . AI women had a higher percentage of HPV infections that were not preventable by HPV vaccination .Compared to White women, AI women were younger (A higher HPV burden and a different HPV genotyping profile may contribute to the high rate of cervical cancer among AI women. Cervical cancer is the second most common cause of cancer-related deaths in women worldwide  and a leInfection with HPV, especially HPV-16 and HPV-18, is the most common cause of cervical cancer . PreventThis study was conducted in two AI reservation clinics in South Dakota operated by the Indian Health Service, and in one urban clinic serving primarily White women located in Sioux Falls, South Dakota. The Indian Health Service is an agency of the U.S. Department of Health and Human Services that provides healthcare to AIs and Alaska Natives. Both reservation sites are large, rural, and extremely economically disadvantaged. According to 2004 data, 38,000 tribal members were living within reservation boundaries at Site 1, and in fiscal year 2002, 4,406 women between the ages of 15 and 64 received services at the Indian Health Service unit at this site , and in The urban site in Sioux Falls is a multi-specialty obstetrics and gynecology clinic serving South Dakota and parts of Minnesota, Iowa, and Nebraska, with approximately 30,000 outpatient visits annually. In 2003, the population of Sioux Falls was 133,834, and only 6% of residents lived below the poverty line . The urbOf note, 99% of women attending the two reservation clinics self-identified as AI and 96% of women from the urban clinic self-identified as White. Hereafter, we refer to the rural sample (Site 1 and Site 2) as AI and the urban sample as White.Before enrollment, all women signed an informed consent form to participate in this study. Both this project and the associated consent forms were approved by the institutional review boards of the University of South Dakota and the University of Washington, as well as by the Aberdeen Area Tribal Review Board and the individual participating tribes. Tribal approval included review by the tribal Health and Human Service Committees and formal resolutions from the tribal councils.Potential candidates for this study included all sexually active women with an intact uterus, aged 18-65 years, who presented for annual gynecologic examinations or other non-malignancy related reasons. Data collection for all White women took place before the Gardasil vaccine was approved in 2007, so that no White participants had prior HPV vaccination. Data collection for AI women took place over a longer time frame because of recruitment issues, so that some AI participants were sampled after vaccine approval. However, the Indian Health Service did not pay for HPV vaccination during this time period, and the women recruited for our study were older than the age range initially targeted for the vaccine (11-12 years) , so we aTwo of the authors trained all clinic staff and providers. Training consisted of a formal explanation of the project and the procedures for collecting and handling study samples, followed by direct observation of interactions with patients. Given budget constraints, clinical commitments, and other assignments, study staff were not always available at clinic sites to enroll patients. When present in the clinics, study staff reviewed patients' charts to assess eligibility, and eligible women were then invited by participating physicians to enroll in the study. Pregnant women were included if their enrolling physicians noted no health risk or contraindication, such as preterm labor or vaginal bleeding. In most cases, a Pap test was obtained first, followed by cervical sampling and completion of surveys. Surveys were administered by a trained staff member in a private setting.All HPV genotypes that cause cervical cancer belong to the Alpha genus. These include HPV-51 (Alpha 5); HPV-56 and HPV-66 (Alpha 6); HPV-18, HPV-39, HPV-45, and HPV-59 (Alpha 7); and HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (Alpha 9) ,29. Of npost hoc analysis that showed efficacy against HPV-31 related CIN 2+ lesions. Our analysis focused only on the four HPV types prevented by Gardasil. Because of scientific uncertainty, we did not consider any potential HPV protection through the reported "cross protection" phenomenon [Two HPV vaccines have been approved in the U.S. The first, Gardasil, was approved in 2007 and prevents infection by HPV types 6, 11, 16, and 18. The second, Cervarix, was approved in 2009 and prevents infection by HPV types 16 and 18, with prescription information citing a enomenon .The study was conducted between December 2006 and November 2008. At each site, four to seven providers collected study samples. Cervical swabs were collected from the cervix under visualization. Each cervical sample was immediately chilled on ice and later cataloged and frozen at -80° C. Samples from the urban clinic were collected daily and taken to the study laboratory. Samples from the two Indian Health Service units were batch-shipped on ice overnight to the same laboratory. After all samples were collected, they were processed by the same technician, and HPV analysis was conducted under the same laboratory conditions with HPV test kits bearing the same lot number.After the DNA samples were defrosted, a digestion solution was added to 1.0 ml of sample to achieve a concentration of 200 micrograms of proteinase K and 0.1% laureth-12, then incubated for 1 hour at 56° C. DNA was precipitated from a 150 microliters aliquot of digested material overnight at -20° C in 1.0 ml of absolute ethanol containing 75 microliters of 5.0 M ammonia acetate. After the precipitated DNA was centrifuged at 13,000 g for 30 minutes, supernatant was aspirated and the remaining DNA was dried overnight at room temperature. The pellet was resuspended in 20 microliters of 10 mM Tris and 1 mM EDTA, then incubated for 15 minutes at 95° C. The integrity of the extracted DNA was confirmed by a standard 1% agarose gel electrophoresis followed by staining with ethidium bromide.The DNA extracts were stored at -80° C until amplification by PCR using the L1 consensus primer system and processed for HPV genotyping as described in a previous study . BrieflyWe categorized age in years  and lifetime number of sexual partners , after checking to ensure that the categorized variables performed as well as the continuous variables in the statistical analysis. We chose this approach to simplify presentation of standardized descriptive statistics, and to allow for easier modeling of potential non-linear associations between age and HPV infection. We defined infection status as not infected, infected by a single HPV type, or infected by multiple HPV types. We measured oncogenic infection with three separate and independent variables indicating Alpha 7 infection , Alpha 9 infection , and infection by oncogenic HPV types not classified as Alpha 7 or Alpha 9 . Based on this information we created a global variable indicating infection by at least one oncogenic HPV type.only by HPV types not prevented by the vaccine), 3) partial prevention , or 4) full prevention .We used two sets of variables to compare each woman's HPV infection profile to the four HPV types prevented by the Gardasil vaccine . First, we created four binary indicators of infection by each of the four types. Next, we created a single composite variable to represent hypothetical protection by Gardasil, assuming a counterfactual scenario in which each woman received vaccination before HPV exposure. This variable comprised four mutually exclusive categories, defined as 1) not infected by any HPV type, 2) no prevention , with the chi-square test to compare the distributions. We anticipated that age and number of sexual partners, which are known risk factors for HPV infection, might be differently distributed between AI and White participants. Therefore, we adjusted HPV infection status and oncogenic infection percentages for these variables by using direct standardization, and we present them with 95% confidence intervals (CI). We also calculated the prevalence of each HPV infection status category by age group, using a non-parametric test for trend to evaluate the correlation of age and any HPV infection separately for each clinic population. To visually represent HPV prevalence patterns, we created a bar chart showing the percentage of patients from each clinic population who tested positive for each of 36 different HPV types.For our second study aim, we calculated the percentage of women who were infected by each of the four HPV types  that are prevented by Gardasil. We calculated percentages to compare the distribution of hypothetical vaccine prevention  by clinic population. All percentage values were adjusted for age and number of sexual partners, and are presented with 95% CI, with the chi-square test to compare distributions between the two populations.All analyses were performed by using Stata version 10 . For chin = 235, White n = 246) were available for analysis. The vast majority of study participants were recruited during annual gynecological examinations. All participants had health insurance of some kind. For AI women, healthcare was provided and paid for by the Indian Health Service; the White women were covered by private or government-sponsored insurance .We collected cervical samples from 258 AI women  and from 252 White women at the urban clinic. Participation rates exceeded 90%, with the total number of refusals ranging from four to seven among the three clinic sites. Some faint positive HPV results required adjudication by a third reader, but all positive results were typable. After excluding three women with missing data for HPV status and 26 women with missing data for age or number of sexual partners, data on 481 women (AI p = 0.01) and reported a higher lifetime number of sexual partners (p < 0.001) than their White counterparts.As shown in Table p < 0.001). Overall HPV infection estimates were 42% for AI women and 23% for White women, with prevalence of multiple HPV infection nearly three times higher in AI women than in White women (19% vs. 7%). AI women also had higher standardized prevalence estimates for infection by Alpha 7, Alpha 9, and other oncogenic types, as well as nearly double the prevalence of infection by any oncogenic HPV type, compared to White women . In addition, AI and White women exhibited different distributions of HPV infection across age categories (Table ptrend < 0.001), whereas no age trend was evident among White women (ptrend = 0.23). Figure One hundred thirteen AI women and 57 White women tested positive for HPV. After standardization for age and number of sexual partners, prevalent HPV infection status differed between the two groups p < 0.00. Overallp = 0.03) and HPV-18 (p = 0.02). The two clinic populations differed significantly with regard to the prevalence of infections by HPV types that are not prevented by Gardasil (p < 0.001). The AI group had a strikingly higher percentage of women with HPV infections that would not have been prevented at all by the vaccine (32% vs. 15%), and a higher percentage of women with infections that would have been only partially prevented