This thesis aimed to describe the epidemiology of HCV in South Australian prisons - prevalence, transmission and risk factors. This thesis also aimed to determine the impact of incarceration on reported risk behaviours. A related objective was to evaluate the epidemiological effectiveness of the ELISA - 3 HCV antibody test using PCR as the gold standard. Finally, this thesis aimed to explore the potential for minimising HCV risk in the South Australian prison population. Methods: Two case note audits were conducted at each of eight publicly operated SA prisons ( in summer and winter ) to identify any documented HCV - antibody test results. Prisoners recruited at entry to prison were offered tests for HCV - antibody and completed a pre - entry risk factor survey. Participants completed additional risk factor surveys and ( if HCV - negative at last test ) underwent further antibody tests at three - monthly intervals for up to 15 months. A sample of participants also provided blood specimens for HCV - RNA testing. Limited stakeholder consultations with prison officers and nurses were also conducted. Quantitative data were analysed using univariate and multivariate techniques. Results: 1347 case notes were audited in summer, and 1347 in winter and an overall HCV prevalence of 42 % was estimated. In both univariate and multivariate analyses, HCV prevalence was significantly higher in female prisoners ( 65 % ), those aged above 28 years ( 48 % ), and in Indigenous prisoners originating from metropolitan areas ( 56 % ). Indigenous prisoners originating from remote areas had significantly lower HCV prevalence ( 20 % ). 666 prisoners were recruited at entry, and 42 % were estimated to be HCV - antibody positive. Three seroconversions were noted in 151 initially HCV - seronegative negative individuals followed up for a median time of 121 days - a rate 4.6 per 100 person years - but community exposure could not be ruled out. Overall agreement between HCV - antibody and HCV - RNA assays was 86 % ( 100% in the HCV negative samples ) - kappa = 0.71. Injecting history was highly prevalent in prison entrants ( 70 % ) and both community and prison injecting ( but not tattooing ) were independent predictors of entry HCV status. Prison history was also independently associated with entry HCV status. Injecting in prison during the study was infrequently reported, but significantly more likely in those testing HCV - antibody positive at prison entry ( risk ratio = 2.48, P = 0.046 ). Stakeholders were most supportive of strategies to increase education and to minimise risks associated with hair clippers, but did not support most other suggested preventive strategies. Other issues related to communicable diseases and infection control were explored in the stakeholder interviews. Conclusions: HCV prevalence in South Australian prisoners is extremely high and may have contributed to a ' ceiling effect ' , minimising the seroconversion rate observed in this population. Injecting is relatively infrequently reported in prison, but more likely in those already infected with HCV. Thus, contaminated injecting equipment represents a significant threat to other prisoners and prison staff. Strategies aimed at reducing HCV risk in prisons, which address the concerns of those expected to implement them, are proposed in this thesis. / Thesis (Ph.D.)--School of Population Health and Clinical Practice, 2006.

This thesis aimed to describe the epidemiology of HCV in South Australian prisons - prevalence, transmission and risk factors. This thesis also aimed to determine the impact of incarceration on reported risk behaviours. A related objective was to evaluate the epidemiological effectiveness of the ELISA - 3 HCV antibody test using PCR as the gold standard. Finally, this thesis aimed to explore the potential for minimising HCV risk in the South Australian prison population. Methods: Two case note audits were conducted at each of eight publicly operated SA prisons ( in summer and winter ) to identify any documented HCV - antibody test results. Prisoners recruited at entry to prison were offered tests for HCV - antibody and completed a pre - entry risk factor survey. Participants completed additional risk factor surveys and ( if HCV - negative at last test ) underwent further antibody tests at three - monthly intervals for up to 15 months. A sample of participants also provided blood specimens for HCV - RNA testing. Limited stakeholder consultations with prison officers and nurses were also conducted. Quantitative data were analysed using univariate and multivariate techniques. Results: 1347 case notes were audited in summer, and 1347 in winter and an overall HCV prevalence of 42 % was estimated. In both univariate and multivariate analyses, HCV prevalence was significantly higher in female prisoners ( 65 % ), those aged above 28 years ( 48 % ), and in Indigenous prisoners originating from metropolitan areas ( 56 % ). Indigenous prisoners originating from remote areas had significantly lower HCV prevalence ( 20 % ). 666 prisoners were recruited at entry, and 42 % were estimated to be HCV - antibody positive. Three seroconversions were noted in 151 initially HCV - seronegative negative individuals followed up for a median time of 121 days - a rate 4.6 per 100 person years - but community exposure could not be ruled out. Overall agreement between HCV - antibody and HCV - RNA assays was 86 % ( 100% in the HCV negative samples ) - kappa = 0.71. Injecting history was highly prevalent in prison entrants ( 70 % ) and both community and prison injecting ( but not tattooing ) were independent predictors of entry HCV status. Prison history was also independently associated with entry HCV status. Injecting in prison during the study was infrequently reported, but significantly more likely in those testing HCV - antibody positive at prison entry ( risk ratio = 2.48, P = 0.046 ). Stakeholders were most supportive of strategies to increase education and to minimise risks associated with hair clippers, but did not support most other suggested preventive strategies. Other issues related to communicable diseases and infection control were explored in the stakeholder interviews. Conclusions: HCV prevalence in South Australian prisoners is extremely high and may have contributed to a ' ceiling effect ' , minimising the seroconversion rate observed in this population. Injecting is relatively infrequently reported in prison, but more likely in those already infected with HCV. Thus, contaminated injecting equipment represents a significant threat to other prisoners and prison staff. Strategies aimed at reducing HCV risk in prisons, which address the concerns of those expected to implement them, are proposed in this thesis. / Thesis (Ph.D.)--School of Population Health and Clinical Practice, 2006.

