Information on the prevalence and type distribution of human papillomavirus (HPV) among Malaysian women is currently limited. The present study therefore aimed to provide an updated estimate on the prevalence and type distribution of HPV among Malaysian women with and without cervical cancer. Total DNA was isolated from the cervical cell specimens of 185 histopathologically confirmed cervical cancer patients and 209 cancer-free healthy females who were tested negative in a recent Pap test. Viral-specific DNA was subsequently amplified with biotinylated primers and hybridized to HPV type-specific probes via a proprietary “flow-through hybridization” process for determination of HPV genotype. It was demonstrated that 83.2% of the cervical cancer patients and none (0.0%) of the cancer-free females were positive for HPV infection. Among HPV-positive subjects, 14 different viral genotypes were observed, namely HPV16, 18, 31, 33, 35, 45, 52, 53, 58, 66/68, 73, 81, 82, and 84/26. A total of 91.6% of the HPV-positive subjects had single-type HPV infections and the remaining 8.4% were simultaneously infected by two HPV genotypes. The most common HPV infections found were HPV16 (35.7%), HPV18 (26.0%), HPV58 (9.1%), and HPV33 (7.1%) single-type infections, followed by HPV16 + HPV18 co-infections (5.2%). The study has successfully provided an updated estimate on the prevalence and type distribution of HPV among Malaysian women with and without cervical cancer. These findings could contribute valuable information for appraisal of the impact and cost-effectiveness of prophylactic HPV vaccines in the Malaysian population. Cervical cancer is the seventh most common cancer worldwide and the fourth most common type of cancer among the female population . IncidenOver the past decades, it has become established that human papillomavirus (HPV) is the central etiologic agent for cervical carcinogenesis . The virHPV infection is preventable through vaccination. Several forms of HPV vaccines are currently available, which include a bivalent form (which targets on HPV16 and HPV18), a quadrivalent form , and a nonavalent 9-valent) form  -valent f,11. For The prevalence and type distribution of HPV in the Malaysian population are incompletely understood at the present. Several previous studies on these aspects in Malaysian population were either outdated , employeThe study received ethical approval from the Human Research Ethics Committee (HREC) of Universiti Sains Malaysia (reference numbers: USMKK/PPP/JEPeM [253.3.(7)] and USM/JEPeM/14100325) as well as the Medical Research and Ethics Committee (MREC) of Ministry of Health, Malaysia (reference numbers: KKM/NIHSEC/08/0804/P12-380 and KKM/NIHSEC/P15-1214). Written informed consent was obtained from all subjects prior to their enrolment into the study.™ Cervical Brush . While collecting the cervical cells, it was ensured that the shorter lateral bristles of the brush were in contact with the ectocervix. The cells obtained were suspended in cobas® PCR Cell Collection Media . A single layer of the cervical cells was then deposited on appropriate glass slides and the cells were examined through a computerized microscope with embedded camera. The residual cells suspended in the cell collection media were used for HPV typing if no abnormality was found in the Pap test. For patients with cervical cancer, cervical cells were collected by using the same method, but no Pap test was performed. Women with a previous history of cervical cancer or any other malignancy were excluded from the study.A total of 394 study subjects  were recruited from (i) Hospital Universiti Sains Malaysia, Kubang Kerian, Malaysia, (ii) Hospital Raja Perempuan Zainab II, Kota Bharu, Malaysia, and (iii) Hospital Sultan Ismail, Johor Bahru, Malaysia, between August 2012 and January 2016. The inclusion criterion for the cervical cancer cases was a clinical and histopathological confirmation of cancer of the uterine cervix. The histopathology analysis was performed on a biopsy specimen by a pathologist. On the other hand, healthy females were included if they were tested negative on a Pap test taken just before their enrolment into the study. The Pap test was performed by first collecting cervical cell specimen from the endocervical canal of the subjects with a sterile PathTeztTotal DNA was isolated from cervical cell specimens by using QIAamp® DNA Mini Kit . First, cervical cells were pelleted by centrifuging 1.5 ml of cell suspension at 13,200 rpm for 5 min. The process was repeated until a cell pellet of approximately 2.0 mm in diameter was obtained. The cervical cell pellet was then washed twice by centrifugation in 1.0 ml of phosphate-buffered saline at 13,200 rpm for 3 min. After the supernatant was discarded, the cell pellet was resuspended in 180 μl of Buffer ATL, and the remaining procedures of DNA isolation were then performed according to the manufacturer’s instructions. The concentration and purity of the DNA obtained were measured by using a spectrophotometer, and the quality of the DNA was checked with agarose gel electrophoresis. The total DNA isolated was subsequently diluted to a concentration of 20 ng/μl with an appropriate volume of nuclease-free water and stored at −20°C until used.HPV-specific DNA present in the total DNA isolated was amplified with biotinylated PGMY primer set supplied in the PCR Kit of GenoFlow HPV Array Test Kit . All reaction volumes and conditions were based on the manufacturer’s instructions. A positive control and a negative control were prepared for each batch of sample run. Amplified HPV DNA was kept at 4°C until used.After PCR amplification, the biotinylated HPV DNA amplicons were hybridized to membrane-coated HPV type-specific probes through a proprietary “flow-through hybridization” technology by utilizing the Hybridization Kit of GenoFlow HPV Array Test Kit . The “flow-through hybridization” and colorimetric signal development procedures were performed in a R2-M Flow-Through Hybridization System , based on the manufacturer’s instructions. HPV genotype was then determined by comparing the patterns of the colorimetric signal developed on the membrane with the reference guide supplied by the manufacturer. The assay was considered valid only if the amplification control (AC) and hybridization control (HC) signals were present, which respectively indicated a successful PCR reaction and probe hybridization.A total of 185 cervical cancer cases and 209 cancer-free healthy females were recruited into the study. The demographic characteristics of the study subjects, including their age and ethnicity, are shown in The prevalence of HPV infection among cervical cancer patients and cancer-free healthy females is shown in The distribution of HPV subtypes among the infected subjects  is shown in In the present study, the prevalence of HPV in cervical cell specimens of cervical cancer patients was determined to be 83.2%. Since HPV has been established as an essential cause of cervical cancer . A preva. [On the other hand, among cancer-free healthy females, no HPV infection was detected in the present study. This observation was contrary to other reports in the Malaysian population. Chong et al.  reported.  demonstr.  showed t. —since ma. [. [. [. [It was also demonstrated in the present work that majority (91.6%) of HPV infections were caused by a single type of HPV. This observation was in agreement with the findings reported by Quek et al. , which s. [.  and Shar. [. [.  reported[. [. [.  , single-type infection of HPV58 (9.1%), single-type infection of HPV33 (7.1%), and multiple-type coinfection of HPV16+18 (5.2%). This observation was slightly different from the prevalence of HPV observed globally  as well .  suggesteInterestingly, the present work was the first study demonstrating the presence of HPV53, HPV73, HPV81, HPV82, and HPV84/26 (the HPV genotyping kit used could not distinguish between HPV84 and HPV26) in cervical epithelium of cervical cancer patients in the Malaysian population. None of the previous studies has reported the involvement of these HPV genotypes among cervical cancer patients and cancer-free healthy females in Malaysia. However, the observation of the present study was not surprising, as infections by all these HPV types have been documented formerly in other populations worldwide . It can N=1), but also in other populations worldwide [In addition to high-risk HPV types, the genotyping kit used in the present work could also identify the presence of low-risk HPV types. The only low-risk HPV type identified in the present study was HPV84/26 (the HPV genotyping kit used could not distinguish between HPV84 and HPV26). Nonetheless, the role of HPV84/26 in mediating cervical carcinogenesis is likely limited, as HPV84/26 was noticed in a very low frequency of cervical cancer specimens not only in the present study , compared with squamous cell carcinomas (80.0%) and cervical cancers of other histopathological types (84.6%). This observation was in contrast with the findings of many other previous studies ,44–46. IThere were few strengths and novelties in the present work. First, the present study employed a relatively large sample size (compared with other previous studies in the same population) for analysis, which could increase the reliability of the study findings. Besides, the present study utilized a HPV genotyping method which can discern diverse subtypes of the virus. Due to these reasons, several additional HPV subtypes  were demonstrated to be present for the first time in the cervical epithelium of Malaysian women.In conclusion, the present study has successfully provided an updated estimation of the prevalence and type distribution of HPV in Malaysian females with and without cervical cancer. These findings could contribute valuable information for appraisal of the impact and cost-effectiveness of prophylactic HPV vaccines in the Malaysian population.


