The main cause of cervical cancer in the world is high risks human papillomavirus infection (mainly represented by HPV-16 and HPV-18), that are associated to the development of malign transformation of the epithelium. HPV prevalence exhibits a wide geographical variability and HPV-16 variants have been related to an increased risk of developing cervical intraepithelial lesion. The aim of this study was to describe DNA-HPV prevalence and HPV-16 variants among a women population from Northern Brazil.Juruti Project, fulfilled an epidemiological inquiry and were screened through a molecular HPV test. HPV-16 variants were determined by sequencing the HPV-16 E6 open reading frame.One hundred and forty three women, during routine cervical cancer screening, at Forty two samples were considered HPV positive (29.4%). None of those had abnormal cytology results. HPV prevalence varied between different age groups (Z(U) = 14.62; p = <0.0001) and high-risk HPVs were more frequent among younger ages. The most prevalent type was HPV-16 (14%) and it variants were classified, predominantly, as European (87.5%).HPV prevalence in our population was higher than described by others and the most prevalent HPV types were high-risk HPVs. The European HPV-16 variant was the most prevalent among HPV-16 positive samples. Our study reinforces the fact that women with normal cytology and a positive molecular test for high-risk HPVs should be submitted to continuous follow up, in order to verify persistence of infection, promoting an early diagnosis of cervical cancer and/or its precursors. Human papillomavirus (HPV) is a large group of epitheliotropic viruses of more than 160 different types. HPVs are very successful infectious agents – inducing chronic infections that have no systemic sequelae, rarely killing the host and shedding large amounts of infectious particles for transmission.High-risk HPVs (hrHPV), which includes HPV type 16 (HPV-16), are etiological agents of cervical cancer-5, beingHPV molecular variants are defined taxonomically based on L1 coding region of the DNA sequence. The comThe aim of this study was to describe HPV infection prevalence and related epidemiological variables, as well as, the HPV-16 variants circulating in a population of women from Northern Brazil.Socio-demographic data was obtained for a total of 143 women. Data analysis revealed that these women had 34 years median age , were married (48%), had basic education as the main school level (58%), had housewife occupation (35%), were not exposed to alcohol (58%) or to tobacco (86%), had no history of STD’s  (84%), had 16 years old  as the median age of the first sexual intercourse and had a median of 10 sexual partners . The higher median observed on the variable number of sexual partners was probably influenced by data related to women that referred to be sex workers (n = 7). When these data was removed from analysis, observed media of sexual partners was 5.5 partners . There was no observed statistical association between the described variables and the occurrence of HPV infection in our population (p > 0.05)  had exhibited no amplification of internal control gene and were considered inadequate for subsequent analysis. Linear Array HPV Genotyping Test®  results revealed that 78 samples were negative  and 42 were positive  for HPV infection. Among the HPV positive samples, the high risk HPV-16 was the most prevalent type (14%), followed by HPV-52 (9.5%) and HPV-45 (7%). Among low risk HPV types HPV-62 (7%) was the most frequent. The others high and low risk HPV types were identified in low frequencies . From those HPV-16 positive samples, six were HPV-16 single infection (14.3%) and five were multiple infections that included HPV-16 (12%) [In the present study we found a 29.4% HPV prevalence between women without abnormal cytology screened by Pap Smear Test, at sé et al, in metasé et al, in meta (24.5%) and to d (24.5%). However (24.5%)-21.A marked variation of HPV prevalence among age groups was observed in our population. Women with less than 25 years and comprehend between 25-34 years were more predisposal to have a positive HPV-DNA test, including for hrHPV types. Some studies in Central and South America have demonstrated a first peak prevalence of HPV infection in women below age 25 and a second peak after age 55,23 whichHPV-16 was the most prevalent type observed in our study (14.3%), followed by HPV-52 and HPV-45. The most prevalent HPV types described in women with normal cytology are HPV-16, -58, -31, -18, -45 and -52, however, it is well documented that this prevalence is lower than that observed among women with HSIL (high-grade lesions) and SCC (squamous-cell carcinoma). HPV-16 Phylogenetic analysis based on E6 sequences of eight HPV-16 isolates allowed the identification of seven European and only one non-European variants. A previous study realized in Belém (Northern Brazil) exhibited a higher prevalence of Asian-American (43.2%) and European (42%) variants among women with cervical cancer. PreviouGenital HPV infection is one of the most common sexually transmitted infections worldwide. It has been estimated that approximately 10% of women worldwide with normal cytological findings carry a detectable cervical HPV infection, depending on the HPV testing technology, study size, and the age groups and geographical region studied. It is nA major limitation of the present study is represented by the modest sample size and by the absence of a follow-up period to confirm persistence of HPV infection or clearance through timeline, as well as, the lack of ethnicity data, which was expected to be associated with HPV-16 variants. Another limitation was related with no amplification of internal control (β-globin gene) between 23 cervical samples on the Linear Array HPV Genotyping Test®. Those results were probably caused by inadequate sample cellularity (yield DNA specimens), as the DNA extraction technique was held as a constant, with DNA isolated using the same commercial kit.Juruti (Northern Brazil) is higher than described by others with the most prevalent HPV types being hrHPVs . The European HPV-16 variant (87.5%) is the most prevalent among HPV-16 positive samples. HPV-16 is defined as a human carcinogenic agent and is associated with cervical cancer among carriers. Despite being descriptive, the present study reinforces the necessity to follow up women with normal cytology, meanwhile, with a positive molecular test for hrHPVs, to determine persistence of infection and to promote early diagnosis of cervical cancer and its precursors.HPV prevalence (29.4%) in a population of women with normal cytology from Juruti Project, a research project designed to investigate the impact of migratory flow on the incidence and prevalence of sexual transmissible diseases. Juruti is a county located in the West Region of the State of Pará that had an estimated population of 34.338 individuals in the year of 2007. The main economical activities of Juruti are agriculture and mining and this county is marked by migratory flow of people from the States of Amazonas (Northern Brazil), Maranhão and Ceará (Northeastern Brazil).Samples were collected from patients included on the Juruti Project were offered systematic cervical cancer screening (Pap smear test) combining conventional cytology through Cytobrush Plus Cell Collector®  for optimal collection of exfoliated cells and a commercialized version of the line blot assay – Linear Array HPV Genotyping Test®  – when they had their routine cervical smear performed in a public hospital. The Juruti Project has been approved by the Instituto Evandro Chagas, statement nº30/05, CAAE 0013.0.072.000-06DC-2008-374. All women were informed and gave their written consent to participate in the study.During fifteen days of March (2007) women participating on the After samples collection cytobrush was immersed into 15 ml falcon tubes with 3ml of phosphate buffer saline  and kept at 4ºC according to the manufacturer’s instructions. At the HPV laboratory the falcon tubes were submitted to manual agitation, followed by cytobrush discard and centrifugation of the liquid per fifteen minutes (1200 rpm). Supernatant was discarded and cell pellet was resuspended in 3 mL of PBS solution.Juruti by the local routine professionals. Cervical specimen DNA extraction was performed according to the Linear Array HPV Genotyping Test® kit protocol , which can detect until thirty seven distinct HPV types in a unique reaction, which includes high and low risk and multiple infections. Only the strips that had an internal positive control test (β-globin gene) were considered proper to genotyping analysis.The conventional cytology results were classified according to the recommendations of Brazilian Ministry of Health and Brazilian Society of Cytology which is based on Bethesda’s definition and werey. The obtained nucleotide sequences of the E6 region were edited and assembled using UGENE software. Sequences were globally aligned and compared with HPV variants sequences available at GenBank database  using MAFFT version 7 software. Phylogenetic tree was constructed and edited using RaxML version 7.0.4 (1.000 bootstraps) and Dendroscope version 3 software’s, respectively.The PCR and sequencing of the HPV 16 E6 gene nt 46-256) was performed as described by Chopjitt et al[6 was perData were analyzed with the software BioEstat 5.3. Fisher The authors declare that they have no competing interests.Instituto Evandro Chagas and is one of the researchers of the Juruti Project. FP participated in the study design and data interpretation. CGMS participated in the study design and data interpretation. RVDS was responsible for study design, for the initial HPV-DNA investigation, is a researcher and member of the HPV group at Instituto Evandro Chagas. All authors read and approved the final manuscript.BPTL performed the genotyping of HPV variants, data and statistical analysis and lead drafting of the manuscript. ESCJ participated in the HPV variant analysis and performed phylogenetic analysis. WAM is member of the HPV at


Data on Human Papilloma virus (HPV) vaccine immune response in sub-Saharan Africa is still sparse yet such knowledge is critical for optimal implementation and monitoring of HPV vaccines. Our primary objective was to evaluate levels of anti-HPV-16/18 antibodies and six other ‘high risk’ HPV (hrHPV) types among the vaccinated and unvaccinated Ugandan girls.We conducted a cross sectional study among AS04-adjuvanted HPV-16/18 vaccinated and unvaccinated school girls aged 10–16 years in Western Uganda using purposive sampling. The vaccinated girls were at 18 months post vaccination. After consenting and assenting, data was collected using interviewer administered questionnaires for demographics and sexual history. Blood was drawn from which serum samples were analysed by the multiplex HPV serology technology to determine anti-HPV antibody levels to HPV-16/18 and six other hrHPV types . The antibody levels were expressed as Median Fluorescent Intensity (MFI).A total of 207 vaccinated  and 197 unvaccinated girls  participated in the study. Sexual activity was self reported among 14/207 (6.8%) vaccinated and 5/197 (2.5%) unvaccinated girls. The MFI levels for HPV-16 and HPV-18 were 15 and 20 times higher respectively in the vaccinated girls than in the unvaccinated girls. HPV-16 mean MFI level was 4691 among the vaccinated compared to 218  among the unvaccinated girls. For HPV-18 the mean MFI level was 1615  among the vaccinated compared to MFI 103  among unvaccinated girls.In addition antibody levels to non vaccine hrHPV types  were all significantly higher in the vaccinated group than in the unvaccinated group (p<0.01).The AS04-Adjuvanted HPV-16/18 vaccinated girls showed a higher level of antibodies to HPV-16/18 and other non-vaccine hrHPV types compared to the unvaccinated girls. This may translate into protection against HPV-16/18 and other hrHPV types. Cervical cancer is the second most common cancer in women worldwide and the primary cause of cancer related deaths among women in the developing countries . More thHuman Papilloma Virus (HPV) is the most common sexually transmitted infection and has been recognized as the necessary cause of cervical cancer . In UganThe recently developed prophylactic vaccines against HPV infection are more than 90% effective in preventing infection and high-grade pre-cancer lesions associated with HPV types included in the vaccine . CurrentTherefore, a cross sectional study was conducted in Western Uganda to compare levels of antibodies to HPV-16/18 and other hrHPV types among vaccinated and unvaccinated Ugandan girls at month 18 post vaccination.The results represent two groups, vaccinated and unvaccinated girls. A total of 375 and 425 girls from the vaccinated and unvaccinated districts, respectively, were present in the schools on the days of the survey. Of these, 211/375 (56.3%) and 200/425 (47%) vaccinated