by the vaccine (5% vs. 2%).Table The primary aims of this study were to examine the prevalence of HPV infection and the distribution of HPV genotypes, as well as the potential benefit of HPV vaccination, in two distinct South Dakota populations. We found that the prevalence of HPV infection among AI women was more than twice that of White women in our samples. Because we are extremely confident that none of the study participants had prior Gardasil vaccination, these differences cannot be attributed, even in part, to discrepancies in vaccine administration or uptake. Increasing age was negatively correlated with HPV infection among AI women, whereas no such trend was detected among the White sample. These findings suggest that efforts to prevent HPV infection must be tailored to the disease burden, the specific HPV prevalence patterns, and the educational and social background of the target community.We previously reported that 22% of AI women in a Northern Plains service unit had HPV infections, a prevalence considerably higher than the 7% observed among AI women in New Mexico  and compThe disparity in HPV prevalence between AI and White women residing in the Northern Plains may contribute to the disproportionately high rate of cervical cancer in the AI population. The Northern Plains incidence rate for cervical cancer in AI women is 11.3 per 100,000, which is 1.5 times higher than in non-Hispanic White women . Recent publications have documented an incidence of cervical cancer among AI women in South Dakota as high as 16.2/100,000, compared to 6.1/100,000 among non-Hispanic White women , and an We also observed different patterns of HPV infection in our two study populations. Overall, AI women showed more variation in prevalent HPV types, and significantly more AI women were infected by HPV types that would not have been prevented by current HPV vaccines. Previous genotyping studies conducted in predominantly White populations have documented that HPV-16 and HPV-18 are implicated in up to 70% of cervical cancer cases in the U.S and worldwide ,33, but Our findings have substantial relevance to public health efforts aimed at improving cervical cancer screening among AI women. The "All Women Count" cancer screening program funded by the State of South Dakota covers Pap testing for eligible women under the age of 30, but likely misses many women with HPV and cervical dysplasia . NotablyThis study is limited in several ways. First, because the study population was a convenience sample drawn from the local service units, our findings may not be pertinent to all AI women living in the Northern Plains of the U.S., and cannot be generalized to other rural or urban populations. Nevertheless, we had an exceptionally high participation rate, and we believe that our samples are an accurate representation of the patient populations, both AI and White, at all clinic sites. Of note, we have additional, unpublished results from 212 AI women living in Montana (ages 16-65), suggesting an HPV prevalence of 37%, very similar to the 42% prevalence observed in our sample of AIs living in South Dakota.Second, our sample size was relatively small compared to many studies of HPV prevalence. Even so, this is the first study to examine HPV prevalence patterns in a high-risk AI population with a geographically localized White comparison group, and despite the small sample size, we were able to detect statistically significant differences between the groups, with important clinical and public health implications.Third, we cannot disentangle the effects of urban residence from race. However, very few White women live on reservations, and only a negligible proportion of AI women were evaluated in the regionally-matched urban clinic, making the selection of samples evenly distributed between race and region virtually impossible.Fourth, we did not use prior receipt of Gardasil vaccination as an exclusion criterion. Nevertheless, we are confident that our results remain unaffected by this limitation, since data on all White women and many AI women were collected before vaccine approval. Further, the AI women who were sampled in 2007-2008 were older than the target age range for HPV vaccination and had no insurance coverage from the Indian Health Service for this purpose. Even in the unlikely event that a few AI participants had prior vaccination, we would expect any resulting bias to be in the direction of less prevalent infection in this group.Fifth, the absence of HPV vaccination among study participants prevented us from considering population differences in vaccine uptake; future research should examine this phenomenon more closely in minority communities.Finally, we did not consider the additional prevention of HPV-31 provided by the Cervarix vaccine. Nevertheless, only four AI women, and no White women, were infected by HPV-31, so the inclusion of Cervarix in this analysis would have added confusion without altering our conclusions.This is the first study of HPV genotyping in a community-based sample of AI and White women. Our results suggest the existence of substantial differences in HPV prevalence and infection patterns among AI and White populations, highlighting the need for research on HPV in high-risk minority groups. If confirmed by future studies, our results might help to explain the documented cervical cancer disparities experienced by AI women. As it is, our findings underscore the need to evaluate vaccine efficacy in this population.AI: American Indian; CI: confidence interval; EDTA: ethylenediaminetetraacetic acid; HPV: human papillomavirus; PCR: polymerase chain reaction.DSG reports that he is a member of the speakers' bureaus for Merck and GlaxoSmithKline, which pay him to give talks on HPV vaccination. None of the other authors have any commercial or other association that might pose a conflict of interest.DCSG participated in the design and coordination of the study, carried out the molecular genetic studies, collected patient samples, and assisted in drafting the manuscript. MCB participated in the design and coordination of the study and collected patient samples. CJM performed the statistical analyses and assisted in drafting the manuscript. DMM collected patient samples and participated in the data analysis. SCC assisted with interpretation. DSB participated in coordinating the study, supervised the overall conduct of the study, and assisted in drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/252/prepub


The presence of IgG antibodies to HPV-16 L1-virus like particles (VLPs) in serum has been reported as a result of persistent exposure to the virus and as a marker of disease progression. However, detection of VLP-specific antibodies in sera does not always indicate a malignant lesion as positive results may also be due to a nonmalignant viral infection. Furthermore, malignant lesions are associated with an increased antibody titer for E6 and E7 proteins. The aim of this study was to develop an ELISA using a novel chimeric virus-like particle (cVLP) encoding an L1 protein fused with a string of HPV-16 E6 and E7 seroreactive epitopes to its C-terminus to be used for detection of HPV-16 specific antibodies in patients with cervical intraepithelial lesion grade 1 (CIN 1).The sera of 30 patients with CIN 1 who also tested positive for HPV-16 DNA and of 30 age-matched normal donors negative for HPV infection were tested for the presence of IgG antibodies specific for either VLP-L1 (HPV-16 L1), gVLP (derived from Gardasil), or cVLP by ELISA. The cVLP-reactive sera yielded two distinct groups of results: (H) reactivity levels that presented very strong cVLP-specific titers, and (L) reactivity levels with significantly lower titers similar to those obtained with VLP-L1 and gVLP antigens. Additionally, the sera that presented the higher cVLP titers closely matched those that had significantly stronger reactivity to E6 and E7 epitopes. Interestingly, the samples with the highest titers corresponded to patients with the higher numbers of sexual partners and pregnancies. On the other hand only 4 out of the 12 sera that harbored antibodies with VLP neutralizing ability corresponded to the group with high cVLP antibody titers.We report for the first time that chimeric particles containing HPV-16 L1 protein fused with E6 and E7 seroreactive epitopes enable much better detection of IgG antibodies in the sera of CIN 1 patients positive for HPV-16 infection than those obtained with VLPs containing only the HPV-16 L1 protein. We also found that the sera with higher cVLP antibody titers corresponded to patients with more sexual partners and pregnancies, and not always with to those with a high neutralizing activity. This novel assay could help in the development of a tool to evaluate cervical cancer risk. Infection of the genital epithelium with human papillomavirus (HPV) is a common sexually transmitted disease, as well as a significant public health burden in developing countries. Most cervicovaginal HPV infections are clinically inapparent and produce no cytological abnormalities. However, persistent infections with certain high-risk HPV strains can cause cervical cancer, which is the second most common cancer in women worldwide and accounts for 250,000 deaths annually . High-riStudies of the immune response to HPV have progressed slowly, due in part to the lack of suitable reagents for immunologic assays. During the last two decades, serologic studies have been important in understanding the natural history of HPV infections and efforts are ongoing to develop reliable genotype-specific assays. In this context, the observation that HPV capsid proteins generated in eukaryotic expression systems self-assemble into virus-like particles (VLPs) has enabled the use of these particles as reagents for studies of the immune response to HPV -9.in situ or invasive cervical cancer [A number of studies have demonstrated that human sera can react with HPV VLPs and that this reactivity is largely HPV- and strain-specific -12. In al cancer ,17. HoweThe presence of antibodies specific for the viral tumor antigens E6 and E7 is considered the best marker for cervical cancer progression. This is because the magnitude of the antibody response is influenced by the stage of disease and the size of tumor mass -20. OptiIn the present study, we developed an ELISA to detect cVLP-specific IgG antibodies in serum samples from patients with incipient lesions such as cervical intraepithelial lesion grade 1 (CIN 1) that were also positive for HPV-16 infection. This ELISA utilizes a novel chimeric VLP (cVLP) composed of HPV-16 L1 and a string of E6 and E7 epitopes fused to the C-terminus . InteresA total of 230 female patients with CIN 1 were analyzed for the presence of HPV DNA by PCR, and only the sera from the 30 patients who tested positive for HPV-16 infection were included in this study. On the other hand, serum samples from 30 women without clinical or molecular evidence of HPV infection were used as negative control for these experiments. The mean age of the experimental subjects was of 32.1(16-43) years while in the normal donors was of 36.4(19-47). The reported mean of sexual partners and pregnancies of the patients was of 1.7(1-5) and 2.5(1-5) respectively, while in the normal donors was of 1.2(1-3) and 1.1(1-3). The sera and clinical data from all participants were taken after their informed consent and according to the ethical and confidentiality requirements concerning work with human samples of the institutions involved.