In order to assess HPV-specific IgG characteristics, we evaluated multiple aspects of the humoral antibody response that will provide insight in the HPV humoral immune response induced by HPV infection and vaccination.Cross-reactivity of HPV-specific antibodies induced by infection or vaccination was assessed with VLP16 or 18 inhibition using a VLP-based multiplex immunoassay (MIA) for HPV16, 18, 31, 33, 45, 52 and 58. HPV16/18 specific IgG1-4 subclasses and avidity were determined with the VLP-MIA in sera after HPV infection and after vaccination. Neutralizing antibodies were determined in a small subset of single-seropositive and multi-seropositive naturally derived antibodies.p<0.0001). IgG1 and IgG3 were found to be the predominant subclasses observed after HPV infection and vaccination. In the small subset tested, the number of single-positive sera with neutralizing capacity was higher than of multi-positive sera.Naturally derived antibodies from single-positive sera were highly genotype-specific as homologue VLP-inhibition percentages varied between 78-94%. In multi-positive sera, cross-reactive antibodies were observed both within and between α7 and α9 species. After vaccination, cross-reactive antibodies were mainly species-specific. Avidity of vaccine-derived HPV-specific antibodies was 3 times higher than that of antibodies induced by HPV infection (Naturally derived HPV-specific antibodies from single-positive samples showed different characteristics in terms of cross-reactivity and neutralizing capacity compared with antibodies from multi-positive sera. Post-vaccination, HPV antibody avidity was approximately 3 times higher than antibody avidity induced by HPV infection. Therefore, antibody avidity might be a potential surrogate of protection. Persistent infection with high-risk (hr) human papillomavirus (HPV) is a necessary event of cervical cancer. Almost all cervical cancers are HPV DNA-positive to at least one of the 15 hr-HPV types that can cause genital infections. Most hr-HPV types cluster together in the α7 and α9 species .After an incidental HPV infection, only 50-70% of the infected individuals develop detectable HPV-specific antibodies in serum mainly directed against the L1 capsid protein of the virus . In contHPV vaccines provide a degree of cross-protection against persistent infection and/or high-grade lesions (CIN2+) attributed to some of these non-vaccine HPV genotypes, particularly HPV31, 33 and 45, but probably not HPV52 and 58 ,7. It isAt present, no immune parameter or antibody concentration has been defined that correlates with protection. While the HPV-specific antibody levels in response to HPV infection or HPV vaccination have been previously described -13, infoSerum samples were available from a large cross-sectional population-based surveillance study (PIENTER-2), performed in 2006-07 in the Netherlands. This serumbank contained samples from individuals 0-79 years of age and at low risk for HPV infection . The surn=121) and multi-positive sera (at least seropositive for HPV16 (n=136) or seropositive for HPV18 (n=91)) of a broad range of antibody concentrations [From this serosurveillance study, a panel of naturally HPV infected individuals was selected that included HPV single-positive n=1 and mulSome infants and children (up to 10 years of age) in the large serosurveillance study were HPV antibody seropositive. To investigate the quality of the antibodies we tested in the PBNA some sera of infants and children, who were part of the previous mentioned selection (n=11).From a prospective cohort study in Rwanda, Africa , a paneln=100). The girls from the HAVANA study received three doses of the bivalent Cervarix® vaccine  at months 0, 1 and 6. The study protocol was approved by a medical ethics review committee of the Faculty of Medicine, VU University Amsterdam (2009/22).From an HPV vaccine monitoring study  in the n=67, HPV18 n=29) and vaccination , isotype-specific mouse anti-human R-PE-conjugated antibodies were used. The mouse anti-human IgG1 monoclonal was used in a 1/500 dilution and the IgG2-4 monoclonals in a 1/100 dilution . Four ‘in-house’ control sera and an ‘in-house’ standard were used on each plate. The ‘in-house’ standard  was calibrated against reference serum of GSK for all the seven HPV types. HPV-specific antibodies were analyzed using the Bioplex system 200 with Bioplex software . Sera were assumed to be IgG seropositive at the following cut-offs determined previously with this assay [HPV-specific IgG antibodies against L1 VLP16, 18, 31, 33, 45, 52 and 58 were measured using a VLP-MIA . Sera weHomologous inhibition of HPV-specific antibodies induced by infection was assessed for single-positive sera with virus-like particle (VLP) 16, 18, 31, 33, 45, 52, and 58, separately. Inhibition of HPV antibodies in multi-positive sera from the Dutch serosurveillance study and sera from vaccinated girls was determined with VLP16 and 18 separately. VLPs were kindly donated by GlaxoSmithKline Vaccines. Sera were pre-diluted 1/100 and 1/200, respectively, and subsequently added to the same volume of PBS containing 10 µg/ml VLPs. Sera, containing vaccine-derived HPV-specific antibodies, were pre-diluted 1/10,000. After an incubation period at RT for 1 hour, all samples were analyzed using the VLP-based multiplex immunoassay (VLP-MIA). The inhibition index was determined as a percentage of the antibody levels post-inhibition in comparison with the antibody levels pre-inhibition, which were set at 100%.n=65, HPV18 n=55) or after HPV vaccination (HPV16/18 n=60) were diluted for HPV16 and 18 separately and adapted to a starting concentration of 0.25 LU/ml .Avidity of HPV16 and 18-specific IgG antibodies was assessed by using a modification of the VLP-MIA for determining HPV-specific IgG antibodies. HPV16 and 18-specific antibody concentrations in sera induced by HPV infection  was used to dissociate low-avidity antigen-antibody binding [4SCN in PBS (pH7.2) was added in a final concentration of 2.5M or only PBS for 10 minutes at RT. Subsequently, sera were washed 3 times with PBS. Residual bound IgG antibodies were detected as described above. The avidity index was determined as a percentage of the remaining IgG levels in the presence of NH4SCN in comparison with the IgG levels after addition of PBS in which the antibody avidity was set at 100%.Ammonium thiocyanate (NH binding . After an=11) and multi-positive (n=10) sera, containing naturally induced HPV16, 18, 31 and 45-specific antibodies, were tested in a pseudovirion-based neutralization assay (PBNA) for the detection of neutralizing antibodies against VLP16, 18, 31 and 45. The PBNA was performed at GSK as previously described [50, when compared to control wells containing only neutralization buffer and HPV pseudovirions). The cut-off of the PBNA was 40 ED50 for HPV16, 18, 31 and 45.Subsets of single-positive (escribed . Serum np<0.05) of geometric mean concentrations (GMCs) of antibodies pre- and post-inhibition or between antibody avidity indices were calculated using a Mann-Whitney test.Data analyses were conducted using Graphpad Prism version 5. Percentage inhibition was calculated using the antibody concentrations pre- and post-inhibition of each tested sample. Subsequently, an average inhibition percentage was calculated. Significant differences . Homologous inhibition percentages for the other 5 single-seropositive HPV types tested varied between 78% and 92%, indicating that these antibodies were type-specific as expected (data not shown).Homologous inhibition of naturally acquired HPV-specific antibodies against a single HPV type amounted to 94.5% for HPV16 and 83.1% for HPV18 , indicating that the antibodies were genotype-specific, was selected from the single- and multi-positive samples. In the single-positive sera from this subset, neutralizing antibodies were found in 5 out of 6 for HPV16 and in 4 out of 5 for HPV18 . In the Naturally derived HPV16-specific antibodies in serum samples of children 1-10 years of age (n=8) showed no neutralizing capacity while from the samples of 3 infants tested (0-6 months of age) in 1 case HPV-specific neutralizing antibodies were found.4SCN treatment. No significant differences in naturally induced antibody avidity were observed between single-positive and multi-positive sera. In contrast, vaccine-derived HPV16 and 18 antibodies showed a significant 3 times higher avidity than naturally induced antibodies, with an avidity index of 86% (95%CI 84-88%) and 84% (95%CI 82-86%), respectively. The avidity indices of the antibodies induced by HPV infection of the vast majority of the single-positive  and multi-positive sera  were lower than the avidity of vaccine-derived antibodies. We observed no correlation between the level of antibody avidity and the number of HPV types for which participants are positive. Also no correlation was observed between antibody avidity indices and the level of HPV16 and 18-specific antibody concentrations after HPV infection and vaccination (data not shown).HPV16 and 18-specific antibodies induced by HPV infections are mainly of low-avidity . On averThe most abundant antibody subclass of IgG induced after HPV16 and 18 infection was IgG1 , followed by IgG3  . Small aHere we describe HPV-specific antibody characteristics after HPV infection and vaccination. Cross-reactivity of HPV antibodies derived after HPV vaccination was mainly species-specific. Naturally induced HPV-specific antibodies from single-positive sera were genotype-specific as expected and tended to be neutralizing. In contrast, antibodies of multi-positive sera were less genotype-specific, cross-reactive, and tended to be non-neutralizing. IgG1 was found the predominant subclass induced after HPV infection and vaccination, followed by IgG3. Moreover, post-vaccination antibody avidity was approximately 3 times higher than after HPV infection.Vaccine efficacy against HPV16 and 18 infection sustains over 8 years post-vaccination  in whichIt is unclear whether naturally derived HPV-specific antibodies can protect against subsequent homologous HPV infection or infection with phylogenetically related HPV types. In women, the protective role of HPV16 serum antibodies induced by HPV infection has been inconsistent in the literature, with moderate protection observed in a limited number of studies ,24-26. PCross-sectional serosurveillance studies ,29 showein vivo genital HPV pseudovirion challenge, that already very low vaccine-derived HPV-specific antibody levels could protect against HPV infections and that these protective antibody levels are far below detection limits of the pseudovirion-based neutralization assay [With the HPV vaccine clinical trials performed so far, a level of HPV-specific antibodies that can be used as a correlate of protection is still not established as too few disease cases occurred within these trials and breakthrough infections could not be clearly distinguished from the reactivation of prevalent HPV infections . Longet on assay . We coulon assay . ApparenOther immune parameters than antibody levels, that might correlate with protection, have not been defined and data on antibody characteristics such as avidity are scarce . AntibodWe found that antibody avidity after vaccination was 3 times higher than after HPV infection. After HPV infection, we observed a wide range of antibody avidity levels in sera of naturally infected individuals reflecting a great biological diversity in individual responses to HPV infection. Naturally induced HPV16 antibodies of low avidity have been associated with possible susceptibility to infection with other HPV types . Higher Our findings on IgG subclasses are in line with Harro et al. reporting that IgG1 was the predominant subclass induced after VLP vaccination . In contIn conclusion, we showed that vaccine-derived antibodies were mainly genotype-specific and cross-reacted only for a smaller part with other HPV types within the species. After HPV infection, single-seropositive antibodies were highly type-specific and neutralizing whereas multi-positive sera were less specific and tended to be non-neutralizing. This might mean that naturally induced HPV-specific antibodies in multi-HPV positive individuals could not or only partially protect against subsequent HPV infection and these individuals are still at risk.Although sample sizes were small, neutralizing antibodies in single-positive sera inclined to be associated with higher antibody avidity indices than non-neutralizing antibodies. Further, we found the avidity of vaccine-derived antibodies to be approximately 3 times higher than after HPV infection. These results imply that the avidity of HPV antibodies might be used as a potential surrogate of protection. However, more studies are needed to establish the role of HPV antibody avidity as a potential surrogate of protection and the use of this immunological tool in sero-epidemiological and vaccine monitoring studies.


