Background: Hepatitis B Virus (HBV) and Human Immunodeficiency Virus (HIV) co-infection in South Africa is estimated between 5-17%; however research determining this prevalence is lacking. With co-infection there is increased risk of liver cirrhosis, end stage liver disease, death as well as higher rates of chronic Hepatitis B infection. Chronic HBV develops in 20% of HIV positive individuals when compared to less than 5% in HIV negative individuals. This also further complicates Highly Active AntiRetroviral Treatment (HAART). Methods: A retrospective observational quantitative, cross-sectional, analytical study was done at the TC Newman Antiretroviral (ARV) centre in Paarl. All adult HIV positive patients that were started on antiretroviral therapy for the time period the new protocol was implemented were analyzed according to their Hepatitis B Antigen (HBsAg) result as well as for any association with gender, CD4 and age. The new protocol stated that all patients who were to start ARV’s had to be tested for Hepatitis B by testing their HBsAg. Results: A total of 498 participants were identified of which 40% were male and 60% were female. The HBsAg positivity rate was established at 7.6%. A higher prevalence was found among men as well as in the age group 50-59 years and those with a CD4 of 50/μL and less. Conclusions: With a prevalence of almost 8% there should definitely be a recommendation towards routine testing of HIV positive patients for Hepatitis B. If not before commencing ART then at least when switching from a regimen containing Lamivudine (3TC) or Tenofovir (TDF) to a regimen not containing these drugs in order to prevent acute flare ups of hepatitis.

M.Tech. (Homoeopathy) / According to the World Health Organisation (2008), an estimated two billion individuals globally, are infected with Hepatitis B (HBV). South Africa reported 864 notified new cases between 2001 and 2004 (Department Of Health, 2005), with an estimated 3-4 million chronic HBV infected black South Africans (Kew, 2008). Kwa-Zulu Natal and Free State were the most affected provinces; while 20-39 years was the most affected age group as of 2005 (Department Of Health, 2005). Workers in the health industry, intravenous drug users and children of women who have Hepatitis B are at the most risk for contracting this disease from blood products and body fluids (Immunization Action Coalition, 2007; Boon et al., 2006), where contraction of the disease can lead to liver cirrhosis, fibrosis and hepato-cellular carcinoma (Highleyman, 2008). The Expanded Program on Immunization (EPI) of the South African Department of Health (2009) suggests vaccination for Hepatitis B should be administered at six, ten and fourteen weeks, or a dose every month for 3 months. Adverse reactions associated with the vaccine include “Guillain-Barre Syndrome, arthritis, demyelinating nervous system disease” (Pratt, 2008) and anaphylaxis (Danis & Halm, 1997). Alternatives that may assist in avoiding such symptoms include: waiting until adolescence to vaccinate (Slonim et al., 2005); only vaccinating high risk groups (Francois et al., 2002); or researching an alternative (Romm, 2001). Homeoprophylaxis is the use of homeopathy to prevent the contraction or development of disease (Zoltan, 2000) and its successful use has been recorded in various disease types and locations. There have been very few studies to show the effect of individual homeopathic nosodes used as prophylactic treatment in their related diseases, with almost none of these utilising any means of serological testing (Bevan-Jones, 2009; Frost et al., 2003; Sheffield, 2006). The aim of this study was to determine the production of the antibody to the surface antigen of Hepatitis B (anti-Hbs) due to Hepatitis B 12cH nosode administration.   Forty-three participants ranging, in ages 18 to 65 years, who tested negative for the presence of anti-HBs, took part in this four week long, double-blind, placebo controlled study. Participants were randomly placed into either the Verum or Placebo group, each group receiving four lactose powders to be taken weekly for four weeks. The Active group received lactose powders medicated with Hepatitis B 12cH, whereas the Placebo group received lactose powders medicated with 96% alcohol. Participants underwent a repeat of the serum/plasma antibody testing at the conclusion of the study to determine if there were anti-HBs present in their blood. The results were then statistically analysed using nonparametric testing: Chi-squared independent test, Mann-Whitney test and Sign test. These showed that there was no change measurable effect on the surface antigen of hepatitis B (anti-HBs) of either the Verum (active medication) or Placebo group. Primary preventative medicine is becoming increasingly popular (Kuehlein et al., 2010). Both vaccination and homeoprophylaxis are examples of primary preventative medicine, where the aim is to prevent future disease. Vaccinations encourage the production of antibodies via the activation of T-helper cells and B-lymphocytes, thus providing a template for immunity against future infections (Miller, 2000; Janeway et al., 2001). While the mechanisms of vaccination are well understood, those of homeoprophylaxis are still being investigated. One theory is that nosodes enable the body to overcome diseases. Several studies have been conducted on the effects of nosodes (Bracho et al., Prophylactic vaccination against human papilloma virus infection and disease in women: a systemic review of randomized control trial.; Gosavi et al., 2012; Shuller, 2010) and have shown favourable results in the prevention of diseases associated with those homeopathic nosodes. However, only two studies have investigated the effects that nosodes have on the antibodies of the immune system (Hoover, 2006; Neustaedter, 2002) showing the need for further studies conducted in this area. The study showed that homeopathically prepared Hepatitis B 12cH nosode is not capable of eliciting an immune response that would result in the production of the antibody to the surface antigen of Hepatitis B, and thus not able to provide immunity against Hepatitis B.