®, and contained the serotypes HPV-16, -18, -6 and -11. As expected, the mean absorbance value for the levels of IgG antibodies detected in sera from the CIN 1 patients using either gVLP, VLP-L1, or cVLP were higher than those from normal donors  or a chimeric particle containing HPV-16 L1 and a string of E6 and E7 known antigenic peptides fused to the C-terminus (cVLP). The third type of VLP was derived from the commercial vaccine Gardasils Figure . In the s Figure . Accordis Figure . It is is Figure , Table 1Several studies have shown that infection with HPV induces VLP-specific antibodies with neutralizing ability that are maintained over long periods of time ,23-25. Tin situ or invasive cervical cancer. Moreover, the presence of anti-E6 and anti-E7 antibodies is associated with malignant progression of these cancers [Persistent HPV-16 VLP serum antibodies are associated with an increased risk of developing either carcinoma  cancers ,29. Opti cancers . We desiin vitro or produced in bacteria or baculovirus/insect cell systems. These studies found that the prevalence of anti-E6 and anti-E7 antibodies varies from 16% to 60% in people with cervical uterine cancer, versus only 4% to 20% in normal donors [The integration of HPV DNA increases oncogene expression at various time points of viral pathogenesis and malignant transformation . Additiol donors -37. In tResults from several studies have indicated that HPV infection induces highly strain-specific neutralizing antibodies based on the fact that recipients injected with HPV-16, -18, -31, -52 and -58 VLPs develop neutralizing antibodies specific for homologous HPVs efficiently and with very limited cross-neutralization . FurtherIt would be interesting to repeat the present experiments using a large number of serum samples from patients infected with HPV strains other than HPV-16 and with sera from patients with CIN 1 and cervical cancer. We hypothesize that the use of ELISAs using the cVLPs described herein may be useful in monitoring the pre-existing serostatus of women who have been vaccinated with the recently introduced prophylactic VLP-based vaccines. Furthermore, these ELISAs may be useful in evaluating the vaccine response in women with or without preexisting IgG antibodies specific for viral particles, especially those antibodies specific for high-risk viruses.in situ or invasive cervical cancer [Lastly, we think that the ELISA described herein may prove to be a useful tool in evaluating the risk of cancer progression in women that have high titers of sera antibodies specific for L1 plus E6 and E7 epitopes. Supporting this idea is the fact that longitudinal studies have demonstrated that women with incident infection who have seroconverted were 5 times more likely to have SIL than women who had not seroconverted ,25. Addil cancer ,17, makiThe study population was selected from patients attending the National Center for Clinics of Dysplasias (CENACLID), General Hospital of Mexico, and the Department of Gynecology H.G.Z No. 2-A Troncoso, IMSS, Mexico City, Mexico. Serum samples were collected between February 2008 and July 2010 and informed consent was obtained from all participants. Human material was handled according to institutional experimentation and safety guidelines. CENACLID provides gynecological services to women referred for colposcopy because of abnormal cytology and women who do not have a history of cervical abnormalities but ask for a routine examination. In the present study, women in both groups were analyzed. All women underwent cytological and histopathological analysis of colposcopy-directed biopsies. Two experienced cytotechnologists independently examined all Pap smears. Samples with an inconsistent diagnosis were excluded from the study. Cytology diagnoses were classified according to the Bethesda System while the histopatological one, of the Cervical Intraepithelial Neoplasia Grade I (CIN I), was according to Richardt . Samplesg for 15 min. Serum samples were stored at -70°C until tested. Investigators who performed molecular and immunological assays were not aware of the clinical status of samples. To avoid contamination, cases and controls were tested in separate batches.Blood samples, as well as cervical punch biopsy material or cervical cells adequate for HPV DNA measurement, were obtained both from patients with CIN 1 who were also positive for HPV-16 infection and from negative controls. Cervical cells were collected by gently swabbing the ectocervix with a cytobrush (Digene). Cervical cells and biopsied tissues were placed immediately in tubes containing sterile, contaminant-free PBS (Roche) and processed the same day. Serum was isolated from blood samples by centrifugation at 9000 The HPV Test Hybrid Capture II kit (Digene) was used to detect high-risk HPV strains  or low-risk strains . DNA (250-500 ng in 20 μl) was extracted from cervical cells or tissues and placed in a tube containing 30 μl specimen transport medium. NaOH-based denaturation reagent (25 μl) was added to each sample and the tubes were mixed vigorously and incubated at 65°C for 45 min. Hybridization and hybrid detection were performed according to the manufacturer's instructions. Carrier DNA and plasmid DNA containing the HPV-16 or HPV-11 genome were used as negative and positive calibrators, respectively, and were run in triplicate with each test. An assay was considered valid only when the results from the negative and positive calibrators showed a coefficient of variation ≤15% and the positive-to-negative calibrator mean values ratio was ≥2.0. The cut-off value for positivity was calculated for each assay and was defined as the mean relative light units (RLU) value of the positive calibrator. Samples that were positive for low-risk types were excluded from this study.All reagents used in the isolation and amplification of DNA were purchased from Gibco-BRL. Biopsies were treated with proteinase K as described elsewhere . DNA was® (gVLP). VLPs derived from transgenic plants were purified by using immunoaffinity columns, as described previously [® vaccine to 4B CNBr-Sepharose (Sigma). The columns were washed with 50 vol of washing buffer , followed by 50 vols of the same buffer without NP-40. VLPs were eluted with 100 mM HCl-glycine, pH 4.0. The VLP-containing fractions were neutralized with 100 mM Tris-HCl (pH 9.0) and dialyzed against PBS overnight. The VLPs obtained from transgenic plants was verified by transmission electron microscope, as previously reported [We previously reported the expression in tomato plants of VLPs containing the HPV-16 L1 sequence (VLP-L1) and a chimeric particle (cVLP) containing the HPV-16 L1 sequence and a string of sequences encoding T cell epitopes fused to the C-terminus, which codes for the amino acids 16-30 from E6 and 37-54, 49-57, and 86-93 from E7 . VLPs useviously . Brieflyreported .® vaccine dilutions containing from 10 to 160 ng of HPV-16 VLPs/100 μL were used as standard. All samples were tested in triplicate. Plates were incubated for 16 h at 4°C, washed twice in TBS-0.1% Tween-20, blocked with 2% w/v bovine serum albumin (BSA) in TBS-0.1% Tween-20, and incubated for 2 h at 37°C. Plates were washed four times, and incubated with sera from women vaccinated with three doses of commercial vaccine at 1:500 in blocking buffer. After 2 h at 37°C, plates were washed 6 times and incubated with 1:5000 rabbit-anti-human IgG . Plates were incubated for an additional 2 h at 37°C. After eight washes, alkaline phosphatase substrate (Sigma) was diluted in a 10% diethanolamine solution (pH 9.8) and added to the plates. The absorbance at 405 nm was measured by ELISA plate reader. The concentration of VLPs was calculated by average of three repeats.The quantity of plant-purified VLP-L1/cVLP was measured by ELISA. For this purpose, ELISA plates (Corning) were coated with 100 μl of chromatography-purified VLP-L1 and cVLPs. GardasilThe following antigenic peptides were synthesized by Invitrogen: From HPV-16 E6 protein, 16-30 PRKLPQLCTELQTTI ,45; fromThe specific titers of serum antibodies specific for VLPs and peptides from in subjects with LSIL and negative controls were determined by ELISA. ELISAs were performed with gVLP or VLP-L1 and cVLP obtained from transgenic plants, as well as the antigenic peptide encoding sequences contained in the C-terminus of the cVLPs, were used as antigens in ELISA assays. For this purpose, each type of VLP was diluted in PBS at 1 μg/ml, and the peptides were diluted at 5 μg/ml in sodium carbonate/bicarbonate buffer . ELISA plates were coated with 100 μl of VLPs and incubated for 16 h at 4°C. Plates were then washed four times with TBS containing 0.1% Tween 20 (TBS/Tween 20). Non-specific binding sites were blocked with 200 μl 2% BSA in TBS/Tween-20 for 2 h at 37°C. After washing, 100-μl serum samples (diluted from 1:100) were added in triplicate, and the plates were incubated at 37°C for an additional 2 h. After washing, alkaline phosphatase-conjugated rabbit-anti human IgG (Dako) was diluted in blocking buffer at 1:500 and 100 μl was added to each well. The plates were then incubated for 2 h at 37°C. After washing, alkaline phosphatase substrate Sigma 104 was diluted in a 10% diethanolamine solution (pH 9.8) and added to the plates. The absorbance at 405 nm was measured by an ELISA plate reader. The assay was considered valid only when the coefficient of variation of the triplicates was ≤10%. Additionally, all samples were tested in two wells not coated with VLPs or peptides to define non-specific reactivity. The final ELISA value was calculated by subtracting the non-specific reactivity mean absorbance from the triplicate mean absorbance.To control for inter-assay variation, positive IgG controls selected from a previous study  were incHemagglutination inhibition assays were performed according to Roden et al. . Mouse bAll numerical data are expressed as an average of values obtained ± standard deviation (SD), and all samples were tested in triplicate. The Wilcoxon signed-rank test and Student's t-test were used to compare the mean signal strength (absorbance) of the various groups. All tests were two-tailed and P < .05 was considered statistically significant.The authors declare that they have no competing interests.AMG conceived of the study and wrote the manuscript. MAGL participated in the design and production of VLPs in plants. BWS participated in discussion of results and revision of the manuscript. GPR carried out the molecular studies and cultures of tomato plants. JHM and MTG performed the ELISA assays with VLPs and peptides. KPS collected sera samples of the patients and normal donors. YTG carried out the purification of VLPs. ESO and HS obtained tissue samples and performed PCR analysis for HPV-16 infection. MLMG participated in the design and coordination of the study as well as in discussion of results. All authors read and approved the final manuscript.