and unvaccinated girls assented and had parental consent to participate in the study, respectively  of the girls self reported engaging in penetrative sexual activity. Among those who self reported engaging in sexual activity, 14/207 (6.8%) were vaccinated while 5/197 (2.7%) were unvaccinated girls. Overall, the median age at sexual debut was 11 yrs; range 6–13 years ; HPV-18 (P = 0.1) ; HPV-18 (P = 0.58)  or α9  ,13. LikeIn previous trials, the strong vaccine induced immune response has been proved to protect against persistent HPV-16/18 infections and CIN disease ,10. We aThe high level of immune response to the vaccine seen in this study may support the idea of fewer doses in this age group as has been endorsed by the World Health Organisation (WHO)  though tThe girls were at the same school level in the different schools although there was a significant difference in their age and sexual activity. The vaccinated girls were mainly from around the urban area and tended to be younger. This observation is in line with what is already known that Ugandan children from rural settings tend to start school at an older age than their urban counterparts . FurtherAmong the unvaccinated group, when sexually active girls were compared with those who were not, there was no difference in MFI levels to both HPV-16/18. The sexually active girls may not have been exposed to HPV-16/18 despite the sexual activity. Further, it is known that about 50% of those exposed to HPV infection may not mount an immune response  which maAssociation between sexual activity and levels of antibodies to HPV-16/18 was not assessed for the vaccinated girls as results from some studies  so far hThis study had some limitations. The antibody levels were only assessed at one time point. There was no baseline antibody level from the vaccinated girls before vaccination. However for a similar picture of levels of anti-HPV16/18 after natural exposure to HPV-16/18 a group of girls from a neighboring district who would have been vaccinated at the same time were chosen to participate in this study as a comparative group. This inclusion of the unvaccinated group of girls also enabled a demonstration of a difference in antibody levels which could only be explained by vaccination. The girls were of unknown HIV status. This is the situation that will happen in normal vaccination programs; HIV status will not be exclusion for the HPV vaccine. The method used for HPV serological analysis is not yet widely used to monitor HPV vaccine immunogenicity and comparison with other studies may be difficult, however comparison by trends of antibody levels may be appropriate for future follow up even when different methods are used.The AS04-adjuvanted HPV-16/18 vaccinated girls showed a higher level of antibodies to HPV-16/18 and other non-vaccine hrHPV types compared to the unvaccinated girls.These results support continued use of the prophylactic HPV-16/18 vaccine in Africa where for decades there has been no success with any screening program against cervical cancer, an intervention that has helped to significantly reduce cervical cancer in the developed world.Therefore for African countries, effective HPV vaccination programs seem to be the window of opportunity to reduce cervical cancer mortality and morbidity. By assessing both vaccinated and unvaccinated girls, we are able to provide insight in the immunological response to the HPV-16/18 vaccine as well as provide baseline knowledge of HPV-16/18 and other hrHPV types after natural exposure. It is also evident from this study that some girls initiate sex earlier than expected therefore efforts to vaccinate girls at the earliest WHO recommended age of 9 years  should bWe conducted a cross sectional survey between October 2011-February 2012 among HPV vaccinated and unvaccinated girls at 18 months post vaccination. The study was approved by the higher degrees research and ethics committee (HDREC) of Makerere University College of Health Sciences and finally by the Uganda National Council for Science and Technology (UNCST). Permission was sought from the local government in the districts of study and school authorities. Consent from the parents/guardians of the girls as well as assent from the girls were obtained.The study was conducted within two neighbouring districts of Western Uganda, Ibanda (vaccinated district) and Mbarara (unvaccinated) between October 2011- February 2012. Ibanda district covers an area of 967 square kilometres (373 sq mi) with one county and seven sub counties. It has an estimated population of 242,800 people. The vaccination exercise that was carried out was school based and targeted pupils form primary level 4 at an assumed age of 9 years. Mbarara district covers an area of 1,846.4 square kilometres (712.9 sq mi) with 4 counties and 19 sub counties. It has an estimated total population is about 457,800 people. At the time of the study, Ibanda had 180 primary schools with approximately 23,695 pupils out of which 6,770 (28.5%) pupils had been eligible for HPV-16/18 vaccination in 2010 by the PATH  demonstration project. Mbarara district had 402 primary schools with about 50,307 pupils out which about 11,053 (21.9%) would have been eligible for vaccination in 2010 had the program covered Mbarara district.One sub-county from each district was purposively selected. The selection was based on the population of girls in primary level 5 being at least 500 girls in the sub-county. In addition, the sub-county had to be accessible to the main road so that blood samples could easily be delivered daily to the laboratory for processing.There were 29 schools in the selected sub-county of Ibanda out of which 15(51%) schools participated in the study. The schools were listed according to the number of eligible girls in descending order. Letters introducing the study and requesting for permission to carry out the study in the district schools were sent out to the district leaders. After receiving permission from the district leadership, phone contacts were made to the head teachers briefly explaining the study. Community mobilisers then visited all the schools within each sub county to deliver letters introducing the study and requesting for permission from the head teachers to have their schools participate in the study. A convenient day for the study team to visit the school was agreed upon. The mobilisers carried HPV vaccination registers from the district to verify the vaccination status of the girls who had been vaccinated in 2010. The head teachers were requested to invite the parents/guardians of the girls who had complete vaccine doses according to the register. On the day of the school visit, trained research assistants and the principal investigator (PI) explained the study to the girls and their parents/guardians in separate sessions. The explanations included the objectives of the study and procedures which involved answering a questionnaire and providing a blood sample. After the explanations and responding to questions, written informed consent from parents/guardians (guardians above 18 years) was obtained. In addition, a written assent from the girls whose parents had consented was also obtained. All girls who were fully vaccinated in 2010 and attending schools from within the selected sub-counties were eligible to participate. The girls were consecutively recruited until the desired sample size was realised.Girls were eligible to participate if they were in the same class as the vaccinated ones and not repeaters of the same class. There were 34 schools in the selected sub-county of Mbarara out of which 17(50%) schools were involved in the study. The selection of the schools and visit procedures were similar to those employed in the vaccinated district. However, since the district had not participated in the vaccination program, there were no vaccine registers. Inquiry with the class teachers was made if any of the girls had previously been in the vaccinated district. A school register was used in addition to the information from the girls to verify the class level.In the vaccinated district, girls who were fully vaccinated (received 3 doses) in 2010 with assent and consent participated in the study. In the unvaccinated district only girls who would have been eligible to HPV vaccination at the same time as in the vaccinated district with assent and consent participated in the study. All the girls both vaccinated and unvaccinated answered an interviewer administered questionnaire that included sections on socio-demographic characteristics, vaccination status, general medical history and sexual history. The age was self reported by the girls and recorded as actual age at the time of the survey. Sexual activity was defined as having penetrative vaginal or anal sexual intercourse with the opposite sex. A blood sample was collected from both the vaccinated and the unvaccinated girls. At all times the blood sample was collected under aseptic conditions.mls) of blood were aseptically collected from the cubital vein into a labelled SST™ tube.The study laboratory technician, nurses and PI carried out the blood draws from the girls. In a sitting position, the girl’s non dominant hand was identified. Following identification of the vein in the cubital fossa, the area was cleaned with 70% alcohol swab. Two-three millilitres  in Heidelberg.Antibodies to the L1 proteins of 8 hrHPV types  were analyzed using a multiplex serology method. This method is based on a Glutathione S-transferase capture immunosorbent assay combined with fluorescent-bead technology as described elsewhere -27.http://www.luminexcorp.com) determined the internal bead colour specific for the respective antigen and reporter fluorescence of the beads, reflecting the quantity of antibodies recognizing the respective antigens. For each bead set the reporter fluorescence was expressed as median fluorescence intensity (MFI) of at least 100 beads per set per serum. Final antigen-specific MFI values were calculated by subtraction of Glutathione S-Transferase Fusion Proteins-tag (GST) and an individual bead background value. MFI were obtained for vaccine HPV-16/18 as well as non vaccine high risk HPV (hrHPV) 31, 33, 45, 52, 58. For proteins of HPV types  cut-offs were calculated as mean plus 3 SD of sera from HPV negative controls. The MFI cut-offs were; 373, 200, 303, 200, 200, 200, 297, 275 for hrHPV types 16, 18, 31, 33, 35, 45, 52, 58 respectively.Bead sets carrying different antigens were mixed and incubated with an equal volume of the serum dilutions (2 μl) in a 96 well plate. Human antibodies bound to their respective antigens were stained with a secondary biotinylated goat-anti human immunoglobulin IgG  and the fluorescent reporter conjugate streptavidin-R-phycoerythrin. A Luminex100analyzer  assuming that the common standard deviation is 14263.000 using a two group t-test with a 0.050 two-sided significance level [A sample size of 199 in each group would have 80% power to detect a difference in means of 4024.000  and 288 girls from Ibanda district (Vaccinated).SPSS V20 . At univariate analysis, categorical data were summarized using percentages while continuous variables were summarized by calculating the mean, standard deviation, median, range. Calculation of p values for mean comparison of age was done using Mann–Whitney U test while sexual activity between the two groups was compared using Pearson-chi square and Fishers exact test (p < 0.05significant).The data were analysed using 10 (MFI levels for antibody concentration). We compared the mean of the MFI values between vaccinated and unvaccinated girls with the students t test. The mean of the MFI levels and corresponding 95% CI were calculated. Box plots present the distribution of the MFI values for HPV-16/18 antibodies levels among the vaccinated and unvaccinated girls at month 18 post vaccination. The median of the MFI values for the box plots was compared with the Mann–Whitney U test. In addition effect of age and sexual activity was compared to the median antibody levels among vaccinated girls and unvaccinated girls respectively. Significance level was set at p <0.05.MFI levels of antibodies to HPV-16/18 and other hrHPV types showed skewed distribution therefore the HPV antibody levels were transformed as LogHPV: Human papilloma virus; MFI: Median fluorescent intensity; hrHPV: High risk human papilloma virus; UNCST: Uganda national council for science and technology; HDREC: Higher degrees research and ethics committee; DKFZ: Germany cancer research center; ELISA: Enzyme-linked Immunosorbent Assay; PI: Principal investigator; WHO: World Health Organisation.The authors declare that they have no competing interests.NM, CB, PBN and FMM contributed towards the proposal development, data collection, interpretation of results, and/or writing and revising the manuscript and final decision to submit the manuscript. All authors read and approved the final manuscript.