Hepatitis B virus (HBV) infection is an important global health concern and chronic carriers of the virus are at high risk of developing hepatocellular carcinoma (HCC) and cirrhosis. Current therapies are only partially effective, which emphasises the need for improved treatment strategies. Harnessing the RNA interference (RNAi) pathway as a treatment strategy against HBV has shown great promise. However, there are obstacles that need to be overcome before RNAi-based treatment of HBV infection is realised. These include problems of liver tissue targeting and dose regulation. This study investigated the use of a liver specific and mifepristone-inducible RNA polymerase (Pol) II promoter system for the specific and precise regulation of anti-HBV sequence expression. The inducible system used consists of two expression cassettes; one containing the regulator/transactivator protein and another containing the transgene. Natural primary microRNA (pri-miR) mimics, pri-miR-31/5 and pri-miR-31/5/8/9, were used as anti-HBV sequences. Firefly luciferase gene expression was used to test modulation by the inducible system and to determine optimal induction conditions. The pri-miR-31/5, pri-miR-31/5/8/9 and luciferase encoding fragments were incorporated into the plasmid vector pRS17 that bears the inducible promoter, creating pRS-31/5, pRS-31/5/8/9 and pRS-Luc respectively. Firefly luciferase expression with this system was shown to be inducible and mifepristone dose-dependent. Effective knockdown of HBV gene expression was achieved with both pRS-31/5 and pRS-31/5/8/9 in vitro and in vivo. However, with high vector amounts, similar efficiency in silencing of HBV gene expression was observed in the presence and absence of the inducer mifepristone suggesting leaky expression of the pri-miRs. To confirm this, knockdown studies were carried out with the pri-miR-31/5/8/9-expressing cassette separated from the transactivator cassette. HBV gene expression knockdown was observed with the pri-miR-31/5/8/9 cassette alone confirming leaky expression from the inducible system. Leakiness appears to be as a result of the E1B promoter driving the expression of the pri-miRs in the absence of mifepristone. However, reducing the vector amounts decreased basal expression and improved the inducibility of the system in cell culture studies. Successful propagation of an inducible and liver-specific RNAi-activating expression system will address the difficulty of achieving dose control of RNAi effectors and contribute to advancing the use of RNAi for HBV treatment.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / A 24-year-old pregnant woman arrived at the emergency service at 34 weeks of gestational age with intermittent right upper abdominal pain. An abdominal ultrasound was performed showing signs of hepatopathy with multiple neo-formative nodules with mild ascites and fetal biometry confirmed at 34 weeks gestation. During her hospitalization, an emergency caesarean was induced with favorable result in the survival of the mother and the baby. / Revisión por pares

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Gene therapy has shown potential in alleviating a wide range of diseases, ranging from viral infections to autosomal diseases. One of the obstacles to gene therapy reaching its full potential is the inadequacy of methods to deliver therapeutic nucleic sequences. Current delivery of gene therapy entails use of viral and non-viral vectors. Viral vectors are however associated with drawbacks such as potential toxicity, high cost and labour-intensive production. Thus non-viral delivery alternatives are being developed in an attempt to overcome difficulties associated with nucleic acid delivery for gene therapy. Virus-like particles are potentially very useful delivery vehicles as their production is simple and cost effective, the particle surface is amenable to modification and the capsid interior can be altered to accommodate a variety of cargoes. One such particle is the recombinant HBV capsid, which comprises a single species of protein and is tolerant of amino acid substitutions on the surface exposed immunodominant loops. This study aimed to enhance the vector-like