Cervical cancer (CC) is the second leading cause of cancer death among Filipino women. Human papillomavirus (HPV) vaccination protects against CC. Two vaccines (AS04-HPV-16/18 and 4vHPV) are approved in the Philippines; they were originally developed for a 3-dose (3D) administration and have recently been approved in a 2-dose schedule (2D). This study aims to evaluate the cost-effectiveness of HPV vaccination of 13-year-old Filipino girls, in addition to current screening, in the new 2D schedule. An existing static lifetime, one-year cycle Markov cohort model was adapted to the Philippine settings to simulate the natural history of low-risk and oncogenic HPV infection, the effects of screening and vaccination of a 13-year-old girls cohort vaccinated with either the 2D-AS04-HPV-16/18 or 2D-4vHPV assuming a 100% vaccination coverage. Incremental cost, quality-adjusted life year (QALY) and cost-effectiveness were derived from these estimates. Input data were obtained from published sources and Delphi panel, using country-specific data where possible. Sensitivity analyses were performed to assess the robustness of the model. The model estimated that 2D-AS04-HPV-16/18 prevented 986 additional CC cases and 399 CC deaths (undiscounted), as well as 555 increased QALY (discounted), and save 228.1 million Philippine pesos (PHP) compared with the 2D-4vHPV. In conclusion, AS04-HPV-16/18 is shown to be dominant over 4vHPV in the Philippines, with greater estimated health benefits and lower costs. Cervical cancer (CC), with an estimated yearly incident number of 6,670 cases and 2,832 deaths (year 2012),Cervarix™, an HPV-16/18 AS04-adjuvanted vaccine (AS04-HPV-16/18) protecting against HPV-16 and HPV-18 types; and (2) Gardasil, a HPV-6/11/16/18 L1 virus-like particle vaccine (4vHPV) protecting against 2 non-oncogenic HPV types (HPV-6 and -11) in addition to the oncogenic HPV-16 and -18 types. Both vaccines have high efficacy (around 98%) against vaccine type-related HPV infections.Persistent high-risk human papillomavirus (HPV) infection is a necessary cause of CC, with HPV detected in 99.7% of CC cases worldwide and certain risk factors in the presence of HPV infection may increase the risk of CC.Both vaccines were originally developed for a 3-dose (3D) administration. Different studies with the AS04-HPV-16/18 have shown that 2-dose (2D) vaccination of 9-to-14-year-old girls was immunologically non-inferior to 3D vaccination of 15-to-25-year-old women.The World Health Organization (WHO) recommends HPV vaccination to be included in national immunisation programmes in countries where CC and HPV-related diseases prevention is a public health priority and where vaccine introduction is programmatically and financially feasible and provided that cost-effectiveness of vaccination strategies in the country or region is considered.Information about the value for money, the budget and public health impact of available HPV vaccines in the Philippines may support decisions and choices about the country's vaccination program.The objective of this study is to evaluate the effect of vaccination on CC-related and genital-warts-related disease burden and the cost-effectiveness of the 2D-AS04-HPV-16/18 compared with the 2D-4vHPV for universal vaccination of 13-year-old girls, in addition to the current screening program, in the Philippines.This model, adapted to the Philippine settings, adequately reproduced age-dependent CC incidence  and age-dependent CC mortality . The model also adequately reproduced the age-dependent incidence of genital warts as reported in Japan .The 2D-AS04-HPV-16/18 prevented more CC cases and deaths, screening-detected cervical intraepithelial neoplasia grade 1 (CIN1) and grade 2/3 (CIN2/3) than the 2D-4vHPV at a 100% vaccination coverage rate. The 2D-4vHPV prevented more cases of genital warts than the 2D-AS04-HPV-16/18.The predicted number of life-years and quality-adjusted life years (QALYs) gained, after discounting, were higher with the 2D-AS04-HPV-16/18 than the 2D-4vHPV with a difference of 315 and 555, respectively.The estimated savings in treatment costs as a result of the reduction in CIN  and CC cases with the 2D-AS04-HPV-16/18 would be expected to exceed the estimated savings in the treatment costs from the reduction in genital warts cases with the 2D-4vHPV. Thus, overall discounted treatment costs would be expected to be 228.1 million Philippine pesos (PHP) lower with the 2D-AS04-HPV-16/18 than the 2D-4vHPV for a vaccination coverage rate of 100%.Consequently, the 2D-AS04-HPV-16/18 could be considered dominant, as it would be expected to both improve public health and reduce costs compared with the 2D-4vHPV.The parameters impacting the most on the QALY difference were, in order of importance: the proportion of non-vaccine HPV types in CC, the incidence of HPV oncogenic infection, the vaccine efficacy against non-vaccine HPV types, the HPV-6/11 types distribution in genital warts and the disutility associated with HPV disease . Regarding the cost difference, the order of most-impacting parameters were: the HPV-16/18 types distribution in CC, the cost of vaccine, the screening-associated costs, the vaccine efficacy against non-vaccine HPV types and the cost of treatment of genital warts . The only impact of the change in discount rate from 3% to 1.5% on QALY impact was limited to a switch between the ‘distribution of HPV-16/18 in CC’ and ‘utility loss with HPV disease’ parameters . Regarding the impact on costs, the vaccine efficacy against non-vaccine HPV types and the incidence of oncogenic HPV infection in the general population became more influential. The order of influence of the CC treatment costs and the genital-warts costs are also swapped when changing the discount rate .The results of the probabilistic sensitivity analysis were plotted on a cost-effectiveness plane . WithoutThis study is the first to compare the cost-effectiveness of 2D-AS04-HPV-16/18 vaccine with 2D-4vHPV vaccine, in addition to screening, in the Philippines. The results estimate that the 2D-AS04-HPV-16/18 would generate more QALYs  and be less costly than the 2D-4vHPV, with a probability of 92.2%. This reflects the effect of the higher protection against non-vaccine oncogenic HPV types reported for the AS04-HPV-16/18, which would provide greater protection against CIN and CC compared with the 4vHPV.Our analysis has limitations. As the model is static, it cannot take into account effects on disease transmission and thus cannot quantify any level of herd protection. Administration costs and the cost of logistics and storage for HPV vaccination were not included in the model, due to a lack of currently available data on these costs. This may have under-estimated the total programmatic costs of HPV vaccination. The model also excluded indirect costs, such as lost productivity due to time away from work. It is likely that the estimated lower number of CC cases remaining for the AS04-HPV-16/18 would also result in lower indirect costs. The model assumes a 100% vaccination coverage rate, which may be unrealistic in a real-world setting. However, this vaccination coverage rate was chosen to reflect the maximum possible achievable benefit associated with vaccination. Coverage rates of a different order will result in a linear decline in number of cases and associated cost offset. The resulting incremental cost-effectiveness ratio (ICER), however, is not affected by a different vaccination coverage since the model is fully static.Due to the lack of data on the incidence of genital warts in Filipino women in the general population, we used the best available data from the same region, which in this case were Japanese data. Although the incidence of genital warts was reported to be low in Japan compared with Western countries such as the United Kingdom and the United States, the expert panel who evaluated the input data for this model were of the opinion that these data were the most appropriate to be used for this analysis. The influence of a much higher genital warts burden in the Philippines has not been explored in this analysis due to the lack of reliable data but may have had a relatively strong influence on the results as could be inferred from the one-way sensitivity analysis on the impact of HPV-6/11 distribution in genital warts.The results of this analysis suggest that the addition of 2D-AS04-HPV-16/18 vaccination to the current screening program in the Philippines would generate more QALYs and be less costly than using the 2D-4vHPV.A static Markov cohort model with annual cycles was used to estimate the costs and health benefits of adding HPV vaccination to screening over 95 cycles (lifetime of the cohort). The model has been previously published and was adapted for this study to the Philippine setting by applying country-specific data on epidemiology, screening practice and costs.The model structure, which replicates the natural history of HPV infection, is summarised in Additional File 3. A series of health states represent the natural history of HPV infection. Subjects move between health states at each cycle according to fixed annual transition probabilities. Subjects in the cohort entering the model are assumed to be HPV-naive for oncogenic HPV infection [NoHPV]. At each cycle, a subject may remain in the same health state or become infected with low-risk HPV [HPVlr] or oncogenic HPV [HPVonc]. Subjects infected with low-risk HPV may then develop genital warts [genital wart] or CIN1 [CIN1lr], which may be detected and treated or may spontaneously clear (back to [NoHPV]). Subjects infected with oncogenic HPV may develop CIN1 [CIN1onc], which in turn may evolve into CIN grade 2/3 [CIN23], then progress to persistent CIN2/3 [Persistent CIN23], and then to cervical cancer [Cancer]. Cases of cervical cancer may be treated and cured [Cancer cured], or result in the death of the subject from cancer [Death cancer].Screening was modeled by including a proportion of lesions that are detected by screening, based on the screening coverage and testing sensitivity, and applying different transition probabilities to subjects with detected lesions (‘det’). Subjects with detected lesions at each disease stage are in a different “detected” health state, with higher costs incurred for follow-up and treatment and a lower modified probability of progressing to a more advanced state if treatment is successful than the equivalent undetected state .Vaccination was modeled by modifying the transition probability for becoming infected with oncogenic or non-oncogenic HPV. Vaccination coverage rate was assumed to be 100% to reflect the maximum achievable benefit of vaccination.Philippine specific data were used wherever available. However, there were certain parameters for which no local data were available. In such instances, data from another Asian country were used and validated by a group of key clinical experts who have extensive experience in cervical cancer care and have reviewed them to ensure the data were suitable for use. The group of key clinical experts agreed on the data inputs and assumptions used in the model during a round-table discussion held on July 10, 2013 in Manila, as part of a 2-round Delphi panel with a primary focus on cost data collection.A cohort of 13-year-old girls for the year 2013 was considered in the model . The cohort size was estimated using data from the National Statistical Coordination Board.The transition probabilities for low-risk and oncogenic