Agriculture accounts for ~ 90% of India's fresh water use, and there are concerns that future food production will be threatened by insufficient water supply of adequate quality. This study aimed to quantify the water required in the production of diets in India using the water footprint (WF) assessment method. The socio-demographic associations of dietary WFs were explored using mixed effects regression models with a particular focus on blue (irrigation) WF given the importance for Indian agriculture. Dietary data from ~ 7000 adults living in India were matched to India-specific WF data for food groups to quantify the blue and green  WF of typical diets. The mean blue and green WF of diets was 737 l/capita/day and 2531 l/capita/day, respectively. Vegetables had the lowest WFs per unit mass of product, while roots/tubers had the lowest WFs per unit dietary energy. Poultry products had the greatest blue WFs. Wheat and rice contributed 31% and 19% of the dietary blue WF respectively. Vegetable oils were the highest contributor to dietary green WF. Regional variation in dietary choices meant large differences in dietary blue WFs, whereby northern diets had nearly 1.5 times greater blue WFs than southern diets. Urban diets had a higher blue WF than rural diets, and a higher standard of living was associated with larger dietary blue WFs. This study provides a novel perspective on the WF of diets in India using individual-level dietary data, and demonstrates important variability in WFs due to different food consumption patterns and socio-demographic characteristics. Future dietary shifts towards patterns currently consumed by individuals in higher income groups, would likely increase irrigation requirements putting substantial pressure on India's water resources.  •First analysis of the water used in the production of diets in India using individual-level food consumption data.•The dietary blue (irrigation) water footprint in India is greater than estimates from high-income countries.•Geographic region and socio-demographic factors are strongly associated with dietary blue water footprint. To ensure sustainable and healthy food systems, a combination of consumption- and production-side changes will be required . Many stAnalysing the water use associated with diets is particularly important for India, where ~ 90% of water withdrawal (ground and surface water use) is used for irrigated agriculture, making India the largest user of groundwater in the world . HoweverQuantifying the WF of diets and assessing socio-demographic drivers provides a valuable consumption-side perspective on water resource use and can inform strategies to improve the sustainability of the food system. For example, the approach may help to forecast the potential implications of dietary change on water resource use. The aims of this study were two-fold: first, to quantify the green and blue WFs of typical Indian diets by matching dietary data from a large cross-sectional study of Indian adults to green and blue WFs of food items. Grey WFs are not considered in this study. Due to the importance of the local climate and environment on crop water use, spatial variations in WFs were explored for the two major cereals, rice and wheat. Secondly, to explore the socio-demographic factors associated with dietary blue WF. Blue WFs, although typically much smaller than green WFs, are a particular concern given India's agricultural production is highly dependent on irrigation and availability of groundwater is a significant current issue.22.1Dietary and population data were derived from the Indian Migration Study (IMS) as it provides in-depth data for > 7000 Indian adults. The IMS was conducted during 2005–2007 as part of a pre-existing screening study of cardiovascular disease risk factors among Indian adults. The study used a cross-sectional sib-pair design to study factory workers who had migrated to one of the four following Indian cities – Bangalore, Hyderabad, Lucknow and Nagpur, and their rural-dwelling siblings and co-resident spouses. A 25% sample of urban non-migrants was also recruited. A total of 7067 individuals were included in the final sample with 90% of rural participants and 98% of urban participants living in four states . Full details of the sampling methodology and study design have been reported elsewhere .Dietary intake was measured through an interviewer-administered semi-quantitative food frequency questionnaire (FFQ), assessing consumption of 199 common food items. For the current analysis, the 199 items were aggregated into 36 food groups based on similarity in nutritional content  was calculated using an asset-based survey on 14 items, including quality of house, toilet facilities, land ownership, and source of lighting.2.2www.waterfootprint.org; The WF Network has quantified the WF of crops using a grid-based dynamic water balance model that considers local climate, soil factors, and rates of nitrogen fertiliser use . At the For the present study, state-level WFs of animal products were quantified based on methods from The WF Network database does not report national WFs of seafood products. The majority of the IMS population was situated away from coastlines, and the total intake of seafood (fish and prawns), was small. Therefore, we chose to calculate the WFs based on the WF of fish feed with the assumption that all fish were farmed . Carp ac2.3The 199 IMS food items were matched to products in the WF Network database using author judgement (see Appendix Table A.1). Items were excluded from the analysis if there was no suitable match or they represented < 5% of the food groups' consumption . The WFs of the 36 food groups were calculated from the mean WF of constituent items, weighted by the relative contribution of food items to total food group consumption across the IMS population. The majority of IMS participants resided in urban areas and the location of consumption was unlikely to give a good prediction of the location of production. Thus, in the absence of comprehensive data on interstate trade of major food items, we used a national average value for the WFs of the 36 food groups. However, we maintained a framework that allowed us to investigate spatial variations in WF values and thus the uncertainty of our estimates. Therefore, the WFs of food groups were initially derived at state level and a mean national-level WF for each food group was calculated by weighting the state values by land size . The justification for this approach  is considered in the 2.4R > 0.7) were excluded prior to analysis. The crude associations between socio-demographic characteristics (predictors) and the blue WF of diets (outcome) were assessed using separate mixed-effects linear regression models. Due to the sib-pair clustered design, each model specified the between-pair variation as a family-specific random effect in addition to the fixed effects used for each predictor variable. The reference category was chosen either as the most numerous or the lowest for ordered categorical variables. A combined mixed-effect multiple regression model was used to assess the adjusted relationships with all socio-demographic variables simultaneously. For comparison, each variable was assessed with the same multivariate model, containing each predictor and occupation as a confounder. The variance inflation factor (VIF) was used to assess multicollinearity as a multiple linear regression model. To assess the effect of diet composition  on blue WF, separate analysis was carried out adjusting for total calorie intake in the multivariate model. All statistical analyses were conducted using STATA .The blue and green WFs of IMS participants' diets were quantified by combining individuals' mean daily consumption of each food group with the matched national-weighted WF data. The socio-demographic characteristics assessed for their relationships with dietary blue WF were age, gender, region, rural/urban residency, religion, SLI score and education . Spearma2.5Ethical approval for the IMS was obtained from the All India Institute of Medical Sciences Ethics Committee, reference number A-60/4/8/2004. The present study forms part of the Sustainable and Healthy Diets in India (SAHDI) project which was granted ethical approval by the London School of Hygiene & Topical Medicine (reference number 11509).3The analysis included a total of 6775 participants, aged 17–76 years (mean 41 years). The sample contained more males (57.3%) than females, and the majority of participants were Hindu (91.1%). Descriptive characteristics of the study population are shown in 3.1Detailed dietary characteristics of the IMS population have been reported elsewhere . The mea3.23.2.1The mean ± SD blue WF of diets in the study population was 737 ± 263 l/capita/day, and the mean ± SD green WF of diets was 2531 ± 885 l/capita/day . Rice an3.2.2There were large differences between the WF of food items in India (see Appendix Table A.3 for weighted averages of the 36 food items). Based on the weighted average WF across states, food items with the greatest green and blue WF per g of product were nuts and seeds, poultry and milk products. Wheat had a greater blue WF than rice (i.e. 1.37 l/g vs 0.72 l/g) but a lower green WF (i.e. 0.98 l/g vs 2.07 l/g). The blue WF of fruits and vegetables was relatively low, i.e. 0.25 l/g and 0.09 l/g respectively. However, when considering WFs per kilocalorie, the relationships altered , Fig. 2.The WF of food items varied greatly between states. The blue WF of wheat was highest in the central/western states, whereas the blue WF of rice was highest in north-western and south-eastern states . The gre3.3There were large regional differences in dietary patterns that resulted in substantially different blue WFs . SoutherA total of seven socio-demographic characteristics were assessed for their relationship with dietary blue WF . The unaR = − 140 l/capita/day, 95% CI − 130 to − 151 l/capita/day, p < 0.001). Region was a strong predictor of dietary blue WF, and showed the largest effect size between the groups compared to the other socio-demographic variables. Dietary blue WF was lowest in the south and highest in the western regions . Rural participants had a lower dietary blue WF compared to urban, and Hindus' dietary blue WF was lower than other religions. Socio-economic indicators were associated, with blue WFs increasing with higher levels of formal education and increasing SLI.After adjusting for socio-demographic confounders, there was strong evidence that age was independently and negatively associated with dietary blue WF . The bluR = 110 l/capita/day, 95% CI 104–117 l/capita/day, p < 0.001). Other socio-demographic factors including age, sex, location of residence and standard of living index remained associated with blue WF but effect sizes were modest. Adjusting for energy strengthened the evidence that people from other religions had a higher diet WF than Hindus, suggesting that diet pattern rather than total food consumption is important in this relationship. The association between education level and dietary WF was no longer seen; hence, differences in total energy intake explained the dietary blue WF differences between the groups.Additional adjustment for total dietary energy intakes attenuated many of these relationships, indicating that both the amount and the type of food consumed were associated with dietary blue WF . The rel4This study provides individual-level estimates of the WF of Indian diets, finding them to have a higher blue WF than many other countries. Important differences exist between socio-demographic groups due to food consumption patterns, particularly regionally and between socio-economic groups. These contextual factors should be considered for sustainable diet recommendations and when predicting future water scenarios.4.1The average energy intake in this study population was 2883 kcal/capita/day, with the highest proportion coming from rice and wheat. The average energy intake was greater than National Sample Survey (NSSO) estimates from a similar period, i.e. 2047 and 