Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Hepatitis B virus (HBV) immunisation protocols in much of Africa are based on data from the pre-human immunodeficiency virus (pre-HIV) era that indicated that HBV transmission occurs predominantly horizontally between siblings and play-mates rather than vertically from mother to child. The immunosuppression associated with HIV infection however may release HBV from immune control resulting in higher HBV viral loads, which may increase the risk of perinatal mother to child transmission of HBV. The aim of this study was to determine the prevalence and characteristics of chronic HBV infection in HIV-infected pregnant women compared to HIV-uninfected pregnant women in the Western Cape province of South Africa. Ethical approval was obtained to conduct a retrospective, matched case-control, unlinked anonymous study using residual plasma samples from the 9355 pregnant women included in the Western Cape's 2008 National HIV and Syphilis Antenatal Survey. Samples were tested for HBsAg on the AxSYM (Abbott, Chicago, IL) and confirmed by neutralization. Confirmed HBsAg-positive samples were tested for HBeAg, anti-HBe and anti-HD (Diasorin, Saluggia, Italy) and had HBV viral load and genotyping done. In addition, HBsAg-negative samples were tested for anti-HBc. Samples from 1549 HIV-infected pregnant women were included and matched to the same number of samples from age- and race-matched HIV-uninfected women. Median age of 26 years, parity and education were similar in the two groups. The prevalence of HBsAg was 3.4% for the HIV-infected group and 2.9% for the HIV-uninfected group. HBV DNA loads of greater than 104 IU/ml were detected in 32.1% of HBsAg-positive, HIV/HBV co-infected women, and in 14.3% HBsAg positive, HBV mono-infected women. Among the HIV-infected group 18.9% of HBsAg-positive were HBeAg positive, with a median viral load of 7.93 log10 IU/ml; whilst 15.5% HIV-uninfected women were positive for HBeAg with a median viral load of 6.07 log10 IU/ml. Genotype A was seen in 92.6% of the isolates while 7.4% of the isolates were genotype D. Serum total anti-HBc antibodies that are a marker of past infection were detected in 42.2% of HIV-infected and in 24.1% of HIV-uninfected women that were negative for HBsAg. No positive sample for anti-HD was seen among all HBsAg-positive samples. This data indicates that there is increased exposure to HBV in HIV-infected pregnant women than in HIV-uninfected women and that a greater proportion of HIV-infected pregnant women compared to HBV mono-infected pregnant women may be at increased risk of transmitting HBV to their infants. Further studies are needed to determine the rate of vertical transmission of HBV in the HIV era. / AFRIKAANSE OPSOMMING: Hepatitis B virus (HBV) immunisasie protokolle vir meeste dele van Afrika is gebaseer op data versamel in die era voor MIV. Die data dui aan dat HBV oordrag hoofsaaklik deur horisontale transmissie tussen broers, susters en speelmaats eerder as vertikale transmissie van moeder na kind plaasvind. Die onderdrukking van die immuunstelsel as gevolg van MIV infeksie kan egter lei tot 'n verhoogde risiko van perinatale HBV oordrag van moeder na kind. Die doel van hierdie studie was om die voorkoms en karakter van chroniese HBV infeksie in MIV-positiewe swanger vroue te vergelyk met die van MIV-negatiewe swanger vroue. Etiese goedkeuring is verkry om 'n retrospektiewe, deursnee-, ongekoppelde anonieme studie uit te voer wat gebruik maak van oorblywende plasma monsters van 9355 swanger vroue wat ingesluit is in die Wes-Kaap 2008 Nasionale MIV en Sifilis Voorgeboortelike Opname. Die monsters was getoets vir HBsAg antiliggame (AxSYM, Abbott, Chicago, IL) en bevestig deur neutralisasie toetse. Positiewe monsters was getoets vir HBeAg en anti-HBe (Diasorin, Saluggia, Italië). HBV viruslading en genotipering was ook op HBsAg positiewe monsters gedoen. Die HBsAg negatiewe monsters was getoets vir die teenwoordigheid van anti-HBc. Monsters van 1549 MIV-positiewe swanger vroue was ingesluit in die studie. Dieselfde aantal monsters van MIV-negatiewe vroue, met ooreenstemende ouderdom en etnisiteit, was ingesluit as kontroles. Die gemiddelde ouderdom van albei groepe was 26 jaar. Pariteit en opvoeding was dieselfde in albei groepe. Die voorkomssyfer van HbsAg was 3.4% in die MIV-positiewe groep en 2.8% in die MIV-negatiewe groep. HBV DNS ladings van meer as 104 IU/ml was waargeneem in 32.1% van die MIV-mede-geinfekteerde vroue en in 14.3% van die MIV-negatiewe groep. In die MIV-positiewe groep was 18.9% vroue HBeAg positief, met 'n gemiddelde virale lading van 7.93 log10 IU/ml, terwyl 15.5% MIV-negatiewe vroue positief was vir HBeAg met 'n gemiddelde virale lading van 6.07 log10 IU/ml. In ons studie was 92.6% van die monsters genotipe A en 7.4% genotipe D. Toatale anti-HBc antiliggame, 'n merker van vorige infeksie, was gevind in 42.2% van MIV-mede-geïnfekteerde vroue en 24.1% van MIV-negatiewe vroue wat negatief was vir HBsAg antiliggame. Data van ons studie dui op 'n verhoogde risiko vir vertikale HBV transmissie van MIV-positiewe moeders na hul babas. Verdere studies word benodig om vas te stel of vertikale transmissie van HBV van MIV-positiewe vroue na hul babas plaasvind. / Wellcome Trust / Poliomyelitis Research Foundation

