3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The response to natural RSV infection in HIV-infected children compared to HIV-uninfected children(2004-10-11) Sorour., Gillian. Ann.Respiratory syncytial virus ( RSV ) is the most important virus causing acute respiratory tract infections in children and is an important cause of morbility and mortality. HIV- infected children are also at an increased risk of recurrent and more severe RSV infections. There are few studies looking at how HIV-Infected infants respond immunologically to natural RSV infection.Item The role of CCL3 and CCL4 encoding genes in control of HIV-1 infection(2017) Mncube, SizananiTwo CC chemokine ligands that bind the CCR5 receptor (major coreceptor used by HIV-1), namely CCL3 and CCL4, are encoded by the CCL3 and CCL4 genes found in 2 copies per diploid genome and their non-allelic variants, CCL3L and CCL4L, respectively, which are found in variable copy numbers. CCL3L and CCL4L gene copy numbers lower than the population specific median have been associated with various HIV-1 related outcomes including increased susceptibility to HIV-1 infection, faster rate of progression to AIDS and high viral loads. These associations have however been controversial and the controversy has in part been attributed to technical challenges of accurate high copy number determination. In this study we investigated the association of the variable copy number genes, CCL3La and CCL3Lb (the sum of which is CCL3L) and CCL4La and CCL4Lb (the sum of which is CCL4L) and previously identified CCL3 haplotypes (Hap-A1, Hap-A3 and Hap- 2SNP) in natural control of HIV-1 infection in the South African black population. The study was conducted on antiretroviral (ARV) naive HIV-1-infected controllers (HICs, N=52), comprised of elite controllers (ECs, N=11), viraemic controllers (VCs, N=30) and high viral load long term non progressors (HVL LTNPs, N=11) and HIV-1-infected progressors (N=74). HIV-1 controllers were also categorised and analysed on the basis of their viral loads as individuals with less than 400 RNA copies/ml (<400 HICs, N=20) and greater than 400 RNA copies/ml (>400 HICs, N=32). Droplet digital PCR (ddPCR), which is a much more accurate method for determination of high copy numbers compared to real-time PCR, was used to determine CCL3L and CCL4L gene copy numbers. We developed and validated ddPCR assays for CCL3L copy number determination and used previously developed ddPCR assays for CCL4L copy number determination. CCL3L and CCL4L gene copy numbers were compared between HICs, HIC subgroups and progressors, individually or in combination as continuous variables or stratified around the population median. CCL3 haplotypes were determined using previously designed CT shift real-time PCR assays and these were compared between HICs, HIC subgroups and progressors. We found CCL3La copy number to be equal to CCL4L copy number in all individuals (with the exception of one individual who had 8 copies of CCL3La and 7 copies of CCL4L), thus any associations involving these genes would mirror each other and were designated as CCL3La/CCL4L. CCL3L and CCL4L copy numbers compared as continuous variables did not yield any significant differences between HICs, HIC subgroups and progressors. However, a strong trend of higher representation of individuals with high CCL3La/CCL4L copy numbers was seen in VCs (p=0.07) compared to progressors. When individuals were grouped and compared according to copy number > population median (high) and ≤ population median (low), significant overrepresentations of individuals with CCL3La/CCL4L copy number > population median were seen when comparing VCs (p=0.03, OR=0.39) and <400 HICs (p=0.01, OR=0.21) to progressors. Comparing the total controller group (HICs) to progressors, however only showed a strong trend of overrepresentation of individuals with CCL3La/CCL4L copy number > population median (p=0.05, OR=0.4). Comparison of HICs, HIC subgroups and progressors having high CCL3L and/or high CCL4L copy numbers revealed a significant difference in VCs (p=0.04, OR=0.28) compared to progressors, with VCs having a higher representation of individuals with high CCL3L and/or CCL4L. Combined associations were however more significant when comparing individuals with high CCL3La and/or CCL4La with HICs (p=0.01, OR=0.38), VCs (p=0.02, OR=0.31) and <400 HICs (p=0.01, OR=0.21) having a higher representation of individuals with high CCL3La and/or CCL4La compared to progressors. Given that the CCL3L and CCL4L gene copy numbers occur in different combinations in different individuals, we next explored the specific patterns of copy number possession by assigning pattern identifiers (IDs) to each individual based on the number of copies of CCL3L, CCL3La and CCL3Lb or CCL4L, CCL4La and CCL4Lb. The 651 CCL3L pattern (i.e 6 copies of CCL3L, 5 copies of CCL3La and 1 copy of CCL3Lb) was overrepresented in ECs (p=0.02, OR=0.17) and <400 HICs (p=0.04, OR=0.26) whereas a 633 CCL4L pattern was overrepresented in HICs (p=0.03, OR=0.07) and >400 HICs (p=0.03, OR=0.06) with a combined 550541 CCL3L and CCL4L pattern being overrepresented in VCs (p=0.02, OR=0.05). Moreover, we found a CCL3La and CCL3Lb copy number difference of 2 to be underrepresented in HICs (p=0.03, OR=3.58) compared to progressors, with a copy number difference of 5 being overrepresented in VCs (p=0.02, 0.29) when compared to progressors. No significant differences were seen when CCL3 Hap-A1, Hap-A3 