Dissertations / Theses on the topic 'Hepatitis viruses Prevention'

Background: Hepatocellular Carcinoma (HCC) is the third most common cause of cancer related death worldwide and is often diagnosed by measuring serum Alpha-fetoprotein (AFP); a stand-alone biomarker with limited diagnostic proficiency. To compensate for this, AFP is commonly used in conjunction with high performance imaging and radiological methods. However, as the burden of HCC is predominantly in the developing world where such technologies are not readily available, it is imperative that efforts are made to pursue the discovery of novel, high performance, easy to measure and robust biomarkers. With the aim of improving on the diagnostic ability of AFP, our project focuses on the study of plasma proteins as identified by Mass Spectrometry (MS) in order to investigate differences seen in the respective proteomes of controls and subjects with liver cirrhosis (LC) and HCC. Methods: Matrix Assisted Laser Desorption Ionization Time-of-Flight MS (MALDI-TOF MS) was first attempted on weak cation exchange (WCX) fractionated plasma in a pilot selection of forty subjects. On the main case-control group, quantitative MS analysis using liquid chromatography electro spray ionization quadrupole time-of-flight (LC-ESI Q-TOF) was conducted on 339 subjects using a pooled expression profiling approach. Enzyme-linked immunosorbent assays (ELISA) and 1 and 2Dimentional electrophoresis methods were performed to validate and detail candidate protein levels and modification patters in individual and pooled subjects. The human plasma used for the MS based protein discovery experiments was collected as part of a five year Liver Cancer Case-control Study (Gambia, West Africa). A smaller set of samples from subjects who formed a spectrum of non-liver disease controls, LC and HCC were obtained from the Jos University Teaching Hospital (JUTH) in Nigeria and ELISA and gel electrophoresis assays conducted on them to confirm the trends and differences seen in the Gambian subject set. Results: Bioinformatic evaluation of MALDI-TOF data highlighted peak masses 2444m/z, 2583m/z and 2559m/z to have high diagnostic abilities based on area under curve (AUC) statistics of >0.75. Of these polypeptide fragments, one was identified as the plasma glycoprotein, alpha chain fibrinogen. Results from the large-scale label free discovery experiments indicated twenty-six proteins to be differentially expressed between the three subject groups. These prospective markers include proteins previously linked to HCC as well as novel candidates, namely glutathione peroxidase 3, serum amyloid p, carboxypeptidase N and complement factors I and H which have not been implicated in the context of HCC diagnostics. Direct measurement of Hemopexin (HPX), alpha-1-antitrypsin (α1AT), apolipoprotein A1 (Apo A1) and complement component 3 (CC3) levels confirmed their change in abundance in LC and HCC versus control patients. Further biochemical characterization of glycosylated HPX isolated from glycoprotein enriched plasma sample pools showed evidence of isoelectric point shifts, indicating differential glycosylation patterns in high mannose structures of HPX which may be disease stage linked. The direct measurements of HPX, α1AT, Apo A1 & CC3 conducted on the independent Nigerian subject group also confirmed much of the trends reported from the Gambia Liver Cancer Study (GLCS) plasma. Conclusions: The independently validated, significant changes in the quantitative expression of ApoA1, α1AT, CC3 and HPX could be exploited for development into high-performance affordable assays, usable in the diagnosis and monitoring of HCC and LC patients. The unique signatures observed for most of these proteins, from liver disease free controls to LC and HCC suggest their involvement in independent pathways. As such, combining some or all of these four markers within a diagnostic panel could offer a much-needed boost in robustness and accuracy for AFP. The differences in the processing and molecular weight separation of these proteins also offers a novel inroad into biomarker identification. These suggested disease specific signatures could with further study offer highly specific biomarkers able to discern the key stages that predispose individuals to hepatocarcinogenesis. Impact: This is the first MS based discovery and extensive validation study on West African subjects whose primary cause of HCC are the Hepatitis B Virus (HBV) and fungal toxins.

Thesis (MPH))University of Limpopo (Medunsa Campus), 2011.
Introduction: Hepatitis B virus (HBV) is a highly infectious virus responsible for considerable morbidity and mortality world wide. Chronic HBV carriers can transmit HBV parenterally in a hospital setting putting healthcare workers (HCWs) and their patients at risk of infection. Aim and objectives: This study aimed to investigate knowledge, attitudes and practices towards prevention and control of HBV amongst nurses, doctors and laboratory personnel. Objectives were to determine: (a) the knowledge; (b) the attitudes; (c) the practices of nurses, doctors and laboratory personnel; (d) if there are any associations between (1) knowledge and practice, and (2) attitudes and practice; (e) the predictors of HBV vaccination uptake. Materials and Methods: This was a cross-sectional descriptive study. Self-administered questionnaires were distributed to doctors, laboratory staff and nurses at Princess Marina Hospital. Results: Two hundred questionnaires were distributed and a total of 117 were returned, giving an overall response rate of 58.5%. More doctors had good knowledge (38.9% [7/18]), followed by 20% (4/20) of laboratory staff and 11.4% (9/79) of nurses. Most staff (100% [20/20] of laboratory staff; 97.5% [77/79] of nurses; 94.4% [17/18] of doctors) had positive attitudes. More laboratory staff (100 [20/20]) displayed good practices, followed by nurses (94.9% [75/79]); and lastly doctors (88.9% [16/18]). There were no significant associations between knowledge or attitudes and practices. Vaccination was inadequate, with 50.9% (59/116) of HCWs having received at least one dose, and of these only 61% (36/59) receiving all 3 doses. Needle stick injuries occurred in 31.6% (37/117), while 33.9% (39/115) reported blood or body fluid splashes. None of the HCWs accessed PEP after exposure. Being a laboratory worker (OR: 148.4) or doctor (OR: 125.7) were the only predictors of vaccination uptake. Conclusion: There is need to increase knowledge of HCWs, vaccination availability, vaccination uptake, PEP, and reduce the exposures of HCWs.

