Infections by HIV increase the risk of acquiring secondary viral and bacterial infections and methods are needed to determine the spectrum of co-infections for proper treatment. We used rolling circle amplification (RCA) and Ion Proton sequencing to investigate the vaginal microbiome of 20 HIV positive women from South Africa. A total of 46 different human papillomavirus (HPV) types were found, many of which are not detected by existing genotyping assays. Moreover, the complete genomes of two novel HPV types were determined. Abundance of HPV infections was highly correlated with real-time PCR estimates, indicating that the RCA-Proton method can be used for quantification of individual pathogens. We also identified a large number of other viral, bacterial and parasitic co-infections and the spectrum of these co-infections varied widely between individuals. Our method provides rapid detection of a broad range of pathogens and the ability to reconstruct complete genomes of novel infectious agents. Massively parallel sequencing (MPS) provides an unprecedented opportunity to detect known viral and bacterial pathogens and identify novel infectious agents. The throughput of MPS makes it possible to study also highly complex human microbiota, such as that of the vaginal environment. The complexity of the vaginal environment is due both to the hormonal cycles, and resulting changes of the mucosa, and the multitude of sexually transmitted pathogens. Infections that compromise immune system function, such as with human immunodeficiency virus (HIV), have a strong effect on the vaginal microbiota. HIV infection increases the risk for opportunistic infections by bacteria, viruses, fungi and parasites and the development of several types of cancers  and recurring respiratory tract infections. Antiretroviral (ARV) treatment can restore the CD4+ count, but HIV infected individuals still have an increased risk of acquiring secondary infections. For patients with a reduction of immune system function, information on the viral, bacterial and parasitic co-infections may be critical for proper clinical management and choice of therapy.MPS has been employed for genotyping of HPV in cervical cell samples by sequencing of amplicons1237MPS technologies differ substantially in sequence read length, turn-around time from sample preparation to sequence generation, and cost. For the clinical usefulness of MPS, the cost per sample and the turn-around time are particularly important. The massively parallel semiconductor sequencing of Ion Proton is amendable to automation in preparation of sequencing libraries and has rapid sequence generation, making it suitable for analysis of clinical samplesClinical characteristics of the 20 women are summarized in 5. This may indicate large differences in HPV titer between types. Across the 20 samples the most highly abundant type was HPV 58, followed by a number of other high-risk HPVs  of HPV infections detected by genotyping  and viral titer  (The sequence-based identification of HPV types was compared to genotyping using the Roche Linear Array HPV Genotyping Test and the notyping . However = 0.73) . Thus, tde novo assembly of remaining reads. This resulted in the identification of two novel HPV types, denoted HPV X and Y, and an assembly of their complete genome sequences  viruses, chromosomal and extrachromosomal elements (plasmids and transposons) from pathogenic, potentially pathogenic and non-pathogenic bacteria, as well as parasites in the samples  and others occurring in only a single sample   . We furtabundant . As seenClostridium being the most common match among the women. Sample S16 showed the highest abundance of unknown bacterial sequences. The majority of contigs in S16 contained ORFs that showed resemblance to Clostridium, while others were assigned to other species including Veillonella, Lactobacillus and many more. This demonstrates that S16 carries an infection of a bacteria with a presently undescribed genome. However, it remains unclear whether all the sequences in S16 comes from one single infection, or if there are in fact multiple infections of uncharacterized bacteria in the sample. In conclusion, our results show that the RCA-Proton method can be used not only for the analysis of known viral, bacterial and parasitic infections, but also for the genome assembly and initial classification of previously uncharacterized infectious organisms.For some samples, there remained a substantial number of long and high-quality sequence reads after the analysis of non-HPV infections see . These rThe RCA-Proton method enabled us to study the infection burden in HIV positive women at a new level of resolution. The prevalence of HPV is known to be substantially higher in HIV infected individualsTorque teno, SEN-V and JC viruses were detected. Torque teno is asymptomatic, SEN-V associated with post-transfusion hepatitis, and the JC virus asymptomatic but may be activated in immunosuppressed individuals and result in damage to the brain and kidneys. Among the bacterial infections are human pathogens such as Streptococcus pneumoniae, Neisseria meningitides and Neisseria gonorrhoeae. Enterobacter agglomerans is an opportunistic pathogen and cause wound, blood, and urinary tract infections in immunosuppressed individuals, and Bacteroides fragilis is involved in 90% of anaerobic peritoneal infections. Streptococcus agalactiae is found in the gastrointestinal flora of humans and if transferred to a neonate can cause serious streptococcal infection . Gardnerella vaginalis can cause bacterial vaginosis in some women. We also identified the parasite Trichomonas vaginalis, which is a flagellated protozoan causing trichomoniasis.The RCA-Proton assay resulted in a broad screening of the infectious burden of these women and identified a large number of DNA molecules from co-infecting bacterial plasmids, viruses and parasite genomes. Except HPV, infections by Clostridium, a genus of Gram-positive bacteria that include species of common free-living bacteria as well as important pathogens. It should be noted there are many uncertainties in the prediction of the closest related organism for these unknown DNA sequences, and these results should be seen as a preliminary rather than a final classification. Although further bioinformatics and biomedical analyses are required to confirm the genomic origins of these sequences and elucidate the role of these previously unknown infections in human disease, our results shows that the RCA-Proton method can be used as a first step in identifying novel pathogens.Our results also showed the presence of additional infectious agents, for which the genome sequences have not been determined. In several samples it was possible to reconstruct large pieces of DNA that did not match any known reference sequence. The analyses of protein-coding domains revealed that many of these sequences represent additional, previously uncharacterized, bacterial infections. The most common infection showed 50–75% amino acid similarity to The RCA based sequencing has many benefits but there are also some limitations with this approach. One of the main limiting factors is that RCA requires deeper sequencing as compared to 16S rRNA sequencing for metagenomic sequencing of bacterial communities. This is because the RCA method amplifies larger parts of genomes instead of just a single amplicon, and thus requires more reads to identify the bacterial species present in a sample. Moreover, the RCA reaction generates a relatively high proportion of reads originating from the host genome, thereby increasing the need for deep sequencing even further. When it comes to analyses of HPVs, the RCA method is not suitable for studying HPV integration into the human genome. Since small circular genomes are preferentially amplified in the RCA reaction the integrated HPVs will typically not be sequenced at high coverage.in vitro genetic screening is the most powerful approach to assess the microbiota. For MPS to be suitable for such analyses it has to be economically feasible to use. By barcoding of samples prior to Ion Proton sequencing, the price tag per sample can be reduced substantially. Further increases of the sequence throughput, such as that predicted for the Ion Proton chip PII, and the use of a higher number of barcodes, would make it possible to analyze as many as 100 samples in a single run, dramatically reducing the cost per sample. The run time on the Ion Proton instrument is only a few hours, and it is therefore possible to complete the process from sample preparation to generation of medically actionable data, in a few days. In summary, application of the latest MPS technology, such as the RCA-Proton method employed here, is likely to reveal a number of known and novel co-infections occurring in many infectious diseases. The pattern of co-infections in the present study varied dramatically between women, with a unique spectrum of viral, bacterial and parasitic co-infections present in each sample. Further studies of the co-infection profile will be important in order to determine their importance for proper clinical management of individual patients.We have shown that the RCA-Proton method can be used for comprehensive profiling of the infection burden in vaginal samples. Since many bacteria and viruses are difficult or impossible to isolate and grow in aerobic conditions or outside their host or community, The cervical samples are from a study conducted at the Empilisweni centre, Gugulethu, Cape Town, South Africa, conducted from 2006 to 2009. Study participants were recruited to Manyanani clinic with their sexual partner. The Research Ethics Committee of the University of Cape Town approved all aspects of the investigation (reference: 258/2006) and informed consent was obtained. All experiments were carried out in accordance with the approved guidelines. Samples were collected and stored as described131415HPVIR real-time PCR assay as describedHMBS); GenBank accession no. M95623.1) and the three other reactions to detect and quantify HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. The threshold for a positive HPV type was set at 10 copies per PCR. Similarly, as a threshold for inclusion in the study a copy number of 10 genomic equivalents were used. HPV copies per cell (titer) were calculated by dividing HPV copies per sample by copies of HMBS.The samples were HPV typed using two different systems. Samples S1–S6 and S8–S20 were typed using the Roche Linear Array HPV Genotyping Test. This line-blot assay individually identifies 36 HPV genotypes see . All sam2O in a total volume of 50 μl, as describedWe used a version of the rolling circle amplification (RCA) method designed to more specifically target human papillomavirus (HPV)Sequencing of RCA products was performed using the Ion Proton Sequencer (Life Technologies). A total of 500 ng of each sample was fragmented using the Covaris S2 system with a target peak of 150 bp. End repair and adaptor ligation were performed by the AB Library Builder System (Life Technologies). The samples were amplified, purified with Agencourt AMPure XP beads (Beckman Coulter Genomics) and size selected with a target peak of 220 bp . Library size and concentration were assessed on a Bioanalyzer High Sensitivity Chip (Agilent Technologies) and the samples were diluted to a concentration of 12 pM. The 20 samples were barcoded and pooled together in four sets of five samples each before template preparation with the Ion PI Template