This thesis aimed to describe the epidemiology of HCV in South Australian prisons - prevalence, transmission and risk factors. This thesis also aimed to determine the impact of incarceration on reported risk behaviours. A related objective was to evaluate the epidemiological effectiveness of the ELISA - 3 HCV antibody test using PCR as the gold standard. Finally, this thesis aimed to explore the potential for minimising HCV risk in the South Australian prison population. Methods: Two case note audits were conducted at each of eight publicly operated SA prisons ( in summer and winter ) to identify any documented HCV - antibody test results. Prisoners recruited at entry to prison were offered tests for HCV - antibody and completed a pre - entry risk factor survey. Participants completed additional risk factor surveys and ( if HCV - negative at last test ) underwent further antibody tests at three - monthly intervals for up to 15 months. A sample of participants also provided blood specimens for HCV - RNA testing. Limited stakeholder consultations with prison officers and nurses were also conducted. Quantitative data were analysed using univariate and multivariate techniques. Results: 1347 case notes were audited in summer, and 1347 in winter and an overall HCV prevalence of 42 % was estimated. In both univariate and multivariate analyses, HCV prevalence was significantly higher in female prisoners ( 65 % ), those aged above 28 years ( 48 % ), and in Indigenous prisoners originating from metropolitan areas ( 56 % ). Indigenous prisoners originating from remote areas had significantly lower HCV prevalence ( 20 % ). 666 prisoners were recruited at entry, and 42 % were estimated to be HCV - antibody positive. Three seroconversions were noted in 151 initially HCV - seronegative negative individuals followed up for a median time of 121 days - a rate 4.6 per 100 person years - but community exposure could not be ruled out. Overall agreement between HCV - antibody and HCV - RNA assays was 86 % ( 100% in the HCV negative samples ) - kappa = 0.71. Injecting history was highly prevalent in prison entrants ( 70 % ) and both community and prison injecting ( but not tattooing ) were independent predictors of entry HCV status. Prison history was also independently associated with entry HCV status. Injecting in prison during the study was infrequently reported, but significantly more likely in those testing HCV - antibody positive at prison entry ( risk ratio = 2.48, P = 0.046 ). Stakeholders were most supportive of strategies to increase education and to minimise risks associated with hair clippers, but did not support most other suggested preventive strategies. Other issues related to communicable diseases and infection control were explored in the stakeholder interviews. Conclusions: HCV prevalence in South Australian prisoners is extremely high and may have contributed to a ' ceiling effect ' , minimising the seroconversion rate observed in this population. Injecting is relatively infrequently reported in prison, but more likely in those already infected with HCV. Thus, contaminated injecting equipment represents a significant threat to other prisoners and prison staff. Strategies aimed at reducing HCV risk in prisons, which address the concerns of those expected to implement them, are proposed in this thesis. / Thesis (Ph.D.)--School of Population Health and Clinical Practice, 2006.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Schistosomiasis is a parasitic disease, severe, chronic waterborne and development, whose etiologic agent is Schistosoma mansoni. It occurs in 74 countries with 207 million people infected and 700 million in risky areas. In Brazil, data indicate a prevalence of eight million. The Schistosomiasis can be exacerbated when patients are carriers of the Hepatitis B Virus (HBV) and C (HCV), resulting in simultaneous evolution of both pathologies. In Brazil, the prevalence of co-infection with HBV/HCV and S. mansoni found in studies ranged from 13.6% to 40% for HBV and 0.5% to 19.66% for the HCV. On this basis, this research aimed to identify the prevalence of serological markers of HBV and HCV and risky factors in individuals carrying the S. mansoni in the Santa Maria neighborhood, Aracaju, state of Sergipe. We conducted a cross-sectional epidemiological study. Data were collected for each patient by means of a questionnaire investigation. This questionnaire included variables identifying the subject of the research participants and variables that included the epidemiological risky factors for infection with HBV or HCV. We collected serum samples of research participants and forwarded to the laboratory testing to identify serological markers of hepatitis B and C: Total Anti-HBc IgG, Anti-HBs, HBsAg and Anti-HCV. All procedures performed using the technique of chemiluminescence immunoassay using the ARCHITECT assay for each serological marker following the protocols established by the laboratory equipment manufacturer SYSTEM ARCHITECT. In addition, we carried out the analysis of spatial distribution of co-infection in the district through the program using the TerraView Kernel intensity estimation. It was found that 16 individuals had contact with HBV (9.41%), one of these was positive for HBsAg. Only Thirty-two samples (18.82%) were positive for the marker Anti-HBs. Three samples were positive for anti-HCV (1.76%), and also a positive for Anti-HBc. The main risks of HBV and HCV infection were related to parenteral interventions of health services, as well as sexual activity without condom use in the case of HBV. Spatial analysis of cases of co-infection (Schistosomiasis and Hepatitis) allowed the visualization of areas of higher concentration of these infections, as well as those that are exposed to different degrees of risk of transmission. The survey results allow to offer, the municipal health services, a tool to facilitate the understanding of the spatial distribution of schistosomiasis and hepatitis (B and C) in Santa Maria neighborhood. Although our values are above the estimated prevalence for the Brazilian population and the Northeast, we can t infer that the individuals with Schistosomiasis are more susceptible to infection with HBV or HCV, since the risky factors were the means of risky transmission of causative agents of hepatitis and not infected with S. mansoni. / A Esquistossomose Mansônica é uma doença parasitária grave, de veiculação hídrica e evolução crônica, cujo agente etiológico é o Schistosoma mansoni. Ocorre em 74 países, com 207 milhões de pessoas infectadas e 700 milhões em áreas de risco. No Brasil, dados indicam uma prevalência de oito milhões. A Esquistossomose pode ser agravada quando os pacientes são portadores dos Vírus das Hepatites B (VHB) e C (VHC), resultando na evolução simultânea de ambas as patologias. No Brasil, As prevalências da co-infecção VHB/VHC e S. mansoni encontradas nos estudos realizados variaram de 13,6% a 40% para o HBV e de 0,5% a 19,66% para o HCV. Com base nisso, esta pesquisa objetivou identificar a prevalência de marcadores sorológicos do VHB e VHC e os fatores de risco em indivíduos portadores do S. mansoni no Bairro Santa Maria, Aracaju/SE. Foi realizado um estudo epidemiológico do tipo transversal. Coletaram-se dados de cada paciente por meio de um questionário investigativo. Este questionário contemplou variáveis de identificação do sujeito participante da pesquisa e variáveis epidemiológicas que incluíram os fatores de risco para infecção pelo HBV ou HCV. Foram coletadas alíquotas de soro dos participantes da pesquisa e encaminhadas para a realização das análises laboratoriais para identificação de marcadores sorológicos dos Vírus das Hepatites B e C: Anti-HBc Total IgG, Anti-HBs, HBsAg e Anti-HCV. Todos os procedimentos realizados utilizaram a técnica de Imunoensaio Quimioluminescente através do Ensaio ARCHITECT para cada marcador sorológico seguindo os protocolos de análises laboratoriais estabelecidos pelo fabricante do equipamento ARCHITECT SYSTEM. Além disso, realizou-se a análise espacial da distribuição da co-infecção no bairro através do programa TerraView utilizando o estimador de intensidade Kernel. Constatou-se que 16 indivíduos tiveram contato com o HBV (9,41%), desses um foi positivo para HBsAg. Apenas Trinta e duas amostras (18,82%) foram positivas para o marcador Anti-HBs. Três amostras foram positivas para Anti-HCV (1,76%), sendo uma também positiva para Anti-HBc. Os principais riscos de infecção pelo HBV e HCV foram relacionados às intervenções parenterais dos serviços de saúde, assim como à atividade sexual sem uso de preservativo, no caso do HBV. A análise espacial dos casos de co-infecção (Esquistossomose e Hepatite) permitiu a visualização de áreas de maior concentração dessas infecções, assim como as que são expostas a diferentes graus de risco de transmissão. Os resultados da pesquisa possibilitam oferecer, aos serviços municipais de saúde, um instrumento que facilite a compreensão da distribuição espacial da Esquistossomose e Hepatites (B e C) no bairro Santa Maria. Embora nossos valores sejam acima da prevalência estimada para a população brasileira e da região nordeste, não podemos inferir que os indivíduos portadores da Esquistossomose são mais susceptíveis à infecção pelo HBV ou HCV, uma vez que, os fatores de risco foram as vias de risco de transmissão de agentes causadores de hepatite e não a infecção pelo S. mansoni.

No description available.