HPV infection were obtained from published sources or calibrated,Screening coverage was set at 7.7% of women aged 18–69 y.No published cost data were available for the Philippines. Therefore, cost data were obtained from a 2-round Delphi panel. The expert panel consisted of 6 members from Manila and Quezon City in the Philippines and was held in Manila on May and July 2013.All costs related to the screening, treatment of pre-cancerous lesions and genital warts were provided as point estimates for both private and public healthcare settings in an urban area, by the members of the expert group. An average of private and public estimated costs was used for the base-case analysis.CC treatment costs were based on an unpublished costing study performed by a member of the expert group in a hospital based in Manila.The obtained cost figures were provided by experts during the first meeting and thereafter discussed by all experts through e-mail exchange and finally endorsed by all experts during a second meeting. All costs represent direct medical costs and were calculated on an annual basis for the year 2012–2013. Cost data are shown in The model uses vaccine effectiveness against CC on incident oncogenic HPV infection and vaccine effectiveness against genital warts on incident low-risk HPV infection. These were thereafter adjusted for each lesion.i) with the proportion of each HPV type within each type of lesion (%HPVi), according to the following equation:A proxy for overall vaccine effectiveness (VE), including protection against non-vaccine HPV types, was calculated by combining the vaccine efficacy against each HPV type . These data are the most representative of vaccine efficacy among girls before the onset of sexual activity and are therefore the most relevant to the vaccination of 13-year-old girls in the Philippines.The vaccine efficacy was assumed to be the same for both the 2D vaccines as non-inferiority in the immune response has been observed with the 2D and 3D schedule for the AS04-HPV-16/18v.Since country-specific utility data for the Philippines were lacking, published disutility data from other evaluations of HPV vaccination were applied in this analysis.The model was validated against observed local/regional epidemiological outcomes. The CC incidence was retrieved from the 1998–2002 cancer registry data in Manila as reported by the Philippine Cancer Society.In the base case, all costs and outcomes were estimated non-discounted and discounted at 3.5% per year, as recommended by the Philippine Ministry of Health.The analysis was conducted from the perspective of the Philippine government. Only direct medical costs  were included.The base-case analysis compared vaccination of 13-year-old girls with either the 2D-AS04-HPV-16/18 or the 2D-4vHPV, in addition to screening. Vaccination coverage was assumed to be 100% with all subjects completing the full vaccination course.The model estimated the lifetime number of cases of CC, CIN1, CIN2/3 and genital warts, associated costs and QALYs for a 13-year-old girls cohort vaccinated with either the 2D-AS04-HPV-16/18 or 2D-4vHPV. Incremental cost, QALY and cost-effectiveness were derived from these estimates.Cost-effectiveness thresholds from 1xGDP (gross domestic product)/capita ( = “highly cost-effective”) to 3xGDP/capita ( = “cost effective”) were used, as recommended by the World Health Organization (WHO).Both one-way and probabilistic sensitivity analyses were conducted.One-way sensitivity analyses evaluated the effect on the results of variability in the model parameters. The key parameters were varied by ±20% from the base-case values used except for vaccine efficacy and cost parameters. For vaccine efficacy, the reported 95% confidence intervals were used see . For theA specific sensitivity analysis explored the effect of using a discount rate of 1.5% for costs and outcomes as recently recommended by the National Institute for Clinical Excellence (NICE) in the United Kingdom for healthcare interventions with long-lasting health benefits.Risk . Distributions were assigned to input parameters by using a normal distribution when confidence intervals were reported and uniform distribution when no range was available . A total of 10,000 iterations were sampled from the assigned distribution.A probabilistic sensitivity analysis was conducted using Monte Carlo simulation with the software package @


We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing, and CLC Genomics Workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV-negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall type-specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology. Papillomaviridae. More than 200 genotypes are recognized based on the sequence of the approximately 8-kbp circular genome, with variants in each genotype based on sequence relatedness  are double-stranded DNA viruses in the family atedness , 2. DeteV genome , 4. StudV genome , 6.Next-generation sequencing (NGS)-based methods have the promise to improve the spectrum of HPV genotypes detected. Some NGS methods used PCR with consensus primers targeting the L1 region for library preparation, restricting regions of the genome that could be analyzed –13. HPV –17–2 in pool 1 (samples 1 to 16) and 2 (samples 17 to 32), respectively. All raw reads from both pools passed the default filtering of the Illumina BCl2fastq V1.8.4 software . The average number of reads per sample in pool 1 was 8,538,564, and that from Caski at 100 ng (sample 11) with ∼500 HPV16 copies/cell dominated, comprising 21.9% of the total  ranged from 93 to 100% . Samples with 100 ng genomic DNA  generated a mean of 7,406 reads  that, using L1-S1 stringency, mapped to the globin reference (human beta-globin [HBG] nucleotides [nt] 2041 to 3480) with an average depth of coverage ranging from 132.2 to 779.5  . The numWe evaluated mapping results for the 8 control samples  in termsEnriched whole-genome sequencing (eWGS) data from the remaining 24 samples were analyzed using criteria set in the control samples to assign WGS HPV results without knowledge of prior HPV data. Comparing eWGS typing results with prior Linear Array (LA) results, type-specific HPV detection was at least partially concordant in 18 of 24 samples (75%) . GenotypWhile some LA types were not detected by WGS in this small sample set , other LA types were detected in some but not all samples (HPV18 and -66). In some instances, eWGS detected types but did not meet the criteria for number of reads or depth of coverage. For example, in sample 25, eWGS failed to call HPV66 and HPV18, which were detected by LA, but 1,478 reads mapped to HPV66 with 99% of the genome covered but failed because the average depth of coverage (18.9) was just below the cutoff of 20. HPV18 had a subthreshold of 234 reads mapped with 71% genome coverage. In sample 20, eWGS failed to call HPV72; although 1,412 reads mapped to HPV72 with 87% genome coverage, the average depth of coverage of 17.7 was below the cutoff of 20.In sample 29, LA detected HPV84 but eWGS detected HPV114. HPV84 and HPV114 have 84% identity over the whole genome, suggesting the possibility of misclassification based on genomic fragments. To explore this, sample 29 reads were mapped with HPV84 as the only reference sequence. While no reads mapped to HPV84 under stringent conditions, under L0.5-S0.8 conditions 62,203 reads mapped to HPV84 with 94% of the genome covered. HPV53 was not identified in any of the 4 samples that were LA positive for this type; however, HPV53 sequences were found in one of four samples  if mapping was done under less stringent conditions (L0.5-S0.8).AJ812226.1) with 85% coverage. As the full-length reference sequence for HPV64 is not available and HPV64 has been reclassified as a subtype of HPV34, the eWGS results were assigned to HPV64 based on post hoc assessment, giving partial concordance with LA results.LA includes probes for HPV64, which has been reclassified as a subtype of HPV34. For sample 17, 16,120 reads mapped to HPV34 with 16% coverage (compared to the whole genome), whereas 1,736 reads mapped to a 458-bp HPV64 L1 partial sequence  using RNA baits of the Agilent SureSelect target enrichment technology. The target enrichment method avoids the limitations of targeting only limited areas of the genome, minimizes the potential for PCR bias, and significantly increases the potential to increase the number of types identified in a single assay. A recent report using Roche NimbleGen DNA bait-based target enrichment largely focused on understanding the mechanistic signatures of integration of HPV types  in cervical carcinomas , with liThe custom RNA bait library exhibited excellent performance in terms of the fraction of genome coverage, percentage of on-target reads mapped to predicted HPV types, uniformity of coverage for the types that we evaluated, and an observed average level of target enrichment 184,483) by a factor of nearly 5 log. Our results support the concept of specific capture and whole-genome sequencing of viruses from clinical samples through target enrichment technologies 4,483 by , 24. SeqThe mapping stringencies and threshold for the number of mapped reads required to avoid bleed-through of nonspecific HPV signals was established using 8 control samples . The most stringent L1-S1 mapping conditions, along with a cutoff of ≥1,000 mapped reads, average coverage of ≥20, and fraction of genome covered of greater than 50%, allowed reliable sequence assignment. The control cell lines, Caski (∼500 HPV16 copies/cell), HeLa (∼50 HPV 18 copies/cell), and SiHa (1 to 2 HPV16 copies/cell), vary in copy numbers of HPV and were analyzed at two concentrations of input DNA (100 ng and 10 ng in 50 μl). In each case, the number of mapped reads under stringent conditions roughly correlated with copy number, and type assignment could be made with an input of 10 ng DNA. In agreement with previous reports using other methods , 26, theThe bleed-through of reads from dominant sequences in adjacent clusters has been reported in multiplexed samples sequenced using the Illumina platform , 28. ThiWe applied the mapping stringency and cutoff determined with the control samples for determination of HPV types in 24 samples blinded to their HPV status. The HPV types determined by the eWGS were 100% concordant with LA in the 9 plasmid samples, but concordance was lower in the cervicovaginal samples . Nine ofSome HPV types detected by LA were not detected by eWGS in this small sample set . HPV84 was also reported as discordant between NGS and LA in earlier studies , 15. FurIn conclusion, we developed and provided results from the initial evaluation of a non-PCR whole-genome sequence-based approach which is possibly close to a gold standard for broad-spectrum HPV genotype determination. The method relies on a custom-made RNA bait library that showed excellent performance in terms of genome coverage, percentage of reads mapped to predicted HPV type, uniformity of coverage, and the level of target enrichment. Further optimization and evaluation of this eWGS method for HPV detection is required in terms of cost per sample and throughput. Additional studies with samples including more types and optimization of sequencing conditions to minimize the bleed-through are needed. Studies evaluating the reproducibility and lower limits of detection are planned. Finally, it should be noted that despite enrichment with RNA bait by a factor of 5 log, 75% of the total reads (average) were human (off-target), with the exception of the sample with the highest viral load (100 ng Caski), in which 36% of total reads were off-target (data not shown). This suggests adjustment in the conditions for on-target enrichment could enhance results. Future laboratory and bioinformatics efforts are focused on determining the sensitivity and reproducibility of this HPV genotyping method, along with expanding its application for HPV detection and genotyping in samples from a variety of anatomical sites and development of a bioinformatics pipeline for automatic determination of HPV types and variants. This methodology is conceptually suitable for detecting both known and unknown HPV types through adjusting stringencies at the level of hybridization to RNA bait and reference mapping parameters. The current reference mapping stringency used to detect known HPV genotypes would likely miss unknown types.DNA extracted from three cells lines  known to include HPV16  and HPV18  served as HPV-positive controls, and water and human placental DNA  were included as HPV-negative controls. A panel of coded samples was prepared so that testing and analysis could be performed in a blinded fashion. The blinded set included 9 residual samples from past WHO validation panels  and 15 residual DNA extracts from anonymized cervical/vaginal samples previously typed using the Linear Array . Two of the cervical/vaginal extracts were negative for the 37 genotypes targeted by LA, and the other 13 samples were selected to include multiple HPV genotypes . DNA was quantified by fluorescence-based PicoGreen assay , and volume was adjusted with a buffer (pH 8.0) composed of 10 mM Tris-HCl and 1 mM EDTA so that a final concentration of 100 ng/50 μl was used for library preparation. The exception was the water blank (0 ng DNA) and cell line samples that were also prepared at 10 ng DNA to test the effect of reduced DNA input on sequencing results. The final number of samples sequenced was 32: 8 control samples  and 24 blinded samples .http://pave.niaid.nih.gov/) as of September 2014 (http://www.ncbi.nlm.nih.gov/). The 191 HPV reference genomes included in this study  ranged in size from 7,095 to 8,104 bp and were distributed phylogenetically into 5 genera . The complete reference sequences were used for RNA bait design as well as for sequence mapping.We retrieved the whole-genome HPV reference sequences for all 188 HPV genotypes that were available in the PapillomaVirus Episteme (PaVE) database . The custom biotinylated RNA bait library (HPV and HBG) was synthesized at Agilent Technologies. Upon request to the authors, the custom-designed ID may be shared with those who want to synthesize these probes through Agilent Technologies.Agilent Technologies Inc.  designed a custom RNA bait library, using eArray software, comprised of 120-base RNAs complementary to one strand of the whole genomes of all 191 HPV genotypes at 2-fold coverage (60 bases overlapping). The resulting 23,941 RNA sequences comprised a bait library size of 1.442 Mbp. The bait library sequences were subjected to BLAST  search against the NCBI human genome database, and 21 fragments were identified to have homology to human sequences. Removing these 21 fragments from RNA bait synthesis resulted in 100% coverage for 182 HPV genotypes and 99.17% to 99.25% coverage for 9 HPV genotypes , for an overall design coverage of 99.96%. A BLAST search of the final synthesized bait sequences against the full NCBI database identified 15,669 unique hits, with 99.94% being specific to HPV sequences. The other 10 hits (0.064%) shared identity to chimpanzee, feline, and macaque papillomaviruses. As an internal control for the quality of samples, we included 12 RNA fragments covering nt 2041 to 3480 of the coding sequence of the human beta-globin (HBG) gene , optimized for 100 ng DNA . The reference sequences of 191 HPV types/subtypes and HBG that were used for bait design were imported into CLC Genomics Workbench for mapping.The analysis was directed to HPV detection and typing, so duplicate reads were not removed. We used reads with 0 mismatches in the index sequence for analysis in this report. Two parameters, read lengths (L0.5 or L1) and similarity scores (S0.8 or S1.0), were used to adjust mapping stringency. BAM  files were analyzed to generate mapping statistics by CLC Genomics Workbench. Three additional parameters were evaluated in the process of differentiating signal from noise: number of reads mapped to the HPV type-specific reference sequence, average depth of coverage for the mapped sequences, and fraction of HPV genome covered by mapped reads. HPV types in 8 control samples detected by eWGS with different mapping stringency and acceptance parameters were recorded prior to unblinding expected HPV results from 24 blinded samples. Subsequent matching of HPV genotype calls by WGS to expected results was used to evaluate performance of the assay and analysis.http://www.strand-ngs.com/) using BAM files generated by CLC Genomics Workbench under L1-S1 mapping stringency. The mean and the standard deviation (SD) of the average mapping depths among the bins were calculated. The uniformity of coverage was calculated as the percentage of bins with coverage within the average read depth ± 2 SD for all bins. The target enrichment factor for a sample was calculated using the following formula: /(HPV genome size/human diploid genome size) , and uniformity of coverage. To evaluate the uniformity, the reference sequences were divided into bins consisting of 300 bp, and the average mapping depth within each bin was calculated by Strand NGS software (me size) . HPV and


Movement planning is thought to be primarily determined by motor costs such as inaccuracy and effort. Solving for the optimal plan that minimizes these costs typically leads to specifying a time-varying feedback controller which both generates the movement and can optimally correct for errors that arise within a movement. However, the quality of the sensory feedback during a movement can depend substantially on the generated movement. We show that by incorporating such state-dependent sensory feedback, the optimal solution incorporates active sensing and is no longer a pure feedback process but includes a significant feedforward component. To examine whether people take into account such state-dependency in sensory feedback we asked people to make movements in which we controlled the reliability of sensory feedback. We made the visibility of the hand state-dependent, such that the visibility was proportional to the component of hand velocity in a particular direction. Subjects gradually adapted to such a sensory perturbation by making curved hand movements. In particular, they appeared to control the late visibility of the movement matching predictions of the optimal controller with state-dependent sensory noise. Our results show that trajectory planning is not only sensitive to motor costs but takes sensory costs into account and argues for optimal control of movement in which feedforward commands can play a significant role. The dominant theory of how movements are planned suggests that a task specifies a motor cost in terms of effort and accuracy and the motor system chooses a movement to minimize this cost. The pre-eminent theory is that this results an optimal feedback controller that both generates the movement and can correct online for disturbances that arise due to factors such as noise. However, current formulations of the optimal feedback control problem have assumed that sensory information has noise independent of the action, or noise that simply scales with movement speed. In reality, visual and proprioceptive noise of limb position vary substantially with the state of the body. We show that including such variation in sensory feedback qualitatively changes the optimal solution so that feedback control alone is no longer optimal for the task and an additional feedforward controller must be used. To test this idea, we develop a novel motor learning paradigm in which we can vary the sensory noise on visual feedback of the limb and show that human participants adapt to such a perturbation to produce complex reach paths which are well accounted for by our model. Despite the abundance in the degrees of freedom of the human body, our movements occupy a very small subset of all possible movements, showing remarkably stereotypical patterns of motion for a given task. There is a long history which posits that such stereotypy arises from each possible movement inducing a cost and the selected movement is the one that minimizes the cost  until the system reaches the final state xn. We define a quadratic cost induced by the controller:Q to be zero and R to be diagonal. The optimal controller u =  is a control sequence that minimizes the cost:We formulate a finite horizon stochastic optimal control problem for the above system. Starting from a distribution of the initial state  utTRut),where th studies ,37, we ay and is represented with the following linear dynamical system:yt) and the expected (ηt). The gain Kt is called the Kalman filter gain. As we assume that all the parameters from Eqs ut and Kt, the optimal control and estimation problem requires computing an optimal controller ut and a Kalman filter gain Kt for each step.Since the system has uncertainty, the optimal controller contains a feedback term, which means that control for each step is determined based on an estimate of state. As the state is not directly observable, optimal control is accompanied by an optimal state estimator. In LQG, the optimal state estimator updates the estimated state ut and Kt. While the classical solution regards these two procedures to be independent—and this is actually guaranteed by the separation theorem [u and K until convergence. The algorithm is guaranteed to reduce the total cost for every iteration and therefore is expected to converge to the optimal solution. The original derivation [Our algorithm is based on the work of Todorov . In acco theorem  for addirivation  deals wilt) terms, while traditional LQG assumes a purely feedback controller (i.e. lt = 0). Full details of the derivation with the resultant update rules for the controller (Lt and lt) and the Kalman gains and the model of two dimensional arm movement and corresponding parameters used in our simulation are provided in Importantly, the resultant controller contains both feedback . In this 24 dimensional parameter space, we defined a hypercube spanning each dimension and sampled 105 parameter sets from a uniform distribution inside the hypercube. The range of the hypercube in each dimension was set to ±50% of the original values for non-zero parameters and fixed ranges for the parameters set to zero . For each sampled parameter set, we calculated the predicted trajectories for the 12 movements of Experiment 1.To examine the robustness of the optimal control model’s predictions to the choice of model parameters, we examined its sensitivity to parameter variation. The model has eight parameters: two for the state-dependent sensory noise  Full details of the derivation with the resultant update rules for the optimal controller and the Kalman gains. 2) Specific properties of the optimal controller for a system with decoupled dynamics. 3) The model of two dimensional arm movement and corresponding parameters used in our simulation. 4) Sensitivity and convergence analysis.(PDF)Click here for additional data file.


