Three hundred and twenty-five women were analyzed. One sample was used for conventional cytology and another sample was collected using BD SurePath™ kit for HPV tests. Eighty samples (24.6%) were positive for HPV gene by PCR-Multiplex and were then submitted to PCR-RFLP and PapilloCheck® microarray. There was a genotyping agreement in 71.25% (57/80) on at least one HPV type between PCR-RFLP and PapilloCheck® microarray. In 22 samples (27.5%), the results were discordant and those samples were additionally analyzed by DNA sequencing. HPV 16 was the most prevalent HPV type found in both methods, followed by HPVs 53, 68, 18, 39, and 66 using PCR-RFLP analysis, and HPVs 39, 53, 68, 56, 31, and 66 using PapilloCheck® microarray. In the present study, a perfect agreement using Cohen's kappa (κ) was found in HPV 33 and 58 (κ=1), very good for HPV 51, and good for types 16, 18, 53, 59, 66, 68, 70, and 73. PCR-RFLP analysis identified only 25% (20/80) HPV coinfection, and PapilloCheck® microarray found 62.5% (50/80). Our Cohen's kappa results indicate that our in-house HPV genotyping testing  could be applied as a primary HPV test screening, especially in low income countries. If multiple HPV types are found in this primary test, a more descriptive test, such as PapilloCheck® microarray, could be performed.Persistent human papillomavirus (HPV) infection is an essential factor of cervical cancer. This study evaluated the analytical performance of restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) assay compared to PapilloCheck Cervical cancer was responsible for an estimated 530,000 diagnoses and 266,000 deaths in 2012, the most common type of gynecological tumor worldwide . From thThe introduction of HPV vaccine is stimulating developed countries to change their cervical screening program from cytology to HPV testing as a primary screening test –10. H® microarray and restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) assay are HPV tests based on different methodology that could be applied in screening programs. PapilloCheck® microarray is based on PCR amplification of a fragment of approximately 350pb from the E1 region of HPV genomes. The kit simultaneously detects 24 different HPVs  . The two methods are different in sensitivity, specificity, and costs. This study evaluated the analytical performance of PCR-RFLP assay compared to the PapilloCheck® microarray for HPV identification.PapilloCheck and 82) . PCR-RFL® microarray. For discrepancies of HPV results, DNA sequencing of L1 amplified fragments was performed.From November 2011 to March 2013, 990 women were attended for cervical screening in public clinics in São Miguel do Oeste, SC, Brazil. Three hundred and twenty-five of those women accepted to participate in our study. Two samples were collected from each woman. One sample was used for conventional cytology for routine diagnosis. Another sample was collected with BD SurePath™ kit for HPV tests. The routine cytological analysis was performed by Papanicolaou staining, analyzed and classified according to the 2001 Bethesda system: NILM , ASC-US , ASC-H , LSIL , HSIL , and ICC  . The abs® DNA mini Kit   , phenol–Germany) , and ammGermany) . The besg for 5 min at 25°C. The preservative fluid was removed and cells were resuspended in 180 μL sterile phosphate buffered saline (PBS). Subsequently, 20 µL of Proteinase K (Qiagen) and 180 µL of AL buffer (Qiagen) were added to these cells, vortexed and heated at 56°C under agitation (1200 rpm) for 1 h and at 90°C for another hour. Two hundred microliters of 2 M ammonium acetate  were added to the cell lysate, submitted to an ice bath for 5 min, and centrifuged at 12,000 g for 4 min at 25°C. Then, the supernatant was transferred to another tube and 600 µL of isopropanol (Sigma) was added. The cells lysate was homogenized for 20 inversions and centrifuged at 12,000 g for 4 min at 25°C. The supernatant was discarded; the pellet was washed with 1 mL of 70% ethanol (Sigma) and centrifuged at 12,000 g for 4 min at 25°C. The supernatant was removed and the pellet was maintained at 60°C until complete evaporation of the ethanol. DNA was suspended in 50 µL of elution buffer Tris-EDTA and stored at –20°C until further use. DNA quality control was determined by NanoVue spectrophotometry .DNA was extracted from cervical cytology vials . A 1-mL aliquot was pelleted by centrifugation at 12,000 2, 200 μM of dNTP (Life Tecnologies™), 2U Taq Platinum DNA Polymerase , and 5.0 μL of DNA. The target DNA was amplified by PCR  and reaction was carried out with a denaturation step at 95°C for 10 min, 40 cycles of 1 min at 95°C, 1 min at 55°C, 1 min at 72°C,and final extension at 72°C for 10 min. Plasmids containing HPV-33 L1 gene were used as positive control and DNase- and RNase-free water was used as negative control in all amplifications.Multiplex PCR with PGMY09/11 and PCO4/GH20 primers  was perfPstI , HaeIII , DdeI (Promega), and RsaI (Promega).HPV DNA positive samples were submitted to a PCR reaction with PGMY09/11 primers to perform PCR-RFLP for HPV genotyping. Reaction was performed according to Nobre et al. , with th® microarray  5μL of DNA eluate was used in the PapilloCheck® microarray for each reaction. Specimens containing the target DNA are hybridized to specific oligonucleotide probes immobilized on a DNA chip and detected by the binding of a Cy5-dUTP labeled oligonucleotide probe to the tag sequence. The DNA chip was scanned by the CheckScanner apparatus at 532 and 635 nm wave lengths. This test detects HPV genotypes 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53.56, 58, 59, 66, 68, 70, 73, and 82. In addition, human ADAT1 gene  was used as an internal control to assess the quality of the DNAAll positive HPV PCR-Multiplex samples were submitted to PapilloCheck® PCR Purification kit  or with QIAquick Gel Extraction kit (Qiagen), according to the manufacturers' instructions. Automated DNA sequencing was performed in an ABI 3730 Genetic Analyzer sequencer . The accuracy of the DNA sequencing was evaluated through the CAP3 program based on Phred quality score . HPV type was identified based on >90% sequence homology over 449–458 nucleotides.® microarray, Kappa test was performed and the reference values adopted were determined as proposed by Altman , and participants provided written informed consent to the study protocol.The cytological diagnosis from the 325 sexually active women , included 313 (96.3%) women with NILM and 11 (3.4%) with some cytological abnormality as follows: 4 ASCUS, 4 LSIL, 3 ASC-H. All samples with abnormal cytology were HR-HPV positive .® microarray. Of those, 22 (35.0%) were additionally analyzed by DNA sequencing.All 325 samples were positive for β-globin gene (control) and 80 samples (24.6%) were positive for HPV gene by PCR-Multiplex. Those 80 samples were then submitted to PCR-RFLP and PapilloCheckFrom the 80 HPV positive samples, 70 (87.50%) were genotyped by PCR-RFLP and 10 (12.5%) were inconclusive. The PCR-RFLP method identified 28 different HPV types. From those, 17 were classified as HR-HPV  and 11 were classified as LR-HPV . In HR-HPV group , HPV 16 ® microarray genotyped 72 (90.0%) samples, and 8 (10.0%) were not identified. The PapilloCheck® microarray identified all 24 HPV types described by the manufacturer. In HR-HPV group , the results were discordant. All discordant results were additionally analyzed by DNA sequencing. From those 22 samples, 10 were inconclusive by PCR-RFLP. Eight samples were not identified by PapilloCheck® microarray. In one sample, HPV 68 was identified by PCR-RFLP, but HPV 39 by PapilloCheck® microarray. Two samples were positive for HPV 62 by PCR-RFLP. HPV 53 was detected in one sample and HPV 39 and 43 in another sample by PapilloCheck® microarray. HPV 61 was found in one sample by PCR-RFLP and HPV 53 and 44/55 by PapilloCheck® microarray. HPV 32 was revealed by PCR-RFLP in one sample and HPV 56 in another sample by PapilloCheck® microarray (® microarray. Multiple HPV infections were detected in 7 samples using PapilloCheck® microarray.There was genotyping agreement in 71.25% (57/80) on at least one HPV type between PCR-RFLP and PapilloCheckcroarray . From th® microarray, genotyping was performed by PCR-RFLP and/or DNA sequencing: HPV 89 (three samples), HPV 61 (two samples), and HPVs 74, 83, 84 (one sample each). All HPV types revealed by PCR-RFLP were in concordance to DNA sequencing  of those positive samples revealed discrepancies between PCR-RFLP and PapilloCheck® microarray.The present study found 24.6% 80/325) positivity for HPV gene by PCR-Multiplex. Compared with other Brazilian reports that applied PCR-RFLP, discrepancies between regions were present. Miranda et al.  positivi 48% in R® microarray. HPV 16 was the most prevalent HPV type found in Brazilian studies that applied PCR-RFLP analysis , very good for HPV 51, and good for types 16, 18, 53, 59, 66, 68, 70, and 73. PCR-RFLP analysis identified only 25% (20/80) HPV coinfection, and PapilloCheck® microarray found 62.5% (50/80). Results indicated that our in-house HPV genotyping testing  could be applied as a primary HPV test screening, especially in low income countries. Probably, the small number of patients limited our results. However, if multiple HPV types are found in this primary test, a more descriptive test, such as PapilloCheck® microarray, could be performed. Currently, there is no gold standard for HPV typing (In the present study, a perfect agreement was found for HPV 33 and 58 (V typing ,40 and t


Our next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it’s diagnostic performance has not been tested in cases with multiple concurrent HPV infections.We conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections.p < 0.05). The NPV for all types was 92.5% (95% CI: 88.4–95.4). The NPV and specificity were similar for high and low risk HPVs . The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA .In total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2–91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk  as compared to low risk HPV   contains supplementary material, which is available to authorized users. Identifying type-specific infections of human papillomavirus (HPV) is fundamental for understanding carcinogenic risk in epidemiologic studies, particularly in high burden areas such as sub-Saharan Africa. We recently developed a next-generation sequencing (NGS)-based HPV genotyping assay that shows 92% concordance on type-specific infections with the Roche Linear Array (LA) in cases with head and neck or cervical cancer . The ass0 C. A 10-μL aliquot of the purified DNA was amplified using the PGMY 09/11 L1 consensus primer system which co-amplifies 37 HPV genotypes and a human β-globin internal control target. Both high- and low-risk HPV genotypes were detected using the LA . High-risk HPV included 13 type specific infections: 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, and 68  and the negative predictive value [)*100] for any HPV, and separately for high-risk HPV and low-risk HPV with the associated 95% confidence intervals. To estimate the ability of the NGS assay to correctly identify those with and without HPV genotypes, we calculated the sensitivity [)*100] and the specificity [)*100] for any HPV, and separately for high-risk HPV and low-risk HPV with the associated 95% confidence intervals. Two-sample tests of proportions were used to compare the performance of the NGS-HPV assay for high and low risk HPV infections relative to the LA. Analyses were performed using Stata Statistical Software: Release 13 .p = 0.04). The