Host-pathogen interactions in Hepatitis C Virus infection: Deciphering the role of host proteins and microRNAs Hepatitis C virus (HCV) is a positive sense single stranded RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV genome consists of a single open reading frame flanked by highly structured 5‟ and 3‟ untranslated regions (UTRs) at both ends. Unlike cellular mRNAs, HCV RNA translation is independent of the cap structure and is mediated by an internal ribosomal entry site (IRES) present in the 5‟UTR. HCV replication begins with the synthesis of a complementary negative-strand RNA using the positive strand RNA genome as a template catalyzed by the NS5B RNA dependent RNA polymerase (RdRp). The de novo priming of HCV RNA synthesis by NS5B occurs at the very end of the 3‟UTR. The 3‟UTR is organized into highly structured regions namely the variable region, poly U/UC region and the 3‟X region. These regions contain cis-acting elements that determine the efficiency of viral replication. In addition, the interaction of trans-acting factors with the 3‟ UTR is also important for regulation of HCV replication. HCV 3‟UTR interacts with several cellular proteins such as the human La protein, polypyrimdine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) and Human antigen R (HuR). However, the molecular basis of regulation of viral replication by these proteins is not well understood. Many proteins that are hijacked by HCV as well as other cytoplasmic RNA viruses, such as La, PCBP2, HuR and PTB are RNA binding proteins (RBPs). They are involved in post transcriptional regulation of cellular gene expression. Thus the subversion of these proteins by the virus can affect their normal physiological functions. In addition to proteins, recent reports also describe the involvement of non-coding RNAs including microRNAs (miRNA) and long non coding RNAs (lncRNA) in HCV infection. miRNAs can either directly bind to the HCV genome and regulate its life cycle or indirectly modulate the expression of host proteins required by the virus. miRNAs that are differentially regulated in virus infected tissues or body fluids of infected patients can also serve as biomarkers for diagnosis of various stages of the disease. Hence, it was planned to study the role of host proteins and miRNAs in the HCV life cycle and pathogenesis to have novel insights into the biology of HCV infection. Riboproteomic studies have identified several host proteins that directly interact with the 5‟ and/or 3‟UTRs of the HCV RNA. One of the RNA binding proteins that predominantly interact with the 3‟UTR of HCV RNA was found to be HuR. In the present study, we have extensively characterized the interaction between HuR and HCV 3‟UTR and studied its functional implications in HCV life cycle along with other host factors. Characterizing the HCV 3’UTR–HuR interaction and its role in HCV replication HuR is a ubiquitously expressed member of the Hu family which shuttles between the nucleus and cytoplasm in response to stress. Whole genome siRNA knockdown and other studies have suggested that HuR is essential for HCV replication. However, the molecular mechanism of its involvement in this process was not clear. We observed that siRNA mediated knockdown of HuR reduces the HCV RNA and protein levels. Immunofluorescence studies indicated that HuR relocalizes from the nucleus to the cytoplasm in HCV infected cells. Through confocal microscopy and GST pulldown assays, we have demonstrated that HuR co localizes with the viral polymerase, NS5B and directly interacts with the NS5B protein. Membrane flotation assays showed that HuR is present in the detergent resistant membrane fractions which are the active sites of HCV replication. In addition to the interaction of HuR with the viral protein NS5B, we also characterized its interaction with the viral RNA. Direct UV cross linking assays and UV cross linking immunoprecipitation assays were performed to demonstrate the interaction of HuR with the HCV 3‟UTR. The RRM3, hinge region and RRM1 of HuR were found to be important for binding. Further, we observed that HuR competes with PTB for binding to the 3‟UTR when cytoplasmic S10 extracts or recombinant proteins were used in UV cross linking assays. In contrast, the addition of HuR facilitated the binding of La protein to the HCV 3‟UTR in the above assays. Competition UV cross linking assays indicated that both HuR and PTB bind to the poly U/UC region of the 3‟UTR while La binds to the variable region. HuR and La showed higher affinities for binding to the 3‟UTR as compared to PTB in filter binding assays. Since HuR and PTB interact with the same region on the 3‟UTR and HuR showed ~4 fold higher affinity for binding, it could displace PTB from the 3‟UTR. Next, we investigated the roles of HuR, PTB and La in HCV translation and replication in cell culture using three different assay systems, HCV sub genomic replicon, HCV bicistronic SGR-JFH1/Luc replicon as well as the infectious HCV full length RNA (JFH1). Results clearly indicated that HuR and La are positive modulators of HCV replication. Interestingly, PTB facilitated HCV IRES mediated translation but appeared to have a negative effect on HCV replication. The positive effectors, HuR and La showed significant co localization with one another in the cytoplasm in immunofluorescence studies. GST pulldown and coimmunoprecipitation experiments indicated protein-protein interactions between HuR and La but not between HuR and PTB. Through quantitative IP-RT assays, we demonstrated that the overexpression of HuR in HCV RNA transfected cells increases the association of La with the HCV RNA while HuR knockdown reduces the association of La with the HCV RNA. Previous studies