2021 kcal/capita/day for rural and urban adults, respectively . It is p4.2The mean green WF of diets in this Indian population was 2531 l/capita/day, with the highest contributor being vegetable oils (19%). The mean blue WF was 737 l/capita/day with the greatest contribution from wheat (31%). There are currently no comparable middle-income country data available, although the combined green and blue WF (3268 l/capita/day) is similar to a South European vegetarian diet (3176 l/capita/day), lower than the average South European diet (5364 l/capita/day) but much higher than other estimates for European diets . The meaIn this study, the foods with the highest WF per g and per kcal were poultry products, which relates to the WF of feed and the feed conversion efficiency. When considered per g, the foods with the lowest WFs were fruits, which is consistent with global averages . HoweverKharif (monsoon) season with higher water availability, while wheat is grown in the Rabi season when irrigation (blue water) substitutes the lack of rainfall  may be more susceptible to erratic rainfall, whereas those with a higher blue WF (e.g. wheat) may contribute to groundwater depletion and be susceptible to groundwater scarcity.Importantly, there are large variations in the WF of crops and their relative blue or green water use due to climate and agricultural practices. For example, paddy rice has a high green WF as it is grown in the rainfall . Phenolo4.3Socio-demographic groups were significantly associated with dietary blue WF, due to the amount and type of food consumed. Region was the strongest predictor of dietary blue WF, which is important to consider in terms of India's current water situation. The blue WF of diets in the north was ~ 1.5-fold greater than those in the South, i.e. 846 l/capita/day compared to 611 l/capita/day, and groundwater scarcity is most widespread in the north of India. Dietary change could be an important adaptation to limited groundwater resources, for example substituting wheat and rice by other cereal crops with lower water demand (e.g. sorghum and millet).Associations between dietary blue WF and other socio-demographic factors were less pronounced than those for region, but still significant. Females and older participants had a lower dietary blue WF, mostly due to lower total energy intake. Hindus consumed less animal-source foods and their diets had lower blue WFs. Urban diets had a greater blue WF than more traditional, rural diets, even when accounting for total energy, and dietary blue WF increased with greater standard of living. Previously, urbanisation and increasing socio-economic status has been linked to the nutrition transition in India . This is4.4The dietary intake data are likely to include inaccuracies as FFQs can introduce measurement error and over-estimate food consumption . PreviouThe WF estimates for the food groups are subject to assumptions that will have introduced measurement error. The majority of participants were from urban areas so their location of residency was considered a poor predictor of the location of food production. Cereal grains, for example, are traded across long distances in India including via the nationally-coordinated government procurement, storage and redistribution activities . ReliablFinally, these findings are not fully generalizable to India. The IMS survey was not intended to be nationally representative, but to assess the effects of rural-to-urban migration; therefore, the sample includes a greater proportion of urban dwellers than is found across India. Additionally, we have mainly focused on blue WF, which although particularly relevant for India due to high irrigation use and water scarcity concerns, does not capture relative green water use.4.5This study provides an insight into the dependency of Indian diets on water resources, finding greater blue WFs than previous estimates from high income countries. These dietary WF estimates can be used to assess potential water scenarios as food consumption patterns change. Dietary blue WF varies significantly between regions and future investigation should focus on improving our understanding of the water demands of local diets. Analysis at refined geographical and social levels will help inform policy by identifying realistic dietary changes based on culturally acceptable limits, as well as providing evidence to effectively target interventions and incorporate sustainable diet recommendations into existing nutrition programmes.Additionally, it is possible to consider the impact of blue water use at the local level using Life Cycle Assessment-based methods that include relative blue water scarcity . Future Linking diets and WFs demonstrates the dependence of our food systems on water resources, and frames understanding from a consumer point of view, which could encourage behaviour change . It will


IL-6 -174 G>C, IL-8 +396 G>T, andTGF-β1 +869 G>C and +915 G>C polymorphisms were associatedwith susceptibility to HPV infection in women from north-east (Pernambuco) Brazil. Weanalysed 108 healthy uninfected women (HC) and 108 HPV-positive women with cervicallesions. Genetic polymorphisms were assessed using Sanger sequencing and polymerasechain reaction-restriction fragment length polymorphism. Comparison of thedistribution of the genotypic and allelic frequencies of the IL-18 +396 T>Gpolymorphism between HPV infected woman an uninfected controls showed that the GGgenotype and G allele were both more frequent in the HC group, and were associatedwith protection from HPV infection . Individuals from the control groupcould have previously had HPV infection that was spontaneously eliminated; however,it was undetectable at the time of sample collection. Based on our findings, wehypothesize that the IL-8 +396 G>T polymorphism could interferewith susceptibility to HPV infection, by modulating the ability of immune system tofight the virus.Human papillomavirus (HPV) infections are strongly associated with the development ofcervical intraepithelial neoplasias and invasive cervical cancer. Polymorphisms incytokine-encoding genes and behavioural cofactors could play an important role inprotecting an individual against viral infections and cancer. Here, we investigatedwhether  Cervical cancer represents a significant public health problem causing a great impactworldwide; every year approximately 528,000 new cases and 266,000 deaths  are expected . In BrazAlthough the incidence of genital HPV infections is high, most of these infections aretransient and do not lead to cervical intraepithelial neoplasia (CIN) or cancer, suggestingthat other factors such as the immune system as well as the host genetic background couldinfluence disease risk .HPV infection promotes immune cell migration to the dermis. In the squamous epidermis,macrophages, Langerhans cells (LC), T lymphocytes, dendritic cells (DC), natural killercells (NK), and B-lymphocytes play important roles in the immune response to infection. HPVinfection could induce the immune system to become more tolerant to the infection, therebycreating a microenvironment susceptible to further infection, facilitating CIN progression.These local immune responses seem to play an important role in the natural history of HPVinfection of the uterine cervix. Cytokines are important regulators of HPV transcriptiondue to their important role in the defence against HPV infection, through modulating viralreplication . PolymorThe discovery of a TH17 variant has improved the understanding of inflammatory processes.TH17 cells are linked to chronic neutrophilic inflammation and can be induced todifferentiate by transforming growth factor β1 (TGF-β1) and interleukin-6 (IL-6).Therefore, TGF-β1 and IL-6 can also induce the differentiation of TH9 and TH22 cells,respectively (both linked to tissue inflammation) .IL-6expression and on serum protein levels , presents severalsingle nucleotide polymorphisms (SNPs), one of which is localised in the promoter region, and is associated with variations in TGF-β1 gene located on the long arm of humanchromosome 19 (19q13). The +869 T>C (rs1982073) SNP is located at codon 10 of exon 1 andresults in a leucine-to-proline substitution, whereas +915 G>C (rs1800471) is located atcodon 25 and results in an arginine-to-proline change. The C variant allele of codon 10, aswell as the wild-type G allele of codon 25, is associated with increased production ofTGF-β1  andseveral promoter, intron, and 3′UTR SNPs have been identified (IL-8 +396 G>T (rs2227307)polymorphism has been associated with respiratory diseases caused by viruses (Interleukin-8 (IL-8), the first chemokine discovered, has pro-inflammatory activity, and isproduced as part of immune or acute inflammatory reactions, as well as during chronicinflammation; this cytokine functions to attract and activate neutrophils in inflammatoryregions . TheIL-entified . The IL-IL-6,IL-8, and TGF-β1 genes with the risk of HPV infection,we evaluated whether IL-6 (-174 G>C), IL-8 (+396G>T), and TGF-β1 (+869 T>C and +915 G/C) functional genetic variantswere related to HPV susceptibility, by studying HPV infected and uninfected women fromPernambuco, Brazil.Since few studies have been performed correlating SNPs in Patients - A hospital-based cross-sectional study was performed aimedat analysing IL-6, IL-8, and TGFβ1 gene polymorphismsin HPV-infected women with cervical lesions and invasive cervical cancer (ICC). Onehundred and eight women aged 17-68 years, with a mean age of 33.9 ± 10.1 years,presenting with either low-grade squamous intraepithelial lesion (LSIL) or high-gradesquamous intraepithelial lesion (HSIL), were selected from the outpatient clinics of theLower Genital Tract Pathology Clinic at the Women’s Healthcare Centre of the ProfFernando Figueira Institute of Integrated Medicine, . Patients were selected by spontaneous demand from January 2009 to December2010. Patients were included in the study if there was no discrepancy betweencytological abnormality and histological diagnosis made at the first visit. All patientswere initially assessed by colposcopy and subsequently cervical smears were collected.Histological diagnosis was made according to the Associação Brasileira de Genitoscopia, with no history of lesions or neoplastic disease asevaluated by the physician, and testing negative for HPV infection, were used ascontrols.Informed written consent was obtained from the women, and the women were informed of thebackground of the study, risks and benefits, and voluntary nature of participation(CEP/CCS/UFPE Nº 355/08).Clinical samples - Cervical smears were obtained using cytobrushes.Each cytobrush was packed in a TE buffer solution and maintained at -20ºC until analysis.DNA extraction - Genomic DNA extraction was performed from 300 µL ofcervical smear, from each study subject, using the Wizard Genomic DNA Purification kit following the manufacturer’s instructions. The analyses wereexecuted in Laboratory of Genetic, Biochemistry and DNA Sequencing (LGBS) of RuralFederal University of Pernambuco.HPV DNA detection - Human papillomavirus DNA was detected from DNAextracted as previously described using two polymerase chain reaction (PCR) steps. Thefirst was with MY09/11 external primers (MY09 5′-CGTCCMARRGGAWACTGATC-3′ and MY115′-GCMCAGGGWCATAAYAATGG-3′) and the second was with GP05+/06+ (GP5+5′-TTTGTTACTGTGGTAGATACTAC-3′ and GP6+ 5′-GAAAAATAAACTGTAAATCATATTC-3′) as internalones. These two primers pairs are most widely used for the detection of genital HPVs. Anegative control containing only digestion buffer was included for every five samples toprevent and detect carry-over between samples.IL-6, IL-8, and TGF-β1 SNPs genotyping - IL-6 andTGF-β1 polymorphisms were amplified from the same DNA utilised forHPV detection, using specific primers (IL-6 -5′-TTGTCAAGACATGCCAAAGTG-3′ and 5′-TCAGACATCTCCAGTCCTATA-3′ and TGF-β1- 5′-TTCCCTCGAGGCCCTCCTA-3′ and 5′-GCCGCAGCTTGGACAGGATC-3′) which flankedthe polymorphisms. PCR was performed following standard protocols from the literature.Briefly, after amplification, the amplicons were submitted to a sequencing