sensitivity of the NGS assay was higher for high risk HPV as compared to low risk infections (p = 0.0001). Interestingly, the NGS HPV assay never detected any of the low risk HPV types 84 and 89. If we exclude these two types, the sensitivity of the NGS assay becomes 78.6%, 95% CI: 59.0–91.7. The NPV for all types was 92.5% (95% CI: 88.4–95.4) and the NPV and specificity were similar for low and high risk HPVs . Overall, the NGS HPV assay detected an additional 10 HPV genotypes that were not among the 37 genotypes found on the LA  , the median number of high risk HPV was 5 (IQR: 5–7), and the median number of low risk HPV was 4 (IQR: 2–4) (Fig. Our study confirms that the NGS assay was able to detect multiple HPV infections, particularly the high risk type-specific infections. The NGS assay detected 90% of the same high risk HPV as the LA and its sensitivity or true positive rate for high-risk HPV was 93%. More specifically, the NGS assay can detect HPV-52, a high risk HPV that is underdiagnosed on the LA because it cross reacts with other type-specific infections. For the true negative infections, the NGS assay was able to detect 93% of the same types not detected on the LA. The NPV and the specificity were similar regardless if they were low or high risk infections. These findings are consistent with an earlier study that found that the additional eight forward broad-spectrum primers and two backward primers significantly improved the ability of this primer set to detect multiple infections [The Ion Torrent NGS assay may not be as sensitive for detecting all HPV type-specific infections as the new multiplex type-specific HPV E7 PCR system , 12, butThere is a possible limitation to our study. We oversampled specimens that had a large number of high-risk HPV infections which may have reduced the number of low-risk types included in the total sample. This may in turn have affected our estimates of the positive predictive value and sensitivity of the assay to detect low-risk HPV infections. Still, in larger studies, the capability to detect multiple oncogenic type-specific infections is more relevant to understanding risk factors for progression to cancer.Despite this limitation, our study demonstrates that the NGS assay is comparable to the LA in detecting multiple high-risk HPV infections. The NGS assay could provide a diagnostic tool for large epidemiologic studies evaluating the natural history of HPV and identifying those most at risk of developing HPV-associated malignancies. The reagent and material cost per sample is significantly lower with the NGS assay, although that will have to be balanced against the capital investment in the Ion Torrent platform.


We are pleased that our publication  inspiredIn addition, the PCR methodology is difficult to evaluate. The second paragraph states “In this study, we conducted seven PCRs according to Buehring’s group , 4 and oIt is plausible that the reported lack of detectable BLV infection in Chinese women with breast cancer could relate to lower consumption of bovine food products in China compared to the USA. However, this conclusion cannot be firmly made based on the methodology presented in this publication; further research on this topic is needed.


A second generation HPV vaccine has been developed for the prevention of anogenital cancers and precancerous lesions of the cervix, vulva, vagina, anus and of genital warts due to nine HPV types.We estimated the annual burden of these diseases attributable to the nine HPV types compared to HPV types from first generation vaccines in women and men in Europe.Incidence rates from the IARC database, cancer registries, the literature and Eurostat population data were used.The burden attributable to the HPV types targeted by both vaccines was estimated by applying the relative contribution of the respective HPV types from epidemiological studies.In 2013, the number of new anogenital HPV-attributable cancers was 44,480 with 39,494 of these cases related to second vs. 33,285 to first generation vaccine types.Among the 284,373 to 541,621 new HPV-attributable anogenital precancerous lesions 235,364–448,423 and 135,025–256,830 were estimated to be related to second and first generation vaccine types, respectively.The annual number of new genital warts was 753,608–935,318, with 90% related to HPV6/11.These data demonstrate how the large public health impact that was achieved by the first generation HPV vaccines could be further increased by second generation vaccines. The International Agency for Research on Cancer (IARC) has identified twelve HPV types as carcinogenic to humans: HPV16/18/31/33/35/39/45/51/52/56/58/59 ® 9 (Sanofi Pasteur MSD)/Silgard® (Merck Sharp & Dohme), and the bivalent HPV vaccine, Cervarix® . Both vaccines have reassuring safety profiles, as demonstrated in clinical trials, and are indicated for the prevention of cervical, vulvar and vaginal premalignant lesions and cervical cancer related to HPV16/18. The quadrivalent HPV vaccine is also indicated against premalignant anal lesions and anal cancer and protects against low-risk HPV6/11, which are responsible for about 90% of genital warts Two HPV vaccines have been licensed so far in Europe: the quadrivalent HPV vaccine, Gardasilptrend<0.0001) p=0.003) As it takes several decades for HPV infection to progress to cancer, it will be some time before the real effects of current HPV vaccination programs will be seen in terms of a reduced incidence of HPV-related cancers. In the meantime, in countries like Australia, where vaccination coverage is high, a reduction of 77% in the prevalence of infections with the HPV types targeted by the vaccines has been observed in young women ® 9), which aims to protect against the seven high-risk HPV types (HPV16/18/31/33/45/52/58) most frequently responsible for cervical cancer development worldwide, and the low-risk HPV types (HPV6/11) responsible for about 90% of genital warts. Thus, the nine-valent vaccine is designed to protect against five ‘new’ high-risk HPV types (HPV31/33/45/52/58) that are not targeted by the quadrivalent or the bivalent vaccine. The vaccine is indicated to protect against premalignant lesions and cancers affecting the cervix, vulva, vagina and anus caused by vaccine HPV types as well as genital warts caused by specific HPV types.After HPV16/18, high-risk HPV31/33/35/45/52/58 are the six most frequently detected HPV types in invasive cervical cancer worldwide In a Phase III study, the nine-valent vaccine prevented 97% of high-grade precancerous lesions of the cervix, vulva, and vagina caused by the five new high-risk HPV types (HPV31/33/45/52/58) The aim of this study was to estimate the annual burden of selected cancers, precancerous lesions, and genital warts attributable to the HPV types targeted by the second generation nine-valent HPV vaccine Gardasil 9®  in women and men in Europe in 2013, and to compare this to the estimated annual burden of the same lesions related to the HPV types targeted by the first generation HPV vaccines.22.1The Cancer Incidence in Five Continents (CI5) database, available on the IARC website The information in CI5 Volume X was obtained from national cancer registries for Belgium, Bulgaria, Croatia, Cyprus, the Czech Republic, Denmark, Estonia, Finland, Iceland, Ireland, Latvia, Lithuania, Malta, the Netherlands, Norway, Slovenia, Slovakia, and Sweden. The information in CI5 Volume X was obtained from regional cancer registries for Austria, France, Germany, Italy, Poland, Portugal, Spain, Switzerland, and the United Kingdom .Fig. 1SeTo ensure that national populations were adequately represented in countries where only regional cancer registries exist, we assessed the geographical coverage and distribution of these registries.Five of the 32 countries selected did not have data available in CI5 Volume X, as they did not have reliable cancer registry data: Greece, Hungary, Liechtenstein, Luxemburg, and Romania. We excluded Lichtenstein from our analysis, but for the remaining four countries we extrapolated the age-specific average cancer incidence rates of neighboring countries, or from cancer registries in the same area as the countries selected. The choice of countries used for extrapolation was the same as that in the Globocan database In conclusion, our results are referring to a geographical region of 31 European countries.2.2The following HPV-related cancer sites were selected: cervix (ICD-10 code C53), vulva (C51), vagina (C52), and anus . We estimated the mean annual number of new cancers at these sites in the selected countries based on the sex- and age-specific cancer incidence data available in CI5 Volume X The number of new cancers attributable to HPV overall, to HPV16/18, and to HPV16/18/31/33/45/52/58 was then estimated by applying the corresponding cancer site-specific HPV prevalence. Data on HPV prevalence were extracted from the most relevant published cancer site-specific data. To avoid overestimating the prevalence of individual HPV types due to multiple infections, different adjustment methods, which are described in the cited paper for each cancer site, were used.There are other cancers known to be associated with HPV, including penile cancer in men and a subset of head and neck cancers in both sexes. We did not estimate the burden of these cancers in this work, as the currently licensed HPV vaccines don’t have an indication for cancers at these anatomical sites.Moreover, the data currently available did not allow us to calculate the burden associated with the seven high-risk HPV types targeted by the nine-valent vaccine. Robust studies to estimate the prevalence of HPV in these cancer sites, and more specifically the attributable fraction of the seven high-risk HPV types targeted by the nine-valent vaccine, are still ongoing.2.3We performed a literature review to retrieve robust age-specific incidence data for cervical pre-invasive neoplasia CIN2+ , vulvar intraepithelial neoplasia grades 2 and 3 (VIN2/3), and vaginal intraepithelial neoplasia grades 2 and 3 (VaIN2/3) in Europe.Nygard et al. estimated the incidence rates of CIN2+ (including CIN2/3 and AIS) per 100,000 woman years in 2004–2006, the period prior to the availability of the quadrivalent vaccine. The estimates were presented by age group and were age-standardized using the European standard population. Data were available for four Nordic countries: Denmark, Iceland, Norway, and Sweden. Moreover, we had access to incidence data on CIN2+ for the period 2009–2011 from the French cancer registry of the Alsace Region, where an organized screening program has been in place since 1994 in Bas–Rhin and since 2001 in Haut–Rhin (unpublished data). Based on the data from these five countries we estimated incidence rate ranges for CIN2+ by age group, which we then extrapolated to the combined female population of the 31 included countries. The study by Nygard et al. We then estimated the number of CIN2+ (including CIN2/3 and AIS), VIN2/3, and VaIN2/3 cases attributable to the HPV types targeted by the nine-valent vaccine and the quadrivalent vaccine, respectively. For CIN2+ we applied the site-specific overall HPV prevalence, the relative contribution of HPV6/11/16/18, and that of HPV6/11/16/18/31/33/45/52/58, respectively. As no specific European data for CIN2+ have yet been published, the authors of the recently published paper As no age-specific incidence data are available for anal intraepithelial neoplasia grades 2 and 3 (AIN2/3), we used sex-specific, age-standardized incidence rates from the Danish Registry of Pathology 