Theses and Dissertations (Virology)Theses and Dissertations (Virology)http://hdl.handle.net/10386/9832024-03-29T15:05:50Z2024-03-29T15:05:50ZAssessing the impact of hepatitis B immunization in over 5 year olds from selected province of South AfricaAmponsah-Dacosta, Edinahttp://hdl.handle.net/10386/11592017-11-16T10:03:12Z2014-11-06T00:00:00ZAssessing the impact of hepatitis B immunization in over 5 year olds from selected province of South Africa
Amponsah-Dacosta, Edina
Introduction: The hepatitis B virus (HBV) causes a serious type of liver disease referred to as hepatitis B, which is associated with various fatal sequelae following the onset of chronic infection. As a result of the global burden of chronic HBV infection, HBV-related mortality is currently estimated at 620 000 annual deaths worldwide (Hwang and Cheung, 2011). The World Health Assembly (WHA) recommended in 1992 that the hepatitis B vaccine be incorporated into national immunization programmes universally, especially in the hyperendemic regions of the world, in order to curb the global burden of hepatitis B (WHO, 1992). Accordingly, South Africa introduced the hepatitis B vaccine into the national Expanded Programme on Immunization (EPI-SA) in April 1995. Almost 17 years later, South Africa has not conducted any nationwide serosurveys to monitor the population impact of the hepatitis B vaccine. Instead, a number of field and laboratory studies have been conducted only in vaccinated children within the first 5 years of life and as such reports on the short¬term impact made by the hepatitis B vaccine in the country have largely relied on these studies (Tsebe et al., 2001; Schoub et al., 2002; Simani et al., 2008). The aim of the current study therefore was to assess the population impact made by the hepatitis B vaccine post its introduction into EPI-SA using an age stratified, cross-sectional study. The objectives were to compare the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa, determine the influence of HIV infection on the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa by performing a subset analyses, and lastly, to perform molecular characterization of hepatitis B surface and polymerase genes in HBV DNA positive individuals.
Materials and Methods: This was an explorative and descriptive retrospective, cross¬
sectional study based on recently tested and stored blood samples from the NHLS
fi
Diagnostic Laboratory in the Department of Virology. For the purpose of this study, these
samples were obtained after Ethics approval, and two target populations identified based on the year (Le. 1995) the hepatitis B vaccine was introduced into EPI-SA; a post-vaccination population consisting of 605 blood samples from individuals aged 1-15 years and a pre¬vaccination population consisting of 601 blood samples from individuals aged 16-25 years. The post-vaccination population was further stratified by age as follows; 1-5,6-10 and 11-15 years, in order to assess immunity and chronic carriage of HBV across the different age groups. All samples were tested for the following primary serological markers; HBsAg, anti¬HBc and anti-HBs, to determine the prevalence of HBV chronic carriage, past HBV exposure and immunity to HBV infection respectively. Samples were further assessed for the
vi
incidence of acute HBV infection by testing for IgM anti-HBc. All serological testing was performed using the Elecsys@ 2010 Immunoassay System. Samples with serological evidence of infection or exposure to HBV were selected and screened for HBV DNA using a real time PCR assay to determine the prevalence of active HBV infection within this group. Study subjects with records of their HIV status, either positive or negative, were also pooled for subset analyses in order to determine the influence of HIV infection on immunity and chronic carriage of HBV. Finally, samples positive for HBV DNA were subjected to molecular characterization of the hepatitis B surface (S) and polymerase (pol) genes.