Betapapillomavirus, species β2, and is most closely related to HPV-145.We report the cloning and characterization of a novel human papillomavirus (HPV), now officially recognized as HPV-174, isolated from a cutaneous squamous cell carcinoma. HPV-174 has a genome organization characteristic of cutaneous HPV types, clusters to the genus  Betapapillomavirus (β-PV) have been found in association with various benign and malignant cutaneous lesions, including nonmelanoma skin cancer in immunocompromised individuals; however, the role of these viruses in tumor formation is still unclear  from the genus  unclear . Current species . We repo-PV broad-range nested PCR  was obtained using βsted PCR . A refer6-GGT), two putative TATA boxes (TATAAA) of the E6 promotor, and the putative polyadenylation site (ATATAA) necessary for processing viral late gene mRNA transcripts  and five early  and two late (L1 and L2) genes but no E5 open reading frame (ORF). Multiple binding sites for transcriptional regulatory factors  were identified within the LCR genomic region using SIGSCAN software V4.05 ( protein . The HPVHF930491.The complete genome sequence of HPV-174 is available in the ENA, GenBank, and DDBJ databases under the accession number


Human Papillomavirus (HPV) in this disease. The purpose of this study was to determine the prevalence of HPV in breast cancer.The etiology and the molecular mechanisms related to breast carcinogenesis remain poorly understood. Some recent reports have examined the role of Sixty one fresh frozen breast cancers samples were analyzed. Samples were tested for HPV by PCR, and products were automatically sequenced. Findings were correlated with clinical and pathological characteristics.2 test). Sequence analysis in a subgroup of cases indicates the prevalence of low risk HPV11, followed by high risk HPV16. We found no HPV transcriptional activity.The HPV DNA prevalence in the breast cancer samples was 26% (16/61). Clinical parameters were not statistically associated with HPV presence (p>0.05 χThe present study demonstrated for the first time in Argentina the presence of HPV in a proportion of the malignant breast tissues. This finding suggests that HPV may have a biological significance in breast carcinogenesis. Breast cancer is the most frequently diagnosed malignancy in women in many populations in vitroCurrently a large number of infectious agents have been described to either cause or contribute to specific human cancers Human papillomaviruses (HPVs) species, family Papillomaviridae, genus Alphapapillomavirus, are small DNA viruses that infect epithelial cells of the skin and mucosa. HPV is the causal agent of cervix-uterine cancer and anogenital malignancies. However, HPV has been detected in extragenital tumors such as oral, esophageal, tonsillar, laryngea and lungs Considering the controversial reports on the association of HPV in breast carcinomas, the aim of this study was to explore HPV presence in this neoplasia among a female Argentinean population. Also to examine HPV genotype, particularly those of high risk and correlate their presence with clinical characteristics. This study demonstrated for the first time in Argentina the presence of HPV in a proportion of the malignant breast tissues, with a prevalence of low risk HPV11, followed by high risk HPV16.The study was conducted on 61 fresh tissue biopsies of breast carcinoma, collected without any preselection criteria from the pathological archives of the Pathology Service of M. Villegas de Martinez Hospital. Tumors, which were typed according to the American Joint Committee on Cancer, included: 43 invasive ductal carcinomas, 10 invasive lobular, 2 papillary, 2 tubular, 1 adenocystic, 1 mucinous, 1 aprocrine and 1 comedocarcinoma. Patients’ age ranged from 35 to 92 years (median age 57 years).This study has the approval of the Institutional Review Board and the Ethics Board of both M. Villegas de Martinez Hospital and Ricardo Gutierrez Children Hospital and is also in accordance with the Helsinki Declaration of 1975. A written informed consent was obtained from every patient.DNA was extracted from tumor fresh biopsies using QIAamp DNA Mini Kit  following manufacturer’s instructions.5′-CTTATACTAAATGTCAAATAAAAA-3′ generate 140 to 150 bp fragment of the L1 region of the virus The presence of HPV DNA sequences was verified by amplification with two sets of primers. A first round with degenerated primers MY09, 5′-CGTCCMARRGGAWACTGATC-3′ and MY11, 5′-GCMCAGGGWCATAAYAATGG -3′, amplifying 450 bp long fragment in highly conserved region in L1 gene; and a second round with consensus primers GP5+, 5′-TTTGTTACTGTGGTAGATACTAC- 3′ and GP6+, 5′-GAAGAGCCAAG GACAGGTAC-3′ and 5′ -CAACTTCATCCACGTTCACC-3′, were incubated with the template DNA. The presence of a 260- base pair (bp) amplification product served as a control to monitor the amplification ability of a single copy gene.In a separate reaction tube, a set of primers for the beta-globin gene, HPV PCR products were separated by electrophoresis in 2% agarose gel stained with ethidium bromide and purified with QIAEXII gel extraction kit (Qiagen GmbH) according to manufacturer’s instructions. These purified PCR products were directly sequenced using Big Dye Terminator v3.1 kit  in an automated Genetic Analyzer 3130×l (Applied Biosystems). HPV sequences were compared with published ones of known HPV types. At least two independent sequencing reactions were performed with the inner primers to confirm each sequence.CAGAGCTGCAAACAACTATAC 3′, rev 5′ AGTGGC TTTTGACAGTTAATA C 3', E7 HPV16 fwd 5′ GACAAGCAGAACCGGACAG 3′, rev5′ ATTCCTAGTGTGCCCATTAACA 3′, E6 HPV18 fwd 5′ GCGACCCTACAA GCTACCTGA T 3′, rev 5′ GCACCGCAGGCACCTTATTA 3′, E7 HPV18 fwd 5′ TGTCACGAGCAATTAAGCGACT 3′, rev 5′ CACACAAAGGACAGGGTGTTCA 3′.Total RNA in fresh biopsies was extracted using Trizol according to manufacturer instructions. In order to confirm efficient extraction, the quality of RNA from each sample was assessed by RT-PCR amplification of the ubiquitously expressed phosphoglycerate kinase gene (PGK), which acted as a control to ensure that only RNA was amplified (247 pb fragment), along with the absence of contaminating DNA . Good quality RNA samples were chosen and 2 ug were used for cDNA synthesis using Superscript II RT kit  according to the manufacturer’s instructions. Amplification was performed with specific primers (kindly given by Dr Luis Jave Suárez) for E6 HPV16 fwd 5′ Statistical analysis was performed using GraphPad Prism 4 software . Fisher’s exact test or Chi square tests (χ2) were used for statistical analysis when appropriate.Patients’ clinical characteristics and HPV association are shown in In Latin American population, Cantu de León et al described a 29.4% of HPV association with Mexican breast carcinoma 2 test), in accordance with previous reports In this report, clinical parameters such as tumor histology, axillary lymph node status or estrogen and progesterone receptors were not statistically associated with HPV presence , particularly of the cervix, HPV 16, 18, 31, 33, 35, 45, and minor types. The most important players of cervical cancers are HPV 16, found in 50–70% of cases, and HPV 18, found in 7–20% of cases HPV types in Latin American breast carcinoma patients, displayed prevalence for high risk HPV. In fact, in Chile only the HPV-16 genotype was present in positive breast carcinomas without co-infections with other HPV genotypes HPV analysis performed so far in this report enables exclusively evaluation of the presence of viral DNA, but do not inform on the possible viral activity or infection productivity. Therefore, evaluation of E6 and E7 mRNA in high risk genotypes may represent a good marker for HPV involvement in malignant transformation. In fact, a strong association between high risk mRNA-HPV presence and risk of neoplastic progression in cervical lesions has been described Although the route of transmission for the virus has not been determined, women positive for both breast and cervical cancers were found to be infected with the same HPV type in both tumors The present study demonstrated for the first time in Argentina the presence of HPV in a significant proportion of the malignant breast tissues analyzed. Presence of low risk types, together with the absence of transcriptional activity for high risk types suggest that HPV presence may have a biological significance in breast carcinogenesis as a contributing factor to tumor development, but not as the tumor driven force. It should be pointed out that these observations need additional studies to be substantiated, especially by monitoring future breast cancer incidence amongst women vaccinated against high risk HPV type. Meanwhile, the present results may represent an important issue in virus-associated cancer, which represent potential tools for the development of specific therapies leading for patients with breast cancer.


Papillomavirus (HPV) infection is particularly burdensome for women infected with human immunodeficiency virus (HIV), which increases their risk of developing cervical lesions and cancer (CC). We conducted a molecular study of the distribution of cervical HPV genotypes and the risk factors for this infection in HIV-infected Brazilian women.Human 3 (37.6%) had a two-fold greater risk of HPV infection than those with > 350 cells/mm3 (26.4%). HPV was associated with parity ≥3, hormonal contraceptive use and current smoker. HPV infection occurred with high frequency (46.6%) but a low frequency of cervical abnormalities was detected (7.30%), mainly low-grade squamous intraephitelial cervical lesions (LSIL) (84.6%). A high frequency of multiple HPV infections was detected (23.0%), and the most frequent HPV genotype was HPV-72 (6.7%), followed by −16, -31 and -51 (6.14% each).Cervical and endocervical samples for Papanicolaou screening and HPV detection were collected from 178 HIV-infected women using highly active antiretroviral therapy (HAART) of Maringá city/Brazil. Risk factors were assessed using a standardized questionnaire, and the data regarding to HIV infection from medical records. HPV was detected by polymerase chain reaction (PCR), and genotyping using PCR-restriction fragment length polymorphism analysis. HIV infection was well controlled, but women with a current CD4+ T lymphocyte count between 200–350 cells/mmWe showed that HAART use does not protect HIV-infected women from HPV, but appear to exert some protection against cervical lesions development. This study provides other important information about risk factors and cervical HPV in HIV-infected women, which can contribute to planning protocols. It is estimated that in 2020, cervical cancer (CC) will be diagnosed in over 665,035 women worldwide, and 357,852 will die as a result . The frePapillomavirus (HPV) and CC has been well established [The association between persistent high-risk (HR) Human ablished . HPV infablished . In Brazablished . While tablished ,7.Studies have unanimously showed that HIV-infected women are more commonly infected with non-16 and −18 HR-HPV genotypes, such as 52 and 58 . Given tConsidering that epidemiological data from different populations are required for public policies addressing CC prevention in HIV-infected women and that few studies from Brazil and Latin America have collected these data, we conducted a molecular study of the distribution of cervical HPV genotypes and the risk factors associated with this infection in HIV-infected Brazilian women.In total, 178 HIV-infected women using HAART, aged 18 to 66 years, who attended the Specialized Assistance Service (SAE) for sexually transmitted diseases (STD)/AIDS of Maringá city/Southern Brazil, from April 1 to October 30, 2011, were included. The inclusion criterion required that the women had been diagnosed twice with HIV/AIDS using different methods and using HAART. The exclusion criteria were previous hysterectomy, current or recent pregnancy, age younger than 18 years, and no history of sexual activity.Of the 424 HIV-infected women enrolled in the SAE, 100 were excluded, and a total of 324 were eligible for the study. The sample size was calculated with an HPV prevalence of 50%, confidence interval of 95%, error estimate of 5%. With an increase of 10% for possible participant losses, the total sample size was fixed at 178 randomly selected women.The women were interviewed using a standardized questionnaire to