STK11 gene, which has been found frequently mutated in HPV-related cervical carcinoma, has been analyzed in HPV-positive and HPV-negative invasive penile cancers to establish its mutational status and the possible correlation of HPV infection with specific genetic alterations.Human papillomavirus (HPV) infection accounts for about 40-50% of all cases of penile carcinoma suggesting that other factors, including host genetic status, are involved in neoplastic transformation. In this perspective, STK11 gene by quantitative real-time PCR. Ratios of potentially deleted and non-deleted exons were indicative of specific loss of STK11 coding regions. DNA samples of 5 cancer cases were subjected to standard PCR amplification of STK11 exons 1 to 9 and analyzed for somatic mutations by direct nucleotide sequencing analysis.Genomic DNAs extracted from 26 cases of penile squamous cell carcinoma were analyzed for genetic alterations in the exons 1 to 9 of STK11 exon 1 and 2 were identified in 2 out of 14 HPV-positive (14.3%) and 1 out of 12 HPV-negative cases (8.3%). Complete nucleotide sequencing analysis of exons 1 to 9 showed a single nucleotide change upstream the exon 2 coding region in 1 out of 5 penile carcinoma samples.Heterozygous deletions of STK11 gene are rare events in penile cancer. Moreover, no significant association was observed between STK11 alterations and HPV infection in these tumors.The present results suggest that single nucleotide mutations and/or deletions of  Penile cancer is a rare malignancy in Western Europe and the United States with age-standardized incidence rates (ASR) of 0.1-1.5 per 100,000 men. In Africa, Asia and South America, however, the estimate ASRs are significantly higher with peaks of 2.8 and 3.7 per 100,000 men, in Uganda and Brazil, respectively[The most common histological type of penile carcinoma is the squamous cell carcinoma (95%) which comprises several subtypes such as usual squamous cell carcinoma48-65%), basaloid (4-10%), warty 7-10%), verrucous 3-8%), papillary (5-15%) and mixed carcinoma (9-10%). Althoug-8%, papi5%, basal-10%, verSTK11 is a 23 kb tumor suppressor gene, mapped to chromosome 19p13.3, which encodes for the serine/threonine kinase 11 (STK11) also known as liver kinase B1 (LKB1) or renal carcinoma antigen NY-REN-19[STK11 gene is widely expressed in embryonic and adult tissues and the encoded STK11 kinase is an essential regulator of chromatin remodeling, cell cycle arrest, p53-dependent apoptosis, Wnt signaling, cell polarity and energy metabolism[STK11 gene are associated with Peutz-Jeghers syndrome (PJ)[Several studies have reported genetic alterations in tumor suppressor genes and oncogenes, in both HPV-positive and HPV-negative penile cancers, which may have a critical role in tumor carcinogenesis,10. STK1Y-REN-19. The STKtabolism. Germ-liome (PJ), an autoome (PJ)-18. PJ pSTK11gene have been frequently found in several sporadic tumors, including HPV-related cervical cancer[STK11 mutational status in penile carcinoma.Somatic mutations of l cancer-25. No sSTK11 gene in HPV-positive and HPV-negative penile cancers to possibly establish a relationship between HPV infection and genetic alterations involved in cancer progression.The aim of the present study was to analyze genetic alterations and single nucleotide mutations in exons 1 to 9 of This study included DNA samples extracted from liquid-nitrogen frozen specimen of penile squamous cell carcinoma (n = 6) from Black Ugandan patients and paraffin-embedded penile squamous cell carcinoma (n = 20) from Caucasian Italian patients. These samples were previously characterized in terms of histology, DNA quality, HPV genotypes, HPV16 variants and viral integration status,26. The STK11 gene were independently amplified by real time polymerase chain reaction (PCR) using primer sequences listed in Table STK11 exons were amplified in a total volume of 25 μl containing iQ SYBR Green Supermix containing 50 mM KCl, 20 mM Tris-HCl, pH 8.4, 0.2 mM of each dNTP and 25 units/ml iTaq DNA polymerase, 3 mM MgCl2, 10 nM SYBR Green I , 3 μmol/L of each primer, and 100 ng - 500 ng of genomic DNA. All experiments were performed on the CFX96 Real Time System . The specificity of amplification was confirmed applying dissociation analysis starting at 65°C.Exons 1 to 9 of STK11 gene in the unknown samples. All PCR reactions were set up in duplicate.A 5-log dilution series of genomic DNA, extracted from the cell line PC23, were amSTK11 regions were verified by calculating the ratios (R) of potentially deleted and non-deleted exons. Values of R between 0 and 0.3 and between 0.4 and 0.7 are assumed to be indicative of homozygous or heterozygous deletions, respectively; values of R between 0.8 and 1.2 indicate no changes of the copy number in the two exons.Deletions of STK11 gene were independently amplified by polymerase chain reaction (PCR). All primer sets, described in Table 2, 20 pmol of each primer, 200 μM of dNTPs mix and 1.25 units of thermostable Taq DNA polymerase . DNA was amplified in a Perkin-Elmer GenAmp PCR System 9700 thermal cycler with the following steps: an initial 2 min denaturation at 94°C, followed by 36 cycles of 30 s at 94°C, 30 s at the annealing temperature specified in Table. STK11 exons 1–9 from 5 samples were subjected to automated direct nucleotide sequence analysis at Primm Laboratories  using the same primers used for PCR amplification. STK11 mutations were sought by comparison analysis with the NG_007460.1 reference sequence present in the GenBank.Exons 1 to 9 of STK11 deletions were identified in 2 out of 14 HPV positive (14.3%) and 1 out of 12 HPV-negative cases (8.3%)  and twelve HPV-negative 46.2%) penile squamous cell carcinoma samples were analyzed for genetic alterations by real-time PCR using specific primer sets designed to individually amplify exons 1 to 9. While exons 3 to 9 were always amplified, exons 1 and 2 were not efficiently amplified in 3 out of 26 (11.5%) samples. All 26 samples were subjected to a further quantitative real-time PCR using primer pairs specific for exon 1 (test) and for exon 7 (reference) to determine an absolute quantification of genomic equivalents of exon 1 and exon 7 in each sample. A 5-log dilution series of genomic DNA, extracted from the cell line PC23, was also amplified in the same reaction set to obtain an amplification standard curve  total samples analyzed  of STK11 somatic mutations in both HPV-positive and HPV-negative penile squamous cell carcinoma cases, in order to verify a potential role of this tumor suppressor gene in penile tumorigenesis. The results obtained in our study showed that STK11 exon 1 and 2 are deleted in 11.5% of penile cancers. In particular, heterozygous deletions of STK11 exons 1 and 2 were observed in 14.3% of HPV-positive and in 8.3% of HPV-negative tumors, indicating that HPV status has no effect on the genetic alteration of STK11 gene.The knowledge of genetic alterations in penile cancer is limited. In this study we investigated STK11gene have been identified in many sporadic cancers and derived cell lines, including HPV-related cervical carcinoma. In particular, Wingo et al. showed that up to 20% of cervical cancers harbored somatic mutations in STK11 coding regions, of which approximately one-half were single nucleotide substitutions or small deletions while the other half consisted of larger monoallelic or biallelic deletions[STK11gene was observed in SiHa and HeLa cell line[Genetic alterations of eletions. Moreoveell line,25.STK11 in 5 penile carcinoma cases showed no somatic mutations in none of the 9 coding regions. A single nucleotide substitution was identified in the intron 2 of one cancer case, however it seems to have no effect on the expression of the protein. Somatic mutations, affecting conserved residues within the kinase domain or intron/exon junctions of STK11, were previously identified in 4 out of 6 cases (66.7%) of cervical adenocarcinomas and 2 out of 10 cases (20%) of squamous cell carcinomas, indicating that single nucleotide substitutions are frequent events in a specific histological subtype of cervical cancer[The nucleotide sequence analysis of exons 1 to 9 of l cancer.Very few studies are available on somatic mutations of other genes in penile carcinoma. A recent report showed a dysregulation of phosphatidylinositol 3-kinase and Ras pathways through somatic alterations of the PIK3CA, HRAS and KRAS genes in 11 out of 28 (39%) penile cancer samples, without any statistically significant difference between HPV-positive and HPV-negative cases.Other studies analyzing genetic alterations in TP53 gene of penile carcinomas reported mutations in fractions varying from 8% to 33% of the cases-35. FurtSTK11 gene are rare events in penile carcinoma suggesting that STK11 genetic alterations do not have a relevant role in the pathogenesis of penile cancer.The present study has the limitation to analyze a modest sample size, but has the advantage to include cancer cases from high incidence (Uganda) and low incidence  geographic regions. The results obtained so far allow to conclude that single nucleotide mutations and/or deletion of HPV: Human Papillomavirus;STK11: Serine/threonine kinase 11;LKB1: Liver kinase B1.The authors report no competing of interests.MLT and FMB were responsible for the overall planning and coordination of the study. LB contributed to the data analysis. SL carried out the histopathology evaluation of the cases. CA was responsible for specimen processing, DNA analysis and with MLT compiled and finalized the manuscript. All authors read and approved the final manuscript.