properties of the HBV virus-like particle by including amino acid substitutions on the particle surface. These substituted residues in turn provided a conjugation site for tropic and immuno evasive moieties. It was found that lysine substitutions resulted in poor conjugation to the capsid surface, whereas substituted cysteine residues resulted in almost all protein-moiety conjugates forming. In order to introduce lysine and cysteine substitutions, a novel method of cloning into the HBV was generated. In doing so, complicated procedures associated with cloning into the immunodominant loop of the HBV capsid have been alleviated. Ligands containing galactose were utilised on the surface of both the HBV capsid and liposomes to confer hepatotropism. The presence of the galactose moieties on the surface of the HBV capsid prevented indiscriminate cellular uptake in cultured cells, however did not improve hepatotropism. Galactose ligands on the surface of liposomes did improve transfection efficiency, however they required a short linker distance between liposome surface and galactose group. The inclusion of galactose in liposome formulations also provided a means to deliver siRNA to the liver of transgenic HBV mice. It is believed that with alterations to the ligand structure, it is possible to provide HBV capsids with hepatotropism in future experimentation. This study demonstrated that the exposed external surface of the HBV capsid is amenable to convenient conjugation, which potentially facilitates immune evasion and conferring of defined tropism.

Despite evidence that viral hepatitis poses a significant risk to public health, universal vaccination has not yet been implemented. The risk for viral hepatitis infection is particularly high among injection drug users and other individuals who do not attend regular health care visits. Jails provide a structural opportunity to vaccinate these high risk individuals. HAV and HBV vaccines administered on an accelerated three week schedule could dramatically decrease the lifetime risk for contracting viral hepatitis among jail detainees. Assuming that 75% of detainees would accept vaccination, 33% have previous exposure to HAV, 25% have previous exposure to HBV, and independent future healthcare costs were US $317,000, the US health care system would save $12 per individual with a vaccinate upon entry program in comparison to no intervention. This savings translates into an economic benefit amounting to about US$ 5,000,000 saved if all new jail inmates in a given year were immunized. A vaccination upon entry program for HAV/HBV in jails should be widely implemented with coordination between the corrections system and public health agencies to reduce the growing cost of viral hepatitis infection.

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

Thesis (DMed)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background and Objective: The most common cause of severe proteinuria/nephrotic syndrome (NS) in children worldwide is minimal change disease (MCD). This is also the pattern observed in white and Indian children in South Africa (SA). By contrast, black and mixed race/coloured children of Southern Africa in the 1960s to 1990s were shown to have a different pattern of NS. One of the main differences was the frequency of hepatitis B virus (HBV) associated glomerulonephritis, usually membranous glomerulonephritis (MGN). The objective of this project was a clinicopathological study of this subgroup of nephrotic children to document the disease further and in particular to seek correlations between pathological and clinical features including prognosis. A central focus was to document the detailed ultrastructural examination of the renal biopsies of these children and to correlate the spectrum of pathological features with demographic, clinical, laboratory and prognostic features. The hypothesis was that the clinicopathological features of HBV MGN in children differed substantially from idiopathic MGN in general (children and adults) and also from HBV MGN in adults and that HBV MGN in children should be viewed as a distinct disease. Patients and methods: The childhood (12 years and younger) patient cohort