Serving military personnel are more likely to smoke, and to smoke more heavily, than civilians. The aim of our study was to examine whether veterans have an increased risk of a range of smoking-related cancers compared with non-veterans, using a large, national cohort of veterans.We conducted a retrospective cohort study of 57,000 veterans resident in Scotland and 173,000 age, sex and area of residence matched civilians. We used Cox proportional hazard models to compare the risk of any cancer, lung cancer and other smoking-related cancers overall, by sex and by birth cohort, adjusting for the potential confounding effect of socioeconomic deprivation.p = 0.008). Other smoking-related cancers occurred in 737 (1.31 %) veterans compared with 1883 (1.09 %) non-veterans . A significantly increased risk was observed among veterans born 1950–1954 for lung cancer and 1945–1954 for other smoking-related cancers. The risk of lung cancer was decreased among veterans born 1960 onwards. In comparison, there was no difference in the risk of any cancer overall , whilst younger veterans were at reduced risk of any cancer .Over a mean of 29 years follow-up, 445 (0.79 %) veterans developed lung cancer compared with 1106 (0.64 %) non-veterans (adjusted hazard ratio 1.16, 95 % confidence intervals 1.04–1.30, Military veterans living in Scotland who were born before 1955 are at increased risk of smoking-related cancer compared with non-veterans, but younger veterans are not. The differences may reflect changing patterns of smoking behaviour over time in military personnel which may, in turn, be linked to developments in military health promotion policy and a changing military operational environment, as well as to wider societal factors. In 1950, Doll and Hill published epidemiological evidence of the link between smoking and lung cancer which had been postulated by Müller in 1939 . By 2004In the worldwide military context, there has been much anxiety about the carcinogenic potential of substances such as chemical warfare agents, ionising radiation, defoliants, industrial chemicals and the smoke from the destruction of industry and infrastructure to which military personnel may have been exposed in the course of their duties . AlthougThe Scottish Veterans Health Study provided an opportunity to examine rates of all cancer and smoking-related cancer in a large cohort of UK military veterans, irrespective of exposure to conflict, in comparison with civilians with no record of service, in order to determine whether veterans are at increased risk.The Scottish Veterans Health Study is a retrospective cohort study of all 56,570 military veterans resident in Scotland who were born between 1945 and 1985 and who were registered with National Health Service (NHS) Scotland both before and after service, and a comparison group of 172,753 individuals with no record of service matched 3:1 for age, sex and postcode sector of residence (mean population 5000). The study cohort and methods have been described elsewhere . DemograThe electronic NHS record provided dates of entering and leaving the Service for veterans. The maximum period of follow-up was from 1 January 1981  to 31 December 2012. The data extract was pseudo-anonymised and approval for the study was granted by the Privacy Advisory Committee of the Information Services Division of NHS Scotland.http://www.scotland.gov.uk/Topics/Statistics/SIMD). The SIMD has been used to derive quintiles of regional socioeconomic status (SES) for the Scottish population; ranging from 1 (most deprived) to 5 (least deprived). We used postcode of residence to categorize the cohort participants according to these quintiles.In Scotland, there are 6505 datazones, based on postcode of residence, with a mean population of 800. The Scottish Index of Multiple Deprivation (SIMD) for each datazone is derived from information on income, employment, health, education (including skills and training), housing, crime and access to services , stomach , oesophagus , oropharynx and larynx , bladder  and kidney , at any position in the record. Cox proportional hazard models were used to examine the association between veteran status and risk of any cancer, lung cancer and other smoking-related cancer , using age as the time dependent variable, first smoking-related cancer as the failure time and death (if no smoking-related cancer) as the censor time. The a priori rejection level was set at 0.05. Cox proportionality assumptions were tested using methodology based on Schoenfeld residuals [After data cleansing, 56,205 (99.3 %) veterans and 172,741 (99.9 %) non-veterans were included in the analysis. Of the 56,205 veterans included in the study, 5235 (9.2 %) were women, reflecting the gender balance of the Service population. The mean period of follow-up was 29.3 years, with a total of 6.7 million person-years of follow-up among veterans and non-veterans combined.There were 3588 (6.38 %) cases of any cancer in the 56,025 veterans during the period of follow-up, compared with 11,560 (6.69 %) cases in the 172,741 non-veterans. The difference was not statistically significant overall. Among those born prior to 1960 there was no significant difference between veterans and non-veterans, whereas among those born from 1960 onwards, there was a significantly lower risk of any cancer among veterans  veterans had a diagnosis of lung cancer compared with 1106 (0.64 %) non-veterans, equating to an incidence of 3.47 per 10,000 person-years among veterans compared with 2.04 among non-veterans, rate ratio (RR) 1.70, 95 % CI 1.52–1.90. For veterans aged over 50 years, there were 12.29 cases per 10,000 person-years compared with 10.02 for non-veterans, RR 1.23, 95 % CI 1.08–1.39. The Cox proportional hazard model for men and women combined showed a statistically significantly increased risk of lung cancer among veterans both in the univariate model, hazard ratio (HR) 1.22, 95 % CI 1.09–1.36, p = 0.002) than veterans who had completed at least the minimum term of military engagement  (Table Early Service Leavers (ESL) showed a greater increase in risk of lung cancer compared with non-veterans  and multivariate analyses . The hazard ratios were increased for both men and women but were only statistically significant for men (Table There were 737 1.31 %) other smoking related cancers in veterans compared with 1883 (1.09 %) in non-veterans. In men, the numbers were 713 (1.40 %) and 1796 (1.18 %) respectively and in women they were 24 (0.46 %) and 87 (0.42 %) respectively. There was an incidence of 5.66 per 10,000 person-years for other smoking-related cancers in veterans compared with 3.47 in non-veterans, RR 1.63, 95 % CI 1.49–1.77. In the Cox proportional hazard models, including both men and women, veterans were at significantly higher risk in both the univariate , there was a non-significant reduction in the likelihood of receiving a prescription for NRT among veterans, adjusted OR 0.96, 95 % CI 0.91–1.02, p = 0.159.Among older study participants (born 1945–1959), veterans were more likely to have received a prescription for nicotine replacement therapy than non-veterans, adjusted odds ratio (OR) 1.41, 95 % CI 1.35–1.48, The Scottish Veterans Health Study demonstrated that although overall rates of cancer showed no differences between veterans and non-veterans, the veterans were at increased risk of developing lung cancer and other smoking-related cancers compared with civilians with no record of service matched for age, sex and area of residence. The increase was only apparent for veterans born before 1960, and was greatest in those born between 1950 and 1954. For veterans born from 1960 onwards there was a non-significant reduction in risk in lung cancer compared with non-veterans, whilst the reduction in risk for all cancers in this group was statistically significant. Both male and female veterans demonstrated an increased risk of smoking-related cancer, although the results were only statistically significant for men. There were peaks of incidence of lung cancer associated with having joined the Armed Forces during or immediately prior to a period of heightened operational activity. The increase in risk was highest in those with the shortest service. Our finding of increased prescribing of NRT for the older veterans compared with non-veterans suggests that smoking was more prevalent in older veterans and may account for the higher prevalence of smoking-related cancers although as we had no data on occupational exposures, other contributory factors cannot be ruled out.The increased risk of smoking-related cancers in veterans who entered the Armed Forces in the 1960s and early 1970s is consistent with earlier studies on smoking prevalence in military personnel. In the first formal study of UK military smoking rates, Richards and Crowdy reported that the prevalence of smoking among junior soldiers aged 15–19 years (born 1940–1944) was 17-20 % higher than the general population prevalence quoted by the Tobacco Manufacturers’ Standing Committee. The prevalence of regular smoking ranged from 51 to 58 % at age 15 years to 79–80 % at age 18–19 years, with the greatest increase taking place during the 16th year .Higher rates of smoking in military personnel than civilians have also been described in a number of other armed forces. In 1986, Schei examined changes in smoking behaviour as a result of military service in 2105 Norwegian conscripts aged 18–25 years. Of those who were non-smokers on enlistment, 7.8 % had started smoking during their service, whilst of those who were already smokers, 55.7 % reported smoking more heavily during their military service. The