Background: Human papillomavirus (HPV) is estimated to play an etiologic role in 40–50% of penile cancers worldwide. Estimates of HPV prevalence in U.S. penile cancer cases are limited.Methods: HPV DNA was evaluated in tumor tissue from 79 invasive penile cancer patients diagnosed in 1998–2005 within the catchment areas of seven U.S. cancer registries. HPV was genotyped using PCR-based Linear Array and INNO-LiPA assays and compared by demographic, clinical, and pathologic characteristics and survival. Histological classification was also obtained by independent pathology review.Results: HPV DNA was present in 50 of 79 (63%) of invasive penile cancer cases. Sixteen viral genotypes were detected. HPV 16, found in 46% (36/79) of all cases (72% of HPV-positive cases) was the most prevalent genotype followed equally by HPV 18, 33, and 45, each of which comprised 5% of all cases. Multiple genotypes were detected in 18% of viral positive cases. HPV prevalence did not significantly vary by age, race/ethnicity, population size of geographic region, cancer stage, histology, grade, penile subsite, or prior cancer history. Penile cases diagnosed in more recent years were more likely to be HPV-positive. Overall survival did not significantly vary by HPV status.Conclusion: The relatively high prevalence of HPV in our study population provides limited evidence of a more prominent and, possibly, increasing role of infection in penile carcinogenesis in the U.S. compared to other parts of the world. The annual, average age-adjusted incidence of invasive penile cancer in the U.S. is less than 1 per 100,000, which represents less than 0.5% of all cancers in men , 3. HumaWe conducted a study to evaluate the genotype-specific HPV distribution in invasive penile cancer cases from the U.S. and to compare the viral status of cases by demographic, clinical, and pathologic characteristics. This evaluation was part of a larger initiative by the Centers for Disease Control and Prevention (CDC) to examine HPV genotype distribution among anogenital and select head and neck cancer sites for cases diagnosed in the United States prior to the implementation of widespread HPV prophylactic vaccination . This stin situ components were excluded.Formalin-fixed paraffin-embedded tissue specimens from the 90 cases were prepared at central laboratories servicing each registry or medical facility following a uniform protocol. As previously detailed, one representative block from each case was prepared using procedures to minimize the risk of sample-to-sample contamination . Section2O, otherwise following the manufacturer’s protocol. The reverse line-blot hybridization was performed with an automated platform; Beeblot instruments . Samples with negative or inadequate LA results were re-tested with the INNO-LiPA HPV Genotyping Assay , which detects 29 HPV types including three types not covered by the Linear Array  and performs a generic probe to identify other HPVs not on the array (HPV X). The assay was performed according to the manufacturer’s specifications using an Autoblot 3000  for the line-blot procedure. Both typing assays included an endogenous positive control for the presence of amplifiable DNA. Samples negative for HPV and for the control probe in both assays were considered inadequate. Of the 83 penile cancer cases genotyped, 4 yielded inadequate results and were excluded.Tissue from the remaining 83 cases was genotyped. One suitable tissue sample from each case was processed as previously described using high temperature assisted tissue lysis  and autoA total of 79 cases were retained in the statistical analyses. Deidentified demographic , clinical , pathologic , and outcome data  were available from each registry. Stage was based on the SEER staging classification system . A largep-value of 0.05 was considered to be statistically significant. Overall HPV prevalence was based on the detection of one or more HPV genotypes in tumor tissue. Multiple genotypes detected in a case were not counted more than once in prevalence estimates. HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were considered high-risk genotypes  invasive penile cancer cases. A total of 16 genotypes were detected (Table n = 53), basaloid SCC (n = 3), warty SCC (n = 3), mixed basaloid-keratinizing SCC (n = 4), mixed warty-keratinizing SCC (n = 1), and verrucous SCC (n = 1); 13 remained SCC NOS (n = 13).Demographic and clinical characteristics of the 79 invasive penile cancer cases study cases in the study sample were comparable to that of all registry cases diagnosed over the same time period (data not shown). Study cases spanned a wide age range; 33–100 years (mean 70.9 ± 14.8). Whites comprised 56% of cases followed by blacks (24%), and Hispanics (17%). The large majority of cases were diagnosed in 2004–2005 (86%). Most cases were localized, early stage tumors (67%), and of moderately differentiated grade (51%). Thirty-two percent of tumors were of the glans or prepuce. A prior history of cancer was reported in 43% of cases; information on type of cancer was not available. Based on registry data, 61 of the 79 (76%) cases were histologically classified as SCCs not otherwise specified (SCC NOS), 17 were keratinizing SCC and 1 verrucous SCC. NOS cases consisted of those without histological subtype documented in the pathologic record. Following independent pathologic review, cases were re-classified as keratinizing SCC (p = 0.05). In order to evaluate the possible influence of the age of specimens on the detection of HPV, we compared the number of samples with negative or inadequate results generated from the linear array test requiring retesting with the second assay by period of diagnosis. Four of 11 cases diagnosed in 1998–2003 were derived by LiPA compared to 10 of 67 cases diagnosed in 2004–2005 (p = 0.09).The detection of HPV (any genotype) in penile cancers did not significantly vary by age, race/ethnicity, population size of geographic region, stage, histology, grade, penile subsite, or cancer history Table . Of penip-value = 0.37)  Figure . SurvivaThis is the first study of HPV prevalence in invasive penile cancer cases drawn from diverse regions of the U.S., including those with large Hispanic, black, and Asian populations. Although not a representative sample of the entire U.S. population, cases came from seven cancer registries covering defined geographic regions of the country, including five registries with statewide coverage. The overall HPV prevalence of 63% in this U.S. sample is higher than estimates of HPV prevalence in penile cancers worldwide , 3, 5. TOur findings agree with previous studies demonstrating HPV 16 to be the predominant genotype in penile cancers , 6, 7. HRegional and racial/ethnic disparities in the incidence of penile cancer have been observed in the U.S. with higher incidence in the South and among blacks and Hispanics and lower incidence in the West and among Asian-Pacific Islanders , 25, 26.We also observed no significant differences in HPV status by stage, histology, grade, or penile subsite. Notably, nearly all basaloid and warty tumors, including mixed subtypes, were HPV-positive. This is consistent with previous studies demonstrating a higher prevalence of HPV in these histological types , 4, 7.The incidence of invasive penile cancer in the U.S. has significantly declined over the past several decades , 26. TheHuman papillomavirus was detected more frequently in the recently diagnosed cases. A recent study reported that the prevalence of HPV in U.S. oropharyngeal cancers significantly increased over calendar time from 1984 to 2004 . It was In patients with oropharyngeal cancer HPV tumor positivity predicts a favorable outcome, including overall survival and disease-free survival –36. Few INK4a and E6/E7 mRNA, may provide important insight into the clinical relevance of viral detection in penile cancers (As with other PCR-based studies, we cannot be certain that the HPV DNA detected was present and causal in the tumor. HPV infection could have been present in nearby tissue. Evaluation of other molecular markers of HPV, including p16 cancers .Forty-eight percent of penile cancers were positive for HPV 16 or 18, which are included in current vaccines. HPV vaccination has demonstrated high efficacy for the prevention of genital HPV infection and external genital lesions in males . NonetheThe relatively high prevalence of HPV in our study population provides limited evidence of a more prominent and, possibly, increasing role of infection in penile carcinogenesis in the U.S. compared to other parts of the world. Population differences in the prevalence of sexual risks, circumcision, and other factors influencing HPV acquisition and persistence may account for these results. Our findings would be bolstered by future U.S. studies which include larger, population-based samples, which are derived from patients diagnosed with penile cancer over a wider time period.Brenda Y. Hernandez has received consultation from Merck and Co., Inc., for activities unrelated to this research project. The other co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glossina swynnertoni, a vector of both human and animal trypanosomiasis in open savannah in Tanzania and Kenya, and in neighbouring conservation areas used by pastoralists. The goal was to determine the most practical device/material that would induce the strongest landing response in G. swynnertoni for use in area-wide population suppression of this fly with insecticide-impregnated devices.Here we set out to standardize long-lasting, visually-attractive devices for swynnertoni in all situations, catching up to 19 times more flies than pyramidal traps. Baiting with chemicals did not affect the relative performance of devices. Fly landings were two times higher on 1 m2 blue-black targets as on pyramidal traps when equivalent areas of both were covered with adhesive film. Landings on 1 m2 blue-black targets were compared to those on smaller phthalogen blue 0.5 m2 all-blue or blue-black-blue cloth targets, and to landings on all-blue plastic 0.32–0.47 m2 leg panels painted in phthalogen blue. These smaller targets and leg panels captured equivalent numbers of G. swynnertoni per unit area as bigger targets.Trials were conducted in wet and dry seasons in the Serengeti and Maasai Mara to measure the performance of traps and targets of different sizes and colours, with and without chemical baits, at different population densities and under different environmental conditions. Adhesive film was used as a simple enumerator at these remote locations to compare trapping efficiencies of devices. Independent of season or presence of chemical baits, targets in phthalogen blue or turquoise blue cloth with adhesive film were the best devices for capturing G. 2 cloth targets show promise as cost effective devices for management of G. swynnertoni as they can be used for both control (insecticide-impregnated cloth) and for sampling (rigid plastic with insect glue or adhesive film) of populations.Leg panels and 0.5 m Glossina swynnertoni is restricted to open savannah in north-western Tanzania and south-western Kenya, where it is a vector of both human and animal trypanosomiasis in wildlife reserves and in neighbouring conservation areas used by pastoralists. Despite the challenge to minimize disease transmission through effective management of the vector in the presence of abundant wildlife reservoirs, little has been done to test the efficacy of modern tsetse traps or targets for controlling G. swynnertoni. We made field tests in the Serengeti and Maasai Mara to determine the most visually-attractive, long-lasting and practical object that induces the strongest landing response in G. swynnertoni. Fly landings were twice as high on 1 m2 blue-black targets as on pyramidal traps when equivalent areas of these devices were covered with adhesive film. Furthermore, blue leg panels in either cloth or plastic and blue or blue-black-blue cloth targets under half the size of traditional targets captured tsetse at equivalent numbers per unit as the latter. These smaller targets and leg panels show promise as cost-effective devices for management of G. swynnertoni populations as they can be used for both control (insecticide-impregnated cloth) and monitoring of this species (rigid plastic with insect glue or adhesive film). Glossina swynnertoni Austen  is restricted to open savannah in north-western Tanzania and south-western Kenya, extending from Tarangire in the south through Manyara to the Serengeti plains, and into the Maasai Mara in the north G. swynnertoni is in the savannah or morsitans group of tsetse. Considerable progress has been made in developing visually-attractive control devices such as traps G. swynnertoni since initial tests were conducted in Tanzania in 1991–1993 G. swynnertoni, like G. morsitansG. swynnertoniG. swynnertoni outside of the works of Ndegwa & Mihok 2 black sticky target. Unlike Glossina pallidipes Austen , baiting traps with attractants such as acetone, 1-octen-3-ol, phenols and/or cow urine does not result in large increases in catch Savannah tsetse are attracted to artificial objects of modest size G. swynnertoni. The trials were based on existing trap/target/bait technology following a similar experimental approach throughout Africa G. swynnertoni for future use in area-wide population suppression of this fly with insecticide-impregnated