Dissertations / Theses on the topic 'Papillomaviruses Diagnosis'

En utilisant une technique originale de détection et de quantification des HPV16 et 18 par PCR duplex, ce travail de thèse illustre la signification, l’intérêt et les limites de l’utilisation clinique de la mesure de la charge virale pour ces deux types d’hrHPV. Si nous n’avons pas démontré de réelle signification de la charge virale en HPV18, il n’en est pas de même pour l’HPV16 dont la charge virale augmente avec la sévérité des lésions constatées. Néanmoins, l’extrême variabilité des charges virales mesurées limite son utilisation en pratique clinique. Après un frottis cervico-utérin (FCU) anormal, une charge virale seuil en HPV16 à 3,0x106 copies par millions de cellules permet la prédiction optimale de la présence d’une CIN2+ (spécificité : 91 % et sensibilité : 58,2 %). Cette valeur seuil est particulièrement performante pour les patientes ayant un FCU de bas grade (spécificité : 96,4 % et sensibilité : 88 %). Si la charge virale en HPV16 et 18 ne semble pas être prédictive de la clairance virale chez les jeunes femmes de moins de 30 ans ayant un FCU normal, elle l’est chez les patientes HPV16 positives ayant une colposcopie normale malgré un FCU équivoque ou de bas grade (spécificité : 86,7 % et sensibilité : 85,7 %)
Using duplex PCR technique for the detection and quantification of HPV16 and 18, this work investigates the significance, value and limitations of the use of HPV16 and 18 viral load quantitation in routine clinical practice. Although HPV18 viral load was not found to be of any clinical relevance, HPV16 viral load was found to significantly increase with the severity of cervical lesions. However, the wide range of viral load observed strongly limitates its use in routine clinical practice. After an abnormal cervical cytology, a HPV16 viral load cut-off of 3.0x106 copies per million cells allows for the best prediction of CIN2+ (91% specificity and 58.2% sensitivity). Such cut-off is particularly efficient in case of low grade abnormal cytology (96.4% specificity and 88% sensitivity). Although HPV16 viral load does not appear to predict for HPV16 clearance in women under 30 with normal cytology, such prediction was observed among women with normal colposcopy following equivocal or low grade cytology (86.7% specificity and 85.7% sensitivity)

Currently, the most common strategy employed to detect DNA sequences is PCR (Polymerase Chain Reaction). Nevertheless, in the last few years research on DNA biosensors has increased significantly. Such sensors represent an alternative to PCR in the detection of specific DNA sequences, once they exhibit fast response, low limits of detection, and require simpler sample preparation. The development of a biosensor for detection of DNA from Human Papillomavirus type 18 is reported. To immobilise DNA probe onto indium-tin oxide (ITO) electrodes, a silanisation was carried out using 3-Aminopropyltryethoxysilane (APTES). Silanisation was studied and optimised using ultra-violet absorption spectroscopy, atomic force microscopy, fluorescence microscopy, and cyclic voltammetry. After immobilisation, the hybridisation with target sequence is detected by changes in surface properties of ITO electrode by Cyclic Voltammetry and Electrochemical Impedance Spectroscopy, using the Ferri-Ferrocyante redox couple. The detection of synthetic target sequence was performed in the range of 12.5 to 100 nM, and 300nM for PCR products. The sensor did not show significative response for non-complementary sequence at 50 nM. This sensor can be applied for fast and low cost detection of HPV genetic material at nanomolar levels.
A estratégia mais empregada atualmente na detecção de sequência de DNA é a PCR (Reação em Cadeira da Polimerase). Contudo, nos últimos anos, a pesquisa em biossensores de DNA tem aumentado significativamente. Estes sensores representam uma alternativa a PCR na detecção de sequências específicas de DNA, uma vez que exibem resposta rápida, baixos limites de detecção e requerem preparação simples da amostra. Nesta dissertação descrito o desenvolvimento de um biossensor para a detecção do DNA do Papilomavirus Humano tipo 18. A fim de imobilizar a sequência de captura de DNA em eletrodos de óxido de estanho e índio (ITO), realizou-se uma silanização usando 3-Aminopropiltrietoxisilano (APTES). A reação de silanização foi estudada e otimizada através das técnicas de Espectroscopia de Absorção Ultravioleta, Microscopia de Força Atômica, Microscopia de Fluorescência e Voltametria Cíclica. Após a imobilização, a hibridização com a sequência alvo é detectada através de alterações nas propriedades de superfície do eletrodo através de Voltametria Cíclica e Espectroscopia de Impedância Eletroquímica, usando o par redox Ferri-ferrocianeto. A detecção da sequência alvo sintética foi realizada no intervalo de 12.5 a 100 nM, e para o produto de PCR, 300 nM. O sensor não demonstrou resposta significativa para sequência não complementar a 50 nM. Este sensor pode ser aplicado na detecção rápida e de baixo custo de material genético do HPV a níveis nanomolares.

Human Papillomavirus (HPV) is the cause of cervical cancers (among these, adenocarcinoma, AdCa) and is associated to a subgroup of oropharyngeal carcinomas (OPSCCs). Even if the risk for cancer development is linked to the infection by some viral genotypes, mainly HPV16 and 18, viral DNA alone seems not to be sufficient for diagnosis. Moreover, the role of the virus in OPSCCs has not been totally clarified yet. In the first part of the thesis, the performances concerning viral genotyping in clinical cervical samples of a new pyrosequencing-based test and a well-known hybridization-based assay have been compared. Similar results between the methods have been obtained. However, the former showed advantages in detecting intratype variants, higher specificity and a broader spectrum of detectable HPV types. The second part deals with the evaluation of virological markers (genotyping, viral oncoproteins expression, viral load, physical state and CpG methylation of HPV16 genome) in the diagnosis/prognosis of cervical AdCa and HPV-associated OPSCCs. HPV16 has been confirmed the most prevalent genotype in both the populations. Interestingly, the mean methylation frequency of viral DNA at the early promoter showed the tendency to be associated to invasion for cervical AdCa and to a worse prognosis for OPSCCs, suggesting a promising role as diagnostic/prognostic biomarker. The experiments of the third part were performed at the DKFZ in Heidelberg (Germany) and dealt with the analysis of the response to IFN-k transfection in HPV16-positive cervical cancer and head&neck carcinoma cell lines to evaluate its potential role as new treatment. After 24h, we observed increased IFN-b expression which lead to the up-regulation of genes involved in the antigens presentation pathway (MHC class I and immunoproteasome) and antiviral response as well, in particular in cervical cancer cell lines. This fact suggested also the presence of different HPV-mediated carcinogenic pathways between the two anatomical districts.
Il Papillomavirus umano (HPV) è causa dei carcinomi della cervice uterina (tra cui adenocarcinomi, AdCa) ed è associato ad un sottogruppo di tumori dell’orofaringe (OPSCCs). Nonostante il rischio di sviluppo di tumore sia associato all’infezione da parte di alcuni genotipi virali, principalmente HPV16 e 18, il DNA virale da solo sembra non essere sufficiente in campo diagnosico. Inoltre, per tumori orofaringei il ruolo del virus non è ancora del tutto chiaro. Nella prima parte della tesi, sono state confrontate le performance riguardo la genotipizzazione di HPV su campioni clinici cervicali di una tecnica innovativa, basata su amplificazione e pirosequenziamento, e una di routine, basata su amplificazione e ibridazione inversa. Lo studio ha evidenziato performance simili tra le due metodiche, sottolineando per il sequenziamento una maggiore specificità e capacità di rilevare varianti intratipo. Nella seconda parte sono stati analizzati marker virologici (genotipizzazione, espressione delle oncoproteine virali, carica virale, stato fisico e metilazione del genoma di HPV16) in funzione dei dati clinici disponibili, per un possibile impiego nella diagnosi/prognosi di AdCa cervicali e OPSCCs HPV-associati. HPV16 si è confermato il genotipo prevalente in entrambe le popolazioni. La frequenza di metilazione nel promotore precoce virale ha mostrato una tendenza ad essere associata ad invasione negli AdCa, e ad una prognosi peggiore negli OPSCCs, emergendo come il più promettente marker diagnostico/prognostico. La terza parte, svolta presso il DKFZ di Heidelberg (Germania), ha visto l’analisi della risposta alla transfezione di IFN-k in linee cellulari tumorali HPV16-positive della cervice uterina e della regione testa-collo, per valutarne l’impiego terapeutico. Dopo 24h, è stato osservato un incremento dell’espressione di IFN-b e, di conseguenza, una up-regolazione dei geni coinvolti nella presentazione antigenica (MHC classe I ed immunoproteasoma) e nella risposta antivirale, specialmente nelle cellule cervicali, suggerendo la presenza di diversi meccanismi patogenetici tra tumori HPV-positivi dei due distretti anatomici.

