Genital human papillomavirus infection is the most common sexually transmitted virus in the United States, with almost 20 million Americans currently infected and an additional 6.2 million becoming newly infected each year. Women rely strongly on their health care providers to educate and comfort them regarding this distressing diagnosis. This study will use an online, self-completion questionnaire to obtain women’s opinions regarding their health care providers’ performances concerning the initial consultation after learning of their HPV diagnosis. Findings from this study provided insight as to how women prefer receiving information from their health care providers concerning their diagnosis of HPV. In addition, study findings provide suggestions for improving clinical practices regarding HPV consultations in the Tri-cities area of East Tennessee.

Introduction : Depuis 2007, la vaccination contre les infections à papillomavirus humains (HPV) est recommandée en complément du dépistage du cancer du col utérin (CCU). Cependant, au vu de la faible couverture vaccinale en France, l’impact épidémiologique de la vaccination est discuté, ainsi que le choix de la population cible et les moyens déployés pour son adhésion à la recommandation. Cette thèse propose des données et des outils originaux pour l’évaluation et l’optimisation de l’impact de la vaccination HPV en France. Pour les aspects quantitatifs, une modélisation de la transmission de l’infection à HPV appuyée sur des données détaillées décrivant les partenariats sexuels dans la population générale est nécessaire. L’exploration du lien potentiel entre la participation au dépistage du CCU des femmes précaires et leur choix d’administrer le vaccin HPV à leurs filles, l’appréciation de l’acceptabilité de la vaccination à partir des réseaux sociaux, et l’évaluation médico-économique de la pertinence de l’extension de la vaccination aux hommes, sont déterminants pour parfaire le ciblage des populations à atteindre.Méthodes : Nous avons développé une plateforme de modélisation destinée à l'étude des contacts sexuels et de la dynamique de transmission des infections par les HPV à partir des données de l’enquête Contexte de la Sexualité en France. Grâce à des modèles de mélange de lois, nous avons identifié des classes latentes d’activité sexuelle, permettant de définir des profils à risque d’infections sexuellement transmissibles. Ensuite, nous avons interrogé les femmes consultant au sein du Centre de Prévention et d’Éducation pour la Santé de Lille, ayant au moins une fille éligible à la vaccination HPV, sur leurs attitudes vis-à-vis du dépistage du CCU et de la vaccination. Puis, nous avons analysé les opinions exprimées par les internautes sur le forum en ligne d’un site d’information en santé, concernant la sécurité, l’efficacité et la perception du vaccin HPV. Enfin, nous avons réalisé une revue systématique des études médico-économiques relatives à l’extension de la vaccination HPV aux hommes.Résultats : Les simulations sur la plateforme de modélisation ont permis de reproduire les données de prévalence des infections à HPV mais les résultats restent sensibles aux hypothèses sur les comportements sexuels qui présentent des incohérences entre les hommes et les femmes. Cinq classes latentes d’activité sexuelle ont été identifiées pour les hommes ainsi que pour les femmes. Le cluster correspondant au niveau d’activité sexuelle le plus élevé représente 3,3% chez les femmes et 4,8% chez les hommes. Par ailleurs, le statut vaccinal des filles ne diffère pas selon le profil de dépistage de leur mère. L’argument majoritairement rapporté par les mères pour justifier la non-vaccination de leurs filles concerne le manque d’information, surtout parmi celles qui ne se dépistent pas. De plus, les opinions négatives, exprimées sur le forum de discussion en ligne, sont passées de 28,6 % des avis exprimés en 2006 à 42,2 % en 2013. Les arguments avancés par les « anti-vaccinaux » concernent la sécurité du vaccin et la perception de la vaccination. Enfin, les modèles médico-économiques montrent que l’extension de la vaccination aux hommes est très rarement une stratégie coût-efficace. Néanmoins, la vaccination ciblée des homosexuels masculins semble être la stratégie optimale du point de vue éthique et médico-économique.Discussion : La plateforme de modélisation des contacts sexuels constitue le socle de l’évaluation de l’impact de la vaccination HPV. La surveillance des forums de discussion en ligne permet le monitoring de l’acceptabilité de la vaccination et le ciblage des actions d’information. L’optimisation de couverture vaccinale nécessite la mise en place d’un programme organisé de vaccination des jeunes filles. À défaut d’une implémentation en milieu scolaire, les centres de prévention offrent une alternative intéressante. / Introduction: Since 2007, prophylactic vaccination against human papillomavirus (HPV) has been recommended in addition to cervical screening in French women. However, given the low vaccine coverage in France, the epidemiological impact of the vaccination is debated, as well as the choice of the target population and the means to ensure compliance with the recommendation. This doctoral thesis provides original data and tools for the evaluation and the improvement of the impact of HPV vaccination in France. For quantitative aspects, modelling HPV transmission based on the best data describing sexual partnerships in the general population is essential. The investigation of potential links between participation to cervical screening of deprived women and their choice of vaccinating their daughters, the appraisal of vaccine acceptability through social media and the cost-effectiveness evaluation of the relevance of extending the HPV vaccination program to include males are key elements to improve the focus on targeted populations.Methods: We developed a modelling platform to study the dynamics of HPV transmission, using data from Social Context of Sexuality, the latest national French sexual behavior study. Using finite mixture models, we identified latent classes of sexual activity to define profiles of partner acquisition with age, likely to have different risks of sexually transmitted infections. Then, we asked women attending the Centre for Preventive Medicine and Health Education of Lille, who had at least a daughter eligible for HPV vaccination, about their attitudes towards cervical screening and HPV vaccination. Next, we explored sentiments about HPV vaccine safety, efficacy and perceptions, spontaneously expressed by web users on the online discussion forum of a French-speaking health information website. Finally, we performed a systematic review of the cost-effectiveness studies about extending HPV vaccination to include males.Results: Simulations from the modelling platform reproduced HPV infection prevalence observed in France. Nevertheless, results were sensitive to assumptions about sexual behavior, with discrepancies between men and women. Five latent classes of sexual activity were identified in men and in women. The cluster describing the highest level of sexual behavior represents 3.3% in women and 4.8% in men. Besides, daughters’ vaccination profile did not differ with their mothers’ profile of participation to cervical screening. The main reason for not vaccinating their daughters reported by mothers was lack of information, especially for those non-compliant with cervical screening recommendations. Moreover, negative sentiments, reported by the health website forum, evolved from 28.6% of total opinions in 2006 to 42.2% in 2013. The arguments expressed by “anti-vaccine” postings involved most often vaccine safety and negative vaccine perceptions. Finally, cost-effectiveness analyses show that extending the HPV vaccination program to include males is rarely found to be a cost-effective strategy. Nevertheless, the targeted vaccination of men having sex with men seems to be the best strategy from ethical and cost-effectiveness points of view.Discussion: The modelling platform of sexual contacts represents the basis of the evaluation of HPV vaccination impact. The surveillance of online forums enables the monitoring of vaccine acceptability and hence the targeting of preventive messages. Improving the HPV vaccine coverage requires offering girls and young women an organized vaccination program. In the lack of a school-based vaccination program, Centres for Preventive Medicine and Health Education offer an interesting alternative.