2.4To estimate the annual number of new genital warts cases we used the methodology previously described 33.13.1.13.1.1.1The estimated annual number of new cervical cancers in 2013 was 34,708  in the 31 selected European countries combined. It is generally accepted that HPV infection is necessary for the development of cervical cancer 3.1.1.2The estimated annual number of new vulvar cancers was 9544 . Given the overall HPV prevalence in vulvar cancer in Europe of 19.3% (95% CI: 16.7–22.0) 3.1.1.3The estimated annual number of new vaginal cancer cases was 2171 (95% bound: 1676–2684). Of these cases, 1544 (95% bound: 1046–2107) are estimated to be attributable to HPV, assuming an overall HPV prevalence in vaginal cancer of 71.1% (95% CI: 63.2–78.1) in Europe 3.1.1.4The estimated annual number of new anal cancer cases was 4562 (95% bound: 3875–5265) among women in Europe, irrespective of HPV status. Given the overall HPV prevalence in anal cancer of 87.6% (95% CI: 81.6–92.1) 3.1.23.1.2.1Age-standardized incidence rates of CIN2+ (including CIN2/3 and AIS) in the five countries for which data were available ranged between 138.8 (Norway) and 183.2 (Iceland) per 100,000 woman-years (Appendix A). Incidence was very low in the youngest age group (<20 years) in the four Nordic countries considered  All CIN is HPV-related, with HPV6/11/16/18 accounting for 23–25% of CIN1, 38.4–39% of CIN2, and 58% of CIN3. The HPV types targeted by the nine-valent vaccine account for 46–51% of CIN1, 71–74.3% of CIN2 and 85–90% of CIN3 (worldwide data) 3.1.2.2The age-standardized incidence rates of VIN2/3 in the three Nordic countries for which data were available ranged between 4.8 (Norway) and 8.8 (Iceland) per 100,000 woman-years (Appendix B). Based on the age-specific incidence data from these countries, the estimated annual number of new VIN2/3 cases in Europe was estimated at between 13,886 and 27,592. Of these cases 86.9% (95% CI: 82.6–90.4) are believed to be HPV-related, with HPV16/18 accounting for 82.2% (95% CI: 77.2–86.6) and HPV16/18/31/33/45/52/58 accounting for 94.4% (95% CI: 91.0–96.9) of HPV-positive cases 3.1.2.3Age-standardized incidence rates of VaIN2/3 in the three Nordic countries for which data were available ranged between 0.9 (Norway) and 1.3 (Iceland) per 100,000 woman-years (Appendix C). Based on age-specific incidence data from these countries, the estimated annual number of new VaIN2/3 cases in women in Europe ranged between 2549 and 4719. Of these cases, 95.8% (95% CI: 91.8–98.2) are expected to be HPV-related, with HPV16/18 accounting for 64.1% (95% CI: 56.6–71.2) and HPV16/18/31/33/45/52/58 accounting for 77.6% (95% CI: 70.6–83.3) of HPV-positive cases 3.1.2.4Based on the age-standardized rate of 0.58  for AIN2/3 in women 3.1.3The most robust data on the incidence of genital warts come from Germany and the United Kingdom. Based on these studies, a lower incidence estimate of 142.0 per 100,000 woman-years 3.23.2.13.2.1.1The estimated annual number of new anal cancers in men in Europe was estimated at 2729 (95% bound: 2195–3279), irrespective of HPV status. Given the overall HPV prevalence of 87.6% (95% CI: 81.6–92.1) in anal cancer 3.2.23.2.2.1Based on the age-standardized rate of AIN2/3 in men of 0.43 3.2.3The most robust data on the incidence of genital warts come from Germany 4Our estimates demonstrate the high burden of cancer and precancerous lesions associated with the HPV types targeted by the new nine-valent HPV vaccine (HPV6/11/16/18/31/33/45/52/58) that are expected to occur every year in women and men in Europe. Overall, 39,494  of the 53,714  new HPV-related cancers of the cervix, vulva, vagina, and anus in women and men in Europe are expected to be associated with the high-risk HPV types included in the nine-valent vaccine, versus 33,285  associated with high-risk HPV16/18. This represents an additional 6210 cancer cases (95% bound: 4892–7981), corresponding to a relative increase of 19% .Fig. 3EsThe proportion of cervical cancer attributable to the seven high-risk HPV types targeted by the nine-valent vaccine is similar worldwide. The combined relative contribution of these seven high-risk types in Europe was 89.0% (95% CI: 87.5–90.3) compared to worldwide values of 89.4% (95% CI: 88.8–90.1), which range from 84.6% (95% CI: 81.9–87.1) in Central America to 95.5% (95% CI: 91.2–98.2) in North America Furthermore, the estimated annual number of new cases of precancerous lesions occurring in women and men in Europe  was 286,423 to 545,558, with 284,373 to 541,621 HPV-positive cases. Of these, 235,364 to 448,423 cases are associated with HPV6/11/16/18/31/33/45/52/58 versus 135,025 to 256,830 associated with HPV6/11/16/18. The five new HPV types 31/33/45/52/58 were thus associated with an additional 100,339 to 191,592 cases per year, corresponding to a relative increase of 75% . We did As previously mentioned, there are other cancers known to be associated with HPV, including penile cancer in men and a subset of head and neck cancers in both sexes. The currently licensed HPV vaccines don’t have an indication for cancers at these anatomical sites and up to now efficacy of the vaccines against these cancers has not been demonstrated. However, it can be expected that future studies based on routine use of the vaccines may demonstrate their efficacy against head and neck cancers and penile cancers.Based on the above described methodology we estimated the overall burden of head and neck cancers in women and men and penile cancer in men in Europe. 17,676 new head and neck cancer cases were estimated to occur every year in women, with 2089 and 1915 related to second and first generation vaccine types, respectively. For men the number of head and neck cancers was 80,062, of which 7911 were related to second and 7144 cases to first generation vaccine types. The burden of penile cancer was 4096 this region with 1070 and 932 cases related to second and first generation vaccines, respectively (work in progress). These data are preliminary, as the key studies estimating the prevalence of HPV at these sites, and more specifically the attributable fraction of the seven high-risk HPV types targeted by the nine-valent vaccine, are still ongoing. The data currently available in the literature are very heterogeneous, as only a subset of head and neck cancers is HPV-related, and many other risk factors exist, like alcohol and tobacco consumption, which differ from region to region. Moreover, differences in the results of HPV prevalence between studies might be due to contamination and the variability of HPV detection methods. Studies considering more accurate biomarkers of oncogenic HPV activity, such as E6/E7 mRNA or p16INK4a in addition to HPV DNA prevalence, are still ongoing.Recurrent respiratory papillomatosis is also highly associated with HPV. It is a very rare disease, which is difficult to treat and has high recurrence rates. However, incidence data in Europe are scarce 4.1A short-term prediction method was used to estimate the number of new cancer cases in 2013 from the most recent data collected from 2003 to 2007. Therefore these estimates were accurate only if the incidence rates of the cancers under study remained stable over time. In the case of increasing incidence, they would slightly underestimate the expected number of cases, and the opposite would be true in the case of decreasing incidence.As mentioned above, the CI5 database contains national cancer incidence rates for 19 European countries. Eight of the countries included in this report had only regional incidence rates available, which were extrapolated to the entire country. Although we assessed the geographical coverage and distribution of these regional registries, other factors could vary and influence regional incidence rates. For the remaining four countries no robust regional or national data were available. We thus extrapolated the mean incidence data from surrounding cancer registries to these countries, but we had no means to check the robustness of this method. Therefore the results should thus be interpreted with particular caution.For some cancer sites additional tests such as p16 or mRNA had been performed to confirm causality in the presence of HPV DNA (lack of data availability). Indeed, the mere presence of HPV is insufficient to prove causation, as the infection may be transient and not related to the carcinogenic process. Therefore our application of previously published HPV prevalence to an estimated number of new cancer cases may have yielded an overestimation of cases attributable to HPV.Our calculations were based on the relative contribution estimates extracted from one study per cancer site. However, for each cancer site we selected the study with the most robust design and a large sample size, and that provided individual data allowing weighting for multiple infections. We used only European data and, when available, adjusted data rather than crude data. Sex-specific data for HPV prevalence in anal cancers were available, but we decided not to use it, as sample size by sex was not sufficient to obtain meaningful, precise estimates. In any case, there were no sizeable differences between the data for both sexes. It was not possible to compare the extracted data to results from meta-analyses, as to our knowledge no meta-analysis contains information about multiple infections in precancerous lesions, and thus the relative contribution of individual HPV types, taking multiple infection into account  could not be estimated. A literature search that we performed in parallel showed large differences in the prevalence of the different HPV types between studies and regions. However, it is not clear if these differences are due to real differences in the distribution of HPV types among European women, or to differences in HPV detection methods. Furthermore, most of the published studies contained no information about individual multiple infections or had low sample sizes. The calculation of a robust relative contribution of specific types was thus not possible based on these data, as it would have provided less meaningful results.The method used to calculate the estimated annual number of new CIN cases also has some limitations. For CIN2+ we based our estimations on data extracted from one published epidemiological study covering four Nordic countries and one French study (unpublished). To calculate the estimated annual number of new CIN2+ cases in women in Europe we extrapolated the lower and the upper mean value of each age group to the population of each included European country. However, the incidence of CIN may vary throughout Europe, not only due to regional differences in the prevalence of HPV, but also due to different screening and prevention methods.In Iceland for example, screening starts at age 20, with a 2-year interval until age 69. This might explain the high incidence rates observed in the youngest age groups and lower incidence rates in the age group over 50 in Iceland compared to the other four countries .We used data from countries with well-organized screening, thus reflecting the real high burden of precancerous lesions. By extrapolating these data to countries without or with less organized screening, we overestimated the detected burden in those countries. However these lesions do exist, even if not detected, and have a high potential to develop to cancer if not recognized and treated: 5% of CIN2 and more than 12% of CIN3 progress to invasive cancer Another limitation is that our estimations of CIN2+ were based on estimates of the relative