In vivo staining is a simple, inexpensive, and fairly sensitive method. Involved 120 patients  stained by Methylene Blue (MB). The results of MB uptake were compared with a simultaneous biopsy of these lesions. Pathologically confirmed precancers and cancers were the positive targets of this screening, while hyperkeratosis without dysplasia and no evidence of malignancy were sorted as negative subjects of screening. The results revealed sensitivity of 91.4%, specificity of 66.6%, positive predictive value 97.7% and negative predictive value 33% leading to diagnostic accuracy of MB stain to 90%. We state that MB staining is useful diagnostic tool in community oral cancer screening programmes for high-risk individuals.Oral cancer is one of the most common neoplasm’s and is ranked eighth in the cancer incidence worldwide. Early detection is of critical importance because survival rates markedly improve. The online version of this article (doi:10.1186/2193-1801-2-95) contains supplementary material, which is available to authorized users. Oral cavity cancer accounts for approximately 3% of all malignancies and is a significant worldwide health problem Kademani . Most orin vivo staining is advocated as a simple, inexpensive and fairly sensitive method  has been used to detect gastric, prostrate and bladder cancers  and Oral cancer were made on basis of clinical examination. Patient’s more than or equal to 15 yrs of age, with the habit of smoking and/or tobacco consumption were made part of the study. Patients with age less than 15 years, immunocompromised states, with premalignant conditions, and patients with diabetes or other systemic disorders were excluded from the study. Ethics committee approval from the college ethics committee was taken for the smooth conduct of the study. (CPGIDSH/09). Controls were subjects with clinically normal appearing oral mucosa. The subjects were made to sit comfortably on the dental chair and were thoroughly examined under artificial illumination. The clinical examination was done and the relevant data was entered into the proforma.Methylene blue dye system had 2 solution bottles. The dye rinse solution (Bottle A) had 1% methylene blue, 1% malachite, 0.5% eosin, glycerol, and dimethylsulfoxide. Pre- and post-rinse solution (Bottle B) had 1% lactic acid, and purified water.The application of methylene blue was as follows. The patients were directed to rinse their mouth with 1% lactic acid & distilled water for 30 seconds to remove food debris and excess saliva and to provide a consistent oral environment. The mucosa of the target area was gently dried with gauze and power air spray to ensure that the lesion was not being contaminated with saliva. The dye was directly applied on the lesion with help of cotton bud  Oral Precancerous: Mild, Moderate and Severe Dysplasia B) Oral Cancer: Well, Moderately and Poorly differentiated Squamous Cell Carcinoma C) Lesions neither precancerous nor malignant: Hyperkeratosis without dysplasia and No evidence of malignancy. The results were then deduced and were statistically analyzed using the various statistical tests given below and the final results were drawn.The pathologically proven OSCC and precancerous lesions were the targets of screening and the concerned is presented by number and percentage of the same. The results of positive/negative uptake of methylene blue in each lesion were correlated with the histopathological diagnosis. Statistical analysis was performed, including sensitivity, specificity, positive and negative predictive values. The association of methylene blue uptake and pathologic diagnosis among the precancers/ OSCC group, lesions neither premalignant nor malignant group, nor normal group were analyzed using Fisher’s exact test Wilson . A p val120 subjects  were enrolled in this study. The patient’s ages (patient group) ranged from 15 yrs to 80 yrs, with the ratio of male to female being 5:1. Both the groups gave history of tobacco consumption. The suspected lesions were distributed over the buccal mucosa (n = 84), palate (n = 6), tongue (n = 4), floor of the mouth (n = 2). In the control group, as methylene blue dye was not used to examine the oral cavity, it was necessary to verify that the dye would not be retained on normal mucosa. The results demonstrated that there was no retained dye in the control group.The Clinical and Histological characteristics of the various study Groups are shown in Table Specificity suggests the proportion of pathologic benign lesions, neither precancerous lesions nor cancers, which are correctly identified as negative staining of methylene blue. In our study, 4 out of 6 cases with no dysplasia or malignancy showed negative staining; thus, the specificity was 66.6%. The results of staining with methylene blue for all lesions correlated well with the pathologic diagnosis and are summarized in Table Out of 100 lesions, 88 (92%) lesions retained methylene blue stain while 12 (8%) lesions failed to retain the stain. Out of these 88 methylene blue stained lesions, 86 were histologically proved as having either dysplastic or carcinomatous change, while 2 (2%) were diagnosed as hyperkeratosis without dysplasia / no evidence of malignancy. Out of 12 unstained lesions 8 were histopathologically diagnosed as carcinomatous while 4 were diagnosed as hyperkeratosis without dysplasia / no evidence of malignancy respectively. The sensitivity of methylene blue in determining dysplastic and carcinomatous changes was determined as 91.4% while the specificity was determined as 66.6%, the positive predictive value 97.7%., the negative predictive value 33.3%. The overall diagnostic value of methylene blue stain in distinguishing premalignant and malignant lesion 90% The p value less than <0.001 which is indicates that these results are stastically very significant.Oral Cancer is very common in a country like India due to habits of smoking and chewing tobacco containing pan masala. Tobacco consumption is the main etiologic factor inducing carcinogenesis in oral mucosa. WHO indicates a 500 patients increase in cancer by 2025 of which 220 will be due to tobacco use. A survey by WHOSEA  has indicated that almost 50% of oral cancer in men and 25% cases of women in India are believed to be tobacco-borne  false positives were 0 and specificity was 100% although our control group was having normal mucosa but the flaw in our experimental design was that these subjects were not having any type of tobacco chewing or smoking habits, as we cannot screen individuals with these habits and no lesions because performing biopsy in normal mucosa will be unethical. Thus we can say that this study has established methylene blue as a diagnostic agent  for early detection of cancerous and precancerous lesions. This dye will be convenient to substitute for toluidine blue because of its non-toxicity and low cost. However pathology report based on biopsy will always remain gold standard to exactly and accurately diagnose the lesion before a treatment modality is determined Mashberg .Staining should be routinely used as a method to assist the choice of biopsy site and in the follow up of premalignant lesions and in the experienced hands marginal demarcation of the malignant lesions enables an intervention method to be adopted earlier for the diseases, which carries a high rate of morbidity and mortality. Further study is recommended to study the exact binding mechanism of methylene blue in precancerous, cancerous, benign and normal mucosa to establish this dye as the efficient diagnostic tool for diagnosing and differentiating between different pathological conditions.


Background. Low-Risk Human Papillomavirus (LR-HPV) genotypes 6 and 11 cause genital warts. This study investigated the association of LR-HPV infection with male circumcision (MC). Methods. We used data from the South African MC trial conducted among young men. Urethral swabs, collected among intervention (circumcised) and control (uncircumcised) groups, were analyzed using HPV linear array. Adjusted LR-HPV prevalence rate ratio (aPPR) and Poisson mean ratio (aPMR) of number of LR-HPV genotypes were estimated using log-Poisson regression, controlling for background characteristics, sexual behaviour, and HIV and HSV-2 statuses. Results. Compared to controls, LR-HPV prevalence and mean number of genotypes were significantly lower among the intervention group  and , resp.). Mean number of LR-HPV genotypes increased with number of lifetime sexual partners and decreased with education level and consistent condom use. Conclusions. This study shows a reduction in LR-HPV infection among circumcised men. Human Papillomavirus (HPV), a small DNA virus, is the most common sexually transmitted infection in the world. HPV prevalence in Africa is disproportionably high, ranging from 22.1% to 46.0% among women . More thLow-risk HPV (LR-HPV) genotypes 6 and 11 cause around 90% of genital warts (GWs), also called condylomata acuminate . In Sub-There is evidence that multiple HPV infection may have important clinical and epidemiological implications. Infection with multiple HPV genotypes has been associated with concurrent anogenital warts –13, acquHence, the analysis of LR-HPV infection in terms of number of LR-HPV genotypes detected in urethral swabs is important since being infected with one genotype may be different from being infected with two or more genotypes.A recent observational study has suggested that HPV prevalence was reduced among circumcised men compared to uncircumcised men , whereasThe primary objective of this study was to investigate the effect of MC on LR-HPV infection by considering both LR-HPV prevalence and the number of LR-HPV genotypes detected among young men. The secondary objective was to identify other risk factors of LR-HPV infection. Data used were collected during the MC randomized controlled trial (RCT) conducted in Orange Farm (South Africa), which demonstrated a partially protective effect of MC on the acquisition of HIV .Technical details of the Orange Farm MC RCT (ANRS-1265) have been published elsewhere . The triThe current study uses data collected during the 21-month visit. Participants were included in the trial regardless of their HIV and HSV-2 status. The sample includes all the men, among the trial participants, who reported for the 21-month follow-up visit between March 7th, 2005 and November 24th, 2005, and among whom urethral swabs were collected during this period. The selection has been described in further details in a previous publication . Five huμL of the kit lysis buffer for 30 minutes at room temperature. The MagNa Pure external lysis protocol was used to extract DNA from the lysis buffer into a 100 μL eluate. 