Several meta-analyses confirmed the five most prevalent human papillomavirus (HPV) strains in women with and without cervical neoplastic diseases are HPV16, 18, 31, 52, and 58. HPV16/18 are the predominant oncogenic genotypes, causing approximately 70% of global cervical cancer cases. The vast majority of the women studied in previous analyses were from Europe, North America, Asia, and most recently Latin America and the Caribbean. Despite the high burden of cervical cancer morbidity and mortality in Africa, a robust meta-analysis of HPV genotype prevalence and distribution in African women is lacking.We systematically searched 14 major databases from inception to August 2013 without language restriction, following the Meta-Analysis of Observational Studies in Epidemiology and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Seventy-one studies from 23 African countries were identified after screening 1162 citations and data abstracted and study quality appraised from 195 articles. HPV type-specific prevalence and distribution was estimated from 17,273 cases of women with normal cervical cytology; 1019 women with atypical squamous cells of undetermined significance (ASCUS); 1444 women with low-grade squamous intraepithelial lesion (LSIL); 1571 women with high-grade squamous intraepithelial lesion (HSIL); and 4,067 cases of invasive cervical carcinoma (ICC). Overall prevalence of HPV16/18 were 4.4% and 2.8% of women with normal cytology, 12.0% and 4.4% with ASCUS, 14.5% and 10.0% with LSIL, 31.2% and 13.9% with HSIL, and 49.7% and 18.0% with ICC, respectively. Study limitations include the lack of adequate data from Middle and Northern African regions, and variations in the HPV type-specific sensitivity of different genotyping protocols.To our knowledge, this study is the most comprehensive assessment of the overall prevalence and distribution of HPV genotypes in African women with and without different cervical neoplasias. We have established that HPV16/18 account for 67.7% of ICC cases among African women. Based on our findings, we highly recommend the administration of existing prophylactic vaccines to younger women not infected with HPV16/18 and an increase in HPV screening efforts for high-risk genotypes to prevent cervical cancer.Review registration: International Prospective Register of Systematic Reviews CRD42013006558. Cervical cancer is the leading cause of mortality and years of life lost due to cancer in Africa  and predTo date, several global meta-analyses on the distribution of HPV genotypes have confirmed the five most prevalent strains in women with normal cytology ,15 and cA comprehensive meta-analysis of HPV genotype prevalence and distribution among a large sample size of African women is needed to inform local, national, regional, and global policy to curb the spread of cervical cancer. In May 2013, the Global Alliance for Vaccine and Immunization (GAVI) announced the availability of HPV vaccines (Gardasil and Cervarix) for a reduced price of $4.50 per dose to low-income countries meeting the eligibility criteria . With clTo our knowledge, this systematic review and meta-analysis of HPV genotype prevalence and distribution in Africa is the largest ever performed and includes 25,463 African women. The goal of this study was to establish the overall and type-specific prevalence and distribution of HPV genotypes among women with normal cervical cytology, neoplastic cervical lesions, and cancer in all five World Health Organization (WHO) regions of Africa.See S1 Table). The investigators wrote a protocol and registered it with the International Prospective Register of Systematic Reviews (identification number CRD42013006558) in November 2013 . Similarly, HPV type-specific prevalence by cytology or histology was also calculated for those testing positive for the specific HPV type by cytological category according to the 2001 Bethesda System ,35. HPV Statistical analysis was conducted as described by Ciapponi et al. , using S2) statistic was used to quantify the heterogeneity between studies.The computed proportions were arcsine square root transformed using the Freeman-Tukey method to stabilize the variance . The metAdditionally, a meta-regression using grouped logistic regression analysis adjusted for age-group was performed to further identify the possible sources of heterogeneity. We then computed lesion-specific age-adjusted prevalence for any HPV and HPV16 for each region. We then used LSMEANS statement with the ILINK options in the SAS LOGISTIC procedure to back transform log-odds into proportions and generate 95% confidence intervals.No institutional review board approval was required for this study.Fig 1.A total of 1162 citations were retrieved and 195 full articles were reviewed. Data was abstracted from 71 epidemiological studies with individual-level data meeting the inclusion criteria. The study flow diagram is shown in S4 Table). Fifty-six studies reported multiple infections with any HPV type-specific. A total of 30,444 women from 23 African countries were recruited to participate in the various studies included in the systematic review. Beta globin tests were done on 27,915 sample specimens. Finally, HPV DNA testing was done on 25,463 sample specimens from women who had a positive beta globin test result. Their distribution by cytological type and geographical region is given in Table 1.Data were collected from studies conducted between 1968  to 2010 Out of 24,643 women tested for any HPV infection, 17,273 had normal cytology; 1,019 were classified as ASCUS; 1,444 were diagnosed with LSIL; 1,571 were HSIL-positive; and 4,067 had ICC. In addition, a total of 346 women were reported as HSIL or higher (either HSIL or ICC), 505 were classified as abnormal without separation into a specific lesion grade, and 257 were of an unspecified low lesion type. The last three categories were expunged from further analysis.Table 1). Middle Africa was the least represented region, including only 30 women from two studies . South Africa  contributed the largest study size followed by Tanzania , Senegal , Nigeria  and Kenya . Morocco, Cote d’Ivoire, and Algeria each contributed over 1,000 women to the study. Each of the remaining 15 countries had fewer than 1,000 women included in the study. Two studies [Based on the WHO classification of geographic regions of Africa, Eastern Africa had the highest number of women included in the study 10,246), followed by Western Africa , Southern Africa , and Northern Africa   classification according to the World Bank Table 3and S3 Table. There were no statistically significant differences in all the sub-group analysis for any HPV in women with normal cervix, neoplasia or ICC. Age-adjusted prevalence for any HPV and HPV16 categorized by region and lesion type is provided in Table 4.The meta-analysis of HPV type-specific prevalence by lesion types according to the Bethesda classification of women with normal cervical cytology and cervical neoplastic lesions  are presented in 2 = 88.8%, 43 studies; HSIL I2 = 81.5%, 33 studies; LSIL I2 = 93.3%, 23 studies; ASCUS I2 = 98.5%, 15 studies; and Normal I2 = 99.1%, 42 studies, all p-values <0.001. Random effects age-group adjusted meta-analysis using logistic regression analysis were performed for every HPV genotype.Heterogeneity was found to be substantial, ICC: ITable 1). HPV16, 52, 35, 18, 58, 51, 45, 31, 53 and 56 were the ten most common genotypes in women with normal cervical cytology in descending order (Table 2).Systematic review included 42 studies testing for cervical HPV infection in 17,273 women with normal cytology. This is the highest number of African women with normal cervical cytology ever included in a single HPV prevalence meta-analysis study. Most of the women tested for HPV infections were from Eastern Africa , followed by Western Africa , Southern Africa  and Northern Africa . A total of 138 individuals were from studies that contained mixed countries . Type-specific HPV16 and 18 were higher among women with normal cervical cytology from Southern Africa compared to other regions at 9.9% and 5.8% respectively . No study representing women from Middle Africa met the inclusion criteria.There was wide variation in any HPV infection rates based on region, with Southern Africa (57.3%) having the highest prevalence, followed by Eastern Africa (42.2%), Western Africa (27.8%), and Northern Africa (12.8%) (2 = 97.2%), closely followed by the age group 15–24 years with 48.2% . Age groups 35–44 and 45–54 years had HPV prevalence of 36.1%  and 31.6% , respectively, all p-values <0.001 (Table 3). Further lesion-specific age-adjusted prevalence for any HPV and HPV16 for each region is provided in Table 4.Overall, 39 studies stratified eligible women screened for HPV infection according to age groups. Analysis of these studies revealed that the age group 25–34 years had the highest overall HPV prevalence at 50.5% .To date, only one meta-analysis of HPV type-specific prevalence among women from Africa with ASCUS and LSIL exists . HoweverTable 2). The prevalence of type-specific HPV and distribution in women diagnosed with ASCUS for each geographic region is presented in Fig 2, in the second panel from the left.HPV16, 52, 18, 35, 45, 51, 66, 53, 39 and 58 were the ten most common genotypes in women with ASCUS (Table 1) Africa regions. HPV16, 35, 52, 18, 53, 56, 58, 51, 45 and 66 were the top ten most common genotypes in women with LSIL (Table 2). HPV type specific prevalence and distribution by region for LSIL is summarized in .For LSIL, data from a total of 1,444 women from 23 studies were abstracted. A majority of the patients were from Eastern (n = 605), followed by Southern (n = 521), Western  and Middle (n = 8) . HPV type-specific prevalence and distribution by region for HSIL is summarized in . Western Africa reported the highest prevalence of HPV16 and 18 at 33% and 22.2% respectively for HSIL. In Eastern and Southern regions, HPV16 prevalence was tied at 28%, followed by HPV 52 (16.5%) and HPV 35 (15.1%), respectively.HPV16, 18, 35, 52, 58, 33, 31, 53, 45 and 66 were the ten most frequently detected genotypes in HSIL cases (2 = 0.0%) as shown in Table 3.Overall, 30 studies stratified eligible women screened for HPV infection by age. Analysis of these studies revealed that the age group 45–54 years had the highest overall prevalence of any HPV for HSIL at 87.1% . A majority of the ICC cases were from the Eastern region , followed by Western (n = 951), Northern (n = 517), Southern (n = 457) regions, and only three patients were from the Middle Africa region. Two hundred and thirty-five patients were from mixed regions.A total of 4,067 cases from 43 studies were included in the ICC systematic review analysis (Table 2). While HPV16, 18, 35, and 52 were the four most common types in normal cytology through HSIL lesions; in ICC tissues HPV45 replaced HPV52 as the third most common genotype (Table 2). Together HPV16, 18, and 45 accounted for 77.7% of all ICC cases in African women.HPV16, 18, 45, 35, 33, 52, 31, 58, 51 and 68 were the top ten most frequently detected genotypes among ICC cases , 18.0%  and 10.0% , respectively. HPV16 and 18 varied by region; in descending order: Northern , Eastern (54.4% and 21.0%), Southern (46.8% and 18%), and Western (44.1% and 11.7%) regions of Africa.HPV type-specific prevalence and distribution by region is presented in Comprehensive data on the geographic distribution of specific HPV genotypes in women with all grades of cervical diagnosis are crucial for estimating the baseline burden of disease, the future impact of HPV vaccines on cervical cancer prevention, and the identification of optimal HPV screening tools. To the best of our knowledge, our report is the largest meta-analysis to date of HPV genotype distribution and prevalence in women from Africa by region, lesion type, age group, publication year, and PCR primers for genotyping. Seventy-one studies matched the inclusion criteria and were abstracted for systematic review and meta-analysis. Each of the five sub-regions of African countries (n = 23) was fairly represented with the exception of the Middle