Results: Following serological screening, immunity to HBV infection was found to be significantly (p<0.001) higher (56.7%) in the post-vaccination population than in the pre-vaccination population (15.5%). Within the post-vaccination population alone, immunity was found to wane with increasing age from 76.1 % in those 1-5 years of age to 50.0% in those 6-10 years and 44.2% in those 11-15 years of age. Chronic carriage on the other hand was significantly (p=0.008) reduced in the post-vaccination population with 1.5% HBsAg prevalence as compared to 4.0% in the pre-vaccination population. Within the different age strata, chronic carriage increased with increasing age (0.5% in 1-5 years; 1.3% in 6-10 years; 2.5% in 11-15 years). Overall, no acute HBV infection was detected within the post-vaccination population, while a 14.6% prevalence rate of acute HBV infection was found for the pre-vaccination population. From the subset analyses, immunity was found to be significantly (p<0.001) higher in the HIV uninfected population as compared to the HIV
infected population; 82.5% versus 22.0% in the post-vaccination population and 26.7% versus 0% in the pre-vaccination population, while chronic carriage was found to be higher in
the HIV infected population than in the HIV uninfected population. Following molecular characterization of the HBV S gene, it was revealed that the majority of the viral isolates
were genotype A, with only 1 genotype D isolate found. A number of notable amino acid
i$
variations were also detected within the antigenic region of the HBsAg of viral isolates,
inCluding the K122R, N131T, T143S, and E164D mutations.
Conclusion: Introduction of the hepatitis B vaccine into EPI-SA has shown remarkable success in children under the age of 5 years. Overall, immunity and chronic carriage of HBV within the post-vaccination population has been greatly impacted by hepatitis B immunization. Within the HIV infected population, susceptibility to HBV infection remains a cause for concern. Finally, although amino acid variations within the viral HBsAg are present, vaccine escape-related mutants appear to be rare or even absent within the South African population.
vii
Thesis (M Med (Virology))-- University of Limpopo (Medunsa Campus), 2013
2014-11-06T00:00:00ZEpidemiology of rotatirus diarrhoea in children under five years years of age from selected healthcare facilities in SwazilandMaphalala, Gugu Petunia.http://hdl.handle.net/10386/11542017-11-16T10:03:13Z2014-01-01T00:00:00ZEpidemiology of rotatirus diarrhoea in children under five years years of age from selected healthcare facilities in Swaziland
Maphalala, Gugu Petunia.
Background: It has been established that rotaviruses are the main cause of acute gastroenteritis in children worldwide, resulting in more than 453 000 deaths, with a high mortality still occurring in African countries and Asia. In Swaziland, diarrheal diseases are a common cause of morbidity and mortality among children <5 years of age. Approximately 10% of hospitalised Swazi children die due to diarrhoea every year. Through financial assistance from the World Health Organization (WHO), many African countries have conducted a lot of rotavirus disease studies. In Swaziland, the epidemiology of rotavirus infection is unknown due to lack of data. Thus, the study’s aim was to examine the epidemiology and characterize rotavirus strains in children <5 years of age, hospitalised and attending the outpatient departments of public and private healthcare facilities in Swaziland.
Materials and methods: A total of 745 diarrheal stool specimens were collected from children <5 years of age from April 2009 to December 2010. Group A rotavirus antigen was detected using a commercially available enzyme immunoassay (EIA) kit (ProSpectTM, Oxoid Ltd, UK). Polyacrylamide gel electrophoresis (PAGE) was used to determine the electrophoretic pattern of rotavirus strains. The P and G genotypes were established by reverse transcription polymerase chain reaction (RT-PCR) and multiplex hemi-nested PCR amplification of the VP4 and VP7 genes respectively, using type-specific primers. Sequencing was performed on 35 specimens to confirm the circulating genotypes. The phylogenetic tree and similarity distances between genotypes were constructed using the neighbour joining method and the Kimura two-parameter model package in the MEGA version 5.05 software program.
Results: Group A rotavirus was detected at 13.3% in 2009 (based on samples collected from April to December) and 23.4% in 2010 (based on one year collection) from children <5 years of age hospitalized and attending outpatient departments. The rotavirus infection was more frequently detected in the age group 0-11 months (22.2%). Gender did not play a major role in rotavirus infection, because both male (20.8%) and female (18.8%) children were equally affected. Of the children that were admitted in the hospital, 33.3% were affected by rotavirus infection compared to those attending the outpatient departments (13.5%). The rotavirus infection was observed during the cooler, drier months of the year. The three most predominant G and P genotypes detected were G2P[4] (30.4%), followed by G1P[8] (15.5%) and G9P[8] (8.8%). A significant number of uncommon rotavirus strains (32.4%), mixed infections (8.8%) and nontypeables (4.1%) were also detected. The circulating genotypes detected were classified into lineages and sub-lineages defined by phylogenetic analysis of nucleotide sequences. The Swaziland strains were found clustering with known African and global strains from the GenBank.