50 μL of the eluate was used for screening (Roche Amplicor HPV test), and the other 50 μL eluate was used for genotyping (Roche Linear Array Genotyping test). This standardized PCR-based method can detect 37 genotypes of HPV [Detailed laboratory methods for HIV-1 and HSV-2 testing have previously been reported , 30. Ures of HPV , 32. FifRoche Linear Array Genotyping test was used to detect 23 HPV genotypes which are classified as nononcogenic. In this paper, all of the following 23 genotypes are characterized as LR-HPV: 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 67, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6108. A more conservative approach was used, which considered a subset of 11 genotypes , excluding any genotype having been labelled as of intermediate risk in the literature or not listed in the LR-HPV genotypes list –35.We used multivariate log-Poisson regression to analyze both LR-HPV positivity and number of LR-HPV genotypes detected for each participant. Results are formulated in terms of prevalence rate ratios (PRRs) of LR-HPV positivity and Poisson mean ratio (PMRs) of the mean number of LR-HPV genotypes, respectively, comparing men of the intervention group with men of the control group. The intention-to-treat (ITT) and as-treated (AT) adjusted PRRs (aPRRs) and PMRs (aPMRs) were estimated controlling for the following covariates: ethnic group, age, education, number of lifetime sexual partners, condom use in the past 12 months, as well as HIV and HSV2 statuses at the 21-month visit. Analyses were repeated using as an outcome the subset of 11 LR-HPV genotypes. Because HIV is reduced by MC and is associated with HPV infection , we assehttp://www.clinicaltrials.gov/ under the number NCT00122525.The trial was conducted with the understanding and the written consent of all participants. The research protocol was reviewed and approved by the University of the Witwatersrand Human Research Ethics Committee  on February 22nd, 2002 (protocol study no. M020104). The trial was also approved by the Scientific Commission of the French National Agency for AIDS Research , and authorization was obtained from the City of Johannesburg, Region 11, on 25 February 2002. This trial has been registered at The 1753 participants from whom a urethral swab was collected at the 21-month visit and analysed were included in the analysis. The mean (median) durations in days of follow-up among the intervention and control group were 653 (637) and 648 (637), respectively. P = .04) and reported more frequently having had a sexual partnership in the last year. Level of education, living with a partner, number of lifetime sexual partners, and consistent condom use were not statistically different between the two groups. HIV and LR-HPV prevalences at the 21-month visit were significantly lower in the intervention group in comparison with the control group. The mean number of LR-HPV genotypes was 0.25 (95%CI: 0.21–0.30).ρ = 0.67, P < .001). Among HR-HPV-infected men, 56.2% (95%CI: 50.7–61.6) were infected with LR-HPV. Among LR-HPV-infected men, 85.9% (95%CI: 81.1–90.6) were infected with HR-HPV. The number of HR-HPV and LR-HPV genotypes were correlated .The prevalence of LR-HPV infection was significantly lower among intervention group participants compared to control group participants .Results remained unchanged when the more restrictive definition of 11 LR-HPV genotypes was considered, with a twofold decrease of the mean number of LR-HPV in the intervention group compared with the control group . Prevalent HIV and HSV-2 infections at the 21-month visit were associated with an increased number of LR-HPV genotypes . Conversely, mean number of LR-HPV genotypes was lower for participants having completed the primary level of education  and those reporting consistent condom use . Mean number of LR-HPV genotypes significantly increased with the number of lifetime sexual partners  were excluded from the analyses, the effect of MC on number of LR-HPV remained unchanged . This implies that the effect of MC on LR-HPV is independent of the effect of MC on HIV.When participants who HIV seroconverted during follow-up . This suggests that a possible imbalance between groups due to a differential follow-up had no effect on the association of LR-HPV infection with MC.The aPMRs were almost identical when the analyses were adjusted for the propensity score in addition to the other covariates , we found a strong independent association between LR-HPV urethral infection and MC. LR-HPV infection was analysed as a prevalence rate and in terms of number of LR-HPV detected in urethral swabs, because it was assumed that being infected with one genotype was different from being infected with two or more genotypes , 13, 20.The mechanisms by which circumcised men are less likely to be infected with HPV could be due to a reduced acquisition of new infection or an increased clearance of pre-existing infection, because the absence of foreskin may reduce the risk of autoreinfection at the urethral site . This study has some limitations, primarily the lack of data on HPV status at baseline, so the causality of MC on reduced LR-HPV infection cannot be rigorously demonstrated. However, MC was randomly assigned, and controlling for the propensity score didnot affect the results. A second limitation is that participants were not blinded, so they might have changed their sexual behaviour according to their randomization group. Lastly, detection of LR-HPV was performed on urethral swabs, which are known to miss infections compared with detection in the glans, corona sulcus, or penile shaft , 39. ThuTo date, little was known on the association of MC with LR-HPV. Few observational studies have studied this association but results remained inconsistent , 40, 41.In accordance with observational studies, our study reveals some other risk factors of LR-HPV, such as the number of lifetime sexual partners , 42, 43 MC, an immediately available and cost-effective method, was proven to reduce HR-HPV infection , 27. Hen


Oncogenic human papillomavirus (HPV) infection prevalence is required to determine optimal vaccination strategies. We systematically reviewed the prevalence of oncogenic cervical HPV infection among Canadian females prior to immunization.We included studies reporting DNA-confirmed oncogenic HPV prevalence estimates among Canadian females identified through searching electronic databases  and public health websites. Two independent reviewers screened literature results, abstracted data and appraised study quality. Prevalence estimates were meta-analyzed among routine screening populations, HPV-positive, and by cytology/histology results.Thirty studies plus 21 companion reports were included after screening 837 citations and 120 full-text articles. Many of the studies did not address non-response bias (74%) or use a representative sampling strategy (53%).Age-specific prevalence was highest among females aged < 20 years and slowly declined with increasing age. Across all populations, the highest prevalence estimates from the meta-analyses were observed for HPV types 16  and 18 . The highest prevalence occurred among HPV types 16 and 18, contributing a combined cervical cancer prevalence of 65.9%. Further cancer protection is expected from cross-protection of non-vaccine HPV types. Poor study quality and heterogeneity suggests that high-quality studies are needed. Human Papillomavirus (HPV) is one of the most prevalent sexually transmitted infections in the world . Over 10For nearly half a century, cervical cancer prevention programs consisted mainly of cervical cancer screening for early detection of pre-cancerous lesions . AlthougBaseline prevalence data is necessary to inform optimal prevention programs and evaluate current and future prevention strategies ,22. SincA systematic review protocol was compiled based on guidelines from The Cochrane Collaboration  and the Studies reporting DNA-confirmed  or polymerase chain reaction (PCR)) HPV prevalence estimates among Canadian females who were not previously vaccinated against HPV were included. Authors of studies not reporting the sample size were contacted and the study was excluded if the sample size could not be obtained. Inclusion was not limited by study design, publication status, year of dissemination or language of dissemination.In order to ensure reliability, a training exercise was conducted prior to commencing the screening process. Two independent reviewers screened the search results for inclusion using a pre-defined relevance criteria form, obtained the full-text of potentially relevant articles and screened them to determine inclusion. Discrepancies were resolved by discussion or the involvement of a third reviewer.Medical Subject Headings and text words related to the prevalence of HPV cervical infection among Canadian females were used to search MEDLINE , EMBASE , The Cochrane Library (Wiley interface), and POPLINE . The electronic database search was supplemented by conducting a targeted search of Canadian public health websites , websites of organizations that produce guidelines , and vaccine manufacturer websites . Furthermore, general Internet searches were conducted in Google and the first 60 unique hits were scanned. In addition, the reference lists of included studies were scanned, the authors' personal files were searched, and HPV experts in public health and industry were emailed to ensure that all potentially relevant data was obtained.An experienced information specialist conducted all of the literature searches. The search strategy for the main (MEDLINE) search is presented in Additional File A draft data abstraction form was developed, piloted, and modified as necessary. Two reviewers abstracted all of the data using the standardized data abstraction form, independently. Discrepancies were resolved by discussion or the involvement of a third reviewer.The data abstracted included study characteristics, , participant characteristics , HPV detection method , PCR primers , number of HPV positive, as well as the overall HPV prevalence. As done in previous reviews of HPV prevalence, -28 we foFour of the included studies directly compared prevalence results for different tests  or methods  -32. For A commonly used and validated quality tool for observational studies does not exist . As suchStudies reporting the age-specific prevalence of HR HPV infection were plotted and variation due to timing of data collection, HPV detection methods, and study location was noted. In order to ensure consistency across studies, only studies that collected samples in routine screening  were included in the plot. These results were described narratively and not combined via meta-analysis to view the wide heterogeneity observed across studies.HPV prevalence among routine screening populations was defined as the proportion of individuals who were positive for HR HPV infection divided by the total population tested for HPV infection. HPV type-specific prevalence among routine screening