<p>Oncogenic human papillomaviruses (HPVs) are implicated in 99.7 % of cervical cancer cases but require the co-operation of other factors. To investigate potential genetic risk factors we have typed the HLA class II DRB1 and DQB1 loci in 478 women diagnosed with cervical carcinoma in situ and in 608 age-matched controls. Quantitative measurements of HPV 16, HPV 18/45 and HPV 31 were obtained. The DRB1*1501 and DQB1*0602 alleles were found to increase the risk of HPV 16 infection. Carriers of DRB1*1501 and DQB1*0602 were also shown to have an increased risk of a higher viral load compared to non-carriers. The DRB1*1301 and DQB1*0603 alleles were found to protect from HPV 18/45 and 31 infections as well as resulting in a lower viral load in carriers compared to non-carriers. Women with a high HPV 16, 18/45 or 31 viral load were more prone to long-term infections and women with a low HPV 16 viral load were more prone to short-term infections. Carriers of DRB1*1501 and DQB1*0602 alleles were also shown to have an increased risk of long-term infections compared to short-term infections. We also tested if an HPV susceptibility locus found for epidermodysplasia verruciformis (EV) was also linked to HPV susceptibility in cervical cancer. We did not find any linkage to this locus in a set of 77 families, each with at least three cases diagnosed with cervical carcinoma in situ. Other potential risk factors tested were HPV 16 E6 variants together with a p53 codon 72 polymorphism and HLA class II alleles. We found an association between the E6 L83V variant and the HLA DR4-DQ3 haplotype, as well as an increased frequency of Arg homozygosity of p53 in women infected with the L83V variant. These results show that alleles at HLA class II loci represents risk factors for persistent HPV infection and thereby also contribute to the risk of development of cervical carcinoma <i>in situ</i>.</p>