OT2 200 kit on the OneTouch 2 instrument (Life Technologies). The templated Ion Spheres for each pool of five samples were loaded onto a PI.v1 chip and sequenced on the Ion Proton using the Ion PI Sequencing 200 kit (400 flows). Quality trimming was performed to ensure high quality throughout the whole reads, using the default settings in Torrent Suite v3.4.2. The quality trimming removes low-quality base calls at the 3′ end of the reads.http://pave.niaid.nih.gov/)de novo assembly using Newbler v2.6. Among the assembled sequence contigs, we screened for novel HPV genomes by applying two filtering criteria: i) a contig length close to those of known HPV-types, i.e. between 7000 and 9000 bp and ii) a blastx search should identify the HPV proteins L1, L2, E1 and E2. Novel HPV genomes detected using this approach were added to the references sequence after which the process was repeated until no additional HPV types were detected. This resulted in the identification of two novel HPV types, denoted HPV X and HPV Y.Our analysis pipeline is based on successive rounds of mapping and subtraction of mapped reads . First, The abundance of each HPV type was estimated from the Ion Proton reads by measuring the median coverage over the HPV references. Single nucleotide polymorphism (SNP) calling was performed by the Variant Caller in Torrent Suite v3.4.2, and the resulting SNP information was used to determine sequence variation for each HPV. Only those HPV types detected in samples with sufficiently high read depth were used for this analysis (90% of the bases were required to have ≥10× coverage). For each HPV type and sample satisfying the coverage criteria, all homozygous SNP calls were extracted. A distance matrix was computed for each HPV type based on the number of SNP differences between samples infected by the same HPV. The distance matrixes were used as a basis for a clustering analysis (using custom R scripts) to obtain number of distinct variants for each HPV. A distance of at least 30 SNPs was required for two HPVs belonging to the same type to be classed as different sequence variants.de novo assembly (using Newbler) of all reads not mapped to any HPV (including HPV X and Y) or the human reference genome (hg19) for each of the samples  resulted in the detection of 62 long contigs (of length at least 4 kb) in six of the samples, which could not be matched to the NCBI nucleotide sequence database using blastn. Predicted open reading frames (ORFs) encoding proteins of at least 100 amino acids (aa) were extracted from the contigs using CLCbio. The predicted ORF sequences were matched with blastx to identify conserved protein domains. For each contig, the organism showing the highest total number of ORF hits was considered to be the evolutionarily closest species.Reads that could not be mapped to the human reference, to HPVs or to other co-infections were used to search for additional infections with unknown reference genomes. A A.A., T.M., Z.M., A.L.W. and U.G. designed the study. T.M., S.H., C.L., J.H.L. and I.G. developed the workflow for sample preparation and sequencing. A.A., I.B. and T.M. performed the data analysis and results were interpreted together with A.L.W. and U.G. A.A. and U.G. wrote the main manuscript text. All authors reviewed the manuscript.Supplementary information


Prevalence of both cervical cancer and Human Immunodeficiency Virus (HIV) infection are very high in India. Natural history of Human Papilloma Virus (HPV) infection is known to be altered in HIV positive women and there is an increased possibility of persistence of HPV infections in this population. Therefore, this study was conducted to understand the epidemiology and circulating genotypes of oncogenic HPV among HIV positive and negative female population in West Bengal, India.In this hospital-based cross-sectional study, 93 known HIV positive females attending a pre-ART registration clinic and 1106 HIV negative females attending a Reproductive and Child Health Care Clinic were subjected to study. Cervical cell samples collected from the study population were tested for the presence of HPV 16, 18 using specific primers. Roche PCR assay was used to detect other specific HPV genotypes in the cervical cells specimens of HIV positive cases only.Prevalence of HPV 16, 18 among HIV positive females  was higher than HIV negative females . About 53% (23/43) of cases with oncogenic HPV were infected with genotypes other than 16, 18 either as single/multiple infections. HPV 18 and HPV 16 were the predominant genotypes among HIV positive and HIV negative subjects respectively. Oncogenic HPV was not found to be associated with age and duration of sexual exposure. But the presence of HIV was found to a statistically significant predictor oncogenic HPV.The currently available HPV vaccines offer protection only against HPV 16 and 18 and some cross- protection to few associated genotypes. These vaccines are therefore less likely to offer protection against cervical cancer in HIV positive women a high percentage of who were infected with non-16 and non-18 oncogenic HPV genotypes. Additionally, there is a lack of sufficient evidence of immunogenicity in HIV infected individuals. Therefore, prevention of cervical cancer in HIV positive women must be focused towards early detection of oncogenic HPV & cervical cytological abnormality followed by an appropriate treatment. Cervical cancer is the most common malignancy among Indian women . India cIt was a hospital-based cross-sectional study. The study aimed to compare the prevalence of HPV-16 and 18 among HIV positive and HIV negative subjects with associated socio-demographic variables. The former was selected from the patients attending the pre-ART registration clinic of School of Tropical Medicine Hospital , Kolkata. A total of 93 ART naïve, known HIV positive females participated voluntarily in this study. Written informed consent was obtained from all of them. Interview with a pre-tested questionnaire was used to collect their information on socio-demography and relevant sexual behavior. Interview was followed by collection of cervical cells from external os and adjoining ecto-cervical area with the help of a cervical cyto-brush and disposable vaginal speculum to avoid transmission of STIs among them.A total of 1106 HIV negative females attending a Reproductive and Child Health (RCH) Care Clinic in a Southern part of West Bengal area were taken as controls. The controls had similar socio-economic status as that of cases as evidenced by their income, occupation & education. Informed consent was taken from all of them. Brief interview was followed by collection of cervical cells (as described in collection of specimen from cases). The major reason for their attending the clinic was for routine reproductive health care counseling or for getting treatment for some common gynecological ailments. Pregnancy was excluded from both cases and control subjects. Collected cervical specimens were dipped into cold phosphate-buffered saline (PBS) and transported to the laboratories of the Department of Human Genetics, Indian Statistical Institute (ISI) and National Institute of Cholera & Enteric Diseases (NICED), Kolkata, for HPV DNA testing. At ISI, HPV 16, 18 specific primers were used to detect these 2 genotypes  in cervp-values are two-sided. The significance level for the predictors to stay in the model was 0.05.Statistical analyses were performed using SAS  software. Chi-Square Test for Homogeneity and two sample T test were performed to compare demographic variables of the independently chosen study samples. The associations of potential risk factors with HIV/HPV infection were estimated by means of odds ratios (OR) and 95% confidence intervals (95% CI). All reported Ethical clearance was obtained from the ethical committee of the National Institute of Cholera & Enteric Diseases (NICED) before initiation of the study.A total of 93 HIV positive women and 1106 HIV negative women participated in this study. Cases and controls did not differ significantly with respect to mean age, religion and parity , prevalence of oncogenic HPV was 46.2% (n = 43) and prevalence of HPV 16 and/or 18 was 32.2% (n = 30). Among HIV negative study population prevalence of HPV 16 and/or 18 was 9.1% (n = 101) . Thus, HIV positive females were almost 5 times more at risk of acquiring oncogenic HPV co-infection than HIV negative females.Figure Figure Figure On univariate analysis, the significant predictors of HPV infection were HIV, age, and duration of sexual life. Though other demographic predictors such as education level, religion and socio-economic status have been found to be confounding factors in other published study, these factors did not seem to be significant confounders in our study. Next, both bivariable and multivariable logistic regression analysis were conducted to further analyze data. In bi-variable logistic regression Wald chi-square test statistic and p value was used. In bi-variable models HIV was a significant predictor of HPV . Neither age  nor duration of sexual life  were found to be statistically significant as predictors of HPV. Next, we conducted multivariable logistic regression to find out if multicollinearity or synergism existed between the three predictors . Chi square (likelihood ratio) and p value were used as test statistics. After adjusting for age and duration of sexual life, the OR for HIV was 4.9  i.e. the odds of having HPV infection were 4.9 times higher among HIV sero-positive women than HIV sero-negative women. Age and duration of sexual life were not statistically significant predictors of HPV after adjusting for HIV status and duration of sexual life, and HIV status and age, respectively .Distribution of the oncogenic HPV in HIV infected subjects is shown in Figure About 17% (n = 15) of the cases were infected by multiple oncogenic strains of HPV of which 8 were infected with dual genotypes, 6 were infected with triple genotypes and only 1 was infected with 4 genotypes. HPV 16 and/or 18 were present in 13 out of 15 multiple genotype infected cases.In this study it was observed that the prevalence of HPV 16 and or18 among HIV negative female population to be 9.1% which is comparable to findings from various other studies conducted in India. -16. ThisGenerally HPV prevalence in women shows a peak at younger ages and then declines over the age of 30 years ,8. Even In HIV positive females, prevalence of HPV 16 and/or18 showed a rising trend till 40 years after which it declined. Since this was a cross-sectional study it was impossible to determine whether HPV infection followed HIV or otherwise. But earlier studies have shown that presence of HIV infection facilitates the development and persistence of HPV infection. This may be because HIV infection lowers body immunity. So, co-infection with HIV can be a probable explanation for acquisition of new HPV infections later in life, delayed clearance of HPV with age and reactivation of latent infections acquired earlier in life due to a gradual loss of immunity. The decline of HPV prevalence beyond 40 years may be influenced by less number of subjects caused by death due to AIDS, increased immunity following receiving ART etc.Duration of sexual activity had opposite influence on HPV 16 and/or18 prevalence among HIV positive and negative subjects. HPV showed rising trend with increase in duration of sexual activity in HIV positive females whereas it showed a decline with increase in duration of sexual activity among