populations was defined as the proportion of females testing positive for the specific HR HPV type among all of those testing for HPV infection. These analyses only included studies of females engaged in routine screening.HR HPV prevalence and HR HPV type-specific prevalence was also calculated for those testing positive for HPV infection. These analyses included studies of females engaged in routine screening, as well as those returning after a previous abnormal cytological test .HPV type-specific prevalence by cytology or histology was defined as the proportion of individuals testing positive for the specific HR HPV type broken down by the cytological and/or histological category according to the 2001 Bethesda System . Thirty study reports ,35,38-62Data were collected from 1960  to 2007 Overall, 74% 17/23) did not address non-response bias, 53% (16/30) did not use a representative sampling strategy , 17% (5/30) did not use adequate timing of sampling , and 17% (5/30) did not use sensitive sampling techniques (e.g. hybridization). Seven studies out of 30 were not relevant to the non-response bias and response rate methodological quality items because they included data from cancer biopsies  and the prevalence for each of the other HR types was less than 5%. Among those classified with LSIL, the three most prevalent types were HPV-16 (28.3%), HPV-31 (7.3%), and HPV-18 (5.8%). Among those classified with HSIL, the three most prevalent types were HPV-16 (54.0%), HPV-31 (14.2%), and HPV-18 (7.9%). Among those with confirmed cervical cancer, the highest prevalence was observed for HPV-16 (48.8% [95% CI: 34.0-63.6%]) ,43,61, fWe conducted a post hoc sensitivity analysis of the meta-analysis results for key study characteristics including study location, study design, HPV detection method, sample size, and methodological quality. Differences in prevalence estimates across the included studies were not identified (data not shown).To our knowledge, this is the most comprehensive systematic review for a single country. Other HPV prevalence reviews included worldwide data and grouped all countries in North America together ,28,65. THPV prevalence data are required to provide information related to baseline HPV burden of disease when implementing vaccination programs. All of the included studies were either conducted prior to HPV vaccination or did not include vaccinated individuals. These data can be used to evaluate current HPV vaccination program in the future, including vaccine impact on HR genotype prevalence. The data are also useful for health economic evaluations and epidemiological modeling research. HPV is not a reportable disease so baseline prevalence data could only be obtained by synthesizing highly heterogeneous individual studies. A limited number of studies provide population-based estimates of HPV prevalence and only one provincial based study  was avaiThe results can inform optimal vaccination program scheduling. Our findings indicate that HPV prevalence was highest among females aged less than 20 years. Some countries (such as Canada) have publicly funded vaccination programs targeting pre-adolescent and adolescent girls and our results support this approach. High age-specific HR HPV prevalence up to the age of 30 years stresses the need of evaluating the impact of catch-up vaccination programs in these older age groups.Our results indicate that the prevalence of HR HPV varies among different subgroups of the population with indications of higher prevalence in those at greater risk of being under-screened. In our review, socially disadvantaged individuals  had the greatest HPV prevalence. Although declines in incidence and mortality due to routine screening have occurred,  false-neThe HPV vaccines protect against oncogenic strains HPV-16 and -18. These two HPV types contributed a combined prevalence of 12% for HR HPV infection and 66% of cervical cancer, which potentially can be prevented by the vaccines assuming 100% efficacy and long-lasting immunity. These estimates are slightly lower than previous reviews that reported this estimate as being approximately 70%,  indicatiOur results are similar to previous reviews of HPV prevalence. The overall HR HPV prevalence observed in our systematic review was 27.1% among screening populations, which is lower than the prevalence for any HPV infection in HIV positive women (36.3%) reported in a previous review, as expected . In addiWe found that multiple infections occurred in 17.1% of the included prevalence estimates. Multiple infections occurred in 11.2% of the prevalence estimates in a review of invasive cervical cancer  and 11.9Compared to different regions throughout the world, HPV prevalence in Canada is moderate. One review found that the highest prevalence of HPV among middle-aged women (35-50 years) was observed for Africa, Central and South America, and the United States (approximately 20%), while a lower prevalence was observed in Asia, Australia, Europe, Middle East, and Canada (approximately 15%) . FurtherLimitations of this systematic review include that the meta-analyses were based on each HPV type individually so may include concomitant infection. Data were also pooled across studies using different tests to measure the presence of HPV  and these tests vary in their sensitivity and specificity. For example, HC-II has been found to be a more sensitive test versus HC-I . The metPopulation-based, high-quality, HPV prevalence studies are warranted. These studies should report age-specific prevalence, overlap between single and multiple infection, and HPV type-specific infection. A recently published prevalence study from the United Kingdom is an excellent example of such a study . These dPrevalence data of cervical human papillomavirus infection is necessary to inform optimal prevention programs and evaluate current prevention strategies. Previous HPV prevalence reviews included worldwide studies and focused on HPV prevalence among cervical cancer, HIV or healthy individuals. To our knowledge, this is the most comprehensive systematic review for a single country and the data can be used in health economic evaluations and epidemiological modeling research to help inform public health policy. Our results show that the highest prevalence occurred among females < 20 years of age, yet prevalence remained high among women up to 30 years of age.ACT, CHN, and SC have been paid consultants for vaccine-related products from GlaxoSmithKline, Canada. VG has received consultancy fees from GlaxoSmithKline and Merck Frosst Canada. AA is an employee of GlaxoSmithKline Biologicals. The funding agreement ensured the authors' independence in designing the study, interpreting the data, writing, and publishing the report.ACT designed and coordinated the study, participated in data collection and analysis, and drafted the manuscript. CHN participated in data collection and analysis and helped to draft the manuscript. VG helped to draft the manuscript. AA helped to draft the manuscript. BP conceived of the study, participated the study design and coordination, and helped to draft the manuscript. SB participated in data collection and analysis and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/235/prepubSearch strategy for MEDLINE. Search strategy used in systematic review.Click here for fileMethodological quality and risk of bias tool. Tool used to assess methodological quality of studies included in systematic review.Click here for fileBethesda classification system. Classification system for cervical cancer used in analysis of results.Click here for fileMethodological quality of the included studies. Results of the methodological quality tool.Click here for file


Human papillomaviruses (HPVs) are responsible for an enormous global burden of genital disease. HPV is annually associated with 500,000 new cases of cervical cancer and 250,000 cervical cancer deaths worldwide. There are more than 130 HPV genotypes that have been recognized from various clinical lesions. HPV types 16 and 18 are found in the majority of cervical cancer cases. The association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. HPV genome codes eight early or regulatory proteins (E1-E8) and two late or capsid proteins (L1/L2). Two HPV oncogenic proteins, E6 and E7, are consistently co-expressed in HPV-expressing cervical cancers and are important in the induction and maintenance of cellular transformation. Therefore, immunotherapy targeting E6 and/or E7 proteins provides an opportunity to prevent and treat HPV-associated cervical malignancies. Effective therapeutic HPV vaccines should generate strong E6/E7-specific T cell-mediated immune responses. Currently, we have focused on different vaccine modalities including DNA vaccines, protein vaccines, live vaccines and the combined approaches  against HPV infections. In our studies, various strategies were applied to enhance DNA vaccine potency including the utilization of adjuvants especially heat shock proteins  and delivery systems such as polyethyleneimine (PEI), polymer-peptide hybrid (PEI600-Tat conjugate) and also electroporation. At first, the level of humoral and cellular immune responses were compared by using HPV16 E7 + Gp96 co-injection as DNA/DNA and prime-boost (DNA/ protein) immunization strategies in C57BL/6 mice model. Assessment of cellular immune responses against E7 antigen indicated that co-delivery of naked DNA E7 + Gp96 plasmid is immunologically more effective than E7 alone and induces Th1 response [in vivo. Indeed, PEI600-Tat/E7DNA complex at certain ratio induced IFN-γ response slightly more than that GP96 adjuvant [Leishmania tarentolae, as a recombinant live HPV vaccine candidate expressing HPV16 E7 and evaluate its ability to elicit E7-specific T cell responses in mice model as compared to E7 DNA vaccine. Moreover, a preventive and therapeutic DNA vaccine has been generated using HPV16 E7 co-linked to N-terminal fragment of GP96 and chemokines delivered by chemical system (PEI600-Tat conjugate) and/or physical method (electroporation). Altogether, combining new and improved immunotherapeutic strategies are crucial for enhancing long-term immune responses in patients.Furthermore, in another study we demonstrated that PEI600-Tat conjugate is efficient to improve immune responses adjuvant . In our