prevalence of daily smoking was 51 % among army conscripts during service compared with a population prevalence of 37.2 % in men aged 19–21 and 35.4 % in men aged 22–24 years in the general population. Living in dormitories where smoking was permitted resulted in 90.8 % being exposed to environmental tobacco smoke .We were unable to obtain data on smoking prevalence in our cohort and therefore used NRT prescribing as a novel proxy measure for relative prevalence in veterans and non-veterans. Although fewer studies have been conducted on smoking prevalence in veterans than in serving personnel, a consistent pattern of higher prevalence persisting into post-service life has been demonstrated, predominantly in the US. The first major study to compare smoking rates in veteran and non-veteran civilian populations was the 1987 US National Medical Expenditure Survey, a questionnaire-based study which gathered data from 36,400 people in 15,000 US households; 3372 male and female veterans were compared with 18,606 male and female non-veterans. Seventy-seven per cent of veterans were ever-smokers of cigarettes compared with 47 % of non-veterans. Thirty-five per cent of veterans were current cigarette smokers compared with 28 % of non-veterans, the mean age at smoking cessation for those who had quit being 39 years for veterans and 40 years for non-veterans . Brown aFeigelman also examined data on 698 male veterans and 1505 male civilians gathered between 1977 and 1991 for the US General Social Survey and found that 47.6 % of veterans smoked compared with 40.7 % of civilians. The author concluded that higher rates of smoking in serving military personnel led to lifelong elevated rates of smoking, and that this also generated an environment in which spouses and children of military personnel were more likely to smoke, thus increasing the long-term adverse public health impact of military smoking . A studyThe overall increased risk of lung cancer and other smoking-related cancers in veterans in our study is also consistent with the findings of other studies. In a major study of the long-term health of US World War II and Korean War veterans followed up for 20–50 years, Bedard et al. calculated an implied mortality rate for lung cancer of 2.2 per 1000 for veterans compared with 1.2 per 1000 for non-veterans and estimated that 35–58 % of the excess veteran lung cancer deaths were attributable to military-induced smoking . WatanabThe reduced risk for cancer overall in younger veterans in our study is consistent with an Australian cohort study which followed up veterans of the first Gulf War from 1991 to 1998 and found a standardised incidence ratio of 71 (95 % CI 37–137) for all cancers compared with the general population. The veterans had a mean age of 27 years at entry to the study in 1991 . Follow-Our finding of a higher incidence of smoking-related cancer in older veterans who joined the services at times of major operational activity is consistent with reports of increased smoking on operations. The US military Millennium Cohort Study, a 21-year prospective study, has demonstrated that operational deployment is associated with an increased risk of both smoking initiation in previous never-smokers and a resumption of smoking in ex-smokers, and that the risk is further increased with exposure to combat . There iIn our study, the oldest veterans commenced their service in 1960 as junior soldiers, although they would not have become eligible for deployment until the age of 18, in 1963. UK military operations in the 1960s included Aden (December 1963–November 1967) and Northern Ireland (from August 1969), so those who joined from the mid-1960s (born 1950 onwards) and completed training were highly likely to have seen operational service. Operations in Northern Ireland continued through the 1970s and did not conclude until 2007, although there were major shifts in operational tempo over that period, the earliest period (up to and including 1972) generally having been the most intense. Our finding of the highest risk of smoking-related cancer in veterans born 1950–1954 suggests that this early period of intense operational activity was associated with increased rates of smoking. This is borne out by the pattern of peaks of incidence of lung cancer in those who joined for service during or shortly before the periods of high-intensity operations. Taken together with our finding that ESL demonstrated a greater increase in risk than those with longer service, there is a strong indication that the most junior personnel were at highest risk, consistent with Lynch’s conclusion that smoking habits, and hence risk profile for smoking-related ill-health, are determined in the first few years of Army life . It alsoThe Cold War (approximately 1947–1991) resulted in many people spending long periods engaged in training and exercises in Germany, where tobacco was readily available tax-free. By the late 1970s when the veterans born from 1960 were entering service, not only had there been changes in operational tempo but there was also an increasing emphasis on military health and fitness. The Falklands War (April–June 1982) involved around 28,000 UK military personnel but was a brief conflict and may have had a smaller impact on military smoking rates than the more sustained campaigns, although Fig. p < 0.001) [In our study, the hazard ratios were statistically significant for increased risk of smoking-related cancer in veteran men but not in women. It is likely that small numbers of cases in women, who constituted only 9 % of the veteran study population, provided insufficient statistical power to demonstrate significance. Studies on smoking behaviour in serving UK military women are sparse, whilst none have been identified on smoking in UK female veterans. In a study of military healthcare workers deployed to Iraq, Boos and Croft reported no differences in smoking rates between men and women , whilst < 0.001) . Notwith< 0.001) .The strengths of the present study are that it was based on a large cohort covering the whole of Scotland with 30 years follow-up. The cancer diagnosis was taken from hospital admission, cancer registration and death records, and is therefore likely to be both reliable and reasonably complete in respect of those events occurring within Scotland. The use of record linkage to analyse individual level data directly derived from health records allowed a robust cohort study design to be employed. The results were able to be matched or adjusted for potential confounders including sex, deprivation and geography. It was possible to do subgroup analysis by sex, and the ability to study birth cohort effects and incidence by year of entry to service contributes to the understanding of factors influencing veterans’ health. The use of a Nelson-Aalen plot at Fig. A limitation of the study is the loss to follow-up of subjects due to migration away from Scotland, which cannot be quantified. There were no follow-up data prior to 1 January 1981 but since the oldest of the cohort were only aged 36 years at that date and the landmark entry point was age 40 years, and few smoking-related cancers would be expected to develop prior to that age, we considered the effect of immortal time bias to be negligible. As the oldest of the cohort was aged 67 years at the end of follow-up and 95 % were under age 65 years, many smoking-related cancers would not yet have developed by the end of the study period. For the veterans, we have not been able to link to military health records and thus cancers developing during service will not have been captured until medical retirement and return to NHS care. However as most personnel leave the Armed Forces by age 40 years, the landmark entry point will have minimised any risk of loss of information. No information was available on personal lifestyle risk factors which could have modified the risk of cancer. Therefore, receipt of an NRT prescription was used as a proxy for relative smoking prevalence, as it is unlikely that there is a systematic difference between NRT prescribing rates for veterans and non-veterans other than as a reflection of smoking rates. Veterans with Reserve service only could not be identified from NHS records and have therefore have been included with the non-veterans. This would have had the effect of weakening any observed differences arising as a result of military service. No information was available on the service to which a veteran had belonged ; smoking rates are known to differ between the services with Royal Air Force personnel smoking less than those of the other two services [Veterans born between 1950 and 1954 are at significantly higher risk of lung cancer and other smoking related cancers than their civilian counterparts. This is likely to be due to higher rates of smoking during service and their persistence into civilian life and may be linked to heavier smoking during operational deployment in the mid-1960s and early 1970s. The highest risk is in those with the shortest service. There is no increased risk of lung cancer or other smoking-related cancer among younger veterans, who may have benefited from military health promotion activity aimed at reducing smoking, as well as the overall reduction in smoking prevalence in the general population.Ethical approval was given by the Privacy Advisory Committee (PAC) of NHS Scotland, PAC Reference 27/12.Individual consent was not required as this was a secondary data study using a pseudo-anonymised database extract.Not applicable.The Scottish Veterans Health Study remains in progress and the data are not currently available for access.