HIV negative females. But, when adjusted for both age and duration, HPV prevalence showed a steady rise till 30 years of age in cases and controls when duration of active sexual life was less than equal to 5 years. However, when duration was more than 5 years, HPV declined with age till 30 years in both HIV positive and negative females. This may probably be due to development of immunity against HPV with higher duration of sexual life. So, duration of sexual life is perhaps a greater determinant of development of immunity and clearance of HPV than age alone till 30 years even in the presence of HIV. After 30 years, even with higher duration of sexual life HPV showed a constant prevalence with increase in age in HIV negative subjects whereas in the HIV positive group HPV increased with age till 40 years beyond which it again declined. In this study we were unable to compare these trends with those in population above 30 years of age but duration of sexual life less than equal to 5 years since none of the subjects belonged to this group. However, in multivariate analysis neither age nor duration of sexual life were found to be significant predictors of HPV infection. HIV co-infection was the only significant predictor of HPV infection and HIV positive women were almost five times more likely to be infected by oncogenic HPV compared to HIV negative women.Previous studies in India have reported circulating strains of oncogenic genotypes other than HPV 16 and/or18 in the general population . But adeWith the advent of ART, HIV positive women tend to live longer. Since cervical cancer generally develops in older age groups, therefore in the era of ART, there is an increased chance of cervical cancer cases among HIV positive women because of longer survival period. Information on interaction between HIV and HPV is therefore important to understand individual influence of each virus against the other. The currently available HPV vaccines offer protection only against HPV 16 and 18 and some cross-protection to few associated genotypes. Moreover, there is lack of evidence of immunogenicity in HIV infected individuals following receiving HPV 16/18 vaccines. These vaccines are therefore less likely to offer protection against cervical cancer in HIV positive women who harbor a high percentage of other oncogenic HPV genotypes. Therefore in HIV positive women early detection of oncogenic HPV & cervical cytological abnormality followed by an appropriate treatment is possibly the best approach at present.The authors declare that they have no competing interests.KS: made substantial contributions to conception, design, implementation and overall supervision of all the steps of this study as Principal investigator. RP: made substantial contributions to acquisition, analysis and interpretation of data and have been involved in drafting the manuscript. BB: have made substantial contributions to acquisition and editing of data. BS: have made substantial contributions to acquisition and editing of data. SB: have made substantial contributions to acquisition and editing of data. SS: carried out the HPV DNA testing using HPV 16, 18 specific primers. PPM: coordinated and supervised laboratory work including quality assurance. SC: carried out the HPV DNA testing using HPV 16, 18 specific primers and also conducted Linear Array HPV Genotyping Test (Roche) as chief of laboratory.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/72/prepub


Surveillance designed to detect changes in the type-specific distribution of HPV in cervical intraepithelial neoplasia grade 3 (CIN-3) is necessary to evaluate the effectiveness of the Australian vaccination programme on cancer causing HPV types. This paper develops a protocol that eliminates the need to calculate required sample size; sample size is difficult to calculate in advance because HPV’s true type-specific prevalence is imperfectly known.A truncated sequential sampling plan that collects a variable sample size was designed to detect changes in the type-specific distribution of HPV in CIN-3. Computer simulation to evaluate the accuracy of the plan at classifying the prevalence of an HPV type as low (< 5%), moderate (5-15%), or high (> 15%) and the average sample size collected was conducted and used to assess its appropriateness as a surveillance tool.The plan classified the proportion of CIN-3 lesions positive for an HPV type very accurately, with >90% of simulations correctly classifying a simulated data-set with known prevalence. Misclassifying an HPV type of high prevalence as being of low prevalence, arguably the most serious kind of potential error, occurred < 0.05 times per 100 simulations. A much lower sample size (21–22 versus 40–48) was required to classify samples of high rather than low or moderate prevalence.Truncated sequential sampling enables the proportion of CIN-3 due to an HPV type to be accurately classified using small sample sizes. Truncated sequential sampling should be used for type-specific HPV surveillance in the vaccination era. Infection with human Papillomavirus (HPV) is recognised as the main cause worldwide of both cervical cancer-3 and itUltimately, the success of such programs will be measured by the extent to which they reduce cancer incidence, but shorter term changes in benign and pre-cancerous lesions caused by HPV can be used to assess their impact. In AustKey indicators in the early evaluation of an HPV vaccination program are provided by monitoring the distribution of HPV types. It is important both to track the expected decline in prevalence of the types that the vaccine protects against, and to monitor the prevalence of oncogenic types that are not the target of current vaccines. HoweverNmin), make an initial estimate of prevalence of the characteristic of interest , and then continue to obtain specimens and recalculate the prevalence estimate until predetermined statistical criteria have been met.The sequential sampling approach to monitoring the prevalence of particular characteristics in a population has been used extensively in agriculture and industry, as well as in public health, where lot quality assurance sampling methods are particularly important-18. We dNmax) Stopping rules are commonly represented visually as pairs of lines on a graph delineating an area which, if breached, mean that sampling should be stopped and a classification made  were calculated using Wald’s formulae[The formulae,18,20:α and β are the nominal probabilities of a false positive and false negative, p1 and p2 are thresholds defining low and high prevalence and q1 and q2 are equal to 1 - p1 and 1 - p2[Nmax, used for truncation is determined arbitrarily[In equations-3α and βnd 1 - p2. The maxitrarily.The sequential sampling approach can be used in any population, but in the context of evaluating the impact of HPV vaccination, it is most relevant for CIN-3. Monitoring HPV prevalence in normal cervical specimens, or lower grade CIN, is known to detect a high proportion of infections that are transient or lesions that will regress, and areα) and false negative (β) probabilities of 0.01 and 0.025, exceeding commonly used standards of α = 0.05  and 1-β = 0.8 .We used thresholds of (a) less than 5% for an HPV type, designating low prevalence; ( b) 5-15% positive, designating moderate prevalence; and ( c) greater than 15% positive, designating high prevalence. In Australia only HPV 16 may occur in more than 15% of CIN-2 and 3; other types are evenly divided between those associated with ≤5%  and 5 -15%  prevalence in CIN-2 and 3 specimens[Nmin) of 20 specimens was considered large enough to make a classification in many cases. The choice of starting sample size does not affect the eventual classification of prevalences[Nmax = 60). Myatt and Bennett[Based on prior experience in other fields, a starting sample size  but for this paper the code was translated into plain C for improved efficiency.We used simulation to assess how well the sequential sampling approach worked in classifying HPV types as follows. First, we generated synthetic data representing samples of CIN-3 cases, each with a different proportion positive for an HPV type). We generated 1,000 data sets under two different scenarios: one in which all prevalences between 0.00 and 1.00 were equally likely (uniform distribution); and another in which the most likely prevalence (mode) was 0.05, or 5%, with a linear decay to 0.00 at the lower limit and 1.00 at the upper limit (triangular distribution). The use of an upper limit of 1.00 is legitimate because very large proportions of some samples may be positive for high prevalence types – HPV 16 in particular has been detected in up to 95% of specimens in some studies. The uselow, PCmod and PChigh) and the sample size required to make each classification. PClow, PCmod and PChigh were then plotted against the true prevalence in each data set, to show the relationship between the probability of classifying a data-set as being either of low, moderate or high positivity and the true proportion positive in the data set. Variation in the sample size collected was summarised using tables. Graphs were created using SigmaPlot 8.0 .For each of the 1,000 data sets, we summarised the result of the 10,000 resampling iterations by first calculating the proportion of times the prevalence was classified as low, medium or high  than implied by the 20% type II error rate often used as a benchmark in sample size calculations. A much lower average sample size (21–22 versus up to 48) was required to accurately classify high prevalence samples. Truncated sequential sampling represents a practical, efficient method of conducting HPV type-specific surveillance in the absence of accurate, prior information about type specific prevalence, and is especially efficient at classifying the likely prevalence of more common HPV types.Linkage Project (LP0883831)  includes contributions from CSL Biotherapies Ltd  and Victorian Cytology Service Inc, which monitors cervical screening in Victoria and the National HPV Vaccination Register. BD has received honoraria from CSL Biotherapies and is a member of an advisory board for GlaxoSmithKline and CSL Biotherapies. AJH has no conflicts of interest to declare.EKW devised the research idea, conducted and analysed the computer simulations, and wrote the manuscript. AJH, AMAS, DJP and DGR assisted with the development of methods and edited the manuscript. JK and BD provided epidemiological advice on the biology of HPV infection and current surveillance protocols and edited the manuscript.This paper was funded from the following sources: the Australian Government Department of Health and Ageing; Australian Research Council (ARC) Linkage Project (LP0883831) which included contributions from CSL Ltd and Victorian Cytology Service Inc. The views expressed in this publication do not necessarily represent the position of the Australian Government.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/12/77/prepub