obtain socio-economic and demographic information, obstetric and gynecologic history and data on their sexual behavior. Data regarding HIV infection were obtained from SAE medical records. A single nursing contacted all of the women, administered the questionnaire and collected the cervical samples. This project was approved by the Committee for Ethics in Research Involving Humans at the State University of Maringá (UEM)/Paraná, Brazil (nº 085/2011).HpyCH4V [Ecto/endocervical samples were collected with an Ayre’s spatula and cytobrush for cervical cytology (Papanicolaou screening) and polymerase chain reaction (PCR); the samples were suspended in 1 ml of 0.9% NaCl solution and stored at -20°C. The cytological smears were sent to the Clinical Cytology Laboratory of UEM and were graded according to the Bethesda System . GenomicHpyCH4V  was perf2-square and Fisher’s exact test. Some variables of interest with p < 0.20 were selected for logistic regression analysis. A crude odds ratio (OR) and 95% confidence interval (CI) were calculated. A p-value < 0.05 was considered significant.Statistical analysis was performed using Open Source Epidemiologic Statistics for Public Health/OpenEpi, Version 2.3.1. All variables were expressed as absolute and relative frequencies. For univariate analysis, categorical variables were compared with HPV infection by χ3 and 26.4% with > 350 cells/mm3) and low current viral load (58.4% < minimum limit and 38.8% between the minimum limit and 100 copies/ml).Most HIV-infected women showed excellent control of the HIV infection, based on HAART proper use (79.2%), high current CD4+ T lymphocyte count (37.6% with 200–350 cells/mm3 (p = 0.06) , not undergoing Pap screening in the last 3 years (p = 0.03), current smoking (p = 0.01), previous smoking (p = 0.05) and current CD4+ T lymphocyte count < 200 cells/mm, not und3  and between 200–350 cells/mm3  , hormonal contraceptive use , current smoking , and current CD4+ T lymphocyte count < 200 cells/mmA total of 7.3% (n = 13) of the women showed cervical abnormalities, of which 84.6% (n = 11) were low-grade squamous intraepithelial cervical lesions (LSIL) and 7.7% (n = 1) for both atypical squamous cervical cells (ASC) of undetermined significance (ASC-US) and ASC could not be excluded a high-grade squamous intraepithelial cervical lesion (ASC-H) (Table The frequency of HPV was 46.6% n = 83), and in 23.0% of participants (n = 41) multiple HPV genotypes were detected. Low-risk (LR)-HPV was detected in 14.6% (n = 26), and HR-HPV was detected in 32.0% (n = 57) of the women (p < 0.01). LR and HR-HPV showed similar frequencies in single and multiple infections . However, in multiple HPV infections, HR-HPV was more frequent than LR-HPV in both women with normal cervical cells (NILM)  and those with LSIL  (p = 0.01). HPV infection occurred most commonly in women with NILM  (p = 0.022) ; HPV 66 and 58 3.4% each); HPV 59 and 82 (2.8% each); and HPV 56 and 69 (2.3% each). The most commonly detected LR-HPV genotypes were HPV 72 (6.74%); HPV 61 (3.9%); and HPV 11, 70, 74 and 83 (2.3% each)  had a two-fold greater risk of HPV infection than those with > 350 cells/mm3 (26.4%). However, our results also showed that although the frequency of HPV was high (46.6%), a low frequency of cervical abnormalities was detected (7.30%), mainly LSIL (84.6%). Recently, it was reported that HPV frequency was much lower (6.7%) in HIV-uninfected women in the city in which this study was conducted [In the present study, we found that although HIV infection was well controlled, women with a current CD4+ T lymphocyte count between 200–350 cells/mmonducted . TherefoHPV infection was positively associated with parity number ≥ 3, hormonal contraceptive use and smoker. A relationship between these risk factors and CC has been reported in HIV-uninfected women ,15. Our Multiple HPV infections were frequently detected, occurring in half of the HPV positive women; similar findings have already been widely reported ,10,14. IIn conclusion, we acknowledge that we did not include HIV-uninfected women for comparison. However, we have provided very important information about the risk factors associated with HPV frequency and genotypes in HIV-infected women, and this information can contribute to planning protocols for CC prevention in these patients. It should be noted that the STD/AIDS program of the Brazilian Ministry of Health distributes HAART free of charge and ensures its use. This program also provides comprehensive care to HIV-positive patients, which certainly contributed to the low frequency of cervical lesions (most were LSIL) we observed. This study can serve as a model for the populations of other Latin American countries.HPV: Human papillomavirus; HIV: Human immunodeficiency virus; HR-HPV: High-risk HPV; LR-HPV: Low risk HPV; CC: Cervical cancer; SAE: Specialized Assistance Service; STD: Sexually transmitted disease; AIDS: Acquired immunodeficiency syndrome; PCR: Polymerase chain reaction; LSIL: Low-grade squamous intraepithelial cervical lesions; ASC-UUS: Atypical squamous cervical cells of undetermined significance; ASC-H: Atypical squamous cervical cells could not be excluded a high-grade squamous intraepithelial cervical lesion; OpenEpi: Open source Epidemiologic Statistics for Public Health; OR: Odds ratio; CI: Confidence interval; HAART: Highly active antiretroviral therapy.The authors declare that they have no competing interests.All of the authors contributed to the manuscript. SCR-B, RPS, FG, and ALPA searched the literature and prepared the manuscript. SCR-B, MDBC and SMP collected the biological samples from the women. MELC, ALPA and FG wrote the manuscript. MELC, RPS, FG and ALPA participated in methodology design and execution. MMTI performed the experiments related to cervical cytology. RGS and AAFG performed the statistical analysis. SCR-B, MDBC and SMP contributed to the statistical analysis and design of the study. MDBC, SMP and MELC were involved in revising the manuscript critically for important intellectual content. MELC revised the final version of the manuscript and provided information and suggestions. All of the authors read and approved the final draft of the manuscript.


Background. Mounting evidence suggests an association between human papillomavirus (HPV) and HIV acquisition. This study aimed to explore this association among South African female sex workers (FSWs). Methods. We used data from 88 HIV-negative FSWs who participated in a vaginal gel (COL-1492) trial. Cervicovaginal rinse samples, obtained before HIV-seroconversion, were genotyped into high-risk (HR-) and low-risk (LR-) HPV. HIV-adjusted hazard ratios (aHRs) and 95% confidence intervals (CI) were estimated using Cox survival analysis. Results. HR- and LR-HPV prevalences were 70.5% (95% CI : 60.5–79.2) and 60.2% (95% CI : 49.9–70.0), respectively. Twenty-five women HIV seroconverted. Controlling for background characteristics and other sexually transmitted infections, HIV aHR increased by a factor of 1.7  for an increase of one unit of the number of HR-HPV genotypes. Conclusions. HIV seroconversion among FSWs is associated with genital HR-HPV infection. Further investigation is warranted, including testing the possible protective effect of available HPV vaccines on HIV acquisition. HIV prevalence in South Africa is one of the highest in the world, with over 17% of adults infected with HIV . In SoutA number of sexually transmitted infections (STIs), notably HSV-2, have been identified as key biomedical risk factors for the heterosexual transmission of HIV , 6. AmonIn recent years, the association between HIV and genital human papillomavirus (HPV), one of the most common STI worldwide, has generated much interest. In South Africa, genital HPV is thought to affect about 21.0% of women with normal cytology , and HPVMounting evidence suggests an association between HPV serostatus and HIV infection. Several studies conducted among sub-Saharan African women indicate that HIV seropositivity is strongly associated with higher HPV prevalence , 16–23, More recently, an association between HPV infection and HIV acquisition has been suggested, but to date, only few studies have been conducted among women to test this hypothesis –30. The Data were obtained from South African FSWs from the Kwazulu-Natal Midlands, who were participating in a multicentre, randomised, placebo-controlled, triple-blinded, phase 2/3 efficacy trial of COL-1492, a low-dose nonoxynol-9 microbicide. The trial was funded by the United Nations Programme on HIV/AIDS (UNAIDS), and conducted between September 1996 and June 2000. In brief, 187 healthy, nondrug using, not pregnant, HIV-negative South African FSWs were recruited by the Medical Research Council of Durban from truck stops along the major highway between the port city of Durban and the commercial capital Johannesburg and enrolled in the trial. At the screening visit, comprehensive information on the trial was provided and written informed consent was obtained. Participants were randomly assigned to a nonoxynol-9 containing gel (COL-1492) or the same placebo gel without nonoxynol-9. Data on demographics, background characteristics and sexual behaviour were collected at baseline. The primary endpoint was incident HIV-1 infection. The retention rate in the study at 48 weeks was 88%. Due to issues with processing, HPV sample data were unavailable for 83 women, but their baseline characteristics did not differ significantly from those of the remaining 104 women . DetailsTrichomonas vaginalis, Candida albicans, Neisseria gonorrhoeae, Chlamydia trachomatis, and syphilis were also obtained as described elsewhere [Samples used for the present study were cervicovaginal rinses (CVRs), which were collected and tested for HPV. Theses samples were obtained at baseline, which started in 1996, in December 1998 (Followup A) and in December 1999 (Followup B). The full follow-up schedule of the study participants has been previously described , 33. Fullsewhere . In the current study, we included the 88 women who had at least one HPV result, obtained before HIV conversion for those who seroconverted.The detailed testing procedures have been previously described , 32. HIV testing was performed with sensitive antibody ELISA  and confirmed by line immunoassay . Seroconversion dates were established using p24 antigen detection ELISA and a DNA PCR assay. β globin control. As recommended by the manufacturer, β globin negative samples were not included in the analysis [HPV DNA was extracted from the collected cervical cells using a Qiagen DNA mini kit . HPV typing was performed with a reverse line blot assay , according to a published method . This asanalysis . Sampleshttp://image.thelancet.com/extra/02art3365webmethods.pdf. Laboratory testing procedures for other STIs have been previously described  and are Trichomonas vaginalis, Candida albicans, Neisseria gonorrhoeae, Chlamydia trachomatis, and syphilis). The variables age, length of sex work, and number of clients were dichotomized based on their respective median values.HIV-adjusted hazard ratios (aHRs) and 95% confidence intervals (CIs) were estimated with survival analysis (Cox model), using as covariates the following baseline characteristics: number of HR-HPV genotypes (referred to as number of HR-HPV in the text), number of LR-HPV genotypes (referred to as number of LR-HPV in the text), age , intervention group (placebo or Nonoxynol-9), condom use , anal sex (yes or no), length of sex work , number of clients per week  and status of other STI , the effect of LR-HPV on HIV incidence was first tested by controlling for HR-HPV. Similarly, the effect of HR-HPV on HIV incidence was first tested by controlling for LR-HPV.Because infections with HR-HPV and LR-HPV were correlated : 19 to 30). Participants had been engaging in sex work for a median duration of 2.0 years , and their median number of clients per week was 20 . Median duration of participation in the study was 2.5 years (IQR: 1.7 to 2.8). Other background characteristics and STI status are indicated in For the analyses, we used the first available HPV testing, which was obtained at screening for 33 women , in December 1998 for 44 women  for whom HPV data at screening were unavailable, and in December 1999 for 11 women  for whom HPV data were unavailable at screening and in December 1998. The HR- and LR-HPV prevalences were 