Cervical cancer lesions represent a major threat to the health of the women worldwide. Human Papillomavirus (HPV) is responsible for 99.7% of cervical cancer cases, the infectious etiology giving the possibility of preventing cervical cancer by vaccination. The most aggressive HPV types are 16 and 18, which cause about 70% of cases of invasive cancer. The vaccination is recommended to the girls aged 11–12. The diagnosis and the treatment of cervical preinvasive disease allow the doctor to prevent the development of the invasive disease. Cervical cancer and precancerous cervical lesions constitute a major health problem for the women. The clinical and molecular studies have identified the HPV as the main cause of cervical preneoplastic lesions and cervical cancer. Almost all cervical cancers contain genetic material from the high–risk HPV types. The screening has dramatically reduce the incidence of the cervical cancer. However, in the developing countries where the access to the medical services is poor, the cervical cancer has a very high incidence, being in the second place as a cancer related cause of death . The treThe history of the HPV began in the seventies when zur Hausen isolated the type 6 from the warts lesions. Since then, HPV is recognised as the etiologic agent of the cervical cancer. Until now, there are over 40 viral strains implicated in the pathogenesis of cervical cancer.HPV belongs to the family of the Papovaviridae. The viral infection is a necessary factor for the appearance of the cervical neoplasia, but it is not enough, requiring also particular host and environmental factors. The HPV infection seems to be implicated in 99.7% of the cervical cancer. Virtually all cervical cancers (over 99%) contain the genes of high–risk HPV, especially 16, 18, 31 and 45. Most of HPV infections regress spontaneously, especially those in adolescents and young women .There are over 200 types of HPV, divided in three categories: the high–risk types (generating HG–SIL lesions and invasive neoplasia)–16, 18, 31, 33, 35, 45, 52, 56, 58, 59, probably high–risk types– 26, 53, 66, 68, 73, 82 and low–risk –6, 11.The vaccine against the infection with HPV has made possible the cervical cancer primary prophylaxis. The secondary prophylaxis was found also very effective. The cervical dysplasia cure is accurately assessed by the combination of negative PAP smear and negative DNA–HPV test.The majority of HPV infections are subclinical and transitory (>80% disappear within 2 years) due to the cellular immune response. After infection, HPV is latent for 2–12 months. The cellular immune response starts in about 3 months from inoculation of virus and eliminates or suppresses it down to undetected levels.In some subjects, the infection can cause warts (condyloma) and low–grade cervical lesions. Sometimes, in certain cases, the high risk HPV types 16, 18, 31, 45) can be persistent and progress to high–grade lesions and cervical cancer in a few years [1, 45 canHPV infection means the viral particles replicate in the mature keratinocytes. Active infection occurs when the virus invades the basal epithelial layer. Persistency of infection means the HPV infected the stem epithelial cells. Viral replication needs the oncoproteins E6 and E7 which bind the tumor supressor genes p53 and pRB. The initiation of oncogenic process begins when viral DNA was integrated in the host genome . Viral tUntil the age of 50, it is estimated that over 80% of sexually active women became HPV infected at some moment in their lives. Approximately 75% of the new HPV infections appear between the ages 15 and 24. Most of these infections are transient and disappear spontaneously . HIV coinfection, which suppresses the cellular immune response, represents a severity factor. The time from the first HPV infection, persistency, preneoplastic lesions and cancer is estimated to be 13–15 years.Diagnosis of preneoplastic lesions is based on cytology, colposcopy and the final diagnosis is established by histopathological examination of the tissue sample.The Bethesda System and its 2001 revisions aim to simplify Papanicolaou (Pap) smear reporting and to make it more reproducible. The main types of preneoplastic lesions described are: atypical squamous cells of undetermined significance (ASCUS); atypical squamous cells, cannot exclude high–grade squamous intraepithelial lesion (ASC–H); low–grade squamous intraepithelial lesion (LSIL), high–grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma, atypical glandular cells (AGC), endocervical adenocarcinoma in situ (AIS) and adenocarcinoma  Table .Acetowhite changes represents the white zone that appears after treatement of the cervix with acetic acid (3–5%). The acetic acid does not affect the mature epithelium rich in glycogen, but colours the dysplasic epithelium in white because of the high protein content. The metaplasic epitelium is very thin–unlike CIN which becomes white–so it will become gray and translucent. Leukoplakia (white epithelium after acetic acid aplication)–hyperkeratosis–is the most frequent cause of HPV infection. Punctations (a zone of red dots–dilated capilaires which ends to the surface) in well circumscribed areas indicates an abnormal epithelium, most frequent CIN. Mosaicism  is associated with high grade lesions–CINⅡ/Ⅲ [Colposcopy identifies a series of lesions associated with preneoplastic cervical lesions . –CINⅡ/Ⅲ .In mild dysplasia (CIN Ⅰ) only few cells in the basal third of the epitelium are abnormal, while in moderate dysplasia (CINⅡ) the abnormal cells involve about 2/3 of the thickness of the surface lining of the cervix. In severe dysplasia or carcinoma in–situ (CINⅢ) the entire thickness of the basal epithelium is abnormal. Spontaneous regression rate of CIN Ⅰ is about 60–85%.The regression usually solves in about 2 years. All CINⅡ and CIN Ⅲ lesions need treatment. The progression CINⅡ to CIS is 20% and invasion in about 5% of patients .The characteristic of the cervical cancer is its infectious etiology and the possibility of preventing the cancer through vaccination against HPV, the central factor implicated in pathogenesis.Speaking about prevention strategies, we can separate them in three categories:Primary prevention –prophylactic vaccination and modification of risk factors .Secondary prevention –early detection and treatment of the preneoplastic lesions .Tertiary prevention–cervical cancer treatment and postsurgical follow–up .Two vaccines are at the moment licensed in USA and Europe: a bivalent one and a tetravalent one. Both of them are using the same principle: they contain particles identical to the viral capsid, without any viral DNA, lacking the infectivity or the oncological risk. The empty protein shells are composed of major L1 capsid proteins which are specific for each HPV type . The tetThere are a few categories for whom the management is a little bit different because of their characteristics: adolescents (aged <20), pregnant women, postmenopausal women and immune–compromised women. Adolescents have a higher prevalence of the HPV infection, more frequent minor lesions and a very low risk of cervical invasive cancer compared to older patients, because most of the HPV infections disappear spontaneously in 2 years of time and have little short term significance. Therefore, the colposcopy should not be performed as a first line investigation. During pregnancy, endocervical curettage is forbidden, while the colposcopic guided biopsy is indicated for HSIL or when invasive cancer is suspected. Colposcopy is a reasonable investigation for the low risk pregnant women .High risk DNA–HPV testing, repeated cervical smear and colposcopy are all acceptable methods in the management of ASC–US lesions for the patients aged over 20 years. The patients ASC–US and DNA–HPV negative for the high risk types will be followed up at 12 months. Patients with ASC–US and DNA–HPV positive should be treated as the patients with LSIL and evaluated by colposcopy. Endocervical curettage is preferred for the patients without colposcopic lesions or with unsatisfactory colposcopy. The management after colposcopy for ASC–US/DNA–HPV positive patients, which do not have CIN diagnosed, consists in repeating the DNA–HPV test every 12 months or repeating Pap smear every 6–12 months. DNA–HPV testing is not recommended earlier than 12 months. Repeating cytology at 6 months is indicated until two consecutive negative results are obtained, then normal follow–up is recommended . ColposcExcisional diagnostic procedures are not recommended in the treatment of the patients with ASC–US as an initial diagnosis, in the absence of CIN Ⅱ/Ⅲ histopathological diagnosis.The management of ASC–H implies the colposcopic evaluation from the beginning. If there is an evident colposcopic lesion, biopsy is recommended. For the patients without CIN Ⅱ/Ⅲ, the follow–up is acceptable by testing DNA–HPV every 12 months or repeating cytology every 6–12 months. If DNA–HPV testing is negative or two consecutive smears are negative, return to normal follow–up is recommended.Colposcopy is recommended from the beginning for LSIL cytology. Endocervical curettage is preferred for the patients without colposcopic lesions or with unsatisfactory colposcopy. After colposcopy, for the patients with LSIL without histopathological CIN Ⅱ/Ⅲ, the recommended attitude is DNA–HPV testing at every 12 months or repeated cytological at 6–12 months. If the DNA–HPV testing or two successive cytological results are negative, normal follow–up is recommended. If HPV DNA tesing is positive or at least one cytological testing result is ASC–US or higher, colposcopy have to be repeted.For HSIL lesions the risk for preinvasive disease is 70–75% and for invasive disease is 1–2% . So, colFractioned curettage  and colposcopy are recommended for the patients with AGC/AIS aged over 40 years old. Also endometrial curettage is to be done for the patients over 40 years old with risk factors for endometrial cancer. DNA–HPV testing is not routinely recommended. The most frequent viral types associated with endocervical adenocarcinoma are 16, 18 and 45.Precursor lesions of the cervical cancer can be treated through local distructive methods or excision. The local distructive methods  are no more a standard therapy because they do not allow the histopathological examination of the specimen.Excisional methods are loop/needle electrosurgical excision procedures, laser excision and cold knife excision. Loop electrosurgical excision (LEEP) can be done as an outpatient procedure, with local anesthesia. Needle electrosurgical excision of the transformation zone (NETZ) has the advantage of excising the lesion as a single piece, easier to be histopathologically evaluated and allowing the optimal tailoring of the specimen to be resected (height and wide) . The useCold knife conization has the advantage of preserving the specimen intact for the histopathological examination and allowing for a good evaluation of the tissue margins. Although the cure rate is the same as with the use of electrocautery, the frequency of complications is double.Total histerectomy, abdominal or vaginal, represents a very rare indication in the therapy of precursor lesions of the cervical cancer. It is the choice when the lesion wasn't totally resected, persistent abnormal Pap smear, lesion extended to the vaginal vault, associated gynecologic pathology, difficult follow–up after conization.