Virus-specific cell-mediated immunity (CMI) plays a role in the outcome of genital HPV infections. To cast further light on the question why most women clear their HPV infection while others develop high-grade cervical intraepithelial neoplasia (CIN), we analyzed HPV16 E2-, E6- and E7 -specific CMI in women who developed CIN during a 10-year follow-up of the Finnish Family HPV cohort.Overlapping 30–35 mer peptides covering the entire HPV16 E2-, E6- and E7 protein sequences were used for defining the lymphocyte proliferation capacity, cytokine production  and numbers of HPV16 -specific CD4+ CD25+ Foxp3+ regulatory T-cells in 10 women who developed CIN, and in 22 control women who tested constantly HPV-negative during the follow-up. HPV-specific CMI was related to the demographic data including sexual behavior, smoking and alcohol consumption.Women with CIN and their controls had similar T-cell mediated immunity against HPV16 E2, E6 and E7 peptide pools. However, nearly fourfold higher T-cell reactivity against common antigens was found in the CIN women than in the healthy donors (p = 0.001). HPV16 E6 stimulation resulted in higher IL-17A secretion in the controls than in the CIN women (p = 0.035). Smoking and use of alcohol affected the T-cell response to common antigens but not to HPV peptides .While both the CIN women and controls exhibited an HPV16-specific CMI, IL-17A might be of importance in HPV induced pathology. The hyper-responsiveness of the CIN patients to common antigens needs further studies. Smoking and alcohol had no effect on HPV-specific CMI.The online version of this article (doi:10.1186/s12967-015-0498-9) contains supplementary material, which is available to authorized users. Currently, nearly 200 different human papillomavirus (HPV) types have been identified infecting either skin or mucosa. The mucosal HPVs are classified as either high- (HR) or low-risk (LR) types based on their oncogenic potential . The mosThe majority of healthy women without any genital HPV disease mount blood T-cell responses against HPV16 E2, E6 and E7 proteins ,13. In pIn the present study, we analyzed the HPV16 E2-, E6- and E7-specific CMI both in women (n = 10) who developed CIN and in those who remained disease free (n = 22) during the entire 10-year follow-up (FU) as participants in the Finnish Family HPV Study. In addition to the genital HPV status, also the oral HPV DNA status and HPV serostatus were known during the FU.The Finnish Family HPV Study is a longitudinal cohort study conducted at the Department of Oral Pathology, Institute of Dentistry, University of Turku and Department of Obstetrics and Gynecology, Turku University Central Hospital. Originally, 329 pregnant women at their 3rd trimester pregnancy and all their newborns  were enrolled in the study between 1998 and 2001, as described previously . The stuThe natural history of genital and oral HPV infection in women participated in the Finnish Family HPV Study has been published earlier ,20,23,24Oral samples for HPV testing were taken during the FU at the baseline and at months 2, 6, 12, 24, 36 and 6.5 years. Samples were taken from the buccal mucosa of both cheeks and upper and lower vestibular areas with a brush  into 80% ethanol and were immediately frozen and stored at -70°C until use. At the last visit, a dentist also performed a careful clinical examination of the oral mucosa. The cervical scrapings for HPV testing were taken with a brush  at the same time points as the oral samples during the 10 years FU. The brushes were placed in a tube with 0.05 phosphate-buffered saline (PBS) with 100 μg of gentamycin and immediately frozen at -20°C and the stored at -70°C until used -Thymidine  per well was added. After 16-18 hours of incubation, the cells were harvested into Unifilter plates (PerkinElmer) using the FilterMateTM Cell Harvester (PerkinElmer). Subsequently, the filter plates were dried and counted on the 1450 MicroBeta + counter (PerkinElmer). The cut-off value for counts per minute (CPM) values was determined by the average plus 3 × SD of the eight medium-only control wells. Stimulation index (SI) was calculated as the average of tested eight wells divided by the average of the medium-only control wells. The proliferative response was defined positive if the CPM values of at least six of the eight wells were above the cut-off value and if the SI was ≥3 [The protocol for LST is described in details recently . The PBMI was ≥3 ,13.The levels of IFN-γ, TNF-α, IL-2, IL-5, IL-10, and IL-17A were determined from supernatants collected from LST at day 6 with the Cytometric Bead Array (CBA) human enhanced sensitivity flex set system  as described previous . The det6 cells/well and the cells were cultured in IMDM containing 10% Human AB serum . More detailed protocol is written in our previous manuscript [Thawed PBMCs were seeded into 24-wells plate at the density of 1.0 × 10nuscript . Cells wnuscript ,31.χ2-test, with the likelihood ratio or Fisher’s exact test for categorical variables. Differences in the means of continuous variables were tested using non-parametric (Mann-Whitney) tests for two independent samples. Bonferroni correction was used in ANOVA with multiple comparisons. All statistical tests were two-sided and considered significant at p-value ≤0.05.All statistical analyses were run using IBM SPSS®  software package . Frequency tables were analyzed using the Additional file Figure T-cell reactivity against HPV16 E6 was rare in both groups; no one had any T-cell response to peptide pool E6.1, and a response against E6.2, E6.3 and/or E6.4 was found only in two of the women with incident CIN (ID2 and ID7)  against the memory response mix (MRM), which was statistically significantly higher among the cases  than in the controls  (p = 0.001)  of the CIN cases, mostly due to the pooled effects of HPV16 E2.1 and E2.2 as also the case with IL-10 and IL-17A secretions.Figure HPV16 -specific CD4+ CD25+ Foxp3+ regulatory T-cells were detected only at low levels in both the cases and the controls  of the CIN women and 38.1% of controls analysis  were smokers. Previous genital warts were recorded in 10% (1/10) of the cases and in 40% (8/20) of the controls, but the difference was not statistically significant. No significant differences were seen either in medication, number of sex partners, different sexual habits, alcohol consumption and STDs between these two groups.We also analyzed the LST response to HPV16 peptides and MRM stratified by smoking, use of alcohol, genital warts, oral sex habits and number of sex partners. Smokers and users of alcohol had significantly higher LST MRM response than the non-smokers or non-drinkers , but no differences were found in CMI responses against HPV16 E2, E6 and E7 peptides ; Group 2, women with cervical HPV16, but her CIN lesion was associated with other or unknown HPV genotype (n = 5); Group 3, women with oral HPV16 and HPV16 -specific antibodies, but no cervical HPV infection (n = 14); Group 4, women with oral HPV16, but neither HPV16-specific antibodies nor cervical HPV16 infection (n = 8). The results of the LST and cytokine analyses stratified by the groups are shown in Table In the present study, we have assessed the HPV16-specific T-cell immunity in the women who developed HPV16-associated CIN during the FU. The results were compared with those of the control women, who tested constantly HPV-negative in their genital samples. Although the number of women was limited, the main strength of this study is the long FU time with detailed data on HPV DNA status, not only of the genital tract but also of oral mucosa. In fact, this is the first study were also the FU data on oral HPV status is reported together with the genital HPV data.To our surprise, the HPV16-specific CMI against E2-, E6- and E7 peptide pools was similar among the women who developed CIN and in those who tested constantly HPV-negative. The FU data showed that the cases and the controls were similar with regard to the presence of either oral HPV16 DNA or HPV16 antibodies in sera. Our results indicate also that these healthy women have been exposed to HPV earlier in their life but the infection has either totally resolved or has never truly developed. Thus, the T -cell responses reported here could be induced by any HPV infection, either resolved or still present, in any site of the body. However, one would expect to find some differences in HPV-specific immunity in the women who developed severe HPV-induced cervical disease as compared to those who remained completely HPV-negative. For example, we recently demonstrated that the clearance of oral high-risk HPV infection is impaired by long-term persistence (24 M+) of cervical HPV infection .In the present study, we also found one interesting association between oral HPV and CMI after re-grouping the women into four groups on the basis of their HPV type in the CIN lesion, HPV serology and oral HPV status. The women with oral HPV16 DNA, but testing HPV-seronegative and being also HPV DNA-negative during the FU (Group 4), had the highest IFN-γ secretion level after stimulation with HPV16 E6.4 peptide pool. One could speculate that productive oral or oropharyngeal HPV infection with E6 expression could result in the local cell mediated mucosal immunity, which also protects from cervical HPV infection. Oral administration of lactobacilli bearing the surface-displayed E6 protein has been shown to induce T cell-mediated cellular immunity and antitumor effects in mice .Importantly, our results also indicated that oral mucosa and its HPV infection as such could not explain the similarity of HPV16-specific CMI in women with and without CIN. Unfortunately, we did not have data on anal HPV status to get a more comprehensive overview on HPV-induced immunity as related to the presence or absence of HPV DNA.Of note is the fact that all patients were treated for their CIN before entry in this CMI study, and their blood samples have been taken after (31-129 months later) diagnosis of the cervical disease. Thus, HPV16-specific CMI might have changed during the FU after CIN treatment. One would even expect to see a better HPV-specific CMI in these women after cone treatment. However, we found no evidence of impaired or improved HPV-specific CMI in these women as compared with HPV-negative controls. In fact, it has been shown before that the number of HPV16 E2 specific T-cells are high regardless the outcome of HPV infection after treatment . AlternaAnother observation of interest was that HPV16 E6 stimulation resulted in significantly higher secretion of IL-17A by T-cells in the controls than in women with CIN p = 0.035). IL-17A has a key role in host defense against infections, and the pathogenesis of some autoimmune and chronic inflammatory diseases. The role of IL-17A in cancer development is controversial . High pr. IL-17A We also found that there is no HPV16 E6.1 specific CMI either in CIN patients or in controls, indicating that this site is not relevant for HPV-induced immunity. As far as we are aware, there are no other studies reporting the CMI response against HPV16 E6.1 peptide pool (amino acid 1-50).Candida Albicans as one of the antigens present in MRM. C. albicans as a component of the normal flora often colonizes the skin and mucosal surfaces of healthy individuals. Underlying acquired immunity to C. albicans is usually present in immunocompetent individuals [C. albicans is capable of triggering an expansion of specific or naïve T-cells [Gardnerella vaginalis was the most frequent agent in women with HPV infection , while in the control group the most frequent agent was Candida . Accordingly, one would expect to find higher MRM response in the controls than in CIN patients. However, slightly higher MRM levels have been reported among the controls than in women with cervical cancer (CC) [Interestingly, the CIN women had T-cell hyperactivity against the common antigens as indicated by the nearly four-fold higher MRM response than in the controls (p = 0.001). One explanation for their higher MRM response in the CIN women might be ividuals . C. albi T-cells ,40. Cand T-cells . Murta acer (CC) ,41. Our We also compared the demographic data with the HPV-specific immunity. Atopic symptoms in childhood have been associated with an increased risk to acquire CC later in life ,43. In tPrevious studies have shown that smoking increases the risk for CC. Several studies have confirmed that smoking increases the risk to contract HR-HPV infections and their persistence ,44,45 anTo conclude, our results show that there are no differences in the HPV16-specific CMI except the higher secretion of IL-17A by the HPV16 E6 in the controls. Interestingly, the CIN women were also hyper-reactive against common antigens which prompt further studies. Smoking can stimulate the reactivity to common antigens but had no direct effect on HPV-specific CMI.