Obwohl der Wildtyp des Hepatitis-B-Virus (HBV) nicht zytopathogen und die Pathogenese der Hepatitis B generell immunvermittelt ist, können in immunsupprimierten Nierentransplantatempfängern mit chronischer Hepatitis B schwere Leberschäden bis hin zu Leberzirrhose und Leberversagen entstehen. Die Entwicklung von Leberzirrhose in den Nierentransplantierten ist assoziiert mit der Akkumulation und Persistenz von komplexen HBV-Varianten mit Mutationen im Core-Promotor / X-Gen, Deletionen im Core (C)-Gen und teilweise zusätzlichen Deletionen im präS-Bereich. Dies lässt eine Rolle der Varianten in der speziellen Pathogenese bei Immunsupprimierten vermuten. In der vorliegenden Arbeit wurden funktionelle Analysen der komplexen Varianten im Vergleich zu Referenz-Wildtypgenomen und Wildtyp-ähnlichen Genomen der Patienten aus der frühen Infektionsphase durchgeführt, um Hinweise auf den potentiellen Beitrag der Varianten zur Pathogenese zu erlangen. Die Analysen erfolgten durch transiente Transfektion der humanen Hepatomazelllinie HuH7 mit repräsentativen HBV-Gesamtgenomen, die aus 2 Patienten während des Krankheitsverlaufs von einer asymptomatischen Infektion hin zur Leberzirrhose isoliert und kloniert worden waren. Trotz einiger Unterschiede im Detail wiesen die komplexen Varianten einen gemeinsamen, drastisch vom Wildtyp abweichenden Phänotyp auf. Dieser war gekennzeichnet durch eine veränderte Transkription mit reduzierten präC- und Oberflächen-mRNAs und verstärkter Expression der prägenomischen RNA, eine starke Reduktion des häufigsten Spleißprodukts der prägenomischen RNA, SP1, eine extrem reduzierte oder fehlende Expression und/oder Sekretion aller Oberflächenproteine und des HBeAg, ein verändertes intrazelluläres Verteilungsmuster des schwach exprimierten Core-Proteins und teilweise der Oberflächenproteine sowie eine erhöhte Replikation und Anreicherung gegenüber Wildtyp-HBV aufgrund einer verstärkten reversen Transkription der prägenomischen RNA. Dieser Phänotyp basierte zum Teil auf den Mutationen in Core-Promotor und C-Gen, wurde jedoch deutlich durch zusätzliche Mutationen in den übrigen Genomabschnitten beeinflusst. Die vielfältigen Veränderungen der Varianten unterstützen ihren vermuteten Beitrag zur Pathogenese. / Although wild-type hepatitis B virus is not cytopathogenic and the pathogenesis of hepatitis B is generally immune mediated, also immuno-suppressed patients, such as renal transplant recipients, with chronic hepatitis B may develop liver cirrhosis and end-stage liver disease. In renal transplant recipients, the development of liver cirrhosis is associated with the accumulation and persistence of complex HBV variants with mutations in core promoter / X gene, deletions in core (C) gene and sometimes additional deletions in the preS region. This suggests a role of these variants in the special pathogenesis in immuno-suppressed patients. In the present work, the complex variants were functionally analyzed in comparison to reference wild-type genomes and wild-type-like HBV genomes from the early asymptomatic phase of infection. For the analyses, representative cloned full-length HBV genomes isolated from 2 patients before and during liver cirrhosis were transiently transfected into the human hepatoma cell line HuH7. In spite of some variations, the complex variants showed a common phenotype, which was drastically altered compared to wild-type. It was characterized by reduced preC and surface mRNAs and increased expression of pregenomic RNA, by a strong reduction of the major spliced pregenomic RNA, SP1, by a partial or complete defect in expression and/or secretion of surface proteins and HBeAg, by an aberrant intracellular localization of the weakly expressed core protein and in some cases of the surface proteins, and by an enhanced replication and enrichment over wild-type HBV due to an enhanced reverse transcription of variant pregenomic RNA. The phenotypic alterations were often based on the mutations in core promoter and C gene but were considerably influenced by the additional mutations in other genomic regions. The multiple functional changes of the variants support their assumed contribution to pathogenesis.