reactionusing the DyEnamic ET Dye Terminator Cycle sequencing kit  according tomanufacturer’s recommendations and were sequenced using a MegaBACE 1000 DNASequencer.IL-8 was amplified from the same DNA utilised for HPV detection usingspecific primers (5 ′-TAAAGGTTTGATCAATATAGA-3′ and 5′-CTTCCTTCTAATTCCAATATG-3′)according to literature, and the genotypes were identified through restriction fragmentanalysis polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) using the ScrFI restriction enzyme. Thirty percent of the samples were sequenced to confirm the results.Statistical analysis - Univariate statistical analysis was performedusing BioEstat 5.0 software. The study was cross-sectional, with independent samplesconsisting of nominal data (genotypes). The influence of polymorphism on the risk fordevelopment of (pre) neoplastic cervical disease was estimated by an odds ratio (OR) anda 95% confidence interval (CI). Allele frequencies were estimated by direct counting.Comparison between genotypic frequencies of patients and control groups was performedusing a χ2 test; a Fisher’s exact test was used to compare the allele frequencies incontingency tables. The OR and their respective 95% CI values were determined. All pvalues ≤ 0.05 were considered statistically significant.Selected characteristics, including age, age of first sexual coitus, parity, smokingstatus, alcohol consumption, and oral contraceptive use of 108 HPV infected women withcervical lesions are summarised in IL-6 (-174 G>C), IL-8 (+396T>G), and TGF-β1 (+869 G>C) and (+915 G>C)polymorphisms in HPV infected women and healthy uninfected controls.IL6 and TGF-β1 (+869 T>C) and(+915 G>C) polymorphism frequencies were in Hardy-Weinberg equilibrium in HPVinfected women and uninfected controls, whereas the IL-8 +396 T>Gpolymorphism showed frequencies not in agreement with the Hardy-Weinberg equilibrium inthe two groups of subjects analysed.IL-6 -174 G>C and TGF-β1 +869T>C and +915 G>C polymorphism alleles and genotype frequencies were notstatistically different between HPV infected women and healthy uninfected controls.Comparison of the IL-8 +396 T>G polymorphism allele and genotypefrequencies between HPV infected woman and uninfected controls showed a statisticallysignificant increase in the frequency of the GG genotype in uninfected subjects comparedto HPV infected women, suggesting an association between this allele and protection fromHPV infection . In addition, the prevalence ofthe G allele was more frequent in HCs than in HPV infected women, thus, suggesting anassociation between this allele and protection from HPV infection .In addition, with the aim of comparing the allele frequencies of the SNPs observed inour study with those of the population from Pernambuco, we inferred the allelefrequencies  for eachpolymorphism based on the equation presented by Essentially, this equation calculates a weighted average of the allele frequencyobserved in the parental populations of Brazil, considering the proportion of eachancestry found in the Brazilian population. Since we had no data regarding thepopulations described in the equation, we used the findings of some related populationswith available data present in the 1000 Genomes Project database(http://browser.1000genomes.org) as proxies.For each polymorphism, we obtained the allele frequencies from European Iberian (IBS),African Yoruba (YRI), and Peruvian South American (PEL) genome databases and combinedthe frequencies considering the admixture of the Pernambuco population, comprisinggenomes resulting from European (59.7%), African (23%), and Amerindian (17.3%)ancestries, as described in IL-6 (-174 G>C) polymorphism; 0.41 for the IL-8(+396 T>G) polymorphism; 0.46 for TGF-β1 (+869T>C); 0.03 for the TGF-β1 (+915 G>C) SNP.Finally, no significant differences in the distribution of IL-6 (-174G>C), IL-8 (+396 T>G), TGF-β1(+869 T>C), and (+915 G>C) polymorphisms were observed between LSIL and HSILgroups (The predicted admixed frequencies for each polymorphism were as follows: 0.22 for theLgroups  in HPV iIL-6 (-174 G>C),IL-8 (+396 T>G), and TGF-β1(+869 T>C) and (+915 G>C) (cytokines encoding genes), on the susceptibility to HPVinfection in women (HPV infected and uninfected) from Pernambuco (Brazil).Since HPV infection is a multifactorial process, depending on environmental and hostgenomic factors, with particular focus on immunoregulatory genes, we analysed thepossible impact of genetic variants, namely IL-6 +174G>C polymorphism and HPV infected women or uninfected controls, or between HSIL andLSIL subgroups; we also did not find any relationship between TGF-β1+869 T>C and +915 G>C polymorphisms and the risk of HPV infection or thedevelopment of LSIL or HSIL. IL-6 and TGF-β1polymorphisms were not associated with the development of cervical cancer in apopulation from Zimbabwe, Africa and HCs (0.35) was reduced in comparison to that predicted by ancestry estimates (0.41).Therefore, more studies are needed to investigate the lower than expected heterozygosityin this population.It is worth noting that both HPV and HC groups were not in Hardy-Weinberg Equilibriumfor the IL-8, a chemokine, acts by attracting granulocytes to sites of inflammation, resultingin neutrophil-mediated viral clearance . IL-8 exIL-8 has been hypothesized to play an important role in ovarian cancer . IncreasIL-8 +396 G/T. The IL-8 +396GG genotype is known to yield higher levels of IL-8 and has been linked to thedevelopment of ovarian carcinoma (IL-8 +396 GG genotype, associated with high levels of IL-8, arebetter able to control HPV infection through the attraction of granulocytes to the sitesof infection. Consequently, this would results in successful neutrophil-mediated viralclearance. Nevertheless, this is a hypothesis that needs to be verified by analysing IL8serum levels, was not possible to analyse in this study. Moreover, theIL-8 +396 T/G polymorphism allele and genotype frequencies assessedin HPV infected and uninfected women were not in Hardy-Weinberg equilibrium and weredifferent from those inferred for the Pernambuco population. Therefore, our findingsshould be considered with caution and thus need to be replicated in at least one othernorth-east Brazilian population.Differential expression of IL-8 could be associated with the presence of functionalpolymorphisms such as the arcinoma . TherefoWe are also aware that the main limitation of this study is the relatively small cohortenrolled; furthermore, HPV high-risk and low-risk genotyping data were not available tous; therefore, it was impossible to evaluate the impact of HPV genotypes on thesusceptibility to cervical lesions as well as their relationship with cytokine-encodinggene polymorphisms.IL-8 +396 G allele and GGgenotypes could play a role in the risk of HPV infection in our study population.Specifically, carriers of these genetic variants, responsible for higher IL-8production, should be able to react better to HPV infection. This work will promptfuture genetic studies regarding the molecular pathogenesis of HPV infection and similaranalyses should be performed to determine whether this polymorphism could serve as aprognostic risk factor for HPV infection.In conclusion, taking into account the fact that individuals from the control groupcould have been previously infected by HPV that was spontaneously eliminated, and basedon our findings, we hypothesize that the


Despite improvement in vaccines against human papilloma virus (HPV), the causative agent of cervical cancer, screening women for cervical precancer will remain indispensable in the coming 30–40 years. A simple test that could be performed at home or at a doctor’s practice and that informs the woman whether she is at risk would significantly help make a broader group of patients who aware that they need medical treatment. Cervical vaginal fluid (CVF) is a body fluid that is very well suited for such a test.Narrative review of cervical (pre)cancer candidate biomarkers from cervicovaginal fluid, is based on a detailed review of the literature. We will also discuss the possibilities that these biomarkers create for the development of a self-test or point-of-care test for cervical (pre)cancer.Several DNA, DNA methylation, miRNA, and protein biomarkers were identified in the cervical vaginal fluid; however, not all of these biomarkers are suited for development of a simple diagnostic assay.Proteins, especially alpha-actinin-4, are most suited for development of a simple assay for cervical (pre)cancer. Accuracy of the test could further be improved by combination of several proteins or by combination with a new type of biomarker, e.g., originating from the cervicovaginal microbiome or metabolome. Considering cervical cancer is the fourth most common female cancer worldwide, it remains a significant global problem , indicatHowever, despite the advancements in HPV vaccination, these vaccines do not cover all hr-HPV types  and are The gradual development of cervical cancer (years) with the occurrence of several precancerous stages and the relative ease with which the tumor can be accessed offer the opportunity to screen population-wide for cervical cancer during prevention campaigns. Cervical cancer screening programs are generally based on the detection of hr-HPV via DNA or RNA assays or on the detection of cytological and/or molecular changes in cervical cells via (immuno)staining methods, such as the Papanicolaou (Pap) smear .High-risk HPV DNA-based PCR tests are currently of high interest because they are 40% more sensitive at detecting a cervical abnormality than cytology , 10. HowCytology-based screening has several other limitations, such as the high intra- and interobserver variability, limited sensitivity, high costs and limited screening coverage –14. CurrINK4a/Ki-67 [INK4a/Ki-67 and L1 capsid protein [The conclusion is that because of the limitations of each of these methods, screening programs are never 100% safe . Moreover, they are subject to oversampling , leading to the treatment of women with clinically irrelevant hr-HPV infections, which increases the costs and possible harm caused by the treatment. Additional triage tests, on a molecular basis, are thus necessary to provide an objective and reproducible basis for the selection of patients with clinically significant disease. Today, the best alternative is dual staining for p164a/Ki-67 –19, but 4a/Ki-67 . Therefo4a/Ki-67  or p16IN protein , are bei protein –25). MorIn 2014, Arbyn et al.  showed tUsing the emerging proteomics technologies that have become increasingly sensitive, our group and others groups have conducted several studies on the identification of the cervical vaginal proteome –65. FuncBecause of the immediate contact of the precancerous or cancerous tissue with the CVF, we expect that the concentrations of important cervical cancer biomarkers will be high in CVF. Unlike plasma, CVF does not contact many other tissues, and its volume is limited (milliliters versus liters). Moreover, the liquid can easily be collected by self-sampling in a non-invasive way using devices for lavages , 66, or Especially in low-resource countries and remote rural areas where mail and transport are much less frequent, the continuous running of an efficient screening program for cervical cancer may demand organizational, financial and logistical efforts , 73 thatA typical example of an LFA test that is already on the marked, is the self-test for HIV (HIVST). According to the WHO such a test used and interpreted by a self-tester can perform as well as an HIV RDT used and interpreted by a trained health worker  althoughIn summary, we believe that CVF is a rich source of information regarding the physiological status of the female genital organs, including the healthy or cancerous state of the cervical region. Components from the CVF could, therefore, be used as the basis for a simple self-test/point-of-care test that, when sufficiently accurate, may overcome current problems with coverage and specificity.Many studies report on the efficiency of HPV DNA testing from self-collected cervical tissue samples, compared to samples collected by a practitioner. In most studies, HPV testing on self- and clinician-sampled specimens is similarly accurate with respect to CIN2+ detection as reported in a large cohort study , 81 or aAs for detection of HPV DNA from cervical vaginal fluid there was a high agreement between the (self-sampled) CVF and the reference smears   and no dUnfortunately, HPV DNA detection requires very specialized equipment ; therefore, it is not suited for a self-test. Furthermore, as mentioned above, the test cannot distinguish between productive and progressive infections, resulting in a low specificity.SOCS1, CDH1, TIMP3, GSTP1, DAPK, hTERT, CDH13, HSPA2, MLH1, RASSF1A, and SOCS2) and reported a correlation of the methylation status with the severity of the cervical lesion, such that invasive cervical cancers could be predicted. Along the same line, Sun and coworkers [With the discovery that global DNA hypomethylation progressively increases in cervical dysplasia and carcinoma , Widschwoworkers  analyzedoworkers . In a hioworkers  investigMicroRNAs have not only been detected in the serum or plasma of patients who are precancerous for cervical cancer  , they haInterestingly, after silencing HPV E6/E7 oncogene expression in HPV-positive cervical cells, such as HeLa cells, a distinct seven-miRNA-signature was identified in the exosomes secreted by the HeLa cells, which was accompanied by significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p . This raHence, it is clear that DNA methylation or RNAs could serve as a CVF biomarker for intraepithelial cancerous lesions; however, analogous to DNA PCR, a methylation-specific or RNA-specific PCR reaction requires skilled people and specialized instruments, making it unsuitable for self-testing.The discovery that carcinoembryonic antigen (CEA), CA19-9 and CA125 were present in the CVF of patients with cervical cancer or with a cervical precancerous lesion led to optimism in the 80 s that these biomarkers could help in detecting cervical cancer or its precancerous stages –99. LateIn a later study , 105, inSince then, many studies were undertaken to identify cervical (pre)cancer protein biomarkers from swab samples or biopsies , 109, yep = 0.001), respectively. ELISA on 28 additional samples showed a discriminatory potential of ACTN4 at 18 pg/ml protein extract between samples from healthy and both low-risk and high-risk HPV-infected women (p = 0.009). Analyzing the ACTN4 concentration in 26 CVF samples originating from longitudinal studies on 9 women who experienced an HPV infection, who had a persistent infection or who cleared the virus showed that the ACTN4 levels correlated with increasing, persisting or decreasing presence of HPV E6 DNA [In a differential proteomics study on CVF samples from six healthy and six precancerous  women, we identified 16 candidate biomarkers  target in the ER-positive breast cancer cell line MCF7, suggested that ACTN4 may play a role in E2-mediated regulation of breast cancer proliferation [Alpha-actinin-4 is predominantly expressed in cellular filopodia and lamellipodia, and as such, it is important for the formation of cell protrusions and migration . Experimferation , 122. Inferation . ACTN4 tN = 43) contained samples originating from healthy women, while the second class (N = 43) contained samples originating from women with small  or larger (HSIL) signs of precancerous tissue or with cancerous tissue. Because the samples came from different hospitals in different volumes, we took the total mass of protein as a reference instead of the sample volume. For this normalization, the ACTN4 concentration was recalculated as pg/mg total protein instead of pg/ml. The resulting ROC curve showed an AUC of 86% , it was not possible to correlate the ACTN4 concentration with different CIN stages. However, clear overexpression of ACTN4 was visible in all cancerous samples. Interestingly, none of the three samples from HIV-1 infected (HPV negative) individuals scored above the cutoff value, suggesting that HIV-1 infection does not interfere with the ACTN4 levels in CVF. Studies in our lab are currently ongoing to examine the correlation of the ACTN4 concentration with the CIN stages and to evaluate the CVF concentration in women infected with additional sexually transmitted viruses, bacteria and protozoa.For a preliminary determination of the sensitivity and specificity of ACTN4 as a cervical (pre)cancer biomarker, we extended our sample pool with 10 CVF samples from the previous cohort , 14 CIN S6K pathway  attacked by the immune system, we hypothesized that ACTN4 is released in the CVF from lysed epithelial cells with many other intracellular factors, including those involved in the development of precancerous lesions and/or cervical cancer. Aberrant concentrations of (some of) these factors in the CVF may indicate there is growing precancerous tissue and, therefore, an increased chance of developing a malignant tumor. Therefore, starting from the protein lists we obtained from the differential proteomics study on CVF from healthy and precancerous patients , proteinway Fig. . The firmotility ; hence, motility –131, andetection . Additioetection , 133. SuLactobacillus species in vaginal and reproductive health, while overgrowth of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as a marked depletion of Lactobacillus were essential for the diagnosis of BV [Over recent decades, a substantial amount of work has been done on the characterization of the vaginal microbiome in relation to several diseases. Two groups , 135 desis of BV . In a reis of BV , it was p < 0.01) elevated in patients with cervical dysplasia and, therefore, may serve as risk-factors for the development of cervical cancer.Several studies have also pointed to a correlation of microbiota with HPV and cervical (pre)cancer. Compared to HPV-negative women, the vaginal bacterial diversity of HPV-positive women is more complex  , and becInterestingly, shotgun microbiota sequencing also showed that the HPV community in healthy woman is much more complex than previously defined, suggesting that co-existing non-oncogenic HPV viruses may stimulate or inhibit the oncogenic virus via viral interference or immune cross-reaction .Although not yet applied for diagnosing HPV or cervical (pre)cancerous lesions, metabolomics data were recently correlated with microbiome data in BV. Alterations in amino acid, carbohydrate, and lipid metabolism were associated with the presence and concentration of specific BV bacteria . AdditioSince CVF is washed away with the first flow of urine, it is expected that especially first-void urine contains most of the CVF components, including the mucus and debris from vaginal and cervical exfoliated cells. This may explain why the first collected part of a urine void collected with a special device  contains more human and HPV DNA than the subsequent parts , 147. MoAlthough their complexity still prevents the development of self-tests or medical practice applications, several biophysical applications are currently being evaluated for high-throughput testing of CVF samples. Fourier transformed infrared spectroscopy (IR) was performed on 25 cervical vaginal lavage specimens from women referred for colposcopy . For theAdditionally, mass spectrometry could contribute to cervical cancer diagnosis. The technology could very well be used in specialized laboratories where samples are collected, and it could complement or replace current methods, such as cytology and immunohistochemistry. However, at present, the main limitation of MS techniques lies in the sensitivity. For instance, to detect alpha-actinin-4, we used a targeted LC–MS technique called multiple reaction monitoring (MRM), but so far we have been unsuccessful because the limit of detection of the LC-Triple Quadrupole system for ACTN4 in CVF was at least ten-fold higher than the cutoff value of 18 pg/ml. Whether CVF contains cervical cancer biomarker proteins that are sufficiently high in abundance for detection by mass spectrometry remains to be elucidated, but at least for CVF labor biomarkers, Brown and co-workers  showed tIn conclusion, several components residing in the cervical vaginal fluid are valuable candidate biomarkers for diagnostic tests for cervical cancer or its precancerous states. Since CVF and CVF-containing first-void urine are appropriate body fluids for use in self-tests or point-of-care tests, we could focus in the future on those biomarkers that lend itself to the development of such tests. Proteins are excellent candidates for this, and may originate from the virus, the tumor, the host immune system or the disturbed microbiome. For this, alpha-actinin-4 may offer a very good starting point, but we still have a way to go. Continued investigation is necessary to define a CVF/urine panel of biomarkers with which we can move forward to a standardized evaluation protocol such as in the PRoBE study design . Moreove


E6 and E7 oncogenes are instrumental in promoting proliferation and blocking differentiation leading to tumorigenesis. Although surgical intervention can remove such tumors, the potential for an etiologic field effect with recurrent disease is real. A downstream effector of E7 oncoprotein, enhancer of zeste homolog 2 (EZH2), is known to promote proliferation and to pose a block in differentiation and in turn, could lead to HPV-induced malignant transformation. However, the EZH2 pathway is amenable to low toxicity therapies designed to promote differentiation to a more benign state and prevent recurrent disease by inhibiting the incorporation of HPV into the genome. This is the first study using clinical specimens to demonstrate EZH2 protein expression in oropharyngeal carcinoma (OPC).Human papillomavirus (HPV) has been identified as an etiopathogenetic factor in oropharyngeal squamous cell carcinoma. The HPV RNAscope® in-situ hybridization technology. Expression of EZH2, Ki-67, and mitotic indices were assessed by morphoproteomic analysis. Biomedical analytics expanded the results with data from Ingenuity Pathway Analysis (IPA) and KEGG databases to construct a molecular network pathway for further insights.The study included eight patients with oropharyngeal carcinoma, confirmed p16INK4a- positive by immunohistochemistry (IHC). The tissue expression of E6/E7 messenger RNA (mRNA) was measured by E6 and E7 oncogenes in p16INK4a- positive oropharyngeal carcinoma was confirmed. EZH2 and its correlates, including elevated proliferation index (Ki-67) and mitotic progression were also present. Biomedical analytics validated the relationship between HPV- E6 and E7 and the expression of the EZH2 pathway.Expression of E6 and E7 oncogenes and the expression of EZH2, Ki-67 and mitotic progression in oropharyngeal carcinoma. The molecular network biology was confirmed by biomedical analytics as consistent with published literature. This is significant because the biology lends itself to targeted therapeutic options using metformin, curcumin, celecoxib and sulforaphane as therapeutic strategies to prevent progression or recurrence of disease.There is morphoproteomic and mRNA evidence of the association of p16INK4a-HPV infection with the  The E7 E6 and E7 oncogenes in oropharyngeal carcinoma; 2. documenting and correlating both the expression of E6 mRNA in such cases with cell cycle progression and the expression of E7 mRNA with p16INK4a, EZH2 and Ki-67 and mitotic progression; 3. confirming the biology of HPV-associated oropharyngeal carcinoma with biomedical analytics; and 4. investigating targeted therapeutic options based on biomedical analytics and preclinical data.The purpose of this report is to address gaps in the knowledge of EZH2 expression in OPC patient specimens by: 1. providing morphoproteomic and mRNA evidence of the association of p16INK4a-HPVinfection with the Eight adult patients  ranging in age from 51 to 72 years were included in this study. The anatomical locations of their biopsy-proven squamous cell carcinomas included palatine tonsil in five and tongue base in three.Data collection and molecular analyses were performed in accordance with the guidelines of the University of Texas McGovern Medical School Committee for the Protection of Human Subjects Institutional Review Board (IRB).RNAscope® technology from Advanced Cell Diagnostics (https://acdbio.com/). Morphoproteomic analysis and biomedical analytics were also performed as part of the molecular analysis in our CLIA and CAP certified Consultative Proteomics Laboratory in order to define the biology of the patients’ tumors, to provide correlative expressions, and to ascertain targeted therapeutic options designed to reduce the progression or recurrence of the HPV-associated oropharyngeal carcinomas.Molecular analyses included in-situ hybridization for the expression of HPV-HR18 E6/E7 mRNA using the RNAscope® 2.5HD Red Assay was performed to evaluate expression in all 8 tissue specimens. The test assayed 18 high-risk HPV serotypes: HPV-HR18 HPV 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82, E6/E7 mRNA. Hs-PPIB was used as a positive control marker for sample quality control (QC) and to evaluate RNA quality in all the tissue samples. Bacterial gene dapB was used as a negative control. Standard pretreatment assay conditions were determined to be optimal for the samples in the study set. All the samples in the study passed QC with strong PPIB expression and no/negligible dapB background. A semi-quantitative scoring system of 0-4 was utilized.Morphoproteomic analysis applies bright field microscopy and immunohistochemistry directed against various protein analytes to define the biology of a neoplastic process. The analysis uncovers etiopathogenetic occurrences that might be responsible for the process development and the propensity for it to recur , 6. Immuwww.ingenuity.com). Since IPA does not include viral species, E6/E7 and their interactions associated with HPV (hsa05203) (“HPV” network) were extracted from the KEGG pathway database (http://www.genome.jp/kegg/pathway.html) and manually added to the ORO network. A “patient” pathway network was also constructed from the median patient scores and linked to ORO-HPV. From these graphs and additional data mining of the National Library of Medicine’s MEDLINE database, a single ORO-HPV network model was constructed using IPA Pathway Designer to represent the key modulation and adaptive responses in the signal transduction processes. Therapies were then linked to the ORO-HPV network model to assess potential benefits.To gain insights into HPV-associated oropharyngeal carcinoma, a standard IPA oropharyngeal pathway network (“ORO”) was constructed from key molecules associated with oropharyngeal carcinoma in the Ingenuity Knowledge Base  with the exception of patient specimens 5 and 6 that scored at 2 and 3, respectively (see Table Nuclear expressions of EZH2 (enhancer of zeste homolog 2) and Ki-67  and mitotic indices (mitotic figures/10 high power fields) by visual estimation on each case revealed a median of 90% and 70% for EZH2 and Ki-67, respectively and a range of 16 to 105 for the mitotic indices  and CDKN2A (p16 INK4a) were present – and affected – in the combined pathway network (not shown.) This combined ORO-HPV network model contained 1086 molecular interactions, or links, in the 3 linked pathways of ORO, HPV, and median patient. Of the 1086 links, 513 bridged between the ORO network and the HPV network. For the patient, the level of CDKN2A affected more molecules associated with HPV than with ORO. In return, more molecules from HPV than ORO affected the patient network, with TP53 and RB1 being the major influencers. 174 molecules from ORO affected HPV and patients; whereas 224 molecules from HPV affected ORO and patientupper left) is a key network point that is activated by E6/E7. Mir-26A can be seen in the upper right hand corner of Fig. The complex ORO-HPV network model was edited to focus on the key molecules: E6/E7 mRNA, EZH2, Ki-67 (MK167) plus related interactions from the National Library of Medicine’s MEDLINE Database. The potential efficacy of sulforaphane and the metformin and curcumin therapies – the latter in part through the upregulation of microRNAs – were graphically demonstrated Fig. . EZH2 waE6 and E7 oncogenes in oropharyngeal carcinoma. The E6 oncoprotein has been shown to bind with and promote the degradation of wild type p53 by activating the ubiquitin ligase E6AP [This report provides morphoproteomic and mRNA evidence of the association of p16INK4a-HPVinfection with the ase E6AP , 9. Simiase E6AP , coincidase E6AP –16. Thisase E6AP  showed tBiomedical analytics confirmed the correlations and the interactive biology of E6/E7 mRNA-associated oropharyngeal carcinoma with the morphoproteomic findings in our study and illustrated the potential targeted therapies of metformin, curcumin, celecoxib and sulforaphane.E6 and E7 [Rationale for the application of these agents against HPV-associated oropharyngeal carcinoma are provided as follows: pharmacogenomic upregulation of microRNAs, miR-26a and miR-101, by metformin and a curcumin analog in preclinical studies correspondingly decreased the expression of EZH2 , 19 and 6 and E7 , 22; cel6 and E7 ; and sul6 and E7 . Lindsay6 and E7 . AlthougE6 and E7 oncogenes and with the expression of EZH2, Ki-67, and mitotic progression in oropharyngeal carcinoma. This biology lends itself to targeted therapeutic options using metformin, curcumin, celecoxib and sulforaphane as therapeutic strategies to prevent progression or recurrence of disease.Our study showed that p16INK4a-HPV infection is associated with the


Women with high-grade cervical intraepithelial neoplasia (CIN) are at increased risk for developing cervical cancer. We examine how women with high-grade CIN perceive their own risk, and about pertinent knowledge concerning human high-risk papillomavirus (HPV), CIN and cervical cancer.All patients who underwent first-time treatment of high-grade CIN (grade 2+) were followed-up at 6-months at the Karolinska University Hospital, Stockholm, Sweden and were invited to participate in the present study. This included completion of a questionnaire examining sociodemographic characteristics, self-perceived risk of cervical cancer without regular gynecologic follow-up, and 14 queries about HPV, CIN and cervical cancer knowledge, inter alia.The participation rate was 96.6%, with 479 women enrolled in this study. Over 75% were age 40 or younger, over half had completed university education. Most were married or co-living with their partner and were gainfully employed. On a scale scored from 10  to 1 (lowest self-perceived risk), 64% rated their risk ≥ 7; almost 30% viewed their risk ≤ 6 and 7.5% did not rate their risk. A Specific Knowledge Scale with six of the queries explained 58.3% of the total variance. Nearly 30% of the women answered four or fewer of the six queries correctly. The Specific Knowledge Scale predicted self-perceived cervical cancer risk  after adjusting for age, income and education. Most of the women with low self-perceived cervical cancer risk did not rate their HPV-related knowledge as good. However, 32 predominantly university-educated women, with low self-perceived cervical cancer risk, considered their HPV-related knowledge good.It is vital to effectively convey accurate information about these patients’ cervical cancer risk, needed preventive and follow-up measures, together with the relevant specific knowledge, for these women at increased risk for developing cervical cancer. Tailored programming to address these knowledge gaps is needed. Among women worldwide, cervical cancer is the fourth most common cause of cancer death . An estith lowest in the EU [Whenever in place, high participation in screening programs through the Papanicolaou test (cytology-based) and subsequent treatment of cervical dysplasia has effectively reduced the cervical cancer mortality –6. An inn the EU , 7. Neven the EU , 9. Cervn the EU .The identification of high-risk human papilloma virus (HPV) as the key etiologic factor for cervical cancer is impacting upon approaches to screening and prevention of this malignancy. Testing for HPV is more sensitive, although often less specific than cytology-based testing for detection of high-grade cervical intraepithelial neoplasia (CIN) , 5. VaccThese considerations are particularly germane for women in whom high-grade CIN has been detected and treated. In a study of 330 women with cervical dysplasia treated with the loop electrical excision procedure (LEEP), a negative high-risk HPV test plus negative cytology at six months were reported to be a reliable test of cure at three-year follow-up . On the In a study from our group of ninety patients followed-up after treatment for CIN with conization via LEEP using a C-LETZ electrode, the presence of high-risk HPV genotype predicted all five cases of residual high-grade squamous intraepithelial lesion/CIN 2+, and the lack of persistent high-risk HPV infection was the most specific indicator of absence of recurrent or residual high-grade disease . A subseEspecially in light of these new and unfolding developments, accurate risk assessment and information concerning HPV and recommended screening follow-up are essential. Women with high grade CIN warrant special attention in this regard. To the best of our awareness, however, knowledge and perception of risk have not been previously investigated in this group of women. The aim of the present study is to examine self-perceived risk of cervical cancer and pertinent knowledge about HPV, CIN and cervical cancer among women treated for high-grade CIN. We will also assess relevant sociodemographic characteristics, HPV vaccination status as well as performing needs assessment regarding how the relevant knowledge could best be conveyed. It is hoped that these efforts will contribute to improving adherence to necessary follow-up among women at increased risk for developing cervical cancer.Patients who had undergone first time treatment of high-grade CIN (grade 2 or higher) and attended the first follow-up 6 months thereafter at the Karolinska University Hospital, Stockholm, Sweden, were the target group for the present study. Each patient received a written invitation letter via postal mail. Brief salient facts about HPV and cervical cancer were included in the invitation letter, as well as contact information for the Attending Gynecologist and Principal Investigator (SA) and for the Research Coordinator (EÖ). The study was presented as entailing completion of a questionnaire with the aim of helping to better prevent cervical cancer plus an HPV self-test. Complete confidentiality and freedom to withdraw from the study at any time without any adverse consequences whatsoever were guaranteed.The clinical research team ensured that the scheduled follow-up appointment was convenient for each patient. With this concerted effort, all the patients who had been first-time treated for high-grade CIN attended 6-month follow-up.At the 6-month follow-up visit the Research Coordinator (EÖ) met with each patient to explain the study procedure. All invited women signed an informed consent form with the choices of agreeing or declining to participate in the study. Approval of the study protocol was obtained by the Ethical Review Board at Karolinska Institute, Stockholm, Sweden .Knowledge of the Swedish language was a requirement for participation in the questionnaire portion of the study, since the questionnaire was in Swedish. A total of 480 women were eligible to participate in the questionnaire portion of the study, of whom 479 signed the informed consent in agreement and were thus included in the present study. In addition, sixteen women who would have otherwise been eligible (i.e. first-time treated for high-grade CIN and attending 6-month follow-up), did not complete the questionnaire due to lack of fluency in the Swedish language. Thus, the actual participation rate was 96.6%. The mean age of the seventeen non-participants was 42 ± 7.8.The questionnaire examined socio-demographic characteristics such as age, civil status, personal annual income, education and employment status. Queries were included concerning knowledge about HPV, CIN and risk of cervical cancer. These knowledge queries were similar, though not identical to those used in a previous study among women attending clinic-based cervical cancer screening in Stockholm . For eacThorough univariate analysis was done, with the distribution of all continuous and semi-continuous variables assessed visually, as well as by examination of skewness and kurtosis. Parametric bivariate analysis was performed insofar as the continuous or semi-continuous variables had both skewness and kurtosis < 1, otherwise non-parametric bivariate analysis was carried out. For bivariate analysis of dichotomous variables, Yates chi-squared analysis was used. Factor analysis was employed to develop the Specific Knowledge Scale, as described in the Results section. Unless otherwise noted, an absolute value of the factor loading > 0.70 was required for inclusion of a component in the Specific Knowledge scale. Binomial logistic regression was performed to compute odds ratios (OR) and 95% confidence intervals (CI) for unadjusted and adjusted models. All statistical analysis was carried out with Statistica 64 software.The socio-demographic characteristics of the cohort are presented in Altogether 42 of the participants reported having been vaccinated. Twenty-four stated that had done so at age 21 or above, fourteen reported having been vaccinated between the ages of fifteen and twenty, and four women did not know or did not answer the query about age at vaccination. Although 395 (82.5%) of the participants stated that they knew that they had not been vaccinated, seventeen women stated that they did not know their vaccination status, and twenty-five did not answer the query about HPV vaccination.Perceptions about risk of developing cervical cancer together with queries concerning knowledge about HPV, CIN and cervical cancer are summarized in Factor analysis of the 14 knowledge queries concerning HPV, CIN and cervical cancer yielded two factors: one with an Eigenvalue of 6.3, explaining 45.3% of the variance, and the other with an Eigenvalue of 1.2, explaining 8.4% of the variance. Five of the queries had an absolute value of their factor loading above 0.70. These are marked in red in the upper panel of We considered these queries to be not only psychometrically suitable, but also to be the most salient from the clinical and public health vantage points. With the latter in mind, we included one additional query whose absolute value of its factor loading was 0.63, namely: Screening is important even if vaccinated. The newly created Specific Knowledge Scale with these 6 factors had an Eigenvalue = 3.5 and explained 58.3% of the total variance. The lower portion of ρ = −0.095, p = 0.039), indicating that the younger women had higher specific knowledge.Age and annual income were correlated , such that the youngest women had the lowest personal gross annual income. The patients who were co-living were younger (mean age = 32.2 ± 7.1), whereas those who were married were significantly older (mean age = 38.1 ± 7.8), . The youngest women also had the lowest self-assessed risk of developing cervical cancer without regular gynecologic follow-up . There was an inverse correlation between age and Specific Knowledge Scale score ; the women with lower income thus had lower specific knowledge about HPV, CIN and cervical cancer.There was a positive correlation between personal gross annual income and educational level , indicating that the women with higher income had a higher level of completed education. The married women also had higher personal annual income . Annual income was positively correlated with self-assessed risk of developing cervical cancer without regular gynecologic follow up . Thus, the participants with lower income perceived themselves to be at lower risk of cervical cancer. There was a direct correlation between annual income and Specific Knowledge Scale score ; the women with lower completed education had lower specific knowledge about HPV, CIN and cervical cancer. The women who did not know that HPV can lead to other cancers had a lower educational level (post-hoc Bonferroni p = 0.03) compared to those who endorsed this statement , F = 3.16, p = 0.02).The participants with higher completed education were more often co-living, whereas those with less completed education were more often single . Education and Specific Knowledge Scale score were directly correlated . Concordantly, the Specific Knowledge Scale score was correlated with perceived frequency of needed gynecologic follow up . The women who considered their own knowledge good had higher scores on the Specific knowledge scale .Perceived frequency of gynecologic follow-up needed to protect the patient from developing cervical cancer was correlated with self-assessed risk of developing cervical cancer without regular gynecologic follow up  than those who stated that they were not vaccinated (mean age = 35.6 years) (p = 0.000). The women who did not answer the query about HPV vaccination were significantly older (mean age = 40.3 years) than each of the other groups of women. The women who did not know their HPV vaccination status had a mean age of 32.9 years.More significant bivariate findings will be reported in the subsection on Needs Assessment. Further analysis of self-assessed risk of developing cervical cancer will be presented in the next subsection.The variable: self-assessed risk of developing cervical cancer without regular gynecologic follow up was dichotomized at 7 or greater  versus 6 or less. The thirty-six patients who left this query blank were placed in the latter category, i.e. as not indicating a realistic assessment of their own risk. The adjusted binomial logistic regression model is presented in As seen in the left column of The participants who would like more information from health professionals and from work or school were younger . On the other hand, women who stated that they would like more information from television or radio, or via postal mail had lower incomes . Women with lower completed education also stated that they would like more information from television or radio .The most robust, strategically and clinically important finding of the present study is that specific knowledge about HPV, CIN and cervical cancer powerfully predicts realistic self-assessment of cervical cancer risk among this at-risk cohort. This finding is independent of age, income and education, the former two of which are also significantly associated with realistic risk assessment. Unrealistic optimism has been identified as an important predictor of lack of intention to participate in early detection programs for cervical cancer and other malignancies . The danBy far the most preferred modality, as indicated by the participants themselves, is that further information be provided by health professionals. This finding suggests that time and resources should be set aside for the health care providers to ensure that all women with high-grade CIN have acquired the needed knowledge to realistically assess their own risk status. This is expected to critically contribute to full adherence to recommendations for further needed gynecologic follow-up. An atmosphere of confidence and trust is the vital precondition to frame the risk assessment in an empowering manner, through objective knowledge, and avoiding the distress that has been reported in association with colposcopy and subsequent HPV testing , 20.The very high participation rate in the present study suggests that such a secure atmosphere was established. The active involvement of the clinical-research team in personally presenting the study to all the eligible women appears to have been very effective. Coherently, in various settings it has been shown that personal contact helps promote women’s enrollment in studies of early cancer detection and prevention , 22.Notwithstanding the nearly complete participation of the invited women, their knowledge about HPV, CIN and cervical cancer risk was overall quite low. This is of major concern and quite surprising given that these women had been screened, visited a gynecologist, had been treated and then followed-up. Nevertheless, they were still not sufficiently informed about the HPV, CIN and cervical cancer. The opportunities to provide information during these visits within the health care system appear to have been very often missed.It is also noteworthy that awareness of the risk for both women and men of developing other HPV-related cancers was the lowest of all the fourteen knowledge queries. Fewer than fifteen percent of the participants endorsed this statement. This small group of women who did endorse the statement were those with higher education compared to the vast majority of the women did not know the answer to this query. This knowledge is particularly important for women with high-grade CIN, for whom, as noted, more intensive follow-up and other measures are recommended due to long-term increased risk of HPV-related carcinomas of the anogenital and orpharyngeal regions . A similThis knowledge is also of general public health importance in promoting adherence to HPV vaccination for both genders. In a recent Swedish study, knowledge about the benefits of HPV vaccination for boys was reportedly low among parents who were offered HPV vaccination for their pre-teenage daughters . In the Among the women who did not appear to have a realistic assessment of their cervical cancer risk, two subgroups were identified. The first, and larger, subgroup was comprised of those did not consider themselves to have good HPV related knowledge. Fewer of these women had completed a university education. In a study from the Appalachian region of the U.S., women referred to colposcopy after abnormal Pap smears were frequently found to have limited understanding of these findings. It was only the women with high health literacy skills who accurately grasped the meaning of these findings. The authors suggested that counseling should be tailored accordingly during the clinical encounter . Such a In contrast, however, we identified a subgroup of women, albeit small, who were generally highly educated, considered their HPV-knowledge to be good, but did not realistically identify their own elevated risk of developing cervical cancer without regular gynecologic follow-up. Underestimation of one’s own cervical risk has been described among some university educated women in the U.S. , 31. On Direct communication by postal mail may be a helpful modality, and was noted to be one of the preferred options, not only among this subgroup of women, but also for the entire cohort. Postal mail communication was also more often chosen among women with lower incomes. An early study on the use of mailed letters that contained tailored, personalized messages based upon medical records showed this strategy to be particularly effective in promoting cervical as well as breast cancer screening among women with low incomes, as well as those from minority backgrounds .It has been noted that in most countries with cervical cancer screening programs, the majority of cervical cancers occur in women who have not been regularly screened . Low incThe age-adjusted incidence of cervical cancer in Sweden is not reported to differ among persons born outside Sweden and those born in the country . With reThe participants in the present study represent quite a unique cohort of women who through participation in cervical cancer screening were diagnosed and treated for high-grade CIN and who attended follow-up. As noted, the study participants were more highly educated and with higher incomes than the overall Swedish population. Sweden as a whole has an organized, population-based cervical cancer screening program, and the cervical cancer mortality rates are among the lowest in the EU . For manEven among women who do attend cervical cancer screening programs in Sweden, on-time screening is often low , 42. ComS1 Appendix(XLSX)Click here for additional data file.


Urine samples processed with the GenoArray assay is an alternative for women who decline to undergo Pap smear even though it is not ideal as the first-line screening option.Human papillomavirus (HPV) is the leading cause of cervical cancer. Urine-based HPV testing offers a simple and non-invasive method because of its increasing acceptance. A total of 164 pairs of cervical swab and urine samples from Thai women who underwent cervical cancer screening were used for HPV testing with HPV GenoArray Diagnostic Kits. The overall concordance percentage for HPV detection in the cervical swab and urine samples was 65.2%. The HPV genotypes most commonly detected were HPV16 and HPV18. An analysis of the urine samples and a second analysis of the cervical swab samples showed that the differences in the overall HPV detection rate between women with normal and abnormal cytology were not significant ( Human papillomavirus (HPV) causes cervical cancer . ApproxiThe incidence of cervical cancer in young women is increasing in many countries due to improved awareness and testing. HPV prevalence in Asia, Africa, Europe and South America varied significantly depending on the population and geographical regions . In EuroThe Papanicolaou (Pap) test is a cost-effective way to screen for cervical cancer. Test results help physicians detect precancerous lesions and determine the course of treatment. Pap test has been shown to reduce the incidence of mortality from cervical cancer . HoweverHPV DNA detection in urine samples presents a feasible alternative to HPV DNA detection in cervical specimens. Urine testing provides an especially simple, non-invasive method for screening . The benMolecular methods for HPV testing have been explored, such as PCR/sequencing , the INNThe research protocol was approved by the Ethics Committee of the National Cancer Institute, Thailand (number EC COA 037/2012), and the Institutional Review Board of the Faculty of Medicine at Chulalongkorn University (number 389/2555). The objective of the study was explained to the patients, and written consent was obtained from all participants. Each specimen was sent for testing anonymously, which only included participant-specific numerical code and age information.All patients underwent the Pap smears test and were subsequently asked to participate in this study. In all, 164 women consented and were willing to provide paired specimens (a Pap smear sample from the cervix and a first-void urine sample). All specimen were recruited between March to December 2014. Specimens were classified into three groups: 95 samples indicating normal cytology, 50 samples indicating low-grade squamous intraepithelial lesions (LSIL), and 19 samples indicating high-grade squamous intraepithelial lesions (HSIL). The ages of the patients enrolled in this study were between 19 and 69 years.Each Pap smear sample (which are the standard samples for HPV genotyping) was evaluated by a specialized cytotechnologist and the results were confirmed by a pathologist. Samples were suspended in the liquid-based cytology (LBC) buffer . Collection of the first-void urine (FVU) samples was performed in a sterile Cell PrepPlus  urine bottle, stored at 4 °C, and processed within 3 days. For each sample, approximately 15 mL of LBC and FVU were centrifuged at 3,000 rpm for 5 min, and the supernatant was removed. The residual 800 µL of the sample suspension containing cell debris was washed and centrifuged at 8,000 rpm for 5 min. The DNA was extracted from the pellets using a DNA Prep Kit  and stored at −20 °C until testing.The extracted DNA from the cervical swab and urine samples was subjected to an HPV genotyping assay using HPV GenoArray Diagnostic Kits  according to the manufacturer’s instructions. This PCR-based assay enables the amplification of 21 HPV genotypes including 13 high-risk types , two probable high-risk types (HPV 53 and 66), and six low-risk or unknown risk types . This assay uses an L1 consensus primer-based PCR and is different from the Linear Array HPV Genotyping Test, which uses MY09 and MY11 primers . After Pp < 0.05.A statistical analysis was performed using SPSS version 17.0 . Pearson’s chi-square test for matched pairs was used to compare the performance of the two types of samples regarding the detection of HPV genotypes. Statistical significance was defined as The mean age of the 164 participants was 45.8 years. Among the women with normal cytology, the mean age was 50.5 years, while among those with abnormal cytology (LSIL or HSIL), the mean age was 41.1 years. Results from the cervical swab samples showed that 39.6% (65/164) were HPV DNA-positive . In totaFor the urine samples, 32.3% (53/164) were HPV DNA-positive , of whicWe next compared HPV detection efficacy between the standard Pap smear samples and the urine samples. Comparison between the cervical swab and urine specimen resulted in the overall concordance of 65.2% (107/164) . In the For multiple HPV infections, the cervical swab-based assays were able to detect more HPV genotypes in each sample. However, in the normal cytology group, for each pair of biospecimens, the most common number of genotypes per sample was one. Similarly, in the abnormal cytology group, 36 of the 69 cervical swab samples (52.2%) and 31 of the 69 urine samples (44.9%) had a single genotype .p > 0.05).An analysis of the urine samples and a second analysis of the cervical swab samples showed that the differences in the overall HPV detection rate between the normal and abnormal cytology groups were not significant . This result suggests that urine represents a viable substitute for cervical swabs. However, the urine samples should be optimized by preventing DNA degradation during extraction and storage, recovering cell-free HPV DNA in addition to cell-associated DNA, processing a sufficient volume of urine, and collecting the first portion of the urine stream in the morning (The HPV DNA analysis of urine samples needs to be developed further before a urine-based assay can replace the Pap smear test. It is possible that a greater amount of urethral cells in the urine samples helped to increase the sensitivity of the test. An analysis of the urine samples and a second analysis of the cervical swab samples showed that the differences in the overall HPV detection rate between women with normal and abnormal cytology were not significant ( morning .Using traditional cytological analysis, it is difficult to determine accurate screening results for HPV-associated anogenital tumors. Therefore, HPV genotyping is an alternative screening method to be used in combination with traditional cytology for identifying patients at high risk of developing squamous cell carcinoma . NowadayThe Linear Array HPV Genotyping Test has been widely used as a standard reference method for evaluating new methods. However, the HPV GenoArray Diagnostic Kit is an alternative technique for studies conducted in resource-limited laboratories because the cost of the HPV GenoArray Diagnostic Kit is lower than that of the Linear Array HPV Genotyping Test and the hybridization time is also lower . MoreoveIn conclusion, the HPV GenoArray assay is an alternative for HPV genotyping using both cervical swab and urine samples, the latter of which is an alternative for women declining to undergo Pap smears. Although it is not the gold standard, utilization of this method is expected to will be increase the number of women who undergo cervical cancer screening.10.7717/peerj.3910/supp-1Data S1Click here for additional data file.