Le cancer du col uterin est un probleme majeur de sante publique, du fait de sa frequence et de son taux de mortalite. Des anomalies precancereuses peuvent etre detectees et traitees grace au depistage de masse, realisable par un frottis cervico-vaginal. Nous nous sommes interesses au diagnostic et au pronostic des lesions de bas grade du col uterin par la recherche conjointe de papillomavirus humain (hpv) et par l'etude de la quantification de l'adn par analyse d'image (ploidie cellulaire). Nous avons demontre clairement l'interet de ces deux etudes complementaires des diagnostics cytologiques et histologiques, dans les lesions de bas grade du col uterin. La detection d'hpv oncogenes et l'appreciation de leur integration potentielle sont des donnees permettant d'apprecier la persistance voire l'evolutivite des lesions. La quantification d'adn par analyse d'images nous a permis d'isoler trois profils principaux de ploidie : le profil diploide, le profil aneuploide compose lui meme de deux profils : aneuploide un pic et multiploide. La presence d'un hpv oncogene seul tout comme celle d'un profil aneuploide seul, ne permet pas toujours de predire l'evolution d'une lesion. En revanche, une lesion de bas grade presentant des hpv oncogenes, de surcroit integres, avec un profil aneuploide mais surtout multiploide, correspond a une lesion potentiellement maligne ; une lesion de haut grade pouvant d'autre part etre situee a proximite. En definitif, l'etude conjointe de la quantification d'adn par analyse d'image et de la detection d'hpv oncogenes, assure une nouvelle et meilleure approche du pronostic de l'evolution des lesions de bas grade mais aussi de haut grade du col uterin.

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 & 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

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Introduction: Genital infection by human papillomavirus (HPV) is considered the most common sexually transmitted infection in several countries. The prevalence of HPV genotypes in women with or without cytological abnormalities may vary according to the studied population and the region. Objective: To detect and identify specific HPV types correlating with sociodemographic/behavioral characteristics and cytological abnormalities present in cervical smears of Maroon women. Methods: This cross-sectional study included 353 maroon women users of the unified health System of the municipalities of Maranhão for screening of cervical cancer. The samples were analyzed for the presence of cytological abnormalities by conventional methods and tested for 37 HPV genotypes by polymerase chain reaction (PCR) with primers PGMY09/11 followed by reverse line blot hybridization performed with the Linear Array HPV Genotyping Test kit by Roche Molecular System®. The association of HPV types and cytological diagnosis was investigated according to the different age groups. Results: HPV infection was detected in 13% (46/353) of the cases. Infection types with high-risk HPV were more frequent (78,3 %, 36/46) than for low-risk HPV (21,7 %, 10/46). Genotypes 68 (24,2 % - 11 /46), 58 (19,8% - 9/46), 52 and 31 (10,8% - 5/46 each), and 62 (8,8% 4/46) were the most prevalent. Only 6,6% (3/46) of the cases were positive for HPV 61. The genotypes 73, 70, 54, 53, 45, IS39 and 18, individually represented 4,4% (2/46) of all cases. The CP6 -108, 84, 72, 71, 66, 59, 56, 55, 51, 39, 33 and 16 genotypes individually represented 2,2% (1/46) of the cases. In women without cytological abnormalities, viral infections, both simple and multiple, were detected in 10,7%(35/328) of the cases. For those diagnosed with an abnormality, the prevalence of HPV infection was 24,0% (1 /46), being higher in high grade squamous intraepithelial lesion (HSIL-75,0 %), followed by atypical squamous cells cannot exclude high-grade squamous intraepithelial lesions (ASC-H, 50%), low grade squamous intraepithelial lesions (LSIL-33,4 %) and atypical squamous cells of undetermined significance (ASC-US, 14,3%). The analysis showed a statistically significant association between HPV infection and the detection of cytologic abnormalities in the age groups between 31-40 years (OR = 7.40, 95 % CI :1.07 -51 ,19 , p = 0.03) and 51 to 60 years (OR = 20.4 , 95 % CI : 1.12 to 704.69, p = 0.03). None of the behavioral variables showed significant association with HPV infection, however the presence of this infection was positively associated with detection of cytologic abnormalities (OR=6,57: IC:2,772-15,606), p=0,001. Conclusion: It is possible that the results of this study are due to characteristics of the study population, geographically isolated, with conservative habits and sexual intercourse only between members of the Quilombo. This study with Maroon women allowed us to delineate the epidemiology of this HPV infection in populations living in Maranhão.
Introdução: A infecção do trato genital pelo Papilomavírus humano (HPV) é considerada a infecção sexualmente transmissível (IST) mais comum em diversos países. A prevalência dos genótipos do HPV em mulheres com ou sem anormalidades citológicas pode variar de acordo com a população e a região estudada. Objetivos: Detectar e identificar os tipos específicos de HPV e relacionar com características sociodemográficas/comportamentais e anormalidades citológicas presentes em esfregaços cervicais de mulheres quilombolas do Maranhão. Metodologia: Este estudo de corte transversal incluiu 353 mulheres quilombolas usuárias do Sistema Único de Saúde dos municípios do Maranhão para rastreamento do câncer do colo uterino. As amostras foram analisadas quanto à presença de anormalidades citológicas pelo método convencional e testadas para 37 genótipos de HPV por reação em cadeia da polimerase (PCR) com os iniciadores PGMY09/11, seguida de hibridização reversa em linhas através do Kit Linear Array HPV Genotyping Test (Roche Molecular System)®. A associação dos tipos de HPV e o diagnóstico citológico foi investigada de acordo com as diferentes faixas etárias. Resultados: A infecção pelo HPV foi detectada em 13% (46/353) das mulheres incluídas. Infecções por tipos de HPV de alto risco foram mais frequentes (78,3%; 36/46) do que por HPV de baixo risco (21,7%; 10/46). Os genótipos 68 (24,2% - 11/46); 58 (19,8% - 9/46); 52 e 31 (10,8% - 5/46 cada) e 62 (8,8% - 4/46) foram os mais prevalentes. Apenas 6,6% (3/46) dos casos foram positivos para o HPV 61. Os genótipos 73, 70, 54, 53, 45, IS39 e 18 representaram individualmente 4,4% (2/46) do total de casos. Os genótipos CP6-108, 84, 72, 71, 66, 59, 56, 55, 51, 39, 33, e 16 representaram individualmente 2,2% (1/46) do total de casos. Em mulheres que não apresentaram anormalidades citológicas, infecções virais, tanto simples quanto múltiplas, foram detectadas em 10,7%(35/328) dos casos. Considerando mulheres com diagnóstico de anormalidade citológica, a prevalência de infecção por HPV foi de 24,0% (11/46), sendo maior nos casos de lesão intraepitelial escamosa de alto grau (high grade squamous intaepithelial lesion)-HSIL-75,0%), seguida por mulheres com diagnóstico de células escamosas atípicas não podendo excluir alto grau (Atypical squamous cells cannot exclude high-grade squamous intraepithelial lesions) ASC-H- 50%), com lesão intraepitelial escamosa de baixo grau (low grade squamous intraepithelial lesion) LSIL- 33,4%) e com diagnóstico de células escamosas atípicas de significado indeterminado (atypical squamous cells of undetermined significance ASC-US (14,3%). Houve uma associação estatisticamente significativa entre a infecção por HPV e a detecção de anormalidades citológicas nas faixas etárias de 31 a 40 anos (OR = 7,40; 95% CI:1,07–51,19, p=0,03) e 51 a 60 anos (OR=20,4, 95%CI: 1,12-704,69; p = 0,03). Nenhuma das variáveis comportamentais analisadas mostrou associação significante com a infecção por HPV, contudo a presença desta infecção foi positivamente associada à detecção de anormalidades citológicas (OR=6,57: IC:2,772-15.606), p=0,001. Conclusão: É possível que os resultados deste estudo se devam às características da população estudada: isolada geograficamente, com hábitos conservadores e relações sexuais apenas entre membros do quilombo. Este estudo em mulheres quilombolas permitiu delinear a epidemiologia desta infecção por HPV em populações residentes no Maranhão.