region, where only two studies were included.Overall, the prevalence of any HPV infection in Africa was higher than other world regions; our meta-analysis determined the prevalence rates in women with normal cervical cytology (29.3%), ASCUS (46.5%), LSIL (74.2%), HSIL (84.8%) and ICC (89.5%) . Our stuNevertheless, the four most common genotypes, HPV16, 18, 52 and 35, were identical from normal cervical cytology through HSIL. In ICC cases, however, HPV45 replaced HPV52 as the third most common genotype. The high prevalence rate of HPV45 in Africa was previously reported among ICC cases ,110,111.While there has been a substantial push toward mass vaccination with either Gardasil or Cervarix , until nUsing the stringent inclusion and exclusion criteria outlined in our meta-analysis, six studies from Northern Africa qualified for data abstraction for women with normal cervical cytology  and ICC (n = 517). Notably, 83.4% of ICC cases were associated with either HPV16 (61%) or 18 (22.4%) in the sub-region, the highest number ever reported from any part of the globe. However, the prevalence of any HPV infection among women with normal cytology was low (12.8%) compared to other regions which ranged between 27.8–57.3%. The prevalence of HPV16 and 18 is 2.9% and 0.9%, respectively. Since most of the women screened for cervical cancers are adults over 18 years of age, low prevalence of HPV16 and 18 in the population suggests that there is a wider window available for delivering HPV vaccine, particularly if it is combined with HPV DNA screening among older women. A recent review on the burden of HPV infections and related diseases in the extended Middle East and Northern Africa reported very low prevalence of HPV infection in women with normal cervical cytology, similar to current findings . HoweverA majority of the population in Northern Africa share similar cultural and religious traditions and known to have more conservative views towards sexual behavior than countries in sub-Saharan Africa. If indeed future studies in Northern Africa clearly indicate that a majority of women in the region with normal cervical cytology have lower risk of any HPV infection even at advanced ages (18–26 years), then it will be important to increase the age of vaccination for Northern African girls up from the current WHO recommended 9–13 years to a much older age . The legThe increasing availability of additional tools to prevent and fight cervical cancer provides an opportunity for all nations to implement cervical cancer control programs that are both cost-effective and easier to deliver or to improve existing programs by saving additional lives and reducing cost . The GAVThe implementation of one lifetime screening at age 35 with either visual inspection with acetic acid or HPV DNA testing would reduce the risk of developing cervical cancer by 25% and cost less than $500 per person per year of life saved . HoweverThe major contribution of our study to the current body of literature is the assessment of 25,463 women from all African regions of whom 46.8% were HPV infected. A further stratification of specific HPV genotype prevalence by disease severity will help identify appropriate tools for HPV genotyping and assess the future impact of HPV vaccines. However, the applicability of this data throughout Africa is limited, since 95% of the studies were obtained from Eastern, Western and Southern African regions, partially attributable to the lack of well-designed studies in the remainder of Africa, especially Middle and Northern Africa. Indeed, quality studies meeting stringent meta-analysis criteria for inclusion were missing from 30 African countries, thus future cervical cancer prevention policies should consider building laboratory infrastructure in these countries.The current WHO recommendations on cervical cancer screening are based on age , howeverSome of the limitations to our study include: (i) the inclusion of cross-sectional studies and their inherent risk of bias; (ii) inclusion of studies that over sampled HIV-seropositive patients in the analysis; and (iii) there may be lack of representativeness of HPV type specific prevalence due to variation in different genotyping methods used and tissue sources. Finally, while we made every effort to identify and include in our meta-analysis all eligible studies, it is possible that we missed some.Although previous research has established the relationship between HIV and HPV infection, the inter-relationship is not fully understood ,102,120.S1 Appendix(DOC)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


The prevalence of HR-HPV types in <CIN1, CIN2 and CIN3+ were 33.7%, 84.4% and 91.6%, respectively. Thirty-eight of 49 (78%) HPV16 positive samples yielded HPV16 sequence information; of which, 32 complete genomes were sequenced and an additional 6 samples were partially sequenced. Phylogenetic analysis and patterns of variations identified 65.8% (n = 25) as HPV16 European (E) and 34.2% (n = 13) as non-European (NE) variants. Classification of disease into CIN3+ vs. <CIN3 indicated that NE types were associated with high-grade disease with an OR = 4.6 . The association of HPV16 NE variants with an increased risk of CIN3+ is consistent with an HPV16 genetically determined enhanced oncogenicity. The prevalence of genetic variants of HPV16 is distributed across different geographical areas and with recent population admixture, only empiric data will provide information on the highest risk HPV16 variants within a given population.HPV16 accounts for 50–70% of cervical cancer cases worldwide. Characterization of HPV16 variants previously indicated that they differ in risks for viral persistence, progression to cervical precancer and malignant cancer. The aim of this study was to examine the association of severity of disease with HPV16 variants identified in specimens (n = 281) obtained from a Cervical Pathology and Colposcopy outpatient clinic in the University Hospital of Espírito Santo State, Southeastern Brazil, from April 2010 to November 2011. All cytologic and histologic diagnoses were determined prior to definitive treatment. The DNA was isolated using QIAamp DNA Mini Kit and HPV was detected by amplification with PGMY09/11 primers and positive samples were genotyped by RFLP analyses and reverse line blot. The genomes of the HPV16 positive samples were sequenced, from which variant lineages were determined. Chi Human Papillomaviruses (HPVs) are double stranded DNA viruses with an 8 Kb episomal genome. The organization of the genome is divided into three functional regions: an upstream regulatory region (URR) that regulates the transcriptional and replication events; an early region that expresses the non-structural proteins , and a late region that encodes the structural proteins L1 and L2 Papillomaviridae family, which includes more than 170 different types of characterized and designated viruses www.hpvcenter.se/html/refclones.html). The papillomavirus members are classified into types based on the DNA sequence of the ORF of the major capsid protein, L1. A new viral type is assigned if the complete genome is cloned and the difference in the L1 nucleotide sequence is at least 10% different than all other classified HPV types HPV belongs to the in situ and cancer including smoking, hormonal contraceptive use, multiple pregnancies and possibly other factors According to the prevalence of specific HPV DNA types in cases of cervical cancers, the anogenital HPVs have been classified into low and high risk types The description and understanding of HPV genome variants is an important area for molecular pathogenesis and for the development of molecular diagnostics for HPV, vaccines and other therapeutic approaches to control and/or eliminate virus-induced diseases. The tumorigenicity of the HPV variants could be different among geographical areas because of population history and host genetics, such as the difference in the distribution of HLA in the population As has been demonstrated by biochemical and biological differences of HPV16 variants and their oncogenic potential changes Cervical smears (n = 281) were obtained during gynecological visits at the Colposcopy outpatient clinic in the University Hospital “Cassiano Antonio Moraes” (HUCAM) in Vitória, Southeastern Brazil, from April 2010 to November 2011. This research obtained approval by the Ethical Research Council of the Center of Health Sciences of the Federal University of Espírito Santo, Brazil, in November 2009; all the participants signed an informed consent.Taq Gold DNA polymerase  and Platinum Taq DNA Polymerase  were utilized as previously described All cytologic and histologic diagnoses were determined prior to definitive treatment and were classified as <CIN3 , n = 257, used as the comparison or control group, and CIN3+ (CIN3 or worse), n = 24, the case group for this study. The classification in control (<CIN3) or case (CIN3+) group was used in the context of the HPV16 variants results. The DNA was isolated using QIAamp DNA Mini Kit  according to the manufacturer’s instructions. The HPV DNA was detected by amplification with PGMY09/11 primers 2 statistics was performed to test the association of HPV16 variants between case and control groups.The PCR product sizes were confirmed by gel electrophoresis, purified using the QuickStep 2 PCR Purification kit  or QIAquick Gel Extraction kit . The amplified fragments were directly sequenced on an ABI Prism Model 377 automated sequencer (Perkin-Elmer Applied Biosystems) in the Einstein DNA Sequencing Core Facility . The sequences of the fragments obtained were assembled using Geneious v6.1.6 The median age of participating women was 38.7 years (SD 10.97). Out of 281 samples, 56% (157/281) were positive for HPV DNA. All of these positive samples were genotyped by RFLP and RLB and HR-HPV was found in 124 samples , from which 32.3% (49/124) were positive for HPV16. Based on cytology results, HR-HPV types were detected in 33.7% (76/225) from <CIN1, in 84.4% (27/32) from CIN2 and in 91.6% (22/24) from CIN3+. HPV16 was found in 14% (35/257) and 58% (14/24) of the samples classified as <CIN3 and CIN3+, respectively (p<0.001).E6) was not associated with CIN3+ , nuclear factor 1 (NF1), octamer-binding protein 1 (Oct1), glucocorticoid/progesterone response element (GRE), specificity protein 1 (SP1), transcription enhancer factor 1 (TEF1), and yin yang 1 (YY1) Increasing studies performed around the world, including Brazil, indicate the relationship between HPV16 variants and higher oncogenic risk is complex Moreover, miss-sense nucleotide mutations theoretically could alter the epitopes targeted by the current HPV vaccine The association of HPV16 non-European variants with CIN3+ is consistent with a genetically determined enhanced oncogenic potential of the NE HPV16. These observations suggest that determination of HPV16 variant lineage has clinical implications. The complete genome sequencing has the goal of allowing the genetics of HPV16 to inform us about differences in HPV biology, and permit continued improvements in phylogenic classification of subgroups with even higher oncogenic risks.The prevalence of genetic variants of HPV16 is distributed across different geographical areas and with recent population admixture, Brazil is an ideal location to study the biology and clinical importance of HPV variants.Figure S1Nucleotide variations compared to the HPV16 reference sequence. The nucleotide positions of detected variations are shown across the top and are indicated by the corresponding nucleotide letter. The absence of variations relative to the prototype is represented by dots, the dashes represents regions without sequence information. 1: nt 1311–1322, a 63 bp insertion of GCGCCATGAGACTGAAACACCATGTAGTCAGTATAGTGGTGGAAGTGGGGGTGGTTGCAGTCA; 2: nt 4192–4193, a 3 bp insertion of TTG; 3: nt 4196–4197, a 3 bp insertion of TTG; 4: nt 7772–7807, a 36 bp deletion of AACTAAATGTCACCCTAGTTCATACATGAACTGTGT.(PDF)Click here for additional data file.Table S1Reference sequences used to perform the alignment for phylogenetic analysis.(PDF)Click here for additional data file.