HPV)‐positive and HPV‐negative oropharyngeal squamous cell carcinomas (OPSCC) and to better understand the functional and biological effects of HPV infection in the premalignant pathway. Twenty‐four consecutive patients with locally advanced primary OPSCC were included in a prospective clinical trial. Fresh tissue samples  were subjected to whole transcriptome analysis and the results validated on the same cohort with RT–quantitative real‐time PCR. In a separate retrospective cohort of 27 OPSCC patients, laser capture microdissection of formalin‐fixed, paraffin‐embedded tissue allowed RNA extraction from adjacent regions of normal epithelium, carcinoma in situ  and invasive SCC tissue. The majority of patients showed evidence of high‐risk HPV16 positivity (80.4%). Predictable fold changes of RNA expression in HPV‐associated disease included multiple transcripts within the p53 oncogenic pathway (e.g. CDKN2A/CCND1). Other candidate transcripts found to have altered levels of expression in this study have not previously been established . Of these, SYCP2 showed the most consistent fold change from baseline in premalignant tissue; aberrant expression of this protein may contribute to genetic instability during HPV‐associated cancer development. If further corroborated, this data may contribute to the development of a non‐invasive screening tool. This study is registered with the UK Clinical Research Network (ref.: 11945).This study was designed to identify significant differences in gene expression profiles of human papillomavirus ( Human papillomavirus (HPV) is strongly associated with the development of oropharyngeal carcinoma. Although over 150 genotypes have been described, HPV16 is considered responsible for ~95% of viral associated cancers at this site.Epidemiological evidence from the USA would suggest that HPV‐associated oropharyngeal squamous cell carcinomas (OPSCC) is rising at an ever‐increasing rate.p53 mutation will normally be present and may contribute to the poorer clinical outcomes observed in this patient cohort.The roles of the two HPV16 oncoproteins E6 and E7 have been studied extensively and include, among others, inhibition of p53 and pRb (retinoblastoma) tumor suppressor proteins.in situ) and, ultimately, invasive cancer.in situ. Prior studies have reported markedly variable HPV prevalence rates due to limitations of size and variable assay techniques.et al. recently published a meta‐analysis of 22 relevant articles, suggesting HPV16 to be present in ~25% of all dysplastic lesions within the oropharyngeal subsite. The same author concluded this to be a conservative estimate due to the inclusion of oral cavity SCC lesions in some of the studies .Oropharyngeal squamous cell carcinogenesis involves progressive transformation of normal epithelium into premalignant tissue (dysplasia/carcinoma The majority ~75%) of patients with HPV+ OPSCC present at an advanced stage (III/IV) due to cystic nodal disease.5% of patin situ (dysplastic) change surrounding invasive carcinoma . Disease stage was classified using the TNM classification of malignant tumors.GSE56142. The trial protocol can be downloaded from the UK Clinical Research Network (http://public.ukcrn.org.uk).This project received formal approval from the National Research Ethics Service Committee of East of England (12/EE/44). After informed consent, 24 consecutive patients with OPSCC donated multiple fresh biopsy samples  at Cambridge University Hospitals National Health Service Foundation Trust between June 2011 and July 2013. A further 27 OPSCC patients were included from a retrospective cohort and assessed for evidence of carcinoma In all prospective fresh biopsy samples, tumor and the adjacent normal tissue were processed for DNA and RNA extraction. A consultant histopathologist with expertise in head and neck pathology reviewed each sample to ensure representative sampling . Whole transcriptome analysis used the Illumina Genome Analyzer IIx machine  and the results were validated with RT–quantitative real‐time PCR (RT‐qPCR) .in situ, and normal epithelial tissue to facilitate RT‐qPCR analysis.The retrospective formalin‐fixed paraffin‐embedded (FFPE) tissue cohort was subjected to laser capture microdissection (LCM). This allowed precise RNA extraction from areas of invasive cancer, carcinoma INK4A immunohistochemistry (IHC), and HPV DNA in situ hybridization.Human papillomavirus stratification methods included: consensus PGMY PCR, type‐specific HPV16 DNA PCR, and E6/E7 mRNA PCR, DNA sequencing, p16INK4a IHC (see below).Oropharyngeal fresh tissue samples from normal and invasive malignant regions (maximum 25 mg) were DNA extracted using a protocol published from this unit.INK4a IHC was carried out on FFPE tissue using a mouse mAb .in situ hybridization consisted of a probe directed against high‐risk HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66 .2O and stored at −20°C. Concentration and purity of DNA was assessed by spectrophotometry. Samples had an absorbance ratio (260/280 nm) in the range of 1.8–2.0, and were diluted with H2O to a concentration of 1–25 ng/μL prior to PCR. The L1 and E6/E7 DNA PCR was carried out as for fresh tissue samples (above).The p16Clinical and histopathological data for all prospective and retrospective subjects are shown in Figures The 24 OPSCC patients provided multiple fresh biopsy samples at the time of diagnostic or therapeutic surgery. All biopsies were selected on the basis of their RNA integrity number after histopathological review.Messenger RNA‐seq cDNA libraries were prepared from ~400 ng total RNA. In brief, mRNA was isolated using polydT oligonucleotides connected to magnetic beads, fragmented using elevated temperature and divalent cations, and converted to cDNA using reverse transcriptase. DNA polymerase I and random primers were then used to convert single‐stranded cDNA into double‐stranded cDNA. This was blunt end repaired with Klenow DNA polymerase and T4 before adenylation of the 3′‐end of the fragment. A final purification step used gel electrophoresis, with fragments cut out in the range 200–300 bp. These fragments were amplified by PCR and sequenced using the Illumina Cluster Station and Genome Analyzer (Illumina). Paired‐end sequence analysis (51 cycles per end) was carried out with primers specific to the ends of the bridge‐amplified cDNA fragments to obtain 51 nucleotides of sequence from each end of all cDNA fragments.anova identified target sequences with significant differential expression between normal and tumor tissue and further stratified by HPV status.Each array on the HumanHT‐12 version 4 Expression BeadChip (GPL10558) targets more than 31 000 annotated genes with more than 47 323 oligonucleotide probes derived from the NCBI Reference Sequence Release 38 and other sources. Raw reads from normal epithelium (control) and tumor samples were processed using the GeneSifter Analysis Edition  pipeline. Expression values for annotated genes were calculated from the aligned data by adding the number of reads linked to all exons and splicing events for a given gene and dividing that parameter value normalized by the total number of mapped reads in a sample. Two‐way ‐actin as a control gene and then with the transcript region of interest. When the optimal primer concentration produced a linear response to input cDNA concentration , samples were analyzed in triplicate for each tested transcript.Validation of expression data by RT‐qPCR analysis used the SYBR green method with an Applied Biosystems ViiA 7 Fast Real‐time PCR system. Primers  were optimized with βspss version 21 . To identify 80% of clinically relevant genes from the Illumina analysis, we based our power calculation on data supplied from Laborde et al.−ΔΔCT technique, as described previously.t was derived for the three replicate analyses of the reference gene (β‐actin), and this was subtracted from the average Ct value from the three replicate analyses for the genes of interest. Expression differences between the HPV+ and HPV− tumors were compared using these normalized ΔCt values and the observed differences subjected to a Student's t‐test. Rates of disease‐free survival (DFS) were estimated by means of the Kaplan–Meier method and were compared by the log–rank test. A multivariate model was developed using Cox regression to investigate the effect of clinical factors on disease outcome .Statistical calculations were carried out using in situ hybridization episomal/integrative staining pattern. Immunohistochemical analysis for expression of p16INK4a was shown for all HPV+ OPSCC samples but also present for 3 out of 10 OPSCC samples categorised as HPV−  and 23/27 of the FFPE samples (retrospective cohort) were classified as HPV+, defined by evidence of HPV16 L1/E6/E7 DNA, HPV16 E6/E7 mRNA, or HPV DNA versus HPV− OPSCC  . The six target transcripts were: CDKN2A and SYCP2 were increased with an average log2 fold change of 1.7  and 1.8 , respectively. Significant decreased expression was found for CRCT1 , CRNN [log2, −2.2; P < 0.01; 95% CI, 1.7–2.9), and DLG2 , HPV+ OPSCC expression levels of in situ  and SFRP1  showed the largest differential expression from normal epithelium to premalignant (carcinoma in situ) tissue. In the same cohort, 70% of patients displayed significantly elevated expression of CDKN2A in the premalignant region . Figure Laser capture microdissection from the retrospective FFPE cohort allowed analysis of log2 fold change in adjacent regions of normal epithelium, invasive malignancy, and carcinoma tu Figs. a,S5,S6. The average DFS for all patients was 43.7 months . For the HPV16+ cohort, the DFS period increased to 47.3 months . Significant expression of SYCP2+  and SFRP1+  cohorts showed an average DFS period of 49.6 months  and 40.1 months , respectively. A multivariate proportional hazards model using Cox regression analysis revealed HPV16, SYCP2, smoking, and physiological performance status to have significant influence on DFS  curve . The area under the ROC curve was found to be 0.86 , indicating a good discriminating power when compared to control subjects. Sensitivity and specificity estimates over a range of cut‐off points suggest optimal results were obtained for the log2 fold range 1.5–3.0 . Similar testing of SFRP1 expression revealed the area under the ROC curve to be 0.64 , indicating a poor discriminating power when compared to controls.To evaluate the prognostic accuracy of versus matched normal tissue. The data obtained were then validated by RT‐qPCR and the results used on retrospective FFPE tissue to investigate premalignant change surrounding areas of invasive carcinoma.In this prospective observational study, we describe the use of mRNA massive parallel sequencing technology to investigate HPV+/− OPSCC tumors To our knowledge, comparatively few studies have investigated expression profiles in HPV‐associated OPSCC,CDKN2A (a proxy marker for HPV infection) in regions of carcinoma in situ relative to normal epithelium. This is higher than the HPV16 estimate (~25%) provided by Jayaprakash et al.et al. (2012) carried out the largest epidemiological study on this topic (~6000 subjects), and estimated the prevalence for high‐risk HPV subtypes to be 6.9%, of which ~1% can be attributed to HPV16.Within our retrospective LCM cohort, 70% of patients showed significantly increased expression of et al. (and largely confirmed in this present study). Available published reports also suggest a higher prevalence of HPV16 within areas of in situ malignant change when stratified by the male sex  and between areas of transformation from normal