Hepatitis C virus (HCV) infects hepatocytes of the liver causing progressive liver disease including; fibrosis, cirrhosis and hepatocellular carcinoma. However, the precise mechanism(s) underlying HCV induced liver injury are poorly understood. Hepatocytes are highly polarized with distinct apical and basolateral membranes separated by tight junctions that maintain a normal liver physiology. We studied the role of HCV infection in driving hepatic injury. Our studies show that HCV infection induces hepatocellular reprogramming via hypoxia inducible factor-1α (HIF-1α) stabilization and increased glucocorticoid receptor (GR) signaling. HIF-1α stabilization promoted epithelial to mesenchymal transition accompanied by reduced polarity and cell adhesion. Whereas GR signaling increased cholesterol synthesis and altered HCV receptor expression. Alterations in hepatocellular biology induced a cellular state conducive for virus entry and replication. Consequently, cells de-differentiate to acquire a malignant phenotype via HIF-1α target genes including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF). In addition, GR transcription induced by glucocorticoid treatment or HCV infection enhanced virus uptake, highlighting the caveat for glucocorticoid immunosuppression post liver transplantation. Importantly, HIF-1α inhibitors and GR antagonist reversed the effects of both transcription factors on virus infection and hepatocellular biology. These findings suggest that HCV potentiate liver injury via indirect mechanisms.

Thesis (MPH)--University of Limpopo, 2010.
Introduction: Hepatitis B infection is a serious blood-borne disease caused by the hepatitis B virus (HBV) which attacks the liver, and is the leading cause of liver cancer and cirrhosis of the liver. HBV can be transmitted through exposure to infected blood and human secretions through needle stick / sharps injuries and splashes. Thus nurses are at high risk for HBV infection. The aim of the study: To investigate the knowledge, attitudes and practices (KAP) regarding the prevention of hepatitis B virus infections, in final year college student nurses in Gauteng province. Methods: A cross-sectional quantitative survey on 350 final year nursing students was conducted in three Gauteng province nursing colleges, using an anonymous self administered questionnaire with questions on knowledge, attitudes, and practices regarding HBV. The data were analysed using SPSS (statistical package for social science studies). Results: Of 350 questionnaires distributed, 312 student nurses returned completed forms (response rate: 89.14% [312/350]). The majority were females (86.8% [270/331]) and were below 31 years of age (30.1% [93/309]). The majority (87.6% [271/310]) had good knowledge of the causes and prevention of HBV. The unvaccinated respondents had fairly low positive attitudes, with a mean, mode and median score of 1 (possible score from -4 to +4). The majority (79% [244/310]) practiced good compliance with universal precautions of, and the majority (64.9% [202/311]) were vaccinated. College A displayed significantly higher knowledge (p<0.001), positive attitudes (p=0.001) and safer practices (p<0.001) than college B and C.

Hepatitis C virus (HCV) is a major human pathogen and the leading cause of cirrhosis and liver cancer worldwide. HCV entry is clathrin- and pH-dependent, and requires CD81, Scavenger receptor BI (SR-BI), and the tight junction (TJ) proteins Claudin-1 and Occludin. Primary HCV strains cannot be efficiently cultured in vitro, making the evaluation of potential antiviral compounds in a biologically relevant context extremely difficult. Despite being suitable for high-throughput screening, most cell-based reporter assays rely on the secretion of serine alkaline phosphatase and thus do not allow the selection of HCV infected cells, or the screening of patient samples to identify cell culture infectious viral strains. We aimed to develop a cell-based reporter assay, which utilizes the viral NS3/4A protease to cleave and activate a fluorescent reporter protein constitutively expressed in Huh-7.5 hepatoma cells. HCV tropism is restricted to the liver, where hepatocytes are polarized and form TJ, which are indispensable for normal liver functionality. We demonstrate that in confluent cells, SR-BI and Claudin-1 expression is increased and that HCV entry is enhanced when cellular contact is established. Furthermore, cell junction formation and SR-BI overexpression, respectively, accelerated virus entry, suggesting a key role for SR-BI in HCV internalization. The mechanisms underlying HCV-associated hepatic injury are poorly understood, however, it is thought that HCV may disrupt TJ integrity, thus compromising hepatocyte polarity and function. We demonstrate that the HCV structural proteins modulate the expression and localization of TJ proteins, leading to their redistribution to cytoplasmic vesicles with possible consequences for TJ integrity in vivo.

Thesis (D Phil. (Medical Virology))--2008.