Although female sex workers (FSWs) are a well-known high-risk group for Human Papillomavirus (HPV) infections, few tailored intervention programmes for HPV have been established worldwide. The lack of reliable data on the prevalence of HPV and related cervical lesions hampers the establishment of evidence-based intervention programmes.The objectives of this study were to describe the prevalence of high-risk Human Papillomavirus (hrHPV) infections and abnormal pap smears in FSWs compared to a control group in Antwerp, Belgium.HPV genotyping and cytology data were analysed from routine Pap smear tests that were collected from both FSWs and the general population (1334 samples for each group) between June 2006 and June 2010. Within the laboratory database, all FSWs were matched 1:1 for age and testing date to determine the ORs of hrHPV genotypes, DNA and cytology outcome.th and 7th most frequent genotype at 5.0 and 2.5 % in the FSW and control groups, respectively.The prevalence of hrHPV DNA in FSWs was 41.7 % compared to 19.8 % in the age-matched controls with an overall OR of 2.8 (95 % CI: 2.3–3.4). Significant differences were observed in all age groups, and the most significant differences were observed in the cohort under 21 years of age . Significantly more cervical lesions were observed in FSWs, particularly in the 17- to 21-year old age group (OR for LSIL or HSIL: 10.3 (95 % CI: 3.2–33.8). In both groups, HPV 16 was the most prevalent at 12.1 and 6.6 % in the FSW and control groups, respectively. HPV 18 was the 8FSWs have a significantly higher prevalence of hrHPV and more abnormal Pap smears than does the general population in Antwerp, Belgium. The hrHPV prevalence in FSWs is similar to that reported in the literature. The need for tailored intervention programmes should be investigated further. Worldwide, cervical cancer is the fourth most frequent type of cancer in women and causes an estimated 270,000 deaths every year . High-riIn Belgium, the most common hrHPV genotypes are type 16 (3.7 %), 31 (3.0 %), 51 (2.3 %), 53 (2.1 %) and 59 (1.7 %), followed by HPV type 18 (1.5 %) [Although female sex workers (FSWs) are a well-known high-risk group for HPV infections, few tailored intervention programmes for HPV have been established worldwide. The lack of reliable data on the prevalence of HPV and related cervical lesions hampers the establishment of evidence-based intervention programmes. In addition, a direct comparison with an age-matched control group is often difficult due to a lack of data, and the use of different laboratory tests makes comparisons among studies difficult. However, the major hurdle is that many FSWs start sex work when they are younger than the screening age recommended by the respective national cervical cancer screening programmes, and therefore, there often is no readily available control group among younger age cohorts.The main aim of this cross-sectional survey is to determine the prevalence of hrHPV and HPV genotype distributions among FSWs in Antwerp. In addition, the OR for hrHPV DNA positivity, HPV genotypes and cytology compared to the general population are investigated.The study was performed in accordance with the Helsinki Declaration, procedures established by Belgian law and the guidelines of ICH-GCP. Ethical approval was obtained from the medical ethical board of Antwerp University (Ref: B30020072416).http://www.ghapro.be/en/index.html). This nested case-control study was based on the results of cervical cytological screenings and hrHPV testing of all FSWs visiting the Ghapro clinic between June 2006 and June 2010. This data was selected from the laboratory database of the Laboratory for Clinical and Molecular Pathology , Antwerp, Belgium. All samples were processed in this laboratory. For women tested on multiple occasions during this period, only the first screening results were included.The Antwerp health house for prostitution (Ghapro) offers sex workers free and anonymous screenings for sexually transmitted infections (STIs) as well as advice and education on sex work-related issues . All FSWs were matched 1:1 for age, testing date, and location (Antwerp region). As with the FSWs, only the first set of results during this testing period was taken into account for the controls.Pregnant women were not screened. Women with immunodeficiency conditions, such as HIV, and women without a reported age or date of birth were excluded from the study.Cervical cells were collected using the Cervix-Brush® Combi . After collection, brush heads were transferred directly into alcohol-based preservatives . All vials were transported to the Laboratory for Clinical and Molecular Pathology, Antwerp, Belgium. Thin-layer slides were prepared with the robotic BD PrepStain™ Slide Processor  . CytologDNA isolation from liquid-based cytology was performed as previously described . A cliniData analysis included descriptive statistics, cross-tabulations, and graphical representations using SPSS, version 22. Forest plots were created in Excel, version 2013. Odds Ratios (OR) of this nested case-control study are calculated by conditional logistic regression using COXREG in SPSS. All statistical tests were conducted at a 5 % significance level, and 95 % confidence intervals were computed.A total of 2106 samples from FSWs between June 2006 and June 2010 were tested. Of these, 753 were excluded because they were samples from FSWs who had already been tested earlier in the study period. In addition, 3 FSWs were excluded due to HIV positivity, and 15 were excluded because age was not available. The 1334 controls were selected from 200,736 data sets from women living in the Antwerp region. The analysis was performed on samples from 1334 FSWs and 1334 age-matched controls.The average age of the FSWs was 29.10 years . The number of FSWs in each age group is shown in Fig. Nationality was known for 1,254 FSWs (94 %). The majority  were European. Table p <0.00001). The OR for hrHPV in FSWs compared to the controls was 2.8 (95 % CI: 2.3–3.4). Figure The overall hrHPV prevalence for FSWs (41.7 %) was significantly higher than for the control group (19.8 %) , HPV-31 (8.9 %) and HPV-52 (7.0 %). The most common hrHPV genotypes in the controls were HPV-16 (6.6 %), HPV-51 (4.5 %) and HPV-31 (4.0 %).p = 0.11) in the occurrence of multiple infections (2 or more) between groups, with 40.6 % in the FSW group and 34.8 % in the control group. Only FSWs younger than 21 years of age had significantly more multiple infections (2 or more) than did the controls, at 51.0 and 27.2 %, respectively (p = 0.04).Both groups (FSW and controls) had mostly single infections  and duo-infections . Small percentages of both groups (FSW and controls) had 3 or more hrHPV infections . There was no significant difference (n = 149) (p < 0.05). In FSWs younger than 21 years old, a prevalence of 64.4 % was observed with an OR of 10.3 (95 % CI: 5.0–21.2). The group between 36 and 40 years of age showed a significantly higher hrHPV prevalence among FSWs than controls, with an OR of 5.3 (95 % CI: 2.4–11.9).Figure p <0.05). In addition, significantly more ASCUS  and LSIL  were diagnosed in FSWs than in controls, with ORs of 2.8 (95 % CI: 2.0–3.7) and 2.9 (95 % CI: 2.2–4.0), respectively.Figure n = 3), ASC-H  (n = 6) or HSIL  (n = 27) was detected. In cases with LSIL and ASCUS, we found hrHPV prevalences of 91 % and 83 %, respectively. The hrHPV prevalence in FSWs with normal cytology was 26 %. Looking at the different age groups in table p <0.001).In FSWs with AGC   n = 7 was detTo our knowledge, this is the largest nested case-control study (2 N = 2668) describing hrHPV genotyping in FSWs . OverallN = 61) with the general population (N = 84) and found that the HPV prevalence in FSWs was significantly higher (34.4 % in FSWs and 22.8 % in controls) [A 2013 literature review and analysis of the results of 35 HPV-prevalence studies in FSWs reported a median overall prevalence of HPV infections in FSWs of 42.7 %, with a range of 2.3 to 100 % . This isontrols) .In both groups Fig. , the mosThe decreasing prevalence of hrHPV with age that we observed in the control group confirms what is found in the literature , 18, 19.Lactobacillus gasseri) are associated with an increased clearance of detectable HPV [In addition to frequent exposure to multiple sex partners, we want to emphasize the frequent use of vaginal douching in this group. This procedure is known to alter the vaginal microbiome and to frequently cause bacterial vaginosis (BV) . Based oable HPV . The oftMost HPV studies in FSWs describe hrHPV prevalence, but only a few papers also provide cytological or histological results. Two studies reporting on both cytology and histology in FSWs found more cellular abnormalities in FSWs than in the general population, which confirms our findings , 24. In p < 0.0001).Regarding HPV vaccination, the current guidelines for older women may need to be refined. It has been clearly demonstrated that HPV vaccines are most effective when administered before exposure to HPV because they have no therapeutic effects and protect against HPV types not yet acquired at the time of vaccination , 27. ForThe health benefits of vaccination in this high-risk group, which has significantly more HPV infections, more cervical lesions and more exposure to non-vaccinated males, may be substantially different than in women from the general population. It seems obvious to investigate, in line with other occupational health immunisations, whether it would be beneficial to offer catch-up vaccinations for this specific at risk population.In Flanders, free vaccinations for one age-cohort of girls between 10 and 13 years of age have been offered since 2010 through a school vaccination programme. In addition, a free catch-up vaccine is offered up to age 18. In the first year of implementation (the 2010–2011 school year), 3 doses of the vaccine were administered to 83.5 % of the 11 year old girl population . This mehttp://www.ghapro.be/en/index.html), where 65 different nationalities are registered. We only included FSWs who came for routine screening for cervical cancer and not on FSWs in general, although the test is available if necessary. Furthermore, the selection of the controls with perfect case control match for age and testing date is a unique feature of this study. A bias could have occurred only in the youngest age cohort because there are no standard screening guidelines for this cohort, and we may have selected proportionally more sexually active girls. In Belgium, a number of treating physicians and gynaecologists do screen young girls (<21 years) during consultations for oral contraceptive prescriptions. In addition, many physicians screen young girls after childbirth. In both cases, these women were sexually active, and any selection bias would generate a higher prevalence in the youngest control group and potentially mask the specific impact of sex work at young ages. A number of physicians and gynaecologists routinely take pap smears starting at the age of 21, and this should be a representative control group. These data clearly confirm the previously reported increased hrHPV prevalence and increased number of abnormal cervical smears in this high-risk population.To our knowledge, this is the largest study on HPV genotyping in FSWs. The city of Antwerp has 510,000 inhabitants representing more than 160 different nationalities as well as a major port. This diversity is also reflected in the population of FSWs visiting the health house . It is not clear whether this is due to possible differences in the level in the cervix from multiple washings, the application of creams, or other factors. However, the collected samples still contained enough material to adequately detect an HPV infection. Even if this influenced the presented results, it would have led to an underestimation of HPV infection in FSWs, again masking the impact of sex work on HPV prevalence and abnormal cytology.This study confirms that FSWs have an increased hrHPV prevalence as well as an increased prevalence of premalignant lesions. This effect is very pronounced in the under 21 years group. To protect this higher-risk population, specific prevention and control measures may be appropriate, including adapted HPV screening and vaccination programmes. Tailored prevention and control interventions that take into account the multiple occupational contacts of FSWs will also lead to a direct benefit for the general population.