was 309 children with severe proteinuria/nephrotic syndrome who presented at Tygerberg Hospital (TBH) over a 21 year period from 1974-1995, including 67 children from Namibia. The study group was 71 children with HBV MGN who were followed up to 2005. The comparative adult group was 45 adults with MGN of whom 12 had HBV MGN and 33 idiopathic MGN. (A comparison could not be made with idiopathic MGN in childhood as this centre only had 2 such patients during the study period.) Demographic, clinical, laboratory and renal pathology data were collected, compared and correlated. Results: HBV associated MGN was the most frequent cause of NS in the Namibian subgroup, 25/67 (37%) and the third most frequent, 71/309 (23%) in the childhood cohort as a whole. The MGN group was 86% (71/83) of the total HBV childhood nephrotic cohort, by far the dominant subgroup. The average age of the 71 children with HBV MGN was 6.0 years (range 2-12 years) at presentation and boys comprised 80% of the group. Hepatitis B envelope antigen (HBeAg) was identified in the serum of 87% of children tested. Laboratory features different from idiopathic MGN included more prominent haematuria, mildly raised serum transaminases and more frequently lowered serum C3 and C4 levels. Light microscopic examination of renal biopsies showed mesangial proliferation in all patients but with minimal glomerular sclerosis and interstitial disease. On ultrastructural examination mesangial and subendothelial deposits were common and prominent as was mesangial interposition. The MGN of HBV in children therefore frequently showed mesangiocapillary glomerulonephritis (MCGN) features in addition to the subepithelial deposits of MGN. The subgroup of 23 whose renal biopsies displayed severe mesangial interposition in addition to the subepithelial deposits of MGN were termed the mixed HBV MGN-mesangiocapillary GN group. Virus like bodies and tubuloreticular inclusion bodies were both found in more than 80% of biopsies of childhood HBV MGN. HBeAg was identified in the subepithelial deposits in the glomeruli. This was the first time this feature was demonstrated in Africa. The 46 South African children with HBV MGN showed a cumulative remission rate of 25% at 2 years and 52% at 4 years. Seven of the children (10%) of the total cohort developed chronic renal failure (CRF). Age of 6 years and above at presentation and severe mesangial deposits on biopsy correlated with fewer remissions and poorer outcome. In 3 patients the interval between the diagnosis of HBV MGN and the onset of CRF was more than 19 years with the longest being 23 years. The 358 cases of childhood HBV MGN from Southern Africa constitute 37% of the reported childhood patients. Comparative data A comparison was made between the 71 children with HBV MGN, 12 adults with HBV MGN and 33 adults with idiopathic MGN. The main differences were that both HBV MGN groups included only coloured and black patients and were more predominantly male while the idiopathic MGN group included all races. In the HBV patients, haematuria was more frequent and severe, liver enzymes were frequently raised and C3 more frequently reduced than in the idiopathic cohort. Both groups of adult MGN patients had normal C4 levels while the childhood HBV MGN group had reduced C4 levels. The immune complex pattern in both of the HBV MGN adult and childhood groups on biopsy was similar with more mesangial and subendothelial deposits as well as mesangial interposition than the idiopathic group. Despite this similarity between the two HBV groups, both adult groups showed more glomerular sclerosis and interstitial disease than the childhood group. The clinical outcome of the children’s cohort was better than the other 2 groups with remission (52%) more frequent at 4 years (p<0.01) and better renal and patient survival. Including the 83 cases from this series, at least 1243 renal biopsy proven cases of HBV MGN have been reported in the English literature; children (80%) and adults (20%). The male gender predominance in both age groups for HBV MGN is similar (children 79%; adults 84%) and significantly greater than for idiopathic MGN. Conclusions: The findings confirm that HBV MGN in children