Conclusion: The findings of this study reveal that group A rotaviruses are the etiological agents of severe diarrhoea in children under 5 years in Swaziland. The diversity of rotavirus strains that were detected highlights the importance of introducing the rotavirus vaccine in the country. The currently licensed vaccines may confer protection against the circulating strains detected in this study. Data on the burden of rotavirus disease in Swaziland will be used to convince the Ministry of Health and policy makers in the country to advocate for the introduction of the rotavirus vaccine. This is the first data on the epidemiology and characterization of rotavirus strains in Swaziland; therefore there is a need for continuing with the surveillance of rotavirus in the existing sentinel sites to determine the impact of rotavirus infection over time. It is also essential to continuously monitor the rotavirus strains circulating among Swazi children.
Thesis (MSc (Medical science in medical virology)) -- University of Limpopo
2014-01-01T00:00:00ZAssesing the impact of hepatitis B immunization in over 5 year olds from selected provinces of South AfricaAmponsah-Dacosta, Edinahttp://hdl.handle.net/10386/10652017-11-16T10:03:10Z2014-02-13T00:00:00ZAssesing the impact of hepatitis B immunization in over 5 year olds from selected provinces of South Africa
Amponsah-Dacosta, Edina
Introduction: The hepatitis B virus (HBV) causes a serious type of liver disease referred to as hepatitis B, which is associated with various fatal sequelae following the onset of chronic infection. As a result of the global burden of chronic HBV infection, HBV-related mortality is currently estimated at 620 000 annual deaths worldwide (Hwang and Cheung, 2011). The World Health Assembly (WHA) recommended in 1992 that the hepatitis B vaccine be incorporated into national immunization programmes universally, especially in the hyperendemic regions of the world, in order to curb the global burden of hepatitis B (WHO, 1992). Accordingly, South Africa introduced the hepatitis B vaccine into the national Expanded Programme on Immunization (EPI-SA) in April 1995. Almost 17 years later, South Africa has not conducted any nationwide serosurveys to monitor the population impact of the hepatitis B vaccine. Instead, a number of field and laboratory studies have been conducted only in vaccinated children within the first 5 years of life and as such reports on the short¬term impact made by the hepatitis B vaccine in the country have largely relied on these studies (Tsebe et al., 2001; Schoub et al., 2002; Simani et al., 2008). The aim of the current study therefore was to assess the population impact made by the hepatitis B vaccine post its introduction into EPI-SA using an age stratified, cross-sectional study. The objectives were to compare the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa, determine the influence of HIV infection on the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa by performing a subset analyses, and lastly, to perform molecular characterization of hepatitis B surface and polymerase genes in HBV DNA positive individuals.
Materials and Methods: This was an explorative and descriptive retrospective, cross¬
sectional study based on recently tested and stored blood samples from the NHLS
fi
Diagnostic Laboratory in the Department of Virology. For the purpose of this study, these
samples were obtained after Ethics approval, and two target populations identified based on the year (Le. 1995) the hepatitis B vaccine was introduced into EPI-SA; a post-vaccination population consisting of 605 blood samples from individuals aged 1-15 years and a pre¬vaccination population consisting of 601 blood samples from individuals aged 16-25 years. The post-vaccination population was further stratified by age as follows; 1-5,6-10 and 11-15 years, in order to assess immunity and chronic carriage of HBV across the different age groups. All samples were tested for the following primary serological markers; HBsAg, anti¬HBc and anti-HBs, to determine the prevalence of HBV chronic carriage, past HBV exposure and immunity to HBV infection respectively. Samples were further assessed for the
vi
incidence of acute HBV infection by testing for IgM anti-HBc. All serological testing was performed using the Elecsys@ 2010 Immunoassay System. Samples with serological evidence of infection or exposure to HBV were selected and screened for HBV DNA using a real time PCR assay to determine the prevalence of active HBV infection within this group. Study subjects with records of their HIV status, either positive or negative, were also pooled for subset analyses in order to determine the influence of HIV infection on immunity and chronic carriage of HBV. Finally, samples positive for HBV DNA were subjected to molecular characterization of the hepatitis B surface (S) and polymerase (pol) genes.