Background and Objectives: South Africa is one of the countries highly affected by human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections. Some drugs (e.g. lamivudine) used as part of combination antiretroviral regimens for HIV treatment have dual activity against HBV and HIV. Despite high infection rate with both viruses, routine screening for HBV before initiation of treatment for HIV is not yet a standard practice. This study undertook to investigate: (1) the burden of HBV co-infection in HIV-positive patients enrolling for highly active antiretroviral therapy (HAART) at Dr George Mukhari hospital, (2) the impact of anti-HBV containing HAART regimens on HBV during the management of HBV/HIV co-infected patients, (3) the co-evolution of HBV and HIV drug-resistant strains, and (4) the correlation of HBV genotypes with response to anti-HBV containing HAART regimens. Study Population and Methods: To investigate the burden of HBV/HIV co-infections, a cohort of 192 HIV patients who were candidates for ARV treatment at Dr George Mukhari hospital were studied by screening for HBV serological markers (HBsAg, anti-HBs and anti- HBc) (Elecsys 2010, Roche Diagnostics) and HBV DNA with an in-house nested PCR assay targeting HBV polymerase gene. Quantitation of HBV DNA positive samples was performed with Roche Cobas Taqman HBV test 48 assay. To investigate the impact of lamivudine-containing HAART regimens on HBV during the management of HBV/HIV co-infected patients, as well as the coevolution of HBV and HIV drug-resistant strains, a total of 78 patients were studied. HBV virological response against lamivudine containing-HAART regimens [1a (lamivudine, stavudine and efaverenz); 1b (lamivudine, stavudine and neviripine)] was measured (Cobas Taqman HBV test 48, Roche diagnostics). HBV direct sequencing targeting HBV polymerase gene was performed on all baseline samples (n=78) and additional samples collected at various time points (n = 45). Direct sequencing was also performed on 30 HIV baseline samples targeting the HIV reverse transcriptase and protease genes (Spectru-Medix SCE 2410 Genetic Analysis System and ABI PRISM® 3100 Genetic Analyzer version 3.7). To explore the genetic diversity of HBV and HIV strains circulating in Pretoria and surrounding areas, as well as the correlation of HBV genotypes with response to lamivudine-containing-HAART regimens in co-infected patients, all baseline and follow-up HBV and HIV sequences were analysed, compared and correlated with treatment. Sequence alignments and phylogenetic studies for both HBV and vi HIV were conducted with MAFFT, Mega 4 and neighbour joining phylogenetic trees generated with the PHYLIP programme. Results: Three significant findings were observed in this study. Firstly, the majority of South African HIV patients enrolling for HAART were exposed to HBV infection and either had acute or chronic HBV infections. A total of 63.0% of patients were found to have one or more HBV markers, with 40.6% having detectable HBV DNA as an indication of replication. The study also detected 22.9% with positive HBsAg, and 23% of 77% HBsAg-negative patients having occult hepatitis B infection. Secondly, HBV/HIV co-infected patients do benefit during the management of HIV infections with lamivudine-containing HAART regimens. A total of 68.4% of patients responded to HAART, with undetectable HBV DNA during 18 to 24 months of follow-up. A total of 91.3% of HIV patients also responded to HAART with an undetectable HIV viral load during 6 to 12 months of follow-up. However, a total of 18% of patients had persistent HBV DNA, yielding various HBV virological responses against lamivudine containing-HAART regimens. This proportion of patients poses a question regarding the management of HBV and HIV coinfections, as guidelines on the use of HAART with anti-HBV activity from developed countries, may not necessarily be followed in developing countries. The results further showed that baseline drug-resistance was more frequent with HIV than HBV in this cohort of patients. The following HIV primary drug resistant mutants were observed: nine major NRT's primary mutants, M41L (1/30), E44A (1/30), V75M (1/30), F77L (1/30), V118I (1/30), M184V (1/30), L210S (1/30), T215Y (1/30) and V90I (1/30), and five major NNRT’s primary mutants were also detected, K103N (3/30), Y318CFSY (1/30), E138Q (1/30), P225H (1/30) and K238T. However, all followup samples had undetectable HIV viral load. In contrast to HIV, only one patient was detected with HBV mutant, M204I, at baseline. The mutant reversed to wild type during 6 months and other follow-up (12, 18 and 24 months). Finally, this study indicated that the HBV genotype A is still the most prevalent genotype circulating in South Africa. Of the 78 HBV sequences, 77 were genotype A and 1 sequence was genotype G. This is the first report from Africa of the detection of HBV genotype G. HIV subtype C remains the predominant prevailing subtype in South Africa. HBV genotype or HIV subtype C was not observed to influence any treatment outcome following treatment with vii lamivudine-containing HAART regimens. The study also indicated that patients on lamivudinecontaining HAART regimens do benefit not only by suppressing HIV and HBV viral load, but also improving immunity (i.e. CD4 cells count increases). Conclusion: Overall, the present study highlights the need for screening HBV before initiation of any HAART containing anti-HBV regimens in HBV/HIV co-infected patients. It necessitates the use of molecular assays for effective laboratory in diagnosis of occult HBV infections in HIVpositive patients, especially in developing countries where these assays are not widely available. While lamivudine-containing HAART regimens do benefit both HBV and HIV patients in co-infected individuals, however, whether HBV virological response is temporary or sustained is unknown at this stage. What is certain is that these patients require an effective monitoring programme as (1) a small percentage experience variable HBV virological responses (partial, reactivation, or no response), and (2) hepatitic flares are likely to develop if HAART is terminated (e.g. by patient), or the current HAART regimen is switched to another regimen without anti-HBV activity. HBV genotype A remains the dominant genotype in South Africa, but novel genotypes can be detected. HIV subtype C was found to be the prevalent subtype. HBV genotype or HIV subtype C were not seen to influence any treatment outcome following treatment with lamivudine-containing HAART regimens. Recommendations: HIV patients should be screened for HBV before initiation of anti-HBV containing HAART regimens. The screening of HBV in HIV patients is also important since some drugs included as part of HAART (e.g. nevirapine) may cause hepatotoxicity and exacerbate HBV infections leading to increased morbidity and mortality due to liver complications. Immunization and immune boosters of HIV patients with low (< 10IU/L) or no immunity against HBV should be done as this could be beneficial, although these patients may not respond optimally, or their immunity may wane faster due to immunocompromised status. Monitoring of both HBV and HIV resistant strains should be conducted for timely detection for the occurrence of multiple resistant mutations, which could limit future therapeutic option for both viruses.