The ability of the host to trigger apoptosis in infected cells is perhaps the most powerful tool by which viruses can be cleared from the host organism. To avoid elimination by this mechanism, human papillomaviruses (HPV) have developed several mechanisms that enable the cells they infect to elude both extrinsic and intrinsic apoptosis. In this manuscript, we review the current literature regarding how HPV-infected cells avoid apoptosis and the molecular mechanisms involved in these events. In particular, we will discuss the modifications in intrinsic and extrinsic apoptotic pathways caused by proteins encoded by HPV early genes. Many of the current efforts regarding anti-cancer drug development are focused on directing tumor cells to undergo apoptosis. However, the ability of HPV-infected cells to resist apoptotic signals renders such therapies ineffective. Possible mechanisms for overcoming the resistance of HPV-infected tumor cells to anticancer drugs will be discussed. Human papillomaviruses (HPV) are small, double stranded DNA viruses that infect epithelial tissues, including those of the anogenital tract. Of the more than 100 different identified types of HPV, about 40 are involved in genital tract infections. Some types of HPV cause warts, while others can increase the risk of malignancy and lead to cancer of the cervix, other anogenital regions, and the head and neck areas . PersistThe HPV genome is divided into an early region (E), a late region (L) and a non-coding long control region (LCR). The E region encodes seven non-structural proteins: E1, E2, E4, E5, E6, E6* and E7, while the L region encodes two structural proteins: L1 and L2, which code for the major and minor capsid proteins, respectively. Early genes are responsible for modulating epithelial cell function so as to favor virus production. For example, their gene products modulate keratinocyte differentiation, promote viral DNA replication and segregation, and inhibit viral clearance by the immune system. All early HPV proteins perform multiple functions, and act by interacting with a variety of cellular partners ,4. TheseApoptosis, also known as programmed cell death, plays an important role in development, cellular homeostasis and the pathophysiology of many diseases . ApoptosApoptosis is triggered through the extrinsic pathway when specific receptors are activated by their corresponding ligands. The majority of these “death” ligands belong to the TNF (tumor necrosis factor) family, and their corresponding cell surface “death” receptors to the TNF receptor family. TNF-alpha binds to TNF receptor 1 or 2 (TNF R1 and TNF R2), Fas ligand to Fas, and TRAIL (TNF-related apoptosis-inducing ligand) binds to Death Receptor (DR)1, DR2, DR3, DR4, and DR5. Other receptors within this family include FADD-like interleukin-1 beta-converting enzyme (FLICE) and TNF-like weak inducer of apoptosis (TWEAK) . AdaptorThe classic intrinsic apoptotic pathway, also referred to as the mitochondrial pathway, can respond to a variety of external stimuli such as DNA damage, radiation and osmotic stress ,21. ActiFollowing infection of host cells, a virus typically employs a number of different mechanisms to avoid the host immune response and to initiate and maintain efficient production and spread of its progeny. In most cases, a host organism will attempt to initiate apoptosis in order to eliminate infected cells. For this reason, many viruses, especially the DNA viruses, encode a variety of viral proteins that can inhibit or delay these protective actions, often by targeting key cellular proteins . For exaHPV infects cells in the basal layer, below the surface of the epithelium, and carries out a life cycle that is closely tied to the differentiation program of the host cells. During the early stages of infection, the episomal viral genome is usually present at fewer than 100 copies per cell, and functions as a slowly replicating plasmid. Shortly after infection and uncoating, low-level expression of early genes  as directed by the early promoter, enables maintenance of the episomal state of the HPV genome ,34,35,36As cells move up through the layers, they begin to proliferate. The ability of E6 and E7 to suppress apoptosis and to alter the function of factors involved in cell-cycle regulation, respectively, facilitates prolongation of the proliferative stage of keratinocyte differentiation ,38. DuriAmplification of viral genomes begins in the proliferative layers, and an increased level of expression of all the early viral gene products, including E4 and E5 ,41,42, iBoth the E4 and E5 proteins participate in the late viral functions, although details of these mechanisms are not yet entirely defined. It has been shown that E5 plays a subtle role during the productive stage of the HPV16 life cycle ,47. WhenApoptosis is an important mechanism through which the host can eliminate infected cells, and the ability to avoid apoptosis can therefore enhance virus survival. In addition, apoptosis plays an important role during keratinocyte differentiation . For thevia p53-independent mechanisms [E2 functions as a viral transcription factor, and also plays a role in HPV expression and viral replication. It has a transcriptional activation domain near its N-terminus and a DNA binding domain near the C-terminal end. These two regions are separated by a flexible hinge . In earlchanisms . In addichanisms , and haschanisms . Intereschanisms . Other rchanisms . It should be noted that these pro-apoptotic functions of E2 were demonstrated in experiments utilizing transient over-expression, and that the levels of E2 resulting from this approach are much higher than would be found under more physiological conditions. During the HPV life cycle, expression of E2 is typically quite low in keratinocytes at early stages of differentiation, and may not be sufficient to induce apoptosis. In later stages when E2 expression is higher, the production of E4 may reduce the pro-apoptotic activity of E2. Alternatively or in addition, this pro-apoptotic activity of E2 may coincide with apoptosis induced during normal keratinocyte differentiation. During carcinogenesis, often triggered by HPV integration, the activity of E2 is likely to drop because integration frequently disrupts the E2 region. HPV E5 is considered an oncogene because it can induce transformation in murine fibroblasts and human keratinocytes . In addiHPV E6 is an essential oncoprotein that has been widely studied over the last two to three decades. E6 is a small protein, consisting of 151 amino acids and presenting two atypical zinc fingers with motifs that contain two cysteines (Cys-X-X-Cys). The p53 tumor suppressor is the first-described and best-known target of HPV E6 . p53 actIn addition to p53, E6 also interacts with other partner proteins that play a variety of roles in the cell . FollowiIn each of these cases, E6 functions to reduce cell death by compromising the integrity of cellular pathways leading to apoptosis. In the context of the virus life cycle, the prevention of apoptosis is critical because it protects infected cells from elimination by the immune system, and therefore enables them to continue serving as hosts. This ability may also prolong the early stages of the life cycle. During carcinogenesis, the expression of E6 is usually increased, resulting in cells that do not undergo apoptosis and that are then susceptible to E6 and E7-mediated transformation. One recent study found that E6-mediated cervical cancer relies on the continuous expression of E7 along with constitutively expressed E6, and that continuous expression of E7 is required for the maintenance of cervical cancer and precancerous lesions in transgenic mice .E6*, a splice isoform corresponding to approximately the N-terminal half of E6, has significance in both the viral life cycle and in carcinogenesis. Early work suggested that the purpose of the splicing events that produce the HPV16 E6 splice variants was to facilitate translation of E7 . HoweverHPV E7 is also a well-known oncoprotein with multiple functions, including the inhibition of differentiation and the activation of cell cycle progression. E7 acts by releasing active E2F, thereby enabling transcription of several genes involved in DNA synthesis . The priThe association between HPV and human cancer has been studied for more than three decades. HPV-induced cervical cancer is the second most common cancer and the fifth leading cause of cancer-related deaths among women worldwide ,102. CerRecently, two HPV vaccines, Gardasil (MSD) and Cervarix (GSK), have been developed and approved for the prevention of infection with either two or four types of HPV. These vaccines are therefore predicted to reduce the incidence of cervical cancer. In Phase III randomized clinical trials, for example, Cervarix was able to prevent 90% of moderate and severe precancerous cervical lesions associated with HPV-16 and 18 . HoweverSurgical treatment is often employed to cure cervical cancer in the early stages, but it is not always easy to use this approach in head and neck cancers or in laryngeal papillomatosis, due to the need to maintain the normal structure of the airway and to avoid pulmonary spread. One option currently used to treat recurrent respiratory papillomatosis is endolaryneal laser surgery. Following surgical excision, however, HPV-associated cancers frequently return, especially in the immunocompromised population. Combined chemoradiotherapies have been assessed, with variable results . TopicalTo expand and enhance the limited options currently available, several laboratories have begun examining the possibility of using molecular strategies to inhibit HPV ,112. ForCurrently, therapeutic approaches that rely on activation of either the intrinsic or the extrinsic apoptotic pathways are unlikely to be helpful for HPV-associated diseases due to the ability of high-risk E6 proteins to subvert both extrinsic and intrinsic apoptotic pathways by mediating the rapid degradation of signaling molecules (p53 in the case of the intrinsic pathway and FADD and caspase 8 in the case of the extrinsic pathway). As a result of these E6 activities, therefore, engagement of either the extrinsic or the intrinsic apoptotic pathways does not result in the transduction of the intended death signal because the mediator molecules are missing. Therefore, if any of these apoptosis-inducing signaling pathways are to be used as tools for the elimination of HPV-associated malignancies, it will be necessary to restore the missing signaling molecules.+ cells to inducers of apoptosis, and could therefore make certain existing cancer treatments available to those suffering from HPV-associated malignancies. Progress has been made in the search for inhibitors of the E6/E6AP interaction; this approach has the potential to re-activate the intrinsic apoptotic pathway. First, the essential binding motif through which E6 and E6AP interact was identified and verified [in vitro and cell-based assays, several compounds with these characteristics were identified; such compounds have the potential for development into new therapeutic agents for treatment of HPV-associated cancers [An approach based on inhibiting the ability of E6 to mediate the rapid degradation of its cellular partners has the potential to re-sensitize HPVverified ,115,116. cancers .It may also be possible to target the extrinsic apoptotic pathway. For example, activation of the TRAIL-mediated, extrinsic apoptotic pathway shows promise in the treatment of several types of tumors. TRAIL-based therapies have elicited significant interest in recent years, largely due to their apparent ability to kill tumor cells while sparing most normal cells. Previous studies have reported on the effectiveness of both TRAIL and α-TRAIL R1 and R2 in combination with other sensitizing agents such as doxorubicin, bortezomib, adriamcin, 5-fluorouracil, irinotecan hydrochloride, paclitaxel, carboplatin, gemcitabine, cisplatin and radiation in both cell and animal model systems ,120,121.