INK4a expression in tumor tissues. When tumors originate from hidden anatomical sites, this method can be challenging. A non-invasive and cost-effective alternative to biopsy is therefore desirable for HPV-16 detection especially within a community setting to screen at-risk individuals.Human papilloma virus-16 (HPV-16) infection is a major risk factor for a subset of head and neck squamous cell carcinoma (HNSCC), in particular oropharyngeal squamous cell carcinoma (OPSCC). Current techniques for assessing the HPV-16 status in HNSCC include the detection of HPV-16 DNA and p16INK4a status, in 82 HNSCC patients using end-point and quantitative polymerase chain reaction (PCR).The present study compared detection of HPV-16 DNA and RNA in salivary oral rinses with tumor p16INK4a-positive tumours, 39  had oral rinse samples with detectable HPV-16 DNA, using end-point and quantitative PCR. No HPV-16 DNA was detected in oral rinse samples from 40 patients with p16INK4a negative tumours, yielding a test specificity of 100 %. For patients with p16INK4a positive tumours, HPV-16 mRNA was detected using end-point reverse transcription PCR (RT-PCR) in 24/40 , and using quantitative RT-PCR in 22/40 . No HPV-16 mRNA was detected in oral rinse samples from the p16INK4a-negative patients, yielding a specificity of 100 %.Of 42 patients with p16We demonstrate that the detection of HPV-16 DNA in salivary oral rinse is indicative of HPV status in HNSCC patients and can potentially be used as a diagnostic tool in addition to the current methods.The online version of this article (doi:10.1186/s12885-016-2217-1) contains supplementary material, which is available to authorized users. Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are the most common types of head and neck squamous cell carcinoma (HNSCC), accounting for 263,900 new cases and 128,000 deaths worldwide [In recent decades, the overall incidence of HNSCC is in the decline in the developed world due to a reduction in the consumption of tobacco. However, there is a concomitant increase in the incidence of OPSCC attributable to human papilloma virus (HPV) infection, in particular the high risk HPV (HR-HPV) strain HPV-16 . About 4INK4A in HPV-infected tumor cells [INK4A in HNSCC tumor biopsies has been shown to serve as a surrogate marker to identify HPV infection in tumor. Tests that measure HPV DNA or RNA directly in tumor samples have also been reported recently, using in situ hybridization (ISH) or PCR for detection of HPV DNA, and RT-PCR and RNA ISH for HPV-related RNA [HPV-positive OPSCC has genetic alterations that are a direct result of HPV oncoproteins, E6 and E7, which inactivate the tumor suppressor gene products, p53 and Rb respectively. During immortalization of host cells, the E7 protein of HR-HPV binds to Rb, resulting in the compensatory over-expression of the tumor suppressor gene p16INK4a - positive HNSCC.Oral fluids have been shown to contain different analytes such as hormones, steroids, antibodies, growth factors, cytokines, chemokines and drugs, which may reflect local and systemic disease states –10. TheyINK4a immunohistochemistry (IHC). p16INK4a IHC was performed at the pathology laboratory of the Princess Alexandra Hospital, Woolloongabba, Australia using CINtec Histology Kit  according to manufacturer’s instructions. p16 INK4a status of tumor sections was assessed and established by qualified pathologists unaware of the HPV oral rinse results.Subjects in the current study were recruited from patients treated in the Ear, Nose and Throat (ENT) Department of the Princess Alexandra Hospital in Woolloongabba, Queensland, Australia between 2013 and 2015. This study was approved by the University of Queensland Medical Ethical Institutional Board (HREC: 2014000679), Queensland University of Technology Medical Ethical Review Board (HREC: 1400000616) and by the Princess Alexandra Hospital Ethics Review Board (HREC: HREC/12/QPAH/381). All patients with a primary cancer of the oral cavity and oropharynx or patients with loco-regional metastasis with oral/oropharyngeal origin who agreed to sign the information and consent form were enrolled. Demographic data and risk factors associated with oral and oropharyngeal cancers were collected by a patient questionnaire. A pathology report was included for each patient, which contained pathological staging of the tumor, histopathological classification and HPV status based on p16g for 10 mins at 4 °C. Cell pellets were resuspended in sterile PBS for DNA extraction or Qiazol  for RNA extraction and stored at−80 °C until further processing.Oral rinse samples were collected by having the patients swish and gargle for 1 min with 10 mL 0.9 % saline. Samples were immediately frozen on dry ice and transported to the laboratory for processing. Samples were then thawed, centrifuged at 1000 × g for 10 min at 4 °C and the aqueous phase was collected. Chloroform (200 μL) was added to the aqueous phase, vortexed for 5 min followed by centrifugation at 10,000 × g for 10 min at 4 °C. The aqueous phase was collected and an equal volume of isopropanol was added for RNA precipitation overnight at −20 °C. RNA was pelleted by centrifugation at 10,000 × g at 4 °C for 20 min, washed with 1 mL of 70 % ethanol and centrifuged again at 10,000 × g for 5 min at 4 °C. Supernatant was removed and the samples were air dried for at least 30 min. The RNA pellet was re-suspended in 15 μL RNase- free water. DNA and RNA samples were assessed for purity and quantified on a Nanodrop 1000 Spectrophotometer .Oral exfoliated cell pellets were resuspended in sterile PBS and DNA was extracted using the QIAmp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA was extracted from oral exfoliated cell pellet resuspended in Qiazol as described previously . BrieflyFor the detection of HPV-16 DNA in oral rinse samples, we used end-point PCR method as well as quantitative PCR (qPCR). Specific primers were used for the amplification of a region spanning the E6 and E7 genes of the HPV-16 genome  and primFor qPCR detection of HPV-16 DNA, specific primers were used for the amplification of a region spanning the E7 gene of the HPV-16 genome   and 0.2 μM of each primer in a total volume of 10 μL. qPCR was run on ABI Viia7  with the following conditions: 10 mins of denaturation at 95 °C; 40 cycles of: 95 °C (15 s), 60 °C (60s). To discriminate primer specific amplicon from primer dimers or unspecific PCR products, we also performed melt curve analysis with the following conditions: 95 °C (15 s), 60 °C (60s), 95 °C (15 s).Standard curves were developed for HPV-16 E7 using serial dilutions of Caski-derived DNA with 66, 13.2, 2.64 and 0.528 ng DNA. Caski cells are known to have 600 viral copies per genome (6.6 pg DNA/genome) [Total RNA up to 1 μg) was treated with two units of DNase I  in 1x DNase Buffer (New England Biolabs) in a total volume of 10 μL to remove genomic DNA. The digestion mix was incubated at 37 °C for 10 min followed by inactivation at 75 °C for 10 min before cooling to 4 °C. To evaluate whether contaminating genomic DNA impacted on detection of HPV-16 RNA, 1 μL of selected RNA samples were subjected to cDNA synthesis without the addition of the iScript reverse transcriptase (Biorad) in a total volume of 20 μL. We then ran a PCR for GAPDH and observed no amplification products, showing that the isolated RNA was free from genomic DNA contamination after DNase treatment. DNase treated RNA was added to a cDNA synthesis reaction containing 1 μL of iScript reverse transcriptase (Biorad) and 1x iScript reaction mix in a total volume of 20 μL. The cDNA synthesis mix was incubated at 25 °C for 5 min, 42 °C for 30 min followed by enzyme inactivation at 85 °C for 5 min before cooling to 4 °C. Specific primers were used for the amplification of a region spanning the E6 gene of the HPV-16 genome and primers for a housekeeping gene (GAPDH) was run in parallel to normalize the amount of cDNA input (Table  μg was tSpecific Taqman primers (Life Technologies) have been designed for the amplification of a region spanning HPV-16 E6 and E7 genes  were p16-negative based on IHC analysis for p16INK4a by qualified pathologists. p16INK4a-positive tumors were predominantly found in the oropharyngeal site (90.5 %), with 69 % found to be in stage IV. Whereas p16INK4a-negative tumors were equally spread across the three major sites  with 35 % of patients in stage IV.We investigated 82 patients diagnosed with HNSCC in the oral cavity, oropharynx, nasopharynx, hypopharynx, larynx, salivary gland, throat/neck and cervical lymph node . 39 of the 42 patients with p16INK4a-positive tumors had a detectable level of HPV-16 DNA in the oral rinse samples, yielding a test sensitivity of 92.9 % and positive predictive value (PPV) of 100 % and negative predictive value (NPV) of 93 %  was also found using qPCR HPV-16 DNA  tested positive for HPV-16 DNA in salivary oral rinse samples  is crucial in tumor initiation and progression . TherefoINK4a-positive tumors .We isolated RNA of sufficient quality for PCR analysis from 80 of 82 samples . HPV-16 specific E6/E7 mRNA was detected in 22 out of 40  with the presence of HPV DNA in tumors [INK4a is an indirect marker for HPV status which is widely used by clinical pathology laboratories around the world due to its low technical costs compared to ISH and PCR based tests [Immunostaining of tumor sections for the HPV-16 surrogate marker, p16n tumors . Howevered tests . TherefoINK4a-positive patients. This is consistent with a previous report by Koskinen et al [The sensitivity of our HPV-16 DNA test is 92.9 % in salivary oral rinse samples collected from p16en et al  which shen et al . Anotheren et al . More reen et al . The impen et al . Anotheren et al .INK4a positivity compared to HPV-16 DNA. One possible reason may be that not all of the patients with HPV-16 DNA display E6/E7 mRNA expression in their tumors. Another possible cause may be due to the unstable nature of RNA in salivary oral fluids, which may have contributed to the modest test sensitivity [The initiation and maintenance of an HPV-driven carcinoma requires viral oncogene expression . Therefositivity . Prior tINK4a instead of direct HPV-16 biomarkers such as HPV DNA or RNA. This was due to the lack of ISH or PCR analysis as part of the routine diagnostic for HPV at the Princess Alexandra Hospital, where the tumour samples were processed and analysed. Limited availability of tumour samples also hampered our ability to assess tumour HPV-16 status of the patients using direct HPV markers. However, given that up to 92.9 and 60 % of the patients with p16INK4a-positive tumours had detectable levels of HPV-16 DNA and RNA respectively in their oral fluid, as well as the lack of false positive in the p16INK4a-negative patients, strongly indicate that p16INK4a positivity in tumour is associated with HPV-16 infection in the current study.We acknowledge the limitations of the current feasibility study, which include a limited sample size and results may not be generalizable to the population at large. In addition, the tumour HPV-16 status of patients was classified based on the evaluation of the surrogate marker, p16From the perspective of translation of salivary oral rinse HPV-16 assay into a routine clinical diagnostic tool, it is important to consider different origins of cells being tested in oral fluid, including tumor cells, healthy exfoliated cells and immune cells . The natWe conclude that PCR detection of HPV-16 DNA in oral rinse can serve as a diagnostic tool in HPV-16 positive HNSCC patients in addition to the current diagnostic methods. With further studies, the detection of HPV-16 in salivary oral rinse can potentially facilitate early detection of pre-cancerous lesions that may warrant further monitoring and intervention.