Human papillomavirus (HPV) vaccination targets high-risk HPV16/18 that cause 70% of all cancers of the cervix. In Australia there is a fully-funded, school-based National HPV Vaccination Program which has achieved vaccine initiation rate of 82% among age-eligible females. Improving HPV vaccination rates is important in the prevention of morbidity and mortality associated with HPV-related disease. This study aimed to identify factors and barriers associated with uptake of the HPV vaccine in the Australian Program.Between 2011 and 2014, females aged 18–25 years, living in Victoria, Australia who were offered HPV vaccination between 2007 and 2009 as part of the National HPV Vaccination Program, living in Victoria, Australia were recruited into a a young women’s study examining effectiveness of the Australian National HPV Vaccination Program. Overall, 668 participants completed the recruitment survey, which collected data of participants’ demographics and HPV knowledge. In 2015 these participants were invited to complete an additional supplementary survey on parental demographics and attitudes towards vaccinations.In 2015, 417 participants completed the supplementary survey (62% response rate). Overall, 19% of participants were unvaccinated. In multivariate analyses, HPV vaccination was significantly associated with their being born in Australia (p<0.001), having completed childhood vaccinations (p<0.001) and their parents being main decision-makers for participants’ HPV vaccination (p<0.001). The main reason reported for HPV non-vaccination was parental concern about vaccine safety (43%). Compared with HPV-vaccinated participants, those unvaccinated were significantly more likely to be opposed to all vaccines, including HPV vaccines (p<0.001) and were less likely to consider vaccinating their own children with all vaccines (p = 0.033), including HPV vaccines (p<0.001). Overall, 61% of unvaccinated participants reported that a recommendation from GPs would increase HPV vaccine acceptance.Attitudes towards general health, vaccinations in general, as well as HPV vaccines are important in HPV vaccine uptake. Long-term monitoring of the knowledge, attitude and beliefs towards HPV vaccination in the community is critical to ensure a continued high uptake of the vaccine and success of the program. Persistent infection with high-risk human papillomavirus (HPV) types HPV16 and HPV18 cause 70% of all cancers of the cervix worldwide, and a portion of cancers of the vagina, vulva, anus, penis and head and neck . In 2007In 2013, data from the National HPV Vaccination Program Register (NHVPR) showed that 86% of Australian adolescent girls, aged 12 to 13 received at least one vaccine dose, with 77% receiving all three doses, whilst 64% of those aged 20 to 26 received at least one dose . High vaPublished research exploring attitudes, knowledge and socio-demographic factors associated with uptake of the HPV vaccine in high-income countries are mostly US-based and yield inconsistent findings –18. In ABetween September 2011 and December 2014 women aged 18–25 years, living in Victoria, Australia were recruited into the Vaccine Against Cervical Cancer Impact and Effectiveness (VACCINE) Study, through targeted advertisements placed on the social media website Facebook  as previFor the current study, VACCINE participants who agreed to be contacted for additional research and who were confirmed as being vaccinated or unvaccinated by the NHVPR were invited to complete a supplementary survey (n = 1154). Participants were categorised as being “vaccinated” if they had received three doses of the vaccine, as recorded in the NHVPR. Participants were categorized as being “unvaccinated” if they self-reported not receiving the vaccine and the NHVPR had no record of any HPV vaccine dose being delivered. Those with fewer than three doses were excluded from the supplementary survey. Participants who self-reported that they had received the HPV vaccine overseas and therefore did not have a NHVPR record were excluded from the study.Responses to two web-based surveys, including the original 2011–2014 VACCINE study survey and a supplementary survey administered in this study were collected. The former collected information on participants’ demographics, sexual history, cervical screening history and HPV knowledge , while tUnivariate and adjusted logistic regression analysis were performed with Odds Ratios (ORs) and 95% confidence intervals (CI) generated to identify factors associated with vaccination. Factors included age at program commencement, country of birth, completion of childhood vaccinations, maternal and paternal countries of birth. All variables that were associated with vaccination at p<0.100 in univariate analysis were included in the initial multivariable model. A final multivariable model was obtained by performing backward elimination of statistically non-significant variables, each time assessing for confounding and co-linearity, until only statistically significant variables remained. Descriptive statistics were used to examine reasons for HPV non-vaccination in unvaccinated participants. Childhood socioeconomic status (lower or upper 50th centile classified as more or less disadvantaged) was derived from the Australian Bureau of Statistics Index of Relative Socioeconomic Disadvantage for each individual’s residential postcode. Childhood residential area (major city or) was based on the Accessibility/Remoteness Index of Australia (ARIA) classification, 26. ChiBetween 2011 and 2014 1,154 women who were eligible to receive the free HPV vaccine during the 2007–2009 catch-up period were recruited into the VACCINE study. The women in this study aged 18–25 years in 2011 to 2014 would have been 11–21 years of age at the commencement of the program and therefore had access to the vaccine for free through either the school based or catch up program. Overall, 124 unvaccinated and 544 fully vaccinated participants who consented to be contacted for future studies were invited to the 2014 supplementary survey. Of these, 417 completed the survey . RespondOverall, 337 (81%) participants were fully HPV-vaccinated and 80 (19%) were unvaccinated. Of the vaccinated participants, 68% received their first vaccine dose at school and 32% at a general practice (GP). The median (interquartile range) age at HPV vaccination for the first dose was 16 years (IQR: 15–18), with no significant difference in age between school- and GP-vaccinated participants (p = 0.262). Compared with unvaccinated women, those HPV-vaccinated were significantly more likely to be born in Australia (<0.001), be non-smokers (p = 0.014) and have private health insurance (p = 0.007). HPV-vaccinated and unvaccinated participants were not different with respect to age at first sex (p = 0.310) and lifetime number of sexual partners (p = 0.530). However, HPV-vaccinated participants were more likely to use contraception (p = 0.039) and have had a Pap smear within the last two years (p = 0.073); however, the latter did not reach significance .The results of univariate and multivariable analyses are presented in Participants were asked to complete an HPV knowledge test and report their most trusted sources of HPV information. Overall, 97% of participants knew HPV causes cervical cancer and 99% knew regular Pap smears were required after HPV vaccination . Two thiNext, participants were asked to report their agreement with a number of statements about vaccination on a seven-point Likert scale. The results of this are presented in Unvaccinated participants were asked to report their reasons for why they believed they were not vaccinated. The most commonly reported reasons included parental concern about vaccine safety (42.5%), parental perception of their daughter being at low risk of HPV infection (22.5%) or having a needle phobia (21.3%). Fifteen percent (15.0%) of unvaccinated participants reported practical barriers to HPV vaccination, including being absent at school and forgetting to bring their signed consent forms on the day of vaccination. Only 5.0% reported parental belief that HPV vaccination promoted promiscuity as the reason for not being vaccinated . When stUnvaccinated participants were asked to report factors that would encourage them to get the HPV vaccine . The mosIn this study of 417 young women living in Victoria Australia, we investigated attitudes, knowledge and factors associated with receipt of the HPV vaccine in the National HPV Vaccination Program. Compared with the vaccinated, unvaccinated women were more likely to be smokers and were less likely to use contraception. Unvaccinated women were also more likely to report neutral or negative attitudes towards the HPV vaccine and vaccinations in general. Sexual behaviour and HPV knowledge did not differ between the two groups. Independent factors associated with receipt of the vaccine in the school-based program included being Australia born and having completed childhood vaccinations. Among unvaccinated women, parental concerns about vaccine safety and perceived low risk of HPV infection for their daughters were predominant reasons for non-vaccination. The results of the study emphasize the need for long-term monitoring of the knowledge, attitude and beliefs towards HPV vaccination in the community to ensure high uptake of the vaccine and success of the program.In Australia, those offered HPV vaccination through the school-based program are provided with information on the vaccine at the same time of receiving the consent form . DespiteStudies on HPV vaccine acceptability have reported parents’ health behaviours, beliefs and knowledge about HPV are significant predictors of parental intent for vaccination , 32. In Among unvaccinated women who reported neutral or negative attitudes towards vaccinations, the majority took a neutral position. Research into parental opinions on immunisation have identified a continuum of views on vaccine hesitancy ranging from unquestionable acceptor through hesitancy to refuser , 37. AccPrimary health care provider recommendation remains a key strategy to increase HPV vaccination rates and promote vaccine acceptance , 40. In In line with previous literature, this study showed that being born overseas was associated with lower HPV vaccine uptake . This isThis study has several limitations. First, the retrospective nature of the study design means that it may be subjected to recall bias. Furthermore, the views of parents were reported by the participants rather than the parents themselves and may therefore be inaccurate or misconstrued. Second, reasons for non-vaccination are likely to be different in the catch-up program than in the school program. However in our study, the proportion of women who were aged 18 years and over at the time of the catch-up program was small (23%). Therefore, we had limited power to investigate factors associated with HPV vaccination in this group. Third, we did not demonstrate an association between geographic remoteness and uptake of the HPV vaccine. While this is consistent with published Australian data which shows a relatively equal uptake across urban and regional areas, lower vaccine completion rates have been reported among residents of remote areas . It is lIn summary, attitudes towards health, HPV infection and vaccinations may impact on the success of the HPV vaccination program. It is important for public health campaigns to continue to emphasise the efficacy and safety of the HPV vaccinations as a preventative health measure. Furthermore, long-term monitoring of the knowledge, attitude and beliefs towards HPV vaccination in the community is critical to ensure a continued high uptake of the vaccine and success of the program into the future.S1 File(XLS)Click here for additional data file.