Background. HPV vaccination may prevent thousands of cases of cervical cancer. We aimed to evaluate the understanding and acceptance of the HPV vaccine among adolescents. Methods. A questionnaire was distributed to adolescents at health clinics affiliated with a large urban hospital system to determine knowledge pertaining to sexually transmitted diseases and acceptance of the HPV vaccine. Results. 223 adolescents completed the survey. 28% were male, and 70% were female. The mean age for respondents was 16 years old. Adolescents who had received the HPV vaccine were more likely to be female and to have heard of cervical cancer and Pap testing. Of the 143 adolescents who had not yet been vaccinated, only 4% believed that they were at risk of HPV infection and 52% were willing to be vaccinated. Conclusions. Surveyed adolescents demonstrated a marginal willingness to receive the HPV vaccine and a lack of awareness of personal risk for acquiring HPV. Approximately 20 million people in the United States are infected with genital human papillomavirus (HPV) . An estiIt is predicted that HPV vaccination will prevent thousands of cases of cervical cancer worldwide. In addition, the emotional stress and economic burden associated with abnormal Papanicolaou test results and the treatment of preinvasive cervical lesions will be greatly reduced. The quadrivalent HPV vaccine  was found to have 97% efficacy at preventing HPV 16 and/or 18 related cervical intraepithelial neoplasia 2 or 3, adenocarcinoma in situ, and cervical cancer [ Until recently, studies of women and young adults have shown poor levels of knowledge about HPV, Papanicolaou smear testing, and cervical cancer , 5. Curr Continuing, several studies indicate that most parents, especially with proper knowledge of HPV and the vaccine, are willing to accept the HPV vaccine for their children –12. Heal The purpose of this study was to evaluate the understanding and potential acceptance of the HPV vaccine by adolescents aged 13 to 18 years at adolescent health clinics affiliated with New York Presbyterian Hospital. Although previous research shows that most adolescents, parents, and health care providers are willing to accept universal vaccination for themselves, their attitudes and knowledge regarding the vaccine have yet to be evaluated. Improved knowledge of adolescent understanding and acceptance of the HPV vaccine would help practitioners provide appropriate and useful information to parents, providers, and adolescents when making the decision to vaccinate. Approval for this study was received from the institutional review board. We administered a questionnaire to all adolescent patients (defined as between 13 and 18 years of age) awaiting annual well-patient medical examinations in adolescent health clinics affiliated with Weill Cornell Medical College and Columbia University Medical Center. These health clinics provide affordable, comprehensive services to a diverse patient population including underserved populations from each of the five boroughs in the New York City area. The adolescent patient was provided the questionnaire in English and/or Spanish. We could not predict the number of adolescent patients who would be interested in participating in this study, and therefore prior to study initiation we defined the survey collection period as September 1, 2007 through February 1, 2008 with the goal of collecting the maximum number of surveys possible. The research personnel emphasized that participation was voluntary, anonymous, and without incentive for questionnaire completion. The questionnaire consisted of two parts; the first part elicited the adolescent's demographic information and knowledge pertaining to sexually transmitted diseases and vaccinations. When the first part was completed, the adolescent was provided with a second part that contained current facts about the HPV vaccine and cervical cancer, followed by questions specifically designed to ascertain adolescent acceptance of the vaccination. Adolescents whose primary language was neither English nor Spanish were excluded from the study since the questionnaire was self-administered and available only in English and Spanish. χ2 and Fisher's exact tests were used for comparison of proportions between “HPV vaccine status” and 15 other variables of interest  and 143 participants (willingness to be vaccinated) provided a mean power >99.9% for detecting a difference between both whether the adolescent had been vaccinated and whether the adolescent was willing to receive the HPV vaccine and the respective variable, using 2-tailed test with statistical significance defined as P < 0.05.Data were analyzed using commercially available software . A series of rest see  and “Wilrest see . A P valOf the 223 adolescents who completed the survey, 28% were male and 70% were female (2% did not respond to the question). The mean age of respondents was 16 years old (range 13–18). The demographics of adolescents including in this study are displayed in Of the 223 adolescent surveyed, 33% stated that they had received the HPV vaccine, 64% had not had the vaccine, and 3% where not sure. Among the 143 adolescents who had not yet been vaccinated, 52% were willing to get the HPV vaccine, 12% were not willing, and 36% were unsure. Of the 143 adolescents who had not yet been vaccinated, only 4% believed that they were at risk for being exposed to HPV.P < 0.001). HPV vaccination status was not significantly associated with language, race, religion, or having younger or older siblings who had received the HPV vaccine. HPV vaccine status was associated with knowledge of cervical cancer , knowledge of Pap screening (43% who had heard of Pap screening were vaccinated), and having had other vaccines (73% of those who had received other vaccines were vaccinated) . Female respondents were more likely to have received the HPV vaccine than male respondents  .Among the adolescents who had not yet received the HPV vaccine, 58% of females and 42% of males were willing to be vaccinated; however this difference was not statistically significant. In addition, 53% of Hispanic adolescents and 56% of African American adolescents were willing to accept vaccination. There was no association with willingness to receive the vaccine and gender, race, religion, language, having vaccinated siblings, knowledge of cervical cancer, Pap screening, sexually transmitted disease, or any other studied variable.The current study reveals only a marginal willingness of teenagers to accept the HPV vaccine. The majority of data to date suggest that young women are interested in vaccines that prevent HPV and other sexually transmitted infections. However, most of these studies have been conducted with women and men of 18 years of age or older. Holcomb et al. found that women are interested in learning about HPV, how the virus is transmitted, and how women can prevent becoming infected . In 2001Many of the adolescents stated that they would seek advice from a parent or physician when deciding whether or not to accept the HPV vaccine. Interestingly, we found no significant association between having had the HPV vaccine or willingness to receive the vaccine and the level of education of the adolescent's parents. There was also no association between having younger or older siblings who had been vaccinated and having had the HPV vaccine or willingness to receive the vaccine. Many studies have shown that most parents are willing to accept the HPV vaccine for their children. A recent study, postlicensure of the vaccine, evaluated mothers of children in primary and secondary schools in England and found that 75% of mothers would accept the HPV vaccine for their daughter. Acceptance was higher in mothers who had experienced cancer in their families, had older daughters, perceived approval from their husbands/partners, and believed vaccine acceptance would be more normative . Another Healthcare providers are an important source of information for parents and children, and studies show that they generally have a positive attitude towards recommending an HPV vaccination. One study of 207 fellows of the American College of Obstetricians and Gynecologists found a fairly positive disposition towards recommending the HPV vaccine, with a mean rating for vaccine recommendation of 79 out of 100 . Similar Previous studies have shown that knowledge of personal risk is generally limited with regard to sexually transmitted infections. A study of sexually active heterosexual college students found that personal risk of contracting Acquires Immune Deficiency Syndrome was estimated to be significantly lower than risk for each of a set of hypothetical persons who varied in degree of similarity to respondents . In a st Study limitations should be considered when interpreting the present findings. Participants were recruited from a hospital-based adolescent health clinic serving a racially and ethnically diverse, predominantly low-income, population. As a result, the findings may not be applicable to all adolescents in the United States. In order to include 13 and 14 years old in this study at an adolescent health clinic, we were unable to ask certain direct questions, including “Are you sexually active?”. The answer to this question may have provided more information regarding age of sexual activity in the current study population and would allow direct comparison of these results with a personal risk perception question. In addition, because the time frame was not stated explicitly in the question regarding personal risk, it is unclear whether participants were rating their risk of HPV infection at present, in the immediate future, or over their lifetime, which would also affect the accuracy of their ratings. It is clear that education will play an important role in the implementation of an HPV vaccination program. In this study, only 53% of adolescents who had not yet received the HPV vaccine were willing to be vaccinated, and lack of education likely contributes to this hesitancy. Healthcare providers must be proactive in educating and discussing benefits of vaccination. Based on factors affecting vaccine acceptability, HPV vaccine programs in the United States should emphasize high vaccine effectiveness, the high likelihood of HPV infection, physician recommendations, and address barriers to vaccination. It is important to educate patients that the vaccine is most effective prior to sexual activity and HPV exposure. However, the vaccine does not replace routine screening for cervical cancer or the need for education about continued cervical cancer screening. Thus, it is essential for healthcare providers to offer guidance about the ongoing need for screening. Future studies will be necessary to evaluate public health issues that arise once the HPV vaccination programs have been established more extensively.


Plasmodium parasites degrade hemoglobin resulting in ferriprotoporphyrin (IX) accumulation; toxic to the parasite [During the intra-erythrocytic stage, parasite .in-vitro studies [β- Hematin, a synthetic polymer made from ferriprotoporphyrin- IX is structurally, chemically and spectroscopically identical to purified hemozoin is used in  studies .in-vitro inhibition of β-hematin [Salvia officinalis).Resistance to Chloroquine, highlights the need for new drugs. Earlier attempts showed the effect of pyrimidine derivatives in -hematin , and cis-hematin . We concPlant materials, collected from areas around Jerusalem, were dried at room temperature; leaves and stems separately grinded. Extraction was by soaking 5gm of dried plant parts in 40 ml of 35% ethanol or ultrapure -water; left standing for about 24-hours. Extracts were then filtered using MN615- Φ 90 nm filter paper, rotary evaporated at 50°C then lyophilized to constant weight. Stock solutions were prepared in water.Amodiaquine for positive control. The final precipitate of β-hematin dissolved in 200µl of 0.1 M NaOH to give alkaline hematin for spectroscopic quantification at 405 nm.The procedure was according to , ultra-pThe efficiency of sage leaf extracts compared to controls is shown below. Each absorption value is the average of five experiments. The mechanism of inhibition is probably through formation of a complex between active compounds in these extracts and ferriheme; this complex prevents the formation of β-hematin.The extract is a natural product and has been used in folk medicine with no reported toxicity.Although the results for the extracts are lower than for the positive controls we must take into account the fact that the extracts are crude, we are already working on isolating the ingredients; results will be published in the near future.