Oropharyngeal HNSCCa is frequently associated with HPV infection. DNA-based Immunotherapy with plasmids encoding HPV16 and HPV18 E6/E7 antigens has been shown to generate robust immune responses in women with HPV-driven high-grade cervical dysplasia. We hypothesize that HPV-specific immunotherapy with INO-3112 (VGX-3100 + INO-9012) in patients with HPV-associated HNSCCa will generate robust immunity which may contribute to disease stabilization or regression.Eligibility for this prospective Phase I/IIa trial included adults with HPV-positive (assessed by p16) HNSCCa, ECOG PS 0-1, and adequate organ function. Patients (pts) are enrolled into two cohorts. In Cohort 1, pts receive INO-3112 pre and post-surgery. In Cohort 2, pts receive INO-3112 after completion of cisplatin based chemoradiation. INO-3112 (6mg of VGX-3100 plus 1mg of INO-9012) is delivered IM followed by electroporation with the CELLECTRA® device, once every 3 weeks for a total of 4 doses. Pts are followed for 2 years. Primary and secondary endpoints are safety and immune responses. Exploratory endpoints include: anti-tumor effect and progression-free-survival. Assessment of post-immunotherapy surgical specimens is being done to evaluate vaccine-induced lymphocyte infiltration in tumor.As of June 2015, 19 pts have been enrolled. Complete safety data is available for 13 pts. Cohort 1: n=3, Cohort 2: n=10; 12 males; median age 57.7 years (range 39-76); cancers at base of tongue=6, tonsil=6, soft palate=1; never smoker=5, median follow-up is 104 days. INO-3112 was well tolerated with no Grade 3 or higher AEs. The most common AEs were injection site pain (n=11), local erythema (n=4) and hematoma/swelling . Two subjects had Grade 3 lymphopenia at baseline and no worsening during the trial. There was a Grade 2, unrelated SAE of post-surgical procedure hemorrhage. Enrollment and correlative analysis are ongoing; among samples tested to date, as compared to baseline, 4 of 5 evaluable pts showed elevated anti HPV16 and 18 E6/E7 antibody titers. Nine of 10 evaluable pts exhibited increased HPV-specific cellular responses by IFN-gamma ELISpot. Seven of 8 evaluable pts had HPV-specific CD8+ T cell activation concurrent with increased lytic proteins (granzymes and perforin) by flow cytometric analysis.These interim results demonstrate that this DNA-based immunotherapy (INO-3112) can safely generate HPV-specific CD8 T cell immunity in patients with HPV-related HNSSCa. All tested pts had positive cellular immune responses in at least one assay.This study (NCT02163057) is co-sponsored by Inovio and the Abramson Cancer Center at the University of Pennsylvania (5P30CA016520-39).ClinicalTrials.gov identifier NCT02163057.


Objective. Nowadays numerous ancillary techniques detecting HPV DNA and mRNA compete with cytology; however no perfect test exists; in this study we evaluated classification and regression trees (CARTs) for the production of triage rules and estimate the risk for cervical intraepithelial neoplasia (CIN) in cases with ASCUS+ in cytology. Study Design. We used 1625 cases. In contrast to other approaches we used missing data to increase the data volume, obtain more accurate results, and simulate real conditions in the everyday practice of gynecologic clinics and laboratories. The proposed CART was based on the cytological result, HPV DNA typing, HPV mRNA detection based on NASBA and flow cytometry, p16 immunocytochemical expression, and finally age and parous status. Results. Algorithms useful for the triage of women were produced; gynecologists could apply these in conjunction with available examination results and conclude to an estimation of the risk for a woman to harbor CIN expressed as a probability. Conclusions. The most important test was the cytological examination; however the CART handled cases with inadequate cytological outcome and increased the diagnostic accuracy by exploiting the results of ancillary techniques even if there were inadequate missing data. The CART performance was better than any other single test involved in this study. Cervical cancer (CC) is the third most common cancer and the fourth leading cause of cancer death in females worldwide . More thCC is known to be caused almost always by human papillomavirus (HPV) infection which is the commonest sexually transmitted infection worldwide. About 100 types of HPV virus that can infect humans have been identified. Among them, 15 are oncogenic and can cause CC. Improved understanding of HPV infection and the natural history of cervical neoplasia have resulted in the addition of the HPV DNA test along with the Pap test and frequently a competing test.Nowadays, ancillary techniques for CC screening are available. These include HPV DNA typing and mRNA identification of the viral E6/E7 oncogenes that are linked to oncogenic activation. Among them, mRNA typing with nucleic acid based amplification (NASBA)  and mRNASeveral published studies in the literature attempted to clarify the role of each technique as a unique test to substitute the Pap test –7, 9–17.Computer science and artificial intelligence enabled the development of computer assisted systems for the support of clinical diagnosis or therapeutic and treatment decisions. Various classification techniques such as neural networks –29, discAmong the various decision support techniques (CARTs) is an attractive statistical approach to extract knowledge from data as they are straightforward to construct and easily understandable by physicians. The application of these systems produces simple decision algorithms linked with probabilities that can be promising to define triage rules and perhaps give a better understanding of the disease.The aim of this study was to investigate the potential role of CARTs applied on various diagnostic variables measured in the modern cytopathology laboratory and to build algorithms for the triage of individual cases. Special focus was given to design the study as pragmatic as possible: thus, (1) cases from two different parts of the country were selected, (2) a part of the cases were considered to be negative as no histological confirmation could be obtained due to ethical reasons, and (3) inadequate test results  were included as this is the cytopathology laboratory reality.Our study involved the 3rd Department of Obstetrics and Gynecology, the Department of Cytopathology and the 2nd Department of Pathology, all three hosted in “Attikon” University Hospital, Medical School of Athens University, and the Department of Obstetrics and Gynecology of University Hospital of Ioannina City. The study was approved by the University Hospital Ethics Boards and participating women signed an informed consent (ICON) form to allow use of their epidemiologic, diagnostic, and ancillary test data.All cytological and ancillary examinations were based on ThinPrep liquid based cytology (LBC) material obtained before colposcopical examination. The smears were routinely prepared for cytological examination and the remaining material in the ThinPrep vial was used for additional evaluation of biomarkers related to the HPV lifecycle. The smears were assessed by experienced cytopathologists. Histological material was obtained during colposcopy and/or during treatment by conization. The obtained histological samples were fixed and prepared according to standard histopathology protocols.The cytological findings for each woman were formulated according to the revised Bethesda classification system (TBS2001 system) , 39.The histological diagnosis was the golden standard and was used as the target category of each woman. Punch biopsies were performed by experienced colposcopists (in practice for more than 10 years) as part of the study protocol. The three-tiered cervical intraepithelial neoplasia grading system was used for reporting histological diagnosis. Clinically negative (CN) cases were included in the study. These were defined as CN if the cytology, colposcopy, and the CLART Human Papillomavirus 2 HPV DNA test see  were allIn relation to the HPV lifecycle biomarkers we used (a) HPV DNA typing using the CLART Human Papillomavirus 2 (GENOMICA) kit for the simultaneous detection of 35 different HPV genotypes by PCR amplification of a fragment within the highly conserved L1 region of the virus ; (b) NASWithin the clinical laboratory, it is not infrequent that an ancillary test produces invalid results or the biological material that remains in the vial is not adequate to perform additional tests; therefore, it is not guaranteed that there are available sets of such data for all women participating in the study. Additionally parous details were not available for all women as such data often were not considered important and referral forms were incomplete.Our target was to classify each woman into one of the following categories: (a) <CIN2, which included the histologically negative and CIN-1 cases as well as the CN cases, and (b) ≥CIN2, which included the histological categories: CIN-2, CIN-3, SCC, and ADENO-CA.For each case, a vector of 50 variables was created ; this haThe CART model was created using IBM SPSS Statistics 19 for Windows . The CART algorithm is possible to be configured and use a specific feature at the first node of the tree; however, in this study CART was allowed to select as first test the test with the highest overall accuracy. To assess the performance, various statistical measures were extracted: specificity, sensitivity, positive and negative predictive value (PPV and NPV), false positive and false negative rates (FPR and NPR), and overall accuracy (OA).In total 1006 histologically confirmed cases  and 18 adenocarcinomas (ADENO-CA))) were included in this study and additionally 619 CN cases. The correlation of the cytological versus the histological outcome of our material appears in For the construction of the CART model, the CHAID algorithm was used; the CART architecture was 20-5-10; that is, each parent node was forced to 20 or more vectors, and each terminal node had more than 5 vectors, and the tree depth was not allowed to grow more than 10 levels. The system was pruned to obtain simpler forms, the significance level for splitting a node was set to 0.05, the chi-square statistic was based on the likelihood ratio and the significance values were adjusted using the Bonferroni method, resplitting of merged categories within one node was allowed, the age and flow percentage intervals were set to 10 , and finally the maximum number of iterations was 500.The user starts from the top node and examines the value of the proposed characteristic, in our case the cytological examination outcome.According to the value the user navigates to the appropriate node; in our case if the cytological examination is inadequate, we examine the flow result and a negative result leads the user to a probability for the case to be benign equal to 93.9% while if there is no result or the outcome of flow cytometry is positive then the user may examine the existence on HPV subtype 16 within the mRNA test, and interestingly a negative result provides a probability for malignancy equal to 93.8% as 15 out of the 16 cases with this profile were ≥CIN2 in histology.During navigation to the tree, the user may choose to stay in the proposed node if he/she is satisfied by the risk danger (probability) otherwise may examine the next proposed feature to find a more accurate result.The steps are repeated down to the terminal nodes if the user is not satisfied from the proposed risk levels from the previous parent nodes.The CART architecture appears in In a second example, the user may start with a negative cytological examination result; according to the lab performance, the probability for such a case to be positive is very small (0.9%). However, if more guarantees are required for this result, an HPV DNA test may be performed, a positive outcome on HPV subtype 16 reduces the probability of a case to be less than CIN-2 from 99.1% to 66.7% , and a negative or invalid result