Immunochromatography (IC) is widely used to detect target molecules in biological fluids. Since this method can be performed without a special technique or device, IC is a convenient way to assess the existence of antibodies or pathogens such as viruses and bacteria, simply and quickly. In this study, we established an IC method to detect serum antibodies against oncogenic human papillomavirus (HPV)-16 and HPV-18 L1 proteins using recombinant L1 proteins produced by silkworms as antigens. Infection of oncogenic HPVs is a major risk factor of cervical cancer, which is one of the most common cancers in women worldwide. We first measured blood sera of two groups by magnetic beads enzyme-linked immunosorbent assay (MB-ELISA). For the first group, sera were collected prospectively from young women who planned to receive HPV vaccination. The second group consisted of children under 20 years of age, non-vaccinated healthy women, vaccinated healthy women, dysplasia, cervical intraepithelial neoplasia III, and cervical cancer patients. We confirmed that standard vaccination doses significantly increased serum HPV antibody concentrations, and the level was sustained at least more than 30 months after vaccination. In contrast, an increase in antibody concentration was not observed in patients with precancerous cervical changes and cervical cancer. We next measured the samples in both groups using the IC method we originally developed, and found that the measurement values of IC highly correlated with those of MB-ELISA. The simple and quick IC method would be a useful tool for rapid monitoring of L1 specific antibody levels in a non-laboratory environment. With less than one drop of serum, our IC can easily detect serum HPV-16/-18 antibodies within 15 minutes, without the need for electronic devices or techniques. Human papillomavirus (HPV) has a non-enveloped capsid and a circular double stranded DNA genome. The global prevalence of HPV infection is estimated at around 11–12% . Cervica®) containing HPV-16 and -18 antigens, and the quadrivalent vaccine by Merck (Gardasil®) containing HPV-6, -11, -16, and -18 antigens, have been well studied for almost a decade [The first two HPV vaccines on the market, the bivalent vaccine by GlaxoSmithKline  [Available prophylactic HPV vaccines utilize the recombinant HPV major capsid L1 protein as an antigen that forms capsid-like multimers by intrinsic activity, which is referred to as a virus-like particle (VLP) ,27. VLPsy (PBNA) ,29,30. Ty (PBNA) . The tity (PBNA) –33. ThesIn this study, we developed an immunochromatography (IC) method to measure serum anti-HPV-16/-18 Ab concentrations, in addition to our originally developed magnetic beads ELISA (MB- ELISA). Our IC method successfully measured Abs in the serum of vaccinated individuals and a cohort including cervical cancer-related entities, with correlated results measured by MB-ELISA.This study was approved by the Ethics Review Board of Tokyo Metropolitan Institute of Medical Science (IRB#17–13), Mie University (IRB#2388), and Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital (IRB#1061). All serum samples were obtained from women who provided signed informed consent. In the case of children, we used the surplus specimen, which was collected for their treatment with signed informed consent by their parents.®  and Gardasil® , were purchased from the manufacturers. World Health Organization (WHO) international standard HPV-16  and HPV-18 antibodies  were purchased from NIBSC . Normal anonymized human female sera, which were collected under an IRB approved protocol for de-identified, were purchased from Complex Antibodies, Inc.  and were used as a control serum which showed no immunoreactivity against the Cervarix® antigen based on our prior ELISA.TMB peroxidase substrate and stop solution were purchased from Kirkegaard & Perry Laboratories, Inc. . One-Step NBT/BCIP (nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate) was purchased from Thermo Fisher Scientific Inc. . ZyMAX™ horseradish peroxide (HRP)-labeled goat polyclonal anti-human IgG (H + L) was purchased from Life technologies . Human papillomavirus vaccines, Cervarix® . HPV-16 L1 (GenBank Accession No. GQ423063) and HPV-18 L1 (GenBank Accession No. AY383628) genes were synthesized by Integrated DNA Technologies, Inc . These genes were then ligated into pDock-His_pM01 vectors to obtain recombinant transfer vectors. This transfer vector was co-transfected with Bombyx mori nucleopolyhedrovirus (BmNPV) DNA [B. mori-derived cell line (BmN) [g) for 1 h at 4°C. The supernatant was applied to Dock Catch Resin (Sysmex Corporation), and both proteins were eluted with buffer B . The purified proteins were concentrated with Amicon Ultra-4 30 k units .HPV-16 and HPV-18 L1 proteins were expressed in the silkworm-Baculovirus system produced by ProCubeNPV) DNA  into a Bne (BmN) . After 7® in 50 mM carbonate buffer (pH 9.6) at 4°C overnight. After blocking with PBS containing 1% BSA , 1,000-fold diluted two rabbit polyclonal antisera to Cervarix®, was added and incubated at room temperature (r.t.) for 1 h. After washing the wells 3 times with PBS containing 0.1% Tween 20 (Sigma-Aldrich), HRP-conjugated anti-rabbit IgG  was added and incubated at room temperature (r.t.) for 1 h. After washing the wells 3 times, o-phenylenediamine dihydrochloride (Sigma-Aldrich) was added to the wells and incubated for 5 min at r.t., followed by the addition of 1 M H2SO4. The absorbance was measured at 490 nm using the ARVO X5 .F96 Maxisorp Nunc-Immuno plates  were coated with purified HPV-L1 recombinant proteins or Cervarixet al. [Proteins obtained from pupae infected with recombinant baculovirus containing HPV-16 L1 or HPV-18 L1 were analyzed by western blotting according to the method of Towbin et al. . The pro2PO4-Na2HPO4, 0.05% Tween 20, 10% goat serum, pH 7.4). Next, 50 μL of each serum sample diluted at 1:100 in assay buffer was then added. Antigen-free wells were set as negative controls. After incubation for 2 hrs, the beads were captured on a 96-well Magnetic-Ring Stand  for 5 min, and washed 4 times with wash buffer . After washing, HRP conjugated-goat anti-human IgG (H + L) Ab, diluted 1: 5,000, was added and incubated for 30 min. The plate was then washed 4 times with the wash buffer. The reaction was started by adding Sure Blue TMB microwell peroxidase substrate, and the absorbance was measured at 450 nm using the microplate reader Benchmark . All reactions were performed at room temperature. To correct the difference of OD value among plates, we employed an in-house standard sample for every measurement.MB-ELISA was performed as previously described  with the2 value for HPV-16 was 0.994 and 0.9801 for HPV-18. Based on these equations, we calculated the international unit of our in-house reference serum: the values were HPV-16; 197 U/mL, and HPV-18; 138 U/mL. Using these values, OD values were converted to WHO international units (IU/mL) by comparing to an in-house reference serum in addition to the plate compensation.To compensate for the variation in ELISA values among plates, we used one vaccinated sample that demonstrated high Ab concentrations of both HPV-16 and -18 as a reference for every assay. Moreover, to provide objectivity, WHO international standard HPV-16 antibodies (code: 05/134) and HPV-18 antibodies (code: 10/140) were used . These WHO standards were set as 1, 0.5, 0.25, and 0.125 U/mL and MB-ELISA was performed. The linear standard curves and the linear equations were obtained by plotting values . The R2) was provided by breaking through the small pouch. The dry AP substrate (BCIP), fixed on the membrane, was mixed with conjugation buffer after opening the reservoir. Anti-HPV antibodies in serum samples were captured by the immobilized HPV-16 or HPV-18 antigen on the IC membrane and were detected by a color change indicating an enzymatic reaction. Scores of the color strength was leveled from 0 (no color) to 8 (maximum density) by visual inspection according to the control color sample card.IC chips were prepared as previously described, with some modifications . BrieflyDifferences were evaluated by Kruskal-Wallis one-way ANOVA followed by Dunn’s post hoc multiple comparison in indices of clinical samples. Pearson’s correlation coefficient was used to assess correlations between HPV-16 and HPV-18 in MB-ELISA. Spearman’s correlation coefficient was used to assess correlations between IC and ELISA. Data are expressed as means ± standard deviation. Statistical analysis was performed by GraphPad Prism  and SigmaPlot .S1 Fig). The expression of dock- and His-tagged HPV-16 L1 and HPV-18 L1 recombinant proteins was confirmed by western blotting .To detect Abs against HPV-16 L1 and HPV-18 L1 in serum from vaccinated individuals, these genes were cloned and then expressed in the silkworm-baculovirus system as described in the Materials and Methods section . We next checked their immunoreactivities by using the WHO HPV type-specific serums in ELISA . Both antigens showed type-specific immunoreactivity, however, it was likely that non-negligible backgrounds were observed. To reduce the backgrounds, we employed magnetic beads method as previously described [Fig 1D).We also confirmed these antigens by ELISA using two independent polyclonal rabbit antisera immunized with Cervarixescribed . The resTable 1) and we prospectively collected their sera from peripheral blood at three time-points including at pre-vaccination and at two time-points  post vaccination .We first examined whether the anti-HPV-16 L1 and anti-HPV-18 L1 Ab concentrations increased in serum after vaccination in individuals who did not receive previous HPV vaccination. Twenty healthy women were enrolled  and at the second time point , suggesting parallel effects of each antigen on the clinically used vaccines.We measured the concentration of these sera by MB-ELISA and compensated for varying values among plates by using an in-house reference sample. We then plotted converted OD values with the WHO international unit (IU/mL) as described in Materials and Methods and in Table 2).We next validated the increased serum levels of anti-HPV Abs in vaccinated individuals from other hospitals in addition to the samples, which included patients with cervical cancer and cervical cancer-related diseases. We examined the collected samples consisting of 6 groups including cervical cancer and cervical cancer-related entities in addition to another group of vaccinated individuals .The concentrations of serum anti-HPV-16 and anti-HPV-18 Abs were measured by MB-ELISA . These concentrations were kept high for more than 15 months after vaccination in both Cervarix®  . Baculoviruses obtained using the BmN cell line with BmNPV were injected into silkworm pupae. Recombinant L1 proteins were purified using the dock-tag purification system.Synthesized L1 genes of HPV-16 and HPV-18 were subcloned into the pDock-His_pM01 vector ((TIF)Click here for additional data file.S2 FigA). Direct ELISA showed the expected dual immunoreactivities of WHO international standard serum but not of control serum against Cervarix. Asterisk (*) represents p < 0.05, one-way ANOVA. B). Standard curve and calculated equation using WHO standard L1 Abs.(TIF)Click here for additional data file.S3 FigAppearance of the IC for detecting HPV-16 L1 and HPV-18 L1 Abs in (TIF)Click here for additional data file.