HLA) genes and serum cytokine expression in this pathology. In the current study, we examined the associations of HLA alleles and haplotypes including Class I  and II  with serum levels of cytokines interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-10 and IL-17 as well as risks of HPV infections, lesions and CC among admixed Brazilian women. HLA polymorphisms were associated with an increased risk or protection from HPV, lesions and CC. Additionally, we demonstrated a potential association of a HLA class I haplotype (HLA-B*14-C*08) with higher IL-10 cytokine serum levels in cervical disease, suggesting an association between HLA class I and specific cytokines in cervical carcinogenesis. However, larger studies with detailed HPV types coupled with genetic data are needed to further evaluate the effects of HLA and CC by HPV genotype.Only a small proportion of women who are exposed to infection with high-risk human papillomavirus (HR-HPV) progress to persistent infection and develop cervical cancer (CC). The immune response and genetic background of the host may affect the risk of progression from a HR-HPV infection to lesions and cancer. However, to our knowledge, no studies has been conducted to evaluate the relationship between variability of human leukocyte antigens ( Cervical cancer (CC) is currently the fourth leading cause of cancer mortality in women worldwide, despite the existence of highly effective prevention and screening methods . It is aThe activation of an effective immune response against virus-infected and tumor cells involve the antigen presentation to specific T cells. Human leukocyte antigens (HLA) play an important role in this process by selecting peptides generated from the processing of foreign antigens and exposing them on the surface of the cell for T cell recognition . HLA claHLA genes and serum cytokine expression in the HR-HPV cervical infections to our knowledge.Depending on the stimulus of activation, T-helper lymphocytes may differentiate with at least three subtypes, each one producing their own pattern of cytokines: T-helper 1 (Th1), T-helper 2 (Th2) or T-helper 17 (Th17). Cytokines are known to have an important function in defending against HPV infection, regulating viral transcription and modulating viral replication ,19,20,21HLA alleles and haplotypes, including HLA Class I  and Class II , with serum levels of cytokines IL-6, TNF-α, IL-10 and IL-17 as well as the risk of HPV infections, cervical lesions and invasive CC among Southern Brazilian women of highly admixed descent of a geographic area with a high incidence of CC. Our main finding was higher IL-10 serum levels in women carrying the HLA-B*14-C*08 haplotype, which is related to protection against HR-HPV infection and severe cervical lesions/CC. This suggests a possible association between HLA class I and specific cytokines in cervical carcinogenesis.In the current study, we examined the associations of The mean ages of the 124 women included in the study were similar among the different groups used for statistical analyses. The average ages were as follows: 33.29 years for women with a negative histological diagnosis for intraepithelial lesions or malignancy (NILM); 31.04 years for women with low-grade squamous intraepithelial lesions (LSIL); and 36.1 years for women with high-grade squamous intraepithelial lesions (HSIL) or CC.n = 29, 31.2%), followed by HPV-18 , HPV-51 and HPV-82 , HPV-33 and HPV-45  in addition to others (frequency lower than 5%). A total of 39 HPV genotypes were detected: 17 HR-HPV, 21 low-risk HPV (LR-HPV) and 1 undetermined-risk HPV (UR-HPV). Of the 124 women included, 78 (62.9%) were HPV-positive. Of these HPV-positive women, 16 (20.5%) had only LR-HPV, 48 (61.5%) had only HR-HPV, while14 (18.0%) had multiple HPV infections with LR-HPV, HR-HPV and/or UR-HPV genotypes. HPV-16 was the most prevalent in the overall population , HLA-B*44 , HLA-C*07 , HLA-DRB1*07 , HLA-DQA1*01  and HLA-DQB1*03   . The HLAHLA allelic groups and haplotypes were compared, with HPV positivity and different cytological findings examined to detect possible associations. Associations of HLA allelic groups and HPV infections or LSIL, HSIL and CC that were significant before Bonferroni correction were grouped as shown in HLA-B*07  and HLA-DQB1*03  allelic groups were more frequently found in women with HPV infections and LSIL/HSIL/CC, respectively. In contrast, the frequency of HLA-B*14 and HLA-C*08 allelic groups was decreased in women who had HR-HPV infections and LSIL/HSIL/CC .The frequencies of HLA haplotypes and HPV infections were grouped as shown in HLA haplotypes were associated with increased risk: HLA-B*15-C*03-DQB1*03 for HPV infections ; HLA-A*02-B*40 and HLA-A*03-DQB1*06 for HR-HPV infections ; and HLA-A*02-C*03 and HLA-A*03-DQB1*06 for HPV-16 infections .Statistically significant associations of HLA haplotypes were associated with decreased risk (protection): A*11-B*35-C*04-DRB1*01-DQA1*01-DQB1*05 , A*11-B*35-C*04-DQB1*05 , A*11-B*35-C*04 , A*11-B*35-DQB1*05 , and A*11-C*04-DQB1*05  for HPV infections; HLA-A*01-B*08-C*07-DQB1*02 and B*08-C*07-DQB1*02 for HR-HPV infections ; and HLA-A*01-B*08-C*07-DQB1*02 also for HPV-16 infections   .HLA haplotypes with LSIL and HSIL/CC were also grouped, as shown in HLA haplotypes were associated with increased risk: HLA-B*15-C*03-DQB1*03  and C*03-DQB1*03  for LSIL and HSIL/CC. In contrast, the following HLA haplotypes were associated with decreased risk (protection): A*11-B*35-C*04-DRB1*01-DQA1*01-DQB1*05 , A*11-B*35-C*04-DQB1*05 , A*11-B*35-C*04 , A*11-B*35-DQB1*05  and A*11-C*04-DQB1*05  for LSIL and HSIL/CC.Statistically significant associations of p = 0.0459)  regardless of cytological findings and HPV status  and against HSIL/CC  as previously described.With respect to the cytokine profile, IL-6 serum levels were higher in women with HSIL/CC than in those with LSIL  . FurtherV status . The HLAHLA alleles and haplotypes, including HLA Class I  and HLA Class II , with serum levels of cytokines IL-6, TNF-α, IL-10 and IL-17 as well as the risk of HPV infections, cervical lesions and invasive CC among Brazilian women of highly admixed descent. To our knowledge, this is the first study to demonstrate a potential association between HLA polymorphisms and serum cytokine levels. Our main finding was an association of higher IL-10 serum levels with the HLA-B*14-C*08 haplotype, which showed a protective effect for HR-HPV infections and for HSIL/CC. Additionally, higher IL-6 serum levels were associated with HSIL/CC. Finally, the HLA-B*07 and HLA-DQB1*03 allelic groups as well as the HLA-B*15-C*03-DQB1*03, HLA-A*02-B*40, HLA-A*02-C*03 and HLA-A*03-DQB1*06 haplotypes were associated with increased risk of HPV infection and/or for LSIL/HSIL/CC. In contrast, the HLA-B*14 and HLA-C*08 allelic groups as well as the HLA-A*11-B*35-C*04-DRB1*01-DQA1*01-DQB1*05 and HLA-A*01-B*08-C*07-DQB1*02 haplotypes were associated with protection against HPV infections and LSIL/HSIL/CC. However, considering that the total number of samples included in the study was limited in order to minimize the risk of population–stratification bias, larger studies with detailed HPV types coupled with high quality HLA data are needed to further evaluate the effects of HLA and CC by HPV genotype.In the present study, we analyzed the associations of HLA genes and cervical carcinogenesis has been hypothesized due to the high degree of polymorphism of HLA genes, which confers different abilities of immune-recognition and clearance of infections. Thus, this would result in different degrees of susceptibility to HPV and neoplastic progression [HLA alleles and haplotypes were associated with an increased risk of or protection against HPV, cervical lesions and cancer. Additionally, we demonstrated a potential association of HLA class I polymorphism with higher IL-10 cytokine levels in cervical disease.HPV is necessary but not sufficient to cause CC, which supports the hypothesis of an influence of additional factors in carcinogenesis associated with HR-HPV . An assogression ,15,16,17gression . Our resHLA-B*14:02 and HLA-C*08:02 alleles were previously associated with an increased risk of LSIL compared to HSIL or CC [HLA-B*14 and HLA-C*08 alleles or the HLA-B*14-C*08 haplotype. Reinforcing our evidence of protective effects of these HLA alleles and haplotype, HLA-B*14-C*08 was potentially associated with higher serum levels of IL-10, independently of HPV infection or cervical abnormalities status. IL-10 is known as an important regulator of the immune system, which attenuates exaggerated immune responses that can lead to deleterious tissue lesions by inhibiting the production of proinflammatory cytokines by both antigen-presenting cells (APCs) and CD4+ T cells [+ T cells as well as with increased expression of IFN-γ [HLA-B*14-C*08 haplotype may be involved in the clearance of HPV infections by stimulating cytotoxic T cells (CTLs) against HPV-infected cells. Furthermore, the protection from cervical disease progression may be due to the anti-inflammatory and anti-angiogenic effects of IL-10. However, more studies involving cytokine polymorphisms are needed to clarify the link between IL-10 and HLA in order to assess this hypothesis.The IL or CC . Our dat T cells ,30. IL-1 T cells ,30,31,32In relation to other cytokines studied, IL-6 serum levels were significantly higher in women with HSIL/CC than in those with LSIL. These findings are in agreement with other studies in which IL-6 promoted CC growth by inhibiting apoptosis and increasing neo-angiogenesis ,33,34.HLA-B*07 allelic group was more frequent in HPV infected women, with this same allelic group associated with increased risk of HPV infection in other reports [HLA-DQB1*03 