About 15 types of human papillomavirus (HPV) are classified as high-risk based on their epidemiological link with cervical cancer. These HPV types have deferent degrees of oncogenicity and their distribution among cervical precancers and cancers varies ethnogeographically. HPV58 is rare worldwide but being found more commonly in East Asia.A high prevalence of HPV58 among squamous cell carcinoma has been reported from China  and other countries in East Asia including Korea (16%) and Japan (8%). HPV58 ranks the third in Asia overall, but contributes to only 3.3% of cervical cancers globally. The reasons for a difference in disease attribution may lie on the host as well as the virus itself. HLA-DQB1*06 was found to associate with a higher risk of developing HPV58-positive cervical neoplasia in Hong Kong women, but not neoplasia caused by other HPV types. An HPV58 variant  commonly detected in Hong Kong was found to confer a 6.9-fold higher risk of developing cervical cancer compared to other variants. A study involving 15 countries/cities has shown a predilection in the distribution of HPV58 variant lineages. Sublineage A1, the prototype derived from a cancer patient in Japan, was rare worldwide except in Asia.HPV58 accounts for a larger share of disease burden in East Asia, which may be a result of differences in host genetics as well as the oncogenicity of circulating variants. These unique characteristics of HPV58 should be considered in the development of next generation vaccines and diagnostic assays. Human papillomavirus (HPV) plays a necessary, though insufficient, role in the development of cervical cancer, which is the third most common cancer in women worldwide, just following breast and colorectal cancers ,2. It haPapillomaviruses have a small double-stranded DNA genome of about 8 kb long. To date, more than 120 types of HPV have been well characterized, of which about 40 types can infect the genital tract . About 1®, GlaxoSmithKline Biologicals) [HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 are regarded as high-risk types [ogicals) . TherefoSince mid 1990s, a few studies have reported a higher prevalence of HPV58 among cervical squamous cell carcinoma cases in the southern and eastern parts of China -13. WithThe reason for a higher prevalence of HPV58 in East Asia is still not fully understood. The development of a clone of tumour cells from an HPV-infected epithelium is a multi-step process involving multiple factors in which host genetics is likely to be a main determinant. Our previous study on Hong Kong women has observed a positive association between HLA-DQB1*06 and HPV58-positive cervical intraepithelial neoplasia III/invasive cancers . Of noteWe have also observed that an HPV58 variant commonly found in Hong Kong was epidemiologically associated with a higher oncogenic risk . This vaWhile studies from Hong Kong suggest that the host genetic factors and oncogenicity of circulating variants may play a critical role in the screwed ethnogeographical distribution of HPV58, further large-scale studies incorporating data from places with high and low prevalence of HPV58 are crucial to verify the observation and further explore the association. In this regard, it is instrumental to have a universally accepted classification system for HPV58 variants. Recently, two independent studies have reached the same conclusion and set an essential backbone for future studies ,25. The The disease impact attributed to HPV58 varies ethnogeographically. The prevalence of HPV58 among cervical cancers found in East Asia is much higher than that reported from other parts of the world. This could be a result of differences in host genetic background as well as the circulation of HPV58 variants with different oncogenicity. These unique characteristics of HPV58 should be considered in the development of next generation vaccines and diagnostic assays.The author declared that he has no competing interests.


Balaenoptera musculus spp) are an endangered species found in the Southern and Indian Oceans. Here two recognized subspecies of blue whales and, based on passive acoustic monitoring, four “acoustic populations” occur. Three of these are pygmy blue whale (B.m. brevicauda) populations while the fourth is the Antarctic blue whale (B.m. intermedia). Past whaling catches have dramatically reduced their numbers but recent acoustic recordings show that these oceans are still important habitat for blue whales. Presently little is known about the seasonal movements and degree of overlap of these four populations, particularly in the central Indian Ocean. We examined the geographic and seasonal occurrence of different blue whale acoustic populations using one year of passive acoustic recording from three sites located at different latitudes in the Indian Ocean. The vocalizations of the different blue whale subspecies and acoustic populations were recorded seasonally in different regions. For some call types and locations, there was spatial and temporal overlap, particularly between Antarctic and different pygmy blue whale acoustic populations. Except on the southernmost hydrophone, all three pygmy blue whale acoustic populations were found at different sites or during different seasons, which further suggests that these populations are generally geographically distinct. This unusual blue whale diversity in sub-Antarctic and sub-tropical waters indicates the importance of the area for blue whales in these former whaling grounds.Understanding the seasonal movements and distribution patterns of migratory species over ocean basin scales is vital for appropriate conservation and management measures. However, assessing populations over remote regions is challenging, particularly if they are rare. Blue whales ( Balaenoptera musculus subspp., (Linnaeus 1758) was abundant in the Southern Hemisphere. In only a few decades, the largest living animal was hunted to the brink of biological extinction B.m. intermedia) and the smaller pygmy blue whale (B.m. brevicauda) B.m. indica, by some based on the original description by Blyth (1859). Although there is little morphometric evidence for this subspecies designation, the indica name should have precedence over brevicauda if all these populations are considered to be pygmy blue whales Prior to intensive commercial whaling, the blue whale, It is presumed that during the period of legal whaling in the Southern Hemisphere (1904–1964), most of the blue whales killed were the Antarctic subspecies. Pygmy blue whales were only discovered in 1959–60 Most of our current knowledge of blue whale distribution in the Indian and Southern Oceans is derived from long-term passive acoustic monitoring. Passive acoustic monitoring has been used successfully in many oceans of the world to study the presence, migratory movements and feeding behavior of blue whales e.g. . This meAntarctic blue whales are found largely south of 60°S in the austral summer where they feed, and then likely spread out over a broader range during the austral winter Pygmy blue whales are rare at latitudes south of 60°S Euphausia superba) exploit the presence of abundant phytoplankton near ice edges, continental shelves and dynamic frontal regions Blue whales are known to concentrate in regions of high primary and secondary productivity year-round The Indian Ocean is clearly important habitat for at least four blue whale populations but little is still known about the seasonal movements and degree of overlap of these populations, particularly in the center of the Indian Ocean. In this study, we analyzed more than one year of passive acoustic recording from each of three sites located at different latitudes of the Indian Ocean basin to examine the geographic and seasonal occurrence of different blue whale acoustic populations to better understand their distribution.Three autonomous hydrophone packages were deployed near the French territories in the Southern and Indian Oceans from October 2006 to January and April 2008 Blue whales worldwide produce stereotyped calls with high intensity, low frequency and long duration. They also produce downswept short duration (1 to 4 s) and frequency modulated calls (90 to 25 Hz) named D calls www.birds.cornell.edu/brp/). The Antarctic blue whale call template consisted of one complete call. Only single units of each pygmy blue whale calls were used in those templates because they were usually above ambient noise level. We selected the third unit of the Sri Lanka call; the second unit of the Madagascar call, and the harmonic at 70 Hz of the second unit of the Australian call . In 2007, 4,532 calls were detected at MAD from March through June, with peaks in April and May, while only half as many calls were detected at SWAMS from December to May . Madagascar calls predominantly occurred in autumn . The distribution of call detections by season was quite different between the two sites: at NEAMS nearly 68% of calls were detected in winter while at SWAMS over 70% were detected in autumn  and for relatively short periods of the year. In 2007, four times more calls were detected at SWAMS  than at NEAMS , and over a longer period, January to June versus March to June, respectively . The din autumn . No AustThe vocalizations of the different blue whale subspecies and populations were recorded seasonally in three different areas of the Indian Ocean. For some call types and locations, there were spatial and temporal overlaps, particularly between Antarctic blue whale calls and the different pygmy blue whale calls. Except on the southernmost hydrophone (SWAMS), the different calls of the three pygmy blue whale acoustic populations were found at different sites or during different seasons, further suggesting that these populations of pygmy blue whales preferentially occupy different regions of the Indian Ocean, in addition to being acoustically distinct. Overall, when placed in the context of previous work , the results here show the distribution of subspecies and populations at basin scales and emphasize the importance of the Indian Ocean for blue whales .Historically, Antarctic blue whale distribution has been defined by a migration pattern between high-latitude summer feeding areas and low-latitude wintering areas Sri Lanka blue whales are presumed to be resident in the northern Indian Ocean Madagascar calls were only recorded in autumn and winter months at the westernmost station (MAD) and from late summer through winter months at the southernmost station (SWAMS). This seasonal pattern of Madagascar calls matches all other reports of acoustic detections in the Indian Ocean to date . MadagasAustralia-type calls were recorded at relatively few locations in the Indian Ocean . No AustThere were similarities in the seasonal detections of Australia and Madagascar pygmy blue whale calls, but they appeared to be geographically separated between the eastern and western Indian Ocean basins, respectively, as previously suggested . Both MaBecause we detected individual calls, rather than using an energy ratio method, which detects the acoustic energy from an unknown number of calls, it is likely that the detected calls were from animals that were relatively close to the instruments. In a comparison of the two methods on a same data set, Širovic et al (2009) In the vicinity of the southernmost station (SWAMS), four acoustic populations of blue whales use these sub-Antarctic waters at different times of the year, but sometimes overlap in space and time. Madagascar and Australia pygmy blue whales co-occur at sub-Antarctic latitudes during the end of summer and autumn months. This is similar to the pattern reported in the southwest part of the basin One of the biggest gaps in knowledge in blue whale acoustic ecology is a lack of understanding of the relationship between the number of calls and the number of calling whales. An increase in the number of calls from one month to another may be the result of an increase in the number of calling whales, or that calling whales stay in the detection area during a longer period of time, or that individual whales may increase their call rates seasonally. Regardless, the high number of calls highlights the importance of the sub-Antarctic Indian Ocean for Antarctic blue whales either as a wintering ground or as a final destination before moving southwards to the ice edge of the Antarctic continent.The easternmost station (NEAMS) is roughly 1500 km to the northeast of SWAMS, at the edge of the northern sub-tropical front. Here, the detection patterns of blue whale calls are clearly different. The acoustic diversity was less since no Madagascar pygmy blue whale calls and only few Australia type calls were recorded. However, some Sri Lanka type calls were present here year-round, particularly during austral summer. This area could