70.5%  and 60.2% , respectively. The distribution of the numbers of HR-HPV and LR-HPV is shown in Plinear  trend = 0.76). In contrast, when controlling for the number of LR-HPV, the number of HR-HPV was a risk factor for HIV acquisition . When assessing HIV acquisition as a function of the total number of genotypes, regardless of type, the univariate effect was HR=1.2 , and the multivariate effect was aHR = 1.1 .Among the 88 women, 28.4% (25/88) HIV seroconverted during followup. When controlling for the number of HR-HPV, the number of LR-HPV was not a risk factor for HIV acquisition . The mean (median) duration in years of followup before HIV infection was 2.4 (2.7) for women not infected with HR-HPV and 2.2 (2.5) for those infected with at least one HR-HPV genotype. The effect of HR-HPV status and other covariates on HIV acquisition, and the distribution of HIV cases, are indicated in As shown in P = 0.027). The corresponding multivariate analysis only included covariates having a univariate P value lower than 0.15 (age and education) because of the smaller number of HIV infections and yielded an effect aHR = 1.8 .A sensitivity analysis was conducted to examine the effect of HR-HPV on HIV acquisition among women in the placebo group. The univariate effect was HR=1.9  was very close to the reported estimate from the main analysis.Using the first available HPV testing result for our analysis may have been a source of bias because in doing so, it was assumed that HPV status at baseline and at the two follow-up visits were equivalent. This is not necessarily correct, because the observed association between HPV and HIV acquisition could be time dependent due to the transient nature of HPV infection. To address this issue, it was recommended that the analyses be repeated taking time zero as the date of the first HPV test. Although this approach reduced the power of the analysis because it reduced the total length of followup, the multivariate effect obtained , percentage having been randomized to the intervention group , percentage with at least six years of education , percentage using condoms often , percentage practicing anal sex , average duration of sex work in months , average number of clients per week , prevalence of Trichomonas vaginalis , prevalence of Candida albicans , prevalence of Neisseria gonorrhoeae , and prevalence of Syphilis . However, the difference in the prevalence of Chlamydia trachomatis was borderline . The statistical power for these analyses may have been limited due to the small samples sizes.The 16 (104 minus 88) women who were excluded due to lack of HPV results were compared with the 88 remaining women in terms of background characteristics and STI status, which are listed, While the burden of disease associated with HIV and HPV infection is significant, especially in sub-Saharan Africa, there is limited research on the link between HPV and HIV acquisition. In the present study, we found that HR-HPV was significantly and independently associated with HIV acquisition among South African FSWs and that the risk of HIV infection significantly increased in the event of multiple HR-HPV infections. No association was found with LR-HPV.To our knowledge, only six published observational studies, three conducted among men and three among women, have explored HPV infection and HIV acquisition , 37–39. One study , in whicOur study findings are mostly coherent with these reports; however, comparisons are difficult because of substantial variations in terms of sample population, covariate adjustment and HPV status assessment. For instance, only one study assessed the association among individuals with risky sexual behaviour  but repoSeveral nonexclusive and plausible explanations can account for our findings. Firstly, HIV and HPV are sexually transmitted viruses which share the same transmission route and behavioral risk factors. However, the association observed remains largely significant even after controlling for sexual behavioral covariates although residual confounding cannot be excluded. Secondly, it could be argued that HIV-HPV coinfected male partners may have shed HR-HPV more intensively than HIV-negative male partners. Indeed, in the case of a depressed immune system, HPV lesions are more likely to be dysplastic , and immSeveral factors could account for a causal association of HR-HPV with HIV acquisition. first, their association is very strong, as shown in observational studies. Secondly, causality is biologically plausible: HPV could disrupt the local and systemic immune systems and predispose to HIV infection , either This study has several limitations: First, its observational nature does not allow the interpretation of the findings as a causal association between HR-HPV status and HIV acquisition. Secondly, we did not obtain sets of samples collected at the same time for all participants although only a small proportion were not included because of missing data. Thirdly, HSV-2 status, anal HPV sampling, and cervical cytology were not available, and thus their effects on the observed association could not be tested. In particular, HSV-2 has been hypothesized to facilitate HIV acquisition , and couDespite these limitations, these findings indicate that the hypothesis of a facilitating effect of HR-HPV on HIV acquisition requires further investigation using longitudinal data. The validation of this hypothesis could necessitate testing the potential protective effect of available HPV vaccines on HIV acquisition.


Although cervical cancer is the leading cancer in Cambodia, most women receive no routine screening for cervical cancer and few treatment options exist. Moreover, nothing is known regarding the prevalence of cervical HPV or the genotypes present among women in the country. Young sexually active women, especially those with multiple sex partners are at highest risk of HPV infection. We examine the prevalence and genotypes of cervical HPV, as well as the associated risk factors among young women engaged in sex work in Phnom Penh, Cambodia.We conducted a cross-sectional study among 220 young women (15–29 years) engaged in sex work in different venues including brothels or entertainment establishments, and on a freelance basis in streets, parks and private apartments. Cervical specimens were collected using standard cytobrush technique. HPV DNA was tested for by polymerase chain reaction (PCR) and genotyping using type-specific probes for 29 individual HPV types, as well as for a mixture of 10 less common HPV types. All participants were also screened for HIV status using blood samples. Multivariate logistic regression analyses were conducted to assess risk factors for any or multiple HPV infection.The prevalence of cervical HPV 41.1%. HPV 51 and 70 were the most common (5.0%), followed by 16 (4.6%), 71 (4.1%) and 81 (3.7%). Thirty-six women (16.4%) were infected with multiple genotypes and 23.3% were infected with at least one oncogenic HPV type. In multivariate analyses, having HIV infection and a higher number of sexual partners were associated with cervical HPV infection. Risk factors for infection with multiple genotypes included working as freelance female sex workers (FSW) or in brothels, recent binge use of drugs, high number of sexual partners, and HIV infection.This is the first Cambodian study on cervical HPV prevalence and genotypes. We found that HPV infection was common among young FSW, especially among women infected with HIV. These results underscore the urgent need for accessible cervical cancer screening and treatment, as well as for a prophylactic vaccine that covers the HPV subtypes present in Cambodia. Cervical cancer is the third most common cause of cancer among women worldwide, and it is more frequent in developing countries where population-level screening programs are generally unavailable . In CambGenital human papillomavirus (HPV), the most common sexually transmitted infection (STI) worldwide, is the key causal agent in cervical cancer ,4. More Cervical HPV prevalence and genotypes differ substantially from one population to another worldwide. Although cervical cancer is the leading cancer in Cambodia, nothing is known regarding the prevalence of HPV infection or the genotypic distribution in the country. As in other parts of the world, epidemiological studies in Southeast Asia has shown that the distribution of cervical HPV genotypes differ across populations, and oncogenic genotypes other than HPV-16 and −18 are frequent . Two comAlso of importance in planning clinical and public health responses is an understanding of the factors associated with acquiring cervical HPV infection. Factors detected so far include higher number of sexual partners, early age at first sexual intercourse, smoking, oral contraceptive use, inconsistent condom use, and other STI infections, including HIV -17. The To date, nothing is known regarding cervical HPV prevalence, genotypic distribution, or risk factors in the Cambodian female population. To inform policy and guide further research, we undertook a study to examine cervical HPV genotypes and the associated risk factors among young FSW working in Phnom Penh, Cambodia.Cross-sectional data were obtained from a single baseline visit, in the Young Women’s Health Study 2 (YWHS-2). This prospective study, conducted from August 2009 to August 2010, investigated HIV/STI, sexual behaviors and drug use among women engaged in sex work in Phnom Penh, Cambodia; methods described previously in detail . In brieor engagement in transactional sex  in the last three months. Eligible women who were interested in the study were invited to a community location used by various sex-worker organizations, where they were provided with comprehensive study information. A group informed consent process including oral review of the written informed consent, with explanations and opportunities for questions and answers, was conducted. Women who consented to participate were provided with appointment cards to present to the YWHS clinic site. Free transportation was offered to facilitate attendance at scheduled study visits. All women negative for HIV and not pregnant were offered a three-dose regimen of a HPV quadrivalent vaccine (GardasilTM).A convenience sample of women engaged in sex work was recruited through informational meetings, neighborhood outreach, and referrals by participants. Inclusion criteria for the study were: being female, aged 15–29 years, ethnic Cambodian, reporting at least two different sexual partners in the last month A structured paper questionnaire was administered in Khmer by trained interviewers in a private room covering socio-demographic characteristics, occupational and sexual risk, alcohol and drug use exposures. Client-centered risk reduction counseling was provided, and participants were offered testing for HIV and HPV. HIV results were disclosed to participants and women infected with HIV received counseling and were referred for free medical evaluation and treatment, where indicated.Cervical specimens were collected by a medical doctor using a standard cytobrush technique and transported to University of California at San Francisco for HPV testing. HPV DNA was tested by polymerase chain reaction (PCR) and genotyping was conducted using type-specific probes ,26. MY09(TM) HIV rapid HIV test  and the Clairview HIV 1/2 STAT-PAK . HIV-positive and discordant samples were confirmed by HIV-1 immunoblot. HIV specimen testing occurred at the Cambodian National Institute of Public Health (NIPH) laboratory.HIV serology with venous blood was performed using two rapid tests; Uni-Gold Recombigen The study protocol was reviewed and approved by the Cambodian National Ethical Committee, the Committee on Human Research at UCSF and the University of New South Wales Human Research Ethics Committee.Outcome variables were “any HPV infection” and “multiple HPV infection” (two or more genotypes). Sociodemographic variables included age, marital status, education and number of children. Based on sex work activities in the past 30 days, women were classified as working in either 1) entertainment establishments  or 2) working as freelance FSW in streets and parks or in brothels. The promulgation of anti-sex-trafficking and sexual exploitation laws enacted in 2008 has resulted in the closure of brothels and the displacement of many FSW to entertainment establishments or