in our laboratory have shown that La helps in HCV genome circularization. The addition of HuR significantly increased La mediated interactions between the 5‟UTR and the 3‟UTR of HCV RNA as monitored by 5‟-3‟ co precipitation assays, suggesting a possible mechanism by which cooperative binding of HuR and La could positively regulate HCV replication. Taken together, our results suggest a possible interplay between HuR, PTB and La in the regulation of HCV replication. Studying the role of HuR- associated cellular RNAs in HCV infection HuR belongs to the category of mRNA turnover and translation regulatory proteins (TTR-RBPs), which are capable of triggering rapid and robust changes in cellular gene expression. HuR plays a role in several post transcriptional events such as mRNA splicing, export, stability and translation. In the present study, we have investigated the possible consequences of relocalization of HuR on cellular processes in the context of HCV infection. We observed that 72h post transfection of infectious HCV-JFH1 RNA, there is an increase in the mRNA levels of some of the validated targets of HuR including the vascular endothelial growth factor A (VEGFA), dual specificity phosphatise 1 (MKP1) and metastasis - associated lung adenocarcinoma transcript (MALAT1). IP-RT assays demonstrated that the association of HuR with VEGFA and MKP1 was higher in HCV-JFH1 RNA transfected cells as compared to the mock transfected cells indicating that increase in HuR association could probably help in stabilization of these mRNAs. Interestingly, we observed that the association of HuR with the lncRNA MALAT1 decreases in the presence of HCV RNA, while its RNA levels increased. Earlier it has been reported that MALAT1 interacts with HuR and was predicted to interact with La. We confirmed the interaction of both HuR and La proteins with MALAT1 RNA in vitro and in the cell culture system. Results from our time course experiments suggest that relocalization of HuR and La upon HCV infection might decrease their association with the nuclear retained MALAT1 RNA leading to significant reduction in MALAT1 RNA levels at the initial time points. However at later time points, MALAT1 was found to be unregulated through activation of the Wnt/beta-catenin pathway as demonstrated using a chemical inhibitor against β-catenin. Since MALAT1 is a known regulator of epithelial mesenchymal transition (EMT) and metastasis, we further studied the physiological consequence of the observed increase in MALAT1 levels upon HCV infection. Cell migration and cell invasion studies suggested that the knockdown of MALAT1 led to the inhibition of HCV- triggered wound healing and matrigel invasion and also rescued the down regulation of E-Cadherin protein levels, an EMT marker. Our study highlights the importance of the lncRNA, MALAT1 in HCV infection and suggests its possible involvement in HCV induced HCC. Investigating the role of miRNAs in HCV pathogenesis and replication miRNAs can also regulate HCV infection and pathogenesis in multiple ways. It is known that under disease conditions, there is aberrant expression of intracellular as well as circulating miRNAs. We have investigated the expression profile of 940 human miRNAs in HCV infected patient serum samples to identify the differentially regulated miRNAs. miR-320c, miR-483-5p and the previously reported miR-125b were found to be upregulated in the serum of cirrhotic and non-cirrhotic HCV infected patient serum samples. All three miRNAs were also unregulated in the cell culture supernatant of HCV infected cells as well as within the HCV infected cells. miR-483-5p was specifically enriched in the exosomes isolated from patient serum samples. Knockdown of miR-320c and miR-483-5p did not have significant effect on HCV replication while knockdown of miR-125b affected HCV replication through regulation of one of its target genes, HuR. We observed that with time, miR-125b levels in HCV-JFH1 RNA transfected cells increase while the HuR protein levels decrease. Using luciferase reporter constructs, we demonstrated that the decrease in HuR protein levels is indeed mediated by miR-125b. Mutations in the target site of miR-125b in the HuR 3‟UTR prevented the down regulation of luciferase activity. Next we tested the effect of silencing miR-125b on HCV replication. Knockdown of miR-125b prevented the reduction in HuR protein levels but with no significant effect on HCV replication. It appeared that the HuR protein already present in the cytoplasm could be sufficient to support HCV replication. Hence similar experiments were carried out in cells depleted of HuR using either siRNA against HuR or a chemical inhibitor of nucleocytoplasmic transport of HuR, Leptomycin B. We observed that when the intracellular levels of HuR are reduced using either of the two approaches, there is a decrease in HCV replication. This is in accordance with the results obtained in the first part of the thesis. However when miR-125b was silenced in HuR depleted cells, we noticed an upregulation in the HuR protein levels by western blot analysis and a consequent increase in HCV RNA levels as quantified by qRT-PCR. From our findings, we can conclude that miR-125b mediated regulation of HuR plays an important role in HCV replication. We hypothesize that this could be a cellular response to HCV infection to which the virus responds by inducing protein relocalization. Altogether, these studies outline the importance of host factors including cellular proteins and non-coding RNAs in the regulation of HCV life cycle and pathogenesis. Results reveal the mechanistic insights into how HCV infection triggers host defense pathways, which are evaded by the virus by counter strategies.