be a possible wintering ground for this population in addition to that hypothesized for Crozet Islands The northernmost station (MAD) is in sub-tropical waters 2400 km away from the southernmost station and 2500 km west of the easternmost station. The acoustic diversity here is generally poor. This area is not exploited by Australia pygmy blue whales, and perhaps the instrument is at the edge of the distribution of this population. More surprisingly, no Sri Lanka pygmy blue whale calls were detected here; this population may be distributed in a more northerly and easterly manner in the Indian Ocean. Madagascar pygmy blue whales were detected exclusively during autumn months. Unsurprisingly, fewer Antarctic blue whales were detected in these sub-tropical latitudes although the number of calls increased during winter months. During winter, Antarctic blue whales may move northwards and are widely dispersed at different latitudes in the Indian Ocean. However, the year-round presence of calls, especially in this area, far away from the main Antarctic feeding grounds, suggests that some individuals remain at lower latitudes possibly to exploit food resources present in the region. It is also possible that individuals are moving between the Antarctic and sub-Antarctic within a season such that, rather than year-round occupancy of some individuals, there is year-round occupancy of some part of a broader population. The age, reproductive status or individual condition may explain these geographic differences but there are no data to support this at present.Blue whale sightings in the Indian Ocean are rare and difficult to obtain, and long-term passive acoustic recorders provide most of the recent information about the seasonal occurrence and movements of these animals over this remote and large area. As more information from long-term passive acoustic recorders in other regions and over longer time periods is gathered, the importance of the Indian Ocean as year-round habitat for at least four acoustic populations of blue whales, including the Critically Endangered Antarctic blue whale


Currently, population pharmacokinetic (PK) studies of anti-malarial drugs are designed primarily by the logistical and ethical constraints of taking blood samples from patients, and the statistical models that are fitted to the data are not formally considered. This could lead to imprecise estimates of the target PK parameters, and/or designs insufficient to estimate all of the parameters. Optimal design methodology has been developed to determine blood sampling schedules that will yield precise parameter estimates within the practical constraints of sampling the study populations. In this work optimal design methods were used to determine sampling designs for typical future population PK studies of dihydroartemisinin, the principal biologically active metabolite of oral artesunate.Optimal designs were derived using freely available software and were based on appropriate structural PK models from an analysis of data or the literature and key sampling constraints identified in a questionnaire sent to active malaria researchers . The derived optimal designs were then evaluated via simulation-estimation.The derived optimal sampling windows were 17 to 29 minutes, 30 to 57 minutes, 2.5 to 3.7 hours and 5.8 to 6.6 hours for non-pregnant adults; 16 to 29 minutes, 31 minutes to 1 hour, 2.0 to 3.4 hours and 5.5 to 6.6 hours for designs with non-pregnant adults and children and 35 to 59 minutes, 1.2 to 3.4 hours, 3.4 to 4.9 hours and 6.0 to 8.0 hours for pregnant women. The optimal designs resulted in acceptable precision of the PK parameters.The proposed sampling designs in this paper are robust and efficient and should be considered in future PK studies of oral artesunate where only three or four blood samples can be collected. Though lts from -24 were lts from  appearedlts from  and [24]lts from  and [24]ted from  and [24]ted from  and [24]lts from ,24 and [et al. [falciparum malaria. The BSV for V/F was extremely large, which is likely due to V/F soaking up the between-subject variability of ak, which was not estimated. To account for at least some between-subject variability of a kin the design, the BSV of a kwas set to that of the non-pregnant adults. To compensate for the inclusion of the BSV for ak, the population value for the BSV of V/F was reduced. It is therefore possible that alternative sets of PK parameter values exist for this group, so as previously noted sampling should be varied within the windows and as more PK data become available from pregnant women the optimal design will be re-evaluated. In addition, if the investigator expects to use an analytical method for measuring DHA concentrations that is less sensitive than that used in [The pregnant women design was based on models obtained from McGready et al. , which w used in , it is rFor all designs, the empirical %RSEs were consistently higher than the expected %RSEs from POPT. This observed difference may have been due to the way covariates (i.e. weight and age) were dealt with in POPT and the simulation-estimation procedure. In POPT, likely values for age and weight were assumed (i.e. weight for the adult designs and a range of age/weight combinations for each age group of the design with children). However, for the simulation-estimations, distributions were assumed for age and weight based on these likely values. Though this difference in procedures may have contributed to the consistently higher empirical %RSEs, the empirical %RSEs were still acceptable for both the PK parameters (<25%) and BSVs (≤55%). Furthermore, the %RSEs from the population Fisher Information matrix are lower bounds, so it is not surprising that the empirical %RSEs were larger. Therefore the derived optimal designs should be robust to plausible values of age and weight, and as more information is collected on the effect of these covariates on the PK parameters the designs can be re-evaluated.The optimal sampling schedules proposed in this work were based on the key constraints of three to four blood samples per patient, a minimum of 15 minutes between consecutive samples and a sample size of 60 individuals. Thus they are the optimal sampling times assuming these key constraints. However, it is recommended to take additional blood samples, if at all possible, and if convenient within the sampling windows. Additional samples will provide even more information for the estimation of the model parameters and may assist with the exploration of other plausible structural PK models and/or parameter values.The use of optimal design methodology provided an analytical yet flexible framework to determine robust and efficient designs for future population PK studies of DHA following oral AS for all target populations, including pregnant women and children.Current dosing recommendations for oral AS provide a resistance selection opportunity in patients with low drug concentrations and high parasite burdens, which are often children and pregnant women . More poKMJ: Kris M Jamsen; SBD: Stephen B Duffull; JT: Joel Tarning; NL: Niklas Lindegardh; NJW: Nicholas J White and JAS: Julie A Simpson.The authors declare that they have no competing interests.JAS and KMJ conceived the project. JAS and NJW conducted the survey and KMJ, JAS and SBD implemented the models in NONMEM and the designs in POPT. KMJ wrote the first draft of the manuscript. SBD, JT, NL, NJW and JAS revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.This file explains the mathematical details of applying allometric scaling and maturation to PK parameters, the Bateman model and the Dost model. The results from the evaluation of the naïve designs are also displayed [isplayed -31.Click here for file


Only a small portion of HPV 16 infections persist and can lead to cervical intraepithelial lesions and cancer. Factors that favour HPV persistence versus clearance are still poorly understood, but several studies have suggested that HPV intra-type variants may influence persistence and clinical outcome. The aim of this study was to assess the possible association between HPV 16 variants and the risk for viral persistence in the general population of France.One hundred and forty two women infected with HPV 16 with normal cytology, without previous treatment for cervical lesions, and with a valid second follow-up visit 4 to 16 months later, were selected from patients participating in routine cervical cancer screening in the Reims HPV Primary Screening Cohort Study. HPV intra-type variants were determined by sequencing the HPV 16 E6 open reading frame, and were compared for viral persistence at the second visit using odds ratios (OR) to estimate relative risk.Although no statistically significant differences in risk for persistence were observed by the HPV 16 variant lineage, European variants containing the polymorphism 350 T (EUR-350 T) appeared to persist more often than those containing 350 G (EUR-350 G) .No strong differences were observed in the risk of viral persistence for the HPV 16 variants that predominate in France. High-risk (HR) human papillomaviruses (HPVs), are the etiological agents of cervical cancer . Even thA recent proposal to define major HPV variant lineages is by approximately 1.0% difference between full genomes of the same HPV type, with differences of 0.5-0.9% designated as sub-lineages . NaturalThe most common individual polymorphism within the European lineage is T350G in the E6 oncogene, which leads to an amino acid change Leucine to Valine (L83V). Thus, the European lineage can be divided into isolates containing 350 T (EUR-350 T) or 350 G (EUR-350 G), even though this polymorphic position does not seem to define phylogenetic lineages . SeveralThe aim of this study was to assess the possible association between the genetic variants of HPV 16 and the risk of persistent HPV infection in the general population in France.Sequencing results were obtained for a total of 142 women infected with HPV 16 with normal cytology and a valid follow-up visit. The follow-up visits ranged from minimum 4.0 months to maximum 16.0 months . The majority of the baseline infections belonged to the European lineage , with EUR-350 T and EUR-350 G accounting for 40.8% and 51.4% of the isolates, respectively. C109T (n = 3) was the only other single nucleotide polymorphism (SNP) found in more than one EUR isolate  showed a persistent infection at the second visit. The proportion of HPV 16 infections that persisted was slightly higher for EUR-350 T (67.2%) than EUR-350 G (56.1%) , although this difference did not reach statistical significance. Adjustment for age and time to second visit did not materially alter this finding    with those included in a previous report on HPV 16 variants from this same cohort between 1997 and 2003 .HPV 16 E6 variants were analysed from DNA stored from the first HPV 16-positive normal cytology sample. DNA was extracted from liquid-based cervical samples, collected in PreservCyt Medium , with the EZ1 DNA Tissue Kit on the EZ1 instrument , according to the manufacturer’s instructions. Samples were lysed overnight using proteinase K at 56°C and DNA was eluted in a 200 μL volume.HPV 16 DNA presence was screened initially by a specific HPV 16 E6/E7 Taqman real-time polymerase chain reaction (PCR) and confirmed by either the Linear Array HPV Genotyping Test  or the Inno-Lipa Extra HPV Genotyping Test .The PCR and sequencing of the HPV 16 E6 gene was performed as described by Gheit et al. 2011 . If amplThe HPV 16 infection was defined as persistent if the woman was still HPV 16-positive at the next follow-up visit. Relative risks for persistence were compared between EUR-350 T, EUR-350 G, and non-European lineages, using odds ratios (OR) and corresponding 95% confidence intervals (CI). ORs were adjusted for age (<28 and 28+ years) and the duration between two visits (<8 and 8+ months).HR-HPV: High Risk Human Papillomavirus; CIN: Cervical Intra-epithelial Neoplasia; EUR: European; As: Asian; AFR: African; NA/AA: North-American / Asian-American; PCR: Polymerase Chain Reaction; SNP: Single Nucleotide Polymorphism; OR: Odds Ratio; CI: Confidence Interval.The authors declare that they have no competing interests.VD and CC are responsible for the Reims screening study and research databases and participated in the study design. IC performed the HPV variants testing and lead drafting of the manuscript. TG developed the HPV variant testing platform and performed variant testing. MT developed the HPV variant testing platform and participated in the data interpretation. JC participated in the data analysis and performed statistical analysis. GC participated in the study design and interpretation. SF participated in study interpretation. All authors read and approved the final manuscript.