contribution of HPV observed in a population of young women (aged 16 to 26 years), who may have more multiple infections than older women. The burden of precancerous lesions is most important in the younger age group, and in the absence of age-specific relative contribution estimates, it seemed appropriate to use the data for all age groups combined.Finally, it has to be considered that all results on the incidence of cancers and cervical precancerous lesions in this study have been estimated in a post-screening context.The reporting of VIN2/3 and VaIN2/3 might not be exhaustive as there is no dedicated screening program, thus the reported burden is probably underestimated. Differences in the incidence of these lesions were observed between the different countries and it remains unclear to what extent the observed discrepancies can be explained by the completeness of registries, differences in clinical practice and management, or different background risk. Compared to a study from the Netherlands 5A total number of 53,714 new cases  of cancers of the cervix, vulva, vagina in women, and anus in women and men, were estimated to occur in Europe every year, of which 44,480  were estimated to be HPV-positive. Of the latter, 89%, i.e., 39,494 cases  were estimated to be related to HPV types 16/18/31/33/45/52/58 versus 75% to HPV16/18, corresponding to 33,285 cases .Additionally, the estimated annual burden of CIN2+, VIN2/3, VaIN2/3 in women, and AIN2/3 in women and men ranged between 286,423 and 545,558 new cases per year, of which 82% were related to HPV6/11/16/18/31/33/45/52/58 , corresponding to 135,025 to 256,830 cases.In addition to cancers and precancerous lesions, between 753,608 and 935,318 new annual genital warts cases were estimated to occur in women and men in Europe, of which 90%  were estimated to be related to HPV6/11.The relative increase in the number of new cancers attributable to HPV16/18/31/33/45/52/58 compared to HPV16/18 was 19%. For precancerous lesions this increase was 75% when comparing HPV6/11/16/18/31/33/45/52/58 and HPV6/11/16/18These data demonstrate how the large public health impact in the prevention of cancer that was achieved by the first generation HPV vaccines could be further increased by the second generation nine-valent HPV vaccine, due to additional cancer prevention and notably the prevention of precancerous lesion in women.SH and GDF are employees of Sanofi Pasteur MSD; JJB has been a member of the scientific advisory board of MSD Sanofi-Pasteur and GSK, has received speaker’s fees and travel grants from MSD Sanofi-Pasteur and GSK, and research supports from MSD Sanofi-Pasteur; FS was employee of Sanofi Pasteur MSD at the time of study initiation; LA received occasional travel grants to attend scientific meetings from MSD and Sanofi Pasteur MSD; SDS received travel grants from MSD, GSK and Qiagen and unrestricted research grants through ICO from Merck & Co. Inc. and Glaxo Smith Kline.; XC has received speaker honoraria, travel grants to attend scientific meetings from SPMSD, and research funding through ICO to undertake HPV studies from Merck & Co. Inc., Glaxo Smith Kline, and Sanofi Pasteur MSD.SH contributed to the study design, literature research, data analysis, interpretation of findings and drafting of the manuscript. JJB contributed to the data collection and interpretation of findings. GDF contributed to the study design, and interpretation of findings. FS contributed to the study design, data-analysis, interpretation of findings, and critical editing of the manuscript. LA contributed to the interpretation of findings. SDS contributed to data collection, interpretation of findings, and critical editing of the manuscript. XC contributed to the study design, data collection, interpretation of findings, and critical editing of the manuscript. All authors critically reviewed the manuscript and approved the final version.


The Swedish National Cervical Screening Registry collects and evaluates comprehensive, nationwide health data to optimise organised cervical cancer prevention. Since all cervical cancer specimens are saved in biobanks, population-based data from the specimens should be available for analysis and linkage with other health information.E6/E7 regions of HPV16/18.We identified all cervical cancers diagnosed in Sweden during 2002–2011 (4254 confirmed cases) and requested the tissue blocks to retrieve human papillomavirus (HPV) genotype data using general primer PCR with Luminex genotyping and real-time PCR targeting the E6/E7 region.We obtained blocks from 2932/4254 (69%) of cases. Valid HPV genotyping data was retrieved for 2850 cases (97%). The most common type was HPV16 (60%), followed by HPV18 (19%), HPV45 (7%), HPV31 (3%), HPV33 (2%), HPV52 (2%), HPV39 (1%), HPV70 (1%), HPV56 (1%), HPV35 (1%), HPV58 (1%) and HPV59 (1%). Ninety-six percent of all HPV-positive cases had a single infection. Eighty-nine cases were HPV-positive only when testing for the HPV16/18-We present one of the largest series of HPV-genotyped cervical cancers to date. The systematic collection of cervical cancer HPV genotyping data by the screening registry will facilitate prevention and monitoring of HPV type-specific disease burden. Most countries that organise cervical screening programmes also have comprehensive screening registries that can provide the evidence base for evaluation and improvement,2 but modes of operation of these registries vary and the rate of innovation to evaluate new methods for data capture and analysis can also be variable.Human papillomavirus (HPV) infection is the major risk factor for cervical cancer.www.nkcx.se); despite this, more than 450 women are diagnosed with cervical cancer every year.3 The Swedish National Cervical Screening Registry (NKCx) collects and evaluates comprehensive data on cervical screening, as a basis for evidence-based optimisation of cervical cancer prevention in Sweden.4 As part of an initiative to promote excellence in the operation of quality registers in healthcare, the screening registry piloted systematic, population-based HPV genotyping of cervical cancers and importing of resulting data. For all cervical cancers diagnosed in Sweden during a 10-year period, 2002–2011, we requested the corresponding archival diagnostic blocks and subjected the delivered blocks to HPV genotyping. Consecutive and population-based HPV genotyping data of cervical cancers will establish a baseline of the HPV type-specific disease burden as basis for optimising cervical cancer prevention strategies and enable a later comparison after the switch to primary HPV screening and/or implementation of new vaccination strategies.Sweden has had organised cervical screening since the 1960s, with very high population coverage by international standards (participation rate 82%) . The medical charts of all 4533 registered cancers were retrieved from the respective hospitals and subjected to a detailed review by an experienced gynaecologist. Primary, invasive cancers of epithelial, cervical origin were confirmed in 4254 cases and clinical data including histopathology, FIGO cancer stage and treatment were collected -blocks for HPV-genotyping. The Regional Ethical Review Board determined that, due to the population-based nature of the study, informed consent from study participants was not required (EPN-Dnr: 2011/1026-31/4) and collection of the samples for histology review and HPV-typing was also allowed (EPN-Dnr: 2012/1028/32).Access to the samples was requested from 25 different biobanks that serve as regional administrative units. Samples are stored in 30 local pathology laboratories (for Stockholm county the oldest cases are stored in a long-term storage facility). All biobanks approved access to the diagnostic slides. Twenty-two biobanks also approved the application for FFPE-blocks but three biobanks declined. In total, five biobanks never sent any blocks. All diagnostic slides were reviewed by an expert pathologist. In total, 2954 blocks from 2932 patients were received for sectioning. Information on histopathology type was collected and the FFPE block with the highest tumour to healthy tissue proportion and the most severe morphology was chosen for HPV genotyping (one block per patient).5 The first and last sections for each case were stained with haematoxylin and eosin (H&E-staining) for later re-review. In-between each case-block, a blank-block was sectioned as contamination control. All sections, blank-blocks and case-blocks, were extracted with a xylene-free method6 and HPV genotyped using PCR with modified general primers (MGP)-PCR (primer targeting L1) and hybridisation with type-specific probes in Luminex, as previously described.8 Forty-two beads, encompassing 37 different HPV-types, three HPV variants, and two ‘universal’ HPV probes, were included in the Luminex. If the case was HPV-negative, extracted material from both the blank-block and case-block was diluted 1/10 and re-tested.All cases were sectioned at an accredited laboratory, HistoCenter, Inc. in Gothenburg, Sweden, according to a contamination-proof procedure, as previously described.9 The blank-block had to be negative for both HPV and beta-globin and the case-block positive for beta-globin. Cases that were beta-globin negative were classified as inadequate samples and were not analysed (n = 4). Cases with a contaminated blank block were not analysed (n = 11). Cases with a valid HPV negative result (both undiluted and diluted 1/10) were re-reviewed by an expert pathologist to confirm presence of invasive cervical cancer tissue, as previously described.5 In total, 44 cases were not confirmed to contain invasive cervical cancer tissue in the block and were excluded from the study.Blank-blocks and the matched case-blocks were treated in exactly the same way during the whole process. All samples were analysed for beta-globin by quantitative real-time PCR to confirm sample adequacy, as previously described.E6/E7 regions of the two most common oncogenic HPV types, HPV16 (primer targeting E7) and HPV18 (primer targeting E6).12 Briefly, 1 µL DNA was used in both assays with a total volume of 25 µL. Both PCRs used a standard curve with a known concentration of 1–10,000 copies/µL.Cases that were HPV-negative in genotyping but whose tumour material was confirmed to contain invasive cervical cancer tissue were analysed for the In order to estimate the proportion of the current tumour series that might have been prevented by current HPV vaccines, we calculated the proportion of tumour HPV types covered by the bivalent HPV vaccine, quadrivalent HPV vaccine and nonavalent HPV vaccine, respectively. In this calculation, we only considered single infections and did not consider multiple infections, or potential cross-protection.The case ascertainment started with all 4533 cervical cancers registered in the Swedish Cancer Registry over 10 years. Following the detailed medical chart review, 4254 (94%) cases with primary, invasive cancers of epithelial, cervical origin were confirmed. Blocks were obtained for 2932 of 4254 requested blocks (69%). Valid HPV genotyping results were obtained for 2850/2932 cases (97%). The remaining 82/2932 cases (3%) were excluded due to the following reasons: 23 cases because they had more than 1 block, 15 cases because they had no result due to technical issues, and 44 cases because invasive cervical cancer was not confirmed in the re-review of the sectioned tissue block. The HPV genotyping data thus comprised 2850/4254 (67%) of all cervical cancers in the country during the 10-year period investigated.When considering single infections only, the most common HPV genotype was HPV16 , followed by HPV18 (19/16%), HPV45 (7.1/6.1%), HPV31 2.8/2.4%), HPV33 (1.9/1.6%), HPV52 (1.6/1.4%) and HPV39, HPV70, HPV56, HPV35, HPV58 and HPV59 (<1% each). Only one HPV type was present in 2364/2850 (83%) of all cases with valid results. Multiple infections were present in 92/2850 cases (3%) and no HPV infection was detected in 394/2850 cases (14%)  and HPV18 (E6 gene) using quantitative real-time PCR. Eighteen percent (89/483) of these cases were positive for E6/E7 of HPV16 and/or HPV18.The slides from the sectioning were re-reviewed by the study pathologist for the 527/2850 (18%) of cases that were negative after HPV genotyping. No invasive cervical cancer tissue was found in 44/527 (8%) of these cases and therefore, they were excluded after this re-review. The remaining 483/527 (92%) negative cases were analysed for HPV16  in the bivalent vaccine were found in 79% of all HPV-positive  cervical cancers. The proportion of tumours positive for the four types in the quadrivalent vaccine  was also 79%. The types in the nonavalent HPV vaccine  were found in 94% of all HPV-positive cases and 78% of all cases  is another important strength. The alternative strategy to select the block that tests positive in the case of multiple blocks creates a systematic bias and should be avoided.Although all pathology biobanks were (in principle) open access, not all blocks from case patients were actually sent. A number of blocks were not sent due to quality issues such as lack of tissue left in the block, or the diagnostic slides we required for review having been discarded. Furthermore, 636/4275 blocks (~15%) were not sent because the biobank did not approve our application for access. Three of the 22 counties approved the application but did not send any blocks. Some offered to instead provide sections from the block if they performed the sectioning themselves. As mandated by European Union purchasing laws for services worth as much as the amount of sectioning required by this study, we requested sectioning services through a formalised purchasing procedure and were therefore legally unable to use other sectioning services than from the company that had won the tender. Also, a central, accredited laboratory for sectioning is crucial in order to avoid variability in sectioning procedures. Ordinary H&E staining of the first and last section before the sections that were HPV genotyped enabled verification of whether the sections tested did indeed contain cancer tissue.Limitations of our study include that some diagnostic FFPE-blocks might have been sectioned so many times that no cancer tissue is left in the block that was delivered to us. Pathologist services to re-review the entire study of almost 6000 slides, first and last for all cases, was too expensive and we therefore decided, already at the outset, to re-review only HPV-negative specimens. This could conceivably have resulted in that some positive HPV-tests derive from specimens where cancer tissue was not present. However, only a small proportion (8%) of the HPV-negative specimens that we did re-review were excluded because of lack of cervical cancer tissue and since specimens positive for a virus with tropism for epithelium should have contained at least some epithelial tissue, the proportion of false positives is likely to be even lower in the full material. The fact that very few tumour specimens contained more than one HPV type also suggests that presence of HPV types originating not from the cancer but from adjacent benign epithelium is likely to be very low.13 Cervical cancer cases that appear to have lost HPV DNA seem to constitute a specific subgroup of cervical cancers with a different biological behaviour (worse prognosis).15 Possibly, some or all of the HPV genome may have been lost with increasing progression of the tumour resulting in that the HPV L1 region was no longer detectable. Indeed, the proportion of HPV-positive cancers increased when also testing for the E6/E7 region. Future efforts should be directed at testing whether parts of the HPV genome may still be present in these apparently HPV-negative cancers.Some cervical cancers in this study were HPV DNA negative, even though HPV is considered a necessary risk factor and virtually all cervical cancers in situ are HPV-positive.16 found that more than 2/3 of ICC and half of HSIL could be prevented by vaccines protecting against HPV16 and 18, a finding which we confirm.When estimating the effect of eliminating HPV vaccine types from the population, the proportion of all cervical cancers that were found to contain vaccine types  constitutes a lower bound of the estimated effect and the proportion of HPV-positive cancers that contain vaccine types  constitutes an upper bound of the estimated effect. A large meta analysis17 but estimation of the possible effects of cross-protection was considered to be beyond the scope of this paper. Similarly, formal predictions of number of cases prevented for each year after vaccination would require a more comprehensive age and period effect analysis. The problem with assigning causality on cases with multiple infections has been extensively discussed and several strategies to approach the problem have been proposed.18 However, none of these approaches are entirely satisfactory. Therefore, as 96% of our cases were only positive for a single HPV type, and consideration of multiple infections did not change which conclusions on type distribution, we decided to focus our analysis on single infections only.Cross protection seems to vary between the vaccines and elimination of HPV types merely by cross-protection is conceivable,We present one of the largest series of HPV-genotyped cervical cancers to date. The systematic collection of cervical cancer HPV genotyping data by the screening registry will facilitate prevention and monitoring of the HPV type-specific disease burden.


This study reports six HR-HPV types’ infection prevalence discriminated by species and multiple infection in unvaccinated Colombian female adolescents, as well as some factors modulating the risk of infection. HPV DNA for six high-risk viral types was identified in cervical samples taken from 2,134 12–19 year-old females using conventional generic and type-specific PCR. Binomial logistical regression analysis was used for modelling HR-HPV infection and multiple infection risk. The interaction between variables in a stepwise model was also included in such analysis. Viral DNA was detected in 48.97% of the females; 28.52% of them had multiple infections, HPV-16 being the most frequently occurring type (37.44%). Cytological abnormality prevalence was 15.61%. Being over 16 years-old (1.66: 1.01–2.71 95%CI), white ethnicity (4.40: 1.16–16.73 95%CI), having had 3 or more sexual partners (1.77: 1.11–2.81 95%CI) and prior sexually-transmitted infections (STI) (1.65: 1.17–2.32 95%CI) were associated with a greater risk of HPV infection. Having given birth was related to a higher risk of infection by A7 species and antecedent of abortion to less risk of coinfection. Where the females in this study came from also influenced the risk of infection by A7 species as female adolescents from the Andean region had a lower risk of infection (0.42: 0.18–0.99 95%CI). The presence of factors related to risky sexual behaviour in the study population indicated that public health services should pay special attention to female adolescents to modify the risk of infection by high-risk HPV types and decrease their impact on this age group. The human papillomavirus (HPV) causes cervical cancer (CC), this being one of the five most frequently occurring types of cancer according to the International Agency for Research on Cancer (IARC). It is the second most frequently occurring type of cancer in Colombia, mainly affecting reproductive-aged females . In addiVariable HPV prevalence has been reported in young females –6; howevThis study was aimed at estimating six HR-HPV types’ infection prevalence, as well as the sociodemographic and behavioural factors, which might be associated in the target, risk group . No convincing data regarding Colombia is currently available for providing new means of detection or improving P&P programmes, treatment and/or the timely control of cervical abnormalities in female adolescents.This was a cross-sectional study involving female Colombian adolescents (12–19 years-old) who were voluntarily attending CC P&P programmes provided by the health services of 19 municipalities/cities in Colombia between March 2007 and May 2012. The females included in the study represented four geographically distinct regions of Colombia , having different characteristics regarding relief, climate, vegetation and soil. There were also cultural, economic and social differences. The Caribbean region was represented by females from Barranquilla (Atlántico department), Cartagena (Bolívar department), Guajira department and Santa Marta ; the Pacific region was represented by females from Quibdó (Chocó department), Jamundí , Popayán (Cauca department), Tumaco and Ipiales (Nariño department). Bogotá , Soacha, Girardot and Guaduas (Cundinamarca department), Apartadó (Antioquia department), Bucaramanga (Santander department) and Chaparral (Tolima department) are part of the Andean region, whilst Villavicencio (Meta department), Leticia (Amazonas department) and Mocoa (Putumayo department) provided females from the southern region.The females in the study agreed to answer a questionnaire concerning sociodemographic data, sexual behaviour, smoking, family planning method, parity (# of deliveries > 20 weeks) and having a background of STI. A cervical sample was then taken which was used for the Papanicolaou test (Pap test) and for the molecular detection of HPV. Pap test results were reported according to the Bethesda system; females having an abnormal result had a colposcopy .β-globin gene with GH20/PC04 primers [The methodology used for viral extraction, detection and typing has already been reported , 13; cyt primers . Three s primers , 15. The primers . HR-HPV  primers . Positiv primers .Prior to the start of the study, a psychological evaluation verified minors’ understanding, reasoning and logical capability (Colombian guidelines / resolution 008430/1993) . Females2 test or Fisher’s exact test depending on the values observed in each category.For analysis purposes, multiple infections were taken as the presence of at least one viral type. Percentages were reported for categorical variables, along with their respective 95% confidence intervals (95%CI); medians with interquartile ranges (IQR) were used for continuous variables . Bivariate analysis was used for finding differences in percentages for females having or without HPV infection, i.e. χCervical lesion frequency was analysed regarding age, as was multiple infection frequency, infection by specific type regarding the degree of lesion reported in cytology .Binomial logistical regression analysis was used for modelling the risk of HR-HPV infection, infection by A9 and A7 species and infection by more than one viral type. Crude and adjusted odds ratios (OR) were reported, together with 95%CI. The models were adjusted for age, region, ethnicity, monthly income, civil state, the amount of sexual partners, STI incidence, a background of births and /or previous abortions, contraceptive method used and whether the participant smoked. A stepwise model was used for assessing association and interaction between variables (0.15 as p.e and 0.2 as p.r).Stata 12 software was used for all statistical analysis (0.05 significance level).β-globin) could not be amplified  in their samples. Generic viral identification, followed by type-specific identification, was used on the remaining 2,134 females. It was found that 1,045 young females were positive for HPV (48.97%: 46.83–51.11 95%CI); 70.62% (n = 738: 67.75–73.37 95%CI) of HPV positive females had single infections whilst 28.52% (n = 298: 25.79–31.36 95%CI) were positive for more than one HR viral type.This study initially involved 2,253 12 to 19 year-old females; females were excluded if the control gene , followed by HPV-18 (14.99%: 13.50–16.58 95%CI). The infecting viral type was not established in 9 HPV positive females (0.86%) since they were negative for the types identified in this study , suggesting another HPV type.Mean age at the start of sexual relationships was 15 years-old ; no statistically significant differences were observed between females having and without HPV . Females stating that they had had 3 or more sexual partners had a greater percentage of infection compared to females having had 0 to 2 sexual partners (61.09%: 54.32–67.55 95%CI compared to 38.46%: 32.01–45.22 95%CI); this was a statistically significant difference and further statistically significant differences were found for females having and lacking a background of STI (p = 0.000).Some degree of cervical lesion was identified by Pap test in 250 females (15.61%: 13.86–17.48 95%CI). Atypical squamous cells of undetermined significance (ASC-US) were found in 39.60% of the females, low-grade squamous intraepithelial lesion (LSIL) in 57.20% and high-grade squamous intraepithelial lesion (HSIL) in 3.20%. The following variables were related to a greater risk of infection when using logistical regression analysis for modelling the risk of infection by HR-HPV types: age >16 years, white ethnicity, having had 3 or more sexual partners and a background of STI. When determining risk by species, the risk of infection by A9  was associated with being over 16 years-old, as was a background of STI, whilst a background of births and STI conferred a risk of infection by A7 species (HPV-18 and -45). Factors such as previous abortions and coming from the Andean region appeared to be related to less risk of infection. When modelling the risk of coinfection in the study population, it was observed that births were associated with a greater risk and previous abortions with lower risk .The stepwise model suggested that HR-HPV infection risk variables remained significant, including interaction terms for determining whether these had an effect on the model: being over 16 years-old (adjusted OR 1.64: 1.01–2.64 95%CI), white ethnicity (adjusted OR 3.95: 1.07–14.60 95%CI), ≥ 3 lifetime sexual partners (adjusted OR 1.75: 1.11–2.75 95%CI) and a background of STI (adjusted OR 1.60: 1.15–2.22 95%CI). Stepwise models were constructed considering the other outcomes (infection by species A9 and A7 and coinfection); it was found that the aforementioned variables maintained their association with outcomes (data not shown).HPV prevalence was estimated in a female Colombian adolescent population and the factors which might have modulated the risk of infection were evaluated. It was found that age, ethnicity, the amount of sexual partners, a background of STI and previous births and abortions modulated the risk of HPV infection, infection by types from A9 and A7 species and infection by more than one viral type .Overall HPV infection prevalence varied according to geographical region, age and cervical abnormality –6. ConsiThe females participating in the study came from 18 municipalities/cities outside Bogotá which have reported high infection rates ; these rThe highest rates of infection worldwide have been found in females under 25 years-old (up to 80%) , 5; infeWorldwide 11%-12% HPV prevalence has been reported in females lacking cervical lesions; however, such prevalence increases proportionally with lesion severity  and invasive cervical cancer (ICC)) , 6 Tabl. As expeRegarding ethnicity, it was found that being white was associated with a greater risk of infection by HR-HPV types (67.57%: 50.21–81.99 95%CI); however, further studies are needed for determining these females’ ancestry and thus confirm such association. It was also found that this group of females was more likely to be exposed to HPV infection risk factors  and a background of STI (45.94%)). Curiously, it was also determined that the region they came from influenced the risk of infection by types from the A7 species; females from the Andean region had a reduced risk. This might partly be explained by most of the population in this region being mestizo (91.83%: 90.23–93.25 95%CI) and the lowest A7 type infection percentage was found for this ethnicity (16.92%: 15.19–18.77 95%CI).Previous studies have described the role of viral variants in CC, specifically depending on region and ethnicity , 24. PosEarly onset of sexual activity has been related to a greater amount of sexual partners during adolescence and in young adults, thereby favouring acquiring other STI and, in some cases, pregnancy at an early age , 28, 29.IARC studies in Lyon, France and by the Institut Català d’Oncologia (ICO) in Barcelona, Spain, have shown that the early onset of sexual relationships and pregnancies in adolescents increase the risk of developing CC due to early exposure to carcinogenic viral types, a longer period of adolescents’ susceptibility to HPV infection, the hormonal influence of steroids against HPV infection and host immune response during pre-adolescence and adolescence .A mixture of columnar, squamous and metaplastic epithelium is found in the cervix of adolescents and young females; the squamous-columnar junction (transformation area where HPV infection tends to cause CC) is exposed, facilitating stratified epithelium base layer exposure to HPV regarding minimum trauma . HormonaEarly onset of sexual relationships and pregnancy at an early age in females from developing countries usually occurs (as in this study) at around 15 years of age. Early maternity has been associated with a greater risk of CC, attributed to trauma during the first birth  as lesioAn inverse association between the risk of HPV infection and a background of abortion was identified, this having been previously reported in Mexican females . AbortioChlamydia trachomatis, Trichomonas vaginalis and Candida spp. promote cell proliferation, tissue debridement, free radical and cytosine release and facilitate colonisation, thereby improving microorganism virulence, increasing host susceptibility, facilitating HPV entry and reducing its elimination [The relationship between STI and HPV infection is based on the inflammatory effects of other pathogens. It has been described that alterations and damage to cervical epithelium produced by pathogens such as mination . The premination .Studies have indicated that multiple infections favour the persistence of a single viral type, whether due to inter-genotype competition or a more effective type-specific immune response triggered by other viral types; this means that most cases of CC are caused by a single viral type . The forA study dealing with an adolescent population provides relevant information for improving P&P and diagnosis programmes considering adolescents’ exposure to various factors such as those related to sexual behaviour, ethnicity and region of origin since they affect the risk of infection and coinfection.These results suggested that the risk factors reported for HPV infection and developing CC could vary when discriminated by infecting species, viral type and multiple infections in an unvaccinated population such as the one studied here. This highlights the importance of having different aged populations during analysis for establishing individual risk and the influence of background and associated factors. The findings showed that adolescents’ risky sexual behaviour is a key factor modifying HPV infection dynamics. Encouraging delay regarding the start of sexual life would reduce the risk of HPV infection and also the risk of contracting other STI. The foregoing, together with other P&P strategies and the appropriate use of contraceptive methods  aimed atDespite young females having a lower relative risk of acquiring CC, there is a slight risk of non-participation. It has been demonstrated that a lack of opportunity for prevention increases risk by reducing this population’s commitment to self-care and participating in the healthcare programmes available in each region , 40. Beh


Glucose-6-phosphate dehydrogenase (G6PD) is a rate limiting enzyme of the pentose phosphate pathway and is closely associated with the haemolytic disorders among patients receiving anti-malarial drugs, such as primaquine. G6PD deficiency (G6PDd) is an impending factor for radical treatment of malaria which affects the clearance of gametocytes from the blood and subsequent delay in the achievement of malaria elimination. The main objective of this study was to assess the prevalence of G6PD deficiency in six malaria endemic districts in Southern Nepal.® and Care Start™). Equal proportions of participants from each district (n ≥ 200) were enrolled considering ethnic and demographic representation of the population groups.A cross-sectional population based prevalence survey was conducted in six malaria endemic districts of Nepal, during April–Dec 2013. A total of 1341 blood samples were tested for G6PDd using two different rapid diagnostic test kits  and Care Start: male 8.4%; 44/521 versus female 4.5%; 37/820 (p = 0.003)]. G6PDd was higher in ethnic groups Rajbanshi  and Tharu  (p = 0.006), major inhabitant of the endemic districts. Higher prevalence of G6PDd was found in Jhapa  and Morang districts  (p = 0.031). In a multivariate analysis, male were found at more risk for G6PDd than females, on Binax test  and Care Start test .The higher prevalence of G6PDd in certain ethnic group, gender and geographical region clearly demonstrates clustering of the cases and ascertained the risk groups within the population. This is the first study in Nepal which identified the vulnerable population groups for G6PDd in malaria endemic districts. The finding of this study warrants the need for G6PDd testing in vulnerable population groups in endemic districts, and also facilitates use of primaquine in mass supporting timely progress for malaria elimination. Plasmodium vivax, and each year more than a hundred million suffer from clinical infection . The male preponderance of G6PD deficiency can be attributed to the X-linked inheritance in G6PD gene . The higThis study is the first study of its kind in Nepal to estimate G6PD deficiency prevalence in different ethnic groups within population, particularly residing in malaria endemic areas of Nepal. In India, G6PD deficiency was first reported by Baxi et al. in 1963 and the prevalence rate varied from 0 to 27% in different caste, ethnic, and linguistic groups .In this study, the highest G6PD deficiency prevalence was detected in Rajbanshi ethnic group  on Care Start test (p = 0.006). Rajbanshi is one of the main ethnic group inhabitants of the Terai  believed to have an innate resistance to malaria. Following a successful control of malaria through IRS  in early 1960s by National Malaria Eradication Office (NMEO), a large and heterogeneous non-Rajbanshi population has inhabited the area in recent years. By analysing NMEO records, prevalence of residual malaria cases has nearly been seven times lower among Rajbanshi compared to sympatric non-Rajbanshi . This miMost of the districts in this study are bordering India, and ethnic groups such as Rajbanshi, Tharu, Satar, Dhimal and Mushar are the major inhabitants in those districts. In Nepal, status of G6PD deficiency in these ethnic groups is not clearly understood, although a few local studies have indicated some prevalence in the mentioned districts and populations. In some studies, hereditary anaemia such as sickle cell blood and glucose-6-phosphate dehydrogenase (G6PD) deficiency were found at a high rate among the Rajbanshi people , 35.The current study findings showed that G6PDd is more prevalent in Rajbanshi ethnic groups compared to others by enzymatic chromatography method. The findings of this study are consistent with the findings from India where G6PDd prevalence ranged between 2.3 and 27% . In anotThe prevalence of G6PD deficiency varied between the endemic districts in this study. Jhapa (9.8%) and Morang (8%) showed the highest prevalence of G6PDd among six districts (p = 0.031). The heterogeneity of G6PDd in this study can be explained in part by the presence of Rajbanshi ethnic population in these districts. In addition, Rajbanshi ethnic groups have been identified as one of the vulnerable groups as they are linked with the marriage within the siblings of the same family.More than 80% of vivax malaria with predomination of G6PD Mediterranean type occurs in Southeast Asia. Person having red cell G6PD activity <30% of the normal mean has G6PDd and such person experience haemolysis after administration of primaquine. Heterozygote females with higher mean red cell activities may still show haemolysis. As Care-Start and BinaxNow test kit are qualitative G6PDd screening test, they may still miss to detect intermediate G6PD activity in some of heterozygote females. This is one of major limitation of the study , 9, 10. Most of the phenotypic tests detect G6PDd only in hemizygous male & homozygous female . The cytP. vivax cases. Knowing the G6PDd status gives leverage to use 14 days primaquine in G6PD normal patients, while weekly primaquine under close clinical monitoring/medical supervision with ready access to blood transfusion services in G6PD deficient cases. In absence of appropriate G6PDd testing facilities and lack of knowledge on the prevalent genotypes and severity, it will be prudent to err on the safe side. Rational use of such radical treatment facilitates the country in moving forward for timely malaria elimination.Prevalence of G6PD deficiency in Nepalese population varies in ethnic groups, strongly recommending need of G6PDd testing before the start of primaquine for each confirmed


This is the first description of dynamic changes occurring in the abnormal rate of cervical cancer screening as a result of positive changes in national HPV vaccination rates.Japan’s governmental recommendation of HPV vaccine has now been suspended for more than 4 years. In and before 2013, the targets of 20-year-old cervical cancer screening were females born in and before 1993, i.e., those who could not have received HPV vaccination because it was not yet publicly introduced. The targets during 2014–2019 are, or will be, those born in 1994–1999, i.e., those who came of age during a period with the highest HPV immunization rate. We analyzed the statistical data for each birth year, for the cumulative HPV vaccination rates achieved as of age 16, and for the corresponding results of cervical cancer screening at age 20. The rate of abnormal findings in cervical cytology increased slightly from 3.68% in 2010 (birth year: 1990) to 4.35% in 2013 (birth year: 1993); however, it dynamically dropped to 2.99% in 2014 (birth year: 1994) and 3.03% in 2015 (birth year: 1995). In total, the rate of abnormal findings in cervical cytology was 3.96% in 2010–2013, but significantly dropped to 3.01% in 2014–2015 ( Japan’s public subsidy for human papilloma virus (HPV) vaccination started in 2010, permitting girls aged 13 to 16 to be immunized for only a small out-of-pocket fee. Even though it was not a school-based program, a high vaccination rate, of around 70%, was achieved across the entire country3. By April of 2013, HPV vaccination had nationally become a routine immunization for all girls aged 12–16.The prevalence and the mortality of cervical cancer in Japan, especially for women in their twenties and thirties, are both steadily on the rise4. That suspension is now more than 4 years old, despite that the World Health Organization’s (WHO’s) Global Advisory Committee on Vaccine Safety (GACVS) has stated (concerning the curcumstances in Japan) that “Policy decisions based on weak evidence, leading to lack of use of safe and effective vaccines, can result in real harm.”5. Subsequent to MHLW’s recommendation withdrawal, the immunization rate among girls has plummeted, and the degree of protection against HPV-associated cancers for young women now differs greatly across Japan depending on the year the female was born - relative to the initial successful public HPV vaccination program period - and to this ongoing interruption of that program.Unfortunately, at around this same time, complaints of patients with widespread pain or movement disorders occurring after HPV immunization began to circulate, and the national news media soon began to question the vaccine’s safety. As a result, within two months the Ministry of Health, Labor and Welfare (MHLW) announced a suspension of their official recommendation for routine HPV immunization until appropriate information about HPV vaccine can be provided to the public7. However, as a result of media-caused vaccine fears, and lack of an official recommendation, HPV vaccination has ground almost to a halt. By birth year, only 4% of girls born in 2000, who became vaccination-eligible in 2013, the year of recommendation withdrawal, have been vaccinated as of this date. But even that dismal rate has become worse, with 1% of girls born in 2001, and almost 0% of girls born after 2001, having been immunized7. Previously, taking into account the female’s birth year - relative to these three periods  - we had predicted HPV-16/18 infection rates for 20-year-old girls8. According to those predictions, the relative risk of infection for females born from 1994 to 1999 approached 0.3.The HPV vaccine, first publicly introduced into Japan in 2010, was not used for girls born in and before 1993, who were 17 or older at the time. During the height of the public vaccination program (2010–2013), roughly 70% of targeted eligible girls, those born between 1994 and 1999, were successfully fully immunized using public subsidyThose girls are now older than 20, which is the standard starting age for cervical cancer screening in Japan. In Japan, all females aged 20 or older can undergo subsidized public cervical cancer screening every 2 years; those who miss their targeted year can undergo the exam the next year. In the present study, we analyzed the statistical data for each birth year, for the cumulative HPV vaccination rates, and for the corresponding results of cervical cancer screening at age 20, to evaluate effectiveness of previous introduction of HPV vaccine, which will provide strong evidence the Japanese government needs to reconsider a resumption of their recommendation for the HPV vaccine.In and before 2013, the targets of 20-year-old cervical cancer screening was for females born in and before 1993, i.e., those who could not have received the HPV vaccination with public subsidies because it was not yet publicly introduced. The targets during 2014–2019 are, or will be, those born in 1994–1999, i.e., those who came of age during a period with the highest HPV immunization rate (of around 70%).In the present study, we analyzed the statistical data for each birth year, for the cumulative HPV vaccination rates achieved as of age 16, and for the corresponding results of cervical cancer screening at age 20. Data was obtained from seven local governments in Japan: Iwaki, Kawasaki, Otsu, Takatsuki, Osaka, Matsuyama, and Fukuoka. The total population of these cities is around 6–7% of that of Japan. We analyzed for annual changes in the rates of abnormal findings in cervical cytology of 20-year-old females, in association with the HPV vaccination rate of the corresponding birth year. Complete data for both 20-year-old cervical cancer screening results for 2010–2015 (birth years 1990–1995) and overall rates of HPV vaccination were available in four of the seven cities. In the present analysis, for the females who did not receive cervical cancer screening at the age of 20, the screening results from the following year, at age 21, were included. In the present study the data for cumulative vaccination and cervical cancer screening were both aggregated; however, individually, vaccination histories did not correspond to cervical cancer screening results. This is a limitation of the present study.Fisher’s exact test was used to analyze the difference of the rates of abnormal findings in cervical cytology between the periods of 2010–2013, whose targets (birth year: 1990–1993) did not have a chance for HPV vaccination, to 2014–2015, whose targets (birth year: 1994–1995) were the generation of girls with high vaccination rates. The level of statistical significance was set at p = 0.05.This study was approved by the Institutional Review Board and the Ethics Committee of the Osaka University Hospital.Japan’s governmental recommendation of the HPV vaccine has now been suspended for more than 4 years. It is timely that we evaluate the clinically relevant results from Japan’s previous introduction of the HPV vaccine. This is the first clear description of dynamic changes occurring in the abnormal rate of cervical cancer screening as a result of positive changes in national HPV vaccination rates, providing strong and credible evidence that the Japanese government needs to reconsider a resumption of their recommendation for the HPV vaccine.The rate of abnormal findings in cervical cytology increased slightly, from 3.68% in 2010 (birth year: 1990) to 4.35% in 2013 (birth year: 1993); however, the rate dropped dynamically in 2014 (birth year: 1994) to 2.99%, and held steady, 3.03%, in 2015 (birth year: 1995) in association with the public introduction of HPV vaccine   did not have a chance for HPV vaccination, to 2014–2015, whose targets (birth year: 1994–1995) were the generation of girls with high vaccination rates ranging from 63.9 to 74.7%. The rate of abnormal findings in cervical cytology (ASC-US+) was 3.96% (330/8333) in 2010–2013, but dropped to 3.01% (99/3291) in 2014–2015. This difference was statistically significant  in the generation of girls with high vaccination rates. In the present study, the data of cumulative vaccination and cervical cancer screening were aggregated ones, and did not correspond each other.To the best of our knowledge, this is the first such clear description of the dynamic changes of the abnormal rate of cervical cancer screening as a result of positive and negative changes in HPV vaccination rates. In Japan, the standard starting age for cervical cancer screening is 20, which is among the earliest in the world, leading to this study’s earliest finding of this dynamic change. We have found more strong evidence that the government’s decision in 2010 to start public subsidies for the HPV vaccine has critically lowered the cervical cancer risk of its citizens by the present population-based study than previous vaccinated v.s. unvaccinated females studies4, and the suspension is now more than 4 years old. During this period, the results of the nation-wide epidemiological study on safety of HPV vaccine funded by MHLW has already been reported that there was a certain number of unvaccinated patients with similar diverse symptoms observed after HPV immunization14. However, the governmental recommendation is still on suspension in Japan.Due to alleged severe adverse events occurring after HPV immunization, MHLW announced a suspension of their official recommendation for routine HPV immunization until appropriate information about HPV vaccine can be provided to the publicOur results, hopefully, will become the evidence the Japanese government needs to reconsider a resumption of their recommendation for the HPV vaccine. Otherwise, we foresee a looming unfortunate outcome for an entire generation of our young women. By 2019, the females born in 1994–1999 - the highly HPV vaccinated generation - will be the targets of cervical cancer screening, at the age of 20. However, in 2020, almost all females born in 2000 will reach 20 without any protective HPV vaccination, and this is/will be due entirely to the continued suspension of the governmental recommendation. If and when that recommendation is restarted, we still predict a delayed deflection in the rate of abnormal findings in cervical cytology due to current prolonged dramatically decreased HPV immunization rate following the governmental announcement of its suspension of recommendation. This cohort of unprotected women will play out as increased cases of cervical and other types of HPV-associated cancers in the coming years.8. Although elsewhere around the world cervical cancer is effectively being prevented with the HPV vaccine, it is still challenging here in Japan. This must change, soon.As resumption of recommendation delays for years, the relative risk of additional birth years will recapitulate the risk of the times before the vaccine was introduced


In French Guiana, cervical cancer is the second most frequent cancer in females. The objective of the present study was to describe the prevalence of HPV infections in women with normal cervical cytology living in the remote villages of French Guiana.Before the study, the study team communicated in the remote villages on the importance of screening. All women from the target population were offered to participate. They signed informed consent during inclusion and then had a concomitant HPV-test and cervical smear. Only women with normal cytology and a good quality smear were analyzed. The detection of HPV-DNA was performed using the GREINER-BIO-ONE kit.Overall, 27.2% of women with normal cervical cytology had a positive HPV-test. There was a U-shaped evolution of prevalence with women over 50 years having the highest HPV prevalence, followed by the 20 to 29 years group. The most prevalent HPV genotypes were HPV 53(3.52%), 68(3.33%), 52(2.59%), 31(2.22%) and 16 (1.85%). The proportion of HPV 16 among HPV-infected women was 6.8%.HPV prevalence in cytologically normal women was very high. The most prevalent genotypes were very different from what is usually described in the world, and notably in South America. Cervical cancer is caused by the Human Papilloma Virus. In French Guiana, the standardized incidence rate of cervical cancer is 30.3 per 100,000 women. It is the second most frequent cancer in females . These fFrench Guiana is covered by primary forest and has a population of 240,000 inhabitants, mostly living along the coastline. However, part of the population lives in the interior of French Guiana and has reduced access to care when compared to persons living in the main cities. Studies have shown that persons start their sexual life early and have a greater number of sex partners than reported in mainland France. This presumably favors the sexual transmission of this virus .Vaccination against HPV is now recommended in France, and thus in French Guiana  . The oppIn French Guiana, efforts to conduct organized screening have been implemented in coastal areas, but not in the interior where isolation and lack of staff make it more difficult. Throughout the world, a number of studies have described the prevalence and risk factors of HPV in cytologically normal women –12. SeveIn French Guiana, weconducted a study between 2012 and 2014 to determine the prevalence of HPV infection in women aged 20–65 living in the remote villages along the border rivers between French Guiana and Suriname or French Guiana and Brazil. Women had both a cervical smear and an HPV test. The objective of the present report was thus to describe the prevalence and risk factors of HPV infections in women with normal cervical cytology, and to compare these results with other regions of the world.The cross sectional study took place between December 2012 and September 2014. The study population consisted of women aged 20–65 years having previously had sexual activity living in the remote villages on the Maroni and Oyapock rivers. Among these women only those with normal cervical cytology (Bethesda 2001 criteria) were included in the analysis.Women with a history hysterectomy, pregnant women (>3 months pregnant) were not included. Women with abnormal cervical cytology were not included. Smears of insufficient quality  were excluded.The detection of HPV DNA was performed using the GREINER-BIO-ONE kit at the Virology laboratory in Fort de France University Hospital. This kit allows the identification of high risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 and low risk genotypes 6, 11, 40, 42, 43, 44, 55.A short questionnaire was used to collect socio economic and demographic data, and the gynecological and obstetrical history. Vaccines against HPV being theoretically available since 2006  or 2007, all women were asked if they had been vaccinated against HPV.A study coordinator first went to the study sites to meet the key opinion leaders, local authorities . Communication was performed in all villages in order to sensitize the local populations about the public health problem of cervical cancer before beginning the inclusion of study patients. Health center workers were also informed and the logistical conditions of the health centers were assessed to ensure that the study was feasible. Three weeks before the research started, radio messages were aired several times a day in local languages informing the population about when the study would take place in the village. A study coordinator first went to the study sites to meet the key opinion leaders, local authorities. Three weeks before the research started, radio messages were airedseveral times a day in local languages informing the population about when the study would take place in the village. When the study team arrived, persons wishing to do an HPV test and cervical cytology came to the health center. The project was explained to them, the questionnaire was then filled and samples were taken using the ThinPrep® kits. Samples were stored in a cooler until the end of the mission. The samples were then sent to Fort de France Hospital’s Virology laboratory, in Martinique, where an automated method was used for DNA extraction and genotyping. Sample DNA extraction was performed using a minimum of 2 ml in a liquid phase, followed by PCR for amplification, and genotyping using a kit that could discriminate between 20 genotypes,  and identify multiple infections.When the HPV test was positive and when cytology was negative, a gynecological follow up was recommended to verify if HPV positivity had disappeared or if cytological lesions had appeared.For study participants, the HPV test and cytology were free of charge. All included subjects gave written informed consent to participate in the study and for the publication of its results. Regulatory and ethical approval were given by the Comité d’Evaluation Ethique de l’Inserm (CEEI), approval n° 12–064; the Comité Consultatif sur le Traitement de l’Information en matière de Recherche dans le domaine de la Santé (CCTIRS), n° 12.310; and the Commission Nationale de l’Informatique et des Libertés (CNIL), n° 912,459.Prevalence was obtained by dividing the number of women with normal cytology infected by at least one HPV genotype (high grade or low grade) by the total number of women with normal cervical cytology. For specific genotypes, prevalence was obtained by the number of women infected with normal cytology by that specific genotype by the total number of women with normal cervical cytology. The direct standardization method was used to obtain the standardized HPV prevalence (using the world population as a reference population). The sample size required to detect a 30% prevalence with a 5% margin of error and a 95% confidence interval and an estimated population of 10,000 would be 313 persons, a figure which was exceeded.Risk factors associated with HPV prevalence were determined using Generalized Linear Models using the log link and Poisson family. The Pearson goodness of fit test was used. The data was analyzed using STATA 13 software .Overall, a total of 643 women were included, of which 540 women had normal cytology. Sexual relations generally started at a young age, with 19.1% percent of the surveyed women having had their first sexual relation before the age of 15 years.N = 147, 27.2%) of women with normal cytology had a positive HPV test. Table one shows a high HPV prevalence notably for the high risk viruses. The overall age-standardized prevalence was 28.9%. Table A large proportion N = 47, 27.2%Table p = 0.037.Overall, 115 women (21.3%) had single HPV infections, 23 (4.3%) had double infections, and 9 (1.7%) had 3 or more different HPV genotypes. Having more than one HPV genotype was more frequent in women over 50 years of age (11/20) relative to those <= 50 (21/95) (crude OR = 2.5 (95% CI 1–6.4), Table The apparent variation in the relative importance of different HPV genotypes requires further research including studies from regions where data is lacking, and reanalyzing existing data to better understand the observed patterns.  The publOverall, the observed crude and standardized (28.9%) HPV prevalence among cytologically normal women in this French territory was among the highest reported in the world. It was notably much higher than in other areas of South America . However, the published reviews in the group of cytologically normal women in South America , 14 did These differences may reflect epidemiologic differences in transmission chains in these remote areas populated with ethnically homogenous populations with little admixture. A possible explanation for these differences in small isolated populations could be the founder effect, were a few individuals have spread these HPV genotypes. Immunologic differences between these ethnic groups  could aThe originality of this epidemiologic situation may result from the very singular situation of the isolated populations: different ancestral origins of the Maroon and Amerindian populations living in adjacent villages, with early sexual debut, rare sexual encounters between ethnic groups, different immunological susceptibilities, and different pools of HPV viruses. These results reemphasize  the poteHPV prevalence in cytologically normal women was very high. The most prevalent genotypes were different from what is usually described in the world, and notably in South America beyond the Amazon basin. In a region where cervical cancer is frequent, vaccination against HPV and screening of women are important, but they must be adapted to the local epidemiology and made affordable for the populations.