Nasopharyngeal carcinoma (NPC) accounts for 0.6% of all cancers worldwide with the highest prevalence in South East Asia, Southern China and Northern Africa but the disease is uncommon in Europe with an annual incidence in this region of less than 1 per 100 000. Although the Epstein-Barr virus (EBV) is a well known causative agent in NPC, recent reports have implicated oncogenic Human Papillomavirus (HPV) in a subgroup of these tumours. The recent striking rise of oropharyngeal carcinoma has been attributed to HPV, but little is known about the prevalence and clinical significance of the virus in NPC. The aim of this study was to determine the prevalence of oncogenic HPV in NPC from tissue archives of two head and neck cancer centres in the UK.Samples were available for 67 patients with clinically validated NPC. The detection of high-risk HPV was carried out by screening all cases for p16 using immunohistochemistry and HPV DNA by polymerase chain reaction (PCR) using GP5+/6+ primers. All cases with p16 over-expression or positive for HPV by PCR were then examined by high-risk HPV DNA in-situ hybridisation and genotype analysis by PCR.Eleven cases  showed concurrent over-expression of p16 and evidence of high-risk HPV DNA by in-situ hybridisation; the majority were HPV16 positive. Of these 11 cases, nine occurred in Whites and two in Blacks. Histologically, there were two keratinising squamous cell carcinoma and nine non-keratinising carcinomas (eight differentiated and one undifferentiated). None of the HPV-positive cases showed any co-infection with EBV. There was no statistically significant difference in overall survival outcome between patients with HPV-positive and HPV-negative NPC.The results of this study show that oncogenic HPV is associated with a subgroup of NPCs and is more likely to occur in Whites. However, unlike oropharyngeal carcinoma there was no significant difference in overall survival between patients with HPV-positive and HPV-negative NPC. Nasopharyngeal carcinoma (NPC) constitutes 0.6% of all cancers worldwide but there is a wide geographic and ethnic variation in the incidence rates of the disease . For exaAlthough the Epstein-Barr virus (EBV) has long been implicated in nasopharyngeal carcinogenesis, increasing numbers of reports have identified an association of oncogenic human papillomavirus (HPV) in a sub-group of NPCs -16. WhilTherefore, the aim of this study was to determine the incidence of oncogenic HPV in patients with NPC from two head and neck cancer centres in the UK and to correlate HPV status with patient ethnicity, histological grade, EBV status and overall survival.p=0.01 Chi square) and histological subtype .Of the 67 cases included in this study, there were 6 (8.9%) keratinising squamous cell carcinomas (KSCCs), 20 (29.9%) non-keratinising carcinomas, differentiated type (NK-D), 40 (59.7%) non-keratinising carcinomas, undifferentiated type (NK-U), and 1 (1.5%) was a basaloid squamous cell carcinoma  were positive for EBV. Of these 17 (36.2%), 16 (34.0%), 12 (25.3%) and 2 (4.2%) occurred in Whites, Far Eastern Asians, Blacks and North Africans, respectively. One (2.1%) was a KSCC, 36 (76.6%) were NK-Us, 9 (19.2%) were NK-Ds, and 1 (2.1%) was a BSCC.p<0.0001, Chi square). HPV-positive NPCs were more likely to occur in Whites . Of the HPV-positive NPCs, 9/11 (81.8%) were genotyped as HPV16, 1/11 (9.1%) was HPV18 and there was insufficient amplifiable DNA in the remaining case.Eleven 16.4%) cases demonstrated p16 over-expression, all of which were assigned a maximum H score of 300  . All cas6.4% casep=0.57, log rank; Figure Patients were followed up for a period of 4–120 months . The mean overall survival for all cases was 63.1 months (95% CI 49.6-76.5) whereas the mean overall survival for EBV-negative/HPV-negative, EBV-positive/HPV-negative and EBV-negative/HPV-positive were 47.6 months (95% CI 19.9-75.3), 67.9 months (95% CI 52.1-83.7) and 53.6 months (95% CI 18.3-88.8), respectively. There was no statistically significant difference in the mean overall survival between EBV-negative/HPV-negative, EBV-positive/HPV-negative and EBV-negative/HPV-positive patients  and has been registered with National Health Service Research and Development (RJ112/N247).Patients presenting to two UK head and neck cancer centres  were identified retrospectively from pathology databases over a period of 12 years (2000–2012). Only tumours radiologically confirmed as centred in the nasopharynx without extension into the oropharynx were included in this study. Formalin-fixed paraffin embedded (FFPE) tissue was available from 67 patients of which 48 were males and 19 females (M:F ratio 2.5:1) with a mean age of 48 years (range 12–74 years old). There were 34 Whites (50.7%), 17 Far-Eastern Asians (25.4%), 14 Blacks (20.9%), and two North Africans (3.0%). Patient and tumour characteristic are summarised in Table All cases were independently classified according to WHO histological criteria by two specialist head and neck pathologists (MR and ST) . A consep16 immunohistochemistry (IHC) was performed on all cases using a proprietary kit  on a Ventana Benchmark Autostainer . A tonsil carcinoma with high p16 expression was used as a positive control. The primary antibody was omitted from negative controls. The tests were independently scored by two specialist head and neck pathologists (MR & ST) using a binary scoring system and the recently described H score Figure  18]. Di. Di18]. DNA was extracted from 25μm sections of FFPE biopsies using the QIAamp DNA (FFPE) tissue kit  according to the manufacturer’s instructions. Contamination of samples with extrinsic DNA was minimised by cleaning the microtome with xylene and discarding the microtome blade between each case. PCR reactions were carried out using standard operating procedures to prevent sample contamination. All DNA samples were screened for the human β-globin gene by PCR to verify the presence of amplifiable DNA . DNA froEBV RNA in-situ hybridisastion (ISH) was performed using proprietary reagents  on a Benchmark Autostainer  and visualised using the Ventana ISH/iView Blue detection kit according to manufacturer’s instructions. The Inform EBER probe detects early RNA transcripts EBER-1 and EBER-2. Cells known to contain high numbers of EBER transcripts were supplied by the manufacturer and used as a positive control. An intense blue reaction product in the majority of malignant cells was considered positive. The tests were independently scored by the study pathologists using a binary classification and a consensus agreement was reached for all discordant scores.All cases over-expressing p16 were subject to high-risk HPV DNA ISH using proprietary reagents (Inform HPV III Family 16 Probe (B), Ventana Medical Systems Inc, USA) on a Benchmark Autostainer . The Inform HPV III Family 16 Probe (B) detects high-risk genotypes HPV-16, -18, -31, -33, -35, -39, -45,-51, -52, -56, -58 and −66. Three control samples were used: FFPE CaSki cells , HeLa cells  and C-33A . The high-risk HPV ISH test was scored as positive if there was any blue reaction product that co-localised with cell nuclei. Diffuse nuclear and cytoplasmic  staining and punctate nuclear (‘integrated’ pattern) staining were scored as positive . DNA yield was measured using a Nanodrop spectrophotometer . 200ng of DNA was diluted in 1000 μl of Surepath fluid  and the samples were analysed using the Cobas HPV test on the Cobas 4800 instrument (Roche Molecular Systems Inc. USA).P values below 0.05 were regarded as significant. All data was analysed using SPSS software version 20.The Chi-squared test was used to evaluate the correlation between different groups. Overall survival was defined as the time from diagnosis to the date of death or last follow up. The Kaplan-Meier method was used to estimate time to event curves, and the survival curves were compared using the log-rank test. EBV: Epstein-Barr virus; HPV: Human papillomavirus; IHC: Immunohistochemistry; ISH: In-situ hybridisation; NPC: Nasopharyngeal carcinoma; PCR: Polymerase chain reaction.The authors declare that they have no competing interests.MR was involved in the conception of the study, identified the cases, undertook histological evaluation, carried out genotype analysis, participated in the design and coordination and helped to draft the manuscript. YS undertook DNA extraction, data collection from treatment records and performed statistical analyses. VP undertook data collection and was involved in drafting and critical evaluation of the manuscript for intellectual content. DD performed immunohistochemistry and in-situ hybridisation assays. BA performed PCR assays. LP supported immunohistochemistry and in-situ hybridisation assays. ST was involved in the conception of the study, identified the cases, undertook histological evaluation, participated in the design and coordination and drafted the manuscript. All authors read and approved the final manuscript.


However, since not all patients with HPV+TSCC respond to treatment, additional biomarkers are needed together with HPV status to better predict response to therapy and to individualize treatment. For this purpose, we examined whether the number of tumor infiltrating cytotoxic and regulatory T-cells in TSCC correlated to HPV status and to clinical outcome.Human papillomavirus (HPV) is a causative factor for tonsillar squamous cell carcinoma (TSCC) and patients with HPV positive (HPV+) and regulatory (Foxp3+) T-cells.Formalin fixed paraffin embedded TSCC, previously analysed for HPV DNA, derived from 83 patients, were divided into four groups depending on the HPV status of the tumor and clinical outcome. Tumors were stained by immunohistochemistry and evaluated for the number of infiltrating cytotoxic (CD8+ T-cell infiltration was significantly positively correlated to a good clinical outcome in both patients with HPV+ and HPV- TSCC patients. Similarly, a high CD8+/Foxp3+ TIL ratio was correlated to a 3-year disease free survival. Furthermore, HPV+TSCC had in comparison to HPV−TSCC, higher numbers of infiltrating CD8+ and Foxp3+ T-cells.A high CD8+ cells and clinical outcome indicates that CD8+ cells may contribute to a beneficial clinical outcome in TSCC patients, and may potentially serve as a biomarker. Likewise, the CD8+/Foxp3+cell ratio can potentially be used for the same purpose.In conclusion, a positive correlation between a high number of infiltrating CD8 This increase has been attributed to an increased prevalence of human papillomavirus (HPV) infection + and HPV−TSCC are two different tumor disease entities with different tumor and patient characteristics. Whereas HPV associated tumors, including HPV+TSCC are driven by viral oncogenes, e.g. E6 and E7, and have fewer cellular mutations, HPV−TSCC develop by accumulation of genetic changes mainly caused by environmental factors such as smoking in vitro studies have shown that T-cells isolated from HPV+ cervical cancer can recognize and kill E6 and E7 expressing