Papilomavírus (PVs) são vírus amplamente estudados, sendo enfatizada sua capacidade de infectar os tecidos epitelial e mucoso em diversos animais, incluindo humanos, causando lesões benignas que podem, ocasionalmente, resultar em câncer. Dentre os gêneros que infectam animais, os Deltapapillomavirus têm uma importância veterinária, ecológica e histórica, pois são capazes de infectar seu hospedeiro natural e outros animais. Por isso, esse trabalho contempla todos os tipos virais pertencentes aos Delta-PVs, incluindo sua história. Dentre as proteínas traduzidas pelos PVs, três são consideradas proteínas oncogênicas: E5, E6 e E7. Determinar a estrutura de uma proteína é crucial para a elucidação da sua função, possibilitando aplicações nas áreas de engenharia de proteínas, anotação genômica e desenho racional de fármacos. A estrutura tridimensional da proteína E6 de cada tipo viral pertencente ao gênero Deltapapillomavirus foi determinada por modelagem molecular por homologia. A história evolutiva dessas proteínas foi avaliada com base na geração de árvores filogenéticas e suas propriedades físico-químicas foram analisadas. Além disso, devido ao seu alto grau de conservação, a E6 demonstrou ser útil como um marcador molecular. Apesar de serem consideradas raras, foram observadas lesões papilomatosas em carneiros em uma fazenda do estado de São Paulo. Foi realizado o diagnóstico molecular dessas lesões. Os resultados mostraram pela primeira vez no mundo que, apesar de serem ovinos, o agente causador da papilomatose era um papilomavírus bovino, o BPV2, um Delta-PV. Logo, além de discutir os Delta-PVs, esta tese demonstra na prática a habilidade desse gênero em romper a barreira espécie-específica.
Papillomaviruses (PVs) are widely studied viruses, emphasizing their ability to infect the epithelial and mucosal tissues in several animals, including humans, causing benign lesions that may occasionally result in cancer. Among the genera that infect animals, Deltapapillomaviruses have a veterinary, ecological and historical importance because they are capable of infecting their natural host and other animals. Therefore, this work contemplates all viral types belonging to the Delta-PVs, including their history. Among the proteins translated by the PVs, three of them are considered oncogenic proteins: E5, E6 and E7. Determining the structure of a protein is crucial to the elucidation of its function, allowing applications in the areas of protein engineering, genomic annotation and rational design of drugs. The three-dimensional structure of the E6 protein of each viral type belonging to the genus Deltapapillomavirus was determined by molecular modeling by homology. The evolutionary history of these proteins was evaluated based on the generation of phylogenetic trees and their physicochemical properties were analyzed. In addition, due to its high degree of conservation, E6 has been shown to be useful as a molecular marker. Despite being considered rare, papillomas lesions were observed in sheep on a farm in the state of São Paulo. The molecular diagnosis of these lesions was performed. The results showed for the first time in the world that, despite being ovines, the causative agent of papillomatosis was a bovine papillomavirus, BPV2, a Delta-PV. Thus, in addition to discussing Delta-PVs, this thesis demonstrates in practice the ability of this genre to break the species-specific barrier.

Ce memoire de these concerne l'utilisation de peptides synthetiques pour le diagnostic d'infections virales humaines. L'outil peptide synthetique a ete utilise comme sonde antigenique dans le cadre de tests elisa ainsi qu'a l'aide d'un systeme analytique base sur la technologie de biocapteurs, le biacore#t#m, et comme immunogene chez l'animal, dans le contexte du diagnostic immunochimique de l'infection par les virus vih-1 et pvh oncogenes. Nous avons mis au point un test de capture immunochimique utilisant comme sondes antigeniques des peptides representatifs d'epitopes du vih-1, permettant de reveler la presence d'anticorps anti-vih au debut de l'infection. Nous avons etudie l'antigenicite et l'immunogenicite d'un peptide cyclique chimere, representatif d'une sequence consensus de la boucle v3 de la gpl20 du vih-1. Ce peptide constitue une sonde antigenique tres performante capable de reveler la presence d'anticorps diriges contre differentes souches de vih-1. Les anticorps obtenus en reponse a l'injection du peptide chimere cyclique ont un spectre de reconnaissance croisee tres large, et pourraient etre utilises en tant que sondes dans le cadre de tests immunoenzymatiques pour reveler la presence d'antigenes du vih-1. L'utilisation d'un peptide representatif d'un epitope de la proteine l2 du pvh-33 comme immunogene chez le lapin, a permis la production d'anticorps capables de reconnaitre specifiquement une proteine recombinante l2 du pvh-33 en elisa et wb. Nos resultats demontrent l'existence d'une bonne correlation entre les resultats obtenus a l'aide du biacore et dans le cadre de tests elisa et permettent d'envisager l'utilisation du biacore pour l'analyse et la selection d'epitopes susceptibles d'etre utilises par la suite dans le cadre de tests elisa

Analisamos uma coorte de mulheres no sul do Brasil, com objetivo de identificar associações epidemiológicas para persistência e cura da infecção pelo HPV e realizamos uma metanálise para determinar a acurácia da telomerase nas lesões precursoras do câncer cervical. Métodos: O estudo de coorte foi iniciado em fevereiro de 2003. Foram coletados espécimes cervicais para citologia oncótica e para detecção do DNA HPV na entrada do estudo e no seguimento. O desfecho foi dividido em quatro categorias: (1) persistência, (2) conversão (3) cura. A quarta categoria (referência) eram mulheres negativas no início que permaneceram negativas. Foram usados o teste χ2 de Pearson, regressão logística multinomial e Kaplan- Meier para análise estatística. Para a metanálise foram incluídos estudos que comparavam o teste de telomerase (TRAP) e anatomopatológicos, obtidos por biópsias cervicais para diagnóstico de lesões cervicais. Resultados: A Incidência de HPV foi 12,3%. O HPV16 foi o tipo mais encontrado (18,6%), entre as 501 mulheres do estudo.Trinta e quatro mulheres (6,78%) ficaram persistentemente infectadas pelo HPV, estando essa categoria associada à idade da sexarca inferior a 21 anos (OR = 3,14, IC 95%, 1,43-6,87) e a quatro ou mais parceiros durante a vida (OR = 2,48 IC 95%, 1,14-5,41). No período mediano de 19 meses, 80,7 % das mulheres tinham curado o HPV, a cura foi significativamente associado à cor preta (OR= 3,44 IC 95%, 1,55-7,65), co-infecção com C. trachomatis no arrolamento (OR= 3,26, IC 95%, 1,85-5,76) e história de já ter realizado exame de Papanicolaou (OR= 3,48, IC 95%, 1,51- 8,00). Na metanálise dez estudos foram analisados, os quais incluíram 1069 mulheres. Para lesões intraepiteliais de baixo grau (LIEBG) vs. normal ou lesões benignas, encontrou-se uma positividade do teste da telomerase, sendo que o resultado da odds ratio para diagnóstico (DOR) foi de (DOR = 3,2, IC 95%,1,9-5,6). Nas lesões intraepiteliais de alto grau (LIEAG) vs LIEBG, normal ou benigna: (DOR = 5,8, IC 95%, 3.1-10). )]. Encontrou-se uma DORelevada de 8,1 (IC 95%: 3,2-20) nas lesões de câncer cervical vs LIEAG. Da mesma forma, nas lesões de câncer cervical vs. LIEBG, a razão de chance foi elevada, com uma DOR de 40,9 (IC 95%: 18,2-91). Conclusões: A persistência da infecção pelo HPV foi associada com a sexarca precoce e ao número de parceiros sexuais na vida, sugerindo que estratégias de orientação sexual podem modificar as taxas de persistência do HPV. A associação da cura do HPV com história prévia de realização de Papanicolaou salienta a importância de aprimorar os programas de rastreamento de câncer cervical. Futuros estudos da associação de infecções ginecológicas com cura da infecção pelo HPV são necessários. Na metanálise nossos dados suportam a corrente hipótese da atividade da telomerase como um evento precoce na carcinogênese e que poderia estar associado ao início e à progressão de lesões cervicais.
We analysed a cohort of women in Southern Brazil with the aim to identify epidemiological correlates for persistence and clearance of cervical HPV infection. A quantitative systematic review was performed to estimate the accuracy of telomerase assay in cervical lesions. Methods: A cohort study was started on February 2003. Cervical smears were collected to perform Pap cytology and HPV DNA detection at baseline and during the follow up. The outcome was constructed in four categories (1) persistence of HPV DNA; (2) conversion; (3) clearance of HPV. Pearson’s χ2 test, multinomial logistic regression and univariate analysis using the log-rank test were performed. Meta-analysis studies that evaluated the telomerase test (telomerase repeated amplification protocol) for the diagnosis of cervix lesions and compared it to paraffin-embedded sections as the diagnostic standard were included. Results: Incidence of HPV DNA: 12.3%. HPV16 was the most frequent type (18.6%) among 501 women in the study. Thirty-four women were persistently infected with HPV, which was associated with age below 21 years at first intercourse (OR 3.14, 95% CI, 1.43-6.87) and ≥ 4 sexual partners during lifetime (OR 2.48, 95% CI, 1.14-5.41). In a median period of 19 months, 80.7% of women had clearance of HPV, which was associated with black race (OR 3.44, 95% CI, 1.55-7.65), co-infection with C. trachomatis at baseline (OR 3.26, 95% CI, 1.85-5.76) and history of previous Pap smear (OR 3.48, 95% CI, 1.51-8.00). In meta-analysis ten studies were analyzed, which included 1,069 women. The diagnostic odds ratio (DOR) for a positive telomerase test for Lo-SIL vs. normal or benign lesions was 3.2 (95% CI, 1.9-5.6). The DOR for a positive telomerase test for Hi-SIL vs. Lo-SIL, normal or benign lesions was 5.8 (95% CI, 3.1-10). For cervix cancer vs. Hi-SIL, the DOR for a positive telomerase test was 8.1 (95% CI, 3.2-20.3) and for cervix cancer vs. Lo-SIL, normal or benign lesions, it was 40.9 (95% CI, 18.2-91). Conclusions: Persistence of HPV infection wasassociated with early age at first intercourse and number of sexual partners during lifetime, suggesting that strategies for sexual orientation may modify the rates of HPV persistence. The association of HPV clearance with a history of previous Pap smear screening highlights the importance of improving cervical screening programs. Further studies on the association of gynaecological infections with HPV clearance are needed. In meta-analysis our data support the current hypothesis that telomerase may activate an early event in cervical carcinogenesis, that could be associated with the initiation and progression of cervical lesions.