Thesis (MScMedSc))--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Despite the availability of Hepatitis B virus (HBV) vaccination for over three decades, this infection remains a major public health problem. Whilst the WHO recommends giving a birth dose of the vaccine, in South Africa, routine infant HBV vaccination commences at six weeks of age. This schedule is based on data from the pre-HIV era which showed transmission occurred via the horizontal, rather than the vertical route. In the era of HIV however, maternal HIV co-infection may release HBV from immune control, resulting in higher HBV loads and increasing the risk of vertical transmission. The aim of this study was to determine the prevalence and character of HBV infection in HIV-exposed infected and uninfected infants. Residual plasma samples from routine HIV nucleic acid testing of 1000 HIV-exposed infants aged between 0 and 18 months from the Western Cape were tested. Samples were tested for HBsAg by ELISA (Murex HBsAg Version 3) and confirmed by neutralisation. HBV DNA was quantified using an in-house real-time PCR assay. Infants with HBsAg positive samples were followed up and a blood sample was collected from mother and child. Those HBsAg positive samples were tested for HBeAg/antiHBe (Diasorin) and HBsAg negative samples were tested for antiHBs. HBV DNA was quantified. The surface gene was sequenced and the HBV genotype determined by phylogenetic analysis using HepSEQ (www.hepseq.org.uk). Whole genome sequencing was also performed. Of 1000 samples tested, four samples were positive for HBsAg and/or HBV DNA, indicating a prevalence of HBV transmission of 0.4%. At follow-up, two of three infected infants were positive for HBsAg, with HBV viral loads of greater than 108 IU/ml. The third infant was found to have cleared his infection and the fourth child was lost to follow up. These infected infants had all received HBV vaccination. All four mothers were HBeAg positive. Sequencing analysis showed the HBV strains from the two infants and four mothers belonged to subgenotype A1. The two mother-child paired sequences were identical. The data from this study shows that vertical transmission of HBV infection in HIV-exposed infants from the Western Cape is occurring, despite vaccination. Data from the Western Cape, showing an HBV prevalence of 3.4% in HIV-infected pregnant women, and those presented here suggest a vertical transmission rate of HBV of 12%. This is despite the widespread use of tenofovir and lamivudine in HIV-infected women with low CD4 counts. This study provides data supporting calls to bring HBV vaccination closer to the time of birth. Further work is urgently needed to confirm these findings and to determine the rates of transmission in HIV-unexposed infants. / AFRIKAANSE OPSOMMING: Ten spyte van die beskikbaarheid van die Hepatitis B virus (HBV) inenting vir meer as drie dekades, hierdie infeksie bly 'n groot openbare gesondheid probleem. Terwyl die WGO aan beveel dat'n geboorte dosis van die entstof, in Suid-Afrika, roetine baba HBV inenting op die ouderdom van ses weke gegee word. Hierdie skedule is gebaseer op data van die pre-MIV era wat getoon het dat die oordrag plaasgevind het via die horisontale, eerder as die vertikale roete. In die era van MIV egter, moeder MIV ko-infeksie kan HBV vrylaat van immuun beheer, wat lei in hoër HBV vlakke en die verhoging van die risiko van vertikale oordrag. Die doel van hierdie studie was om die voorkoms en karakter van die HBV infeksie in MIV-besmette en onbesmette babas te bepaal. Residuele plasma monsters van roetine-MIV-nukleïensuur toetse van 'n 1000 MIV-blootgestelde babas tussen die ouderdomme van 0 en 18 maande van die Wes-Kaap was getoets. Monsters was getoets vir HBsAg deur ELISA (Murex HBsAg Version 3) en bevestig deur neutralisering. HBV DNA is gekwantifiseer deur gebruik te maak van 'n in-huis real-time PCR assay. Babas met HBsAg positiewe monsters was opgevolg en 'n bloedmonster is versamel van moeder en kind. Die HBsAg positiewe monsters was getoets vir HBeAg/antiHBe (Diasorin) en HBsAg negatiewe monsters was getoets vir antiHBs. HBV DNA was gekwantifiseer. Die oppervlak gene volgorde en genotipes was bepaal deur filogenetiese analise met behulp van HepSEQ (www.hepseq.org.uk). Die hele genoom-volgordebepaling was ook uitgevoer. Van die 1000 monsters wat getoets was, was vier monsters positief vir HBsAg en of HBV DNA, dit dui op 'n voorkoms van HBV oordrag van 0.4%. By op volg, twee van die drie besmette babas was positief vir HBsAg, met HBV virale vlakke van groter as 108 IE/ml. Die derde baba was gevind dat sy infeksie opgeklaar het en die vierde kind was verlore as gevolg van op volg. Hierdie besmette babas het almal HBV inenting ontvang. Al vier moeders was HBeAg positief. Volgordebepaling analise het getoon die HBV stamme van die twee babas en vier moeders behoort aan subgenotype A1. Die twee moeder-kind gepaarde rye was homoloë. Die data van hierdie studie toon dat die vertikale oordrag van HBV infeksie in MIV-blootgestelde babas van die Wes-Kaap vind plaas, ten spyte van inenting. Data van die Wes-Kaap, wat 'n HBV voorkoms van 3.4% in MIV-besmette swanger vroue, en dié wat hier aangebied is dui op 'n vertikale oordrag koers van 12% van die HBV. Dit is ten spyte van die wydverspreide gebruik van tenofovir en lamivudine in MIV-geïnfekteerde vroue met 'n lae CD4-telling. Hierdie studie bied data wat ondersteunende oproepe van HBV inenting nader aan die tyd van die geboorte bring. Verdere werk is dringend nodig om die bevindinge te bevestig en die pryse van die oordrag in MIV-blootgestelde babas te bepaal. / National Health Laboratory Service Research Trust / Poliomyelitis Research Foundation (PRF) / Harry Crossley Foundation / Stellenbosch University

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

La lutte pour les ressources est un phénomène qui a débuté dès l'apparition d'organismes reproductifs et dont la description a été initiée par Malthus puis remarquablement synthétisée et étendue à la biologie sous le terme d'évolution par Darwin en 1859 dans « De l'origine des espèces ». Si le concept est ancien à l'échelle des sciences biologiques, il continue à caractériser des domaines à l'époque insoupçonnés par son auteur tels que la virologie. En effet, les virus hautement variables tels que le virus de l'immunodéficience humaine (VIH), de l'hépatite B (VHB) et de l'hépatite C (VHC) sont présents sous forme de quasi espèce au sein de leur environnement réplicatif, c'est à dire qu'une multitude de virus génétiquement proche mais distincts coexistent au sein de cet espace qu'ils doivent partager selon les mêmes règles générales que les êtres vivants. Ainsi, lorsque des pressions de sélection s'exercent (immunitaires, antivirales…), une redistribution des variants majoritaires est observé au bénéfice de variants minoritaires mieux adaptés à cet environnement changeant. La modélisation mathématique et informatique de la capacité mutationnelle et la dynamique d'adaptation des variants minoritaires au travers de 6 études de cohortes de patients infectés, par la technique ultra-sensible de pyroséquençage haut débit associée à des logiciels originaux ont permis de mettre en évidence, caractériser et évaluer l'impact de marqueurs diagnostics permettant de prédire la résistance aux antiviraux mais aussi de caractériser de nouvelles cibles antivirales. / Struggle for resources is a worldwide rule which has been first described by Malthus and extended to whole world of living organisms by Darwin in 1859 in “Origin of species”. Today, this concept has been enlarged to virological field, and is particularly adapted to describe highly variable viruses like Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) which have a quasispecies distribution in infected patients characterized by the co-existence of a number of distinct but related viral populations. Selection pressure on viral replicative environment (immune, antiviral drug treatment…), generally lead to a redistribution of the viral quasispecies with an increasing of the best adapted minor viral variants at the expense of major viral populations. Mathematical and bioinformatic modelization of this phenomenon through 6 infected patients cohorts by means of ultra-deep sequencing and an original bioinformatic package allowed discovery, characterization and evaluation of new diagnostic markers that could be used to prevent resistance emergence to antiviral drugs and to characterized new therapeutics antiviral targets.