Background. Haitian immigrant women residing in Little Haiti, a large ethnic enclave in Miami-Dade County, experience the highest cervical cancer incidence rates in South Florida. While this disparity primarily reflects lack of access to screening with cervical cytology, the burden of human papillomavirus (HPV) which causes virtually all cases of cervical cancer worldwide, varies by population and may contribute to excess rate of disease. Our study examined the prevalence of oncogenic and nononcogenic HPV types and risk factors for HPV infection in Little Haiti. Methods. As part of an ongoing community-based participatory research initiative, community health workers recruited study participants between 2007 and 2008, instructed women on self-collecting cervicovaginal specimens, and collected sociodemographic and healthcare access data. Results. Of the 242 women who contributed adequate specimens, the overall prevalence of HPV was 20.7%, with oncogenic HPV infections (13.2% of women) outnumbering nononcogenic infections (7.4%). Age-specific prevalence of oncogenic HPV was highest in women 18–30 years (38.9%) although the prevalence of oncogenic HPV does not appear to be elevated relative to the general U.S. population. The high prevalence of oncogenic types in women over 60 years may indicate a substantial number of persistent infections at high risk of progression to precancer. Haitian immigrant women residing in Little Haiti, a large ethnic enclave in Miami-Dade County, experience the highest cervical cancer incidence rates in South Florida. Between 2007–2009, disease incidence in Little Haiti  was nearly four times higher than that reported for the Miami metropolitan area overall  , HaitianUnderutilization of screening may not solely account for the excess burden of cervical cancer observed among Haitian women. Epidemiologic and virologic studies have shown that persistent cervical infection with oncogenic “high risk” human papillomavirus (HPV) types cause virtually all cases of cervical cancer worldwide , but theThe current study was conducted as part of an ongoing CBPR initiative in Little Haiti, which has been described in previous publications , 3, 9. BPatnè en Aksyon (Partners in Action). This partnership, which involves active participation of community leaders from Little Haiti and an interdisciplinary team of investigators from a large university in the Miami metropolitan area, strives to reduce the excess burden of cervical cancer experienced by Haitian women and to improve the health status of women in Little Haiti. To this end, community health workers (CHWs), who are formally employed by a large community-based organization whose leadership is active in Patnè en Aksyon, play a central role.In Little Haiti, CBPR efforts are governed by a campus-community partnership known as In the present study, female CHWs of Haitian descent (fluent in English and Haitian Kreyol) were trained to recruit study participants and collect data using a standardized manual created by one of the academic partners. As part of the training, each CHW also completed an online certification program for conducting human subjects research (CITI), as mandated by the University of Miami's Institutional Review Board (IRB). The university IRB approved the study. n = 362) and told them about the study. For women who were interested and eligible (n = 290), the CHWs scheduled an interview to have women self-sample for HPV and respond to a short survey.Between September 2007 and March 2008, CHWs recruited participants primarily through the extensive network of the community-based organization (CBO) where they were formally employed, and by canvassing community venues including flea markets, health clinics, and laundromats across Little Haiti to identify women meeting study eligibility criteria . The CHWs approached all women in such venues who appeared to be of Haitian descent and at least 18 years of age  obtain informed consent, (2) teach women how to appropriately self-sample using visual aids, and (3) interview participants about their experience with self-sampling. To monitor adherence to this research protocol, CHWs were required to log the time that they completed each step on data collection forms. Given widespread skepticism about research in Little Haiti, the CHWs spent one hour (on average) explaining to participants the benefits of participation, explaining the purpose of collecting specimens and genotyping those positive for HPV, and assuring women that results would be kept confidential. Following informed consent, CHWs instructed women on how to appropriately use the device using a pictorial brochure that visually demonstrated each step in the process . The parThe CHWs notified all participants of their results, and assisted those with cytological abnormalities and/or infections  in obtaining timely and appropriate followup care. Nearly all women with abnormalities obtained follow-up at a free gynecologic oncology clinic in Little Haiti with the assistance of CHWs.Women self-collected cervicovaginal specimens with the Fournier device, which has demonstrated high concordance with physician-collected cervical specimens and can provide specimens for cytology assays , 14. ThiAccess Genetics, Eden Prairie, MN), which amplifies the highly conserved L1 region of the HPV genome to detect approximately 80 HPV types. Genomic DNA was extracted from ecto- or endocervical epithelial cells obtained via the Fournier device and fixed in alcohol-based liquid ThinPrep™  solution using standard techniques. In brief, cellular material was concentrated and the alcohol preservatives removed by distilled water dilution. Each sample was evaluated for cellularity to determine the dilution volume that equalizes the concentrations across all samples followed by an enzymatic incubation to achieve increased cell membrane permeability. An aliquot of each cellular sample was then combined with Cellerate Access Genetics, Eden Prairie, MN. This mixture was subjected to thermal cycling incubation resulting in protein degradation and the release of purified DNA. Cervicovaginal specimens were genotyped using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay . The resultant fragment band patterns determined the specific HPV types present in each sample. PCR amplification of HPV products used degenerate primers specific for the consensus regions of the L1 gene in the HPV genome. The assay was performed in two parts: HPV detection (presence/absence) and HPV identification (RFLP genotyping). The first reaction included two unique and specific primers, one for the HPV genome, and the other for the For this analysis, oncogenic “high-risk” (HR) HPV types included 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 . All othAmong the 290 women eligible for the study, 246 completed self-sampling and the subsequent questionnaire. Of these, we included 242 women in the present analysis; the four excluded women did not collect adequate specimens for HPV testing. All data were managed and analyzed in SAS version 9.2 for Windows . Frequency distributions of HPV infection and HPV types were calculated for the entire sample and then stratified according cytology result . We also estimated HPV prevalence by age group  according to risk class . To determine factors associated with prevalent HR HPV infection, we calculated odds ratios (ORs) and 95% confidence intervals with unconditional logistic regression models (univariate and adjusted for age). The reference group consisted of women with LR or no HPV. Women with multiple infections were included in the HR HPV group if at least one infection was with a HR HPV type.The vast majority of women (85.1%) were over 30 years of age . Most paP = 0.88).Overall, 21 women (8.7%) had abnormal cytology results, including 14 with atypical cells of undetermined significance (ASC-US), 1 with atypical glandular cells not otherwise specified (AGC-NOS), 4 with low-grade intraepithelial lesions (LSIL), and 2 with high-grade intraepithelial lesions . Of womeP < .01). There was a low prevalence of concomitant sexually transmitted infections (STIs) in our sample including 13 cases of Chlamydia and 1 case of Gonorrhea. There was no significant association between STI infection and any HPV infection, HR HPV infection, or abnormal cytology. There were no significant associations between HR HPV positivity and vaginal infection.These data are the first to estimate the prevalence of HPV and distribution of infections by type and age in Haitian immigrants living in the USA. The crude prevalence of any HPV (20.7%) and HR HPV (13.2%) in this sample was slightly lower than estimates from a representative sample of USA women aged 15–49 years who provided self-collected HPV specimens  . Our estα3 (which includes HPV 61 and 62) and α15 [We found that HR HPV types 53, 82, 52, and 68 and LR HPV types 61 and 62 were the most prevalent in the study population. This type distribution contrasts with findings from a meta-analysis of other USA-based studies, which found HPV 16, 52, 18, 51, and 58 to be the most prevalent types among women with normal cytology . While t and α15 . Thus, tData on the type distribution of HPV in Haiti is currently unavailable , althougAge patterns associated with HPV prevalence vary by population. The prevalence of HPV among Haitian women living in Miami was highest in young women (44.5%), decreasing with age until rising to a second minor peak in women over 60 years (26.1%). A recent meta-analysis suggests that HPV prevalence in the USA declines steadily with age . Data frWe found slightly elevated levels of cytological abnormalities (8.7%) compared to approximately 6% in the general USA population . Still, There are several limitations to this study. Our original study's planned sample size (300) was calculated based on the 95% exact confidence interval approach and good precision . In our study the observed prevalence of HPV for 242 evaluable women is 20.7% with 95% exact confidence interval of 15.7% and 26.3%. Therefore, our study established with high confidence (97.5%) that the true HPV prevalence in the target population is not less than 15.7%. Our study size is sufficiently large to ensure good precision, that is, a 5.3% semiwidth confidence interval, for estimating the HPV prevalence in this population. The sample size was reduced as a result of budgetary restraints and input from key members of the community advisory board. As a part of an ongoing CBPR initiative, it was necessary to balance the achievement of recruitment goals against the logistical constraints imposed by utilizing CHWs, who held roles in both this research study and as employees of various other community-based organizations.Our sample size was small and when we stratified by age group we lost power