A forward projection model was used to estimate the numbers of, both current and former, IDUs who acquired HCV infection and progressed to mild, moderate and severe HCV disease in Glasgow and Scotland between 1960 and 2030. The model was developed initially for Glasgow because more epidemiological information exists for this region, than elsewhere in Scotland, to calibrate model outcomes with local data relating to HCV and its consequence. Insights gained from the model fitting process in Glasgow were then used to extend the model to the rest of Scotland. First, the incidence and cessation of injecting drug use in Glasgow during 1960-2000 were derived through the use of a modified Delphi approach. Instead of the usual iterative process to refine experts’ estimates, the elicitation of IDU incidence and cessation provided an opportunity to combine these data and examine coherence with capture-recapture IDU prevalence estimates. Coherent estimates indicated that incidence (median: 28 to 49) and cessation (1 to 24%) remained low and stable during 1960-1975, rose steeply between 1975-1985 (incidence from 49 to 1,335; cessation from 2% to 6%), and by 2000 there had been a decline in incidence (1,195) but a further rise in cessation (15%). Secondly, stochastic simulation was used to model the transmission of HCV among current IDUs in Glasgow, according to their injecting risk behaviours, and estimate the past incidence of HCV infection. The model that considered higher infectivity during acute viraemia following infection produced seroprevalences (median: 62-72%) and incidences (18-30 per 100 susceptible injector-years) consistent with observed data during the 1990s. The annual number of new HCV infections among current IDUs in Glasgow was estimated to be low during 1960-1976 (median: 10-60), rise steeply during 1960-1976 (median: 10-60), rise steeply during the early 1980s to peak in 1985 (1,120), stabilise during 1991-1997 (510-610) and rise again during 1998-2000 (710-780). Scenario analyses indicated that potentially as many as 4,500 HCV infections (10th and 90th percentiles: 2,400-7,700) had been prevented in Glasgow during 1988-2000 as a result of harm-reduction measures. Scenario analyses also permitted the gauging of changes in risk behaviours required to effect appreciable reductions in the incidence of HCV infection. Incidence can be successfully reduced if IDUs who, unavoidably, share needles/syringes confine their borrowing to one person; with this strategy alone, an estimated 5,300 HCV infections (10th and 90th percentiles: 4,100-6,700) could have been averted in Glasgow during 1988-2000. Such insights will inform those responsible for developing new ways to prevent HCV transmission among IDU populations. Thirdly, linkage of laboratory data on diagnosed HCV antibody positive persons in Scotland to clinical data from hospital and death records provided a unique national epidemiological dataset to estimate the number who had progressed to severe HCV disease.

Hepatitis C Virus (HCV) is a global health problem, with over 170 million infected individuals worldwide. 70-80% of infected individuals develop progressive disease, and approximately 2% of these acquire hepatocellular carcinoma (HCC). HCV entry is dependent on tetraspanin CD81, scavenger receptor BI, and tight junction proteins claudin-1 and occludin. Tetraspanins are involved in multiple biological functions including cell-ECM adhesion and motility. An actin polymerization-dependent cell spread was observed upon ligation of CD81 on hepatoma cells. Importantly, HCV infection perturbed CD81-dependent cell spread, suggesting HCV infection may modulate CD81 function in hepatoma cells. Functional assays demonstrated that CD81 expression and HCV infection promote hepatoma cell motility. These findings allude to a link between HCV infection and associated HCC development. Establishment of a chronic infection demonstrates that HCV can escape from the host adaptive immune responses. We developed an in vitro cell culture system to monitor viral transmission in the presence of neutralizing antibodies (nAb). Separation of producer and target cells ablated nAb resistant transmission, suggesting that cell-cell contact was essential. Furthermore nAb resistant transmission was dependent upon all four co-receptors. These observations confirm HCV immune evasion by cell-to-cell transfer and have major implications for anti-glycoprotein targeted therapies.