No description available.

A infecção pelo HEV é reconhecida como um considerável problema de saúde pública em diversas regiões do mundo. Embora caracterizada como uma infecção benigna com um curso evolutivo autolimitado, recentes estudos têm mostrado sua evolução para cronicidade em indivíduos imunocomprometidos. Além disso, tem sido verificado que nesses indivíduos a infecção crônica pelo HEV pode evoluir para fibrose hepática progressiva, culminando com o desenvolvimento de cirrose. Não existem dados acerca da prevalência da infecção pelo HEV em pacientes infectados pelo HIV no Brasil, onde a circulação deste vírus tem sido demonstrada em diversos grupos de indivíduos imunocompetentes e, até mesmo, em alguns animais provenientes de diferentes regiões do país. Com base nisso, este trabalho teve como objetivo estimar a prevalência de marcadores sorológicos e moleculares da infecção pelo HEV, bem como a padronização de uma PCR em tempo real para a detecção e quantificação da carga viral do HEV na população de soropositivos da cidade de São Paulo. Foram incluídos neste estudo soro e plasma de pacientes infectados pelo HIV (n=354), que foram divididos em grupos de acordo com a presença ou ausência de coinfecção pelos vírus das hepatites B (HBV) e C (HCV). Essas amostras foram coletadas entre 2007 e 2013. Anticorpos anti-HEV IgM e IgG foram pesquisados pela técnica de ELISA (RecomWell HEV IgM/ IgG - MIKROGEN®), e, em alguns casos, confirmados por Immunoblotting (RecomLine HEV IgM/ IgG - MIKROGEN®). Todas as amostras foram submetidas à pesquisa de HEV RNA através da PCR em tempo real padronizada. Cerca de 72% dos indivíduos avaliados pertenciam ao sexo masculino. A média de idade entre a população analisada foi de 48,4 anos. Os anticorpos anti-HEV IgM e IgG foram encontrados em 1,4% e 10,7% dos indivíduos dessa população, respectivamente. Apenas dois pacientes apresentaram positividade simultânea para anti-HEV IgM e IgG. Não houve diferença estatisticamente relevante quanto à presença de marcadores sorológicos nos grupos de estudo. Além disso, foi detectado o HEV RNA em 10,7% das amostras analisadas, entre as quais, seis apresentaram simultaneamente algum marcador sorológico (5 anti-HEV IgG e 1 IgM). A presença deste marcador foi predominante no grupo de pacientes com coinfecção pelo HCV. Através deste trabalho pôde-se constatar, portanto, que o HEV é circulante entre a população de infectados pelo HIV em São Paulo, e que o seguimento desses pacientes se faz necessário dado a possibilidade de progressão para infecção crônica e cirrose / HEV infection is recognized as a significant public health problem in different world regions. Although initially characterized as a benign infection with selflimited course, recent studies have showing its evolution to chronicity in immunocompromised individuals. Furthermore, in these individuals the chronic infection can develop progressive liver fibrosis leading to cirrhosis. There are no data regarding prevalence of HEV infections in HIV- infected patients in Brazil, where the circulation of this virus has been demonstrated in different individuals groups and in some animals from different regions of the country. Based on this, this study aimed to assess the prevalence of serological and molecular makers of HEV infection and the standardization of real-time PCR for the detection and quantification of HEV viral load in HIV-infected individuals in São Paulo. Serum and plasma samples of HIV-infected patients (n=354), collected between 2007 and 2013, were included and organized in groups of co-infection (HIV/ HBV, HIV/HCV and HIV/ HBV/ HCV) and HIV mono-infection. Antibodies anti-HEV IgM and IgG were detected by ELISA (RecomWell HEV IgM/ IgG - MIKROGEN®), and in some cases confirmed by immunoblotting (RecomLine HEV IgM/ IgG - MIKROGEN®). All samples were submitted to research HEV RNA by real-time PCR. About 72% of the patients were male. The mean age of this population was 48.4 years. The anti-HEV IgM and IgG antibodies were found in 1.4% and 10.7%, respectively. Only two patients presented simultaneous anti-HEV IgM and anti- HEV IgG. There was no statistically significant difference in the presence of serological makers among the HIV infection groups. In addition, HEV RNA was detected in 10.7% of samples and six of these samples presented simultaneously a serological maker (5 anti-HEV IgG and 1 IgM). The presence of this maker was more frequent in the co-infection HIV/ HCV group. Through this work, we observed that HEV is circulating among the HIV-infected population in São Paulo, and the monitoring these patients is necessary because of the possibility progression to chronic infection and cirrhosis