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.

Les carcinomes épidermoïdes des voies aéro-digestives supérieures (VADS) se situent au sixième rang des cancers les plus fréquents dans le monde. Ces tumeurs sont liés à deux facteurs de risque : l’intoxication éthylo-tabagique (80% des cas) et l’infection de l’épithélium des VADS par les papillomavirus humain (HPV) à haut risque oncogène (20% des cas). Ces derniers définissent une sous-population de patients de meilleur pronostic. Une des hypothèses actuellement étudiées, afin d’expliquer la survie améliorée des patients HPV positifs, serait une hypoxie moindre dans ces tumeurs. En effet, les tumeurs des VADS sont fréquemment hypoxiques, et l’hypoxie intratumorale est un facteur de mauvais pronostic. Dans une première partie de cette thèse, nous avons entrepris une caractérisation moléculaire de l’hypoxie intratumorale dans les tumeurs humaines oropharyngées en fonction du statut HPV. Il apparaît que les tumeurs HPV positives présentent un statut hypoxique moindre comparées aux tumeurs HPV négatives. Ces tumeurs se caractérisent également par une abondante vascularisation intratumorale, qui pourrait être à l’origine de ce statut hypoxique moindre. Dans une deuxième partie, nous avons étudié l’adaptation à l’hypoxie de la lignée cellulaire HPV négative SQ20B et la lignée cellulaire HPV positive SCC90. De plus, des modèles de xénogreffes ont été établis à partir de ces mêmes lignées cellulaires et ont été analysés du point de vue de l’hypoxie intratumorale. De façon comparable aux tumeurs HPV positives, les xénogreffes obtenus à partir de la lignée SCC90 montre un statut hypoxique réduit comparés aux xénogreffes SQ20B. Les deux lignées cellulaires s’adaptent également différemment en hypoxie in vitro. La réponse à l’hypoxie dans la lignée SCC90 semble plus dynamique. En effet, la lignée SCC90 tente de s’adapter et de répondre à cet environnement hypoxique en induisant de fort niveau d’expression de gènes comparée à la lignée SQ20B
Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common malignancy worldwide. The major risk factors for HNSCC identified are tobacco use and alcohol consumption (80% of all HNSCC), which seem to have a synergistic effect. A subgroup of HNSCCs (20% of cases), particularly those of the oropharynx, is caused by infection with high-risk types of human papillomavirus (HPV). Human papillomavirus HPV-related oropharyngeal squamous cell carcinoma defines a distinct clinical subgroup of head and neck cancer patients with improved prognosis. Currently, one of the several hypothesis studied to account for their improved survival outcomes could be a distinct hypoxia status compared to their HPV-negative counterpart. Indeed, tumour hypoxia is common in solid tumours including head and neck tumours, and hypoxia is a well-known poor prognosis factor. In first part of this thesis, we have performed a molecular characterisation of tumor hypoxia on cohort of oropharyngeal tumours according to HPV status of the patients. The results support the hypothesis that HPV-related tumours display a lesser hypoxia status compared to HPV-negative oropharyngeal tumours. These HPV-related tumours also characterize by an abundant tumour vascularisation, which could be responsible for a lesser hypoxia status. In a second part, we have studied the ability of the adaptation to hypoxia of the HPV-positive SCC90 cell line and HPV-negative SQ20B cell line. Furthermore, HPV-positive and HPV-negative HNSCC xenograft models have been established and have been analysed about tumor hypoxia. Similar to HPV-related HNSCC, tumours-derived HPV positive cell lines display a reduced hypoxic status compared to tumours-derived HPV negative cell lines. The two cell lines adapt also differently to in vitro hypoxia. In the HPV-positive cell line, the hypoxia response pathways could be more dynamics. Indeed, SCC90 cell lines attempt to adapt and to reply to hypoxic environment inducing highly expression of all of the hypoxia related genes compared to SQ20B cell lines

