To evaluate the advantages of cytology and PCR of high-risk human papilloma virus (PCR HR-HPV) infection in biopsy-derived diagnosis of high-grade squamous intraepithelial lesions (HSIL = AIN2/AIN3) in HIV-positive men having sex with men (MSM).This is a single-centered study conducted between May 2010 and May 2014 in patients  recruited from our outpatient clinic. Samples of anal canal mucosa were taken into liquid medium for PCR HPV analysis and for cytology. Anoscopy was performed for histology evaluation.vs. 75.7%) in HSIL screening, with similar positive (PPV) and negative predictive value (NPV) of 20.3 vs. 22.9 and 89.7 vs. 88.1, respectively. Combining both tests increased the sensitivity and NPV of HSIL diagnosis to 100%. Correlation of cytology vs. histology was, generally, very low and PCR of HR-HPV vs. histology was non-existent (<0.2) or low (<0.4). Area under the receiver operating characteristics (AUROC) curve analysis of cytology and PCR HR-HPV for the diagnosis of HSIL was poor (<0.6). Multivariate regression analysis showed protective factors against HSIL were: viral suppression , and/or syphilis infection . HSIL risk was associated with HPV-68 genotype .Anoscopy showed 33.8% were normal, 47.8% low-grade squamous intraepithelial lesions (LSIL), and 18.4% HSIL; 80.2% had HR-HPV. PCR of HR-HPV had greater sensitivity than did cytology (88.8% When cytology and PCR HR-HPV findings are normal, the diagnosis of pre-malignant HSIL can be reliably ruled-out in HIV-positive patients. HPV suppression with treatment protects against the appearance of HSIL. The past few decades have produced an increase in the incidence of anal squamous cell carcinoma (ASCC) due to the increase in risk groups such as men having sex with men (MSM) and immune-depressed individuals especially those infected with HIV . ASCC isThe mucosa of the anal canal has characteristics in common with the uterine cervix, among which is the high susceptibility to the oncogenic effect of HPV . In studCost-effective analysis of implementing a protocol of early diagnosis of dysplastic lesions of the anal mucosa in HIV-positive MSM patients was evaluated by an expert committee , and theHence, we conducted the current study to analyze the role of PCR of HR-HPV in the screening of HSIL in anal mucosa of HIV-positive MSM patients. The secondary objective was to identify the variables associated with the appearance of these lesions.Hospital Universitario Virgen de las Nieves  the ethics committee of which approved the study.This is a prospective cross-sectional study, conducted between May 2010 and May 2014, in a group of 201 HIV-positive MSM patients recruited consecutively into a program of screening, diagnosis, treatment and follow-up of dysplastic lesions of the anal mucosa. The HIV-positive patients were from among those receiving attention in the Infectious Disease Unit of the The inclusion criteria were: adult (≥18 years of age) HIV-positive MSM patients. The exclusion criteria were: females, heterosexual HIV-positive males, and history of anal canal neoplasia at the time of recruitment into the study.The objectives and conditions of the study were explained to the patients at the first clinical visit, and fully-informed written consent to participation in the study was obtained. Clinical, epidemiological and analytical data obtained during the study were codified for anonymity, according to the laws on protection of personal information currently in existence in Spain. All details of the methodology are contained within the manuscript. However, more information can be had from the corresponding author, if needed.The clinical characteristics and laboratory work-up have been described previously .At the clinical visit, 2 mucosa samples were taken from the anal canal with cotton swabs soaked in physiologic saline, and stored in liquid medium for the detection and genotyping of the HPV using the polymerase chain reaction (PCR) technique , and for cytology using the ThinPrep Pap Test . Both samples were sent to the anatomy-pathology laboratory where the same senior pathologist of the research team (JE) carried out the cytology evaluation, validation of PCR methodology, and histology analyses. HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51–53, 56, 58, 59, 66, 68, 73 and 82 were considered high-risk (HR-HPV). Genotypes 6, 11, 34, 40, 42–44, 54, 55, 57, 61, 70–72, 81, 83, 84 and 89 were considered low-risk (LR-HPV). The virus was classified on HPV-18 species as genotypes 39, 45, 59, 68; and of the HPV-16 species as genotypes 31, 33, 35, 52, 58, 67 .Subsequently, anoscopy was performed by the gastroenterologist specialist of the research team (MLdeH), within an interval of between 4 and 12 weeks from the cytology evaluation. A standard endoscope of 9 mm with working channel of 2.8 mm was used, without any image enhancement. A short exploration of 15–20 cm was performed with retrovision maneuver to better visualize the pectinea line. Samples were taken for histology examination using an endoscopic retrograde cholangio-pancreatography (ERCP) catheter. Sampling was from all four quadrants  to access not only the lesions (acetolugol-negative zones) but also ostensibly-normal mucosa. This was to avoid false negatives in anoscopy without amplification system.The cytology classification was that of Bethesda  which clin situ), and invasive carcinoma  . IncludeA value of p<0.05 was considered statistically significant in all the analyses. The statistical software used was the SPSS (version 15.0).There were 201 patients included in the study. Mean age was 37 years. The CD4 nadir was 362 cells/μL, median time of clinical evolution of the HIV infection was around 36 months (interquartile range IQR: 15.3–106.8); 86.6% were on combination ARV therapy over a median period of 26.5 months, at which time there were 724.9 cells/μL CD4, and 71.1% had viral load (VL) <50 copies/mL. The characteristics are summarized in In the anal cytology, we observed that only 38.4% of the patients had normal cytology, the rest having some grade of dysplasia; 51.1% had LSIL, 4.5% HSIL and 6.1% had ASCUS.Also, 80.2% (95%CI: 65–85) had high-risk HPV genotype, 63.5% (95%CI: 50–73) low-risk genotype and 52.3% (95%CI: 33–56) both genotypes. The most-frequently isolated HPV genotypes in anal mucosa of low risk were: genotype 6 (17.3%), genotype 11 (14.7%) and genotype 61 (10.2%); and of high risk were: genotype 16 (31.5%), genotype 18 (14.7%), genotypes 42, 51 and 59 each at 14.2% and 55 and 68 each at 11.2%. Of the 201 anoscopies performed, only 33.8% had normal histology. The rest had some grade of dysplasia, of which 18.4% were HSIL (AIN2 or 3), and 47.8% LSIL (AIN 1) .The sensitivity, specificity, PPV, and NPV of the anal cytology for the diagnosis of HSIL was 75.7% (95%CI: 0.62–0.9), 41.6% (95%CI: 0.34–0.49), 22.9% (95%CI: 0.15–0.3) and 88% (95%CI: 0.81–0.95), respectively. The corresponding values for PCR of HR-HPV were 88.8% (95%CI: 0.79–0.99), 21.8% (95%CI: 0.15–0.28), 20.3% (95%CI: 0.14–0.27) and 89.7% (95%CI: 0.8–0.99), respectively. Combining both tests  the corresponding values of Sen, Spe, PPV and NPV were 100%, 16.2%, 21.5% and 100%, respectively .The probability of HSIL (AIN 2/3) diagnosis in the case of normal cytology was 11.8%; for dysplastic cytology was 22.9%; for PCR of HR-HPV was 20.3%. In the case of normal cytology and absence of HR-HPV, the probability of HSIL was zero.Employing the Kappa Index (KI), we observed significant correlation between abnormal cytology and presence of LSIL  and HSIL  in anal histology, and strong negative correlation (i.e. disagreement) in case of normal cytology and normal histology  .Correlation of PCR HR-HPV and anal histology showed a borderline significant association with the presence of LSIL , and non-significant with HSIL  .The degrees of concordance between the histology findings and the abnormal cytology and PCR HR-HPV were poor in both cases with an area under the receiver operating characteristics (AUROC) curve <0.5 . In case of normal cytology and normal histology, AUROC curve was 0.67 (95%CI: 0.593–7.57) and, in the case of normal cytology without oncogenic genotypes, was 0.616 (95%CI: 0.528–0.704) i.e. in both cases there as good concordance .In the bivariate analysis of the different factors associated with the appearance of HSIL, risk variables observed were: replication of the HIV in plasma with a VL ≥1000 copies/mL (p = 0.029), the presence of genotype HPV-68 (p = 0.01), and HPV-61 associated with other oncogenic virus (p = 0.04). Protective factors observed were: receipt of ARV therapy (p = 0.001), viral suppression , and a longer time since HIV diagnosis (p = 0.001) .Finally, in the logistic regression analysis evaluating the appearance of HSIL, the risk factors identified were: the presence of HPV genotype 68 , the prevalence of which was around 11.2%. The factors protective against the appearance of these lesions were: VL <50 copies/mL , and having had a previous diagnosis of syphilis .vs. 75.7%), with low specificity in both cases (21.8 vs. 41.6%), and similar levels of PPV (20.3% vs. 22.9%) and NPV (89.7% vs. 88.1%). When combining both tests, the sensitivity and the NPV reached 100%. This would preclude the need for anoscopy in those patients with normal cytology and negative PCR HR-HPV. Our findings corroborate the results published previously by other authors proposing the advantage of adding PCR of HPV to the anal cytology in screening for HSIL lesions. Those published results, which are similar to ours, show a low specificity and low PPV but high sensitivity and NPV reaching 100% [In our cohort of HIV-positive MSM patients being screened for HSIL, we observed a higher sensitivity for PCR of HR-HPV than for cytology , which we believe may be due to the low specificity of this technique, and which confers a low capacity to predict the histology grade of lesion in anal mucosa. This could be explained by the type of epithelium in the anus i.e. keratinized malpighian which would tend to obscure the lesions of the anal mucosa. Contrary to the mucosa of the cervix, the anal cytology would not be useful in grading the type of lesion. These results corroborate those of previous studies in which the cytology had low correlation with the histology e.g. approximately 40–50% of the ASCUs or LSIL lesions corresponding to precursor ASCC lesions , 33. In Currently, there are authors who defend high-resolution anoscopy (HRA) as the ideal technique to screen for HSIL . We, on ASCC in HIV patients has special relevance due to its high incidence and, as well, being one of the non-AIDS defining neoplasias, most frequently identified in MSM and women with cervix pathology associated with HPV . Also, iWith respect to HPV infection of the anal mucosa, we observed that 80.2% of our patients had high-risk genotypes, 63.5% had low-risk and 52.3% had both genotypes. The most frequent HPV genotypes isolated were those of low-risk HPV-6 (17.3%), HPV-11 (14.7%), and HPV-61 (10.2%); high-risk HPV-16 (31.5%), HPV-18 (14.7%), HPV-42, HPV-51 and HPV-59 (14.2% each), and HPV-55 and HPV-68 (11.2% each). This high prevalence of anal infection by high-risk and low-risk HPV has been communicated previously in HIV-positive MSM patients, and shown to be significantly associated with abnormal cytology .In the multiple logistic regression analysis we observed, as a factor predictive of HSIL, the presence of the HPV-68 genotype  and, as protectors, viral suppression of HIV, and previous syphilis infection . HIV viral suppression was related directly with the ARV treatment, which confers a protective effect against the appearance of HSIL in our study population of HIV-positive MSM patients. In the period prior to the introduction of ARV treatment (between the years 1985 and 1996) the incidence of ASCC in HIV-positive MSM patients was observed to be around 11 cases/100000 person-years. Since 1997, coinciding with the introduction of ARV treatment, there had been a 5-fold increase in the incidence relative to the previous incidence, with levels reaching around 55 cases/100000 person-years , 14.Treponema pallidum can be a protective factor against the appearance of HSIL could be interpreted as syphilis being a surrogate marker for early diagnosis of HIV and, thus, of the infection by HPV which is encountered in an earlier phase in which the appearance of the dysplastic lesions has not, as yet, occurred (14).That previous infection with vs. 27.9; p = 0.15). We assessed whether association with other high-risk viruses correlated with the appearance of HSIL. We observed that the combination with genotypes HPV-18 and HPV-68 was statistically significantly associated with HSIL . In published studies  independently of the gender or sexual orientation, the genotype HPV-16 constitutes the most prevalent HR-HPV genotype in anal mucosa [The most frequent genotype in our study population was HPV-16 with a prevalence of 31.5%. It was observed to be distributed homogenously between patients with and those without HSIL  such that the patients who presented with this genotype in the mucosa had a 20-fold higher risk of HSIL. This genotype has oncogenic capacity belonging to the C group of the phylogenetic tree of the papilloma virus, similar to genotypes 18, 39, 45 and 59 , 40. TheIn our cohort of HIV-positive MSM patients, PCR of HR-HPV was observed to have a greater sensitivity than cytology for the screening for HSIL. The correlation between cytology and the histology findings was non-significant, and may even provide contradictory results. In the absence of the oncogenic HPV virus, and with normal cytology, the probability of having HSIL approaches zero in our cohort of patients. In the group of HIV-positive MSM patients, based on the prevalence of HSIL, we recommend, irrespective of the center in which the patient is receiving attention, a systematic screening for precursor lesions of ASCC, at least with cytology. When the cytology is normal, we suggest conducting PCR of HPV. If the PCR-HPV findings are of high-risk genotypes, anoscopy should be performed. Anoscopy must be performed always in case of abnormal cytology. Finally, we need to highlight that the principal risk factor associated with HSIL was genotype HPV-68 and, as protector, viral suppression resulting from the administration of ARV therapy.These findings warrant further investigation.


The concept of prognosis dates back to antiquity. Quantum advances in diagnostics and therapeutics have relegated this once highly valued core competency to an almost negligible role in modern medical practice. Medical curricula are devoid of teaching opportunities focused on prognosis. This void is driven by a corresponding relative dearth within physician competency frameworks. This study aims to assess the level of content related to prognosis within CanMEDS , a leading and prototypical physician competency framework.A quantitative content analysis of CanMEDS competency framework was carried out to measure the extent of this deficiency. Foxit Reader 5.1 (Foxit Corporation), a keyword scanning software, was used to assess the CanMEDS 2005 framework documents of 29 physician specialties and 37 subspecialties across the seven physician roles . The keywords used in the search included prognosis, prognostic, prognosticate, and prognostication.Of the 29 specialties six (20.7%) contained at least one citation of the keyword “prognosis”, and one (3.4%) contained one citation of the keyword “prognostic”. Of the 37 subspecialties, sixteen (43.2%) contained at least one citation of the keyword “prognosis”, and three (8.1%) contained at least one citation of the keyword “prognostic”. The terms “prognosticate” and “prognostication” were completely absent from all CanMEDS 2005 documents. Overall, the combined citations for “prognosis” and “prognostic” were linked with the following competency roles: Medical Expert (80.3%), Scholar (11.5%), and Communicator (8.2%).Given the fundamental and foundational importance of prognosis within medical practice, it is recommended that physicians develop appropriate attitudes, skills and knowledge related to the formulation and communication of prognosis. The deficiencies within CanMEDS, demonstrated by this study, should be addressed in advance of the launch of its updated version in 2015. Hippocrunction).The Principles and Practice of Medicine” published twenty-two editions between 1892 and 1988. Osler’s textbook described each disease under seven categories: etiology, presentation, pathology, diagnosis, therapy, prognosis, and complications. Osler’s prognostic formulations took into account symptoms, signs, and diagnostic criteria. Review of a leading contemporary forerunner, “Harrison’s Principles of Internal Medicine”, 12th edition, revealed that only 27% of the diseases had dedicated sections discussing prognosis[Over the past century monumental advances in diagnostic and therapeutic modalities have led to the marginalization of prognosis as a core competency. This trend is reflected by its withering presence within medical literature. The original comprehensive textbook of medicine, written by Sir William Osler 1849 to 1919), “9 to 1919The post Osler era has witnessed the relegation of prognosis to a negligible level of importance. This has occurred in tandem with decreased emphasis, if not denial, of the natural history of disease. In modern times physicians are trained and socialized with a nearly singular objective, to cure. Anything less is regarded as a failure. As a result, there exists a tendency to avoid consideration of prognosis as this would seem to promote nihilism and negativism. Thus, the need to render a prognosis has been deemed redundant, and has led to a noticeable void within medical curricula. Moreover, competency frameworks, which are the main drivers of medical curricular development scarcely touch upon topics related to prognosis. The implications of this trend for patient, family, and society are immense, not the least of which is the tendency for physicians to excessively offer late stage interventions to patients within the final phases of their lives, thus, denying them the opportunity to be managed more appropriately and effectively in a conservative palliative mode of care. In addition, when prognosis is not disclosed, treatment decisions tend to be more paternalistically driven by physicians, often associated with vested interests, rather than being truly patient-centered.foreseeing (computing and formulating the prognosis) and foretelling (disclosing and communicating the prognosis). Hence, prognosis may be viewed as both a science (foreseeing) and an art (foretelling)[At the vanguard of a fledgling renaissance for prognosis in medical practice has been Dr. Nicholas A. Christakis. His award winning book “Death Foretold-Prophecy and Prognosis in Medical Care”, published in 1999, eloquently outlines the deficiencies related to prognosis in clinical care and research together with the serious implications for patients, families and society. Dr. Christakis passionately urges that prognosis be restored as a core competency. His thesis, built upon Hippocratic principals, posits that the process of prognosis comprises two basic components, namely, telling). Thus, aThe impact of Christakis’s work is evidenced by the observation that over the past decade there has been a steep rise in the number of publications related to objective prognostic factors for both quoad vitam and quoad sanantionem. For the purposes of quoad vitam, the TNM  anatomic cancer classification system, and actA paradigm shift to a competency-based approach in medical education is the result of public outcry for improved quality, comprehensiveness, and accountability,20. One This study aims to quantify the level of content related to the domain of prognosis within CanMEDS 2005. It is hoped that by highlighting any deficiencies, attention may be focused on recognizing and restoring prognosis as a core physician competency, both from the perspective of a scholar (foreseeing) and a communicator (foretelling).This study employed the methodology of quantitative content analysis. The entAmong the 29 specialties there were 20 citations of the keyword “prognosis” and one for “prognostic”. Tables  and2. NAmong the 37 subspecialties there were 34 citations of the keyword “prognosis” and six for “prognostic” Table . GeneralTableCanMEDS 2005 is deficient in its content pertaining to the domain of prognosis. Only 26 of the 66 39.4%) combined medical specialties and subspecialties were found to have at least one citation related to the keywords prognosis and prognostic. Overall, there were only 61 citations for the keywords prognosis and prognostic. The distribution of the total citations was skewed as 13 (21.3%) were associated with the specialty of Neurology, and 14 (22.9%) were associated with the subspecialty of General Surgical Oncology. The deficiencies within CanMEDS are further emphasized by the observation that the following specialties and subspecialties had zero citations associated with prognosis despite being heavily involved in the management of terminally ill patients: Radiation Oncology, Internal Medicine, General Internal Medicine, General Surgery, Colorectal Surgery, and Nephrology. Moreover, the following specialties and subspecialties had zero citations associated with prognosis despite being heavily involved in the management of patients who are receiving rehabilitative services: Orthopedic Surgery, Physical Medicine and Rehabilitation, and Rheumatology. These findings are consistent with published data that reports more than 90% of physicians are “reluctant to make predictions” about a patient’s illness% combine. FailureMost of the keyword citations in this study were linked to the competency role of “Medical Expert” (80.3%), while 11.6% were linked with the “Scholar” role, and 8.1% were linked with the “Communicator” role. Although the CanMEDS “Medical Expert” role is an integration of the six other roles, the discussion of a complex and multifaceted issue such as prognosis requires detailed discussion under more than one competency role. Thus, competency frameworks such as CanMEDS must acknowledge that a comprehensive and effective approach to prognosis may only be achieved by articulating the need for objective computation of prognostic estimates through the “Scholar” role, together with skillful disclosure to the patient and/or substitute decision maker through the “Communicator” role. In other words, the “Scholar” role promotes “Foreseeing”, while the “Communicator” role promotes “Foretelling”. Foreseeing may be regarded as a series of scholarly activities that begin with the knowledge translation of available data on prognostic factors, instruments, and tools, followed by the computation of a prognostic estimate. Successful Foretelling is dependent on the physician being effective at “breaking bad news”. Thus, pA useful paradigm for incorporating prognosis into patient-centered decision making is shown in Figure quoad vitam and quoad sanantionem should be discussed. It is also recommended that foreseeing should be discussed within the context of the Scholar role while foretelling should be discussed within the context of the Communicator role. Ultimately, increased attention to prognosis within CanMEDS and other competency frameworks carries the potential to promote medical curricular reform, thus moving towards restoring prognosis as a core physician competency.The provision of a truly patient-centered and ethically sound approach to healthcare is dependent upon prognosis being both formulated and communicated to the patient and/or substitute decision maker. This study has demonstrated deficient levels of content related to the domain of prognosis within the CanMEDS 2005 competency framework. It is recommended that in advance of the launching of CanMEDS 2015, reforms occur that enhance content pertaining to prognosis within the outcome documents of all medical specialties and subspecialties. In order to hold true to the Hippocratic definition of prognosis as a two dimensional construct, both The authors declare that they have no competing interests.VM and PMC designed the study. PMC conducted all of the document scanning, data collation, and analysis. VM drafted the manuscript. VM and PMC read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/14/93/prepub


Some researchers in other regions have recommended human papillomavirus (HPV) vaccination to reduce risk of ovarian cancer, but not in North America, where evidence has previously suggested no role for HPV in ovarian cancer. Here we use a large sample of ovarian cancer transcriptomes (RNA-Seq) from The Cancer Genome Atlas (TCGA) database to address whether HPV is involved with ovarian cancer in North America. We estimate that a known high-risk type of HPV (type 18) is present and active in 1.5% of cases of ovarian epithelial cancers in the US and Canada. Our detection methods were verified by negative and positive controls, and our sequence matches indicated high validity, leading to strong confidence in our conclusions. Our results indicate that previous reports of zero prevalence of HPV in North American cases of ovarian cancer should not be considered conclusive. This is important because currently used vaccines protect against the HPV-18 that is active in ovarian tumors and, therefore, may reduce risk in North America of cancers of the ovaries as well as of the cervix and several other organ sites. Ovarian cancer is the most lethal gynecological malignancy, with mortality within five years exceeding 50%1Most ovarian cancers are epithelial in origin, as with HPV-caused cervical, anal, urogenital, and head-and-neck cancers. It is known that HPV does reach the ovarian epithelium, and is sometimes integrated into the genomes of epithelial ovarian cancersA recent meta-analysisConflicting reports of HPV DNA in ovarian cancers highlight a need both for samples from more patients and for additional detection methods. Here we report the results of a bioinformatics analysis performed on a large cohort (405 cases) based on a different detection method from those of the aforementioned meta-analyses -- using existing RNA sequence data of tumor transcriptomes to search for alignments of cDNA with viral reference sequences.A large set of clinical samples of ovarian cancers was collected and analyzed by The Cancer Genome Atlas Research NetworkE6 in cervical carcinomaIn cervical cancers, HPV gene expression drives oncogenesis. Specifically, the E6 and E7 proteins of high-risk HPV types are involved in cellular transformation and are consistently expressed in cervical cancers7Our positive and negative controls supported the validity of our protocol. As expected, almost all (245 of 251 = 98%) of the cervical tumor samples produced alignments to oncogenes of at least one of the 12 high-risk HPV types. No alignments to low-risk types 6 or 11 were found.Among those cervical tumor samples that tested positive for high-risk HPV oncogene transcripts, we detected 12 HPV types, all of which are considered high-risk types .We used glioblastoma tumor samples as a negative control for HPV RNA. Zero of 156 samples yielded alignments to any of the 14 HPV types we searched for. Thus, we found no evidence of any HPV type in the glioblastoma samples we tested. This also was as expected, because viruses do not normally cross the intact blood-brain barrier.Cervical tumors most often produced alignments to HPV-16. HPV-18 was the second most prevalent type, although our method was not able to reliably distinguish between HPV-18 and HPV-45 in cervical cancers, as most samples that tested positive for one of them, also tested positive for the other. This may be because they are phylogenetically closely relatedAs expected, HPV-positive cervical tumors showed higher expression of viral oncogenes than did HPV-positive associated normal tissue, but with only 3 cases for which data from both cancer and normal samples were available, the difference did not reach statistical significance .E6 or E7 genes. Four of these had concordantly aligned paired reads. Six of 405 cases constituted 1.5% of our ovarian cancer cohort . Among these HPV-positive ovarian cancer samples, all reads aligning to any HPV gene were to type HPV-18. Consequently, the non-zero prevalence of HPV-18 was significantly higher than the zero prevalence of HPV-16 . In contrast to one isolated previous report of its genomic integration in ovarian cancerAmong the ovarian cancer samples we analyzed, 6 of 405 were positive for HPV oncogene expression, with at least one read aligning to the E6 or E7 were 17, 12, 6, 4, 1, and 1. In these samples, we also found reads aligning to HPV-18 genes E1 and E2. Including all these genes, the six positive samples had totals of 63, 47, 16, 8, 2, and 1 read(s) aligning to the HPV-18 genome. Regarding concordancy of the paired end reads, among the six positive samples, there were 17 concordant alignments for HPV-18 oncogenes, including 12 for oncogene E6, and five for oncogene E7.For the six positive ovarian cancer samples, the number of reads aligning to HPV-18 oncogenes Although the prevalence of HPV transcripts in our data from ovarian tumors was not zero, with our sample sizes, its difference from prevalence in glioblastoma tumors did not reach statistical significance .p < 10−4). This may not be due to any difference between the epithelial cells of the two organs, but rather to the nature of the infecting virus, which was primarily HPV-16 in cervical tumors versus exclusively HPV-18 in ovarian tumors. Even within cervical cancers, which HPV-18 is generally accepted as causing−18).We could not determine whether ovarian tumor samples had elevated expression of HPV oncogenes relative to associated normal tissues, because RNA sequence data were not available from those normal tissues. We could, however, determine that total oncogene expression was lower in HPV-positive ovarian tumors than in HPV-positive cervical tumors  . The data were cDNA sequences from primary malignant tumors, generated by Illumina RNA-seq. The ovarian tumors included in this data set were classified as serous cystadenocarcinoma. From the NCI database at https://tcga-data.nci.nih.gov, we used the code in the tissue ID field (“TSS_ID”), to determine what institution had provided the tissue sample. In order to limit our study to North America, we excluded all samples from other regions. The North American sites where ovarian tumor samples were collected are listed in We analyzed tumor RNA transcriptomes for expression of genes of 14 different HPV types. We focused on two well-studied oncogenes of HPV, genes For validation of our methods, we used cervical tumors as a positive control for HPV detection , and glioblastoma tumors as a negative control . In total, we examined cDNA data from 251 cervical tumors, 156 glioblastoma tumors, and 405 ovarian tumors. Each sample came from a different patient. The data as originally downloaded contained duplicate entries for many ovarian samples, but these were omitted from all analyses.https://cghub.ucsc.edu). This database is updated on an ongoing basis, but we used all available sequence data as of Sept. 1, 2014. The downloads were BAM files, with notations of alignment to the human genome. Because we were interested in non-human  genes, we used only the reads that did not map to the human genome.The cDNA sequences from each tumor sample were downloaded from CGHub , maintained by the National Institute of Allergy and Infectious Diseases. Each reference sequence is available from the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov.Before analyses, to filter sequences for quality, we excluded any paired-end read sequence where the sequence for either paired read had more than 3 bases that did not have a phred quality scoreWe created a Bowtie 2 index from all the HPV types considered and then used the following Bowtie 2 command for alignment, where "HPVreferences" is the basename of the Bowtie 2 index we created: bowtie2 -x HPVreferences -1 1_unmapped.fastq -2 2_unmapped.fastq.For each tumor sample, we digitally searched for oncogene transcripts of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. All of these are considered high-risk types except for types 6 and 11Our methods were carried out in accordance with relevant guidelines and regulations. Our research protocol and our access to TCGA data was approved by the TCGA Data Access Committee. All original specimens were obtained from patients with appropriate informed consent, and with approval from the multiple relevant institutional review boards at the fourteen institutions that dealt with the human subjectsThis study was conceived and designed by J.W.P. and R.M.F. Data analysis was carried out by P.A.O. and P.R. Interpretation was performed by R.M.F., J.W.P., P.A.O. and P.R. The manuscript was written by J.W.P., P.A.O., P.R. and R.M.F. The project was supervised by J.W.P. and R.M.F.


Particle Swarm Optimization (PSO) is a meta-heuristic algorithm that has been shown to be successful in solving a wide variety of real and complicated optimization problems in engineering and computer science. This paper introduces a projection based PSO technique, named ProjPSO, to efficiently find different types of optimal designs, or nearly optimal designs, for mixture models with and without constraints on the components, and also for related models, like the log contrast models. We also compare the modified PSO performance with Fedorov's algorithm, a popular algorithm used to generate optimal designs, Cocktail algorithm, and the recent algorithm proposed by . Mixture experiments are widely used in food processing, chemical, manufacturing, agricultural, cosmetics and pharmaceutical industries. For example, , 3 and  analyzedThe aim of this paper is to introduce a popular optimization technique already widely used in engineering and computer science research and modify it to find various optimal designs for different types of mixture models. Particle Swarm Optimization (PSO) techniques have been around for more than ten years, but interestingly haven’t made much impact in statistical applications to date. Our experience reinforces the widely held findings that PSO is a very simple and powerful optimization tool. It requires no assumption of the objective function and has only a few easy-to-use tuning parameters. PSO is intriguing because even though the theory is not fully developed, its repeated successes and increasing widespread applications in various disciplines have even resulted in at least 3 journals that mainly track PSO development and applications in various fields.Section 2 provides statistical setup and briefly reviews general theory for optimal experimental designs before discussing mixture models and their optimal designs. In Section 3, we propose a PSO-based method for finding a variety of optimal designs for different types of mixture experiments. In Section 4 we show that this procedure is efficient for finding various types of optimal or nearly optimal designs for different types of mixture models, including several new models for which optimal designs are not known analytically. Section 5 compares PSO performance with the popular Fedorov-exchange algorithms for finding optimal designs and we conclude in Section 6 with a summary of the advantages of the proposed PSO method over current methods for generating optimal designs.y is the response variable, β is a d×1-vector of unknown coefficients, f(x) is a given d×1 vector of linearly independent regression functions defined on a user-defined compact design space Ω. The error ϵ has zero mean and constant variance and we assume all errors are normally and independently distributed. An approximate design ξ is defined by its design points (xi’s) and the proportions (pi’s) of observations to be taken at these points. Once the sample size n is fixed, either by cost or time considerations, and an optimality criterion is given, the problem is to determine the number (k) of points required, along with the values of xi and pi, i = 1, …, k that optimize the criterion. The implemented design takes roughly npi observations at xi, i = 1 …, k from Ω subject to np1 + … + npk = n and each npi is a positive integer.Our interest is in the general linear model given byξ is measured by its total Fisher information matrix, which is obtained by taking the negative of the expectation of the second derivative of the logarithm of the likelihood function with respect to β. For M(ξ) = Eξ(f(x)f(x)′), which is inversely proportional to the variance-covariance matrix of the estimated parameters β.Following convention, the worth of a design D-optimality for estimating model parameters seeks to minimize the generalized variance using the convex functional Φ(ξ) = −ln |M(ξ)|. Another popular and useful criterion is L-optimality defined by Φ(ξ) = trLM(ξ)−1 and L is a user-selected matrix; if the goal is to minimize the average of the variances of the estimated parameters, we set L = I, the identity matrix whereupon L-optimality reduces to A-optimality. Alternatively, if the goal is to estimate some average of the response over a user-selected region R, one chooses L = ∫Rf(x)f′(x)μ(dx) and μ is a a selected weighting measure over for R. This corresponds to estimating the response surface over R with weights specified by the measure μ with more important parts of R receiving a larger weight. If there is equal interest over the region R, one chooses μ to be the uniform measure on R. We obtain I-optimality when μ is the uniform measure and R = Ω.The design criterion is formulated as a convex function of the information matrix. For example, ξ is D-optimal if f(x)′ M−1(ξ)f(x) ≤ d for all x ∈ Ω, anda design ξ is L-optimal if f(x)′ M−1(ξ) LM−1(ξ)f(x) ≤ trLM−1(ξ) for all x ∈ Ω.a design These are frequently referred to as equivalence theorems or more informally as checking conditions. They are derived from considerations of the Frechet derivatives of the convex functionals at the optimum; the function on the left hand side of each of the above inequality is the directional derivative of the convex design criterion evaluated at the optimum. When the regression model has one or two independent variables, the equivalence theorem can be easily applied to check the optimality of any design graphically. For instance, to check whether a design ξ is D-optimal, we plot the function on the left hand side of the inequality (a) over the design space and determine whether the inequality is satisfied. If it is, the design ξ is D-optimal; otherwise it is not.Given a statistical model and a convex design criterion,  gave us ξ is measured by its efficiency relative to the optimum. Typically, this measure is the ratio of the criterion values of the two designs or some function of this ratio thereof. For instance, the L-efficiency of ξ is trLM(ξL)−1/trLM(ξ)−1, where ξL is the L-optimal design. If this ratio is 0.5, then ξ has to be replicated twice to perform as well as the L-optimal design ξL. To maintain this interpretation, D-efficiency of a design ξ is defined by |M(ξ)M−1(ξD)|d1/ where ξD is the D-optimal design and d is the dimension of the regression function f(x). When the equivalence theorem shows that a design is not optimal, we can also assess its proximity to the optimal (without knowing the optimum) using an efficiency lower bound derived from the equivalence theorem and an examination of the above plot , , , 22], , , 24] an an24] anA- and D-optimal designs for the first order linear models with q factors over Sq−1. Both A- and D-optimal designs coincide and are equally supported on the q vertices of the simplex given by , …, . The A-optimal design for the quadratic mixture model with q ≥ 4 was found by m denote the m-dimensional hypercube. Define the m × 1 vector, xi is a q × 1 vector in q, i = 1, …, k, p ∈ k and define ξ, we define the projection function P:Ξ* → (Sq−1)k × Sk−1 byP is invariant in the sense that ξ has support on ξ is transformed from P.Following convention, PSO can and should always be modified to take advantage the special features of the optimization problem at hand. For our mixture experiments design problems, we found that a more effective way to first optimize over the regular hypercube, and then use a projection function to identify our target optimal design in the search space Ω. To fix ideas, suppose the given mixture model has P in n candidates k design points from Ξ*. We define two notions at each stage of the iteration: let (i) ith particle, i.e. ith particle has ever visited, and (ii) let ξgbest provides the optimal value for the criterion among all the positions that all of the particles have ever visited. The strategy for the ith particle, tth iteration is as follows:t−1)th iteration, wt is the inertia weight, γ1, γ2 are two pre-specified positive constants, and α1, α2 are m × 1 uniform random vectors.Generate a new velocity ith particle isThe next location for the q-simplex using i.e. Project Update the current best for each particle wt+1 = g(wt), where g is a user-selected monotonic decreasing function.Update the inertia weight After updating all particles, P and report ξgbest as our “best” design after a pre-specified maximal number of iterations is reached or when the criterion value does not change much according to some user-specified tolerance level.Our modified PSO algorithm is based on the projection function Sq−1)k × Sk−1. This simplifies and makes the computation more efficient. If we had directly implemented PSO to search for the optimal mixture design, our experience is that some of the sequentially generated particles “flew” outside the simplex and the subsequent work required to ignore them or bring them back to the simplex can complicate and prolong the search for the best design considerably. Because we had expanded the search space from the simplex to the hypercube, multiple solutions can exist but PSO is able to handle problems with multiple solutions  fo fo34] foincludes  and, ..D-optimaposed in  proposedD-optimal designs for the mixture ɛ = 10−6 and 127.4690 seconds of CPU time, the algorithm stopped at the 42th iteration. The Cocktail algorithm generated design ξCocktail−D has 10 points atD-efficiency of the this design relative to ξPSO−D is {det(M(ξCocktail−D))/det(M(ξPSO−D))}1/8 = 1.0000 and so both designs are very close in terms of the D-optimality criterion. our ProjPSO code with 1024 particles and 400 iterations took 57.3281 seconds which is also fewer than that required from the Cocktail MATLAB code.We obtained the MATLAB code for the Cocktail algorithm from Yaming Yu’s web-site and recoded it using the weights exchange algorithm to search for D and c-optimality. This YBT algorithm also requires a user-specified grid before it uses an exchange type approach to optimize the weights of the current design points via the Newton-Raphson method. The points with zero weight are dropped and the new design point to be added is the one that maximizes the directional derivative of the objective function. Thus their algorithm is also sometimes called “optimal weights exchange algorithm”. proposedɛ = 10−6. After 206.225 seconds, the best design found by the YBT algorithm is the same as ξCocktail−D. D-efficiencies and CPU time for these two algorithms using different grid sizes.The stopping criterion for the YBT algorithm is based on the maximal values of the directional derivative of the objective function at all the design points. Following , we recoD-optimal design. However, they almost never are able to find the D-optimal designs because by construction they are dependent on the grid size employed. Further, optimal designs produced from the Cocktail and the YBT algorithms typically have more design points than are needed. For example, the design ξCocktail−D has one pair of points, It is clear from the tables that the OPTEX procedure and the two new exchange algorithms can quickly produce highly efficient designs when we use a small grid size to search for the q = 3. For Becker’s model 2, we used the same setup for comparing the Cocktail algorithm and the YBT algorithm in I-optimalhttp://www.swarmintelligence.org/tutorials.php. Our experience is similar. Following convention, we use the default values in ProjPSO and set γ1 = γ2 = 2 to successfully search for the various optimal designs for our mixture design problems. The only exception was the case when we wanted to find a D-optimal design for the log contrast models where setting γ1 = γ2 = 0.5 seems to work better than the default values with γ1 = γ2 = 2.PSO also compares favorably with other metaheuristic algorithms in one aspect. Many researchers from various fields frequently report that the tuning parameters in PSO seem easy to use and are not as sensitive as those in other metaheuristic algorithms. For example, in genetic algorithms (GA) and simulated annealing (SA), all tuning parameters have to be carefully selected before the algorithms work well, see for example Our experience with ProjPSO suggests that for finding an optimal approximate design, only two parameters in the ProjPSO algorithm seem to matter; the flock size and the number of iterations. The rest of the parameters can be set to their default values. A larger size of randomly generated flock of birds covers a broader range of the search space and so is suitable for more complex and high dimensional problems. A larger number of iterations minimizes the chance of early termination and allows ProjPSO additional time to find the optimum, which it usually does not need for solving our design problems. Our typical value for a flock size is 256 or 512 for more complex models and smaller for less complex models. A typical maximum iteration number that we used is 300.We plan to do follow up work in  and 47]47] and m


Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation. Despite decades of vaccine development, infectious diseases continue to be the major cause of mortality in much of the world. The roadblocks to global vaccination programs, including those in remote regions, are prohibitive costs of goods and delivery, use of needles, waste disposal, cold-chain requirements, and the crowded vaccination schedule. Solutions to many of these problems involve developing efficient vaccines that are stable at ambient temperatures that can be applied needle-free with minimal waste, and that combine vaccine antigens to reduce the number of inoculations. Here we present a case for a skin-targeted, freeze-dried formulation, combination human papillomavirus (HPV) pseudovirus (PsV) vaccine composed of the L1 and L2 capsid proteins of HPV16 and plasmids expressing respiratory syncytial virus (RSV) antigens.+ T cell and antibody responses.Delivery to the skin using microneedles poses an attractive means of immunization. The skin is a potent site of immune induction due to the resident Langerhans cells and dermal dendritic cells poised to initiate immune responses to protect the host . BecauseHPV, most often HPV16, is the primary etiological agent responsible for cervical cancer . While cRSV is the most frequent cause of lower respiratory tract infections requiring medical care in children under 5 years of age, and there is not yet a prophylactic vaccine available ,17. HoweIn order to elicit complex immune responses, gene-based immunization is appealing because intracellular antigen production results in a protein with authentic structure and processing through both the MHC class I and II presentation pathways leads to a full complement of cellular and humoral immune responses. We have previously reported that HPV PsV, containing both the L1 and L2 capsid proteins, can encapsidate plasmids  and delihttp://home.ccr.cancer.gov/lco/target.htm.Empty HPV16 L1 and HPV16 L1/L2 VLPs, green fluorescent protein (HPV16-GFP), Luciferase (HPV16-Luc), F protein (HPV16-F) and M/M2 (HPV16-MM2)-expressing HPV16 PsV were prepared as previously described using the 293TT transfection system, purified by density ultracentrifugation using Optiprep  and infectious units determined by titrating the preparations on 293TT cells ,24. The Microneedle arrays, each consisting of a row of five microneedles, were fabricated from stainless steel sheets  by wet etching. The coating solution was composed of 1% (w/v) carboxymethylcellulose sodium salt , 15% trehalose (w/v)  and 0.5% (w/v) Lutrol F-68 NF  and a vaccine. To increase the coating amount, the HPV-RSV vaccine was concentrated by freeze drying and then reconstituted in coating formulation at the varying concentrations described in each experiment.in vivo. The amount of coated vaccine was consistent within each batch within 10% accuracy  4–6 h prior to microneedle administration. Microneedles were placed for 2 min into the skin on the backs (between shoulders) of anesthetized mice.All animal work was approved by the NIH IACUC. Female 6–8-week-old BALB/c and CB6F1 mice  were housed in the NIAID (VRC) and NCI animal care facilities under pathogen-free conditions and maintained on standard rodent chow and water supplied 8 IU of HPV16-Luciferase (Luc) PsV to determine if they were protected from HPV16 infection [8 IU/array), bifurcated needles (20 punctures) or needle scratching (10 scratches each from top to bottom and left to right) followed by addition of 109 IU of the same preparation of PsV. Luciferase was detected on days 1 thru 7 post HPV administration by intraperitoneally delivering 100 μl of Luciferase substrate , waiting 12 min, then applying 20 μl of the same substrate locally on the skin. After 3 min, images were acquired on an IVIS 100  for 2 min on medium binning. Standardized regions of interest were created around the positive signal and photons were measured using Living Image 3.0 software (Perkin Elmer).For HPV immunogenicity studies, animals received three immunizations, each three weeks apart. Three weeks following the final dose, sera were acquired and animals were cervicovaginally challenged with 10nfection . To meas7 PFU of rAd5-M/M2 [7 PFU of RSV A2 (100 μl) intranasally and evaluating protection as previously reported [For HPV16-F PsV studies, animals received three immunizations and were cervicovaginally challenged with HPV16-Luc PsV as described above. Four weeks after the HPV challenge (seven weeks after the last microneedle immunization) animals were intranasally challenged with RSV. In the HPV16-M/M2 PsV study, animals were primed with HPV and after four weeks, either challenged with RSV or boosted intramuscularly (IM) with 10Ad5-M/M2 . All RSVreported . Bronchoreported , except http://home.ccr.cancer.gov/lco/neutralizationassay.htm and is described in Pastrana et al [A detailed protocol of the HPV neutralization assay can be found at na et al . Briefly− 1 was read at a wavelength of 405 nm every 9 s for 5 min, with the plates shaken before each measurement. The mean mOD min − 1 reading of duplicate wells was calculated, and the background mOD min − 1 was subtracted from the corresponding well.RSV F protein was purified from virus-infected 293 cells , dilutedThis was performed as previously described in Graham et al, 1988 . BrieflyWhole blood (250 μl) was lysed in 1 ml ACK lysing buffer  for 7 min. Samples were washed with 2 ml PBS and centrifuged at 600 x g for 5 min. Lysing and washing was repeated up to three times until the cell pellet was devoid of visible amounts of RBCs. After final removal of the supernatant, samples were stained according to the protocol for the surface and tetramer staining described below.dM282–90 or I-AbM209–223 and antibodies to CD3, CD4 and CD8 and analyzed by nine-color flow cytometry as previously described [Lymphocytes were isolated from right lung with Fico-Lite , and tetramer-stained with Kescribed ,32 usingescribed . Flow Jot-test. All statistical tests were performed using SigmaStat 3.0 for Windows  or GraphPad Prism v5.0 . All plots were generated in Excel  or GraphPad Prism v5.0.Data were analyzed by one-way statistical analysis of variance. Pair-wise comparisons were made using the Holm-Sidak, Dunn’s method, or Student’s in vivo protection.To test the feasibility of generating protective immunity against HPV infection using microneedles, we compared intramuscular injection to microneedle delivery of HPV16 VLPs. An additional intramuscular control using VLPs rinsed from microneedles by vortexing them in a 1.5ml tube containing sterile PBS was included in order to ensure that the conformation-dependent VLP epitopes that induce neutralizing antibodies were not compromised by the freeze drying and microneedle coating process. HPV16 VLP immunization was performed with a high dose (3 μg) and a low dose (0.3 μg) in the absence of adjuvant. Animals were immunized three times, each three weeks apart. Three weeks following the final immunization, sera were collected to measure anti-HPV16 neutralizing antibody (nAb) titers and animals were then intravaginally challenged with HPV16-Luc PsV as a means for determining efficacy of in vivo as demonstrated by protection from cervicovaginal challenge with 108 IU of HPV16-Luc PsV  the immunogenicity of the VLPs remains unaffected by the microneedle coating and application process; 2) there is no dose response signifying that even at 0.3 μg of antigen, the antibody response following microneedle delivery is equivalent to IM immunization with the same or with a one log higher dose; and 3) microneedle delivery is comparable to intramuscular delivery for inducing a nAb response against HPV.In vivo HPV PsV-mediated gene delivery requires both the HPV major (L1) and minor (L2) proteins [8 IU of PsV/array. Microneedles were rinsed into PBS and the L1 content was measured by Western blot and determined to be ~25 ng L1/array . Longer inoculation time points did not increase the efficiency of delivery.We have previously demonstrated that when delivered mucosally, HPV PsV are efficient vectors for gene-based immunization and that 10smid DNA . Prior tge model  and HPV1the cell . Additioin vivo gene delivery to skin, HPV16-Luc PsV-coated microneedle arrays (approximately 108 IU of PsV/array) were used to inoculate animals for 2 min at doses of approximately 1x108 IU, 5x108 IU and 1x109 IU . Uncoated microneedles, bifurcated needle puncture (20x) and needle scraping  served as controls. Beginning 24 h after HPV16-Luc PsV administration, and continuing daily for seven days, animals were imaged for a luminescent signal indicative of successful delivery and expression of the luciferase gene. A luminescent signal could be detected over background in all PsV conditions tested and the transient kinetics of luciferase expression using this method is similar to what has been previously observed in the vaginal challenge model [in vivo to the skin.To evaluate ge model ,33,34. Iin vivo protection from HPV challenge was observed in all but these same two groups . Further, o groups . These dSera acquired on the day of HPV challenge from the animals immunized with HPV16-F PsV described above were evaluated for RSV F-specific antibodies by kinetic ELISA. Mice receiving the highest dose of 10 microneedle arrays all had F-specific antibodies, whereas, at the lower doses, induction of antibody responses was inconsistent . Seven w+ and CD8+ T cell-induced immunity in mice [7 pfu) of recombinant adenovirus serotype 5 vector (rAd5-M/M2) IM. Epitope-specific CD8+ and CD4+ T-cells were measured in lungs 5 days post-RSV challenge. Only the groups immunized with 10 microneedle arrays demonstrated higher increases in CD8+ T-cell and significant increases in CD4+ T-cell responses (p = 0.04) above baseline after challenge (+ tetramer responses were measured in blood fourteen days after boosting with rAd5-M/M2 and significantly higher responses (p = 0.04) compared to HPV-Luc immunized controls were only observed in animals immunized with 10 microneedle arrays  that constantly survey the tissue for foreign antigens ,39. LangImmunization with DNA is the simplest form of gene-based vaccination. While delivery and immunogenicity of naked DNA has been improved using devices such as the Biojector –42, otheAs opposed to mechanical approaches using ballistic force or electrical disruption to improve transduction efficiency, HPV PsV encapsidation provides a biological solution to facilitate DNA delivery. With targeted microneedle delivery to basal epithelium based on HPV tropism, the current approach has potential to facilitate DNA delivery of genes expressing other vaccine antigens. In this study, we showed that microneedle delivery of HPV PsV to the skin can elicit both antibody and T cell responses against RSV antigens encoded by plasmid DNA encapsidated within the PsV. While the responses were relatively modest, a dose response was observed, and, in animals receiving the highest dosage, an immune response was generated that was capable of reducing viral replication. Co-administering an HPV PsV expressing an immune modifier could potentially enhance the B- and T-cell responses. Expression of the modifier would be transient thus diminishing safety concerns associated with the adverse effects of long-term expression or overexpression of immune modifiers.To further optimize this vaccination strategy, the primary focus will be on microneedle design and payload in order to deliver a higher dose of DNA. This can most easily be achieved by increasing the number of microneedles on a patch. The microneedle arrays used in this study were small and limited in their ability to deliver higher doses of vaccine compared to more recently developed technology such as patches and hollow microneedles . Also, dThis study addressed three central issues associated with vaccination using microneedles. First, we found that lyophilized HPV VLP or HPV PsV could be applied to microneedles and remain antigenically stable at room temperature. Second, vaccination with HPV VLP generated a robust and protective immune response, supporting the development of microneedle delivery for HPV vaccination in place of IM injection. Third, vaccination with HPV PsV generated strong, protective immune responses against HPV and induced dose-dependent B- and T-cell responses to RSV antigens encoded by the encapsidated DNA. These data support the future development of combination vaccination strategies in which the HPV PsV vector generates immunity against both HPV and the antigen expressed from the encapsidated plasmid DNA. We conclude that vaccination with microneedles may be an ideal method for HPV vaccination as a VLP, and may allow future development of HPV PsV-encapsidated DNA vaccine concepts that would result in immunity to HPV and additional antigens encoded by the DNA.S1 Fig(A) A complete microneedle device, including an array of five microneedles at the top and a handle for ease of handling at the bottom. Magnified image of (B) five microneedles and (C) a single coated microneedle under fluorescence optics.(TIF)Click here for additional data file.S2 Fighttp://rsbweb.nih.gov/ij/). Samples are as follows: Lane 1- molecular weight marker ; Lane 2—HPV-16 L1 standard 400 ng; Lane 3- HPV-16 L1 standard 40 ng; Lane 4- HPV-16 L1 standard 4 ng; Lane 5- HPV-16 L1 standard 0.4 ng; Lane 6- HPV-16 L1 standard 0.04 ng; Lane 7–10 μl of 10 MN rinse; Lane 8–5 μl of 10 MN rinse; Lane 9–10 μl of 5 MN rinse; Lane 10–5 μl of 5 MN rinse.Amount of HPV L1 protein coated onto each microneedle used in the vaccination experiments was determined by western blot. Ten microneedle arrays and five microneedle arrays from two representative preparations were each rinsed into 50 μl of 1xPBS. Samples were run alongside an HPV16 L1 control standard on a 10% Bis-Tris gel . Blots were probed with CAMVIR-1  and goat anti-mouse-HRP  and developed using Western Lightening Substrate . Blot was imaged on an ImageQuant LAS4000  and quantitated using ImageJ software ((TIF)Click here for additional data file.S3 Fig8 IU of HPV16-Luc was administered and animals were imaged 48hrs later for luciferase activity. The “No PsV” group was not challenged in order to serve as a negative control group for luminescence. Data reported as average radiance; n = 2.Data showing cervicovaginal challenge of animals described in (TIF)Click here for additional data file.


Concerns have been raised that HPV-vaccination might affect women’s cervical screening behavior. We therefore investigated the association between opportunistic HPV-vaccination and attendance after invitation to cervical screening.adj). Women were individually followed for up to 6 years, of which the first and second screening rounds were analyzed separately.A cohort of all women resident in Sweden, born 1977-1987 , and invited to cervical screening, was followed October 2006 - December 2012. Invitations to screening were identified via the National Quality Register for Cervical Cancer Prevention, as was the primary outcome of a registered smear. Vaccination status was obtained from two nationwide health data registers. Hazard ratios (HR) were estimated using Cox regression adjusted for age, education level and income . In the second screening round, attendance was likewise higher in HPV-vaccinated women .Screening attendance after three years of follow-up was 86% in vaccinated women  and 75% in unvaccinated women . The crude HR of screening attendance in vaccinated vs. unvaccinated women was 1.31 (95% CI 1.27-1.35) in the first screening round. Adjustment for education and income reduced but did not erase this difference (HRHPV-vaccination is so far associated with equal or higher attendance to cervical screening in Sweden in a cohort of opportunistically vaccinated young women. Most but not all of the difference in attendance was explained by socioeconomic differences between vaccinated and unvaccinated women. HPV vaccine effectiveness studies should consider screening attendance of HPV-vaccinated women when assessing incidence of screen-detected cervical lesions. Human papillomavirus (HPV) vaccination was introduced in Sweden in October 2006. The quadrivalent HPV vaccine (qHPV) was approved in September 2006 and the bivalent HPV vaccine (bHPV) in September 2007. A nation-wide opportunistic HPV vaccination programme was in effect during 2007–2011 and offered a 50% subsidy towards the cost for each dose prescribed to girls aged 13–17 years .The organized Swedish cervical screening programme issues invitations three years after the latest smear for women aged 23–50 and every five years for women aged 51–60. Repeated annual invitations to screening are issued by regional offices to all women in the population register who have not been screened according to recommendations [HPV vaccination is not therapeutic against prevalent HPV infections , and if A study in Australian university students found that knowledge of cervical screening guidelines was generally poor, but that screening uptake did not differ between vaccinated and unvaccinated women . In contst, 2006 defined study entrance. Women were followed until they attended screening, emigrated, died, or the study period ended on December 31st, 2012—whichever occurred first. Women who emigrated or died before the start of follow up, or had no record of screening invitation, were excluded.The population in this nationwide study comprised an open cohort of all women born 1977–1987, resident in Sweden and invited to cervical screening during the study period. The first issued invitation to screening after October 1Data were collected using the Swedish nationwide population-based health registers. All women born between 1977 and 1987 were selected from the Total Population Register and were linked to the registers described below, using the unique personal identification number (PIN) . InformaThe Swedish HPV Vaccination Register (SVEVAC) and Prescribed Drug Register (PDR) were used to obtain women’s HPV vaccination status and date of vaccination.SVEVAC is maintained by the Swedish Association of Local Authorities and Regions, and holds information on qHPV and bHPV administrations and was launched in parallel with the start of HPV vaccination . ParticiA descriptive analysis on HPV vaccination and socio-demographic status was conducted among women who were eligible for cervical screening but with no record of invitation. In the cohort of women who were invited to screening, the attendance among HPV-vaccinated and unvaccinated women, respectively, was calculated as cumulative incidence proportions over time from invitation. Cox regression was used to estimate hazard ratios (HR) with corresponding 95% confidence intervals (CI) of cervical screening attendance among HPV-vaccinated versus unvaccinated women, with attained age as the underlying time scale. HPV vaccination status was handled as a time-varying exposure. Women were considered to be HPV-vaccinated if they received at least one vaccine dose during the study period for the primary analyses.Attendance was measured over the entire study period, and in the first and second screening round, respectively—the latter to examine potential variations in screening behaviour over time. The first round of screening was defined as attendance during 0–3 years after first invitation during the study period.Entry to the second screening round was defined as whichever occurred first: the first issued invitation after attendance to screening during follow up , or the first issued invitation to screening after three years of non-attendance. Finally, we investigated attendance in the second repeated screening round, in the subgroup of women who attended in the first.The crude main effects model was extended with additional co-variates based on explorative modelling. Adjustments were made for education level and individual income. Education level was categorized into missing; less than high school; high school, and university studies. Women with a missing education level were retained as a separate category, as the women in the study cohort were very young. Furthermore, an association between increasing education level and likelihood of getting vaccinated has been shown in Sweden . The impIn separate sensitivity analyses, women were considered to be HPV-vaccinated only after the full 3 doses. Additional analyses exploring alternate definitions of the first screening round were performed using cut-offs of 2.5, 3.5, and 4 years instead of 3 years. Finally, an analysis was performed including calendar year of study period (2006–2012) as a covariate, to investigate whether the associations varied by study year.Data management was done with SAS statistical software version 9.4 . Statistical analyses were performed with Stata 13 . The study was approved by the Regional Ethical Review Board in Stockholm, Sweden (No. 2012/216-32 and 278/2012-4.1.2) which determined that written informed consent by the study participants was not required.A total of 708,673 women born 1977–1987 were included in the study population . Of thesP<0.0001). Attendance remained higher in the HPV-vaccinated group throughout follow-up  for the entire study period was 1.05 (95% CI 1.02–1.08) in the main effects model  for vaccinated compared to unvaccinated women. When adjusting for income and education, the adjusted HR . However, repeated attendance was higher in HPV-vaccinated women with high school or university education  than in women with high school or university education that were not vaccinated .In this nationwide study, we found that opportunistic HPV-vaccination is associated with an equal or higher attendance after invitation to cervical screening in Sweden. HPV-vaccinated women were also over-represented in the group that obtained a cervical smear without prior invitation. The overall association appeared mainly driven by the higher socioeconomic status of those vaccinated, although our findings by rounds, or in repeated screening, did not appear entirely explained by such factors.The strengths of this observational study derive from the quality of data included: we had complete national follow-up of all invitations sent, all organized and opportunistic smears taken, and the ability to individually link this to registers of socioeconomic status and HPV vaccination status. This enabled a thorough evaluation of potential confounding and effect-modifying factors, as well as sensitivity analyses. The internal validity of our study should thus be high. The main limitation of this study is that women were not randomized to HPV vaccination, but self-selected whether to vaccinate or not and the women who did so constituted a minority of the study cohort (around 4900 women out of around 630 000). For women in this study, the cost of vaccination was unsubsidized due to their age being over 17 and those who got vaccinated would have done so at their own (or a parent’s) expense. Therefore, socioeconomic selection mechanisms were involved, and the relatively few women who actively chose to get HPV-vaccinated might also be more health-conscious, and prone to attend cervical screening from the outset. We have addressed this self-selection to HPV-vaccination in several ways. Firstly, adjustments were made for education level to account for the unequal uptake of vaccination across different levels of education.Furthermore, the study population was restricted to those individuals who were invited to cervical screening as women that spontaneously/opportunistically obtain a cytological smear might be even more health conscious and thus less representative. Furthermore, it should be noted that the vaccine register SVEVAC had an estimated registration coverage of 80–85% during some of the years under study, for women that were vaccinated outside the subsidized vaccination program. This may have resulted in misclassification of the exposure where some HPV-vaccinated women were classified as unvaccinated. Since we found that HPV-vaccinated women were generally more likely to attend screening as compared to unvaccinated women, the effects of such misclassification would have been to slightly increase the screening rate in the comparison group of women classified as unvaccinated. This could mean that the observed positive association between HPV vaccination and screening attendance is somewhat underestimated in size, due to bias towards the null. However, since HPV-vaccinated women were few compared to unvaccinated, such misclassification should be unlikely to have substantially biased our results.We emphasize that these women are not representative of the birth cohorts who are today being HPV-vaccinated as girls age 10–17 years old, nor of women vaccinated through organized programs. Therefore, in terms of external validity, our findings are likely best compared to other opportunistically vaccinated cohorts. It will be equally important to monitor future screening participation in those Swedish females who are now being vaccinated for free as school girls, who may differ in their attitude towards, and understanding of, screening than those vaccinated at a more mature age. Indeed, the Public Health Agency of Sweden is planning for future and regular follow-up of screening rates in HPV-vaccinated girls, to maintain vigilance for the possibility of changing behaviour over time. However, it is still important to perform early studies, such as the present work, of women that are HPV vaccinated, should the outcome have been the reverse and screening attendance reduced.To our knowledge, this is the first nation-wide study to include individual-level data from the whole population and focus on cervical screening attendance after HPV vaccination. To our knowledge, this is also the first study to fully adjust for socioeconomic confounding, report response to invitations, and attendance to a second round of screening. Particularly the latter should be of value, since it is conceivable that HPV-vaccinated women duly attend the first round after vaccination, but then reduce their attendance in the second round due to the double perception of protection from both vaccination and preceding screening. However, this was not the case in our findings.Although there have been theoretical studies on attitudes to screening after vaccination, –14, 20 tIn contrast to their and our findings, though, an Australian study on women vaccinated within the organized program in the region of Victoria recently reported that HPV-vaccinated women age 20–34 had significantly lower rates of 3-year screening attendance, although limitations to the ability of linking registers on the individual level in the study precluded determination of the exact size of the reduction in screening .These data clearly further illustrate the need for comprehensive assessment of screening attendance in different vaccination settings. Possible reasons for the contrast in our results as compared to the Australian study include differences in whether women were vaccinated in an organized or opportunistic setting; how the invitation system to screening is designed, and ongoing background trends in screening participation in general. Indeed, Australia is observing a declining trend in participation among women  whereas Our findings also imply that studies should take increased screening rates in vaccinated women into consideration when assessing the effectiveness against screen-detected cervical cancer precursor lesions, especially in those vaccinated after sexual debut where effectiveness may be compromised. If HPV-vaccinated women participate more extensively in screening, the recorded incidence of screen-detected lesions may be paradoxically somewhat increased, compared to the recorded incidence in unvaccinated—and less frequently screened—women.This potential for detection bias may lead to an underestimation of vaccine effectiveness against cervical lesions in population-based studies of screened populations only, unless the potentially differential attendance rates are adjusted for.In conclusion, opportunistic HPV vaccination was associated with an equal or higher attendance to cervical screening in Sweden in the first wave of self-selected, early adopters of the vaccine. This finding may be of relevance when assessing population-based effectiveness of opportunistic HPV vaccination against screen-detected cervical lesions. Continued regular follow-up of cohorts vaccinated in the free of charge, organized vaccination program is necessary, and regardless of vaccination status, all women will benefit from collective efforts to maintain high fidelity to screening programs.S1 Fig(TIF)Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The association between human cytomegalovirus (HCMV) and glioblastoma multiforme (GBM) is becoming a new concept. However, information on the geographic variability of HCMV prevalence in GBM remains scarce. Moreover, the potential roles of various viruses, such as polyomaviruses and oncogenic viruses, in gliomagenesis remain unclear. Our aim was to investigate the prevalence of HCMV in GBM among Japanese patients. Furthermore, this was the first study that evaluated infection with four new human polyomaviruses in GBMs. This study also provided the first data on the detection of human papillomavirus (HPV) in GBM in the Eastern world.4 HCMV-negative cells. The presence of HCMV and HPV genomes was also assessed by nested PCR. Immunohistochemical study was also carried out to detect HPV-derived protein in GBM tissues.We measured the number of various viral genomes in GBM samples from 39 Japanese patients using real-time quantitative PCR. The tested viruses included HCMV, Merkel cell polyomavirus, human polyomavirus (HPyV) 6, HPyV7, HPyV9, Epstein–Barr virus, human herpesvirus 8, and HPV. Our quantitative PCR analysis led to the detection of eight copies of the HCMV DNA mixed with DNA extracted from 10The viral DNAs were not detectable, with the exception of HPV, which was present in eight out of 39 (21%) GBMs. All HPV-positive cases harbored high-risk-type HPV (HPV16 and HPV18). Moreover, the HPV major capsid protein was detected in GBM tumor cells.In contrast with previous reports from Caucasian patients, we did not obtain direct evidence in support of the association between HCMV and GBM. However, high-risk-type HPV infection may play a potential etiological role in gliomagenesis in a subset of patients. These findings should prompt further worldwide epidemiological studies aimed at defining the pathogenicity of virus-associated GBM. A significant portion of human cancer cases worldwide, an estimated 15%–20%, may be attributed to viral infection . Furtheret al. [In contrast with these findings, several conflicting reports indicated a lack of association between HCMV and GBM –13. A moet al.  collecteIf HCMV infection is an important risk factor for the development of most cases of GBM, it would be worth exploring whether other infectious pathogens coexist with HCMV in these tumors, possibly as cofactors of pathogenesis. For example, several studies suggested that some polyomaviruses, such as the simian virus 40 (SV40) and JC virus (JCV), are associated with GBM , 17. In To detect HCMV DNA, we employed quantitative PCR targeting two different HCMV regions using the primers and probes for major immediate early (IE) and glycoprotein B (gB) genes , 22. TheWe also analyzed the GBM samples for the existence of HCMV DNA by nested PCR with primer sets targeting the HCMV gB gene, as previously reported , 23. LikNext, we tested the GBM samples for the presence of four human polyomaviruses  and three oncogenic viruses . These viral DNAs were not detected, with the exception of HPV, which was detected in three out of 39 GBMs by real-time quantitative PCR targeting HPV16 and HPV18 sequences . The normal brain tissues were obtained during surgery and stocked as FFPE tissues. As a result, HPV DNA was not detected in the non-neoplastic tissues, suggesting that that the HPV positivity resulted from HPV-infected GBM tumor cells.To evaluate the expression of HPV antigen, immunohistochemistry was performed using the mouse monoclonal antibody K1H8 against the HPV major capsid protein , 28. Thi4 HCMV-negative cells. No cases harbored detectable HCMV genomes. Furthermore, nested PCR assays did not detect HCMV in GBM tissues. These results indicate that HCMV is unlikely to play a direct role in the pathogenesis of GBM in cases collected in the south of Japan. Recently, another research group was also unable to demonstrate the presence of HCMV genomes in GBM samples from a population of patients from the central region of Japan [The presence of HCMV in GBM was first reported by Cobbs and colleagues . Since tof Japan . The absof Japan , 31. In of Japan , 32. Thuof Japan . The mosof Japan , 34. A sof Japan . Thus, gTo date, three polyomaviruses, SV40, JCV, and BKV, have been suggested to be associated with the development of brain tumors , 36, 37.et al. [We also searched for the viral DNAs of two oncogenic herpesviruses, EBV and HHV8, in GBMs, with negative results. Recently, Cimino et al.  reportedet al.[HPV is a common oncogenic virus in human. Recently, Vidone et al. reportedThis was the first presentation of data regarding the prevalence of various viruses, including HCMV, human oncogenic viruses , and new human polyomaviruses  in GBMs from an Asian population. In contrast with many previous reports based on Caucasian patients, we were not able to obtain direct evidence to support the association between HCMV and GBM. Furthermore, it is unlikely that GBM is associated with human oncogenic viruses, including EBV, HHV8, and MCPyV. However, our investigations demonstrated the presence of the viral genome and protein of high-risk-type HPVs in a subset of patients with GBMs. These findings should stimulate further large-scale worldwide epidemiological and virological studies aimed at defining the pathogenicity of HPV-positive GBM.This study included 39 Japanese patients with GBM grade IV (22 men and 17 women). The median age of the patients was 68 years . Thirty-one FFPE tumor tissues (denoted as cases 1–31) and eight frozen tumor tissues (cases 32–39) were obtained from patients from the Kochi University Hospital. The patients resided in the Kochi prefecture, which is located on Shikoku Island in southern Japan. This study was approved by the Ethics Committee of Kochi Medical School, Kochi University.For DNA extraction from FFPE tumors, three 5-μm-thick slices were prepared for DNA extraction using a ReliaPrep FFPE gDNA Miniprep System . From frozen tumor samples or cultured cells, DNA was isolated using a QIAamp DNA Mini Kit .RNaseP was used as an internal control, and the PCR mixture without the template DNA was used as a negative control for each experiment.Two hundred nanograms of extracted DNA was analyzed for the detection and quantification of eight different viruses  via TaqMan-based real-time PCR using a StepOne Plus Real-Time PCR System . Real-time PCR reactions and quantitative analyses were performed primarily based on methods described previously , 40–45. After the amplification of DNA by standard PCR or nested PCR, the PCR products were purified with a High Pure PCR Product Purification Kit  and then sequenced directly using an ABI Prism BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies). The sequenced products were analyzed using a model 3130 Genetic Analyzer (Life Technologies). The nucleotide sequences obtained were aligned and edited with BioEdit software .To detect HPV protein, immunohistochemistry was performed on FFPE tissue sections using a mouse monoclonal antibody, K1H8 against the HPV major capsid protein , 28. The


Human papilloma virus (HPV) is considered as the main sexually transmitted etiological agent for the cause and progression of preneoplastic cervical lesions to cervical cancer. This study is discussing the prevalence of HPV and its genotypes in cervical lesions and invasive cervical cancer tissues and their association with various risk factors in women from Varanasi and its adjoining areas in India. A total of 122 cervical biopsy samples were collected from SS Hospital and Indian Railways Cancer Institute and Research Centre, Varanasi and were screened for HPV infection by PCR using primers from L1 consensus region of the viral genome. HPV positive samples were genotyped by type-specific PCR and sequencing. The association of different risk factors with HPV infection in various grades of cervical lesion was evaluated by chi-square test. A total of 10 different HPV genotypes were observed in women with cervicitis, CIN, invasive squamous cell cervical carcinoma and adenocarcinoma. Increased frequency of HPV infection with increasing lesion grade (p=0.002) was observed. HPV16 being the predominant type was found significantly associated with severity of the disease (p=0.03). Various socio- demographic factors other than HPV including high parity (p<0.0001), rural residential area (p<0.0001), elder age (p<0.0001), low socio-economic status (p<0.0001) and women in postmenopausal group (p<0.0001) were also observed to be associated with cervical cancer.These findings show HPV as a direct cause of cervical cancer suggesting urgent need of screening programs and HPV vaccination in women with low socio-economic status and those residing in rural areas. Cervical cancer is one of the common female malignancies, ranking first in developing countries and human papillomavirus (HPV) was recognized as the main sexually transmitted etiological agent for the disease. Since 1981 when a relationship was established between papillomavirus and cervical neoplasia . PresencIndia is a developing country with 17.2% of the world’s total population  . In IndiThis study aimed to determine the prevalence of HPV and its genotypes in different histological grades of cervical specimens of women from Varanasi and adjoining regions. An attempt was also made to find out a correlation of HPV infection with socio-demographic factors forming a definite and effective link with the persistence of virus and disease progression.Patients and data collection A total of 122 fresh cervical tissue biopsies were collected from Sir Sunderlal Hospital and Indian Railways Cancer Institute and Research Centre, Varanasi, between 2008 and 2010. The age range of the patients was 25-80 years (Mean age=53.3). A questionnaire form along with consent was filled by each female participant. Information regarding marital status, age of conception, parity, smoking, menopausal status, diet, place of residence, socio-economic status, history of HPV infection or any kind of disease was recorded. Based on histopathological reports, all the cases were categorized into cervicitis (inflammatory cervix), mild to moderate dysplasia (CIN1/2/3) and cervical cancer (SCC/ADC). Staging of cervical cancer cases was done according to the International Federation of Gynecology and Obstetrics (FIGO) classification. The control subjects included were randomly chosen healthy normal women without any previous history of HPV infection, hysterectomy, cervical neoplasia/ cervical cancer or any other anogenital cancer. Ethical clearance for the present study was taken from the Institutional Ethics Committee.Sample collection and DNA isolationoC for further use. In control subjects, exfoliated cells were collected from ectocervix region in 5ml of pre-chilled PBS. DNA was extracted according to the protocol described previously , chloroform:isoamyl alcohol (24:1 v/v). DNA was precipitated with 1/10 volume of 3M sodium acetate and 0.7 volume of isopropanol, dried and re-suspended in TE  buffer. Purified DNA samples were electrophoresed on 1% agarose gel to check the quality of DNA and its quantity was determined by Nanodrop spectrophotometer .Cervical biopsies were collected in vials kept on ice, immediately transported and stored at -80eviously  with fewDetection of HPV DNA2), 10pmole of each forward and reverse primers, 4µl of dNTP mix , and 0.3U of Taq polymerase . All the sets of PCR reactions were performed along with positive and negative controls. The known HPV positive DNA sample served as a positive control while reaction without DNA served as a negative control. Amplification was performed on thermocycler (Applied Biosystems) and the amplicons were checked on 2% agarose gel giving a product size of 450bp (MY09/11) and 150bp (GP5+/6+) (All the samples were first subjected to β-globin amplification (268bp) to determine the quality of DNA by using PC04/GH20  primer sGP5+/6+) . The seqHPV genotypingThe samples positive for HPV infection were proceeded for genotyping using type- specific primer sets for high risk HPV types 16, 18, 31, 33 and 35 -16. ClonStatistical analysisUnivariate association of socio-demographic factors and clinical parameters with the presence and absence of HPV/HPV16 infection in different cervical histology group was estimated by standard contingency table method using Fisher’s exact or Chi-square tests. Two sided p-value was calculated and considered significant when p≤0.05. All the statistical analysis was done by SPSS statistical software (version 16.0).Out of 122 biopsy samples obtained, 10 samples were of cervicitis, 15 CINs, 92 squamous cell carcinomas (SCC) and 5 with adenocarcinoma (ADC). All the samples were positive for β-globin and suitable for further genotyping.Altogether 10 different HPV genotypes were observed, i.e., HPV6, 11, 16, 18, 31, 33, 35, 45, 58 and 86, all of them were high risk types except HPV6 and 11 (low risk types present in cervicitis and ADC group) and HPV86 (intermediate risk type present in ADC group) . Among sThe prevalence of HPV16 infection showed an increasing trend from 30% in cervicitis to 71.7% in SCC (p=0.03). 11 samples  were negative for HPV infection and 2 remain unidentified due to inadequacy of the DNA amount .The prevalence of single and multiple HPV type infection in cervicitis, CIN, squamous cell carcinoma and adenocarcinoma was shown in . Single Chi-square test and odds ratio showed increasing age (women ≥45 years) as a significantly related factor with cervical cancer  while it was unrelated to the risk of cervicitis and CINs (preinvasive lesions). Further more, the women with high parity (>3) had 3.5 times greater risk for cervical cancer  while it did not show any association with other histologic groups. Investigation revealed that the women in low socio-economic status was significantly associated with the risk of cervical cancer  while those residing in rural areas were associated with the risk of CIN  and cervical cancer  but not with cervicitis. In addition, women in postmenopausal group were at greater risk of developing CINs (p=0.02) and cervical cancer (p<0.0001). Diet, age at first intercourse and religion did not exhibit any relationship with all the three histologic groups.vs urban) in CIN category was found. Women living in rural areas and infected with high risk HPV16 were at increased risk of developing CIN and subsequently, cervical cancer (p=0.07). No other factors showed significant association with HPV 16 infection.HPV16 was observed to be the predominant type in all the groups. When samples were categorized according to high risk HPV16 type and other HPV types (excluding HPV16) along with socio-demographic factors , a signiDifferent epidemiological studies reported HPV as one of the important factors constantly associated with cervical cancer, though alone it was insufficient for the progression of cervical pre-neoplastic lesion to invasive cervical cancer. Although not all the HPV genotypes were equally responsible for the development of pre-neoplastic lesion, some of them present in the anogenital regions belonging to oncogenic category were found constantly associated with the disease.In the present study, the prevalence of HPV and its genotypes was evaluated in the samples of cervicitis (inflammatory cervix), pre-neoplastic lesions (CIN) and cervical cancer. A significant increase in HPV prevalence with increase in severity of the disease (p=0.002) was observed showing HPV as an important risk factor in disease progression .The high prevalence of HPV infection was observed in squamous cell carcinoma patients (95.8%) which was consistent with the previous reports  than CIN (26.6%) in contrast to multiple HPV infections which were observed more commonly in CIN (46.6%) than SCC (30.4%). Such observations were in correlation with previous studies , 23. FIGet al.  with staet al.  in frequency with the increase in severity of the lesion, being highest in SCC (71.7%) and lowest in cervicitis 30%) thus, supporting its importance in the pathogenesis of cervical cancer. The high incidence of HPV16 in squamous cell cervical carcinoma was comparable to the prevalence reported worldwide, i.e., 65.2% in Saudi Arabia . In Indi% thus, sIn our study, HPV18 was the next most frequent genotype detected after HPV16 and both of them comprise of 79% of the total HPV infection in SCC. Earlier findings also suggested that high grade SIL infected with HPV16, 18 or 45 have more probability of progression to SCC which may be due to either of their greater potential to transform non-malignant lesion to malignant one or due to their ability to evade host immune system .We found HPV as the most strongly associated factor in all the histologic groups see  which waAnother aim of our study was to investigate the cofactors in all three histologic groups other than HPV shown in . We obseWe also found high parity (>3) as one of the important risk factors in this population. This may be due to early marriages in India which resulted in higher number of pregnancies. It was reported that high parity leads to four times increased risk of squamous cell cervical carcinoma .We also observed rural residential area as a risk determinant of preinvasive and invasive cervical carcinoma. This has been supported by several studies in India , 37 as wLow socio-economic status was also one of the significant factors found to be associated with cervical cancer in our study population which was in parallel to the other studies , 39. SimWomen in postmenopausal group were at increased risk of developing cervical cancer as observed in our study which was also consistent with the findings in other studies  thus sugHPV16 was the predominant type observed in all the grades of cervical lesion and women infected with HPV16 and living in rural areas were at increased risk of developing CIN (p=0.02) which may later develop into cervical cancer (p=0.07) . In our Our findings suggested the urgent need of HPV and cervical cancer screening programs in women belonging to low socio-economic group and those residing in rural areas to detect abnormal/cancerous cells at initial stage of the development and to provide treatment which could be better responded at early stage of the cancer. Since India alone bear a burden of 1/5 of total cervical cancer cases all over the world and almost 70% of the population in India resides in villages so this study becomes more relevant. Vaccine targeting HPV31, 33, 35 and 58 apart from HPV16 and 18 (covered under Gardasil and Cervarix vaccines) could help prevent the large number of cervical cancer cases. Increasing frequency of HPV/HPV16 infection with increasing stage of the disease shows a direct link of HPV with the disease severity. Since these findings relied on small number of cases, further studies with large number of cases may be of significance to support these findings.