devices.Within the Africa-wide WHO-TDR initiative to develop innovative control strategies for tsetse, we set out to standardize long-lasting, visually-attractive devices for Acacia-Commiphora-Balanites dry savannah woodland in pastoral areas near the Maasai Mara National Reserve in Kenya and deep within the neighbouring Serengeti National Park across the border in Tanzania. Wild hosts remain abundant in the Serengeti but have declined considerably in the Mara in the last few decades Studies were conducted in open G. pallidipes was also present in scattered evergreen thickets. Findings for this species are reported where captures were adequate for analysis.In the Serengeti, three sets of studies took place at Death Valley 2°19′51″ S, 34°49′60″E at an altitude of 1548 m near Seronera. A first set of experiments was conducted in 2009 in the wet season (July) and repeated at the same sites in the dry season (October). In 2010 and 2012, a second and third series were conducted at the same location, both at the start of the dry season (September). In the Mara, studies were undertaken at the Olarro hills 1°25′45.4″S, 35°35′0.9″E at an altitude of 1910 m, and at the Nyonorri hills at 1°27′18.9″S, 35°33′43.5″ E at an altitude of 1877 m. Unbaited and baited trials were conducted at these locations separately in the nominal wet season (June), and then repeated only at one location (Nyonorri) in the subsequent dry season (November) of 2009. The shift in locations was necessitated by the unanticipated use of pyrethroid spray-ons by pastoralists at Olarro.G. swynnertoni is mainly found in remnant woodlands along hillsides that receive moisture from the highlands all year-round. A severe drought was underway during these tests; hence wild hosts were present only in the nominal “wet season”. Vegetation cover was particularly sparse during all seasons in the Mara due to the drought. Cover in the Serengeti was not affected as much, and hence wet and dry season contrasts were more typical of natural climatic cycles in Tanzania.Habitats have been extensively altered by human activities in pastoral areas near the Mara. Hence, 2 (1.5 m wide by 1 m high) divided vertically into equal rectangles of blue and black cloth 2 . Two kinds of slightly larger leg panels were tested in Tanzania. One was 0.47 m2 blue cloth (70 by 64 cm plus 14 by 9 cm legs) and the other was 0.45 m2 blue-painted plastic (90 by 45 cm plus 15 by 15 cm legs). The plastic was 3–4 mm thick and was painted glossy phthalogen blue. All targets were mounted on supports that allowed for limited rotational movement in the wind. The wet season trial in Tanzania occurred under particularly windy conditions.In 2009, three catching devices were tested: standard cloth pyramidal traps 2, TDV, Laval, France) with a reflectance peak at 460 nm as measured with a Datacolor Check Spectrophotometer , referred to hereafter as standard blue cotton, and turquoise blue 65% polyester/35% viscose  with a peak at 480 nm. The phthalogen blue paint on the plastic leg panel had a peak of 460 nm. A 100% polyester black  was used for all devices in all trials described here. To monitor the number of tsetse landing on cloth targets and leg panels, one-sided adhesive film  was stitched with thread to both sides of the trapping devices. However, in 2009 in Tanzania only the lower 60 cm of the targets was covered. Plastic leg panels in Tanzania were coated with a non-setting shiny glue . Transmittance spectra for both adhesives are compared to polybutene in Two blue fabrics were tested: C180 Azur 623 phthalogen blue 100% cotton . For this, adhesive film was also attached to the blue-black fabric of the pyramidal traps to enumerate flies that land but may not be captured. In an additional test only 1×1 m squares of adhesive film on its own (i.e. without targets as a backdrop) were compared to 1 m2 square targets  covered with adhesive film to ascertain whether the adhesive film in itself was attractive. A further set of trials was conducted in Tanzania in 2012 to compare six different two-dimensional cloth targets to evaluate the influence of size, shape and colour combination on fly landing rates. The six devices were: two types of 1 m2 square targets, one divided vertically into equal parts of blue and black material, the other divided vertically into three equal parts of blue-black-blue; two types of 0.5 m2 targets (0.9 m×0.55 m), one divided vertically into equal parts of blue-black-blue and the other all blue, both set up horizontally; and two types of 0.25 m2 square targets (0.5 m×0.5 m) one divided vertically into equal parts of blue-black-blue, the other all blue , 1-octen-3-ol (O), and p-cresol (C)  was used as an attractant for experiments comparing baited devices based on general efficacy for several tsetse species To assess which was the best catching device and the most attractive blue material in the wet and dry seasons at each location, a six-day experiment was carried out to compare six devices in a 6×6 Latin square design of days×sites×treatments, with 3 simultaneous replicates. Trapping positions were always >100 m apart and flies from each device were counted and sexed after 24 hours at each position. The six devices and blue materials tested were: pyramidal traps in standard blue cotton and turquoise blue polyester/viscose; local targets in standard blue cotton and turquoise blue polyester/viscose; leg panels in standard blue cotton (both countries) and turquoise blue polyester/viscose (Kenya only) or plastic leg panels covered with phthalogen blue paint (Tanzania only). The 6 device experiment was repeated using the POCA bait, after the unbaited trials, in the same general area with trapping positions >200 m apart. Flies from each device were counted after 24 hours at each position. The objective was to determine whether baiting changed the performance ranking of the devices/materials.2 blue-black (50∶50) targets covered with adhesive film in 2010. All catching devices were made of standard phthalogen blue cotton and black polyester. Flies caught in the cage of the traps were not included in the total for this comparison. The surface area of adhesive film was the same on both devices, i.e. 2 m2. Traps without attached adhesive film were included as controls to estimate trapping efficiency of the pyramidal device. A 3-day experiment was carried out to compare the three devices in a 3×3 Latin square of days×sites×treatments in four replicates. The trapping positions were always >100 m apart and flies of each sex from each device were counted after 24 hours at each position.To assess the efficiency of 3-d traps versus 2-d targets as landing devices, catches in pyramidal traps with adhesive film on the blue-black fabric were compared to 1 m2 square, two 0.5 m2 horizontal oblongs and two 0.25 m2 square targets; see To assess the influence of size, shape and colour on fly landings, six target types  with adhesive film applied on both sides with a 1 m2 square of adhesive film alone . The two devices were orientated E–W, and a 4-day experiment was conducted following a 2×2 Latin square design of days×sites×treatments in four simultaneous replicates. The trapping positions were always >100 m apart and flies from each device were counted and sexed after 24 hours at each positionTo assess whether the adhesive film on its own was attractive to tsetse and could affect the catching device, a comparison was made between catches of tsetse attracted to a stationary 1 m2, 2, Area-wide population suppression involves consideration of the cost-effectiveness of materials versus deployment and maintenance. Hence, it was important to also quantify catches normalized for the size of each trapping device in the main series of experiments. To derive an empirical adjustment factor for the fact that in 2009, only the bottom 60 cm of the targets in Tanzania was covered with adhesive film, we recorded the heights of flies landing on the blue-black targets in an indicative experiment to test if a standard blue-black target 1 m2,  would caagricolaeG. pallidipes is not mentioned where captures were too low for meaningful analysis.In all trials randomization was set up using design.lsd in the package G. swynnertoni. In both countries and irrespective of season or fabric, sticky targets in the unbaited trials captured more G. swynnertoni than pyramidal traps  covered with adhesive film were the best devices for P≤0.001, . CatchesP≤0.001,  and 1.5–P≤0.001, . The legP≤0.001,  and TanzP≤0.001, . There wP≤0.001, , and sexG. swynnertoni. As before, the sticky targets greatly outperformed the pyramidal trap, with the largest difference in catch in the wet season in Tanzania ×100, G. pallidipes could not be estimated as the trap with adhesive film caught fewer flies than the trap without film.Trap efficiency, i.e. the proportion of flies caught in the trap cage of those that approach at close range, was estimated by dividing the mean daily catch in the cage of the unaltered pyramidal trap by the mean daily catch of the trap with adhesive film on the cloth, i.e. summing flies caught on adhesive film and in the cage. Efficiency for 2 target of adhesive film on its own (unbaited) caught very few tsetse of either species compared to the cloth target covered with adhesive film. This target caught 2% of the detransformed mean daily catch of G. swynnertoni on the cloth target , and 6% of the detransformed mean daily catch of G. pallidipes . Note that the sticky surface area of the cloth targets was twice that of the stand-alone adhesive film target.The 1 m2 blue-black target would catch as many flies as a local blue/black 1.5 m2 Kenyan target as pastoralists were attempting to reduce tsetse in the study area in Kenya when the experiments were conducted and vegetation was also being heavily-grazed. The 1.5 m2 target caught a mean of 5.5 flies versus 3.0 flies on the standard 1.0 m2 target . However, when landing heights were tallied, 101 of 132 G. swynnertoni (77%) landed on the bottom 60 cm of the targets. This information was used to obtain an indicative normalized catch per m2 for the Tanzanian targets in the main experiments of 2009 where only the bottom 60 cm was covered. The detransformed mean catch on adhesive film was multiplied by 1.30 (1/0.77) for flies that could have landed on the upper 40 cm of the target without them landing on the lower 60 cm with adhesive , divided by 3 m2. All other detransformed mean catches were normalized only for the size of the trapping device  and blue-black-blue equal sized vertical stripes (Tanzanian style) both caught very similar numbers of both G. swynnertoni and G. pallidipes which suggests that there is no difference between the two designs for inducing landing , and to 19% and 14%, respectively, on the 0.25 m2 target compared to the 1 m2 targets , i.e. neither colour pattern  nor shape (oblong or square) significantly affects landings by 2, landings on the 0.5 m2 oblong blue-black-blue targets are nearly the same as on the standard blue-black targets for G. swynnertoni and G. pallidipes  and males a slight preference for the blue (55%). In contrast, in G. pallidipes, both sexes showed a strong preference for landing on the blue portion of targets .When the daily landing rates are corrected to an equal target size of 1 mllidipes . These cllidipes . For G. 2 oblong targets (G. swynnertoni catch on the blue-black-blue target was caught by the adjacent transparent target (6 of 464 flies) and the proportion was 6% for the all blue target (15 of 256 flies). In the case of G. pallidipes, only 2% of the total catch on both coloured target types was made on the adjacent transparent target .In the experiment with adjoining adhesive film targets placed next to the 0.5 m targets , only 1%swynnertoni in all situations, catching up to 19 times more flies than pyramidal traps. Baiting with chemicals did not affect the relative performance of devices. When equivalent areas of targets and pyramidal traps were covered with adhesive film, fly landings were twice as high on 1 m2 blue-black targets as on the pyramidal traps. When landings on 1 m2 blue-black targets were compared to those on smaller 0.5 m2 all-blue or blue-black-blue cloth targets and to landings on all-blue plastic 0.32–0.47 m2 leg panels, the smaller targets and leg panels captured equivalent numbers of G. swynnertoni and G. pallidipes per unit area as bigger targets.This study shows that independent of season or presence of chemical baits, targets in phthalogen blue or turquoise blue cloth and covered with adhesive film proved the best devices for capturing G. G. swynnertoni in all situations, catching over 19 times more tsetse than the pyramidal traps. When not baited, large blue-black targets captured 1.5 to 3.7 times more tsetse than much smaller leg panels. Blue leg panels made of either phthalogen blue, turquoise cloth or phthalogen blue-painted plastic nevertheless captured more tsetse than pyramidal traps, or at worst, equivalent numbers. Both targets and leg panels were particularly effective relative to pyramidal traps during the wet season in Tanzania. Of all the experiments, the wet season trials in the Serengeti represented the greatest challenge in terms of attracting flies to artificial devices during peak vegetation cover In both Tanzania and Kenya, and independent of season or the presence of baits, targets covered with adhesive film were the best trapping devices for G. swynnertoni