Introdução: No mundo especula-se que 15 a 20 milhões tenham infecção crônica pelo HDV. No Brasil, a área endêmica de hepatite Delta corresponde aos estados da Amazônia Ocidental, incluindo Rondônia. Hepatite Delta é a mais grave e com mais rápida evolução para cirrose dentre as hepatites virais. Poucos estudos avaliaram os aspectos epidemiológicos, clínicos e laboratoriais de uma coorte de pacientes em nosso país e no mundo. Objetivos: Em uma coorte de pacientes acompanhados em um serviço de referência: 1. Avaliar as características demográficas, epidemiológicas e clínicas; 2. Avaliar a frequência de doença hepática avançada; 3. Avaliar as características da população atendida com idade <=18 anos; 4. Avaliar a acurácia de escores não invasivos (razão AST/ALT, APRI e FIB-4) na determinação dos diferentes graus de fibrose. Métodos: Trata-se de um estudo transversal, descritivo, de uma coorte de pacientes retrospectivamente identificadas no ambulatório especializado em hepatites virais, pertencente ao Centro de Pesquisa em Medicina Tropical do Estado de Rondônia (CEPEM), situado na cidade de Porto Velho, com diagnóstico de infecção pelo HDV. Foram incluídos todos os pacientes com diagnóstico dessa infecção por sorologia (ELISA) ou por biologia molecular (HDV-RNA reagente), matriculados e atendidos neste serviço entre novembro de 1996 a março de 2015. Resultados: Dentre 4.101 pacientes diagnosticado com HBV, 224 (5,5%) apresentavam coinfecção com o HDV, e 205 foram incluídos nas análises. Dentre eles, 132 (64,4%) eram do sexo masculino, com idade média de 35,1 anos. O contato familiar foi o fator de exposição para infecção pelo VHB/VHD mais frequente. A determinação do genótipo do HDV foi obtida em 78 pacientes, destes 74 (94,9%) eram genótipo III e 4 (5,1%) genótipo I. Noventa e dois (44,9%) pacientes apresentavam evidência de doença hepática avançada. Dentre os pacientes incluídos 22 (10,7%) tinham idade <= 18 anos, sendo que 6 (27,3%) apresentavam sinais e sintomas de doença hepática avançada ou fulminante à primeira consulta. Métodos não invasivos foram calculados e comparados à biópsia hepática em 50 pacientes. A razão AST/ALT não teve valor significativo para avaliar fibrose em nenhum dos estágios. APRI e FIB-4 tiveram melhor desempenho para avaliar fibrose significativa (>=F2), com acurácia de 86 e 80, respectivamente. Conclusões: 1. O HDV representa importante agravo de saúde pública em Rondônia com frequência expressiva entre pessoas do sexo masculino e população indígena; 2. A presença da infecção pelo HDV esteve associada a expressivo número de complicações hepáticas e foi frequente causa de óbito na população analisada, particularmente entre adultos jovens; 3. Entre pacientes com idade <= 18 anos a hepatite delta esteve associada a significante morbidade e mortalidade e a falta de adesão dessa população pareceu contribuir para esse tipo de desfecho; 4. A utilização dos métodos não invasivos (APRI e FIB-4) foi capaz de identificar pacientes com fibrose significativa entre indivíduos infectados com HDV na Amazônia brasileira, podendo, apesar de todas as limitações destes métodos servir como alternativa para avaliação de fibrose hepática significativa, na ausência de outros métodos mais efetivos / Introduction: In the world, it is speculated that 15 to 20 million people have chronic HDV infection. In Brazil, the endemic area of hepatitis Delta corresponds to the states of the Western Amazon, including Rondônia. Hepatitis Delta is the most serious and most rapidly evolving cirrhosis among viral hepatitis. Few studies have evaluated the epidemiological, clinical, and laboratory aspects of a cohort of patients in our country and around the world. Objectives: In a cohort of patients followed at a referral service: 1. Evaluate demographic, epidemiological and clinical characteristics; 2. Assess the frequency of advanced liver disease; 3. Evaluate the characteristics of the population served with age <=18 years; 4. To evaluate the accuracy of non-invasive scores (AST/ALT ratio, APRI and FIB-4) in determining the different degrees of fibrosis. Methods: This is a cross-sectional, descriptive study of a cohort of patients retrospectively identified in the ambulatory specialized in viral hepatitis, belonging to the Research Center of Tropical Medicine of Rondônia State (CEPEM), located in the city of Porto Velho. All patients diagnosed with this serological method (ELISA) or molecular biology (HDV-RNA), enrolled in this service between November 1996 and March 2015, were included. Results: Out of 4,101 patients diagnosed with infection by HBV, 224 (5.5%) had coinfection with the hepatitis delta virus, and 205 were included in the analyzes. Among them, 132 (64.4%) were males, with a mean age at the time of enrollment of 35.1 years. Family contact was the most frequent exposure factor for HBV/HDV infection. It was identified seventy-eight patients (94.9%) of genotype III and four (5.1%) of genotype I. Ninety-two (44.9%) patients had evidence of advanced liver disease. Among the patients included, 22 (10.7%) were aged <= 18 years, and 6 (27.3%) had signs and symptoms of advanced or fulminant liver disease at the first visit. Noninvasive methods were calculated and compared to liver biopsy in 50 patients. The AST/ALT ratio had no significant value for evaluating fibrosis in any of the stages. APRI and FIB-4 had better performance to evaluate significant fibrosis (>=F2), with the accuracy of 86 and 80 respectively. Conclusions: 1. The hepatitis delta virus represents an important public health problem in the State of Rondônia, affecting both adults and children, with significant frequency among males and the indigenous population; 2. The presence of HDV infection was associated with a significant number of hepatic complications and was a frequent cause of death in the analyzed population, particularly among young adults; 3. Among patients aged <= 18 years, delta hepatitis was associated with significant morbidity and mortality and the lack of adherence of this population to follow-up seemed to contribute to this type of outcome; 4. The use of the non-invasive APRI and FIB-4 methods were able to identify patients with significant fibrosis among individuals infected with HDV in the Brazilian Amazon, although all limitations of these methods may serve as an alternative for the evaluation of significant hepatic fibrosis in the absence of other more effective methods