is a distinct form of GN which broadens the classical morphologic description of MGN by often including a number of mesangiocapillary GN features. The subgroup of renal biopsies with the most severe mesangiocapillary GN features was classified as the mixed HBV MGNmesangiocapillary GN group. The MGN spectrum as a whole comprised 86% of the HBV positive childhood group. HBV MGN was the most frequent association with NS/severe proteinuria in the Namibian subgroup (37%) and the third largest group (19%) in the SA children. It showed a relatively high spontaneous remission rate but at least 10% of the children developed renal failure. Age of 6 years and above at presentation and severe mesangial deposits on biopsy correlated with fewer remissions and poorer outcome. Extended follow up (more than 15 years) was required to demonstrate renal failure in some patients in the poor outcome group. Urbanisation, associated with lower HBV carrier rates, and HBV vaccination (initiated routinely in 1995 in SA), have already lead to a sharply decreasing incidence of this disease in SA. HBV MGN has been a valuable and possibly unique model of human GN and MGN in particular in that the HBeAg has been identified in both the serum and glomeruli enabling confirmation of the aetiological role of HBeAg. het ’n kumulatiewe remissie koers van 25% teen 2 jaar en van 52% teen 4 jaar getoon. Sewe van die kinders (10%) van die hele kohort het kroniese nierversaking (KNV) ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale neerslae in ‘n biopsie het met minder remissies en ’n swakker uitkoms gekorreleer. Drie pasiënte het meer as 19 jaar na aanvanklike voordoening ooglopende KNV ontwikkel, waarvan 23 jaar die langste interval was. Die 358 gevalle van kinderjare HBV MGN van Suidelike-Afrika maak 37% uit van die gerapporteerde kinder pasiënte. Vergelykende data ’n Vergelyking is getref tussen die 71 kinders met HBV MGN, 12 volwassenes met HBV MGN en 33 volwassenes met idiopatiese MGN. Die hoof verskille was dat beide HBV groepe net kleurling en swart pasiënte ingesluit het en meer oorwegend manlik was, terwyl die idiopatiese groep alle rasse ingesluit het. In die HBV pasiënte was hematurie meer algemeen en erg, lewer ensieme meer dikwels verhoog en C3 meer dikwels verlaag as in die idiopatiese kohort. Beide groepe van volwasse MGN pasiënte het normale C4 vlakke getoon terwyl die kindergroep met HBV MGN verlaagde C4 vlakke bewys het. Die immuunkompleks patroon in biopsies van die HBV MGN volwasse en kindergroepe was soortgelyk met meer mesangiale en subendoteliële neerslae asook meer mesangiale interposisie as in die idiopatiese groep. Ten spyte van hierdie ooreenkoms tussen die twee HBV groepe, het die twee volwasse groepe meer glomerulêre sklerose en interstisiële siekte as die kindergroep vertoon. Die kliniese uitkoms van die kinderkohort was beter as die ander twee groepe met remissie (52%) wat meer algemeen was teen 4 jaar (p< 0.01) en met beter nier- en pasïent oorlewing. Ingeslote die 83 gevalle van hierdie reeks, is ten minste 1243 nierbiopsie bewysde gevalle van HBV MGN in kinders (80%) en volwassenes (20%) in die Engelse literatuur gerapporteer. Die manlike oorheersing in beide ouderdomsgroepe van HBV MGN is soortgelyk (kinders 79%; volwassenes 84%) en betekenisvol meer as vir idiopatiese MGN. Gevolgtrekkings: Die bevindinge bevestig dat HBV MGN in kinders ’n afsonderlike vorm van GN is wat die klassieke beskrywing van MGN verbreed deur die algemene insluiting van ’n aantal mesangiokapillêre GN kenmerke. Die ondergroep van nier biopsies met erge mesangiokapillêre GN kenmerke is as die gemengde HBV MGNmesangiokapillêre GN groep geklassifiseer. Die MGN spektrum in geheel het 86% van die HBV positiewe kindergroep behels. HBV MGN was die mees algemene assosiasie met NS/erge proteïenurie in die Namibiese subgroep (37%) en die derde grootse groep (19%) onder die SA kinders. Die siekte het ’n relatiewe hoë spontane remissiekoers getoon, maar ten minste 10% van die kinders het nierversaking ontwikkel. Ouderdom van 6 jaar en meer by presentasie en erge mesangiale neerslae in ‘n nierbiopsie het met minder remissies en ’n slegter uitkoms gekorreleer. Uitgebreide opvolg (meer as 