Les Papillomavirus Humains (HPV) sont responsables de près de 100% des cancers du col utérin. Récemment, ces HPV sont apparus comme étant aussi la cause de certaines tumeurs des voies aérodigestives supérieures, et particulièrement des carcinomes épidermoïdes de l’oropharynx. En France, la proportion des tumeurs oropharyngées HPV-induites est mal connue, notamment parce que le dépistage viral n’est pas recommandé. De plus, il est difficile d’évaluer la proportion de tumeurs HPV positives dans les tumorothèques car les échantillons tumoraux sont fixés dans du formol puis inclus en paraffine (FFIP), ce qui complexifie les techniques de détection. Nous avons, au cours de nos travaux, testé une méthode de détection des HPV à haut risque oncogène indiquée pour le traitement des frottis en phase liquide. Nous l’avons mise à l’épreuve sur des prélèvements FFIP et comparée à la technique de référence qu’est la PCR (Polymerase Chain Reaction) suivie d’une électrophorèse sur gel. Nos résultats indiquent que cette technique est applicable aux prélèvements tissulaires et apparaît même comme étant plus sensible. En France, deux tiers des patients atteints de tumeurs des VADS sont pris en charge à des stades tardifs. Ceci s’explique en partie par l’absence de dépistage organisé de ces cancers. Nous avons donc mené une étude prospective sur des patients atteints d’une tumeur des VADS afin de tester le frottis oral comme technique de dépistage des cancers mais également des infections par les HPV. Nos résultats indiquent que le frottis a une spécificité proche de celle de la biopsie (94,4%) pour le dépistage des cancers des VADS, mais une moindre sensibilité (66,7%). Cette étude nous a permis de mettre en évidence une tumeur HPV-induite dans 12,2% des cas. Parmi eux, nous avons détecté grâce à un frottis buccal (en zone saine) une infection par un HPV à haut risque oncogène dans 53,3% des cas. L’OMS a classé les HPV comme agents carcinogènes depuis 1995, et a établi que les patientes ayant développé un cancer du col utérin avaient un risque 6 fois plus élevé de développer une autre tumeur HPV-induite. Dans ce contexte, nous avons prévu une étude prospective multi-centrique visant à dépister une infection orale par un HPV oncogène chez des patientes porteuses d’une lésion pré-néoplasique ou néoplasique du col utérin. Le taux de co-infection des deux sites anatomiques est inconnu chez les femmes infectées au niveau génital. Dans la mesure où l’infection orale pourrait être à l’origine d’une seconde localisation tumorale, il semble important d’en connaître la proportion afin de proposer par la suite un suivi particulier aux populations « à risque ». Au-delà des traitements des cancers avérés se pose la question de la vaccination préventive, qui existe contre les HPV 16 et 18 dans la prévention des cancers du col utérin. Le type 16 étant retrouvé dans 90% des tumeurs épidermoïdes de l’oropharynx HPV-induites, l’extension des recommandations vaccinales apparaît comme une nouvelle question de santé publique
The Human Papillomavirus (HPV) are involved in almost 100% of cervical cancers. Recently, HPVs have been recognized as the cause of tumors of the upper aerodigestive tract, especially of squamous cell carcinoma of the oropharynx. In France, the proportion of oropharyngeal HPV-related tumors is unknown, partly because viral testing is not in guidelines. Moreover, assess the proportion of HPV-positive tumors in tumor banks is difficult because the tumor samples were fixed in formalin and embedded in paraffin (FFPE), which complicates detection techniques. We tested a high risk HPV detection method, indicated for liquid based pap smear, on FFPE samples. We compared this technique to the gold-standard : PCR (Polymerase Chain Reaction) followed by electrophoresis. Our results indicate that this technique is applicable to FFPE samples and even appears to be more sensitive. The majority of French patients (2/3) with head and neck consult with an advanced stage of disease. This is explained in part by the lack of organized screening of these cancers, contrary to breast, prostate, cervical, or colorectal cancers. But an early treatment is essential to increase the survival rate. We therefore conducted a prospective study on patients with head and neck tumors to test the oral brushing as screening cancer and HPV detection. We found tumor and/or dystrophic cells in 97.8% of patients with biopsy, and in 88.9% of patients by brushing. Compared with biopsy, our results suggested that smear has similar specificity for HPV detection in tumors (94.4%), but lower sensitivity (66.7%). This study has shown an HPV-related tumor in 12.2% of cases. Among them, we detected by brushing (in healthy area) an oral infection by high-risk HPV in 53.3% of cases. WHO has classified HPV as carcinogenic agents since 1995, and determined that patients who developed cervical cancer are six-times more likely to develop another HPV-related tumor. In this context, we have planned a multicenter prospective study to detect oral HPV infection in patients with a pre-neoplastic or neoplastic lesion of the cervix. Co-infection rate of the two anatomical sites is unknown in women infected with genital level. Insofar oral infection could be the cause of a second tumor location, it seems important to know how much women are co-infected to propose thereafter a special monitoring. The preventive vaccination, which exists against HPV 16 and 18 in the prevention of cervical cancer, is a future perspective. Because HPV 16 is found in 90% of HPV-related squamous cell carcinoma of the oropharynx, extending vaccine recommendations emerge as a new public health issue

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Human papillomavirus (HPV) is considered the central etiological agent involved in the genesis of cervical cancer. The HPV viruses are classified according to their biological niche, oncogenic potential and phylogenetic position. According to the criteria established by the International Committee on Taxonomy of Viruses (ICTV), the various groups of human papillomaviruses that infect the female genital tract are classified phylogenetically in the Alphapapillomavirus genus, including species classified among phylogenetic species 1 and species 15. The main high risk HPV are classified in species 9 (HPV 16, 31, 33, 35, 52, 58, 67), and in species 7 (18, 39, 45, 59, 56, 66, 68 and 70). HPV 16 is the most prevalent type irrespective of diagnosis, principally in more severe lesions. Coinfection with multiple-types HPV is a common finding of many molecular studies. Some HPV types might interact or act synergistically to induce progression. Few studies have investigated the interactions of viral genotypes or species in multiple-type HPV infections. Therefore, the objective of this study was to evaluate the effect of single or multiple-types HPV infections considering also the phylogenetic groups on the prevalence and severity of cervical intraepithelial neoplasia (CIN) among women undergoing colposcopy following a abnormal cervical smear. Methodology: In this analysis, 198 women attending at the colposcopic clinic, because of an abnormal cervical smear were included. Colposcopy was carried out in all cases and biopsies were done in 193 of 198 women included. All specimens were tested for 27 HPV genotypes by Roche s polymerase chain reaction reverse line blot assay. Results: The overall prevalence of HPV in women with an abnormal cervical smear was 86% (171/198). Of the total of HPV-positive women, 45% (77/171) were infected with HPV 16 as a single or multiple-type infections. HPV 31 and 35 were, respectively, the second and third most prevalent types. The prevalence of HPV 16 in high grade cervical intraepithelial neoplasia (CIN2/3) was 52% (40/76) and it was detected in 88.8% (8/9) in cases of invasive carcinoma. The prevalence of type 31 and 35 in high grade CIN was respectively 10.5% (8/76) and 6.6% (5/76). Single HPV infection for any type was significantly associated with neoplastic diagnosis. High grade neoplastic diagnosis (≥ CIN2) was significantly associated with HPV 16 in single or multiple infections. Also, there was significantly association between HPV 16 and others types of specie 9 and high grade neoplastic diagnosis, but no association was observed considering the HPV 16 and other of groups of species 7 or others types. Conclusion: These results indicated that the type 16 is the most important predictor of high grade cervical neoplasia. Multiple-type infections are predictors of high grade cervical neoplasia when the type 16 is present.
O Papilomavírus humano (HPV) é considerado o agente etiológico central envolvido na gênese do câncer cervical. O vírus HPV é classificado de acordo com seu nicho biológico, potencial oncogênico e classificação filogenética. De acordo com os critérios estabelecidos pelo Comitê Internacional de Taxonomia dos Vírus (ICTV), os diversos tipos de HPV que infectam o trato genital feminino são classificados filogeneticamente no gênero Alphapapillomavirus. Esta classificação inclui espécies filogenéticas classificadas entre a espécie 1 e a espécie 15, dentre as quais, as de maior interesse em relação ao potencial carcinogênico são a espécie 9 (HPV 16, 31, 33, 35, 52, 58, 67) e a espécie 7 (18, 39, 45, 56, 59, 66, 68, 70). O HPV 16 é tipo o mais predominante, independente do diagnóstico, presente principalmente nas lesões cervicais mais graves. A co-infecção com múltiplos tipos de HPV é um achado comum em muitos estudos moleculares, contudo, as interações dos genótipos virais ou espécies envolvidas nas infecções por múltiplos tipos de HPV têm sido pouco analisadas. Portanto, o objetivo deste estudo foi avaliar o efeito das infecções simples ou por múltiplos tipos de HPV, considerando também os grupos filogenéticos, sobre a prevalência e a gravidade das neoplasias cervicais. Metodologia: Este estudo de corte transversal incluiu 198 mulheres encaminhadas ao Ambulatório de Colposcopia da Santa Casa de Misericórdia de Goiânia por exame citopatológico anormal. Todas as mulheres foram esclarecidas quanto aos objetivos de estudo e assinaram o termo de consentimento livre e esclarecido. A colposcopia foi realizada em todos os casos e a biópsia em 193 das 198 mulheres incluídas. As amostras foram testadas para 27 genótipos de HPV, por reação em cadeia da polimerase (PCR); em seguida foi realizada a hibridização reversa em pontos da Roche Diagnósticos. Resultados: A prevalência de HPV em mulheres encaminhadas por exame citopatológico anormal foi de 86% (171/198). Do total de mulheres HPV-positivas, 45% (77/171) estavam infectadas por HPV 16 em infecções simples e múltiplas. Os tipos de HPV 31 e 35 foram respectivamente, o segundo e o terceiro mais prevalentes. A prevalência do HPV 16 foi de 52% (40/76) nas neoplasias intra-epiteliais cervicais de alto grau (NIC 2/3) e de 88,8% (8/9) nos casos de carcinomas invasivos. As prevalências dos tipos 31 e 35 em neoplasias intra-epiteliais cervicais de alto grau (NIC 2/3) foram de 10,5% (8/76) e 6,6% (5/76), respectivamente. A infecção simples por qualquer tipo de HPV foi significativamente associada com diagnósticos neoplásicos de alto grau (≥ NIC 2). Os diagnósticos neoplásicos de alto grau (≥ NIC 2) foram significativamente associados com o HPV 16 em infecções simples ou múltiplas, mesmo depois de ajustado pela positividade para DNA de HPV. Houve significativa associação entre o HPV 16 e outros tipos da espécie 9 e os diagnósticos neoplásicos de alto grau (≥ NIC 2), mas não foi observada associação, considerando o HPV 16 e outros tipos da espécies 7 ou outros tipos de HPV. Conclusão: Estes resultados indicam que o HPV 16 parece ser o mais importante preditor de diagnósticos neoplásicos de alto grau. As infecções múltiplas são preditoras das neoplasias cervicais de alto grau quando o HPV 16 está presente.