Results: Following serological screening, immunity to HBV infection was found to be significantly (p<0.001) higher (56.7%) in the post-vaccination population than in the pre-vaccination population (15.5%). Within the post-vaccination population alone, immunity was found to wane with increasing age from 76.1 % in those 1-5 years of age to 50.0% in those 6-10 years and 44.2% in those 11-15 years of age. Chronic carriage on the other hand was significantly (p=0.008) reduced in the post-vaccination population with 1.5% HBsAg prevalence as compared to 4.0% in the pre-vaccination population. Within the different age strata, chronic carriage increased with increasing age (0.5% in 1-5 years; 1.3% in 6-10 years; 2.5% in 11-15 years). Overall, no acute HBV infection was detected within the post-vaccination population, while a 14.6% prevalence rate of acute HBV infection was found for the pre-vaccination population. From the subset analyses, immunity was found to be significantly (p<0.001) higher in the HIV uninfected population as compared to the HIV
infected population; 82.5% versus 22.0% in the post-vaccination population and 26.7% versus 0% in the pre-vaccination population, while chronic carriage was found to be higher in
the HIV infected population than in the HIV uninfected population. Following molecular characterization of the HBV S gene, it was revealed that the majority of the viral isolates
were genotype A, with only 1 genotype D isolate found. A number of notable amino acid
i$
variations were also detected within the antigenic region of the HBsAg of viral isolates,
inCluding the K122R, N131T, T143S, and E164D mutations.
Conclusion: Introduction of the hepatitis B vaccine into EPI-SA has shown remarkable success in children under the age of 5 years. Overall, immunity and chronic carriage of HBV within the post-vaccination population has been greatly impacted by hepatitis B immunization. Within the HIV infected population, susceptibility to HBV infection remains a cause for concern. Finally, although amino acid variations within the viral HBsAg are present, vaccine escape-related mutants appear to be rare or even absent within the South African population.
vii
Thesis ( M Med ( Virology ) )-- University of Limpopo (Medunsa Campus), 2013.
2014-02-13T00:00:00ZFull length genome characterisation of Hepatitis B virus isolates at Dr George Mukhari Hospital in Pretoria, South AfricaMagobo, Rindidzani Edithhttp://hdl.handle.net/10386/6732017-11-16T10:03:11Z2011-01-01T00:00:00ZFull length genome characterisation of Hepatitis B virus isolates at Dr George Mukhari Hospital in Pretoria, South Africa
Magobo, Rindidzani Edith
Introduction: Sub-Saharan Africa is a region with hepatitis B virus (HBV) hyperendemicity with more than 8% HBsAg prevalence. An estimate of two billion people has been reported to carry HBV markers. HBV was associated with about 25% of annual deaths in Africa. HBV possesses a DNA polymerase which lacks proofreading mechanism. This results in highly variability and genetic diversity which poses a challenge for the diagnosis and therapeutic management of HBV infection. High mutation rate of HBV also has great implications on the development of drug resistant mutations. Moreover, HBV diversity represents a challenge for the sensitivity of immunological and molecular diagnostic assays. A number of studies on HBV full length genome have been conducted particularly in developed countries. Limited studies are available in Africa and South Africa. In South Africa, few studies have been done analysing the complete genome of HBV isolates from patients with asymptomatic carriers and fulminant hepatitis B (Owideru et ai, 2001 a; Owideru et ai, 2001 b; Kimbi et ai, 2004; Kramvis et ai, 2002).This study was aimed at characterising the full-length genome of HBV isolates at Dr George Mukhari Hospital, Pretoria, South Africa, with a view of developing a PCR-based technology for amplification and characterisation of HBV strains with different serological profiles. The technology, if successfully developed, will contribute in understanding the molecular mechanisms resulting in various HBV variants or isolates.