The hepatitis C virus (HCV) is highly prevalent among people who inject drugs (PWID) in Scotland and the large majority of new HCV infections occurring in Scotland are within this population group. Harm reduction interventions, mainly sterile injecting equipment provision (IEP) and opioid substitution treatment (OST), to prevent the transmission of blood-borne viruses among PWID, were implemented in Scotland in the late 1980s/early 1990s. More recently, government policy initiatives, particularly the Hepatitis C Action Plan for Scotland, have stipulated the scale-up of these interventions. The overarching aim of this thesis was to investigate the impact of harm reduction interventions on the transmission of HCV among PWID in Scotland. Five secondary objectives were addressed in order to fulfil the main aim: (i) to review the international literature on the effectiveness of IEP and OST in preventing HCV transmission; (ii) to determine the association between self-reported sharing of needles/syringes and incident/prevalent HCV infection; (iii) to determine the association between sharing non-needle/syringe injecting paraphernalia and incident HCV infection; (iv) to determine the incidence of HCV among PWID in Scotland; and (v) to determine the association between self-reported uptake of IEP/OST and incident HCV infection. To address the first thesis objective, a systematic review of the literature was undertaken to identify existing international research evidence (published up to March 2007) for the effectiveness of harm reduction interventions. While HCV was the main outcome of interest, HIV and injecting risk behaviour (IRB) were also considered. A review of reviews approach identified: insufficient evidence that sterile needle and syringe provision (NSP) was effective in preventing HCV transmission; tentative evidence that NSP was effective in preventing HIV transmission; sufficient evidence to support the effectiveness of NSP in reducing self-reported IRB; and little to no evidence on needle/syringe vending machines, outreach NSP or the provision of other injecting paraphernalia (spoons, filters, water) in relation to any of the outcomes. With regard to OST, the findings were: insufficient evidence to show that OST has an impact on HCV transmission; sufficient evidence to support the effectiveness of continuous OST in reducing HIV transmission; and sufficient evidence to support the effectiveness of OST in reducing IRB by reducing the frequency of injection, the sharing of injecting equipment and injecting risk scores. An update to the review of reviews was undertaken to include literature published through March 2011, and found that little changed as a result of additional published reviews: in the main, the evidence statement for the effectiveness of OST with regard to HCV was upgraded from insufficient to tentative. The finding of weaker evidence with regard to biological outcomes (e.g. HCV, HIV), as compared with behavioural outcomes, indicated that low levels of IRB may be insufficient to reduce high levels of transmission, particularly for HCV. The subsequent chapter aimed to address the second thesis objective, by summarising, and exploring factors that explained the variation in, the measure of association between self-reported sharing of needles/syringes and HCV prevalence/incidence among PWID. A systematic review and meta-analysis were undertaken to identify and combine the results of European studies of HCV prevalence (or incidence) among those who reported ever/never (or recent/non-recent) sharing of needles/syringes. Among the 16 cross-sectional studies and four longitudinal studies identified, the pooled prevalence of HCV was 59% among PWID who reported never sharing needles/syringes and the pooled incidence of HCV was 11% among PWID who reported not recently sharing needles/syringes. Random effects meta-analysis generated a pooled odds ratio (OR) of 3.3 (95% confidence interval [CI] 2.4-4.6), comparing HCV infection among those who ever (or recently) shared needles/syringes relative to those who reported never (or not recently) sharing. Differences in pooled ORs were found when studies were stratified by recruitment setting (prison vs. drug treatment sites), recruitment method (outreach vs. non-outreach), sample HCV prevalence and sample mean/median time since onset of injecting. High incidence/prevalence rates among those who did not report sharing needles/syringes during the risk period may be a result of a combination of unmeasured risk factors (such as sharing non-needle/syringe injecting paraphernalia) and reporting bias. Study design and population were found to be modifiers of the size and strength of association between HCV and needle/syringe-sharing. To address the third thesis objective, the risk of HCV associated with sharing injecting paraphernalia (spoons, filters and water) was investigated using data from the 2008-09 and 2010 sweeps in a series of national cross-sectional surveys of PWID in Scotland, collectively called the Needle Exchange Surveillance Initiative (NESI). Logistic regression was used to examine the association between recent HCV infection (anti-HCV negative and HCV-RNA positive individuals) and self-reported measures of injecting equipment sharing in the six months preceding interview. Twelve percent of the sample reported sharing needles/syringes and 40% reported sharing paraphernalia in the previous six months. The adjusted odds ratios (AORs) for sharing needles/syringes (with or without paraphernalia) and sharing only paraphernalia in the last six months were 6.7 (95% CI 2.6-17.1) and 3.0 (95% CI 1.2-7.5), respectively. Among those who reported not sharing needles/syringes, sharing spoons and sharing filters were significantly associated with recent HCV infection (AOR 3.1, 95% CI 1.3-7.8 and 3.1, 95% CI 1.3-7.5, respectively); sharing water was not. This cross-sectional approach to the analysis of the association between sharing paraphernalia and incident HCV infection demonstrated consistent results with previous longitudinal studies. The prevalence of paraphernalia-sharing in the study population was high, potentially representing a significant source of HCV transmission. Addressing the fourth and fifth thesis objectives, a method to determine the incidence of HCV among PWID using a cross-sectional design was applied, and the associations between self-reported uptake of harm reduction interventions (OST and IEP) and recent HCV infection were examined. This was undertaken on data from the first sweep (2008-09) of NESI. Twenty-four recent HCV infections (as defined above) were detected, yielding incidence rate estimates ranging from 10.8-21.9 per 100 person-years. After adjustment for confounders, those with high needle/syringe coverage had reduced odds of recent infection (AOR 0.32, 95% CI 0.10-1.00, p=0.050). In the Greater Glasgow and Clyde region only, there were reduced odds of recent infection among those currently receiving OST, relative to those on OST in the last six months but not currently (AOR 0.04, 95% CI 0.001-1.07, p=0.055). The effect of combined uptake of OST and high needle/syringe coverage was only significant in unadjusted analyses (OR 0.34, 95% CI 0.12-0.97, p=0.043; AOR 0.48, 95% CI 0.16-1.48, p=0.203). The final analysis chapter built on the previous chapter investigating the association between uptake of harm reduction interventions and recent HCV infection, by using data from three sweeps of the NESI survey, undertaken in 2008-09, 2010 and 2011-12. A framework to triangulate different types of evidence – ‘group-level/ecological’ and ‘individual-level’ – was applied. Data on service provision (injecting equipment provision and methadone dispensation) were also collated and analysed.

Hepatitis B virus (HBV) is hyperendemic to southern Africa and parts of Asia where it is a major cause of serious liver disease. Licensed antivirals for chronically infected individuals are only partially effective and approximately one million deaths occur annually as a result ofpersistent infection with the virus. Although RNA interference (RNAi) based gene silencingof HBV has been successfully demonstrated, difficulties with delivery of anti-HBV RNAieffectors remains an obstacle to their clinical use. Recombinant adenoviruses (Ads), amongst the most efficient hepatotropic gene vectors following systemic administration, have been successfully used to deliver expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate andadaptive immunity.To attenuate these effects we used polyethylene glycol (PEG) to modify first generation recombinant Ad (FG Ad) vectors that express an anti-HBV short hairpin (shRNA) sequence. Efficient hepatocyte transduction occurred and expressed shRNAs were processed to generate intended HBV-targeting guides. Inhibition of HBV replication was achieved after intravenous administration of PEGylated or native recombinant first generation Ads (FG Ads) to HBV transgenic mice. Circulating HBV viral particle equivalents (VPEs) remained low for 3 weeks and began to increase after 5 weeks. A second dose of PEGylated anti-HBV Ad caused a less sustained decrease in circulating VPEs, but no silencing after a second dose was observed in animals treated with unmodified vector. Release of inflammatory cytokines was elevated in animals receiving unmodified vectors and only a modest increase in monocyte chemotactic protein-1 (MCP-1) was observed in mice that received a second dose of PEG Abstract Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts. To address concerns about the transient nature of transgene expression by FG Ads resulting from immunostimulation, third generation helper-dependent (HD Ad) were utilised to delivered anti-HBV RNAi effectors. Seven days after intravenous administration of infectious HD Ads to HBV transgenic mice, 80-90% of hepatocytes were transduced and markers of HBV replication were decreased by approximately 95% which was sustained for 8 weeks. HD Ad-induced release of proinflammatory cytokines was minimal in preparations that were enriched with infectious particles. PEGylated HD Ad vectors caused similar anti- HBV effects and may be useful to evade interaction with vector-sequestrating receptors and further attenuate immunostimulation. Collectively these observations indicate that PEG modification of Ads and the use of HD Ads may have utility for delivery of therapeutic HBVsilencing sequences. Future work will focus on improving strategies to avoid immune detection and utilisation of HD Ad vectors for other HBV targeting sequences.

Hepatitis B virus (HBV) is a major global public health burden, with over 350 million people chronically infected. This results in approximately 600,000 liver cancer-related deaths annually. Chronic HBV infections are normally managed with long-term anti-HBV therapeutics, such as reverse transcription inhibitors, which target post-transcriptional viral processes without affecting the cccDNA. Treatment failure however is largely as a result of the stability of this episomal viral DNA. The cccDNA minichromosome serves as a reservoir of HBV DNA and is capable of re-establishing viral replication following withdrawal of treatment. Designer nucleases, like transcription activator-like effector nucleases (TALENs), have recently been used to create double stranded breaks (DSBs) at target-specific endogenous DNA loci. These nucleases are designed as pairs, which upon dimerisation cleave double-stranded DNA. Subsequent activation of the cellular non-homologous end-joining (NHEJ) pathway often results in targeted mutagenesis at the DSB site. As TALENs may be designed to bind to any DNA sequence, they are commonly used as genetic engineering agents. Inactivation of HBV cccDNA, using these engineered TALENs, presents a unique approach to disabling viral replication permanently. To investigate this, a panel of TALENs targeting the core (C), surface (S) and two different polymerase (P1 and P2) regions of HBV cccDNA were generated using a Golden gate modular assembly approach. TALENs were initially tested in two liver-derived cell lines. Firstly as transient co-transfections in Huh7 cells using a HBV replication competent plasmid, followed by long term investigations in HepG2.2.15 cells which model HBV replication in vitro. Inactivation of HBV was determined by measuring markers of viral replication, whilst TALEN-mediated targeted disruption was verified by T7 endonuclease 1 (T7E1) or CELI endonuclease assays and sequencing. In vitro, the S TALEN inhibited HBsAg secretion by 80% in Huh7 cells and 60% in HepG2.2.15 cells. Furthermore, S TALEN-mediated targeted disruption occurred in 35-47% of cccDNA copies, whilst the C TALEN resulted in 11% targeted disruption of cccDNA in without inhibition of HBsAg expression. The P2 TALEN showed no anti-HBV efficacy, however the P1 TALEN inhibited HBsAg expression by up to 60% without any evidence of site-directed cleavage. As this TALEN binding site spans the HBV Enhancer I sequence, knock-down of HBsAg expression is most likely to occur as a result of transient transcriptional repression. To confirm whether permanent repression of HBV transcription could be achieved, a KRAB-based transcription activator-like repressor (rTALE) targeting the HBV pre-S2 promoter was generated. Using an in vitro reporter gene assay, the pre-S2 rTALE inhibited luciferase expression by up to 90%. However this was only achieved using high molar concentrations of the repressor, suggesting multiple rTALEs may improve HBV transcriptional repression. As the S and C TALENs displayed significant anti-HBV efficiency in vitro, they were tested in a murine hydrodynamic injection model of HBV replication. In vivo, the S TALEN inhibited HBsAg secretion by 95% and induced disruption in 77–87% of HBV DNA targets. In addition the C TALEN inhibited HBcAg expression and induced disruption in 78-93% of HBV DNA targets. Additionally, serological analysis showed a reduction in circulating virions and no apparent liver toxicity, as determined by real-time PCR (qPCR) and aspartate transaminase (AST)/ alanine aminotransferase (ALT) liver function tests respectively. Deep sequencing at the S and C TALEN binding sites showed targeted mutagenesis of HBV DNA in samples extracted from murine hepatocytes transfected with TALENs, however wild-type sequences were exclusively detected in mice that had not been treated with anti-HBV TALENs. Furthermore, frameshift deletions were predominantly detected indicating major disruptions in the HBV surface and core sequences. These results indicate that TALENs designed to disable and silence HBV cccDNA are effective both in vitro and in vivo and as such provide a promising therapeutic approach to treat this serious infection.

博士
臺灣大學
預防醫學研究所
98
Background Hepatitis B virus infection is a major health problem worldwide. Understanding the mode of HBV transmission route is important but it is difficult to observe directly so mathematical modeling is an alternative approach. There are multi-level prevention strategies for perinantal transmission including immunization, immunoglobulin injection and maternal lamivudine prophylaxis use in third gestation trimester. Moreover, there is lacking of evaluation, particularly long-term follow-up, of the cost and effectiveness of these prevention strategies simultaneously for the hepatitis B virus infection and its related sequelae. The current thesis focused on three major themes, including (1) modeling the dynamics of HBV infection with a stochastic process making allowance for the mixture proportion of vertical transmission and horizontal transmission; (2) assessing the efficacy of lamivudine use in arresting perinatal transmission through a meta-analysis of literatures; (3) economic evaluation of long-term effectiveness and cost-effectiveness of universal vaccination and lamivudine use. Method For transmission mode, a five-state stochastic model, including susceptible, latent period, active viral replication, carrier and recovery, was use to quantify the dynamics of HBV infection with the consideration of vertical transmission and horizontal transmission. By including three studies on maternal lamivudine use in late pregnancy, a meta-analysis using a Bayesian approach was conducted. A decision analysis was performed to compare total costs and effectiveness between the prevention strategies: universal vaccination and no-vaccination in the year of 1984 and maternal lamivudine use and universal vaccination in the year of 2008. The Markov process was defined as a series of states including acute hepatitis B virus infection, asymptomatic carrier, chronic hepatitis, compensated and decompensated cirrhosis, hepatoma, and death. The incremental cost-effectiveness ratio per quality-adjusted life years gained and acute infection averted calculated at a 3% discount rate. By assigning a series of specific distributions to each parameter, a probabilistic cost-effective analysis using Monte Carlo simulation was conducted to yield 5000 incremental cost-effectiveness ratio replicates. Result In five-state model, the transmission parameter of annual infection rate for susceptible subjects was estimated to be 1.1-3.5% and 0.072-1.112 per year from latent period to active viral replication. Approximately with rate of 0.119-0.184 per year active viral replication may leave as chronic carrier. Annual recovery rates were 1.4-2.4% and 4.8-8.8% for carrier and transient viremia, respectively. Approximately 41.4-80.2% infected subjects were derived from vertical transmission. After allowing for heterogeneity, the efficacy of the maternal lamivudine use statistically significantly halves perinatal transmission compared with the untreated group (RR= 0.52, 95% CI: 0.24-0.94). The effectiveness of a universal vaccination program for reducing hepatocellular carcinoma cases and deaths was approximately 86% compared to no vaccination. The average life years gained per subject as a result of such a universal vaccination was 3.9. The vaccination program dominated over a no-vaccination program (less cost and more effectiveness). Given literature review on the application of lamivudine, the overall efficacy in reducing Hepatitis B virus DNA level was 98.3 % and 48 % in reducing vertical transmission with outcomes measured by HBsAg or Anti-HBc. The incremental cost-effectiveness ratio for prophylactic strategy versus routine active-passive immunoprophylaxis was $47,059 from health care payer perspective whereas dominance has been noted for societal viewpoint. Conclusion Prophylactic lamivudine use can reduce 50% chance of perinatal transmission related to hepatitis B virus infection based on integrated empirical data. The proportion of vertical transmission among all HBV infection varies even in different areas of the same country, which was higher in southern area (80%) and lower in northern area (41%) of Taiwan. The vaccination program dominated over a no-vaccination program (less cost and more effectiveness). The incremental cost-effectiveness ratio for prophylactic maternal lamivudine use versus routine universal vaccination was $47,059 from health care payer perspective whereas dominance has been noted for societal viewpoint. These findings suggest the administration of lamivudine to mothers with high HBV viremia added to current vaccination strategy may improve the vaccination breakthrough and seem to be cost-effective.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2015
A Hepatite C tornou-se um relevante problema de saúde pública. Cerca de 3% da população mundial está infectada, no entanto, a prevalência nas crianças é baixa. Distintamente dos adultos, em que a principal via de transmissão é o uso de drogas injectáveis, actualmente, na idade pediátrica, a transmissão vertical é a principal causa. Na sua maioria, as crianças infectadas pelo vírus da Hepatite C são assintomáticas e apresentam uma progressão geralmente lenta da doença. Ainda assim, alguns grupos seleccionados podem beneficiar de tratamento em idade pediátrica com interferão (IFN) alfa peguilado e ribavarina. A abordagem da Hepatite C na criança pode mudar drasticamente num futuro próximo com a introdução de regimes terapêuticos sem IFN, adequados à populacão pediátrica. Esta revisão tem como objectivo proporcionar informação actualizada sobre a abordagem da Hepatite C na idade pediátrica e perspectivar as diferenças desta infecção no adulto e na criança.
Hepatitis C virus (HCV) infection has become a public health issue. About 3% of the world’s population is affected by HCV. However, prevalence of HCV infection in children is low. Unlike adults, in which the main route of transmission is by intravenous drug use, the most common via of HCV infection in children is, currently, vertical transmission by HCV infected mothers. Nearly all of infected children are asymptomatic, showing a slowly progressive course of disease. Nevertheless, selected children may benefit from treatment with pegylated interferon (IFN)-alfa plus ribavirin. The possibility of IFN-free regiments, suitable for pediatric population, can drastically chance the management of HCV infection in children in the near future. The objective of this review is to provide updated information about the management of HCV infection in the pediatric age group, as well as the differences between the HCV infection in children and adults.

碩士
國立成功大學
護理學系碩博士班
99
Purpose: The theory of planned behavior was applied to frame and determine the correlates, in order to predict the behaviors required for preventing hepatitis C virus (HCV) infection. Method: The cross-sectional design used to survey related information. We recruited 238 residents who were aged above 20, from a remote rural area with HCV endemic in northern area of southern Taiwan. After informed consent given, 193 subjects completed the survey. The response rate was 81.1%. The structured questionnaire, which was well developed and examined for content validity and internal consistency reliability, was used to generate data, collected by a well-trained interviewer in a community activity center. The predictors of behaviors for HCV prevention were determined by stepwise multiple linear regression. Results: After adjusting for age, the indicators and the power estimated including: intentions at 49%, family support at 12.8% and perceived behavioral controls at 1.2% could significantly predict the behaviors for preventing HCV infection, respectively. Conclusion: We suggest that intention be promoted firstly; it would effectively improve residents』 execution of activities related to the prevention of behaviors related to HCV infection. Family support is positively effective as well, in regard to HCV prevention.

Indiana University-Purdue University Indianapolis (IUPUI)
Approximately 2.8% of the world population is currently infected with hepatitis C virus (HCV). Neutralizing antibodies (nAbs) are often generated in chronic hepatitis C patients yet fail to control the infection. In the first two chapters of this study, we focused on two alternative routes of HCV transmission, which may contribute to HCV’s immune evasion and establishment of chronic infection. HCV was transmitted via a cell-cell contact-mediated (CCCM) route and in the form of exosomes. Formation of HCV infection foci resulted from CCCM HCV transfer and was cell density-dependent. Moreover, CCCM HCV transfer occurred rapidly, involved all four known HCV receptors and intact actin cytoskeleton, and led to productive HCV infection. Furthermore, live cell imaging revealed the temporal and spatial details of the transfer process. Lastly, HCV from HCV-infected hepatocytes and patient plasma occurred in both exosome-free and exosome-associated forms and the exosome-associated HCV remained infectious, even though HCV infection did not significantly alter exosome secretion. In the third chapter, we characterized HCV interaction with astrocytes, one of the putative HCV target cells in the brain. HCV infection causes the central nervous system (CNS) abnormalities in more than 50% of chronically infected subjects but the underlying mechanisms are largely unknown. We showed that primary human astrocytes (PHA) were very inefficiently infected by HCV, either in the free virus form or through cell-cell contact. PHA expressed all known HCV receptors but failed to support HCV entry. HCV IRES-mediated translation was functional in PHA and further enhanced by miR122 expression. Nevertheless, PHA did not support HCV replication regardless of miR122 expression. To our great surprise, HCV exposure induced robust IL-18 expression in PHA and exhibited direct neurotoxicity. In summary, we showed that CCCM HCV transfer and exosome-mediated HCV infection constituted important routes for HCV infection and dissemination and that astrocytes did not support productive HCV infection and replication, but HCV interactions with astrocytes and neurons alone might be sufficient to cause CNS dysfunction. These findings provide new insights into HCV infection of hepatocytes and astrocytes and shall aid in the development of new and effective strategies for preventing and treating HCV infection.