OBJETIVO: O objetivo deste estudo foi analisar a concordância entre o esfregaço anal e a biópsia guiada por anuscopia de alta resolução no diagnóstico da displasia anal em pacientes infectados pelo HIV. MÉTODO: Conduzimos uma análise transversal de pacientes infectados pelo HIV submetidos a rastreamento de displasia anal rotineiro. A concordância entre mensurações foi estimada por índice de kappa ponderado através de sistema de avaliação citológica e histológica de três categorias (normal, displasia de baixo grau, e displasia de alto grau). Estimativas de sensibilidade, especificidade e valores preditivos foram calculados através de sistema de avaliação citológica e histológica de duas categorias (ausência de displasia e displasia de qualquer grau). Estimativas foram calculadas também para a detecção de displasia de alto grau. RESULTADOS: No decorrer de um ano, 222 pacientes foram submetidos a 330 esfregaços anais seguidos de biópsias guiadas por anuscopia de alta resolução. Trezentos e onze (311) esfregaços com biópsias concomitantes foram satisfatórios. Considerando-se a histologia como padrão, a freqüência de displasia anal foi de 46%. O índice kappa ponderado para concordância entre o esfregaço anal e a biópsia foi de 0,20. Para detecção de displasia anal de qualquer grau, o esfregaço anal demonstrou sensibilidade de 61%, especificidade de 60%, valor preditivo positivo de 56% e valor preditivo negativo de 64%. Para displasia de alto grau, o esfregaço anal demonstrou sensibilidade de 16% e especificidade de 97%. CONCLUSÃO: Os resultados obtidos no presente estudo, em que comparamos os achados da citologia dos esfregaços com os achados histológicos das biópsias dirigidas pela anuscopia de alta resolução em pacientes infectados pelo HIV permitiram concluir que houve baixa concordância entre eles
Purpose: To analyze the agreement between anal Pap smear and high resolution anoscopy guided biopsy to diagnose anal dysplasia in HIV-infected patients. Methods: Cross sectional analysis of HIV-infected patients receiving anal dysplasia screening as part of routine care. Agreement between measures was estimated by weighted kappa-statistics, using 3-tiered cytologic and histologic grading system (normal, low grade dysplasia, and high grade dysplasia). Estimates of sensitivity, specificity, and predictive values were calculated using a 2-tiered cytologic and histologic grading system (without dysplasia, and with dysplasia of any grade). Estimates were also calculated for the detection of high grade dysplasia. Results: Two hundred and twenty-two patients underwent 330 anal Pap smears followed by high resolution anoscopy guided biopsies in one year period. There were 311 satisfactory Pap smears with concurrent biopsy. Considering histology the standard, the frequency of anal dysplasia was 46 percent (95 percent confidence interval: 40-51 percent). Kappa-agreement between anal Pap smear and biopsy was 0.20 (95 percent confidence interval: 0.10 0.29). Anal Pap smear showed sensitivity of 61 percent, specificity of 60 percent, positive predictive value of 56 percent, and negative predictive value of 64 percent for detection of anal dysplasia of any grade. For high grade dysplasia, anal Pap smear showed sensitivity of 16 percent, and specificity of 97 percent. Conclusion: The present study showed a low concordance between anal Pap smears and high resolution anoscopy-guided biopsy

Indiana University-Purdue University Indianapolis (IUPUI)
In this study we compared the distribution of HPV types in cervical cancer specimens from women living in either Indiana or Botswana. Paraffin-embedded blocks of formalin-fixed cervical cancer specimens were identified from women living in Indiana (n=51) or Botswana (n=171)

The purpose of this study was to obtain information regarding how people are affected by a diagnosis of genital warts/HPV. The psychosocial affects studied included emotional health, social and sexual relationships, feeling's about one's sexuality, safer sex practices and sexual behavior. 147 students (96 females and 51 males) completed the Symptom Check List 90-R (SCL-90-R) and a sexual history and demographic questionnaire. Participants were divided into three groups: students diagnosed with genital warts/HPV (HPV group), students diagnosed with a curable STD (curable group), and students with no diagnosed STD (No STD group). Thirteen students who were diagnosed with genital warts/HPV agreed to be interviewed, and each expressed recurring concerns about fear of transmission, rejection, frustration with the medical establishment and telling future partners. The qualitative findings from this study found that subjects who were older (more than 20-years-old), had more than 2 to 4 sexual partners in their lifetimes, had their first sexual intercourse at an early age (13 to 15-years-old) and only practiced safer sex "sometimes" were more likely to have been diagnosed with either genital warts/HPV or a curable STD than the No STD group. Subjects with genital warts/HPV were more likely to change their sexual behavior after a diagnosis by practicing safer sex "consistently". Some subjects reported that they had stopped being sexually active as a result of their diagnosis with genital warts/HPV. Analysis of variance was used to compare the sample means of the SCL-90-R sub-scales for the three sample groups. The most significant findings were demonstrated when subjects were evaluated by gender. The sub-scales of the curable and genital warts/HPV groups for women were both elevated. This may indicate that "curability" is not a factor affecting the psychological symptomatology of these groups. Based on these findings, the diagnosis of genital warts/HPV and a curable STD requires certain psychological assistance and patient education. People at high risk (those with multiple partners, early age at first intercourse, a history of STDs and alcohol abuse) need to be informed about genital warts/HPV, its prevalence, its virulent nature, and its medical complications. The psychosocial impact of any STD, curable or incurable, is of great importance in treating the person "systemically" to fully address all aspects of the disease.

Tese de mestrado, Biologia (Biologia Humana e Ambiente), 2009, Universidade de Lisboa, Faculdade de Ciências
Cervical cancer is the second most common cancer in women worldwide. Persistent infection by high-risk human papillomavirus (HR-HPV) is considered to play a central role in cervical carcinogenesis. The aim of this work was the implementation and evaluation of new methodologies for early diagnosis of HR-HPV infection. Cervical samples, with or without cytological abnormalities, were tested for HPV DNA by different methods. Four studies were developed, namely, the prevalence of HPV infection among a group of Portuguese women, the evaluation of a new diagnostic system for HR-HPV detection, the implementation and evaluation of real-time PCR methodologies, and a case-study in women infected with HPV 16 and 18. Statistical analyses were done using SPSS version 16.0. Prevalence data showed that genital HPV infection is common among Portuguese women, with an overall positivity of 43.5%. This infection is frequent especially in younger women (25-29 years). The 4 most prevalent HR-HPV were HPV 16 (25.3%), HPV 31 (11.3%), HPV 51 (10.3%), and HPV 66 (9.1%). Evaluation of the new Abbott RealTime HR HPV suggests that this system is efficient, sensitive, specific, robust, reproducible, and suitable for HPV screening, particularly for prediction of premalignant lesions (CIN2+). The real-time PCR methodologies analysed for assessing the viral load, the DNA integration, and the RNAm transcript expression were highly reproducible and accurate for HPV results obtained in the case-study. Results seem to indicate that these methodologies are important for patient management and for cervical cancer prevention in women infected with HPV 16 and 18. In conclusion, the results obtained in this study can help to understand HPV epidemiology prevalence in Portugal and to identify molecular markers that can be used as predictors of premalignant and malignant lesion development, contributing for a decrease in morbidity and mortality rates of women infected by HR-HPV.
Resumo alargado em português disponível no documento