based on their efficacy for sampling G. austeni in Zanzibar G. swynnertoni. Leg panels were 21% of the surface of targets in Kenya and 30% of the surface in Tanzania, but per unit area, captured 1.5 times more flies than the targets in Kenya and the equivalent number to targets in Tanzania. These results with leg panels in 2009 were confirmed in Tanzania in 2012 when similarly sized 0.5 m2 oblong blue-black-blue cloth targets covered with adhesive tape induced landings per unit area at nearly the same level as on the 1 m2 targets for G. swynnertoni. The potential cost-effectiveness of small targets for different tsetse has been demonstrated only very recently for a few tsetse species G. swynnertoni stands out relative to poor results for small all-black targets for two other savannah species G. pallidipes and G. morsitans morsitans in Zimbabwe G. pallidipes2 oblong target in blue-black-blue or all blue cloth induced landings per unit area at nearly the same level as on the 1 m2 targets for G. pallidipes in our trials. This may be related to the predominance of blue in the 0.5 m2 targets tested here, but concurs with earlier findings where doubling the target size doubles the catch for G. pallidipesSmall leg panels that deviate from large square or oblong blue-black fabric targets, G. swynnertoni lives in very open and often windy habitats where visual cues (including colour) may be more important than host odours; this is also manifested in terms of its well-known attraction to large, moving objects 2 in size clearly show promise for trapping G. swynnertoni, particularly as wind damage can be a problem with the larger local targets at many sites. However, 50% of flies captured landed on the black portion of all the targets tested, even those with only one third of the surface area in black. It would therefore be advisable to maintain a black element in visual devices targeting this species, as the presence of adhesive film used in these experiments has been shown to significantly reduce the landing rate on the black section of targets by G. tachinoides and G. palpalis gambiensisG. swynnertoni landing on the black would be higher on unmodified targets. Similar catches with cloth and plastic leg panels also indicate that this strategy can be used for both control (insecticide-impregnated cloth) and sampling of this species (rigid plastic with insect glue or adhesive film). Considering the fall off in landings per unit area on the very small 0.25 m2 targets, it would be inadvisable to employ targets much smaller than 0.5 m2 for control programmes.G. swynnertoni. These results agree with findings for the same fabrics tested in similar devices for several tsetse species in West Africa in situ through a unique dyeing process but now remains in limited production in just a few countries. This has resulted in the ad hoc use of several alternative blue fabrics in tsetse control, some of which are less than optimal for attracting tsetse There was no difference between the performance of any of the same devices made from the different blue materials between seasons and locations. The two blue fabrics chosen for these experiments  were manufactured with only minor differences in fabric texture and with slight but clear differences in blue-green colour. These fabrics, and the equivalent blue paint used on plastic leg panels G. swynnertoni. For a standard 1 m2 blue-black target, catches were twice as high as on the equivalent area of a pyramidal trap for G. swynnertoni, and three times higher for G. pallidipes. The adhesive film used to enumerate tsetse here  in current use for the savannah tsetse G. swynnertoni with traditional tsetse baits Pyramidal trap entry/retention did not appear to be improved by baiting traps with POCA, i.e. baited targets still caught far more tsetse than baited pyramidal traps. As the baited and unbaited trials were sequential, they could not be compared directly. Nevertheless, our results are consistent with previous failures to substantially improve catches of G. swynnertoni, i.e. less than two thirds of the flies that landed on attractive surfaces of the trap ended up being captured in the cage of the trap. This trapping rate is very similar in magnitude (31% efficiency) to that already measured for the S2 trap G. swynnertoni that landed on the panels of the trap were never captured. In contrast to this, the pyramidal trap proved more efficient for G. pallidipes. There has been an underlying deficiency with traps for savannah species of tsetse. An absolute estimate of trapping efficiency is, however, difficult as there are many untested assumptions concerning fly behaviour and counting accuracy near traps that could affect the outcome. In contrast to this, the low number of flies (<2%) landing on transparent 0.5 m2 sticky targets compared to the number alighting on adjoining blue-black-blue cloth targets of the same size and shape suggests that very few G. swynnertoni or G. pallidipes circle the oblong 0.5 m2 targets before landing. Ndegwa and Mihok G. swynnertoni flying in the vicinity of the trap circled within 0–1 m of it, and by covering the blue trap sides with adhesive fly rolls they found that 84% of approaching flies landed on the trap before entering it.As expected from many other studies on savannah tsetse, the pyramidal trap was found to be inherently inefficient as a trapping device for G. swynnertoni is abundant in areas with difficult logistics; it also presents limited options for dealing with as it occurs in protected areas frequented by large numbers of tourists. Simple targets that attract flies, which then land on insecticide-impregnated surfaces, are most suitable in this context. The most practical device for area-wide suppression of G. swynnertoni populations would be a large blue-black, insecticide-impregnated 1 m2 target. Our results show that there is no significant difference between the blue-black and blue-black-blue 1 m2 targets. A number of smaller targets in blue and black or all-blue leg panels with the same surface area would achieve the same result. Although all-blue leg panels would also provide a satisfactory control device a black element in the target is recommended where G. swynnertoni is the target species. The most cost-effective size of these devices and the associated costs of fabricating, deploying and maintaining large targets versus a higher number of small targets or leg panels still need to be determined in field trials. Our findings indicate that targets smaller than 0.5 m2 are not recommended for either G. swynnertoni or G pallidipes. Long-lasting but ultimately biodegradable devices of simple construction could be used to reduce disease transmission in the high-profile wildlife conservation areas of Tanzania and Kenya where G. swynnertoni is the main vector of human and animal trypanosomiasis. Either phthalogen or turquoise blue would be suitable for these visual control devices. Effective control will also require adaptive management Area-wide tsetse population suppression typically requires the deployment of many thousands of devices; devices need to be effective and inexpensive, and ideally should also be maintenance-free


Cervical cancer is the second most common cause of death from cancer in women worldwide, and the development of new diagnostic, prognostic, and treatment strategies merits special attention. Many efforts have been made to design new drugs and develop immunotherapy and gene therapy strategies to treat cervical cancer. HPV genotyping has potentially valuable applications in triage of low-grade abnormal cervical cytology, assessment of prognosis and followup of cervical intraepithelial neoplasia, and in treatment strategies for invasive cervical cancer. It is known that during the development of cervical cancer associated with HPV infection, a cascade of abnormal events is induced, including disruption of cellular cycle control, alteration of gene expression, and deregulation of microRNA expression. Thus, the identification and subsequent functional evaluation of host proteins associated with HPV E6 and E7 oncoproteins may provide useful information in understanding cervical carcinogenesis, identifying cervical cancer molecular markers, and developing specific targeting strategies against tumor cells. Therefore, in this paper, we discuss the main diagnostic methods, management strategies, and followup of HPV-associated cervical lesions and review clinical trials applying gene therapy strategies against the development of cervical cancer. Currently, 500,000 new cases of cervical cancer are diagnosed and 280,000 deaths occur each year worldwide, making cervical cancer the second most common malignancy affecting women worldwide . The higin situ. Molecular target screening should be able to detect HPV-like etiological agents as well as identify precancerous lesions. To detect HPV antigens and determine HPV prevalence in premalignant and precancerous lesions, and to assess the sensitivity and specificity of molecular target screening with respect to cytological, colposcopic, and anatomopathologic methods; immunohistochemical, molecular biology and microarrays techniques have been designed. Laboratory tests can detect HPV DNA and identify HPV genotypes; there are currently several molecular biology techniques used to detect HPV and identify the virus type with which the patient is infected. In summary, HPV nucleic acid can be detected either in cervical smears or biopsy specimens by various methods complementary to traditional cytology. During the last 50 years, screening programs based on conventional cytology have significantly reduced cervical cancer morbidity and mortality , 5. Papa Once the disease has spread beyond the confines of a radiotherapy, chemotherapy, or surgical field, no standard treatment is currently available. However, new molecular targeted drugs being tested in clinical trials might inhibit tumor progression and increase apoptosis, resulting in tumor response or stabilization; furthermore, gene therapy strategies could also be employed for the control of HPV-associated precancerous cervical lesions. In this paper, we discuss the role of HPV in cervical cell transformation and the main methods for the detection of high-risk HPVs, with special attention to application of HPV testing in screening, treatment, and followup of precancerous cervical intraepithelial neoplasia. We also examine the major gene therapy strategies against cervical cancer development that have been tested or are being tested in clinical trials. Currently, over 120 different types of HPV have been identified based on the genotype . The HPV4CGGT that are concentrated within the viral LCR, where they regulate viral oncogene transcription. It has been demonstrated that E2 protein associates in vitro with several cellular transcription associate factors (TAFs), and these events function to activate HPV promoter over a relatively large distance in the viral genome [ The early region (“E”) of the HPV genomes covers more than 50% of the virus genome and contains the E1, E2, E4, E5, E6, and E7 genes that are carefully expressed at the early and late stages of infection. The early proteins E1 and E2 are involved in viral DNA replication and viral RNA transcription, E4 is involved in cytoskeleton reorganization and E5, E6, and E7 are responsible for cellular transformation, and immortalization . It has l genome . Once th∧E4 are products of a spliced transcript of the E1 and E4 open reading frames that can differ in length and/or degree of phosphorylation; some of these isoforms can associate with cellular keratin networks, leading to network disruption [∧E4 protein to generate species that lack the N-terminal fragment, and C-terminal fragments are able to multimerize to form amyloid-like fibers that can lead to accumulation of E1∧E4 and disruption of the normal dynamic of the keratin networks [∧E4 might serve as scaffold, transport, or structural proteins, and E1∧E4 gene viral mutants do not support high-level replication of the viral DNA, suggesting that E1∧E4 is involved in HPV DNA replication [ HPV16 E1sruption . Recentlnetworks . In addilication . HPV E5 oncoprotein interacts with cellular host proteins, and these interactions are important for HPV's biological activity in cell transformation and evasion of the host immune response. Recent studies have highlighted the important role of E5 in cell transformation, tumorigenesis, and immune modulation, thus implicating E5 in pivotal steps of carcinogenesis . For inswaf1/cip1, p27kip1, p53, p300/CBP, p600, TBP, Tyk2, and hTERT have been reported [ An early event in HPV-associated carcinogenesis during HPV DNA integration is a global perturbation of cellular gene expression by the E6 and E7 oncoproteins. The high-risk HPV E6 oncoprotein contains approximately 150 aminoacids and has two zinc finger motives formed by four C-X-X-C motives that are required for E6 functions . One of reported , 23. E6 ∧E2C, functions as a negative regulator for HPV DNA replication and plays a role in the control of viral copy number as well as in the stable maintenance of HPV episomes [ Two new proteins codified in the early gene region have been recently identified, E3 and E8, which are present only in a few papillomavirus types . A fusion protein, E8episomes .