through to dysplastic epithelium (×3).It should still be noted that high‐risk HPV subtypes in normal oral cavity samples have significantly lower prevalence than the estimates reported by Jayaprakesh SYCP2, SFRP1, DLG2, CRNN, and CRCT1. Of these, SYCP2 showed the most significant change from baseline in premalignant retrospective FFPE tissue, superseding the performance of CDKN2A (encoding for p16INK2A).In our prospective cohort of 24 OPSCC patients, RNA massive parallel sequencing data provided a statistically significant association for 223 target transcripts stratified by tumor and HPV status. Gene ontology data revealed the majority of transcripts to have limited clinical relevance but a focused analysis of oncological pathways produced a number of possible transcript candidates e.g. SYCP2 in HPV‐associated tumor tissue has previously been noted in three expression analysis studies.SYCP2 is a testis‐specific human gene and aberrant expression in HPV+ cancers may contribute to the genomic instability induced by high‐risk HPVs and subsequent oncogenic change.SYCP2 as one of three target biomarkers in the development for OPSCC.SYCP2 as a prognostic indicator is also of interest, given our DFS data, however, further research will be required to discern if this is truly independent of HPV16 expression.The elevated expression of INK4a protein is an inhibitor of cyclin‐dependent kinase and has increased expression with elevated levels of HPV E7, however, many units have reported a concern regarding false positive results.INK4a, which may concur with this analysis. At present, stratification of OPSCC tumors by p16INK4a alone is still the preferred approach by the majority of oncology centers. In an era of OPSCC de‐escalation treatments, which are based on a viral etiology, this may incur a risk of undertreating a small proportion of patients falsely considered as HPV+.(48) The use of stepwise algorithms, which combine different HPV assays, may compensate for the limitations of individual tests and should now be considered in clinical settings.The p16P < 0.03). The negative impact of smoking in HPV+ OPSCC has previously been highlighted by several randomized trials with post‐hoc analysis of HPV status.In this study, positive smoking status proved to have a significant adverse effect on DFS, regardless of HPV category . Clearly, further well‐designed prospective studies are required to confirm this data and also to establish if other biomarkers may have future significance.In conclusion, developments in whole‐genome sequencing and mRNA analysis are rapidly creating an opportunity to provide personalized information on genetic and functional aspects of malignant tumors. With regard to HPV+ OPSCC, the investigation of differentially expressed genes in normal, premalignant, and malignant tissue may reveal unique pathways that can explain their different natural history and biological properties. The data from this study reveal The authors have no conflict of interest.Fig. S1. Clinical and histopathological data for retrospective cohort of oropharyngeal squamous cell carcinoma patients. Human papillomavirus (HPV)‐positive status was defined as evidence of HPV16 L1/E6/E7 DNA or episomal/integrative pattern on HPV DNA in situ hybridization +/− p16INK4A expression (>70% tumor cell staining). †Non‐malignant cause of death. ††Malignant cause of death. #Locoregional recurrence. BoT, base of tongue; CRT, chemoradiotherapy; F, female; f/u, follow‐up; IHC, immunohistochemistry; ISH, in situ hybridization; M, male; Mod, moderately differentiated SCC; mths, months; n/a, not applicable; pal., palate; Poor, poorly differentiated SCC; RT, radiotherapy; SCC, squamous cell carcinoma; Well, well differentiated SCC.Click here for additional data file.Fig. S2. Full heatmap of 223 transcripts that are differentially expressed between human papillomavirus (HPV)‐positive and HPV‐negative oropharyngeal squamous cell carcinoma tumors .Click here for additional data file.Fig. S3. Gene ontology data for the 6/223 transcripts with possible oncological relevance.Click here for additional data file.Fig. S4. Reverse transcription–real‐time quantitative PCR data for prospective human papillomavirus (HPV)16‐positive and HPV16‐negative cohorts (fresh tissue). When the optimal primer concentration produced a linear response to input cDNA, samples were analyzed in triplicate for each tested transcript . β‐Actin (ACTB) was used as the housekeeping gene.Click here for additional data file.Fig. S5. Reverse transcription–real‐time quantitative PCR data for retrospective cohort . Formalin‐fixed paraffin‐embedded tissue samples were subjected to laser capture microdissection to enable RNA extraction from representative regions of invasive carcinoma and adjacent normal tissue.Click here for additional data file.Fig. S6. Reverse transcription–real‐time quantitative PCR data for retrospective cohort . Formalin‐fixed paraffin‐embedded tissue samples were subjected to laser capture microdissection to enable RNA extraction from representative regions of in situ carcinoma  and adjacent normal tissue.Click here for additional data file.Fig. S7. Disease‐free survival – multivariable and univariable statistical analysis. A proportional hazards model using Cox regression analysis revealed human papillomavirus (HPV)16, SYCP2, smoking, and physiological performance status to have significant influence on disease‐free survival.Click here for additional data file.Fig. S8. Receiver operating characteristic (ROC) curve to evaluate prognostic accuracy of SYCP2/SFRP1 biomarker expression in regions of human papillomavirus‐positive (HPV+) in situ carcinoma. All control subjects were patients undergoing tonsillectomy for benign pathology . A further analysis incorporated data from the HPV− oropharyngeal squamous cell carcinomas cohort.Click here for additional data file.Fig. S9. Reverse transcription–real‐time quantitative PCR data for the prospective control cohort. Fresh tonsil biopsy samples were subjected to RNA extraction (epithelium and adjacent stroma region).Click here for additional data file.


Blood samples were also tested for HIV and syphilis infections as well as CD4 and CD8 lymphocyte levels. The age range of the participants was 15–24 years and mean age was 18.6(SD 1.4). Vaccine-type HPV-16/18 strains were significantly less prevalent among vaccinated women compared to non-vaccinated women . At type-specific level, significant difference was observed for HPV16 only. Other STIs (HIV/syphilis) were important risk factors for HPV infections including both vaccine types and non-vaccine types. In addition, for non-vaccine HPV types, living in an urban area, having a low BMI, low CD4 count and having had a high number of life time sexual partners were also significant risk factors. Our data concurs with the existing literature from other parts of the world regarding the effectiveness of bivalent HPV-16/18 vaccine in reducing the prevalence of HPV infections particularly vaccine HPV- 16/18 strains among vaccinated women. This study reinforces the recommendation to vaccinate young girls before sexual debut and integrate other STI particularly HIV and syphilis interventions into HPV vaccination packages.The objective of this study was to determine the prevalence and some predictors for vaccine and non-vaccine types of HPV infections among bivalent HPV vaccinated and non-vaccinated young women in Uganda. This was a comparative cross sectional study 5.5 years after a bivalent HPV 16/18 vaccination  pilot project in western Uganda. Cervical swabs were collected between July 2014-August 2014 and analyzed with a HPV genotyping test, CLART Cervical cancer (CC) is the third commonest cancer among women globally, with an estimated 527,624 new cases and 265,672 deaths in 2012 [Persistent infection with sexually transmittable high-risk Human papillomavirus (HPV) is a necessary cause of cervical cancer . SeveralUganda also has a high prevalence of HPV infections among women with normal cervix as well as invasive cervical cancer –12. MoreTo address that, the Bivalent HPV16/18 ASO4-adjuvanted vaccine  was introduced in Uganda in 2008 for young girls as a primary prevention strategy for cervical cancer. In 2008 alone, over 3000 young girls aged 10–18 years who were in primary school grade 5 (P5) were given the Bivalent HPV16/18 vaccine as per the recommended schedule of 0, 1 and 6 months and a dose 3 vaccination coverage of over 95% was achieved . In ordeBivalent HPV immunization has been shown to reduce the genoprevalence of HPV types 16 and 18 in the vaccine trials  and postWe therefore aimed to compare the unvaccinated and vaccinated 15–24 year old women 5.5 years after the bivalent HPV16/18 vaccination  pilot project regarding the genoprevalence and predictors for vaccine and non-vaccine types of HPV infections to provide information on the early impact of bivalent HPV vaccination in Uganda. The findings can inform decisions on groups of girls that should be the primary target for HPV vaccination and catch up or supplementary vaccination campaigns.The ethical approval (number SBS HDREEC-131) for the study was obtained from the Institutional Review Board (IRB) of School of Biomedical Sciences, Makerere University College of Health Sciences, Uganda. The same IRB provided approval for the consent procedure employed for participants under the age of 18 years. Additional research clearance was obtained from Uganda National Council for Science and Technology (UNCST). Informed consents were obtained from each participant aged 18 years and above. For participants below the age of 18 years, assent was obtained from them plus a proxy informed consent from their Teachers because they were self- supporting in boarding schools, not living with their parents at home. These put them in the category of emancipated minors who should be given prompt opportunity to benefit from the medical tests and treatment provided as part of the research. The participants were provided transportation to and from the health facility for sample collection. At the health facility, participants were served a soft drink and were provided the opportunity to receive healthcare services for free.This was a comparative cross sectional study  conducteThe study population was women aged 15–24 years which comprised of bivalent HPV-16/18 vaccinated and non-vaccinated groups of girls. The vaccinated group was selected strictly from the 2008 cohort of bivalent HPV-16/18 vaccination. The total population eligible for the study was 3,459 girls vaccinated in 2008. At the time of vaccination in 2008, the average age of the vaccinated girls was 13 years and range was 9–18 years . Girls wThe sample size was determined by power analysis calculation . The calFrom July 2014 –August 2014, we performed a multi-stage sampling procedure to select the study participants and collect data. We first developed a total list of senior secondary schools where the 2008 cohort of vaccinated girls and their non-vaccinated counterparts were expected to be studying at the time of the study. All the schools were visited by the research team and girls in senior secondary three (S3) to senior secondary six (S6) classes were approached, informed about the study, consented and screened for eligibility. The eligible girls were given appointment to visit a designated clinic at a health facility within the locality for sample collection. On the clinic appointment day, vehicles were sent to transport the girls to and from the health facility in company of their teachers. At the health facility, samples and data