on test for HPV subtype 16 combined with a negative result for flow cytometry practically assures the woman that the probability to have a lesion worse than CIN-2 is negligible, as 568 cases with this profile in our material had less than CIN-2 and no case was found with equal or more than CIN-2 lesion.A third example is related to the cases that are ASC-US or LGSIL in cytology; if the triage is based only on the cytological examination, all these women should be referred to colposcopy. However, in our material 441 cases were <CIN2 and 89 were ≥CIN2 ; thus, the probability to have <CIN2 is 83.2%, and if additional material is available in the vial, then a flow examination may be more helpful for the triage; in particular, a negative result in the flow examination gives a probability for <CIN2 equal to 96.5%, while a positive result indicates that this woman has more chances to harbor ≥CIN2 46.1% , and in The assignment matrix of CART results appears in Specifically in Finally in order to allow a more detailed evaluation of the methodology, in Test Papanicolaou is viewed as the most successful CC test  if it isIn our material the percentage of ≥CIN2 cases in the total of the cases given as ASC-US was 23/169 = 13.61%. Furthermore, the percentage of ≥CIN2 cases in the total of LgSIL cases was 66/361 = 18.28%. Both percentages are in agreement with those reported by other researchers  that ranExploitation of ancillary test data for improvement of cervical intraepithelial lesions is nowadays a hot research topic with important applications. Since 2010, the Hellenic Cervical Pathology (HeCPA) Study Group is working on innovative approaches that use advanced mathematical and computing tools for the exploitation of ancillary tests that are nowadays available. Up to now, preliminary results are presented in the literature , 56. In According to our results, despite the multitude of features entered into the CART model , the traχ2 = 4.027, P = 0.0448). However, for all other comparisons, the differences in the overall accuracy were statistically significant, specifically CART against cytology with ASC-US+ threshold (P < 0.0001), arrays using any type (P < 0.0001), arrays using high risk subtypes (P < 0.0001), arrays for subtype 16 or 18 (P < 0.0001), NASBA for any type (P < 0.0001), and finally p16 (P < 0.0001). In relation to the comparison of the CART model and the cytological examination with threshold ASC-H+, the sensitivity of the CART model (83.28%) was significantly higher than cytology (69.33%); therefore, the proposed method had significantly (P < 0.0001) better performance than all other alternatives.Based on the results, the proposed methodology had superior performance in relation to the overall accuracy (92.00%) than the majority of alternative methods . There wIn relation to the false positive cases, the CART wrongly categorized 74 cases as positive (≥CIN2); from these 12 were negative and 62 CIN-1 in histology, although 50 of these cases were given as ASC-H or HGSIL in cytology , 17 as LThe analysis of false negative cases is more important; the CART model gave 56 false negative cases in total (14 ASC-US, 32 LGSIL, 2 inadequate, 7 negative in cytology, and 1 ASC-H; see In relation to the 61 samples that were inadequate in cytology , 22 wereConcluding the application of the proposed method gave encouraging results and not only could be helpful towards a better management of women for various findings during cytological examination but also provides a flexible technique for the estimation of ≥CIN2 risk. As a result, the proposed method provides a guide towards personalized management and therapeutic decisions, may reduce the overload of colposcopy clinics and unnecessary treatments, and identifies a higher percentage of women at risk of cervical cancer or precancerous lesions.


Currently, there is limited data on the immunogenicity and efficacy of human papillomavirus vaccines in Low and Middle income countries (LMIC). The review aims to summarize the current status from published HPV vaccine safety, immunogenicity and efficacy studies in low and middle income countries (LMIC). Electronic databases (PubMed/MEDLINE and HINARI) were searched for peer reviewed English language articles on HPV vaccination in LMIC that have so far been published from 1st January 2006 up to 30th January 2015. Eligible studies were included if they had used the bivalent (bHPV) or quadrivalent HPV (qHPV) vaccines in a LMIC and investigated safety, immunogenicity and/or efficacy. The main findings were extracted and summarized. A total of fourteen HPV vaccine studies assessing safety, Immunogenicity and efficacy of the bivalent or quadrivalent vaccines in LMIC were included. There are only ten published clinical trials where a LMIC has participated. There was no published study so far that assessed efficacy of the HPV vaccines in Sub-Saharan Africa. From these studies, vaccine induced immune response was comparable to that from results of HICs for all age groups. Studies assessing HPV vaccine efficacy of the bivalent or quadrivalent vaccine within LMIC were largely missing. Only three studies were found where a LMIC was part of a multi center clinical trial. In all the studies, there were no vaccine related serious adverse events. The findings from the only study that investigated less than three doses of the bivalent HPV-16/18 vaccine suggest that even with less than three doses, antibody levels were still comparable with older women where efficacy has been proven. The few studies from LMIC in this review had comparable safety, Immunogenicity and efficacy profiles like in HIC. Overall, the LMIC of Africa where immune compromising/modulating situations are prevalent, there is need for long term immunogenicity as well as surveillance studies for long term clinical effectiveness after two and three dose regimens. Globally, cervical cancer is the 3rd most common malignancy among women with more than 530,000 incident cases and over 275,000 deaths annually . The greHuman Papilloma Virus (HPV), the most common sexually transmitted infection has been recognized as a necessary cause of cervical cancer . There aProphylactic vaccines are composed of virus like particles made with synthetic L1 proteins derived from HPV 6, 11, 16 and 18 that assemble together to form an empty virus-like capsid. The bivalent (Cervarix™) that protects against HPV 16 and 18 and the quadrivalent (Gardasil™) that protects against HPV 6, 11, 16 & 18 have been approved in 2007 and 2009, respectively [To date, at least 110 countries have licensed the bivalent HPV vaccine and over 120 countries have licensed the quadrivalent vaccine . HoweverEven as countries continue to roll out HPV vaccination as a primary strategy to prevent cervical cancer, it will probably be over 20 years before the impact of the vaccine on cervical cancer is realized. Yet in the LMIC, data on safety, immunogenicity, efficacy and long term follow up is still sparse .In this review, we summarize the current status of HPV vaccine safety, immunogenicity and efficacy studies available in LMIC countries and discuss the findings in the context of ongoing advances in HPV vaccine development. We discussed the findings and identified gaps for future research in these countries.To review the current status of HPV vaccine safety, Immunogenicity and efficacy studies in LMIC, electronic databases (PubMed/ MEDLINE and HINARI) were searched for peer reviewed English language articles on HPV vaccination in LMIC that have so far been published from 1st January 2006 up to 31st January 2015. Search terms included HPV vaccination and the name of a LMIC as per World Bank list (July 2014) . StudiesThe titles and abstracts of the articles were reviewed for inclusion. Articles were included if they had used the bivalent or quadrivalent HPV vaccine in a LMIC and investigated safety, immunogenicity and or HPV disease end points. Studies on HPV vaccine delivery, knowledge, attitudes and practice around HPV vaccines, opinions about HPV vaccines were excluded. Information was abstracted from each article on: the first author & year of publication, country of study, study design, study population, vaccine type, schedule of administration, duration of follow up, serological tests to measure the immune response and PCR assays to detect HPV DNA. We also abstracted information of safety-(unsolicited and solicited symptoms), immunogenicity (antibody titers) and efficacy  of the bivalent or quadrivalent vaccines among the populations who were studied.In total, 467 studies were found on searching the databases on published studies of HPV vaccines in LMICs. On searching the titles and abstracts, fourteen HPV vaccine studies were indentified that reported on immunogenicity, efficacy  by Khatun et al. [Similar immunogenicity results were realized from a randomized controlled trial using the bivalent HPV-16/18 vaccine among healthy Bangladesh girls 9–13 years  up to month 7. They also found the vaccine to be highly immunogenic in this older population of women . Girls were randomized to receive HPV-16/18 AS04-adjuvanted vaccine (n = 450) or placebo (n = 226) at 0, 1, and 6 months and followed up to 12 months post 1st vaccine dose. Similar to the studies above, girls < 15 years had a higher immune response than girls aged ≥15 years at month 7 month [In the LMIC of Africa, studies that addressed immunogenicity with the three dose bivalent HPV-16/18 vaccine were conducted in Tanzania, Senegal and Uganda. Sow et al.  publishe 7 month . In this 7 month .n = 298) to measure the impact of malaria infections and helminth infestation on the immunogenicity of the bivalent HPV-16/18 vaccine. They found that parasitic infestations were common and that there was no evidence of a reduction in HPV-16 or HPV-18 GMT at Month 7 or at month 12 follow-up visits among participants with helminths or malaria [n = 211) who had been vaccinated with the bivalent HPV vaccine in a demonstration project. At this time point, the AS04-Adjuvanted HPV-16/18 vaccinated girls showed a higher level of antibodies to HPV-16/18 than those who were not vaccinated [In the same cohort, Brown et al.  conducte malaria . However malaria  conducteccinated . Among tccinated ), which ccinated .Immunogenicity data based on 7 months geometric mean titers to Anti-HPV-16/18 was compared between studies that were purely in a LMIC . Women were randomized to receive placebo (n = 1911) or the quadrivalent HPV vaccine (n = 1908) in order to assess the safety, immunogenicity and efficacy of the quadrivalent HPV vaccine. HPV-6/11/16/18 related incidence of infection of at least 6 months’ duration and cervical and external genital disease  were measured for efficacy. In the per-protocol population (no infection at baseline) efficacy against disease or infection related to HPV-6/11/16 and 18 was 90.5 % (95 % CI: 73.7–97.5) (4 of 1615 cases in the vaccine group versus 41/1607 in the placebo group) and 83 · 1 % (95 %CI: 50.6–95.8) (four of 1601 cases versus 23/1579 cases) against disease or infection related to HPV 16 and 18 alone. In the intention-to-treat population (infection present at baseline) efficacy against HPV-6/11/16/and 18 endpoint was 30 · 9 % (95 % CI: 11.1–46.5) (108/1886 cases versus 154/1883 cases) and against HPV-16/18 was 22 · 6 % (95 %CI 2 · 9−41 · 9) (90/1886 cases versus 115/1883 cases), since infection and disease were present at baseline [Muñoz et al.  conductebaseline . In thisCurrently, there is an on-going phase 3, multinational, double-blind, and randomized controlled trial with the bivalent vaccine, involving 13 countries, with the Philippines as the only participating LMIC  named Vaccine Immunogenicity and Efficacy study (VIVIANE) Skinner et al. . The maiIndividual country analysis of efficacy end points in these studies was not done most probably due to inadequate sample size. Therefore, conclusions of efficacy end points from participating countries individually may not be drawn.Safety of the HPV vaccines was