tumor cells + lymphocytes has been linked to a favorable prognosis in many malignancies, the role of infiltrating T regulatory cells (Tregs) (often defined as Foxp3+ lymphocytes) is often the reverse + and Foxp3+ lymphocytes has, to our knowledge, not been investigated in TSCC in correlation to patient outcome and HPV status.It has been suggested that HPV+ and Foxp3+ T-cells in TSCC in relation to clinical outcome and tumor HPV status, and to establish if these TILs can potentially be used in the clinic as biomarkers either alone, or together with the HPV status of the tumor to predict clinical outcome.The purpose of this study was to examine the presence of tumor infiltrating CD8The study was approved by the Ethical Committee at Karolinska Institutet, Stockholm, Sweden, according to the ethical permissions 2005/431-31/4, 2005/1330-32 and 2009/1278-31/4. Written informed consent was obtained from all participants in the study.The cohort consisted of all patients, diagnosed 2000–2006 with TSCC (ICD-10 C09.0-9), that were treated with the intention to cure at Karolinska University Hospital, Stockholm, Sweden, and had available pre-treatment paraffin embedded tumor biopsies. Treatment consisted of accelerated radiotherapy (RT) or conventional RT  or, in four cases, induction chemotherapy followed by concomitant RT (CRT). After treatment, patients were followed-up by clinical examination every 3 months during the first 2 years and every 6 months the 3rd year.INK4a status (by immunohistochemistry) were obtained from previous studies +, p16INK4a positive (>75% p16INK4a positive tumor cells) (p16+) tumors with a good clinical outcome ; (B) all patients (n=21) with HPV+, p16+ tumors with a poor clinical outcome (defined as relapsed in disease and/or dead of disease within 3 years after diagnosis); (C) all patients (n=11) with HPV−, p16INK4a negative (p16−) tumors with a good clinical outcome; (D) all patients (n=20) with HPV−, p16− tumors with a poor clinical outcome  and HPV−, p16+ tumors (n=4) were excluded from further analysis to obtain homogenous groups and to only include HPV+ TSCC expressing HPV E6/E7, since p16INK4a expression in HPV+ tumors is considered an indication of E7 expression + TSCC were HPV16+ with exception of one tumor with HPV33 and one with HPV56.Data on the presence of high risk HPV types (presence of HPV DNA by PCR) and p16 outcome . A samplA standard streptavidin-biotin peroxidase method was employed on 4 µm formalin fixed, paraffin embedded, sections using the mouse monoclonal antibodies anti-CD8  and anti-Foxp3 . All sections were subsequently incubated with biotinylated secondary anti-mouse antibody  followed by incubation with the avidin-biotin-complex-PO using the VECTASTATIN® Elite® ABC kit (Vector Laboratories) and developed in DAB.+ and Foxp3+ tumour lymphocyte infiltration was evaluated by two researchers, blinded for clinical outcome, each counting the cells in 10 randomly selected high-power fields (40×) per sample. The mean value was reported for each tumor. The ratio of tumor infiltrating CD8+ and Foxp3+ cells was calculated for each individual tumor, the mean of these ratios was then calculated per subgroup.The CD8Tests for comparisons of continuous data were performed using linear regression models. Results were presented as mean differences or odds-ratios together with 95% confidence interval. The comparisons of patient and tumor characteristics between the different groups in Disease-free survival (DFS) was defined as time from the date of diagnosis to the date of the last known occasion that the patient was disease-free or the date of disease recurrence . Death without documented recurrence was censored at the date of death. Kaplan-Meier curves were used to present survival data and the log-rank test was used to compare survival curves. In the multivariate analyze, a Cox proportional hazards model was used to adjust for covariates. Two-sided p-values were reported. These analyses were performed in SPSS .−tumors and good clinical outcome had a lower clinical stage than patients with poor clinical outcome (p=0.005).Details of all patients and their tumors are shown in + TSCC and good clinical outcome , .TSCC patients with a good clinical outcome had a significantly higher number of CD8r tumors , than thr tumors . Further=0.002), . The mea+TSCC had a significantly higher mean number of tumor infiltrating CD8+ lymphocytes compared to HPV−TSCC   . This di+ TILs and DFS in HPV+ TSCC patients, a Kaplan-Meier analysis was performed. HPV+ and HPV-TSCC patients were dichotomized based on the median value (33 and 4.4 respectively) of CD8+ TILs. As demonstrated in + tumors and a high number of CD8+ TIL had a cumulative survival of 84% compared to 55% for patients with low number of CD8+ (p=0.02). Similar cumulative survival trends  were also observed for HPV- patients, although these differences were not statistically significant   (A significantly higher number of Foxp3+ TILs  were obsp<0.001) . When ad, resp.) .+ and Foxp3+ cells was calculated for each tumor. A high CD8+/Foxp3+ cell ratio is an indication that the regulatory T-cells are in a minority and thus less likely to overshadow the function of cytotoxic T-cells. For patients with TSCC a high CD8+/Foxp3+ ratio was positively correlated to a good clinical outcome when adjusted for prognosis (p=0.04)  . However+/Foxp3+ TIL ratio and DFS for TSCC, a Kaplan-Meier analysis was performed. HPV− and HPV+TSCC patients were dichotomized based on a CD8+/Foxp3+ TIL ratio above or below 1. As demonstrated in −TSCC and a high CD8+/Foxp3+ TIL ratio had a cumulative survival of 73% as compared to 28% for patients with a low CD8+/Foxp3+ TIL ratio (p=0.03). Similarly, HPV+TSCC patients with a high CD8+/Foxp3+ TIL ratio had a higher cumulative survival (82%) than those with a low CD8+/Foxp3+ TIL ratio  (To study the correlation between a high/low CD8 p=0.04) .+ and Foxp3+ TILs values for all TSCC, stratified for HPV status, were analyzed for age, sex or clinical stage, no correlation was found (data not shown).When CD8+ and HPV−TSCC, using both univariate and multivariate tests (+TSCC the hazard ratio for a high number of CD8+ cells was 0.27 (p=0.029), while for HPV−TSCC the hazard ratio for a high CD8+/Foxp3+ ratio was 0.27 (p=0.048). To determine whether CD8+ cells and CD8+/Foxp3+ ratio were independent prognostic factors, a multivariate Cox analysis was performed with clinical stage, age and sex as covariates . For HPV−TSCC both CD8+/Foxp3+ ratio and stage was significantly independently correlated to 3-year DFS . Similarly, a high CD8+/Foxp3+ ratio correlated to a 3-year DFS both in the univariate and multivariate analyzes also for HPV+TSCC  (data not shown).Hazard ratios (HR) were calculated separately for HPVte tests . For HPVvariates . For HPV+ T-cell infiltration and clinical outcome for both patients with HPV+ and HPV−TSCC, as well as between a high CD8+/Foxp3+ TIL ratio and disease free survival for both patients with HPV+ and HPV−TSCC. In addition, we demonstrated a correlation both between tumor infiltrating CD8+ and Foxp3+ T-cells to HPV status in TSCC.In this study, we found a significant correlation between a high CD8+ infiltration was more pronounced and influenced prognosis positively in this study for HPV+TSCC, may be an important reason for why the majority of HPV+TSCC have a good clinical outcome in comparison to HPV−TSCC + head and neck squamous cell carcinoma (HNSCC) +, both a high number of CD8+ TIL and a high CD8+/regulatory T-cell ratio were correlated to the absence of lymph node metastasis + T-cell infiltration and cause specific survival + HNSCC was recently demonstrated, although correlation to prognosis was not investigated + TILs and good clinical outcome for patients with TSCC indicates that assessment of CD8+ TILs could be used as an additional prognostic biomarker in combination with tumor HPV status.The fact that CD8+/Foxp3+ T-cell ratio significantly correlated to a disease free survival in both patients with HPV+ and HPV−TSCC. Previous publications have linked Foxp3+ TILs to a worse prognosis in e.g. ovarian and breast cancer + TILs have been linked to a favorable prognosis such as e.g. in colon cancer, Hodgkin lymphoma and HNSCC −TSCC, although possibly to a lesser extent. The assessment of CD8+/Foxp3+ T-cell ratio could thus also be used as a clinical prognostic marker for patients with HPV+ or HPV−TSCC.We also demonstrated that a high CD8+ and Foxp3+ TILs by IHC can be performed at pathology units in hospitals. The HPV status of TSCC is already assessed routinely in some clinics, e.g. at the Karolinska University Hospital in Stockholm, and if used in conjunction with evaluation of CD8+ and Foxp3+ TILs in pathology units, a better prediction of clinical outcome may be obtained. However, the size of the field evaluated and cut-off values have to be precisely defined.Importantly, estimating CD8+TSCC had a significantly higher infiltration of both CD8+ and Foxp3+ cells than HPV−TSCC. This was not unexpected given that HPV+TSCC expresses foreign viral antigens. In fact, the data indicate that, although the immune defense has failed to impede the development of the HPV+TSCC, there is still an immunological difference between HPV+ and HPV−TSCC.In addition, we observed that HPVIt is important to note that the presence of TILs in TSCC is not enough for tumor rejection. However, it has been demonstrated in a mouse model that an intact immune defense is important for a complete tumor clearance upon radiation treatment Recently, many tumor biomarkers have been explored with regard to prognosis for patients with HNSCC There are some limitations in the present study. Firstly, it is a retrospective study with all patients diagnosed with TSCC in Stockholm during 2000–2006 and treated with intention to cure. However, it is important to note that treatment during this period was standardized with conventional/accelerated RT. In addition, previous publications, analyzing the clinical outcome of the patients included in the present study, have failed to demonstrate differences in clinical outcome for patients with oropharyngeal cancers depending on treatment + with a favorable clinical outcome for the patients. Hence, this group was reduced in the present study by randomization. On the other hand, we were not able to demonstrate any significant differences between the study sample and the total material in this group. Another limitation is the few numbers of patients in the HPV− groups, although all patients were included with this feature. To better study the HPV−TSCC population, other medical centers should be included.Secondly, most TSCC today are HPV+ and Foxp3+ cells are targeted against specific tumor associated antigens or assayed for functional CD8+ using e.g. granzyme B. Furthermore, since Foxp3, although at low levels, may be transiently induced in CD4+ and CD8+ T-cells upon stimulation, all Foxp3+ TILs may not be Tregs Finally, we have not assessed if the tumor infiltrating CD8+ and Foxp3+ cells in TSCC display different profiles depending on tumor HPV status and clinical outcome. Thus, combining the presence of CD8+ T-cells and/or CD8+/Foxp3+ cell ratio with HPV status, may be of additive prognostic value and for better individualizing patient treatment.In conclusion, tumor infiltrating CD8