were collected from consecutive girls reaching the clinic until the required sample size was reached. The procedures performed at the clinic included re-screening for eligibility, assignment to study group using the 2008 HPV vaccination register.An interviewer-administered questionnaire was used to obtain data about the participant’s age, address, education level, age at sexual debut, number of sexual partners in the past 3 months, number of sexual partners in the past 1 year, number of sexual partners in a lifetime and condom use level.2. The resultant BMI was categorized into underweight (BMI<18.5), normal weight (BMI 18.5–24.9), overweight (BMI 25.0–30.0) and obese (BMI >30.0) according to WHO criteria [Weight was measured using a standard weight scale. Height was also measured using a standard height scale. BMI was calculated using the formula Kg/mcriteria . The perVenous whole blood samples were drawn from the cubical vein of each participant using 8 mL heparinized or EDTA vacutainer tubes. The blood draws were performed by 2 experienced laboratory technicians.HIV-1/2 testing was performed using HIV-1/2 Rapid Diagnostic Tests (RDTs) in accordance with Uganda national algorithm, which is based on WHO recommendations . The tesMeasurement of CD4 lymphocyte counts was carried out using BD FacsCount®  at the Kiwoko Health Centre IV laboratory, Ibanda Uganda. The laboratory is designated clinical laboratory for Ibanda district and follows national standards for quality control. The BD FacsCount® protocol used for CD4 estimation is internationally acceptable, robust and known to have a low average coefficient of variation . Total lTreponema pallidum which is a common confirmatory test in many algorithms for syphilis testing [Part of the venous blood collected for HIV testing and CD4 estimation was used for the Syphilis testing. Serum obtained from the venous blood at the study site was kept and transported in liquid nitrogen to Immunology Laboratory at Makerere University College of Health Sciences where it was immediately transferred to minus 80°C freezer until testing. The liquid nitrogen tanks ensured cold chain for samples during transportation from fieldwork site to the Laboratory. All the Syphilis tests were performed by an independent Laboratory Technologist (co-author WM) at Immunology Laboratory of Makerere University College of Health Sciences, Kampala, Uganda who was not part of the blood draw team. The Syphilis tests were performed using a commercially available kit SYPHILIS TPHA liquid test . The tests were performed according to the manufacturer’s protocol. The TPHA test is an indirect hemagglutination test for the determination of antibodies specific to  testing .Cervical cells were collected using the CareBrush®  after inserting the right-sized speculum into the vagina and focusing the cervix. CareBrush® is a cytology sampling device manufactured by QIAGEN, Gaithersburg, MD, USA and was approved by US Food and Drug Administration (FDA) to be used for cervical sample collection. After cervical sampling, the brush heads were transferred directly into a vial containing the alcohol-based preservative careHPV collection medium , for storage and transportation to the laboratory. CareBrush® and CareHPV® collection medium (DCM) have been used for cervical sampling and storage in many previous studies in Uganda –32.While in the field, the vial containing the cervical samples were stored and transferred into a liquid nitrogen tank for shipment to immunology laboratory in Makerere University College of Health Sciences, Kampala, Uganda. All samples were shipped from the field to the laboratory every fourth night.® HPV2  which is based on PCR followed by microarray for determination of genotype. CLART HPV2  is a low-density microarray assay based on PCR amplification of genotype specific HPV L1 fragments from 35 individual HPV genotypes , with analytical sensitivity calibrated against known copies of cloned plasmids.Cervical swabs were analyzed with a HPV genotyping test, CLARTTwo hundred and fifty (250) μL of the diluted careHPV sample was spun down , with the supernatant removed and cell pellet re-suspended and incubated with proteinase K for two hours at 56°C. HPV DNA was purified using QIACube with the QIAcube mini kit according to instructions by the manufacturer (QIAGEN).® Human papillomavirus 2 software. All samples returning an invalid outcome were retested, and the second result was considered definitive.Five (5) μl of purified DNA were used for the PCR amplification and 50 μl PCR-reactions were run on PCR-equipment EppendorfMastercycler ep gradient S . Each reaction contained 45 μl reaction mix , 0.75 mM MgCl  together with sample DNA. PCR-program included an initial denaturation step at 95° for 5 minutes followed by 40 cycles of 95° at 30 seconds, 55° for 60 seconds and 72° for 90 seconds. PCR-program ended with a 4° step for 8 minutes. Visualization was performed on the CLART microarray, using 5 μl of the denatured PCR products. Hybridization between the amplicons and their specific probes on CLART results in formation of an insoluble precipitate of peroxidase when adding a Streptavidin conjugate that bind to the biotin-labeled PCR products. Precipitate is analyzed on the Clinical Array Reader  with CLARTCLART HPV2 assay was chosen for HPV genotyping in this study because it has demonstrated performance comparable to Hybrid Capture 2 (HC2) in terms of concordance level, clinical sensitivity and specificity. The agreement between CLART HPV2 and HC2 were both very good concordance levels in the ranges of 98.6–98.8% respectively . The cliData from the laboratory tests and questionnaire were coded, entered and analyzed with Statistical Package for Social Sciences (SPSS) version 22.0 for windows. The primary outcome variable for this study was the binary (positive or negative) HPV test result which was further categorized into any HPV, vaccine HPV-16/18 and non-vaccine HPV strains. The study arms i.e. HPV-16/18-vaccinated and non-vaccinated groups were compared on categorical variables using Chi square statistics or Fisher’s exact tests when frequency of occurrence of the event or status were small, for example <5 . To adjuThe number of participants eligible for the study was 488, those with HIV test results were 412, those with CD4 count results were 410, those with TPHA (syphilis) test results were 418 and HPV test results were 401 . The ageThe participants’ age range was 15–24 years. The HPV vaccinated group was significantly younger and also initiated sexual activity at significantly younger age than their non-vaccinated counterparts . NotablyOut of the 401 cervical samples tested, there were 135 HPV positive cases giving an HPV genoprevalence of 33.7% (95% CI 29.1–38.3). The non-vaccine HPV strains were generally more prevalent than the vaccine HPV-16/18 strains (32.4% 95% CI 27.8–37.0 vs. 3.0% 95% CI 1.3–4.7). After adjusting for age and age at sexual debut in log-binomial regression, both models for any HPV prevalence and vaccine HPV prevalence were not statistical significant improvements from one without such factors .Regardless of the HPV vaccination status, the vaccine HPV-16/18 strains were significantly more prevalent among young women of advanced secondary education, those who tested positive for other STIs (HIV and syphilis) and those with CD4 count <500 cells/ μL. As for the non-vaccine HPV strains, the prevalence were significantly higher among young women from urban address and those who tested positive for other STIs (HIV and syphilis).Generally, the genoprevalence of vaccine HPV-16/18 strains was significantly lower among the bivalent HPV16/18-vaccinated group compared to the non-vaccinated group (p 0.006) . As for Having a bivalent HPV vaccination status was a protective factor for vaccine HPV-16/18 type positivity, while having another STI (HIV/syphilis) was a risk factor . The facHere we report a follow-up study on protective effect of a bivalent HPV16/18 vaccine performed in a group of women aged 15–24 years in Ibanda District, Uganda. The overall 33.7% HPV genoprevalence found in the current study is much lower than the 74.8% from a previous study among Ugandan women of the same age bracket , 36.Our study finding that the genoprevalence of vaccine HPV 16/18 strains combined was significantly lower among the vaccinated group compared to the non-vaccinated group concurs with a number of previous bivalent HPV-16/18 vaccine follow up studies in Germany , Scotlan3, abnormal BMI, multiple lifetime sexual partners and non-condom use when compared to their non-vaccinated counterparts is unique. However, such detailed analysis might be hampered by lack of power in our study. In contrast, the findings from multivariate analysis of our study revealed that a significant risk factor for vaccine HPV-16/18 type positivity was other STIs (HIV/syphilis) as opposed to bivalent HPV vaccination which was a protective factor. As for non-vaccine HPV type positivity, other STI plus women’s number of sexual partners, underweight (BMI<18.5 kg/m2) and low CD4 count <500 cells/mm3 were risk factors. The association between other STI, particularly HIV infection, and HPV positivity observed in our study is consistent previous HPV studies in Uganda [Our study findings that the genoprevalence of vaccine HPV-16/18 strains were not significantly lower among subgroups of vaccinated girls such as those with other STIs (HIV/syphilis), CD4 count <500 cells/mmn Uganda , 36 and n Uganda  were theThe strengths of the current study included inclusion of sexually active young women as confirmed by verbal interview, inclusion of bivalent HPV fully vaccinated group whose vaccination status was first obtained verbally and then confirmed by HPV vaccination register, inclusion of a non-vaccinated comparison group that was statistically not any different from the vaccinated group on key predictors of HPV infections, use of a broad spectrum HPV DNA genotyping assay  that covers 35 HPV genotypes  and laboIt can be argued that the fact that the vaccinated group were younger and initiated sexual activity earlier than the unvaccinated group, they had prolonged exposure and the opportunity to develop persistent HPV infection. These may make age and age at sexual debut important confounders to the association between HPV vaccination status and HPV infection. It should however be noted that the differences in age and age at sexual debut between the vaccinated and unvaccinated group were means of 8 and 7 months respectively, which were too short a time for differential HPV exposure nor persistence. A previous prospective cohort study of Ugandan young women of the same age group (12–24 years) found neither incident HPV infection nor HPV clearance were associated with age after as long as 18.5 months and 226 person-years of follow up . NeverthWe found a significantly lower genoprevalence of vaccine HPV-16/18 strains among the bivalent HPV-16/18 vaccinated (Cervarix®) young women compared to their non-vaccinated counterparts in Uganda 5.5 years after bivalent HPV-16/18 vaccine implementation. Other STIs (HIV/syphilis) persists to be a risk factor for vaccine HPV-16/18 infection despite bivalent HPV vaccination. Our data concurs with the findings of bivalent HPV-16/18 vaccine efficacy trials and other previous bivalent HPV vaccine follow up studies from other parts of the world. This study reinforces the recommendation to vaccinate young girls in adolescence before sexual debut and the need to integrate other sexual health services into HPV vaccination programme targeted at young girls.