Tese de doutoramento, Biologia (Microbiologia), Universidade de Lisboa, Faculdade de Ciências, 2014
A relação entre a infecção pelo Vírus do Papiloma Humano (HPV) e o desenvolvimento do cancro do colo do útero foi estabelecida no final do século XX. Em Portugal, preconiza-se o rastreio do cancro do colo do útero na mulher adulta por constituir uma das neoplasias mais frequentes. Foram recentemente implementados programas de rastreio numa base regional; contudo, o respectivo impacto na redução do número de casos de cancro do colo do útero levará algum tempo. Por outro lado, o recurso a potenciais indicadores de prognóstico, permitirá auxiliar a identificação precoce de mulheres em risco de desenvolver cancro do colo do útero, contribuindo para o estabelecimento de estratégias de prevenção mais efectivas e eficazes. A vacina profiláctica contra a infecção por HPV foi recentemente disponibilizada para administração à população feminina jovem, tendo sido incluída no plano nacional de vacinação. Neste contexto, torna-se importante conhecer os genótipos circulantes na população Portuguesa, de forma a prever o impacto da vacinação (apenas inclui dois ou quatro genótipos de HPV) na infecção por HPV e nas lesões a esta associada. O presente trabalho de doutoramento teve os seguintes objectivos: 1) determinação da proporção da infecção pelos diferentes genótipos de HPV numa amostra da população feminina Portuguesa (obtida por rastreio oportunista), e respectiva associação com o diagnóstico citológico; 2) avaliação dos testes de detecção e genotipagem do HPV relativamente à clínica associada; 3) avaliação de diferentes indicadores de prognóstico, de acordo com o diagnóstico clínico; e, 4) desenvolvimento de um modelo matemático aplicável ao estudo da infecção genital por HPV. A determinação da frequência dos diferentes genótipos de HPV foi efectuada numa população com sinais clínicos sugestivos de infecção por HPV, tendo sido possível estabelecer associações significativas entre as alterações clínicas e a infecção persistente por genótipos de alto risco. Adicionalmente, foi possível identificar os genótipos mais frequentes, nomeadamente os HPV 16, 18, 31, 51, 53 e 66, e verificar que a infecção por HPV é mais frequente nas mulheres até aos 29 anos, sobretudo devido à multiplicidade de contactos e de parceiros sexuais (maioritariamente infecções transitórias). Já nas mulheres com idade superior a 30 anos, a infecção por HPV foi menos frequente mas apresentou maior risco de persistência e, dada a elevada proporção de genótipos de alto risco (mesmo em mulheres com citologia normal – assintomáticas), constituiu, por si só, um importante factor de risco para o desenvolvimento de cancro do colo do útero.Foi efectuada uma caracterização epidemiológica da infecção por HPV, assim como dos programas de rastreio do cancro do colo do útero em Portugal, tendo sido salientadas as especificidades do rastreio em cada região. O desempenho de diferentes testes comerciais de detecção e genotipagem do HPV foi avaliado de acordo com a clínica (sensibilidade, especificidade, valor preditivo positivo e negativo). A compreensão dos princípios e fundamentos de cada metodologia facilita uma correcta apreciação do respectivo desempenho laboratorial em fases distintas da infecção por HPV, assim como da sua aplicabilidade a programas de rastreio. Os testes de detecção do DNA viral, com elevada sensibilidade, revelaram constituir uma boa alternativa à recorrente citologia, enquanto teste de rastreio primário. De facto, a abordagem conjunta de teste HPV com triagem citológica, especialmente nas mulheres com mais de 30 anos, permitirá alargar o tempo de intervalo entre os exames de rastreio, devido ao elevado valor preditivo negativo do teste HPV. Mais, os testes de detecção do DNA viral, que incluem a genotipagem dos HPV 16 e 18 (oncogénicos e frequentemente associados ao desenvolvimento de lesões precursoras de cancro do colo do útero), possibilitam estratificar mais eficazmente as mulheres em maior risco de persistência da infecção viral e respectivas consequências clínicas. Relativamente à detecção de RNAm do HPV, foi avaliada uma metodologia comercial de detecção e genotipagem de transcritos de RNAm para alguns genótipos de HPV de alto risco (NASBA). Esta metodologia permite a identificação precoce de infecções clinicamente relevantes por compreenderem um risco acrescido de desenvolvimento de lesões precursoras do cancro do colo do útero. Verificou-se que a utilização desta metodologia como teste de segunda linha pode aumentar a especificidade do teste de detecção de DNA do HPV nas mulheres infectadas, reduzindo a indicação clínica para colposcopia (que inflige elevada morbilidade e ansiedade na mulher) como método de rastreio das lesões associadas à infecção por HPV, e evitando o tratamento excessivo, ao possibilitar excluir lesões com maior probabilidade de regressão. A utilização de diferentes indicadores de prognóstico da infecção por HPV facilitará a identificação precoce de mulheres em risco de desenvolvimento de lesões precursoras de cancro do colo do útero. Neste contexto, foram avaliadas a carga viral e o estado físico do DNA viral dos HPVs 16 e 18, tendo sido possível determinar uma associação entre o aumento da carga viral do HPV 16 e a gravidade da lesão do colo do útero, pelo que foi considerado como um importante marcador de prognóstico em mulheres infectadas por um dos mais frequentes genótipos de alto risco na população Portuguesa. Para o HPV 18, frequentemente associado ao desenvolvimento de adenocarcinomas (tipo de cancro cervical de difícil identificação citológica), verificou-se que a carga viral é potencialmente preditiva da persistência da infecção.A determinação do estado físico do DNA viral, como metodologia alternativa a procedimentos médicos invasivos (colposcopia e biopsia), foi avaliada em associação com o diagnóstico citopatológico. Durante o processo de carcinogénese viral ocorre integração do genoma viral no genoma da célula hospedeira, por disrupção do gene viral E2 e subsequente sobre -expressão dos oncogenes virais E6 e E7. Foi possível identificar, especialmente para o HPV 18, uma associação entre a presença de formas lineares (maior risco), as lesões precursoras e os casos de adenocarcinoma, o que sugere a utilidade clínica deste indicador de prognóstico para as mulheres infectadas por HPV 18. No caso do HPV 16, a associação entre a determinação do estado físico do DNA viral e o diagnóstico citopatológico não foi tão evidente, pelo que outros mecanismos virais poderão estar associados à transformação maligna que antecede o desenvolvimento de cancro do colo do útero. Por último, o desenvolvimento de um modelo matemático aplicado à infecção genital por HPV incluiu transições entre os diferentes estadios clínicos que correspondem ao processo de carcinogénese viral. Os cenários previstos foram extrapolados a partir da população de estudo (de rastreio oportunista) e posteriormente comparados com uma população Portuguesa de referência (de rastreio organizado), por forma a estimar a evolução e flutuações relacionadas com a infecção por genótipos de HPV de alto risco e respectivas lesões associadas. A utilização eficaz da vacina a nível mundial poderá levar a uma diminuição de casos de cancro do colo do útero na ordem dos 70% (valor estimado de cancros associados à infecção por HPV 16 e 18), decorrente da prevenção vacinal para os referidos HPVs. No entanto, a existência de uma proporção considerável de outros genótipos de alto risco não incluídos nas vacinas disponíveis poderá alterar esta estimativa, pelo que a monitorização constante dos genótipos circulantes de HPV será importante. De facto, estima-se que sejam necessárias algumas décadas até eliminar os casos de infecção associados aos HPV 16 e 18, já que a grande maioria das mulheres já foi exposta à infecção por HPV.
Cervical cancer development has been aetiologically linked to human papillomavirus (HPV) infection. In Portugal, routine screening for cervical cancer has been regionally implemented and was recommended for adult women because it constitutes one of the most frequent malignancies for women aged 15 to 44 years. HPV prophylactic vaccination (which only includes two or four oncogenic genotypes) was made available in the last decade for young girls, but the knowledge of HPV circulating genotypes is crucial for predicting its clinical impact. This PhD thesis comprised the following objectives: 1) assessment of the proportion of HPV genotypes among Portuguese women (opportunistic screening) according to cytological diagnosis; 2) clinical evaluation of several HPV tests; 3) evaluation of some prognostic markers according to the clinical diagnosis; 4) development of a mathematical model applied to genital HPV infection. Portuguese HPV epidemiology and screening programs were characterized, highlighting the specificities of screening in each region. The most frequent genotypes were HPV 16, 18, 31, 51, 53 and 66, and HPV infection was more common in women aged less than 29 years. In women over 30 years, HPV infection was less frequent but tend to persist and to involve high-risk genotypes. HPV DNA tests demonstrated high sensitivity, constituting an alternative to cytology in primary screening, while combined with cytology (especially for women over 30 years) would extend the re-screening testing interval, considering the high negative predictive value of HPV testing. Moreover, HPV DNA tests with concurrent identification of HPV 16 and 18 (most associated genotypes to cervical cancer development) will provide a better risk stratification of women for precancerous cervical lesion development. The detection of HPV mRNA is highly specific in identifying clinical cervical disease, so that its recommendation to reflex testing of HPV DNA-positive women has shown to reduce colposcopy referral, avoiding over-treatment by excluding cervical lesions that would most likely regress (with associated morbidity and anxiety to HPV-infected women). The viral load and physical status of high-risk HPV 16 and 18 were evaluated as prognostic markers in women at risk of developing cervical precancerous lesions. An association between increased HPV 16 viral load and severity of cervical lesion was observed suggesting its prognostic value, whereas for HPV 18, viral load was only predictive of HPV persistency. The presence of linear forms (higher risk) in HPV 18-associated precancerous lesions and adenocarcinomas evidenced the potential clinical utility of viral DNA physical status as a prognostic marker for women infected with HPV 18. The association was not evident for HPV 16, suggesting that other viral mechanisms should be responsible for malignant host cell transformation.Finally, the mathematical model applied to genital HPV infection included transition probabilities between disease states corresponding to different steps of cervical carcinogenesis, and provided scenarios for a Study Population (opportunistic screening), for a Reference Population (routine screening) and for a Hypothetical Population, which were further compared to predict HPV vaccination impact. Estimates of trends and fluctuations associated with high-risk HPV genotype infections and its associated cervical lesions were performed. HPV vaccines may lead to a global decrease of cervical cancer cases of about 70%, considering that most cervical cancers are associated with HPV 16 and 18 infections. However, the existence of a considerable proportion of other highrisk genotypes not included in the currently available HPV vaccines may change this estimate. Thus, the constant monitoring of circulating HPV genotypes remains of particular importance. Furthermore, the vast majority of women have been exposed to HPV infection, alerting to the importance of a continuous follow-up and establishment of public health measures through routine screening, while improving women’s welfare.
Fundação para a Ciência e a Tecnologia (FCT, SFRH/BD/47044/2008)