For HPV 16, 9 variants with nucleotide variations were included into the study. For HPV 31, 33, 18 and 45, nucleotide variations were identified in 6, 4, 2 and 3 variants, respectively. The Bayesian inference and Maximum Parsimony methods were used in order to construct the phylogenetic trees. When types were analyzed independently HPV 16 (European and non-European) and HPV 18 (African and non-African) formed distinct clades. The genomic characterization of HPV variants will be important for illuminating the geographical relatedness and biological differences and for the determination of their risk.The present study explores nucleotide variability, phylogeny and association with cervical neoplasia in high risk HPV types 16, 18, 31, 33 and 45 collected from Greek women. Of the 1894 women undergoing routine cervical cytology examination, 160 samples test positive for single infections of HPV type 16 ( Human papillomavirus 16 or Human papillomavirus 18 [et al. [Papillomaviridae has been expanded and contains 29 genera formed by 189 papillomavirus (PV) types isolated from humans (120 types), non-human mammals, birds and reptiles . According to de Villiers et al. [et al. [Persistent infection of oncogenic HPV types is acknowledged as a major risk factor for the development of cervical carcinoma ,2. Most virus 18 . Accordi [et al.  the class et al.  genomic s et al. . The ICT [et al.  define vAnalysis of HPV variants diversity worldwide is of great importance as the HPV sequence data bases which are created, are useful for epidemiological and evolutionary studies, for the development of accurate diagnostic tests and for efficient vaccine design. The purpose of the present study was to analyze for the first time in Greece the intratype variability of 160 cervical samples positive for HPV 16, 18, 31, 33 and 45 in order to add information on the circulation of HPV variants in Greece.L1 (MY9/11) region was undertaken in 160 positive samples with single infections: 104 for HPV 16, 40 for HPV 31, 7 for HPV 33, 5 for HPV 18 and 4 for HPV 45. According to cytological diagnosis, samples were grouped into five categories: within normal limits (WNL) , Atypical Squamous Cells of Undetermined Significance (ASCUS) , Low grade lesions (LGSIL) , High grade lesions (HGSIL)  and Squamous Cell Carcinoma .Molecular characterization of HPV 16, 31, 33, 18 and 45 variants within the L1 gene (MY9/11 region). Of the 104 isolates of HPV 16 sequenced, 9 representative isolates were selected, because they contained at least one unique nucleotide sequence variation in comparison to other isolates of the same type. The majority of variants (93%) were European and among these the sequence of HM596520 was the most frequent (84.6%)  . From th changes . The iso created . Figure 4.6% . FrL1 (MY9/11) gene and 6 variants were found. The most frequent isolate HPV31 HM596540  . Limited665C . Liet al.  and HPV ®) sample was taken following consent for study participation. The cytologic specimens were originated from different regions all over Greece. In order to explore the intratype phylogeny of the five most oncogenic HPV types, 104 sequences were used for HPV 16, 40 for HPV 31, 7 for HPV 33, 5 for HPV 18 and 4 for HPV 45.All HPV positive samples already typed with single infections for HPV types 16, 18, 31, 33 and 45 were selected from the HPV DNA bank of the Cytopathology Department. A total of 1894 women undergoing routine cervical cytology examination joined the present study that took place in “Attikon” General University Hospital from April 2009 until December 2010. A total of 943 women were negative for HPV. Of the 951 positive samples 448 were found to have multiple infections either with high or low risk HPV types. Of the remaining 503 with single infections, 183 cervical brush specimens were used in the present study as they had single infections with HPV 16, 18, 31, 33 and 45. Of the 183 samples which were positive for single infection, the 160 were both and PCR amplified and sequenced and included in the present study. A liquid-based cytology . Briefly, 1 mL of homogeneous sample was placed to a sterile 1.5 mL microcentrifuge tube and centrifuged for 10 min at 12,000 rpm in order to be in a pellet form. The supernatant was discarded and the pellet was resuspended with 1 mL of sterile water and centrifuged for 10 min at 12,000 rpm. DNA extraction method was followed by adding 180 μL of lysis buffer T1 and 25 μL of proteinase K into the precipitate and incubating the sample in a thermomixer at 56 °C for 1 h and 550 rpm. At the end of DNA extraction method, 100 μL of eluted DNA recovered and stored at −20 °C.DNA was extracted, amplified and analyzed using microarrays method, by CLARTL1 (PGMY9/11) region of the virus. Elimination of false negative results and PCR failure due to several PCR inhibitors present inside the sample, was achieved by using two internal controls: (a) genomic DNA control  control of the amplification reaction (a pair of primers that amplify a 1202 bp fragment of modified plasmid). Hybridization of the amplified PCR product with immobilized matching type-specific DNA probes took place on a low-density microarray tube . The data obtained in each analysis were processed automatically by the system. CLART® HPV 2 kit allowed the detection of infection and co-infection of 35 different HPV genotypes, 20 HR-HPV: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 & 85 and 15 LR-HPV: 6, 11, 40, 42, 43, 44, 54, 61, 62, 71, 72, 81, 83, 84 & 89.Five microliters of eluted DNA were used for the PCR amplification. HPV DNA amplification was achieved by using biotinylated primers designed to amplify a 450 bp fragment within the highly conserved a ≈ 380-bp HPV-specific segment from the L1 (MY9/11) ORF was performed using the sense primer MY09 (nt 6584) 5′-CGTCCMARRGGAWACTGATC-3′ and the reverse primer MY11 (nt 7035) 5′-GCMCAGGGWCATAAYAATGG-3′. Five microliters of DNA were added to 45 μL of reaction mix containing 1× PCR buffer, 0.2 mM dNTPs, 4 mM MgCl2, 0.3 μM of each primer, and 0.025 U/μL of AccuPrime™ Taq DNA Polymerase high Fidelity (Invitrogen). The cycling conditions were as follows: 94 °C for 1 min, 1 cycle; then, 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s, for 35 cycles using the Eppendorf Mastercycler personal thermocycler . This region of L1 gene corresponds in the HPV 16 genome to nucleotides (nt) 6589 to 6969, in the HPV 31 genome to nucleotides (nt) 6509 to 6865, in the HPV 33 genome to nucleotides (nt) 6546 to 6925, in the HPV 18 genome to nucleotides (nt) 6559 to 6944, in the HPV 45 genome to nucleotides (nt) 6577 to 6941. PCR products were subjected to electrophoresis in 1% agarose (Applichem) using 1× TBE buffer (Applichem) and visualized under UV light.Amplification of L1 (MY9/11) gene determined for this study have been deposited in GenBank under the following accession numbers: of HPV 16: HM596508-HM596522, of HPV 18: HM596525, HM596526, of HPV 33: HM596527-HM596530, of HPV 45: HM596531-HM596534 and of HPV 31: HM596535-HM596540.PCR products were purified using commercially available spin columns  and then they were sequenced directly via automated sequencing using both forward and reverse PCR primers. The sequences of L1 (MY9/11) sequences obtained in this study. The alignment and the correction of sequences of L1 gene were done by using the Codon Code Aligner program (v.3.5.2.). Analyses for phylogenetic inference were conducted using three methods: Neighbor-Joining (NJ) Maximum Parsimony (MP) and Bayesian Inference (BI). Nucleotides were used as discrete, unordered characters. Neighbor-joining (NJ) trees were constructed using Kimura two-parameter correction methods of MEGA 4 [et al. 1996 [et al. [et al. [Phylogenetic trees for HPV 16, 31, 33, 18 and 45 isolates were constructed using the variants of f MEGA 4 . To assef MEGA 4 , with hef MEGA 4 . The Bayf MEGA 4  applyingf MEGA 4 . In ordef MEGA 4  was usedf MEGA 4 . In the f MEGA 4  to GenBaL1 MY9/ sequenceL1 MY9/ sequenceL1 genomic region was performed in 160 positive samples with different cytological diagnosis. The viral persistence and the development of high grade lesions associated with specific HPV variants has been the objective of many studies worldwide as genome variations may be relevant to virus infectivity and pathogenicity [In the present study, molecular characterization of HPV 16, 18, 31, 33 and 45 variants within the MY09/11 genicity –23. Thisgenicity –28.HPV 16 prototype (K02718) was isolated from a German cervical cancer patient and was the first one completed sequenced . Based ox2 = 0.036, P < 0.05). Also in 6/6 cases of SCC and in 81.7% of LGSIL included in this study the European (HPV16 HM596520) variant was detected. HPV 31 is considered the fourth most common HPV type in squamous cervical carcinomas and in asymptomatic patients worldwide [L1 (MY9/11) genomic part revealed that the HPV 31 variants detected to circulate in Greece are clustered into 3 different lineages according to Chen [Few studies are available on epidemiology of HPV in Greece –40 and tThe molecular analysis of HPV 33 detected 4 nucleotide substitutions and 1 non-synonymous amino acid change. One of the four nucleotide substitutions a A-to-G at position 6673 was not previously published and was detected in isolate HM596529 making this variant most divergent from the others .Human papillomavirus type 18 (HPV 18) and HPV 45 account for approximately 20% of all cervix cancers . Severalet al. 1996 [et al. 2009 [et al. [In our report, only 5 samples with HPV 18 single infections were collected and 2 variants came up. All 7 nucleotide substitutions and the non-synonymous nucleotide change arose were previously published ,23,48. Tal. 1996  and otheL1 single nucleotide changes were identified. Limitation of the present study should be taken under consideration, as the sample population of HPV 33, 18 and 45 was rather small due to the small number of single infections included in our HPV DNA bank. The phylogenetic analyses were done in an extensive way, using several different approaches. These analyses confirmed the observation that MY part of the HPV L1 gene is informative enough to phylogenetically evaluate high-risk HPVs. Moreover, phylogenetic analyses of Greek isolates and isolates originating from other parts of the world confirmed the previous observations of geographical clustering of HPV 16 and HPV 18 isolates. Finally, it is considered to be of national importance that 84.6% of the samples of HPV16 belong to European variant (HM596520).This is the first Greek study of nucleotide distribution and phylogenesis of HPV 16, 18, 31, 33 and 45, giving important information as 8 new