≈10% of the HPV genome), has no protein-coding function but contains the origin of replication as well as multiple transcription factor binding sites important in the regulation of RNA polymerase II-initiated transcription from viral early and late promoters. HPV L1 capsid proteins in HPV-positive LSIL and HSIL are a major target of the cellular immune response in CIN [ The late region (“L”) of all HPV genomes, comprising almost 40% of the virus genome, is located downstream of the early region and encodes L1 and L2 ORFs for translation of a major (L1) and a minor (L2) capsid protein. Finally, the long control region (LCR), a segment of about 850 bp  viral DNA integration in the host genome; (b) expression of viral proteins ; (c) interactions between E2, E6/E7, and cellular proteins. In general, HPV epithelial infections occur either through mechanical microabrasions or infection of the area, where basal cells undergo a transformation from a columnar to a squamous epithelium. When basal cells are infected, they migrate to the lumen and express the capsid genes L1 and L2. In subclinical infections of low-grade lesions, the viral genome is then replicated as an episome and encapsidated into the nuclei of cells in the upper layer epithelium. Thus, protected viral particles are able to infect new areas of epithelium or be sexually transmitted. In some cases, the infection progresses to high-grade lesions and cervical carcinoma, a process associated with the integration of the HPV genome into the host genome due in part to the loss of the transcriptional repression exerted by the E2 protein. in situ, whereas HPV18 was found only in mixed and integrated forms in these stages of cervical carcinogenesis. HPVs 31, 33, 52b, and 58 appeared most commonly in the extrachromosomal form in all stages of lesions, while the integrated form of these HPV types was present only in CIN 3 and in carcinoma in situ [Invasive cervical carcinoma is preceded by precursor lesions, which are characterized by disturbances of cellular maturation and stratification, as well as atypical nuclei, which tend to promote cellular immortalization and transformation . The tim in situ . Recommendations for cervical cancer screening released in 2012 by the US Preventive Services Task Force (USPSTF) and separately by a partnership among the American Cancer Society/American Society for Colposcopy and Cervical Pathology/American Society for Clinical Pathology (ACS/ASCCP/ASCP) no longer indicate annual screening with cervical cytology but rather screening at three- to five-year intervals with cytology and optional incorporation of testing for high-risk HPV infection. For women aged 21–29, the guidelines indicate screening with cytology  every three years; HPV testing is not recommended for this age group because younger women are more likely to experience transient infections and low-grade lesions that do not progress to precancerous lesions and do not require identification or treatment. Women older than 65 with evidence of adequate negative prior screening and no history of CIN2+ within the last 20 years should not be screened. Screening should not be resumed for any reason, even if a woman reports having a new sexual partner. For women aged 30–65, however, both sets of guidelines indicate screening with a combination of cytology and HPV testing every five years, the “preferred” method, or screening with cervical cytology alone every three years (“acceptable”) . Despite The US Food and Drug Administration (FDA) currently approves three HPV DNA tests for clinical use: Hybrid Capture 2, manufactured by Qiagen; Cervista HPV HR (Hologic); the cobas HPV test (Roche Molecular Systems). The most widely used test is Hybrid Capture 2 (HC2), a second-generation commercial test designed to detect HPV types classified as high-risk. HC2 is a nucleic acid hybridization assay that utilizes an HPV RNA probe mixture containing probes for 13 high-risk HPVs. Samples containing HPV DNA hybridize with the RNA probe and are captured on a plate coated with antibodies conjugated to alkaline phosphatase; when alkaline phosphatase's substrate is added, light is emitted and measured via luminometer. The test's limitations include possible cross-hybridization of low-risk with high-risk HPV types as well as possible false positives in contiguous samples due to chemiluminescent emission of high-load HPV samples . A 2003  An established method for the detection of HPV DNA is PCR amplification with primers derived from consensus sequences of either the conserved E1 or L1 open reading frames of the viral genome. The primer sets MY09/11 and GP5+/GP6+ are both able to amplify a wide range of HPV types. The MY09/11 primers are synthesized with several degenerate nucleotides and constitute a mixture of 25 primers, while the GP5+/GP6+ primers have fixed nucleotide sequences but use a lower annealing temperature during PCR to detect more HPV types . Two-ste Baleriola et al.  has eval Another detection method, the HPV oligonucleotide microarray , was evaluated by An et al.  and Kim  The application of HPV DNA testing alone or in combination with cervical cytology in screening, treatment, and followup of cervical cancer has been the focus a great deal of research in the past two decades. A 2006 meta-analysis of split-sample studies in Europe and North America that compared HPV testing with routine cytology found that HPV testing had significantly higher sensitivity than cytology, while cytology had a higher positive predictive value (PPV), in detecting a cervical intraepithelial lesion of grade II or worse (CIN2+), confirmed via subsequent histologic examination . One stu Several recently published studies have examined long-term outcomes of screening programs that incorporate HPV testing. A large Netherlands-based randomized controlled trial, the Population-Based Screening Study Amsterdam (POBASCAM), determined that women who were screened for HPV DNA (with the GP5+/6+ PCR method) in combination with cytology had fewer cervical intraepithelial neoplasias of grade 3 (CIN3) and fewer cervical carcinomas in a subsequent screen five years later, than those who were screened with cytology alone . The pro High-risk-HPV testing may be particularly valuable in low-resource settings, where the majority (80%) of cervical cancer cases occur, and where the infrastructure does not exist to implement effective cytology-based screening programs. In Colombia, for example, guidelines issued by the National Cancer Institute recommend screening based on a 1-1-3 cytology strategy ; however, coverage rates are low, with one-year coverage of women aged 25–69 ranging from 43.3% in the state of Boyacá to 56.8% in Caldas and three-year year coverage ranging from 66.6% in Magdalena to 81.3% in Caldas . In Sout The technical requirements of most HPV-DNA tests, however, make them impractical for use in low-resource settings. A new HPV test designed specifically for use in developing countries was assessed for clinical accuracy in screening tests in county hospitals in rural China . The car Following histologic identification of cervical intraepithelial neoplasia, surgical treatment is recommended for preinvasive lesions of grade CIN2 or CIN3. The 2006 Consensus Guidelines published by American Society for Colposcopy and Cervical Pathology (ASCCP) and its partner organizations utilize a two-tiered system in which histologically diagnosed CIN1 is classified as a low-grade lesion and CIN2/3 are classified as high-grade precursors . No treain vivo, such as cryotherapy, laser ablation, electrofulguration, and cold coagulation, as well as and excisional methods that remove the affected tissue and provide a specimen for pathological examination; these include cold-knife conization, laser conization, electrosurgical needle conization, and loop electrosurgical excision procedures (LEEPs), also known as large loop excision of the transformation zone (LLETZ). LEEP is now the favored procedure worldwide; although it has not been shown to be more effective than other methods, it has clinical advantages such as less operating time and lower rates of hemorrhage, pain, and infection, and it is less expensive and technically simpler than laser treatment [ Although the risk of CIN2 progressing to invasive cancer is low, diagnosis of CIN2 has limited reproducibility and validity, and thus CIN2 is used as the treatment threshold for safety reasons . Surgicareatment . All of reatment ; howeverreatment . Ablativreatment . Postsurgical followup of women treated for CIN 2/3 with ablative or excisional methods is critical, since treatment failure rates are estimated at 5%–15% across all treatment modalities . Failure If lesions are not identified and treated at the precancerous stages, treatment options are limited for women with metastatic or recurrent cervical cancer. Unfortunately, only up to one third of patients with metastatic and recurrent disease will respond to drug chemotherapy, and these responses are short-lived, on the order of months. Gene therapy strategies targeted to HPV products could represent new treatment options for cervical cancer and also other tumors in which HPV participates as a cancer promoter. Currently, it is well known that HPV E6 and E7 interact with a plethora of cellular proteins, both nuclear and cytoplasmic, and participate in molecular pathways involved in the activation and establishment of the tumor phenotype. Hence, it is feasible to design experimental strategies aimed to block the expression of viral oncoproteins. HPV E6 and E7 oncoproteins are excellent candidates for HPV therapeutic vaccination strategies, and the most of the therapeutic vaccines that have been tested in preclinical and clinical trials focus on interacting with antigen-presenting cells to stimulate cytokine production and T-cell activation. In addition, several efforts are focusing on the development HPV therapeutic vaccines based in attenuated virus and bacterial vectors, HPV peptide and proteins, and specific tumor and dendritic cells, as well as naked plasmid DNA-based vaccines.Listeria monocytogenes encoding HPV16 E7 fused to a nonhemolytic listeriolysin O protein, in patients with persistent or recurrent cervical cancer. Trial US-1093 is a randomized, placebo-controlled study to determinate the safety and efficacy of the VGX-3100 DNA vaccine that contain plasmids targeting E6 and E7 proteins of both HPV16 and HPV18, which are delivered via electroporation to adult women with biopsy-proven HPV16 or HPV18-associated CIN 2 or 3 [N 2 or 3 , 74. It in situ. However, the choice of gene target is the most relevant part on this kind of clinical protocols. In summary, the clinical trial findings will address broad issues about gene therapy vaccines including efficacy, duration of protection, and global impact of vaccination on HPV-related tumors; though these vaccines have the potential to significantly improve cervical cancer outcomes, continued screening will still be required after intervention. Although there is currently limited clinical information about gene therapy in cervical cancer patients, then trials discussed could establish proof of concept; therefore, it could be feasible to use gene therapy  in vitro by administration of specific small interference RNAs (siRNAs) for HPV16/18 E6 and E7 oncogenes in cervical cancer cells in vitro, few protocols have been complemented with animal tumor models with demonstrated eradication of tumors in vivo [in vitro and in vivo [ On the other hand, although many studies have described the induction of cancer cell death in vivo , 76. Thi in vivo –79. When in vivo . The rel Although early stage cervical cancers have a good prognosis with a 5-year survival rate greater than 80%, clinical and epidemiological evidence suggests that the natural history of HPV in young women (aged <30 years) may be such that establishment of a high-grade CIN lesion occurs early in the course (within 2 years) of a high-risk HPV infection. The consequences of HPV infection will depend on the infecting HPV type and site of infection, as well as on host factors that regulate viral persistence, regression, and latency. HPV testing and identification of high-risk strains and multiple infections has potential applications in the screening, treatment, and followup of cervical intraepithelial lesion. The identification and subsequent functional evaluation of host proteins associated with HPV E6 and E7 oncoproteins is the major challenge for their utilization as molecular biomarkers and may provide useful information in understanding cervical carcinogenesis for development of specific targeting strategies against tumor cells. Many experimental HPV vaccine strategies are being developed and tested in preclinical and clinical trials, in combination with immunotherapy and chemotherapy approaches to control of HPV-associated cervical lesions and invasive cancers. The principal factor for the prevention and treatment of cervical cancer is the education of the society, which requires different strategies in developing and industrialized countries –84. The  In conclusion, educational strategies and organized screening programs to detect HPV must be implemented alongside research and development of new therapeutic vaccines infection in order to reduce rates of HPV infection and cervical cancer. New gene therapy and siRNA-based approaches could represent a major step in reducing morbidity and mortality, but more work is required to achieve clinical efficacy at a level that can challenge current therapy.