Cervical cancer is a major problem in the developing world and low- resource settings where standard screening techniques are not accessible. Cervical cancer is one of the few cancers that can be successfully treated when detected early. Therefore, there exists a high clinical need to screen for cervical cancer early. The etiological agent of cervical cancer is the human papillomavirus (HPV), with 70% of cases related to HPV genotypes 16 and 18. I sought to increase access to screening by developing a fully integrated and multiplexed molecular diagnostic assay to extract, amplify, detect, and distinguish HPV16 and HPV18 DNA on a low-cost paperfluidic platform for point- of-care (POC) applications. Isothermal (one temperature) loop-mediated amplification (LAMP) was used to amplify HPV DNA instead of the traditional polymerase chain reaction (PCR) that requires multiple temperatures. The amplified HPV16 and HPV18 DNA were differentially detected on a simple lateral flow strip – similar to that used in a pregnancy test – generating a visible colorimetric readout for each specific genotype. LAMP amplification is difficult to characterize and current methods were insufficient in providing specificity at the level needed for a multiplexed assay. Therefore, a novel characterization strategy was developed based on fluorescence to distinguish positive LAMP amplification products. This workflow used differential fluorescent tags to identify whether HPV16 DNA or HPV18 DNA was present and simplified complex nonspecific LAMP smears to a specific band pattern. After singleplex HPV16 and HPV18 LAMP assays were optimized with the new workflow, the two singleplex assays were successfully combined into one multiplex reaction with 12 primers, a nontrivial feat. Each assay step – DNA extraction, amplification, and detection – was optimized and integrated into a single chip that can control the timing of each step. Several chip configurations were tested to determine the optimal chip form factor, and a small subset of clinical samples were tested to demonstrate feasibility in low-resource settings. With this diagnostic platform, asymptomatic patients positive for HPV16 DNA and HPV18 DNA can be screened more closely, allocating precious resources to those most at risk, a beneficial use in both low-resource settings and the USA.

Indiana University Purdue University Indianapolis
Human papillomaviruses (HPV) are the etiologic agents of most cervical dysplasia and all cervical carcinoma. Integration of high risk HPV into the human genome is thought to be a critical event in the progression from cervical dysplasia to invasive cervical carcinoma. The ability to use molecular assays in the detection and evaluation of HPV integration is essential in informing clinical models for early intervention and therapies. We therefore sought to determine the feasibility of real time-PCR (RT-PCR) as a molecular tool in detecting the physical state, episomal versus integration of HPV 16 and 18 DNA in cervical cancers. Tyramide amplified fluorescent DNA in situ hybridization (FISH) was used to look for evidence of HPV 16/18 integration using formalin-fixed, paraffin-embedded sections of cervical carcinomas. RT-PCR used the ratio of the E2 and E6 genes as a surrogate for determining the physical state of HPV 16 and 18 in 35 infected tissues. Results of RT-PCR showed that 16 cervical specimens (45.7%) contained episomal HPV, 17 cervical samples (48.6%) harbored the integrated form of HPV DNA, and 2 samples (5.7 %) contained both integrated and episomal forms of HPV. Results of the two assays were compared in 25 cervical carcinomas. For 13 of the 25 cervical samples there was an agreement in determining the physical state of HPV. RT-PCR, using the E2/E6 ratio as an assay for HPV integration appears to be promising and may prove to be an essential clinical method in the future.

abstract: Recent studies have shown that human papillomavirus (HPV) plays a role in development of cancers, one of which is head and neck cancer. There is strong and consistent molecular evidence demonstrating that human papillomavirus (HPV) is an etiological cause of these oropharyngeal cancers. Despite the introduction of HPV vaccines, there is still an increase in human papillomavirus associated OPC (HPVOPC) and it is expected that the incidence of head and neck cancer, specifically oropharyngeal cancer (OPC) will increase. The aim of this study is to utilize human papillomavirus (HPV) seropositivity for rapid detection of HPV early specific antigen-antibodies using a lateral flow assay. Human papillomavirus (HPV) 16 proteins of interest, E7, E6 and CE2 were expressed and purified in E. coli for detection of specific antibodies using lateral flow assay because viral and host factors impact the serologic responses to HPV early antigens in HPV-positive oropharyngeal cancer. 17 samples and 5 controls with already known antibody reactivity from ELISA analysis were selected for HPV serologic responses. The lateral flow strip was evaluated for its color band intensity using Image J software. Peak area was used to quantify the color intensity of the lateral flow strip. Out of the 17 samples, 11 (64.7%) showed high antibody levels to E7, 12 (70.6%) showed high Ab levels to E6 and 6 (35.3%) showed high Ab levels to CE2. Correlation coefficient between antibody detection by sight and ELISA for E7, CE2 and E6 were 0.6614, 0.4845 and 0.2372 respectively and correlation coefficient between lateral flow assay and ELISA for E7, CE2 and E6 were 0.3480, 0.1716 and 0.1644 respectively. This further proves patients or samples with HPV 16 oropharyngeal cancer have detectable antibodies to early E7, E6 and E2 proteins, which are potential biomarkers for HPV-associated oropharyngeal cancer.
Dissertation/Thesis
Masters Thesis Molecular and Cellular Biology 2019

abstract: Over the past several decades, there has been a growing interest in the use of fluorescent probes in low-cost diagnostic devices for resource-limited environments. This dissertation details the design, development, and deployment of an inexpensive, multiplexed, and quantitative, fluorescence-based lateral flow immunoassay platform, in light of the specific constraints associated with resource-limited settings. This effort grew out of the need to develop a highly sensitive, field-deployable platform to be used as a primary screening and early detection tool for serologic biomarkers for the high-risk human papillomavirus (hrHPV) infection. A hrHPV infection is a precursor for developing high-grade cervical intraepithelial neoplasia (CIN 2/3+). Early detection requires high sensitivity and a low limit-of-detection (LOD). To this end, the developed platform (DxArray) takes advantage of the specificity of immunoassays and the selectivity of fluorescence for early disease detection. The long term goal is to improve the quality of life for several hundred million women globally, at risk of being infected with hrHPV. The developed platform uses fluorescent labels over the gold-standard colorimetric labels in a compact, high-sensitivity lateral flow assay configuration. It is also compatible with POC settings as it substitutes expensive and bulky light sources for LEDs, low-light CMOS cameras, and photomultiplier tubes for photodiodes, in a transillumination architecture, and eliminates the need for expensive focusing/transfer optics. The platform uses high-quality interference filters at less than $1 each, enabling a rugged and robust design suitable for field use. The limit of detection (LOD) of the developed platform is within an order of magnitude of centralized laboratory diagnostic instruments. It enhances the LOD of absorbance or reflectometric and visual readout lateral flow assays by 2 - 3 orders of magnitude. This system could be applied toward any chemical or bioanalytical procedure that requires a high performance at low-cost. The knowledge and techniques developed in this effort is relevant to the community of researchers and industry developers looking to deploy inexpensive, quantitative, and highly sensitive diagnostic devices to resource-limited settings.
Dissertation/Thesis
Doctoral Dissertation Electrical Engineering 2018