allelic group and the HLA-B*15-C*03-DQB1*03 haplotype was associated with increased risk of HPV infections and of LSIL and HSIL/CC. This finding is in agreement with others that demonstrated the association of HLA-DQB1*03 alleles with increased risk of CC [HLA-A*02-B*40 and HLA-A*02-C*03 haplotypes were associated with an increased risk of HPV and HPV-16 infections, respectively. Previous studies reported conflicting results in relation to an association of HLA-A*02 alleles with increased risk of [HLA-A*03-DQB1*06 haplotype was associated with increased risk of both HPV and HPV-16 infections, as described previously for the DQB1*06 allele [Our results revealed an association between some allelic groups and haplotypes with an increased risk of HPV infections and/or cervical lesions and CC. All associations found for allelic groups were previously described by others, as discussed below. Thus, although significance was lost after Bonferroni correction for allelic groups, we believe these associations may be considered as being possibly valid. The  reports ,22,35,36sk of CC ,39,40,41 risk of ,43 CC an6 allele ,45,46.HLA-B*14 and HLA-C*08 allelic groups in addition to the HLA-A*11-B*35-C*04-DRB1*01-DQA1*01-DQB1*05 haplotype were associated with protection from HPV infections, lesions and CC. Curiously, only the isolated alleles HLA-C*04, DRB1*01, DQA1*01 and DQB1*05:01 were previously associated with a protective effect [HLA-A*11-B*35-C*04-DRB1*01-DQA1*01-DQB1*05 haplotype. Nevertheless, we showed that the HLA-A*01-B*08-C*07-DQB1*02 haplotype was associated with a protective effect, specifically against HPV-16 infections. Otherwise, the alleles DQB1*02:01 and DQB1*02:01/02 were associated with a higher risk of HPV-16 infection [In contrast, we showed an association of allelic groups  and haplotypes with protection from HPV infections and/or lesions and CC. Specifically, the e effect ,47,48,49nfection  and of Cnfection , respectHLA polymorphisms and serum cytokines on HPV persistence. Second, cases of women matching the same ethnic background and residing in the same geographical areas were eligible, in order to minimize the risk of population–stratification bias. As a consequence, a total number of participants included in the study was limited. However, it is reassuring that the frequency of HLA alleles among the women in our study is consistent with findings from other reports from the same state [Some limitations in our study should be noted. First, we have no data on the history of HPV infections and progression of cervical lesions, which limits our interpretations of the influence of me state  and alsome state . Furtherme state . It is wme state ,55. Simime state , furtherA total of 265 unrelated admixed Brazilian women from the state of Paraná in southern Brazil were recruited for the study. All women were referred for colposcopy due to previous abnormal cytological findings (Pap smears) at the colposcopy clinic of the Public Health Hospital in the city of União da Vitória from October 2012 to September 2014.According to Probst et al. , the popConsidering all the criteria adopted, 124 women formed the study population, with an age range from 18 to 67 years and a mean age and standard deviation (SD) of 40.3 ± 10.0 years. The study subjects included: (1) 27 women who underwent colposcopy due to LSIL in referral Pap smears, who had a confirmed histologic diagnosis of cervical intraepithelial neoplasia (CIN) grade I; (2) 42 women who underwent colposcopy due to atypical squamous cells (ASC) but not possible to exclude HSIL (ASC-H) or high-grade SIL (HSIL) in referral Pap smears, who had a confirmed histologic diagnosis of CIN II, CIN III or in situ carcinoma; (3) 7 women who underwent colposcopy due to squamous-cell cervical carcinoma (SCC) in referral Pap smears, who had a confirmed histologic diagnosis of invasive CC; and (4) 48 women who underwent colposcopy due to ASC of undetermined significance (ASC-US) in referral Pap smears, who had a negative histologic diagnosis of intraepithelial lesion CIN or malignancy (NILM).All participants voluntarily agreed to provide biological samples for HPV detection, HLA typing and cytokine determination. They all gave written informed consent before enrollment. This study was approved by the Committee for Ethics in Research Involving Humans at the State University of Maringá (UEM), Brazil, and registered in the National Commission for Research Ethics (CONEP), Ministry of Health of Brazil .In all patients, colposcopy was performed after application of 3% acetic acid. Colposcopically targeted biopsies were taken from the most abnormal area of the cervix. An endocervical curettage was performed if the transformation zone was not entirely visible. Women with a suspicious image penetrating the cervical canal and those in whom colposcopy was unsatisfactory were submitted to cervical conization. The material was fixed in formaldehyde. The morphological diagnosis of cervical biopsies was assessed according to the International Histological Classification of Tumors .According to the Brazilian Cervical Cancer Guidelines, all cases with cytological findings of ASC-H, HSIL and CC should be analyzed by colposcopy and histology, before being properly treated and/or having a follow-up . At colpAn AxyPrep™ Body Fluid Viral DNA/RNA Miniprep Kit  was used for DNA extraction according to the manufacturer’s instructions. The quality and concentration of purified DNA were measured by spectrophotometry .2, 25 mM of each primer and 50 ng of extracted DNA for a final volume of 15 μL was used. Co-amplification of the human β-globin gene was performed as an internal control, using primers GH20 (5′-GAAGAGCCAAGGACAGGTAC-3′) and PC04 (5′-CAACTTCATCCACGTTCACC-3′) under the same conditions as the HPV-PCR. Two types of controls were also included in each reaction: “no-DNA” (negative control) and “HPV-positive DNA” (positive control). PCR products were electrophoresed in 1.0% agarose gel, stained with 1.0 μg/mL ethidium bromide and photo-documented under UV light.A single-target polymerase chain reaction (PCR) method has been in use in our laboratory for several years for HPV detection, which consists of HPV-PCR amplification carried out using primers MY09 (5′-CGTCCMAARGGAWACTGATC-3′) and MY11 (5′-GCMCAGGGWCATAAYAATGG-3′). For the reaction, 2.5 mM of each deoxynucleotide (dNTP), 1 U of Taq DNA polymerase , 0.6 mM of MgClHPV-positive samples were genotyped by PCR-RFLP (Restriction Fragment Length Polymorphism), as described previously . For RFLGenomic DNA from blood samples was extracted using a Mini Spin Plus Extraction Kit  according to the manufacturer’s instructions. The quality and quantity of purified DNA were measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific).® SSO  manufacturer, the purified DNA obtained from blood samples was used to amplify locus HLA Class I  and Class II , before the amplification products were hybridized to sequence-specific oligonucleotide probes (SSO) immobilized on fluorescently coded microspheres. After the hybridization, the samples were read using a LABScan™ 100 flow cytometer  and the results were analyzed in HLA Fusion software version 3.0  to identify possible HLA alleles.As instructed by the LABTypeSerum levels of Th1 (IL-6 and TNF-α), Th2 (IL-10) and Th17 (IL-17) cytokines were measured by a sandwich enzyme-linked immunosorbent assay  and the results were expressed in ng/mL. The lower limit of detection for these cytokines was defined according to the manufacturer’s instructions .http://cmpg.unibe.ch/software/arlequin3). Alleles and haplotypes present in ≥5% of the women were included in the main analyses. To limit the number of comparisons in the haplotype analyses, only the multiloci combinations of greatest interest was explored. OpenEpi v.3.01 software (www.openepi.com) [HLA allele and haplotype frequencies in different groups, with a two-tailed Fisher’s Exact Test or Yates’ corrected chi square. The Odds Ratio (OR) and 95% Confidence Interval (CI) were calculated in all significant comparisons. A Bonferroni correction was used to avoid a type I error.Different variables/groups were evaluated, as follows: (1) Cytological findings: normal (NILM) and abnormal cytology, grouped into LSIL and HSIL/CC (these two most-severe grades were grouped together); and (2) HPV infections: HPV-positive versus HPV-negative, HR-HPV-positive versus HR-HPV-negative and HPV-16-positive versus HPV-16-negative. Allele and haplotype frequencies were calculated using the Arlequin Program v3.1 (available at: epi.com)  was usedt-test for independent samples, using the BioEstat v5.0 program (www.mamiraua.org.br/pt-br/downloads/programas/) [HLA polymorphism and the serum cytokine profile  in carriers and non-carriers of each allelic and haplotype, groups were compared with Kruskal–Wallis analysis of variance or Mann–Whitney test. All p-values less than 0.05 were considered statistically significant.Mean levels of cytokine were compared between different groups by Student’s gramas/) . To analHLA-B*14-C*08 haplotype, which showed a protective effect for HR-HPV infections and for HSIL/CC. Further investigations involving other polymorphisms with longer-term follow-ups and larger sample sizes will be needed in order to determine whether these findings can be repeated in other studies with different populations. Continued examination of the immune response to HPV may lead to the development of novel therapies or effective vaccines against established HPV infections.To our knowledge, this is the first study to demonstrate a potential association of higher IL-10 serum levels with the