also assessed in most of the studies described above , medically significant conditions (MSCs), pregnancies and their outcomes were also assessed , 17, 20.Neuzil et al.  in VietnLaMontagne et al. found similar antibody concentrations at ≥29 months after three doses of HPV vaccine regardless of dose-timing, and that extended schedules did not produce inferior immune responses . The finn = 36), two (n = 145), or three (n = 195) doses. They found that even though immunogenicity with less than three doses did not meet a priori non-inferiority thresholds, antibody levels measured at ≥24 months after last dose were similar to those of adult women who had been followed for more than eight years for efficacy [In Uganda, LaMontagne et al.  investigefficacy .In conclusion, alternative dose schedules have shown non inferior immunogenicity among the 10–13 year old age group when compared to standard dosing schedules in populations where they have been evaluated.In addition to the published clinical trials from LMICs in this review, a total of ten unpublished ongoing, suspended, withdrawn or completed clinical trials were found  within LMIC. India had the largest number (8/10) with six of them using qHPV while two were using bHPV. The qHPV Indian trials are mainly safety and immunogenicity studies among HIV populations (two of them), healthy populations  while another was a vaccine delivery strategy study which was withdrawn. Likewise, the two bHPV vaccine studies are assessing safety and Immunogenicity among HIV positive and negative populations .However, in India, there were media allegations that HPV vaccines caused death of four girls in Northern India. Acting on this information, the government of India suspended two HPV vaccine studies and instituted an inquiry into the safety of both vaccines . One stuIn addition to the Indian studies, two safety and immunogenicity studies were found among pre adolescents in Kenya; HIV positive and negative populations . Results from these trails are yet to be published.We have reviewed results of safety, immunogenicity, efficacy  for both the bivalent and quadrivalent HPV vaccine in LMIC. This review identified ten published clinical trials that involved a LMIC. According to the clinicalTrials.gov (accessed 23rd March 2015), a total of 159 cervarix or gardasil clinical trials have so far been registered, ongoing or completed. Both the published and unpublished clinical trials within the LMICs trials are still few when compared to the HICs. The discrepancy may in part be due to ethical, scientific and logistical challenges that face the conduct of clinical research in LMIC .In this review, we found that only five HPV vaccine studies  have been conducted within the LMICs of Sub-Saharan Africa (SSA), yet, SSA has a high burden of cervical cancer. Additionally, SSA is one of the countries with a high incidence of penile cancers, though overall, its global incidence is low . SSA hasAll the studies in this review that investigated immediate vaccine safety did not record a serious adverse event related to the vaccine. Serious adverse events that have been reported as potential signals have not been confirmed after detailed investigations . The safPublished vaccine efficacy studies that were found in this review where a LMIC participated were multi center studies. Currently, we identified only six clinical trials with cervarix or gardasil that are ongoing or completed within a LMIC . Notably in Sub-Saharan Africa, two Phase three randomized clinical trials that were found  are all from Kenya. The trials are assessing immunogenicity to the qHPV vaccine among HIV-infected adolescents (9–14) while one of them is assessing safety, tolerability and immunogenicity of the qHPV vaccine among healthy women 9–26 years. The other four ongoing or completed trials with cervarix or gardasil are also assessing safety and immunogenicity among healthy and HIV infected Indian populations as well as alternative dose regimens. Results from these trials which are yet to be posted will not feature vaccine efficacy as this was not part of the objectives. Therefore, HPV vaccines efficacy studies within the LMICs should be considered.The few clinical trials that were conducted within the LMIC demonstrated that the 9–15 year old age group mounted a higher immune response when compared to the older age groups (above 15 years). This finding was consistent with what was observed in other clinical trials that were conducted elsewhere . Furtherversus three dose regimen and had a small sample size and therefore was not powered to test the primary hypothesis of non-inferiority. Nevertheless, this study still suggests that even with less than three doses, antibody levels were still comparable with older women were efficacy has been proven [After evidence of non-inferiority of two-dose schedule when compared to the three-dose schedule from clinical trials, WHO currently recommends a two dose schedule at 6-months interval of the bivalent or quadrivalent vaccine for girls vaccinated before age 15 years and a three dose schedule for those aged ≥15 years or those with immune compromising conditions . On alten proven . HoweverSecond generation vaccines are being considered to address some of the limitations faced by LMIC with the current prophylactic vaccines, which include limited valence and dependency on the cold chain among others . So far,The few studies in this review that assessed safety, Immunogenicity and efficacy of the HPV vaccines in the LMIC had a similar safety and Immunogenicity profile like in high income countries. Compared to high income countries, there are much fewer studies on safety and immunogenicity conducted in LMIC and the number of study participants is also lower, so data from LMIC is still limited. Notably, HPV vaccine efficacy data is largely lacking with no efficacy study specifically identified for a LMIC though the available studies have provided evidence of efficacy. Specifically, in Sub-Saharan Africa where immune compromising/modulating situations are prevalent there is need for more robust better designed long term immunogenicity and effectiveness studies , 43.Overall, there is need for evidence of the long term clinical effectiveness and duration of protection after two dose regimens as is elsewhere . This evThe review highlighted the HPV vaccine published trials within the LMIC. The identified research needs are in line with WHO recommendations for future research needs.


K-optimal design). In this paper, we propose a new approach to solve the K-optimal GES design problem for fourth-order tensor-based diffusion profile imaging. The problem is a nonconvex experiment design problem. Using convexrelaxation, we reformulate it as a tractable semidefinite programming problem. Solving this problem leads to severaltheoretical properties of K-optimal design: (i) the odd moments of the K-optimal design must be zero; (ii) the evenmoments of the K-optimal design are proportional to the total number of measurements; (iii) the K-optimal design isnot unique, in general; and (iv) the proposed method can be used to compute the K-optimal design for an arbitrarynumber of measurements. Our Monte Carlo simulations support the theoretical results and show that, in comparisonwith existing designs, the K-optimal design leads to the minimum signal deviation.The design of an optimal gradient encoding scheme (GES) is a fundamental problem in diffusion MRI. It iswell studied for the case of second-order tensor imaging (Gaussian diffusion). However, it has not been investigatedfor the wide range of non-Gaussian diffusion models. The optimal GES is the one that minimizes the variance ofthe estimated parameters. Such a GES can be realized by minimizing the condition number of the design matrix( Diffusion-weighted MRI is a noninvasive imaging technique to probe microstructures in living tissues, for example, the human brain. It involves acquiring a series of diffusion-weighted images, each corresponding to diffusion sensitization along a particular gradient direction. Non-Gaussian diffusion models have gained wide attention among researchers because of their potential ability to resolve complex multifiber microstructures. Özarslan and Mareci  introducK-optimal design. In this paper we solve the problem of K-optimal GES design for HOT-based ADC profile imaging as follows. First, we reformulate it as a nonconvex experiment design problem. Then, by convex relaxation we obtain a tractable semidefinite programming (SDP) problem. The last step is to extract design points (the gradient encoding directions) from the optimal design matrix. Finally, to show the relevance of the proposed design approach, we evaluate our solutions using the rotational variance test and Monte Carlo simulations. Throughout the paper “experiment design” and “gradient encoding scheme (GES)” are used interchangeably. The former is used in the optimization context while the latter is used in the diffusion MRI (dMRI) community.The need for robust estimation of diffusion parameters in a limited acquisition time has given rise to many studies on optimal gradient encoding scheme (GES) design. In the case of the classical second-order model they include –14. Howem ≥ 4) see kl is the element of M10 placed in the kth row and lth column. Carefully note the relationship between q and the original design variables (gis) because this is used in q1 = ∑xi8 and q10 = ∑xi4yi4.The condition number measures the sensitivity of the solution to changes in measurements . Hence,  because (5)κG=κM=s (as in ) seek togi‖ = 1, i = 1,…, N, and solve the problem by the algorithm given in T, M = ∑i=1NaiaiT, and repeat the whole process in N, the optimal condition number of the design matrix is K-optimal GES for N = 6 is listed in udied in . ThereingTDg = gTDg(gTg) implies that the second-order tensor D can be represented by a fourth-order tensor t = , where dij denotes the elements of D. In such cases, the number of free parameters of the fourth-order tensor is reduced to six, and thus it can be estimated using the K-optimal designs for second-order DTI. See N = 6.6 0.5K-optimal GES in comparison to several existing methods. The evaluation framework is adopted from [In this section we evaluate the proposed ted from . More spK-optimal GES has the minimum condition number. References for the competing GESs are also provided in this table.S(gi) is the measured signal and θ, ϕ, and ψ and computing R = Rx(θ)Ry(ϕ)Rz(ψ). To rotate fourth-order tensors, we use the approach in [N = 30, b = 1500 s/(mm2), NMC = 200 , SNR = S0/σ = 12.5, NR = 343 (number of rotations), and t0 = t0i × 10−4, i = 1,…, 10. All tensors used in the evaluation are listed in t01, signal deviation of the K-optimal GES is consistently less than that of the DISCOBALL scheme. The mean value and standard deviation of the K-optimal GES has the minimum mean value of signal deviation (corresponding numbers are denoted by bold font). Considering t01, t08, t09, and t010 the standard deviation of t02 to t07, the K-optimal GES has the maximum K-optimal GES is far better than that of others. Thus, the proposed K-optimal GES is the most favorable choice in all cases.Signal deviation is defined in  to measuroach in . We evalK-optimal, MCN, and Wong schemes produce a nonuniform distribution of points. Regarding the effect of uniform distribution of points, an interesting observation is that uniformly distributed GESs (the Jones and DISCOBALL) have minimum t02 and t09 . The extension to higher order tensors is possible using the same strategy (the dimension of the the information matrix in (n + 1)(n + 2)/2).The proposed approach can be applied in experiment design for other tensors, although the current work focuses on its application and results on fourth-order tensor estimation. In atrix in  will incsi) is zero mean and independent with constant variance. We acknowledge that, in the dMRI context, these noise assumptions may not hold, in general. However, our Monte Carlo simulations in S(gi) and anisotropic tensors), the proposed GES yields the minimum signal deviation and the minimum rotational variance. Moreover, minimizing the condition number of the information matrix is motivated in [In vated in , 12 regavated in .K-optimal design is not unique. This raises several questions including (i) why should we favor 60-point K-optimal design over four repetitions of 15-point K-optimal designs? and (ii) why should we favor a 60-point K-optimal design over a union of 40- and 20-point designs? To answer these types of questions further extensive studies and experiments with real data (or Monte Carlo simulations) are required (as in [As we mentioned above the d as in –32) whic whicK-opK-optimal GES design for HOT estimation can be formulated as a nonconvex experiment design problem. Next, we solved the problem using convex relaxation and semidefinite programming. We also showed that resulting solutions have the following properties: (i) the proposed solution is the globally optimal solution; (ii) the obtained solutions are not unique, in general; (iii) nonzero entries of the optimal information matrix are proportional to the total number of measurements; (iv) odd moments of the K-optimal design must be zero; and (v) union of K-optimal solutions with N1 and N2 measurements leads to the K-optimal design for N1 + N2 measurements. Another advantage of this work is that it establishes a theoretical foundation for the experiment design in even order diffusion tensor imaging.We showed that the