outdoor settings. At the time this study was conducted (2009–2010) most of the brothels were closed and only five women reported working in brothels. Drug and alcohol use variables included recent (last 3 months) amphetamine-type stimulants (ATS) use, recent binge use of drug, number of days of alcohol use and the number of days drunk (past month) . Recent Data were double entered into a database software  and transferred into STATA 11.0  for statistical analyses. Bivariate logistic regression analyses were performed to identify associations between socio-demographics, drug and alcohol use, sexual behaviors, HIV and STI infections and HPV outcomes: 1) “any HPV infection”, and 2) “multiple HPV infection (≥2)”. Multivariate logistic regression models were built according to a conceptual framework where variables were entered as groups in steps : 1) sociThe prevalence of HIV was 15.8% among 220 participants. Cervical HPV DNA was detected in 41.1% (90/220) of women; HPV prevalence was 34.4% (64/186) among HIV negative women and 78.8% (26/33) among those who were HIV positive. Almost a quarter (23.2%) were infected with at least one oncogenic high-risk (HR-HPV) genotype and 17.7% with at least one low risk subtype (LR-HPV). HPV-51  and HPV-16  were the most common HR-HPV genotypes present, followed by HPV-52 and HPV-53 . Among those infected with LR-HPV genotypes, HPV-70 was the most common , followed by HPV-71  and HPV-81   among women engaged in sex work in Phnom Penh. The prevalence of cervical HPV is comparable to that observed in FSW in Japan (48%), Korea (™ and Cervarix™) have been approved by national regulatory agencies and are currently used in several countries. To date, Cervarix™ is the only vaccine that has been approved in Cambodia but access is limited. Our results and those from other epidemiological studies clearly show that currently available vaccines do not cover the most prevalent HR-HPV genotypes found in Cambodia and other Southeast Asian countries. However, some cross-protection with other HPV subtypes has been observed for Cervarix™[In this population of Cambodian FSW, HPV-51 was the most prevalent oncogenic genotype, followed by HPV-16. Other dominant HR-HPV genotypes included HPV-52 and HPV-53, while prevalence of HPV-18 was lower. HPV-70, HPV-71 and HPV-81 were the most frequent low-risk genotypes observed. This is consistent with other epidemiological studies conducted in Southeast Asia where infection with oncogenic genotypes such as HPV-51 and HPV-52 was common . HoweverCervarix™,39. ThesCervarix™.™ was safe and immunogenic in cohorts of HIV-infected children and men infected with HIV [Consistent with previous studies, women infected with HIV were more likely to be infected with any cervical HPV genotype compared with HIV-negative women. ,22,40. Swith HIV ,46.This is the first study to show that recent binge use of ATS is associated with multiple HPV genotypes, even after controlling for sexual behaviors, HIV and STI infection. ATS use has emerged as a potential significant problem among FSW in Cambodia, and Southeast Asia more generally ,47. ATS Women working as freelance FSW or in brothels were at higher risk of being infected with multiple cervical HPV genotypes. Our group and others have shown that these women are more susceptible to HIV and STI infection compared with their counterparts working in entertainment and service venues ,58,59. SOur study has several limitations. The cross-sectional design of this study does not permit us to conclude causality between HPV infection and risk factors studied here with certainty, as experimental design would. Cervical HPV may not necessarily represent total HPV infection, which can also include oral, vaginal/vulvar, and anal HPV. Self-reported data on sexual behaviors and drug use may introduce response bias associated with socially acceptable or desired answers. Measuring condom use with the last partner might not be representative of general condom use behaviors. However, this would result in conservative estimates of risk, biasing our results toward the null. Finally, participants in this study were not sampled probabilistically; as a consequence our rresults may not be generalizable to all young women in sex work in Phnom Penh or Cambodia. Nevertheless, our sample included women from a wide range of sex work venues and captured a wide breadth of this high-risk occupational group in Phnom Penh.Our findings highlight the burden of cervical HPV infection among FSW in Phnom Penh and, in particular the vulnerability of women infected with HIV and those using ATS. Routine screening for cervical cancer is not available for most Cambodian women and few if any treatment options exist upon diagnosis with pre-malignant lesions or cervical cancer. These first results on prevalence of cervical HPV infection and the genotypes present among Cambodian women offer important insights into the need for cervical cancer screening strategies and immunization programs in this country. However, more epidemiological research is needed to examine HPV infection at a population level in Cambodia and, in particular to inform the development of effective prevention interventions for young and high-risk women. Paying for sex is widely accepted among men in Cambodia and the number of women engaged in sex work has grown substantially in the past few years; estimated at 34,193 . FinallyThe authors declare that they have no competing interests.MCC, KP, ESS, NS, KS, JLE, JK, LM and JP designed the YWHS prospective study and contributed to data acquisition. MCC managed the literature search and summaries of previous related studies, conceived the design of the analysis and undertook the statistical analysis. KP, JK, JP and MCC contributed to data interpretation. MCC wrote the first draft of the manuscript and made modifications after comments from the co-authors. All authors contributed with comments and suggestions and have approved the final manuscript.‘On behalf of the Young Women’s Health Study Collaborative .T.M) was generously donated by Merck. The NIH and Merck had no further role in study design; in the collection, analysis and interpretation of data; in the writing and the submission of the paper for publication.The authors would like to acknowledge the coordinated efforts and dedication of the research teams at the National Center for HIV/AIDS, Dermatology, and STDs and the Cambodian Women’s Development Agency. We also want to thanks Maria Da Costa from University of California San Francisco and Dr. Soriyann from the National Maternal and Child Health Center (NMCHC) in Phnom Penh for their help with the laboratory analyses. Finally, we are indebted to all the Cambodian women who agreed to participate in this study and grateful for the privilege to work with them. This work was supported by the National Institute of Health “U01AI0154241”, “1R21 DA025441”, and “1R01NR010995”. M-C Couture received financial support from the Canadian Institutes of Health Research . HPV quadrivalent vaccine (GardasilThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/166/prepub


Vaccination against Human Papilloma Virus (HPV) before sexual debut has been recommended by WHO as a primary prevention strategy against cervical cancer. In Uganda, vaccination against HPV started as a demonstration project among young girls in Nakasongola; and Ibanda districts. Studies have suggested that vaccination against HPV could result in risky sexual behavior and increase the risk of early sexual debut.This study was done to compare the sexual behavior of HPV vaccinated and non vaccinated adolescent girls in two neighboring districts in Uganda; and to assess whether HPV vaccination had any influence on sexual behavior of vaccinated adolescent girls.This was an unmatched comparative study, which used both qualitative and quantitative study methods. It was carried out among 400 primary school girls aged 12 to 15 years in the districts of Nakasongola (vaccinated) and Luwero (non vaccinated). Quantitative data was collected using a questionnaire while qualitative data was obtained using focus group discussions and key informant interviews. The main outcome measure was the number of sexually active girls in each group.Of the 400 girls, 8 volunteered information that they were sexually active, 5(2.5%) from Luwero (non vaccinated) and 3 (1.5%) from Nakasongola (vaccinated), but there was no statistically significant difference between the 2 groups. HPV vaccination was not significantly associated with being sexually active.There was no significant difference in sexual behavior between vaccinated and non vaccinated girls. Human Papilloma Virus (HPV) particularly types 16 and 18, which are sexually transmitted have been shown to be the cause of cervical cancer ,3. VacciThere have been concerns that HPV vaccination will result in early sexual indulgence and risky sexual behavior among vaccinated girls because they might think they are protected against a sexually transmitted infection ,10. StudWe compared the sexual behavior of HPV vaccinated and non vaccinated adolescent girls in two neighboring districts in Uganda. Luwero, the comparison district, was chosen because it is close to Nakasongola and were thought to have similar social and cultural characteristics. This study was done to provide insight into the unknown surrounding behavior change and presumed increase in sexual activity among the vaccinated girls in Uganda. Specifically, the study was done to compare the sexual behavior of HPV vaccinated and non vaccinated adolescent girls in two neighboring districts in Uganda, and find out if HPV vaccination led to increased sexual activity among adolescent girls.We compared the behavior of HPV vaccinated and non-vaccinated primary school girls aged 12 to 15 years in two Ugandan districts, Nakasongola and Luwero. Nakasongola was one of the pilot districts for HPV vaccination campaign initiated in 2008. Luwero was chosen as a comparison district because it is close to Nakasongola, and was thought to have similar socio-demographic and cultural characteristics.Entry criteria included girls aged 12 to 15 years whose parents gave written consent, and who gave assent to the study. Vaccination status was confirmed by reviewing the vaccination register.In each district we randomly selected two rural and one urban sub county. Dating was defined as having a special male friend (boyfriend) whom the girl regularly met privately and spent time with. In each sub county, 2 primary schools were randomly selected to participate in the study, for a total of 6 schools in each district. After obtaining permission from the head teacher, girls were provided a written invitation to give to their parents specifying the day of the study. Interested parents came to school with the child on the day of the study, and written informed consent was obtained from each parent, as was assent from each girl. At each school up to 40 girls were recruited. If they were more, we enrolled consecutively until we reached the required number.Socio-demographic factors including age, class, tribe, religion, presence and level of education of parents and vaccination status of the girls were obtained through self-administered, pretested questionnaire. In addition, a detailed sexual history was obtained from each girl, as well as knowledge and attitudes towards HPV vaccine. Each girl had height and weight measures, and Tanner stage was assessed.The questionnaires (data collection tools) were pretested before the commencement of the study, in a pilot study done in Wakiso district, which validated their use.Data were entered into the computer using EPI-INFO version 3.4.1 and analysed using SAS version 12. Our primary outcome was sexual activity, defined as sexual intercourse any time from the time of HPV vaccination (or any time from 10 years of age for unvaccinated girls) to the time of this study. Logistic regression was used in univariate and multivariate analysis to determine the association between sexual activity and HPV vaccination, as well as other potential confounders. Two sided p values ≤ 0.05 was considered statistically significant.We assumed that 22.6% of enrolled adolescents are sexually active by 15 years of age  and we wIn each district, 3 schools were randomly selected for focus group discussion. Ten girls, from among those whose parents had come, and were not enrolled in the