Made available in DSpace on 2016-12-23T13:49:05Z (GMT). No. of bitstreams: 1 Hepatite_B_Oculta_2012.pdf: 1487094 bytes, checksum: 098e451e5a2e2af112aa8508df956a77 (MD5) Previous issue date: 2012-02-29 / Occult hepatitis B (OHB) is defined as the presence of low levels of HBV DNA in the liver or serum of individuals testing hepatitis B surface antigen (HBsAg) negative. In most cases of OHB, sera are positive for hepatitis B core antibody. The literature contains quite a few studies on the prevalence of OHB in Brazil, as well as in the worldwide population. Such reports, often controversial, demonstrate that the OHB prevalence varies among healthy individuals or patients with diseases unrelated to the liver and patients with chronic liver disease. Despite efforts, it is necessary a better understanding of: the reasons for the persistence of low levels of HBV-DNA in the absence of detectable HBsAg, the potential risk of OHB transmission and its role in the progression and aggravation of some liver diseases. Therefore, it is interesting to know the prevalence of OHB indifferent population samples which allows de monitoring of carriers of the occult infection, followed prospectively in order to try to surprise the possible effects of the presence of low levels of HBV-DNA in these individuals. In this study we investigated the presence of Occult Hepatitis B in peripheral blood obtained from 520 healthy donors of Vitoria, Espirito Santo, with the aim of guiding policies to include or not the sensitive HBV-DNA nucleic acid amplification technique (NAT) screening in blood donations with a detection limit of 54UI/mL. In order to enable the molecular detection we had also developed a method that screens plasma samples in pools which is capable of detecting the presence of HBV in the ratio of 1:40.Through the technique of nested-PCR we found that 0,2% (1/520) had occult HBV in serum samples. Despite the low prevalence of OHB detected in the study, a considerable number of patients with occult HBV infection may not have been detected if the blood units were only tested for serological markers of HBV infection. So it is important to know the prevalence of OHB in one or more additional population groups, in order to follow up carriers of occult infection prospectively to determine possible effects of the presence of HBV-DNA in low concentrations in these individuals / A Hepatite B Oculta (HBO) é definida pela presença do DNA do VHB com carga viral geralmente baixa (<200UI/mL), no fígado ou soro de pessoas com antígeno de superfície (HBsAg) indetectável. Na maioria dos casos de HBO há positividade para o anticorpo contra o antígeno core da Hepatite B (anti-HBc). Na literatura constam poucos estudos sobre a prevalência da HBO no Brasil, bem como na população mundial. Existem relatos, muitas vezes controversos, que reportam frequências variáveis da HBO em indivíduos sem doença ou portadores de doença não relacionada com o fígado e em hepatopatas crônicos. Apesar dos esforços, faz-se necessário ainda uma melhor compreensão das razões para a persistência da baixa viremia do VHB na ausência de HBsAg detectável, do potencial risco de transmissão da HBO e do seu papel na progressão e agravamento de algumas hepatopatias. No estudo investigou-se a presença de HBO em 520 doadores de sangue de Vitória, Espírito Santo, com o objetivo de nortear políticas para incluir ou não a triagem molecular NAT (Nucleic Acid Amplification Technique ) de bolsas de sangue. Para viabilizar a detecção molecular desenvolveu-se também uma metodologia de extração em pool de amostrascapaz de detectar a presença do VHB na proporção de 1:40 com limite de detecção de 54UI/mL. Através da técnica de nested-PCR detectou-se a presença do VHB oculto em 0,2% (1/520) das amostras. Apesar da baixa prevalência de HBO detectada no estudo, um número considerável de portadores da infecção oculta podem não estar sendo detectados, se apenas a pesquisa de marcadores sorológicos da presença do VHB esta sendo realizada nas bolsas de sangue. Sendo assim é importante conhecer a prevalência da HBO em diferentes amostras da população para que se possa ter em mãos portadores da infecção oculta acompanhados prospectivamente a fim de tentar surpreender possíveis efeitos da presença do DNA do VHB em baixas concentrações nesses indivíduos

Made available in DSpace on 2016-12-23T13:56:09Z (GMT). No. of bitstreams: 1 Dissertacao Fernanda Magri de Carvalho PDF.pdf: 4813468 bytes, checksum: f874502e73ece86d6ada6693a89b3fc4 (MD5) Previous issue date: 2009-03-19 / A incidência do carcinoma hepatocelular (CHC) apresenta grandes variações geográficas associadas a diferentes prevalências dos seus fatores etiológicos mais comuns. Embora não se conheça a sua real incidência no Espírito Santo, o CHC é freqüentemente diagnosticado no estado, associado à infecção com os vírus da hepatite B (VHB) e da hepatite C. No entanto, cerca de 40% dos casos não apresentam evidências destes fatores etiológicos. Por essa razão, buscou-se verificar se outro fator aflatoxinas poderia estar envolvido na etiologia do CHC no ES. Nas regiões tropicais, as aflatoxinas são consideradas um importante fator etiológico do CHC, especialmente quando associadas à infecção com o VHB. O objetivo da presente investigação foi estudar a prevalência da mutação específica ser-249 no gene TP53, cuja presença é um indicador indireto de contaminação alimentar com estas micotoxinas e sua relação com a infecção pelo VHB. Foram investigadas 41 amostras de CHC e 74 amostras de cirrose hepática sem CHC, utilizando-se a metodologia de PCR-RFLP e sequenciamento. O DNA do VHB foi pesquisado com iniciadores específicos para seis diferentes regiões do seu genoma. Os iniciadores utilizados foram escolhidos considerando as regiões mais conservadas do genoma viral e englobando os oito genótipos já identificados do VHB. A presença do DNA do VHB foi observada em 46% (19/41) dos casos de carcinoma hepatocelular estudados. A mutação ser-249 TP53 foi observada em 15% (6/41) e 1,5% (1/69) dos casos de CHC e de cirrose hepática sem CHC associado, respectivamente, não havendo correlação estatisticamente significativa com a presença do DNA do VHB. Foram encontradas outras quatro mutações no códon 250 do gene TP53. A prevalência total de 24% (10/41) de mutações ser-249 e mutações no códon 250 nos pacientes com carcinoma hepatocelular possivelmente também associadas à contaminação com aflatoxinas pode indicar a provável exposição alimentar às aflatoxinas no Espírito Santo / The hepatocellular carcinoma (HCC) incidence has large geographic variation, which is associated with the different prevalences of its most common etiological factors. Although its real incidence in the Espirito Santo State is not known, the HCC is frequently diagnosed in ES associated with hepatitis B (HBV) and C virus infections. However, approximately 40% of the cases do not present known etiological factors. Therefore, it was the purpose of this study to verify if another factor - aflatoxins - could be involved in the HCC etiology in ES. In tropical areas, the aflatoxins are considered an important etiological factor of CHC, especially when associated with HBV infection. The aim of this investigation was to study the prevalence of the specific ser-249 mutation in the TP53 gene was studied, that indicates indirect food contamination with aflatoxins and its association with HBV infection. Forty-one HCC samples and seventy-four hepatic cirrhosis samples were investigated, using PCRRFLP methodology and sequencing. The HBV DNA was amplified with specific primers targeting six different regions of its genome. Primers were chosen considering the most conserved regions of the viral genome and encompassing the eight known genotypes of HBV. Presence of HBV DNA was observed in 46% (19/41) of the hepatocellular carcinoma cases studied. The ser-249 TP53 mutation was observed in 15% (6/41) and 1,5% (1/69) of the HCC and cirrhosis cases respectively, not having statistical correlation with the presence of HBV DNA. Other four mutations in TP53 codon 250 were found, yielding a total mutation prevalence of 24% (10/41), considering both ser-249 and codon 250 mutations, possibly also associated with food contamination by aflatoxins, and suggesting an alimentary exposition to aflatoxins in Espirito Sant

A infecção pelo HEV é reconhecida como um considerável problema de saúde pública em diversas regiões do mundo. Embora caracterizada como uma infecção benigna com um curso evolutivo autolimitado, recentes estudos têm mostrado sua evolução para cronicidade em indivíduos imunocomprometidos. Além disso, tem sido verificado que nesses indivíduos a infecção crônica pelo HEV pode evoluir para fibrose hepática progressiva, culminando com o desenvolvimento de cirrose. Não existem dados acerca da prevalência da infecção pelo HEV em pacientes infectados pelo HIV no Brasil, onde a circulação deste vírus tem sido demonstrada em diversos grupos de indivíduos imunocompetentes e, até mesmo, em alguns animais provenientes de diferentes regiões do país. Com base nisso, este trabalho teve como objetivo estimar a prevalência de marcadores sorológicos e moleculares da infecção pelo HEV, bem como a padronização de uma PCR em tempo real para a detecção e quantificação da carga viral do HEV na população de soropositivos da cidade de São Paulo. Foram incluídos neste estudo soro e plasma de pacientes infectados pelo HIV (n=354), que foram divididos em grupos de acordo com a presença ou ausência de coinfecção pelos vírus das hepatites B (HBV) e C (HCV). Essas amostras foram coletadas entre 2007 e 2013. Anticorpos anti-HEV IgM e IgG foram pesquisados pela técnica de ELISA (RecomWell HEV IgM/ IgG - MIKROGEN®), e, em alguns casos, confirmados por Immunoblotting (RecomLine HEV IgM/ IgG - MIKROGEN®). Todas as amostras foram submetidas à pesquisa de HEV RNA através da PCR em tempo real padronizada. Cerca de 72% dos indivíduos avaliados pertenciam ao sexo masculino. A média de idade entre a população analisada foi de 48,4 anos. Os anticorpos anti-HEV IgM e IgG foram encontrados em 1,4% e 10,7% dos indivíduos dessa população, respectivamente. Apenas dois pacientes apresentaram positividade simultânea para anti-HEV IgM e IgG. Não houve diferença estatisticamente relevante quanto à presença de marcadores sorológicos nos grupos de estudo. Além disso, foi detectado o HEV RNA em 10,7% das amostras analisadas, entre as quais, seis apresentaram simultaneamente algum marcador sorológico (5 anti-HEV IgG e 1 IgM). A presença deste marcador foi predominante no grupo de pacientes com coinfecção pelo HCV. Através deste trabalho pôde-se constatar, portanto, que o HEV é circulante entre a população de infectados pelo HIV em São Paulo, e que o seguimento desses pacientes se faz necessário dado a possibilidade de progressão para infecção crônica e cirrose / HEV infection is recognized as a significant public health problem in different world regions. Although initially characterized as a benign infection with selflimited course, recent studies have showing its evolution to chronicity in immunocompromised individuals. Furthermore, in these individuals the chronic infection can develop progressive liver fibrosis leading to cirrhosis. There are no data regarding prevalence of HEV infections in HIV- infected patients in Brazil, where the circulation of this virus has been demonstrated in different individuals groups and in some animals from different regions of the country. Based on this, this study aimed to assess the prevalence of serological and molecular makers of HEV infection and the standardization of real-time PCR for the detection and quantification of HEV viral load in HIV-infected individuals in São Paulo. Serum and plasma samples of HIV-infected patients (n=354), collected between 2007 and 2013, were included and organized in groups of co-infection (HIV/ HBV, HIV/HCV and HIV/ HBV/ HCV) and HIV mono-infection. Antibodies anti-HEV IgM and IgG were detected by ELISA (RecomWell HEV IgM/ IgG - MIKROGEN®), and in some cases confirmed by immunoblotting (RecomLine HEV IgM/ IgG - MIKROGEN®). All samples were submitted to research HEV RNA by real-time PCR. About 72% of the patients were male. The mean age of this population was 48.4 years. The anti-HEV IgM and IgG antibodies were found in 1.4% and 10.7%, respectively. Only two patients presented simultaneous anti-HEV IgM and anti- HEV IgG. There was no statistically significant difference in the presence of serological makers among the HIV infection groups. In addition, HEV RNA was detected in 10.7% of samples and six of these samples presented simultaneously a serological maker (5 anti-HEV IgG and 1 IgM). The presence of this maker was more frequent in the co-infection HIV/ HCV group. Through this work, we observed that HEV is circulating among the HIV-infected population in São Paulo, and the monitoring these patients is necessary because of the possibility progression to chronic infection and cirrhosis

Dans les patients infectés par HBV, les thérapies avec les analogues de nucléos(t)ides (NAs) ou l'interféron α (IFNα) restent inefficaces pour éradiquer l'infection, à cause d'une forme persistante d'HBV, appelée l'ADN circulaire covalent clos (ADNccc), organisé comme un mini-chromosome. Notre but a été de revisiter l'activité anti-HBV d'un panel de cytokines in vitro en utilisant des hépatocytes non transformés, afin d'identifier de nouvelles options immunothérapeutiques. Parmi toutes les molécules testées, l'IFNβ, l'IFNγ, les IFNλ, le TNFα, l'IL-6, l'IL-1β et le ténofovir (ce dernier utilisé comme contrôle positif) ont montré un effet suppresseur sur la réplication d'HBV aussi fort et parfois plus fort que l'IFNα. La cytokine ayant l'effet le plus élevé sur l'ADN total d'HBV (EC50 ≈ 25 pg/mL), sans cytotoxicité, était l'interleukine-1β (IL-1β), qui est naturellement produite par les cellules de Kupffer (KC), les macrophages du foie. De façon importante, les ARNs totaux d'HBV et l'antigène sécrété HBeAg, mais pas HBsAg, ni l'ADNccc, sont fortement diminués par l'IL-1β. Nous avons donc émis l'hypothèse selon laquelle des promoteurs viraux spécifiques su l'ADNccc pourraient être inhibés, même si l'ADNccc n'est pas dégradé. Ensuite, nous avons étudié le mécanisme de l'activité antivirale de l'IL-1β. Nous avons montré que tous les promoteurs d'HBV sembleraient être inhibés par l'IL-1β. En parallèle, nous avons vérifié que l'IL-1β pouvait activer le promoteur de NF-κB, dont la fonction de transcription a été confirmée. Grâce à cette étude, l'IL-1β a été montré comme ayant un effet antiviral très efficace contre HBV in vitro, par l'intermédiaire de la fixation de NF-κB sur l'ADNccc / In HBV-infected patients, therapies with nucleos(t)ide analogues (NAs) or interferon α (IFNα) remain ineffective in eradicating the infection, because of a persistent form of HBV DNA, namely the covalently closed circular DNA (cccDNA), which is organized as a minichromosome. Our aim was to revisit the anti-HBV activity of a panel of IFNs and pro-inflammatory cytokines in vitro using nontransformed cultured hepatocytes of HBV infection, to identify new immunotherapeutic options. Amongst all molecules tested, IFNβ, IFNγ, IFNλ, TNFα, IL-6, IL-1β and tenofovir showed a suppressive effect on HBV replication at least as strong as, but sometimes stronger than IFNα. The cytokine showing the highest effect on intracellular total HBV DNA without any cytotoxicity, was interleukin-1β (IL-1β), which is naturally produced by Kupffer cells (KC), representing the macrophages of the liver. Importantly, total HBV RNAs and secreted HBeAg, but nor HBsAg, neither cccDNA, were strongly decreased. Thus, we hypothesized that even if cccDNA was not degraded, specific viral promoters on cccDNA could be silenced. Then, we investigated the mechanism of IL-1β antiviral activity. We have shown that all HBV promoters were early inhibited by IL-1β. In the meantime, we have verified that IL-1β can induce nuclear Translocation and expression of NF-κB. We also checked NF-κB functionality. Thanks to this study, IL-1β has been found to have very potent antiviral effect against HBV in vitro, through the binding of NF-κB on cccDNA

Les virus des hépatites B et C (VHB et VHC) entretiennent des liens étroits avec le métabolisme lipidique des hépatocytes. Ainsi, la réplication du VHB est dépendante de certains récepteurs nucléaires hépatiques, tels que HNF4α et PPARα, impliqués dans ce métabolisme. L’assemblage des particules virales du VHC dépend lui de la voie de synthèse des lipoprotéines de très faible densité (VLDL) et le virus circule dans le sang sous forme de lipo-viro-particules associé notamment à l’apolipoprotéine B, un composant essentiel des VLDL. Dans ce travail, nous avons d’abord étudié le rôle de FXRα, le récepteur nucléaire des sels biliaires, sur la réplication du VHB. Nous avons montré, in vitro, que les sels biliaires, via FXRα, activaient le promoteur de Core du VHB qui contrôle le niveau de réplication virale. Puis dans l’étude des liens entre les lipoprotéines et le VHC, nous avons montré que l’apoB présente sur certaines particules virales jouaient un rôle important dans l’infectiosité du virus in vitro, et que la protéine Cideb, présente en surface des gouttelettes lipidiques et impliquée dans l’assemblage des VLDL, était impliquée dans l’association du VHC avec l’apoB et influençait l’infectiosité des virions sécrétés. De plus nous avons mis en évidence l’existence de particules sub-virales chez les patients infectés, de nature lipoprotéique mais ne portant que les protéines d’enveloppes du VHC. Tous ces résultats renforcent l’idée d’une adaptation du VHB et du VHC au métabolisme lipidique hépatique. Les bénéfices éventuels qu’en retirent ces deux virus, ainsi que l’existence de possibles thérapeutiques anti-virales ciblant le métabolisme lipidique, restent à explorer / Hepatitis B and C viruses (HBV and HCV) infections are tightly linked with hepatic lipid metabolism. HBV replication depends on specific nuclear receptors, such as HNF4α and PPARα, both implicated in this metabolism. HCV assembly depends on the synthesis of Very-Low-Density Lipoproteins (VLDL), and the virus circulates in the blood as lipo-viral-particles associated in particular with apoB, an essential component of VLDL. In this study, we first studied the influence of FXRα, the nuclear receptor for bile acids, on HBV replication. We showed that, in vitro, bile acids, via FXRα, were able to activate the HBV Core promoter which controls the level of viral replication. Then, in the study of the interactions between HCV and lipoproteins, we demonstrated that apoB, which is associated with a proportion of viral particles, played an important role in HCV infectivity in vitro, and that Cideb, a protein involved in VLDL assembly, was implicated in the association between HCV and apoB and influenced the infectivity of secreted viral particles. Finally, we showed that, besides HCV infectious particles, sub-particles bearing only viral envelope glycoproteins circulated in the blood of infected patients. Interactions of HBV with the metabolism of bile acids, and of HCV with the metabolism of lipoproteins, are two examples of adaptation of a parasite to its host. The potential benefits from these interactions are still to be determined, as well as the possibility to develop anti-viral strategies targeting lipid metabolism

Introduction International guidelines endorse the screening of patients for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Chlamydia trachomatis before assisted reproductive techniques (ART). At present no such guidelines exists in South Africa. At the Reproductive and Endocrine Unit (referred to as “the Unit”) of Steve Biko Academic Hospital, all patients with unknown HIV status are counselled and a blood sample is collected during the initial visit for automated laboratory based HIV screening. These HIV results are not available before semen samples are processed. Furthermore, patients are not screened for HBV, HCV and Chlamydia trachomatis. Couples attending the Unit are of a low to middle socio-economic status and experience financial constraints. Moreover, automated laboratory based assays are expensive to perform. Rapid testing is a cost effective and practical method from screening patients, with a 20–30 minute result turnover time. Until screening at the Unit is improved, the possible identification of semen characteristics that could indicate HIV infection would be a useful tool. Materials and Methods The following rapid point-of-care assays were evaluated: Determine® HIV-1/2 combo test (n=100), Determine® HBsAg test (n=100), DIAQUICK HCV kit (n=74), and the DIAQUICK Chlamydia trachomatis kit (n=30). For profiling, parameters from a basic semen analysis of HIV-positive males (n=60) were compared with HIV-negative males (n=60). Information pertaining to CD4 count, antiretroviral treatment and plasma viral load of HIV-positive males were analysed. Results From all patients included in the study, 8% tested positive for HIV. The risk of a female being HIV-positive was 3.73 times higher than for males. In the pilot study to explore rapid testing for HBV and HCV, 1% and 1.4% of patients tested positive respectively. When testing for Chlamydia trachomatis 31.3% of females, but no males tested positive. Comparing semen profiles, no significant differences were found between samples from HIV positive and negative males or between HIV positive males categorised by CD4 cell count (p>0.05). For the HIV-positive group with a detectable plasma HIV viral load (>40 copies/ml), a significant difference was observed in the semen viscosity (p=0.0460). Significant differences were noted in the sperm motility (immotile sperm p=0.0456, progressive sperm p=0.0192) of patients receiving antiretroviral (ARV) therapy. Discussion and Conclusion The use of rapid testing is an acceptable and feasible option for improving current screening protocols at the Unit. The absence of definite alterations in the semen characteristics of HIV-positive men further motivates the need for a simpler, point-of-care screening protocol. The prevalence of HBV was lower than that reported in the general population of South Africa and further investigation is needed. Although the sample size was small, HCV prevalence was similar to that of the general population. One third of females tested positive for Chlamydia trachomatis. The methodology used was possibly not appropriate for males. This study highlighted the need for guidelines that address the specialised needs of ART clinics in resource-limited and developing countries with a high HIV prevalence. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Obstetrics and Gynaecology / unrestricted