15 jaar) was nodig om nierversaking in sommige van die swak uitkomsgroep aan te toon. Verstedeliking is geassosieerd met laer HBV draersyfers en hierdie faktor saam met algemene HBV inenting in die kinderjare (wat in 1995 in SA begin was), het ’n skerp daling in die voorkoms van hierdie siekte in SA teweeg gebring. HBV MGN is ’n waardevolle en moontlik unieke model van menslike GN en MGN, veral omdat die HBeAg in beide die serum en glomeruli identifiseer kon word om die etiologiese rol van HBeAg te bevestig. / AFRIKAANSE OPSOMMING: Agtergrond en Doelwit: Die algemeenste oorsaak van erge proteïenurie/nefrotiese sindroom (NS) in kinders wêreldwyd is minimale veranderingsiekte. Hierdie patroon kom ook voor in blanke- en Indiër kinders in Suid-Afrika. In teenstelling hiermee is aangetoon dat swart en kleurling/gemengde ras kinders in Suider Afrika tussen die jare 1960s tot 1990s ’n ander patroon van nefrotiese sindroom gehad het. Een van die hoof verskille was die algemene voorkoms van hepatitis B virus (HBV) geassosieerde glomerulonefritis, gewoonlik membraneuse glomerulonefritis (MGN). Die doelwit van hierdie projek was ’n klinies-patologiese studie van hierdie subgroep van nefrotiese kinders ten einde die siekte verder te beskryf en veral om korrelasies te tref tussen patologiese en kliniese kenmerke insluitende prognose. Die gedetaileerde ultrastrukturele ondersoek van die kinders se nierbiopsies en die korrelasie van die spektrum patologiese kenmerke met demografiese, kliniese, laboratorium en prognostiese kenmerke was ‘n sentrale fokusarea. Die hipotese was dat die klinies-patologiese kenmerke van HBV MGN in kinders wesenlik van idiopatiese MGN in die algemeen verskil (in kinders en volwassenes) en ook van HBV MGN in volwassenes, en dat die beeld in kinders as ’n afsonderlike siekte beskou behoort te word. Pasiënte en metodes: Die kinder kohort (12 jaar en jonger) was 309 kinders met erge proteïenurie/nefrotiese sindroom wie in Tygerberg Hospitaal (TBH) behandel was oor ‘n 21 jarige periode vanaf 1974 tot 1995, insluitende 67 kinders van Namibië. Die studiegroep was 71 kinders met HBV MGN wie waar moontlik tot 2005 opgevolg was. Die vergelykende volwasse groep was 45 volwassenes met MGN van wie 12 HBV MGN gehad het en 33 idiopatiese MGN. (’n Vergelyking met idiopatiese MGN in kinders kon nie gedoen word nie omdat hierdie sentrum net twee sulke pasiënte tydens die studietyd behandel het.) Demografiese, kliniese, laboratorium en nierpatologie inligting is versamel, vergelyk en gekorreleer. Resultate: HBV geassosieerde MGN was die algemeenste oorsaak van NS in die Namibiese subgroep, 25/67 (37%) en die derde mees algemeen, 71/309 (23%) in die kinder kohort as geheel. Die MGN groep was 86% (71/83) van die totale HBV kinder nefrotiese kohort en verreweg die oorheersende subgroep. Die gemiddelde ouderdom van die 71 kinders met HBV MGN by presentering was 6.0 jaar (reikwydte 2-12 jaar) en seuns het 80% van die groep behels. Hepatitis B omhullingsantigeen (envelope antigen- HBeAg) is aangetoon in die serum van 87% van die kinders wie daarvoor getoets is. Laboratoriumkenmerke wat van idiopatiese MGN verskil het, het ingesluit meer prominente hematurie, gering verhoogde serum transaminases en meer dikwels verlaagde serum C3 en C4 vlakke. Ligmikroskopiese ondersoek van die nierbiopsies het mesangiale proliferasie in elke pasiënt getoon, maar met minimale glomerulêre sklerose en interstisiële siekte. Met ultrastrukturele ondersoek was mesangiale en subendoteliële neerslae asook mesangiale interposisie algemeen. Die MGN van HBV in kinders het dus dikwels kenmerke van mesangiokapillêre glomerulonefritis getoon bo en behalwe die subepiteliële neerslae van MGN. Die ondergroep van 23 van wie die nierbiopsies erge mesangiale interposisie aangetoon het asook die subepiteliale neerslae van MGN is die gemengde HBV MGN-mesangiokapillêre GN groep genoem. Virustipe liggaampies en tubuloretikulêre insluitingsliggaampies is in meer as 80% van die biopsies bevestig. HBeAg was in die subepiteliële neerslae identifiseer. Dit was die eerste keer dat hierdie kenmerk in Afrika identifiseer is. Die 46 Suid-Afrikaanse kinders

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy