Degree awarded with distinction on 6 December l995. A dissertation submitted to the Faculty of Science, University the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. March. 1995 / Hepatitis B virus (HBV) infection resulting in integration of the viral DNA into host liver cell DNA is associated with the development of hepatocellular carcinoma (HCC). This is indicated by epidemiological trends, molecular studies and studies of animal models infected with viruses closely related to HBv. However, little is known about the mechanism by which the integrated HBV DNA includes HCC despite continuing analysis of the integrated HBV DNA and its surrounding cellular sequences. [Abbreviated Abstract. Open document to view full version]

Ph.D. thesis, Faculty of Health Sciences,University of the Witwatersrand, 2009 / Hepatitis B virus (HBV) infection is endemic in Africa. As many as 98% of black Africans are infected during their lives and about 10% (65 million) have chronic HBV infection, which is the cause of 70-80% of all hepatocellular carcinoma (HCC) cases. Despite this high prevalence of HBV and the high incidence of HCC in Africa, relatively few complete HBV genomes from African HCC cases have been deposited in international data bases. In order to gain a clearer understanding of the role of genetic variants and mutants in the development of HCC, the complete genomes of HBV isolated from southern African HCC patients were amplified and molecularly characterized. HBV DNA was extracted from the serum forty HBsAgpositive HCC patients. Twenty six complete genomes were successfully amplified, cloned and sequenced from nine HCC patients. Phylogenetic analyses of the complete genomes and the individual open reading frames of HBV isolates from the HCC patients, led to the classification of all the isolates within subgenotype A1. No isolates belonging to subgenotype A2 and genotype D were identified, even though these genotypes/subgenotypes have been shown to circulate in South Africa. Three patients contained the uncommon combination of serological subtype ayw1 in the subgenotype A1 strain. This combination has been found previously in South Africa and the Phillipines. Seventy-eight percent of the patients carried HBV strains with the double basic core promoter (BCP) mutation (1762T/1764A), previously shown to reduce HBeAg expression. Furthermore, complete genome sequence analysis has revealed a complex combination of mutations, which include at least three or five of these residues 1753C1762T1764A1766T1768A1809T1812T occurring as the dominant HBV strains isolated from 5/9 HCC patients. These mutations have previously been shown to regulate gene expression at various levels, to enhance viral replication and simultaneously decrease HBeAg expression. All five HBV genomes isolated from one patient contained novel complex BCP rearrangements, which introduced 2 HNF1 and 1 putative HNF3 transcription factor binding sites. These mutations can enhance viral replication and simultaneously abolish HBeAg expression at a transcriptional level. Furthermore, truncated core proteins would be expressed from 4/5 isolates and none would express wild-type HBx. Several mutations were identified in the pre-S/S genes of 2/5 isolates, which would result in the expression of novel 3’ truncated medium surface proteins (MHBst) and large surface proteins (LHBst). The majority of the mutations would contribute to hepatocyte pathogenesis and transformation by activating cell proliferating pathways. Two patients also contained rare HBV variants not previously identified in HBV strains from southern Africa. These included an HBV splice variant and a poly (dA) variant from patient 10 and patient 6, respectively. These variants occurred in combination with other isolates within the respective patients. The envelope genes were characterised in a total of 18 HCC patients, the pre-S gene of HBV contained deletions in 72% of the patients. Deletions across pre- S1/pre-S2, pre-S2 initiation codon mutations with internal deletions, and S gene nonsense mutations were prevalent. Mutated envelope proteins have been shown to accumulate within the hepatocyte endoplasmic reticulum (ER) and are a characteristic histopathological hallmark of HCC known as ground glass hepatocytes. HBV induced ER stress has been shown to dysregulate several cell cycle regulatory pathways, which contribute to HCC. In addition several novel LHBst and MHBst have been described. These potential transactivators require further investigation. The HBV mutations described in this study have been associated with increased risk for HCC. Despite the obvious heterogeneity HBV displays within and between patients, there are common characteristics shared between the HBV variants which emerge during the development of HCC. These include the BCP and pre-C (1753C1762T1764A1766T1768A1809T1812T) mutations and the pre-S/S mutations. These mutations are able to affect HBV replication and gene expression, and may work synergistically to promote liver dysfunction and HCC.

Hepatitis B virus (HBV), the prototype member of the family Hepadnaviridae, is hepatotropic and replicates by reverse transcription. HBV is responsible for the chronic infection of more than 240 million people worldwide, of which 65 million reside in Africa. The nine HBV genotypes (A to I) identified to date, are geographically distributed and exhibit different clinical manifestations and treatment responses. The term occult HBV infection (OBI) refers to a HBV infection in which HBV surface antigen (HBsAg) cannot be detected by conventional serological assays as has been defined by the Taormina expert panel. . HBV and human immune deficiency virus (HIV) are both endemic in many parts of the world and share common transmission routes. Worldwide, 10% of those infected with HIV are also chronically infected with HBV. HIV co-infection has been shown to be a risk factor for the development of OBI in individuals infected with HBV. The aim of this study was to characterize, at the molecular level, HBV from mono-infected and HBV/HIV co-infected individuals in Sudan The objectives of this study were the systematic and comparative analysis of HBV genotype D sequences, available in the public databases; the molecular characterization of HBV from mono-infected Sudanese liver disease patients and from HBV/HIV co-infected Sudanese patients; and the development and testing of bioinformatics tools to explore HBV sequence data generated using ultradeep pyrosequencing (UDPS) and comparison of UDPS results with those obtained from cloning based sequencing (CBS). All available complete genomes of genotype D of HBV from the GenBank database were analyzed. The intra-group divergence of the subgenotypes ranged from 0.8% + 0.5 for subgenotype D6 to 3.0% + 0.3 for subgenotype D8. Phylogenetic analysis of genotype D showed separation into six distinct clusters (subgenotypes D1, D2, D3/D6, D4, D5 and D7/D8), with good bootstrap support. The mean intergroup divergence between subgenotype D3 and subgenotype D6 was 2.6%, falling below the accepted threshold of 4% required to define a subgenotype. This suggests that subgenotypes D3 and D6 are the same subgenotype because they also share signature amino acids. Furthermore, subgenotype D8 is a genotype D/E recombinant, which clusters with subgenotype D7. This analysis provided an update on the classification of the subgenotypes of genotype D of HBV. Although HBsAg seroprevalence in Sudan, a central-African country, is greater than 8%, the only sequencing data for HBV, available prior to our study, was from asymptomatic blood donors, where genotype E predominates, followed by genotype D and subgenotype A2. Ninety-nine HBV-positive liver disease patients were enrolled in our study, including: 15 with hepatocellular carcinoma (HCC), 42 with cirrhosis, 30 asymptomatic carriers, 7 with acute hepatitis and 5 with chronic hepatitis. The surface and basic core promoter/precore (BCP/PC) regions, and the complete genome of HBV were sequenced. Eighty-two percent of the samples from HBV mono-infected liver disease patients were genotyped. Fifty-nine percent were infected with genotype D (74% D1, 10% D2, 3% D3 and 13% D6), 30% with genotype E, 8.5% with genotype A and 2.5% with a genotype D/E recombinant. Patients infected with genotype E had a higher frequency of HBeAg-positivity (29.2%) and higher viral loads compared to patients infected with genotype D. BCP/PC region mutations, including the G1896A mutation, seen in 37% of the HBeAg-negative individuals, could account for the HBeAg-negativity. A total of 358 Sudanese HIV-positive patients were enrolled. HBsAg was detected in 11.7% of the participants, indicating chronic HBV infection. HBV DNA was detected in 26.8% of the participants: 11.7% were HBsAg positive (overt infection) and the remaining 15.1% were HBsAg-negative (OBI). Fifty serum samples from the HBV/HIV DNA-positive co-infected participants were selected for genomic analysis of HBV. Of these, the HBV genotype of 37 was determined. The genotype distribution of HBV isolates from the HBV/HIV co-infected participants did not differ significantly from those from the HBV mono-infected patients: genotype D (46%), E (21.6%), A (18.9%) and a D/E recombinant (13.5%). Compared to the HBV isolates from mono-infected liver disease patients, the frequency of the D/E recombinant and genotype A was higher in HBV/HIV co-infected patients, as was the intragroup divergence of genotype E. No difference in BCP/PC mutations affecting HBeAg expression at the transcriptional and translational levels between genotype D and E was observed. The following mutations could account for the HBsAg-negativity: sM133T, sE164G, sV168G and sS174N. No primary drug resistance mutations were found. Two online bioinformatics tools, the ―Deep Threshold Tool (DDT)‖ and the ―Rosetta Tool‖, were built to analyze data generated from UDPS and CBS of the BCP/PC region of four Sudanese serum samples, infected with either genotype D or E of HBV, from HBeAgpositive and HBeAg negative patients. A total of 10952 reads were generated by UDPS on the 454 GS Junior platform. The Threshold was calculated using DDT based on probability of error of 0.5%. In total, 39 unique mutations were identified by UDPS, of which 25 were nonsynonymous. The ratio of nucleotide substitutions between isolates from HBeAg-negative and HBeAg-positive patients was 3.5:1. From the sequences analyzed, compared to genotype E isolates, genotype D isolates showed greater variation in the X, BCP/PC/C regions. Only 18 of the 39 positions identified by UDPS were detected by CBS. Using the specific criteria, that have been suggested previously, to define genotypes/subgenotypes of HBV, we determined that genotype D has six and not eight subgenotypes. The importance of HBV genotypes in clinical consequences of infection and response to antiviral treatment has led us to characterize HBV genotypes circulating in Sudan. HBV mono-infected patients and HBV/HIV co-infected individuals, were mainly infected with genotype D or E. HBV mono-infected patients, infected with genotype E, had higher HBeAg-positivity and higher viral loads than those infected with genotype D. The ratio of genotype A to non- A, as well as the genotype E intra-group divergence were higher in HBV/HIV co-infected individuals compared to HBV mono-infected individuals. OBI was found in 15.1% HBV/HIV co-infected patients and its clinical relevance remains to be determined. In order to overcome the limitations of Sanger sequencing, which include its high cost and inability to detect minor populations in quasispecies, next generation sequencing techniques have been developed. It was demonstrated that correct analysis of UDPS data required appropriate curation of read data, in order to clean the data and eliminate artefacts and that the appropriate consensus (reference) sequence should be used in order to identify variants correctly. CBS detected fewer than 50% of the substitutions detected by UDPS. This new technology may allow the detection of minor variants between the different genotypes of HBV and provide biomarkers for the prediction of clinical manifestation of HBV and response to antiviral therapy.

by Lau Tze Chin, Gene. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references (Leaves 170-195). / Abstract --- p.1 / Acknowledgement --- p.3 / List of tables --- p.4 / List of figures --- p.6 / List of abbreviations --- p.7 / Chapter Chapter One - --- Introduction --- p.9 / Chapter 1.1. --- Historical Aspects --- p.9 / Chapter 1.2. --- Classification of hepatitis B virus --- p.12 / Chapter 1.2.1. --- Hepadnaviruses --- p.12 / Chapter 1.2.2. --- Comparative properties of hepadnaviruses --- p.13 / Chapter 1.2.2.1. --- Physical properties --- p.13 / Chapter 1.2.2.2. --- Genetic relatedness --- p.15 / Chapter 1.2.2.3. --- Pathogenesis --- p.16 / Chapter 1.3. --- Structural and morphological properties of HBV --- p.17 / Chapter 1.4. --- Molecular biology of HBV --- p.20 / Chapter 1.4.1. --- Molecular structure of HBV --- p.20 / Chapter 1.4.1.1. --- Biochemistry of the virion envelope --- p.20 / Chapter 1.4.1.2. --- The nucleocapsid --- p.21 / Chapter 1.4.1.3. --- Structural features of HBV genome --- p.23 / Chapter 1.4.2. --- Genetic organization of HBV --- p.24 / Chapter 1.4.3. --- Infection cycle of HBV --- p.29 / Chapter 1.4.3.1. --- Viral attachment and internalization --- p.29 / Chapter 1.4.3.2. --- Replication of HBV --- p.30 / Chapter 1.4.3.3. --- Gene expression and regulation --- p.31 / Chapter 1.4.3.4. --- Host-virus DNA interaction --- p.33 / Chapter 1.5. --- Epidemiology and transmission of HBV --- p.34 / Chapter 1.5.1. --- World wide prevalence --- p.35 / Chapter 1.5.1.1. --- HBsAg prevalence --- p.35 / Chapter 1.5.1.2. --- Cumulative rate of HBV infection --- p.35 / Chapter 1.5.1.3. --- Age specific pattern of HBV infection --- p.36 / Chapter 1.5.2. --- Epidemiological pattern of HBV in Hong Kong --- p.37 / Chapter 1.5.3. --- Mode of transmission --- p.38 / Chapter 1.6. --- Clinical outcomes of HBV infection --- p.38 / Chapter 1.6.1. --- Acute infection --- p.41 / Chapter 1.6.2. --- Chronic infection --- p.42 / Chapter 1.6.3. --- Primary hepatocellular carcinoma --- p.43 / Chapter 1.7. --- Laboratory diagnosis of hepatitis B --- p.44 / Chapter 1.7.1. --- The HBV markers --- p.47 / Chapter 1.7.1.1. --- HBsAg and anti-HBs --- p.47 / Chapter 1.7.1.2. --- HBcAg and Anti-HBc --- p.47 / Chapter 1.7.1.3. --- HBeAg and anti-HBe --- p.49 / Chapter 1.7.1.4. --- HBV-associated DM polymerase --- p.49 / Chapter 1.7.1.5. --- HBV-DNA --- p.49 / Chapter 1.7.2. --- Methodology in the detection of hepatitis B markers --- p.50 / Chapter 1.7.2.1. --- Direct detection of HBV and HBV antigens --- p.50 / Chapter 1.7.2.2. --- Serological detection of HBV markers --- p.51 / Chapter 1.7.2.3. --- HBV-associated DNA polymerase assay --- p.51 / Chapter 1.7.2.4. --- Molecular technique for the detection and quantitation of HBV-DNA --- p.52 / Chapter 1.8. --- Antiviral therapy in hepatitis B --- p.52 / Chapter 1.8.1. --- Therapeutic agents for treatment of HBV infection --- p.53 / Chapter 1.8.1.1. --- Steroids --- p.53 / Chapter 1.8.2.2. --- Nucleoside analogs --- p.54 / Chapter 1.8.1.3. --- Interferon --- p.55 / Chapter 1.8.2. --- Clinical trials of interferons --- p.55 / Chapter 1.9. --- Extrahepatic tissue tropism of HBV --- p.62 / Chapter 1.10. --- Objective and design of study --- p.65 / Chapter 1.10.1. --- Objectives of study --- p.65 / Chapter 1.10.2. --- Study design --- p.66 / Chapter 1.10.2.1. --- Cross-sectional study --- p.67 / Chapter 1.10.2.2. --- Longitudinal study --- p.67 / Chapter 2.1. --- Materials --- p.71 / Chapter 2.1.1. --- Patients recruitment and clinical materials --- p.71 / Chapter 2.1.1.1. --- Cross-sectional study --- p.71 / Chapter 2.1.1.2. --- Longitudinal study --- p.71 / Chapter 2.1.2. --- Bacteria] stock --- p.71 / Chapter 2.1.3. --- "Chemicals, equipments and consumables" --- p.72 / Chapter 2.1.4. --- Buffers and solutions --- p.72 / Chapter 2.1.4.1. --- Phosphate buffer saline (PBS) --- p.72 / Chapter 2.1.4.2. --- Leucocyte lysis buffer (X 5)(LLB) --- p.72 / Chapter 2.1.4.3. --- Buffer equilibrated phenol (BEP) --- p.76 / Chapter 2.1.4.4. --- Phenol-Chloroform mixture --- p.76 / Chapter 2.1.4.5. --- 3.0M sodium acetate (pH 5.2) --- p.76 / Chapter 2.1.4.6. --- Tris-EDTA buffer (pH 8.0) (TE) --- p.76 / Chapter 2.1.4.7. --- Stock salmom sperm DNA solution --- p.77 / Chapter 2.1.4.8. --- Tracking dye --- p.77 / Chapter 2.1.4.9. --- Tris-borate electrophoresis buffer (TBE) --- p.77 / Chapter 2.1.4.10. --- Luria-Bertani Broth (LB) --- p.77 / Chapter 2.1.4.11. --- Solution ] --- p.78 / Chapter 2.1.4.12. --- Solution ]] --- p.78 / Chapter 2.1.4.13. --- Potassium acetate buffer (pH 5.4) --- p.78 / Chapter 2.1.4.14. --- Column elution buffer (CEB) --- p.78 / Chapter 2.1.4.15. --- NPMEB solution --- p.79 / Chapter 2.1.4.16. --- Neutralizing solution --- p.79 / Chapter 2.1.4.17. --- Standard saline citrate (SSC) --- p.79 / Chapter 2.1.4.18. --- Denhardt solution --- p.79 / Chapter 2.1.4.19. --- Prehybridization solution (PS) --- p.80 / Chapter 2.1.4.20. --- NETFAP Solution --- p.80 / Chapter 2.1.4.21. --- Heparin solution --- p.81 / Chapter 2.1.4.22. --- Hybridization mix for oligo-nucleotide probe --- p.81 / Chapter 2.1.4.23. --- NEPS solution (pH 7.0) --- p.81 / Chapter 2.1.4.24. --- Restriction endonuclease and buffer --- p.82 / Chapter 2.2. --- Methods --- p.82 / Chapter 2.2.1. --- Sample preparations --- p.82 / Chapter 2.2.1.1. --- Isolation of plasma and peripheral blood leucocytes (PBL) --- p.82 / Chapter 2.2.1.2. --- Extraction of DNA from Peripheral blood leucocytes --- p.83 / Chapter 2.2.1.3. --- Quantitation of Peripheral blood leucocyte DNA --- p.83 / Chapter 2.2.2. --- Preparation of radio-labelled HBV-DNA probe --- p.84 / Chapter 2.2.2.1. --- Plating and selection of bacterial stock --- p.84 / Chapter 2.2.2.2. --- Growth of E. coli HB101 and amplification of pAM6 --- p.84 / Chapter 2.2.2.3. --- Harvesting of E. coli and extraction of plasmid pAM6 --- p.84 / Chapter 2.2.2.4. --- Purification of plasmid pAM6 --- p.86 / Chapter 2.2.2.5. --- Large scale isolation and purification of HBV genome from plasmid pAM6 --- p.86 / Chapter 2.2.2.6. --- Radio-labelling of HBV-DNA --- p.88 / Chapter 2.2.2.6.1. --- Nick-translation of total HBV-DNA genome --- p.88 / Chapter 2.2.2.6.2. --- Multi-primer labelling of total HBV- DNA genome --- p.88 / Chapter 2.2.2.6.3. --- End-labeling of 21-base HBV oligo- nucleotide --- p.88 / Chapter 2.2.2.6.4. --- Determination of labelling efficiency --- p.89 / Chapter 2.2.2.7. --- Purification of labelled HBV-DNA probe --- p.90 / Chapter 2.2.2.7.1. --- Total genomic HBV-DNA probe (pAM6 probe) --- p.90 / Chapter 2.2.2.7.2. --- Oligo-nucleotide HBV-DNA probe (oligo probe) --- p.90 / Chapter 2.2.3. --- Hybridization study of clinical samples --- p.91 / Chapter 2.2.3.1. --- Solution hybridization of sera samples --- p.91 / Chapter 2.2.3.2. --- Spot hybridization of sera samples --- p.91 / Chapter 2.2.3.2.1. --- "Pre-hybridization treatment of sera samples (adapted from Lin et al.,1987)" --- p.91 / Chapter 2.2.3.2.2. --- Pre-hybridization and hybridization of the membrane --- p.92 / Chapter 2.2.3.2.3. --- Washing of membrane --- p.92 / Chapter 2.2.3.2.4. --- Final treatment and autoradiography: --- p.92 / Chapter 2.2.3.3. --- Quantitation of HBV-DNA in the sera samples: --- p.93 / Chapter 2.2.4. --- Assay for serological Hepatitis B marker --- p.93 / Chapter Chapter Three - --- Results --- p.93 / Chapter 3.1. --- Preparation of HBV-DNA probes --- p.95 / Chapter 3.2. --- Radiolabelling of HBV-DNA --- p.95 / Chapter 3.3. --- Hybridization methodology --- p.98 / Chapter 3.4. --- Comparison of the performance of HBV-DNA probes --- p.100 / Chapter 3.4.1. --- Quantitation of serum HBV-DNA --- p.100 / Chapter 3.4.2. --- Comparative hybridization performance of different HBV-DNA probes --- p.105 / Chapter 3.5. --- Clinical application of HBV-DNA probe:Detection of HBV-DNAin serum and peripheral blood leucocytes (PBL) --- p.109 / Chapter 3.5.1. --- Cross-sectional study --- p.112 / Chapter 3.5.1.1. --- Frequency of HBV-DNA detection in relation to different clinical manifestations --- p.112 / Chapter 3.5.1.2. --- Frequency of HBV-DNA detection in relation to the serological status --- p.114 / Chapter 3.5.1.3. --- Distribution of serum and PBL HBV-DNA level in chronic hepatitis B patients in relation to the different HBV-related manifestations --- p.119 / Chapter 3.5.2. --- Longitudinal study of patients with chronic hepatitis B under interferon therapy with prednisolone pretreatment --- p.123 / Chapter 3.5.2.1. --- Features of patients under study --- p.123 / Chapter 3.5.2.2. --- Correlation between the occurrence of HBV- DNA and HBeAg in serum --- p.123 / Chapter 3.5.2.3. --- Outcome of clinical trial: --- p.126 / Chapter 3.5.2.3.1. --- Number of patients responding to therapy: --- p.126 / Chapter 3.5.2.3.2. --- Variation in serum HBV markers during the course of study --- p.128 / Chapter 3.5.2.3.3. --- Change of HBV-DNA statusin peripheral blood leucocytes --- p.134 / Chapter Chapter Four - --- Dicussion --- p.140 / Chapter 4.1. --- Preparation of HBV-DNA hybridization probes --- p.140 / Chapter 4.1.1. --- Source of HBV-DNA --- p.140 / Chapter 4.1.2. --- Raidolabelling of HBV-DNA --- p.141 / Chapter 4.2. --- Hybridization methodology --- p.141 / Chapter 4.2.1. --- Optimization of hybridization conditions --- p.141 / Chapter 4.2.2. --- Comparison of the performance among different HBV- DNA probes --- p.144 / Chapter 4.3. --- Detection of HBV-DNA in clinical serum samples --- p.148 / Chapter 4.3.1. --- Crossectional study of patients with various categories of HBV related diseases --- p.148 / Chapter 4.3.1.1. --- HBV-DNA detection in serum --- p.148 / Chapter 4.3.1.2. --- Detection of HBV-DNA in peripheral blood mononuclear cells --- p.153 / Chapter 4.3.2. --- Longitudinal studies of patients undergoing antiviral therapy --- p.159 / Chapter 4.3.2.1. --- Serum HBV-DNA and HBeAg --- p.159 / Chapter 4.3.2.2. --- HBV-DNA in peripheral blood leucocytes --- p.163 / Conclusion --- p.166 / Future perspectives --- p.168 / References --- p.170

Deng Xue-Long. / "January 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 196-230). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

ABSTRACT In sub-Saharan Africa a causal relationship has been established between hepatitis B virus (HBV) infection and membranous nephropathy (MN), especially in Black children. The most common method of treatment is interferon therapy, which is however, only effective in 30-40% of patients. The reason for this is unclear. The objective of this pilot study was to determine whether mutations in the complete surface gene of HBV isolated from Black children with HBV-associated MN before, during and after treatment with interferon, had any effect on treatment response and vice versa. HBV DNA was extracted from the serum of a responder, reverter and non-responder patient before, during (4 and 16 weeks) and after (40 weeks) IFN treatment. The preS1/preS2/S region was amplified and cloned, and the clones sequenced. Sequence analyses revealed the preS2 region to be the most variable in the reverter and non-responder and HBsAg was the most variable in the non-responder. Phylogenetic analysis showed that the viral population dynamics between the responder strains and the reverter/non-responder strains differed as a result of various mutations found within the surface gene. Thus the presence of mutations in preS2 and HBsAg of the non-responding patients may carry predictive markers for nonresponse but further investigation would be needed to conclusively prove this.

El objetivo del presente trabajo fue determinar la seroprevalencia del virus de la hepatitis C (VHC) y de la B (VHB) en donadores militares y civiles que acudieron al Banco de Sangre del Hospital Naval así como los factores de riesgo asociados. Se trata de un estudio transversal, descriptivo y retrospectivo, cuyos datos epidemiológicos y resultados del tamizaje general se obtuvieron de los Libros de registros del Banco de Sangre e Historias Clínicas en el caso de los donantes militares, en el período comprendido de Enero de 1999 hasta Abril de 2004. El Test de MUREX HBsAg Versión 3 y Hepanostika Anti-HBc UniForm fueron empleados para Hepatis B, mientras que para el virus de la Hepatitis C se utilizó el Test de Elisa de tercera generación. Un total de 7009 donantes fueron registrados entre 1999 y 2004. Se incluyeron en el presente estudio 320, aquellos que presentaban anticuerpos contra el virus C (anti-VHC) y, para el virus de la hepatitis B: Antígeno de superficie (AgSupHBV) y/o Antígeno Core-IgG. Los datos recolectados fueron procesados y analizados en el paquete estadístico SPSS versión 10 para Windows: Prueba de Ji cuadrado o Prueba de Fisher. RESULTADOS: La prevalencia de portadores de VHC y VHB fue de 0.74% y 0.34%, respectivamente. La mayor población de infectados se encuentra entre los 18 y 30 años y corresponde al personal de la sanidad naval para la Hepatitis B y de Infantería de Marina para la Hepatitis C. No se pudo encontrar factores de riesgo relacionados en ninguna de las dos etiologias debido a una tasa de negación del 96,4% del total de donantes. CONCLUSIONES: Los resultados demuestran una baja prevalencia de infección por virus de hepatitis B, pero elevada para la hepatitis C de acuerdo a los reportes nacionales e internacionales. La pobre o nula asociación entre los factores de riesgo y la enfermedad por hepatitis B y C, que estaría en relación a la limitada efectividad y poca utilidad de la AEC. Se hace necesario que el escrutinio, a través de la encuesta de predonación, sea mas completo, diseñando un cuestionario capaz de detectar más factores de riesgo, probablemente determinanates en la transmisión de los virus de la hepatitis B y C. / The aim of the present work was to determine the seroprevalence of the virus of the hepatitis C (VHC) and of the hepatitis B (VHB) in military and civil donors who came to the Bank of Blood of the Navy Hospital as well as the associate factors of risk. It is a question of a transverse, descriptive and retrospective study realized in the Bank of Blood, which information epidemiological and proved from the general tamizaje was obtained of the Books of records of the Bank of Blood and Clinical Histories in case of the military donors, in the included period of January, 1999 until April, 2004. Murex’s Test HBsAg Version 3 and Hepanostika Anti-HBc UniForm were used for Hepatitis B, whereas for the virus of the Hepatitis C there was in use Elisa's Test of third generation. A whole of 7009 donors was registered between 1999 and 2004.Were included in the present study 320, those who were presenting antibodies against the virus C (anti-VHC) and for the virus of hepatitis B: Surface antigen (HBsAg) and/or Antigen Core-IgG. The information was gathered, percentages were obtained and the results were analyzed by means of SPSS 10 Windows version Statistics Program: chi-square or Fisher’s Tests. RESULTS: The prevalence of carriers of VHC and VHB was 0.74% and 0.34%, respectively. The principal infection people are between 18 to 30 years old, and belong to health navy personal for HBV infection and Marine Infantry for HCV infection. It couldn’t find risks factors in relation to the two etiologists, due a negative rate of 96,4%. CONCLUSIONS: The results suggest a low prevalence of infection for VHB but high for VHC infection in the studied population as it are indicate in the national and international reports. The poor or useless association between de risks factors and the illness it could be in relation to a limited effective and a poor utility of de Confidential Autoexclusion Exam. It’s necessary that this exam must be complete and have the property to identify more risks factors on the transmission of HBV and HCV. / Tesis de segunda especialidad

En las dos últimas décadas del siglo pasado, nuestro país atravesó una de las mayores crisis político-social, con una enorme cuota de violencia y muerte y cuya consecuencia socioeconómica de más impacto, fue un acelerado despoblamiento de las áreas rurales donde el fenómeno violentista era mayor, con su consiguiente masiva migración hacia los centros urbanos más importantes del país, principalmente a la capital de la república. Estos inmigrantes en su gran mayoría provenían de los valles interandinos y de la ceja de selva, precisamente las áreas hiperendémicas de hepatitis B. Este fenómeno, asumimos que podría tener un impacto importante sobre la prevalencia de la hepatitis B en la capital, especialmente en los estratos socioeconómicos bajos, como ya se insinuaba en dos reportes de finales de la década de los ’80. La influencia de estos dos fenómenos en todos los cambios sociales en el mundo, y por ello, la migración de decenas de miles de personas del interior del país hacia la capital que hacían prever un cambio en el patrón epidemiológico de la hepatitis B en Lima pasando de endemicidad baja a intermedia, nos ha llevado a pronosticar , un incremento de la prevalencia de la infección por el virus B en adolescentes, y el consiguiente riesgo de transmisión al neonato; además la población adolescente en el año 2003, es en su inmensa mayoría vulnerable a la infección por el VHB, pues las campañas de vacunación contra esta infección que inició el Ministerio de Salud en el año 1996, en los infantes de cero a un año, no alcanzó a los que ahora son adolescentes. Todos estos hechos han motivado nuestro interés en demostrar el incremento de la prevalencia de hepatitis B en este grupo poblacional, así como investigar todos los otros factores de riesgo que puedan contribuir a la transmisión de esta enfermedad. Este es el propósito de esta investigación. / Tesis

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.

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.