Methods: The study design was exploratory. Four stored serum samples collected from HBV infected patients at Dr George Mukhari hospital, Pretoria, were used to develop the molecular technology and test the hypothesis. HBV serology was previously performed targeting 5 HBV serological markers; HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe using Elecsys version; HBV DNA quantification was done using Cobas Amplicor HBV DNA monitor assay, HBV DNA was extracted and subjected to nested PCR assay targeting HBV full length genome as two overlapping fragments: fragment A (1670 bp) and fragment B (1868 bp). The generated PCR products for both fragments were cloned into the pGEM T easy vector and 2 clones were selected from each sample. The plasm ids were purified using Invisorb@ Spin Plasmid Mini Two and the clones were recovered by PCR assay. The sample PCR products and the clone PCR products were purified and sequenced using
SpectruMedix SCE2410 genetic analysis system. HBV genotyping was performed using the NCBI web-based genotyping tool. Phylogenetic analysis was done using MEGA 4 software to confirm HBV genotypes.
Results: Serology results were as follows: All samples were HBsAg positive, Anti-HBs negative, anti-HBc positive and anti-HBe negative. Sample B1121 and sample 6 were HBeAg positive while samples B452 and 5 were HBeAg negative. A total of 12 PCR products were sequenced (4 study samples and 8 clones [2 clones each sample]). In total, 7 HBV full length genome sequences were deduced from this study, with 3 sequences belonging to genotype A, 2 to genotype C and 2 to genotype D.
3 HBV genotypes were detected from this study; genotype A, C and D with subgenotype A2, C1 and D1 respectively. Mutations were observed throughout the genome. In the pre-S/S open reading frame (ORF), the most significant findings were the detection of mutations within the "a" determinant site and major hydrophilic region (MHR). These mutations included Y161F,E164G observed in sample B1121 and B1121C1 belonging to subtype A1; 2 amino acid insertion at aa 161-162 in sample 5 belonging to subtype C1. Drug resistance associated mutations were identified in the polymerase gene, these included M204T and L217R which are associated with adefovir resistance, M204T also resulted in a change from tryptophan (W) to arginine (R) at aa 196 on the overlapping surface gene on sample B452 C1. Basal core promoter (BCP) and pre core/core mutations were detected in study isolates; specifically the BCP double mutation (1762/1764) was seen in 8 isolates which belonged to subtype C1 (5) and D1 (3) and the pre-core stop codon mutations (G1896A) in 4 isolates. (2 belonging to subtype C1 and the other 2 to D1). Other changes observed included a 48 nucleotides deletion in the pre-core gene, 6 nucleotides insertion in the HBx gene of all subtype D1 isolates and a 3 nucleotides deletion in subtype C1 clone.
Conclusion: This study successfully optimised a PCR-based technology for the amplification and characterisation of HBV full length genome. 3 HBV genotypes were detected with subtypes A2, C1 and D1. However, the detected subtypes are rarely detected in South Africa. The detection of subtype A2 may confirm its Southern
African origin. Drug resistance associated mutations were observed in this study. These included the adefovir resistance mutation which the current study confirmed it is a naturally occurring mutation as it was detected in adefovir therapy na'ive patient. The BCP and pre-core/core mutations were detected in genotype C and D isolates; however, their association with serological profile and clinical outcomes could not be deduced. Unique or novel mutations were seen in the study isolates, these included 48 nucleotides deletion in the pre core gene, 3 amino acids insertion in the RNase H and 8 amino acids deletion in the RT domain of polymerase gene. To our knowledge, these mutations have not been identified or reported in the literature. The detection of 6 nucleotide insertion in the HBx gene was reported for the first time in South African isolates. Further analysis is required to ascertain the biological significance of the unique mutations detected in this study.
Thesis (MSc. (Medical Virology))-- University of Limpopo, 2011.
2011-01-01T00:00:00Z