quantitative study were chosen to participate at each school in a group semi-structured focus group. The discussions were tape recorded. Additionally, five key informant interviews were conducted with the District Health Officer, senior nursing officer and the district inspector of schools in Luwero, and in Nakasongola with the district inspector of schools and the HPV vaccination focal person. All were conducted by the principle investigator. These were done to further get information on the sexual behavior of the girls, and to triangulate the quantitative results. The analysis of qualitative data was done manually after transcription. The recordings that were in Luganda were transcribed directly to English by the research assistant. The ones in English were transcribed in English. The research assistant and principal investigator read through the transcripts and the notes that were taken during the discussions to analyse them. This analysis was done according to the preset themes. Direct quotations from the respondents were used in presentation of study findings.The study team adhered to the Relevance Appropriateness Transparency Soundness (RATS) guidelines on qualitative research during the conduct of this study.Ethical approval for the study was obtained from the Department of pediatrics child health, Institutional review board of Makerere University College of Health Sciences, and Uganda National Council of Science and Technology (UNCST) before the study.We enrolled 400 girls aged between 12 to 15 years attending primary school classes 5 to 7 in Nakasongola and Luwero districts in Uganda , although the numbers were small. In terms of physical characteristics, most of the sexually active girls were heavier and were unvaccinated, 5 girls were >42 kg and 3 were 42 kg. There were about equal numbers of sexually active girls across the tanner stages, heights and ages assessed. This is illustrated in Figure The proportion of girls who had ever heard about HPV vaccine, knew how HPV is prevented and knew that the vaccine prevents HPV only was statistically significantly higher in Nakasongola than Luwero. Importantly, more girls in Nakasongola did not think that HPV vaccination would make girls become sexually active. A smaller proportion of girls, 6.5% in Nakasongola reported that they had never heard about HPV vaccine compared to 97.5% in Luwero. Most of the Luwero girls reported abstinence and use of condoms as ways of preventing HPV, while most of the Nakasongola girls reported HPV vaccination Table .Six focus group discussions were held and 52 girls selected from 3 schools in each district for these. A total of five key informant interviews were also held. Data was analyzed in four set themes including; Knowledge of cervical cancer and HPV vaccine, sex education, sexual behavior of girls, and attitude towards influence of HPV vaccination on sexual behavior. This was done to try to triangulate the results from the quantitative study, which relied on self report on sexual behavior.Most of the girls and key informants from Luwero had never heard about cervical cancer or HPV vaccine.“I had never heard about HPV vaccine, I am hearing from you”, said the District Inspector of Schools, Luwero District, but he knew about cancer of the cervix. One girl in one of the schools knew about cervical cancer, because her Aunt had died of cervical cancer, the others did not know.“HPV vaccine prevents cervical cancer”.In comparison to those in Luwero, more girls in Nakasongola and key informants knew about HPV vaccine and cervical cancer. Most of the girls said “our neighbor who was 13 years had sex with a boy and she became pregnant”, (girl from Nakasongola).Across both districts, the girls said they were not engaged in sexual activity but knew of others who were, for example one girl said Girls used to drop out of school in P5, 6 and 7, some were married off due to pregnancy, but it has reduced over the years because of the intervention by police child protection unit, education department and the probation offices”, (key informant from Nakasongola). “Girls in the urban areas generally start sex from as low as 14 years but majority at 18, 17 years. They are seen in antenatal clinic and labour wards. In the rural areas the age is higher, even among those that are well off”, (Key Informant from Luwero).The key informants also reported that young girls do engage in sexual activity. ““some girls need money” . “some think they have grown up”.”some admire their friends’ property and end up going for sex” (13 year old from Luwero). ‘In most cases they are lured by men with gifts, free lifts by bodabodas”, . “In Luwero, poverty used to drive girls to early sex and would get pregnant after the war when people were poor, and there were many orphans but now it is not a major factor.“(Key informant from Luwero).The main reasons why young girls engage in sexual activity were identified as; peer pressure or joining bad groups, need for good things, being lured by men with gifts or free rides by motor cyclists, lack of parents at home, poverty, feeling grown up, and admiring girls who walk to schools with boys. Again, the reasons reported were similar across both districts. Lack of parents, poverty, and feeling grown up were mostly reported by the rural schools. These reasons were elaborated by some girls as follows; “A girl will not start sex because she will know the dangers involved.” (13 year old girl).Most of the participants in Nakasongola had positive attitude towards HPV vaccination. They thought it would not lead girls to initiate sex early or engage in risky behavior. Some girls thought the vaccine protects against other STDs, but believed that it wouldn’t cause bad sexual behavior. They also thought that the current behavior of girls was not contributed to by HPV vaccination, for example, one girl in Nakasongola said some girls don’t listen so even if you immunize them they say I can have sex after all I am immunized.” .A few girls felt that vaccination would increase sexual activity or lead to risky behavior as one said “In Nakasongola, two key informants said initially some health workers thought it would lead girls to have sex early. But after being educated, they no longer thought like that.Parents were worried that their girls would become sexually active’ .One key informant also reported that in one school, when the vaccination programme had just started, some parents had fears that it would increase sexual activity among the girls. But after being educated about the program, they changed their thinking. “some health workers were worried that girls would start sex early because they were taught the reproductive system in detail”.Another key informant said; vaccination comes with education, if the girls are taught, they will not misbehave because they will know the dangers.”Generally, the key informant themselves did not think that vaccination would lead girls to become sexually active early. Participants from Luwero where there was no vaccination had the same thought as one key informant said “Generally the importance of sex education was emphasized by the key informants, and the girls were less likely to have sex early if they were informed.We assessed the influence of HPV vaccination on sexual behavior in by comparing sexual behavior of adolescent girls in two neighboring districts in Uganda, about two years following initiation of the vaccination programme. Our study showed that there was no difference in sexual behavior among HPV vaccinated and vaccinated girls. This was shown by the fact that HPV vaccination was not significantly associated with being sexually active. It was also noted that the unvaccinated girls were more likely to date or be sexually active, 2.5% compared to 1.5% although the difference was not statistically significant. In addition, this result could have been dues to chance, because of the small numbers and the wide confidence intervals. We attributed this to their relative physical maturity compared to the vaccinated girls. We did not compare sexual behavior between the rural and urban schools, but Luwero district is closer to the capital city and this could also explain why unvaccinated girls were more likely to date or be sexually active.Some girls don’t listen so even if you immunize them they say I can have sex after all I am immunized.” With these findings, sexual risky behaviors could occur and should be prevented by education and counseling adolescents during vaccination, which was done.In terms of knowledge, 105/200 (52.5%) of girls from Nakasongola and 82/200 (41%) from Luwero thought that the vaccination did not protect against other STDs, but this was not statistically significant. High percentages 173/200 (86.5%) and 132/200 (66%) respectively thought that vaccination would not make girls become sexually active and this was statistically significant . With this knowledge it would be expected that the girls would not engage in early sex or risky behavior because they know the consequences of such behavior. Although majority of the girls were of the view that HPV vaccination would not enhance sexual activity, a few of them, 14/200 (7.0%) from Nakasongola and 18/200 (9.0%) from Luwero said that vaccination would make girls start engaging in early sexual activity. Their reason for this was that the girls would think they are protected from STDs. This was also cited in one of the FGDs as one girl said, “Parents were worried that their girls would become sexually active”. However with continued understanding about the vaccine, this belief was no longer there. A recent study in USA compared biomarkers of sexual activity between HPV vaccinated and non vaccinated girls aged 11 to 1 2 years. They looked at contraception counseling, testing or diagnosis of an STD, and pregnancy, and found no difference [There is paucity of data in Africa regarding influence of HPV vaccination on adolescent sexual behavior. A study with parents of 8–14 year old school girls in England found that 12% of them thought vaccination would lead to early sexual indulgence or unsafe sexual behavior . AccordiThe finding in our study is similar to that of Bednarczyk et al. . AlthougThe strengths of our study included the fact that the qualitative component was included to get more information on sexual behavior of the girls, and the fact that each girl answered her questionnaire in private from the other. Also, different girls from those who answered the questionnaire participated in focus group discussions to avoid bias. Only female research assistants were used to help ensure the girls would feel free and comfortable.Our study also had some limitations. The study participants were young girls in primary school, who may have feared to answer the questions about personal sexual behavior or give the correct answers. They may have feared to be reported to their parents or teachers, which may have led them to giving us socially desirable responses about sexual their activity. This was minimized by reassurance that the information was confidential and ensuring that each girl answered her questionnaire in private and numbers were used instead of names. Biological markers to confirm previous engagement in sexual activity were not looked for. These could have helped to validate that question; however that was not part of the study. In our study the girls were not matched for age, so most of the girls in Luwero were older and heavier than the Nakasongola group, this could have contributed to the difference in sexual behavior in that there were more sexually active girls un vaccinated girls compared to the vaccinated girls, although the difference was not statistically significant. Studies have shown that older and more physically mature adolescents are more likely to engage in sexual activity ,20,21.There was no difference in sexual behavior between the vaccinated and non vaccinated girls aged 12 to 15 years in Nakasongola and Luwero Districts. From this study, HPV vaccination should be promoted by the stake holders because it does not lead to early sexual debut or risky sexual behavior.The authors declare that they have no competing interests.JCA, SBK, SK and FM designed the study. JCA collected the data and drafted the first draft of the manuscript. All the authors reviewed and approved the final draft of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/155/prepub