PARP-1 is a nuclear enzyme that plays an important role in DNA repair, recombination, proliferation and the genome stability. The PARP-1 Val762Ala polymorphism has been associated with increased risk of developing cancers of the prostate, esophagus and lung. The aim of this study was to determine whether the PARP-1 Val762Ala polymorphism is associated with the risk of cervical carcinoma. MA-PCR was used to genotype the PARP-1 Val762Ala polymorphism in 539 women with cervical carcinoma, 480 women with CIN and 800 controls. The genotyping method was confirmed by the DNA sequencing analysis. The PARP-1 Val762Ala polymorphism was not associated with the risk of CIN. However, women carrying the PARP-1 Ala762Ala genotype were significantly susceptible to cervical carcinoma , and the similar results were also found in squamous cell carcinoma . In HPV positive population, the PARP-1 Ala762Ala genotype was also associated with increased risk of cervical carcinoma . Our results indicate that the PARP-1 Ala762Ala genotype increases the risk of cervical carcinoma. Cervical carcinoma is one of the most common malignant diseases among women with nearly 500 000 women developing this disease each year −/− mice show a higher susceptibility to carcinogenesis induced by alkylating agents(9). There are many identified single nucleotide polymorphism(SNP) in the PARP-1 gene, and some of which are reported to be implicated in carcinogenesis DNA repair pathways exist in all cells for maintaining genome integrity To the best of our knowledge, there is still no research about the association between the SNP of PARP-1 Val762Ala(GTG/GCG) and cervical carcinoma. Therefore, in the current population-based case-control study, we investigated the genotype distributions of the PARP-1 Val762Ala(GTG/GCG) in patients with cervical carcinoma or CIN.All 1019 patients (including 539 cases cancer and 480 cases CIN) and 800 controls were unrelated ethnic Chinese women from Zhejiang Province. Of the 539 cases cancer was consisted of 489 squamous carcinoma, 47 adenocarcinoam, 1 undifferentiated carcinoma, 2 carcinosarcoma; FIGO stage Ia 53 cases, Ib 213 cases, IIa 133 cases, IIb 83cases, III 48 cases, IV 9 cases. Of the 480 cases CIN was consisted of 70 low grade cervical intraepithelial lesion(CIN 1), 410 high grade cervical intraepithelial lesion. The median ages of the cancer, CIN and control women were 45 , 39  and 40.5  years, respectively. In the Hangzhou district, Patients with histologically confirmed primary cervical carcinoma or cervical dysplasia (CIN) at the Women's Hospital School of Medicine at Zhejiang University and the Cancer Hospital of Zhejiang Province were recruited; in the area outside of Hangzhou, a collaborative network of gynecological oncology teams in three areas  of Zhejiang Province was established to identify new cases of cervical cancer or CIN at local hospitals between June 2004 and May 2006. The diagnosis date and tumor histology were recorded for all of the women, and there were no age, stage, histology, or prior therapy restrictions. During the same period, controls were randomly selected from healthy women seen for gynecologic examinations at the Women's Hospital School of Medicine at Zhejiang University. Control selection criteria included no positive cytological findings (TCT: Thinprep cytologic test), no history of cancer and immune disease, age matching to the patients and residence in Zhejiang Province during the gynecological examination. After providing signed informed consent, participants responded to a standard questionnaire about their sexual and reproductive history, including the number of sexual partners, the age at first intercourse, the age at first full-term pregnancy, parities (including full-term pregnancy and abortion at or after 28 weeks) and oral contraception used history. The study was approved by the local Medical Ethics Committees, and informed consent was obtained from all women included in the study(No. 2004002).The blood samples of both patients and controls were collected. Of these, 689 patients  and 410 controls agreed to provide cervix brush-off samples for detection of HPV. The median ages of these HPV detecting cancer, CIN and control group were 42 , 39  and 43  years, respectively.Genomic DNA was extracted from the blood with phenol/chloroform and precipitated with cold ethanol. All genomic DNA samples were dissolved in water and stored at −20°C.5′-TGCTCCTCCAGGCCAAGCT-3′and 5′-TGCTCCTCCAGGCCAAGCC-3′, which differ in only the last base, and the reverse primer was 5′-GAAACGCCCAAAGGTTCT-3′. The PCR product length was 330 bp.The SNP of PARP-1 Val762Ala(GTG/GCG) was determined by Modified polymerase chain reaction-mismatch amplification(MA-PCR). The two forward primers were The PCR was performed in a 25 ul reaction containing ∼50 ng of genomic DNA, 5.0 pmol of each primer, 0.2 mM of each deoxynucleoside triphosphate and 1.0 units of Taq DNA polymerase . PCR was carried out under the following conditions: an initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 45 s, and a final elongation step of 72°C for 10 min. The PCR products were examined by 1% agarose gel electrophoresis, stained with ethidium bromide and visualized with a Typhoon™ 9410 Imaging System . To examine the reproducibility of the results, all samples were tested twice by two technicians and the results agreed for all masked duplicate sets. The results reproducibility of two technicians was one hundred percent.5′-CCACCTGGGTGAGTCTGTCT-3′ and 5′-AGGCCTGACCCTGTTACCTT-3′, which produce a 212 bp fragment. Dye terminator DNA sequencing was performed in both directions using a BigDye Terminator Kit  at the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). PCR products and dye-terminated products were purified by ethanol/sodium acetate precipitation. Comparison of the two methods of analyzing polymorphisms did not identify any discordant pairs. These indicated that the MA-PCR specificity rate of detecting PARP-1 Val762Ala (GTG/GCG) was one hundred percent.To confirm the specificity of the MA-PCR for the polymorphisms, 20% of the samples were randomly selected (n = 200) and sequenced in a blinded manner. The forward and reverse primers for PCR sequencing were HPV infection was identified using the Hybrid Capture II(HC II) assay  using probe B, which contains a pool of full-length RNA probes specific for HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. Cervical sampling for HPV DNA was performed with the Digene Cervical Sampler, and the tests were classified according to the relative light units/positive control ratio, measured as the relative light units of the specimen divided by the mean relative light units of two positive controls.2) test. Logistic regressions were applied to calculate the odds ratio(OR) and 95% confidence interval(CI), for the association between the genotypes and risk of cervical carcinoma. All statistical analyses were carried out using Statistical Package for Social Science software, version 11.5 .Differences in frequencies of the genotypes and alleles between the cases of cervical carcinoma or CIN and controls were evaluated by the chi-square . All genotypic distributions were consistent with that expected in the Hardy–Weinberg model  to nuclear proteins, including histones, X-ray repair cross-complementing factor-1(XRCC-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), p53 and PARP-1 itself +, and crystal structure of the catalytic domain of human PARP-1 reveals that 762Val is located in the fifth helix of the PARP-1 N-terminal regulatory subdomain, facing the pocket of the active site The increased risk of cervical carcinoma in subjects with the PARP-1 Ala762Ala(GCG/GCG) genotype is likely attributable to the reduction of PARP-1 activity. PARP-1 catalyze extensive polymerization of ADP-ribose from its substrate niconamide adenine dinucleotide genotype and increased risk of cervical carcinoma. Our findings suggest that PARP-1 dysfunction may play an important role in the development of cervical carcinoma.


Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma spp., and Ureaplasma spp. were compared in the oropharynx  and urine of male Japanese patients attending a sexually transmitted disease clinic.Oropharyngeal squamous cell carcinoma (OSCC) has shown a gradual increase in male predominance due to the increasing incidence of human papillomavirus (HPV)-associated OSCC. However, the mode of HPV transmission to the oral cavity is poorly understood, and little is known about the epidemiology of oral HPV infection in men. The prevalence rates of HPV, The study population consisted of 213 men aged 16 – 70 years old (mean: 34.4 years old). Oropharyngeal gargles and urine were collected, and sedimented cells were preserved in liquid-based cytology solution. After DNA extraction, β-globin and infectious organisms were analyzed by a PCR-based method. The HPV genotype was determined by HPV GenoArray test.N. gonorrhoeae was more prevalent in oral (15.6%) than urine samples (9.1%), whereas C. trachomatis was detected more frequently in urine (15.9%) than oral samples (4.2%). The detection rates of M. genitalium, M. hominis, and Ureaplasma spp. were 5.2%, 10.3%, and 16.0% in oral samples, and 7.7%, 6.3%, and 19.2% in urine, respectively. There were no significant differences in the detection rates of Mycoplasma spp. and Ureaplasma spp. between anatomical locations. The distribution of HPV types were similar in oral and urine samples, and HPV16 was the most common type. The majority of men with HPV infection in both the oral cavity and urine had concordant oral and urinary HPV infection. The presence of urinary HPV infection was an independent risk factor of oral HPV infection, with an odds ratio of 3.39 (95% CI: 1.49 – 7.71), whereas oral gonococcal infection was inversely correlated with oral HPV infection .β-Globin was positive in 100% and 97.7% of oral and urine samples, respectively. HPV detection rates were 18.8% and 22.1% in oral and urine samples, respectively, suggesting that the prevalence of HPV infection in the oral cavity was similar to that in the urinary tract. Oral HPV infection commonly occurs in sexually active men, and is significantly correlated with urinary HPV infection. Human papillomavirus (HPV) infection is a necessary element for the development of virtually all cases of cervical cancer in women, and hasCervical HPV infection appears to occur frequently within a few years of sexual debut, and its prevalence decreases with increasing age from the peak prevalence in younger women. SexuallNeisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma spp., and Ureaplasma spp. were also examined in oral and urine samples, and we investigated the possible association of oral HPV infection with these infectious organisms.In the present study, we investigated the prevalence of HPV infection and HPV types in the oropharynx  and urine of male Japanese patients who attended a sexually transmitted disease (STD) outpatient clinic, and the concordance of infected HPV types between these two infection sites. In addition, the infection statuses of A total of 213 male Japanese patients who attended the STD outpatient clinic of Kanazawa University Hospital , Ishikawa Prefectural Central Hospital , Dokkyo Medical School, Koshigaya Hospital , Wakatsuki Clinic , and Nakashima Clinic  were enrolled in this study. All participants were men who had sex with women, and homosexual or bisexual men were excluded from the study. The ethics committee of Kanazawa University Graduate School of Medicine approved this study. Oropharyngeal gargle and urine samples were obtained from each subject after obtaining their written informed consent. A clinical diagnosis of urethritis was defined as ≥  5 polymorphonuclear leukocytes per high-power field in collected urethral swabs.g for 10 min, and the sediment was placed into a separate tube containing 2.5 mL of preservative solution for liquid-based cytology  and stored at 4°C until use.To obtain oral specimens, each subject swished 15 mL of normal saline around their oral cavity, gargled with their head tilted back for 10 – 20 s, and then expectorated into a specimen cup. In addition, all urine from one urination for each patient was collected in an individual urine cup, and 15 mL was placed into a separate tube. Each sample (15 mL) was centrifuged at approximately 1500  ×  g for 10 min, and the supernatants were discarded. The cell pellets were washed twice with 300 μL of 10 mmol/L Tris-HCl (pH 8.0). DNA was extracted from the cells using a DNA extraction kit  according to the manufacturer’s instructions. The β-globin gene was first amplified to confirm the adequacy of the extracted DNA in all samples. In β-globin-positive samples, HPV-DNA was tested by modified GP5+/GP6+ polymerase chain reaction (PCR) as described previously[Aliquots of 800 μL of preservative solution containing cell samples were centrifuged at approximately 1500  ×  HPV genotyping was performed using HPV GenoArray Test Kits  according to the manufacturer’s protocol. This assay can be used to determine 21 HPV genotypes, consisting of 14 high-risk HPV types , 5 low-risk HPV types , and two unknown-risk types (53 and CP8304), by flow-through hybridization using HPV-DNA amplified by PCR.N. gonorrhoeae was detected in each liquid-based sample by PCR using specific primers targeting the N. gonorrhoeae orf1 gene[C. trachomatis amplified a 205-bp fragment of the 16S rRNA gene, as described previously, and had sensitivity and specificity of 97.9% and 98.7%, respectively[rf1 gene. For theectively.Mycoplasma genitalium, Mycoplasma hominis, and Ureaplasma spp. (biovars 1 and 2) were detected by multiplex PCR assay as described by Stellrecht et al.[Ureaplasma spp., the 140-kDa adhesion protein gene of M. genitalium, and the 16S rRNA gene of M. hominis, yielding bands of 403 – 448 bp, 282 bp, and 334 bp, respectively[Mycoplasma spp. and Ureaplasma spp. were 87%, 96%, 94%, and 93%, respectively, with an analytical lower limit of detection of <  12.5 colony forming units (cfu) for Ureaplasma spp., M. genitalium, and M. hominis, even if these organisms were mixed together.t et al.,16. ThisP  <  0.05 was taken to indicate statistical significance.The chi-square test was used to compare HPV-positive rates between the two anatomical sites among all 213 patients. Univariate and multivariate analysis using unconditional direct logistic regression analysis for all variables was performed to determine the risk factors for detection of oral HPV infection, and the odds ratios (OR) and 95% confidence intervals (CI) were calculated. The SPSS statistical software package  was used for all analyses, and Herpes genitalis, and Trichomonas was incidentally identified in one patient based on urinary microscopic examination. There were no patients with any pharyngitis-like symptoms.Among the 213 participants aged 16 – 70 years , 91 (43%) had urethritis, comprised of 17 gonococcal, 31 chlamydial, 2 gonococcal and chlamydial, and 41 non-gonococcal and non-chlamydial infections  than in urine samples (9.1%), whereas C. trachomatis was detected more frequently in urine samples (15.9%) than in those from the oral cavity (4.2%). On the other hand, the detection rates of M. genitalium, M. hominis, and Ureaplasma spp. were 5.2%, 10.3%, and 16.0% in the oral cavity and 7.7%, 6.3%, and 19.2% in urine, respectively. The differences in detection rates between oral and urine samples were not significant.Positive amplification of the β-globin gene was observed in all 213 (100%) of the oral samples and in 208 (97.7%) of the urine samples   of 1608 healthy men aged 18 – 78 from the USA, Mexico, and Brazil using the Roche linear array HPV genotyping assay[D'Souza  the USA. HPV infng assay. In adding assay. These pHowever, the prevalence in our subjects (18.8%) was much higher compared with those reported in previous studies, which was likely due to the difference in study populations. Our study was performed in male patients who attended an STD clinic, and therefore they represented a population that would have risk factors for sexually transmitted infection. Indeed, the prevalence of oral HPV infection in women was also reported to differ according to the study population, and its prevalence was higher 23.6%) in women who attended STD outpatient clinics compared.6% in woWe found that type-specific HPV prevalence was similar in oral and urinary samples, and HPV16 was the most common type identified in the present study. In addition, the majority of men with HPV infection in both the oral cavity and urine specimens had concordant oral and urinary HPV infection, suggesting that oral HPV infection in men may also occur through oral and genital contact with women. Indeed, the present study demonstrated that the presence of urinary HPV infection is an independent risk factor for oral HPV infection, with an odds ratio of 3.39.Moreover, single-type HPV infection was frequently identified in the oral cavity, and single HPV16 was the most common type. Many epidemiologic studies indicated that HPV16 is the most common HPV type detected from HPV-associated OSCC,18, whicMycoplasma spp. and Ureaplasma spp. using liquid-based cytology samples of urine[M. genitalium, M. hominis, Ureaplasma parvum, and Ureaplasma urealyticum were detected in 14.5%, 10.9%, 6.5%, and 12.3% of men with urethritis, whereas these species were detected in 3.3%, 2.0%, 4.7%, and 2.7% of controls, respectively[et al. also reported prevalence rates of 1%, 4%, 12%, and 23% for M. genitalium, M. hominis, U. parvum, and U. urealyticum, respectively, in urine samples from 100 asymptomatic healthy Japanese men[M. genitalium has been widely accepted as an agent of STIs, and the differences between these studies may have been due to differences in the sample size and characteristics of the study populations as well as the detection methods used. On the other hand, little information is available regarding the prevalence rates of Mycoplasma spp. and Ureaplasma spp. in the male oral cavity. Sackel et al. reported that M. hominis and Ureaplasma spp. were recovered from pharyngeal samples obtained from 149 (14.3%) and 154 (14.8%), respectively, of 1044 men and women who attended clinics in Boston, USA, which were equivalent to the findings of the present study[M. genitalium, M. hominis, and Ureaplasma spp. in males between the oral cavity and urine samples, suggesting that genital Mycoplasma spp. and Ureaplasma spp. could frequently infect the male oral cavity.We performed another additional study to investigate the possible associations between oral HPV infection and other organisms responsible for sexually transmitted infections (STIs). In a previous study, we investigated the prevalence rates of of urine. M. geniectively. Takahasnese men. These fnt study. There wN. gonorrhoeae, C. trachomatis, and/or M. genitalium are likely to facilitate access of HPV to the basal cells of the urothelium in the urethra. However, multivariate analysis indicated that oral gonococcal infection was a negative factor for oral HPV infection in the present study. One possible reason or this observation is that there were no patients with pharyngitis-like symptoms. Most oropharyngeal gonococcal or chlamydial infections are asymptomatic without inflammation of the oropharynx[N. gonorrhoeae, C. trachomatis, and M. genitalium, were risk factors for the detection of oral HPV infection. Alternatively, the population size in the present study was small, and further large-scale studies will be required to reach more definitive conclusions.It has been reported that the presence of infection in the urinary tract is an independent risk factor for HPV infection. HPV genopharynx, and it N. gonorrhoeae, C. trachomatis, Mycoplasma spp., and Ureaplasma spp., is also suitable for detection of HPV in oropharyngeal gargle samples.In the present study, a liquid-based cytological procedure, which is commonly used in cervical cancer screening, was used for detection of HPV in oropharyngeal gargle and urine samples. Urine is generally considered unsuitable for the detection of HPV due to the poor β-globin detection rate (< 50%). HoweverIn conclusion, oral HPV infection is common in sexually active men, and its prevalence rate was equivalent to that in urine samples. In addition, oral HPV infection was significantly correlated with urinary HPV infection.HPV: Human papillomavirus; OSCC: Oropharyngeal squamous cell carcinoma; STD: Sexually transmitted disease; PCR: Polymerase chain reaction; CIN: Cervical intraneoplasia; STI: Sexually transmitted infection.The authors declare they have no conflicts of interest.All authors made substantial contributions to conception and design, or acquisition of data. KN, KS, and SK were in charge of the analysis and interpretation of all data. KN drafted major portions of the initial manuscript, and KS, TS, and MN helped in writing the final manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/43/prepub