Human papillomavirus (HPV) DNA and p16 expression have been identified to be related to the progression of anal squamous cell carcinoma (ASCC). However, the prognostic relevance of combined detection, particularly HPV-/p16+ and HPV+/p16- signatures, is unknown. A meta-analysis of epidemiologic studies was therefore conducted to address this issue. Data were collected from studies comparing overall survival (OS) and disease-free survival (DFS) / disease-specific survival (DSS) / relapse-free survival (RFS) / progression-free survival (PFS) in ASCC patients with HPV and p16 status. The electronic databases of MEDLINE and EMBASE were searched from their inception till 31 May 2017. Study-specific risk estimates were pooled using a fixed-effects model for OS and DFS/DSS/RFS/PFS. Four studies involving a total of 398 ASCC cases were included in this meta-analysis. The pooled results showed that HPV+/p16+ cancers were significantly associated with improved OS  and DFS/DSS/RFS/PFS . However, patients with HPV-/p16+ or HPV+/p16- do not have a comparably good prognosis compared with HPV+/p16+ patients. The meta-analysis indicated that concomitant detection of HPV-DNA and p16 expression may be of prognostic or therapeutic utility in the evaluation of factors contributing to ASCC. Testing tumor specimens for HPV-DNA and p16 expression might indirectly affect treatment decisions. The incidence rate of Anal squamous cell carcinoma (ASCC) ranges from 1.0 to 2.5 per 100,000 population in a lot of developed countries, which is an rare malignancy of the anal canal and perianal skin area .Even though many risk factors for the development of ASCC have been identified, ASCC is known to be strongly linked with a small double-stranded DNA virus, the human papillomavirus (HPV), known for its role in the development of head and neck squamous cell carcinoma (HNSCC), cervical cancer and other gynaecological cancers –7. The pOne study reported that HPV-positive cervical cancer patients receiving radiotherapy have significantly better survival . Other sINK4a (p16), appeared to be the best validated candidate because of its association with high-risk HPV infection. This cyclin-dependent kinase inhibitor is normally repressed by a phospho-retinoblastoma protein (pRB)/transcription factor E2F complex, but this suppression is inhibited by the high-risk E7 oncoprotein of HPV, resulting in an overexpression of p16INK4a [INK4a detection by immunohistochemistry (IHC) is predictive for a significantly improved response to treatment with RT/CRT and a more favourable prognosis in patients with HPV-associated malignancies [The value for prognostic role of the HPV status has already been studied in ASCC patients. However, the sole detection of HPV DNA may misclassify cancers as being associated with HPV, since it does not prove the overexpression of viral oncogenes and thus the transformation induced by HPV . As a rep16INK4a , 18. Accgnancies –21.Therefore, we conducted the meta-analysis to assess the combined effects of HPV status and p16 expression on overall survival (OS) and disease-free survival (DFS)/ disease-specific survival /progression-free survival (PFS) in patients of ASCC.HRs or CIs. A total of 4 prospective studies was included in the final meta-analysis [86 citations were generated from the search strategy. Through the literature search and browsing titles and/or abstracts, 17 papers were identified as potentially relevant , indicating that the studies included in this meta-analysis were with relatively high quality , which was not significantly correlated with OS. The meta-analysis showed a significant association for OS comparing HPV+/p16+ to HPV-/p16+ cancers the HR (95% CI) being 0.29 (95% CI: 0.13–0.64) from the 3 individual effect estimates. Compared with HPV-/p16- cancers, patients with HPV +/p16+ cancers had significant improved OS , and patients with HPV+/p16- cancers had no significant improved OS   being 0.44 (95% CI: 0.16–1.22) from the 3 individual effect estimates. Compared with HPV-/p16+ cancers, patients with HPV+/p16+ cancers had significant improved DFS/DSS/RFS/PFS , and patients with HPV+/p16- cancers had no significant improved DFS/DSS/RFS/PFS . Compared with HPV-/p16- cancers, patients with HPV+/p16+ cancers had significant improved DFS/DSS/RFS/PFS , patients with HPV+/p16- cancers had no significant improved DFS/DSS/RFS/PFS , and patients with HPV-/p16+ cancers had no significant improved DFS/DSS/RFS/PFS   and HPV-/p16+ tumours (33%), compared only sporadically to HPV+/p16+ tumours (6%) [TP53 mutations are only sporadically found in HPV+ tumours, as the HPV oncoprotein E6 inhibits p53 function by targeting it for ubiquitination and degradation. An additional mutation in TP53 would, therefore, not be necessary for these tumours to evolve. The apparent lower frequency of TP53 mutations in HPV-/p16+ tumours could be explained by aberrations in other tumour suppressor proteins, which could be investigated in future studies. The loss of p53 function was related to resistance to radiotherapy [TP53 mutations). Other tests, such as HPV E6/E7 mRNA tests, can be evaluated in future studies in comparison with combined HPV DNA/p16 detection. However, HPV DNA and p16 tests are easy to perform and widely used, and are therefore accepted candidate prognostic markers.It is important to note that HPV-/p16+ patients do not have a comparably good prognosis as HPV+/p16+ patients, which is probably because the HPV-/p16+ tumors are not induced by HPV. Cancers associated HPV often have a viral sequence integrated into cancer cells genome. E6 and E7 are two early structural genes of the HPV. Through inactivation of p53 and the retinoblastoma protein (pRb), a negative regulator of the cyclin-dependent kinase inhibitor p16 and thus leads to upregulation of p16 , 30, theurs (6%) . It is notherapy –33. It iHR for survival. The limitations including: a) a comparison of all combined HPV-DNA- and p16- subgroups in this meta-analysis is limited due to the few studies included with HPV-/p16+ and HPV+/p16- cancers. b) only English studies were included in the meta-analysis, which might resulting in language bias, and c) the adjusted HRs was only reported in one study, which might introduce residual confounding caused by other prognostic factors.The strengths including: a) the current analysis is the first to study the prognostic impact of HPV status together with p16 expression in ASCCs, and b) we used a strict inclusion and exclusion criterions, fully outcomes of interest (OS and DFS/DSS/RFS/PFS) and an advanced meta-analysis of A systematic search up to 31 May 2017 was conducted in MEDLINE (via PubMed) and Excerpta Medica database (EMBASE) to identify relevant articles. Search terms included “human papillomavirus OR HPV”, “p16 OR CDKN2 OR INK4A”, “anal cancer OR anal neoplasms OR anal carcinoma” combined with “prognosis OR prognostic OR survival”. Additional relevant references cited in retrieved articles were also evaluated.HRestimates with their corresponding 95% CI , and (d) English articles. If the study was reported in duplication, the one published earlier or provided more detailed information was included. Review articles and editorials were included if they contained original data. Abstracts were excluded.All papers were reviewed by two authors (X.T. and H.Q.) independently. Uncertainties and discrepancies were resolved by consensus after discussing with a senior researcher (G.S.). All studies included in the final meta-analysis satisfied the following criteria: (a) patients were pathologically diagnosed as ASCC; (b) OS or DFS/DSS/RFS/PFS as the outcome of interest; (c) reported The quality of each study was evaluated in accordance with the revised ELCWP scoring scale described by Steel . Each itHR estimates for OS or DFS/DSS/RFS/PFS with corresponding 95% CIs. When data for HR was not available, we extracted the total numbers of observed deaths and the numbers of patients in each group to calculate HR. [http://digitizer.sourceforge.net/) from the graphical survival plots when data were only available as Kaplan-Meier curves, [HR was performed by the described method [Two of the authors (X.T. and H.Q.) performed the data extraction from each article and discrepancies were resolved by consensus. For studies meeting our inclusion criteria, a standardized data extraction form was used to extract the following data: the first author's name, year of publication, country of origin, study design, period of enrollment, the length of follow-up, characteristics of the studied population , HPV detection methods, p16 detection methods, and late HR. . Data we curves, . then thd method .HR with 95% CI was used to compute the pooled HPV status combined p16 expression and the OS or DFS/DSS/RFS/PFS in ASCC patients. A fix-effect or random-effect model was used to pool the data, based on the Mantel–Haenszel method [The l method . and thel method . respectQ test  and I2  was used to assess the heterogeneity between eligible studies, which test total variation across studies that was attributable to heterogeneity rather than to chance [Cochrane o chance .P < 00.05 was considered statistically significant.All statistical analyses were performed using STATA version 12 for Windows . A two-tailed In conclusion, concomitant detection of HPV-DNA and p16 expression represents a prognostic marker in ASCC patients. Escalating treatment options for HPV-/p16- cancers and de-escalating therapy for ASCCs with HPV+/p16+ could be considered with the order to generate better outcome and fewer side effects related to treatment. An overexpression of p16 without HPV infection seem to have an unfavorable prognosis and may therefore require intensification of treatment for ASCCs. In this rule, prospective trials are mandatory to further determine the predictive joint-role of HPV-DNA and p16 expression in ASCC patients.