A 9-valent human papillomavirus (HPV) vaccine  was licensed for use in females and males in the United States in December 2014 .http://www.cdc.gov/vaccines/who/teens/downloads/9vHPV-guidance.pdf).In February 2015, the Advisory Committee on Immunization Practices (ACIP) recommended 9-valent HPV vaccine as one of three HPV vaccines that can be used for routine vaccination of females and one of two HPV vaccines for routine vaccination of males. ACIP recommendations were published in a March 2015 report . Additio


This review tackles the issues related to disease burden caused by cervical cancer (CC) and its precursor (CIN) lesions in Brazil. A special focus is given to new technologies with potential to interfere with the development of CC by reducing the high-risk human papillomavirus (hr-HPV)-induced lesions that remain a major public health burden in all developing countries where organized screening programs do not exist. Globally, 85 % of all incident CC and 50 % of CC deaths occur in the developing countries. Unfortunately, most regions of Brazil still demonstrate high mortality rates, ranking CC as the second most common cancer among Brazilian women. Recently, CC screening programs have been tailored in the country to enable early detection of CC precursor lesions and thereby reduce cancer mortality. A combination of HPV testing with liquid-based cytology (LBC) seems to be a promising new approach in CC screening, with high expectation to offer an adequate control of CC burden in this country. Human papillomavirus (HPV) is a non-enveloped small (8000 bp) DNA-virus with circular double-stranded DNA, showing a specific tropism for human epithelial cells both in the skin and mucous membranes , 2. SincHPV is the causal agent, necessary for the development of CC, and identified in practically all cases when highly sensitive detection methods are used. Cancer development is associated with HPV persistence that leads to cellular transformation, disease progression to precancerous lesions and, if uninterrupted, to an invasive cancer , 8.HPV types infecting genital mucosa (in both genders) are subdivided into high-risk (hr) and low-risk (lr) groups. This classification is based on established epidemiological evidence on their association with benign, precancerous and cancer lesions. The hr-HPV are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59; whereas the lr HPVs include 6, 11, 40, 42, 43, 44, 54, 61, 70, 72 and 81. It is now obvious that both hr- and lr-HPV infections do occur in different anatomic sites, inducing either benign, premalignant or malignant lesions [The HPV life cycle is intimately linked with the programmed differentiation process of stratified squamous epithelia , starting from the undifferentiated cells of the basal layer, capable of continuously dividing, and progressing through highly specific cellular events towards fully matured keratinocytes . In principle, HPV life cycle has three major phases. First, infectious virions access the basal layer through a micro-lesion, forming a virus reservoir where the viral genome is conserved as a low–copy number episomal form within the infected cell. Second, the viral genome replication occurs, whereby host cell DNA and the infected cells are divided into daughter cells, which can persist in the basal and parabasal layers and proceed to the division, or move and initiate the differentiation process. In the latter, activation of the late (L) gene expression and vegetative viral DNA amplification begin. This amplification leads to synthesis of a huge number of viral copies , 10, 11.cervical transformation zone (TZ), where squamous epithelium meets the glandular epithelium of the endocervical canal. HPV-induced cell transformation is mediated by the viral E6 and E7 oncoproteins interfering with the cell cycle regulators of the host cells, starting at the basal cells layer and gradually extending to the full epithelial thickness [HPV infection is most easily transmitted in sexual intercourse, and the risk is higher in women with an early onset of sexual activity (due to higher exposure to the virus) as well as in those with impaired immunity –16. Cervhickness , 18. Onchickness , 20.The great majority of sexually active women and men are likely to be infected by HPV at least once in their life-time . In geneAround 70 % of all CC cases are associated with hr-HPV 16 and 18, in contrast to benign genital warts, which are predominantly related to HPV 6 and 11. Of all hr-HPV genotypes, HPV 16 shows the highest probability for persistence, and thus has a major impact on the cancer risk. HPV 18 is the most frequent genotype found in adenocarcinomas , 6. Up tAccording to the estimates for 2012, more than 528,000 incident cases and 266,000 deaths due to CC were detected worldwide –27.Fig. The screening strategy in Brazil is targeted to women aged between 25 and 64 years, repeated every 3 years. Accordingly, the women should have Pap smear taken each three years if she has two consecutive normal smears in the previous screening rounds . In the It is established that an organized population-based screening and treatment of the detected precursor lesions can decrease the disease mortality by 80 %, as shown e.g. in Finland , 21. CC The first national initiative by the Brazilian government to implement a national screening program for CC was taken 40 years ago . Since tSince its introduction, the cytological Papanicolaou smear (Pap test) has been considerate as the method-of-choice in organized screening for CC. When implemented as an organized program at population level, Pap smear screening has resulted in a substantial decrease of both incidence and mortality of CC , 21, 31.Brazil has not implemented an organized population-based CC screening program, and not even a universal system to invite all women to realize these examinations. However, the government has designed a program to identify and control the cytopathology examinations performed. The first examination used to identify CC precursor lesions is the Pap test, targeted to women from 25 years  to 64 years, repeated every 3 years for women who had two consecutive negative smears within five years. However, the guidelines  do not recommend screening for women younger than 25 years by Pap test, mainly because most of these lesions clear spontaneously , 33.Recognition that HPV infection is the necessary cause of CC opened new possibilities for prevention strategies, including the primary prevention with highly effective prophylactic HPV vaccines, and the secondary prevention using highly sensitivity HPV tests, which has significantly improved the performance of the screening programs based on the Pap test –35.Molecular techniques have been considered better than cytology regarding the sensitivity and reproducibility in detecting CIN 2 or CIN 3. Proper treatment of the precursor lesions detected by screening should prevent the development of invasive CC and reduce the incidence and mortality. Some HPV tests used in screening have achieved a 60–70 % reduction in invasive CC incidence as compared with cytology alone . DespiteDuring the past several years, a plethora of novel methods have been developed for the hr-HPV DNA testing in CC screening. The use of HPV DNA testing for oncogenic types has been proposed as a co-test with cytology for screening women above 30 years of age, showing higher accuracy as conventional Pap smear screening , 39–41. The overall acceptance of HPV testing is higher among women who have experience on self-sampling (made at home), as compared with the physician collection . MoreoveFor almost 60 years now, cervical cytology , has been the gold standard in CC screening programs. Based on early detection of CC precursor (CIN) lesions, both tests represent measures of secondary prevention of CC. However, organized screening based on cervical cytology has severe limitations in implementation; extensive training is mandatory, good laboratory infrastructure is necessary, and standardization and quality control are essential , 42, 46.The well-established natural history of HPV infections is the sound basis for the rational use of CC preventive measures based on HPV testing. The single most important goal of CC screening is to reduce mortality. This is achieved by decreasing the incidence of invasive disease by detecting the precancerous lesions, followed by their adequate treatment , 46, 47.Although HPV vaccination of teenagers and young women has proven effective in many countries, the public health strategies to demonstrate effective prevention of CC still represent major obstacles to overcome. The cost-effectiveness analysis for all strategies to reduce CC incidence should involve both the screening and HPV vaccination in an integrated and organized manner. At the moment, the preferable option in Brazil seems to be the use of HPV testing for primary screening, followed by triage of all HPV+ cases with the Pap test. A recent Mexican trial  showed aAccording to WHO, HPV types 16 and 18 are responsible for 70 % of all CC cases worldwide. The currently available vaccines against these two HPV types have the potential of reducing the incidence of cervical and anogenital cancer. Brazil has a population of >64 million women aged above 15 years and at risk to develop CIN and CC, who certainly would benefit from HPV vaccines . In thisWith the widespread implementation of HPV vaccines, HPV infections and HPV-related lesions will decrease substantially, which might increase the rates of false-negative results in cytology, in addition to reduced specificity. The combination of HPV vaccination with HPV testing and cytological triage is suggested to be the optimal algorithm to protect women against CC , 34, 47.The most important applications of HPV testing include i) organized screening programs to optimize an early detection of HPV-induced CC precursor lesions, and, ii) to conduct adequate follow-up of the women who have been treated for high-grade CIN.Some novel technical innovations of HPV testing are particularly designed for remote areas, without compromising the test performance for hr-HPV detection. Certainly self-sampling for HPV testing still needs standardization to make it compatible with the usual sampling techniques. Once successfully accomplished, it has a potential to become a valuable screening tool for large underprivileged populations, thus contributing to alleviating the significant disease burden in these countries.