<p>Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. HPV-16 gene expression is tightly linked to the differentiation programme of the infected epithelium. Expression of the late genes, L1 and L2, encoding the capsid proteins, is delayed until the more terminally differentiated cells. Successful inhibition of HPV-16 late gene expression early in the viral life cycle is essential for persistence of infection, the highest risk factor for cervical cancer.</p><p>The goal of this thesis was to identify regulatory RNA elements and cellular factors that influence RNA processing events, such as alternative splicing and polyadenylation, during late gene expression. For this purpose, transfection of plasmids containing almost the full-length HPV-16 genome into HeLa cells, followed by RNA analysis, was employed. An exonic splicing enhancer (ESE) was identified that firmly supported the use of the E4 3’ splice site. A key regulator of HPV-16 gene expression, the E4 ESE was required for early mRNA splicing and polyadenylation, as well as for inhibition of premature late gene expression. The early polyadenylation signal (pAE) is also an important block of premature late gene expression. An upstream polyadenylation element (USE) was identified in the early 3’ untranslated region that enhanced polyadenylation at pAE, and interacted specifically with the cellular factors CstF-64, hnRNP C1/C2, PTB and hFip1. With the help of adenoviral E4orf4, a protein which causes dephosphorylation of SR proteins, we found that overexpression of SRp30c activated HPV-16 late gene expression by an exon skipping mechanism, and that SRp30c may interfere with early mRNA terminal exon definition.</p><p>This work identified a crucial splicing enhancer, as well as a number of cellular proteins binding to an USE in the early region of HPV-16. Furthermore, the cellular splicing factor SRp30c was shown to play a role in the regulation of HPV-16 late gene expression.</p>

Oncogenic human papillomaviruses (HPVs) are implicated in 99.7 % of cervical cancer cases but require the co-operation of other factors. To investigate potential genetic risk factors we have typed the HLA class II DRB1 and DQB1 loci in 478 women diagnosed with cervical carcinoma in situ and in 608 age-matched controls. Quantitative measurements of HPV 16, HPV 18/45 and HPV 31 were obtained. The DRB1*1501 and DQB1*0602 alleles were found to increase the risk of HPV 16 infection. Carriers of DRB1*1501 and DQB1*0602 were also shown to have an increased risk of a higher viral load compared to non-carriers. The DRB1*1301 and DQB1*0603 alleles were found to protect from HPV 18/45 and 31 infections as well as resulting in a lower viral load in carriers compared to non-carriers. Women with a high HPV 16, 18/45 or 31 viral load were more prone to long-term infections and women with a low HPV 16 viral load were more prone to short-term infections. Carriers of DRB1*1501 and DQB1*0602 alleles were also shown to have an increased risk of long-term infections compared to short-term infections. We also tested if an HPV susceptibility locus found for epidermodysplasia verruciformis (EV) was also linked to HPV susceptibility in cervical cancer. We did not find any linkage to this locus in a set of 77 families, each with at least three cases diagnosed with cervical carcinoma in situ. Other potential risk factors tested were HPV 16 E6 variants together with a p53 codon 72 polymorphism and HLA class II alleles. We found an association between the E6 L83V variant and the HLA DR4-DQ3 haplotype, as well as an increased frequency of Arg homozygosity of p53 in women infected with the L83V variant. These results show that alleles at HLA class II loci represents risk factors for persistent HPV infection and thereby also contribute to the risk of development of cervical carcinoma in situ.