to detect significant predictors of HR HPV infection. While age and exposure to tobacco smoke at home were significantly associated with HR HPV infection among our sample, the significance of other predictors was difficult to discern after adjusting for age. Furthermore, this was not a population-based sample, and results may not be fully generalizable to women living in Little Haiti. Nearly 80% of participants in this study reported receiving a Pap smear in the past three years, which is considerably higher than the 44% indicated by our previous work with women in Little Haiti . We attrDue to cultural considerations raised by our partner community-based organization, we could only collect data on broad age groups, and were limited in our ability to collect data on sexual behavior. Both age and sexual behavior are demonstrated predictors of HPV infection, and our inability to adjust for these in multivariate analysis is a limitation. We also rely on validity assessments of the self-sampling method from other studies, as we were not able to compare self-collected specimens to physician-collected specimens here. We cannot rule out the possibility that women may not have self-sampled correctly.This cross-sectional study is a preliminary investigation into the prevalence and distribution of HPV types among women in Little Haiti, Miami, Florida. Among participants, the burden of vaccine types 6, 11, 16, and 18 was low, but further study will be needed to determine the prevalence of HPV 16 and 18 in Haitian immigrants with cervical cancer. Although the prevalence of HPV and HR HPV does not appear to be elevated among women in Little Haiti relative to the general USA population, we note the unusually high prevalence of HR HPV among women over 60 years, which may indicate a substantial number of persistent infections at high risk of progression to CIN2+. Underutilization of screening is undoubtedly a contributing factor to the high incidence of cervical cancer, but further study is needed to explain the excess cervical cancer burden in this population relative to others in South Florida. We are presently examining common feminine hygiene practices in this community of Haitian immigrants that may physiologically alter the cervix. Changes induced by exposure to particular compounds may reduce a woman's ability to clear infections, placing women at a greater risk of persistent HR HPV infection.Self-collection of HPV specimens has been demonstrated to be acceptable and feasible among women in Little Haiti , and the


T0 and S group). Moreover, patients who received radio and/or chemotherapy had a very high prevalence of HPV semen infection . In conclusion, patients with testicular cancer had frequently altered sperm parameters and higher prevalence of HPV semen infection that were worsened after radio and chemotherapy. Because HPV infection is a risk factor for cancer development and it may further reduce fertility, we suggest screening for HPV in testicular cancer patients at diagnosis and particularly after adjuvant treatments.Testicular cancer represents the more frequent solid tumor affecting males aged 15–35 years. In the last decades, its incidence showed a progressive increased probably due to genetic and environmental factors. Despite exposure to some viruses such as HIV, HCV, EBV, and HPV is frequently related to cancer development, there are no studies aimed to evaluate the possible implication of viral infections in the pathogenesis of testicular cancer. In this study, we analyzed sperm parameters and prevalence of HPV on sperm in 155 testicular cancer patients at diagnosis (T−1), after orchiectomy (T0) and after 12 months from surgery or from the end of adjuvant treatments (T12). All patients showed a significantly higher prevalence of semen infection than controls  and altered sperm parameters both at T−1 and T0. Considering sperm parameters, at T−1 we observed a reduction of progressive motility, and after orchiectomy patients showed a reduction of sperm concentration and count and a further worsening of motility. Thereafter, patients were assigned to three groups on the basis of medical option after surgery: S = surveillance, R = radiotherapy, and C = chemotherapy +/− radiotherapy. At T12, untreated patients had an improvement of sperm parameters while R group and even more C group had a strong decrease of sperm number ( Therefore, patients who underwent chemo and/or radiotherapy were reevaluated after 12 months from the end of treatments. Patients who were followed-up just by surveillance repeated the same analysis after 12 months from surgery.Written informed consent was obtained from all patients, and the study protocol was approved by the local ethics committee. We enrolled 155 consecutive patients affected by testicular cancer candidate to unilateral orchiectomy, who attended the Centre for Human Reproduction Pathology for andrological examination. A medical history including previous circumcision, smoking, and sexual behavior was obtained from each patient. As control subjects, we used a group of 84 healthy proven fertile men. All subjects collected semen for standard semen analysis, detection of HPV DNA and fluorescence Semen samples were obtained by masturbation after 3 days of sexual abstinence. After liquefaction at room temperature, semen volume, pH, sperm concentration, viability, motility, and normal morphology were determined following World Health Organization guidelines for semen analysis WHO, . An aliqThe presence of HPV infection was detected by PCR amplification of HPV DNA with SPF10 primers and then genotyped by the INNO-LiPA HPV Genotyping Extra assay , which can identify the following HPV genotypes: HPV-6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69/71, 70, 73, 74, and 82 , containing biotin-labeled HPV DNA probe . Each slide was covered with a glass coverslip, and the edges were sealed with nail polish to prevent loss of the mixture during denaturation and hybridization. After a simultaneous denaturation of cellular target DNA and HPV DNA probe on a heating block for 5 min at 95°C, hybridization was performed by incubating the samples at 37°C overnight in a humidified chamber. Thereafter, the coverslips were carefully removed and the slides were washed in PBS 1× for 10 min. After 15 min incubation at 37°C with the differentiation reagent (Pan Path), the slides were washed three times in PBS 1×. The biotin-labeled HPV probe was detected by incubation with 1:200 streptavidin Texas Red®  for 40 min at room temperature. After detection, the slides were washed twice in PBS 1×/0.01% Triton and then twice in PBS 1× and mounted with a solution containing 4′, 6-diamidino-2phenylindole (DAPI) and anti-fade . Samples were analyzed using a fluorescence microscope  equipped with a triple bandpass filter set . For each slide, at least 200 spermatozoa were analyzed. Evaluation of nuclear hybridization signals was performed in triplicate by different investigators. In samples that resulted negative during the follow-up, results were confirmed by PCR. When nuclei were completely and homogeneously stained and multiple small spots or single large signals were present, the sperm cells were classified as positive. The method was tested on control slides containing CaSki cells, a human cervical carcinoma cell line with stably integrated and transcriptionally active HPV genomes, which served as a control for the specific probe. Cells smeared on salinated glass slides were fixed with 4% paraformaldehyde in PBS for 10 min. After fixation, cells were subjected to 3–5-min washes in PBS 1× and then dehydrated with 5-min ethanol washes . Cell smears were then air-dried and stored at 4°C until use.Samples containing at least 2 × 10t-test after acceptance of normal distribution with the Kolmogorov-Smirnov test. Comparisons of proportion were performed with a one-sided non-parametric resampling test. P-values (two sided) <0.05 were considered statistically significant.The values shown are the averages of at least three evaluations performed by different operators. Data regarding sperm parameters were presented as mean ± SD while data on semen infection as percentage. Differences between data were determined by two-tailed Student's p < 0.05). After orchiectomy, we found a significant reduction of sperm concentration and count (both p < 0.05 vs. T−1) and a further reduction of sperm motility compared to T−1 and control subjects (p < 0.01). Moreover, sperm volume and morphology were not significantly different in patients compared with controls before and after orchiectomy. Interestingly, the percentage of subjects with HPV infection was higher among patients than controls (9.7% vs. 2.4%). This percentage remained unchanged at T0. After orchiectomy, patients were assigned to three groups based on medical option: S = candidate to surveillance, R = candidate to radiotherapy, and C = candidate to chemotherapy with or without radiotherapy. In Table p < 0.05). In contrast, subjects who had radiotherapy showed no increase in sperm number and reduced sperm motility compared to S group (p < 0.05). Moreover, patients who underwent chemotherapy associated or not with radiotherapy, had a reduction of both sperm number and motility vs. T0 vs. patients from groups S (p < 0.05 and p < 0.01 respectively). In the three groups, we found no significant differences in both ejaculate volume and normal morphology comparing T12 with T−1 and T0. Considering all patients, HPV semen infection was significantly higher in patients at T12 respect to T0 (34.9% vs. 9.7%). By the comparison of different groups, the prevalence of infection resulted 7.7%, 30.8% and 61.5% in S, R, and C groups respectively. Finally, patients treated by chemotherapy had also a higher percentage of HPV infected sperm compared to T0 and to S (both p < 0.05). In Figure Mean age of patients with testicular cancer was 31.2 ± 5.4 years, not different from control subjects (30.8 ± 4.7 years). In Table vice versa. Moreover, our results demonstrated that treatments frequently used in testicular cancer patients and in particular chemotherapy, are able to strongly increase the prevalence of HPV semen infection, probably through their immunosuppressive action. In conclusion, we suggest testing sperm HPV in patients affected by testicular cancer before and particularly after treatments inducing immunosuppression, because this infection can in turn induce cancer in many sites and further reduce male fertility. Further and larger studies are needed to confirm our findings also in patients affected by other tumors.It is of concern, especially during last years, the increasing incidence of reproductive tract diseases and particularly testicular cancer (Richardson et al., The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.