The ability to determine nucleic acid sequences is one ofthe most important platforms for the detailed study ofbiological systems. Pyrosequencing technology is a relativelynovel DNA sequencing technique with multifaceted uniquecharacteristics, adjustable to different strategies, formatsand instrumentations. The aims of this thesis were to improvethe chemistry of the Pyrosequencing technique for increasedread-length, enhance the general sequence quality and improvethe sequencing performance for challenging templates. Improvedchemistry would enable Pyrosequencing technique to be used fornumerous applications with inherent advantages in accuracy,flexibility and parallel processing. Pyrosequencing technology, at its advent, was restricted tosequencing short stretches of DNA. The major limiting factorwas presence of an isomer of dATPaS, a substitute for thenatural dATP, which inhibited enzyme activity in thePyrosequencing chemistry. By removing this non-functionalnucleotide, we were able to achieve DNA read-lengths of up toone hundred bases, which has been a substantial accomplishmentfor performance of different applications. Furthermore, the useof a new polymerase, called Sequenase, has enabled sequencingof homopolymeric T-regions, which are challenging for thetraditional Klenow polymerase. Sequenase has markedly madepossible sequencing of such templates with synchronizedextension. The improved read-length and chemistry has enabledadditional applications, which were not possible previously.DNA sequencing is the gold standard method for microbial andvial typing. We have utilized Pyrosequencing technology foraccurate typing ofhuman papillomaviruses, and bacterial andfungal identification with promising results. Furthermore, DNA sequencing technologies are not capable oftyping of a sample harboring a multitude of species/types orunspecific amplification products. We have addressed theproblem of multiple infections/variants present in a clinicalsample by a new versatile method. The multiple sequencingprimer method is suited for detection and typing of samplesharboring different clinically important types/species(multiple infections) and unspecific amplifications, whicheliminates the need for nested PCR, stringent PCR conditionsand cloning. Furthermore, the method has proved to be usefulfor samples containing subdominant types/species, and sampleswith low PCR yield, which avoids reperforming unsuccessfulPCRs. We also introduce the sequence pattern recognition whenthere is a plurality of genotypes in the sample, whichfacilitates typing of more than one target DNA in the sample.Moreover, target specific sequencing primers could be easilytailored and adapted according to the desired applications orclinical settings based on regional prevalence ofmicroorganisms and viruses. Pyrosequencing technology has also been used forclone-checking by using preprogrammed nucleotide additionorder, EST sequencing and SNP analysis, yielding accurate andreliable results. <b>Keywords:</b>apyrase, bacterial identification, dATPaS, ESTsequencing, fungal identification, human papillomavirus (HPV),microbial and viral typing, multiple sequencing primer method,Pyrosequencing technology, Sequenase, single-strandedDNA-binding protein (SSB), SNP analysis

Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer. HPV-16 gene expression is tightly linked to the differentiation programme of the infected epithelium. Expression of the late genes, L1 and L2, encoding the capsid proteins, is delayed until the more terminally differentiated cells. Successful inhibition of HPV-16 late gene expression early in the viral life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. The goal of this thesis was to identify regulatory RNA elements and cellular factors that influence RNA processing events, such as alternative splicing and polyadenylation, during late gene expression. For this purpose, transfection of plasmids containing almost the full-length HPV-16 genome into HeLa cells, followed by RNA analysis, was employed. An exonic splicing enhancer (ESE) was identified that firmly supported the use of the E4 3’ splice site. A key regulator of HPV-16 gene expression, the E4 ESE was required for early mRNA splicing and polyadenylation, as well as for inhibition of premature late gene expression. The early polyadenylation signal (pAE) is also an important block of premature late gene expression. An upstream polyadenylation element (USE) was identified in the early 3’ untranslated region that enhanced polyadenylation at pAE, and interacted specifically with the cellular factors CstF-64, hnRNP C1/C2, PTB and hFip1. With the help of adenoviral E4orf4, a protein which causes dephosphorylation of SR proteins, we found that overexpression of SRp30c activated HPV-16 late gene expression by an exon skipping mechanism, and that SRp30c may interfere with early mRNA terminal exon definition. This work identified a crucial splicing enhancer, as well as a number of cellular proteins binding to an USE in the early region of HPV-16. Furthermore, the cellular splicing factor SRp30c was shown to play a role in the regulation of HPV-16 late gene expression.

Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill &amp; Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications. / Patogena organismer smittas till värd organismen genom alla möjliga kontaktnätverk och skapar en mångfald olika sjukdomstillstånd. Dock är det fortfarande vanligt förekommande behandlingsbara infektiösa sjukdomar som orsakar den största hälsoförlusten, sett från ett globalt perspektiv. Bill och Melinda Gates Stiftelsen samarbetade med RAND kooperation för att forma “The Global Health Diagnostics Forum”. Deras mål var att etablera och analysera matematiska modeller för vilka effekter en ny diagnostisk metod utrustat för fältarbete skulle ha i utvecklingsländer. Resultaten var häpnadsveckande, med potentiellt miljoner av liv som skulle kunna räddas på en årlig basis. Den etablerade standarden för diagnostik av patogena bakterier har länge varit kultiveringsmedia baserad. Miljö specialiserade biologer har estimerat att mindre än 1 % av alla bakterie arter går att kultivera. Dock erbjuder genetiska analyser potentialen att kunna identifiera alla mikrober från alla de biologiska rikena. Nukleinsyrebaserade diagnostiska metoder har märkbart förbättrats över de senaste årtionden. Nya tekniker erbjuder utökad sensitivitet, selektivitet, sänkta kostnader och parallella analyser av patient prover. Dock är de flesta metoderna begränsade till standardiserade laboratoriemiljöer. För att konstruera en väl fungerande diagnostisk fältutrustning för användning i problem områden, behöver världsledande tekniker identifieras och kombineras. Fokuseringsområdet för denna doktorsavhandling har varit att utveckla och utföra nukleinsyrebaserade metoder för patogen diagnostik. Metoder och experimentella utförande applicerades på två distinkta system i) sökning av antibiotika resistens relaterade mutationer i den patogena bakterien Neisseria gonorrhoeae och ii) genotypning av det cancer orsakande Humana Papillomaviruset (HPV). Den första delen av studien inriktade sig mot utveckling av snabba, direkta och multiplexa Pyrosekvenserings baserade nukleinsyreanalyser. Med förbättrad provprepareringsmetodologi kunde vi detektera multipla HPV infektioner med högre sensitivitet än vad tidigare beskrivits med liknande metodologi. Den andra delen av studien fokuserades på multiplexa nukleinsyre amplifikationer med “Molecular Inversion Probe” tekniken med sista steg Pyrosekvenserings analys. “PathogenMip assay” erbjuder ett komplett detektionsprotokoll för alla kända patogena organismer. Vi introducerar även den nya “Connector Inversion Probe”, en “Padlock Probe” kapabel att genomföra kompletta gap fyllningar för multiplex nukleinsyre amplifiering. / QC 20100624

Developments within the field of genetic analysis have during the last decade become enormous. Advances in DNA sequencing technology have increased throughput from a thousand bases to over a billion bases in a day and decreased the cost thousandfold per base. Nevertheless, to sequence complex genomes like the human is still very expensive and efforts to attain even higher throughputs for less money are undertaken by researchers and companies. Genotyping systems for single nucleotide polymorphism (SNP) analysis with whole genome coverage have also been developed, with low cost per SNP. There is, however, a need for genotyping assays that are more cost efficient per sample with considerably higher accuracy. This thesis is focusing on a technology, based on competitive allele-specific extension and microarray detection, for genetic analysis. To increase specificity in allele-specific extension (ASE), a nucleotide degrading enzyme, apyrase, was introduced to compete with the polymerase, only allowing the fast, perfect matched primer extension to occur. The aim was to develop a method for analysis of around twenty loci in hundreds of samples in a high-throughput microarray format. A genotyping method for human papillomavirus has been developed, based on a combination of multiplex competitive hybridization (MUCH) and apyrase-mediated allele-specific extension (AMASE). Human papillomavirus (HPV), which is the causative agent in cervical cancer, exists in over a hundred different types. These types need to be determined in clinical samples. The developed assay can detect the twenty-three most common high risk types, as well as semi-quantifying multiple infections, which was demonstrated by analysis of ninety-two HPV-positive clinical samples. More stringent conditions can be obtained by increased reaction temperature. To further improve the genotyping assay, a thermostable enzyme, protease, was introduced into the allele-specific extension reaction, denoted PrASE. Increased sensitivity was achieved with an automated magnetic system that facilitates washing. The PrASE genotyping of thirteen SNPs yielded higher conversion rates, as well as more robust genotype scoring, compared to ASE. Furthermore, a comparison with pyrosequencing, where 99.8 % of the 4,420 analyzed genotypes were in concordance, indicates high accuracy and robustness of the PrASE technology. Single cells have also been analyzed by the PrASE assay to investigate loss of alleles during skin differentiation. Single cell analysis is very demanding due to the limited amounts of DNA. The multiplex PCR and the PrASE assay were optimized for single cell analysis. Twenty-four SNPs were genotyped and an increased loss of genetic material was seen in cells from the more differentiated suprabasal layers compared to the basal layer. / QC 20100714

Human papillomavirus (HPV) is widespread in the population and an important concern for public health. HPV-associated benign and cancerous disease is vaccine preventable yet vaccine uptake has been suboptimal. Adolescents are the primary target for vaccination yet their perspective has been inadequately examined. Ontario provides population-based preventive HPV vaccination to adolescent girls yet in the program’s first 2 years only approximately half of eligible girls received it. Effective strategies to improve vaccine uptake are needed. This thesis proposes a theory and ethics-based model to guide health promotion planning for HPV vaccination. Adopting an adolescent perspective, the model is applied and comprises: 1) a systematic review to identify barriers and facilitators to HPV vaccination from the viewpoint of young females; 2) GIS uses for communicating geospatial health information regarding vaccination; and 3) a roadmap for the future including recommendations for guiding principles, research, intervention development, and health policy.