and Hap-2SNP allelic and genotypic frequencies were compared between HICs, HIC subgroups and progressors, however a trend of higher representation of Hap-A1 heterozygosity was seen in >400 HICs when compared to progressors (p=0.09, OR=0.14) or healthy HIV-1 uninfected controls (p=0.09, OR=0.20). Additionally, combined analysis of CCL3 haplotypes and CCL3La copy numbers does not seem to be more significantly associated with natural control of HIV-1 infection compared to CCL3La copy numbers alone, with the exception of CCL3La and Hap-A3 which showed a slightly higher significant association in VCs (p=0.02, OR=0.11) compared to progressors. Overall, our findings suggest that the CCL3 and CCL4 chemokines play a role in natural control of HIV-1 infection, in agreement with previous studies which found high CCL3L copy numbers to be protective against disease progression. However, future studies with larger sample sizes would be beneficial to further elucidate the associations of the CCL3L and CCL4L gene copy numbers with natural control of HIV-1.Item Epidemiology and prevention of sepsis in young infants and the potential impact of maternal HIV infection on neonatal sepsis(2016) Cutland, Clare LouiseIntroduction: Neonatal infections contribute to 25% of all neonatal deaths, which account for approximately 44% of all under-5 childhood deaths globally. Pathogens responsible for sepsis in neonates and young infants can be acquired vertically prior to or during labour, or from the environment (community or hospital). This project evaluated the burden and aetiology of sepsis in neonates and young infants (≤90 days), and explored this association to in-utero exposure to human immunodeficiency virus. The study also included a specific focus on the epidemiology of invasive Group B Streptococcal disease in young infants. Additionally, we assessed the efficacy of intrapartum chlorhexidine vaginal washes for: (i) preventing early-onset neonatal sepsis; and (ii) vertical transmission of potentially pathogenic bacteria to the newborns. Furthermore, we evaluated risk factors for poor outcomes due to neonatal sepsis. Materials and methods: (i) A bacterial surveillance system was established at Chris Hani Baragwanath Academic Hospital (CHBAH) from 2004-2008 to identify young infants with bacterial sepsis hospitalised in the neonatal and paediatric wards. Medical and microbiological records were utilised to obtain clinical and laboratory data. Maternal HIV results were obtained from antenatal testing records or admission records. (ii) A blinded, randomised, placebo-controlled trial of 0.5% chlorhexidine maternal vaginal intrapartum wipes and newborn skin wipes was conducted at CHBAH between 2004 and 2007. Consented, eligible participants were randomised during labour to receive either chlorhexidine vaginal wipes or water external genitalia wipes. Newborns received either chlorhexidine full-body wipes (intervention arm) or foot wipes (control arm). Maternal and infant participants were followed up for admissions during the first month after delivery/ birth. A subset of 5144 maternal participants had an intrapartum lower vaginal swab collected, and skin swabs were collected from their newborns to assess colonisation with potentially pathogenic bacteria (Group B streptococcus, Escherichia coli and Klebsiella pneumoniae). Results: Group B streptococcus (GBS) was the most commonly isolated bacterial pathogen, causing 35.2% of culture-confirmed sepsis in infants ≤90 days, 41.6% of early-onset disease (EOD, 0-6 days), 40.5% of late-onset neonatal disease (LOD, 7-27 days) and 18.7% of young-infant community-acquired disease (YI-CAD, 28-90 days). Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) contribute 16.2%, 12.2% and 3.4% to sepsis in young infants. Overall, incidence (per 1000 live births) of invasive GBS disease was 2.72 (95% confidence interval [95% CI]: 2.46 to 3.01), including an incidence of 1.50 and 1.22, respectively, in infants 0-6 days and 7-90 days of age. HIV-exposed infants were at greater risk of EOD (Relative risk [RR]: 1.69; 95% CI: 1.28-2.24) and LOD (RR= 3.18; 95% CI: 2.34-4.36) than HIV-unexposed infants. GBS serotypes Ia and III caused 84.0% of invasive GBS disease in young infants. Intrapartum chlorhexidine interventional wipes was not efficacious in prevention of any of: (i) vertical transmission of pathogenic bacteria (54% vs. 55%; efficacy -0.05, 95% CI: -9.5 to 7.9) to the newborns; (ii) sepsis in first 3 days of life (3% vs. 4%; p=0.65,); (iii) sepsis in the later neonatal period (both <1%; p=0.4444); or (iv) maternal puerperal sepsis(both <1%; p=0.56). Conclusion: GBS, S. aureus, E. coli and K. pneumoniae are the most commonly isolated bacterial pathogens in neonates and infants ≤90 days old. HIV-exposed infants are at greater risk of GBS sepsis. Intrapartum chlorhexidine intervention was not efficacious in reducing vertical transmission of pathogenic bacteria, neonatal or maternal sepsis. Alternative interventions to prevent sepsis in young infants, including maternal immunisation, need to be investigated in setting such as ours where there is a high prevalence of maternal HIV infection.Item Immune thrombocytopaenia at a central hospital in Johannesburg(2016) Mbao, MelvinBackground. Primary immune thrombocytopenia (ITP) is a rare disease causing significant morbidity. South Africa has a high prevalence of HIV infection which may be associated with immune thrombocytopenia. There is a paucity of clinical, management and outcome data on immune thrombocytopenia in the local South African setting. Objectives. To compare the demographics, clinical presentation, management and treatment outcomes of immune thrombocytopenia in HIV positive and HIV negative patients and to compare the treatment outcomes with established international guidelines. Methods. This was a retrospective comparative study conducted at Charlotte Maxeke Academic Hospital, Johannesburg, from January 2003 to December 2014. Adults (≥ 18 years) with confirmed diagnosis of ITP were included. Hospital charts of eligible patients were reviewed to extract data on their clinical presentation, diagnosis, HIV status, treatment and outcomes. A comparison was made between HIV positive and negative patients. Descriptive analysis was performed on the data and results were presented graphically. The P-value of <0.05 was regarded as significant. Results. A total of 250 patients were screened, of which 154 patients met eligibility criteria for the study. 91% of the patients were female, 58% were HIV negative and 42% were HIV positive. The 25-35 year age-group comprised the highest percentage of HIV positive patients (42%). There was no difference in the presentation of symptoms between HIV positive and HIV negative patients. Response to first line therapy was not significantly different between the HIV positive and HIV negative patients (p=0.1370). The patients who went on second line therapy, showed excellent response with approximately 80% reaching complete response. There was no difference in HIV positive and HIV negative groups. Conclusion. In a large central hospital in a high HIV prevalence setting, there is no significant difference between HIV positive and HIV negative patients in terms of clinical presentation, treatment and outcomes in confirmed patients with immune thrombocytopenia. The management of ITP at the CMJAH is comparable to that of published guidelines.Item The role of CCL4/Mip-1b in HIV infection(2015) Bharuthram, AvaniThe controversy surrounding the findings that copy number variation (CNV), of the CCL3 encoding genes, influences HIV-1 infection and disease progression has been in part attributed to the variable results obtained from methods used for copy number evaluation. Like CCL3, the genes encoding the CC chemokine CCL4, also a natural ligand of the CCR5 receptor, are found to occur in population-specific multiple copy number and have been shown to play a protective role against HIV-1. In this study, we evaluated the standard method of quantitative Real-Time PCR (qPCR) and Droplet Digital PCR (ddPCR) for CCL4L gene copy number determination. The CCL4 encoding genes are CCL4, occurring in two copies per diploid genome (pdg), and the non-allelic CCL4L genes, comprised of CCL4L1 and CCL4L2, which are both found in multiple copies pdg. Copy number of CCL4L, CCL4L1 and CCL4L2 was determined in a cohort of HIV-1-uninfected individuals from the South African Black (n=23) and Caucasian (n=32) population groups using qPCR and ddPCR, with the addition of another 30 black individuals to the ddPCR cohort. A stronger correlation between the number of CCL4L copies and the sum of CCL4L1 and CCL4L2 copies generated by ddPCR (r=0.99, p<0.0001) compared to qPCR (r=0.87, p<0.0001) was observed. Real-Time qPCR exhibited greater inaccuracy at higher copy numbers which is particularly relevant to our cohort of Black individuals who have a higher range of CCL4L copies (3-6) compared to Caucasians (0-4) and a higher population median (4 and 2, respectively). Medians and ranges of CCL4L1 (Black: 2, 0-4, Caucasian: 0, 0-2) and CCL4L2 (Black: 2, 1-5, Caucasian: 2, 0-3) were also higher in the Black population. Droplet Digital PCR was shown to be a far superior method to qPCR for assessment of CCL4 gene copy number variation, the accuracy of which is essential for studies of the contribution of variable gene copy number to phenotypic outcomes of host infection and disease course. We further used the CCL4L copy number data to examine variation of these genes with reference to measured stimulated CCL4 production by the same individuals. Although significant differences in the copy number range medians and patterns of distribution of the genes CCL4L1, CCL4L2 and combined CCL4L were observed between the two populations, on a whole, the two populations do not differ significantly with respect to CCL4 production. Caucasian females however had a higher level of protein production per copy of CCL4L than Black females. When stratifying production based on population specific copy number median, Black individuals showed a decreased level of protein production at a CCL4L copy number below the median, although this was not maintained when the CCL4L1 and CCL4L2 genes were analysed individually. CCL4 copy number and production data was compared to data generated for CCL3 in the same cohort. CCL4 chemokine levels were significantly higher in both the Black and Caucasian populations and Black individuals had a higher number of gene copies of the CCL3L genes giving rise to functional CCL3 protein than the CCL4L genes producing functional CCL4 protein. These results highlight genetic differences between divergent populations, differences in distribution of CCL4 and CCL3 encoding genes and protein production, and suggest an intricate regulation of the CCL4L encoding genes. In order to investigate the role of CCL4 in HIV-1 control, we next assessed variation in the numbers of CCL4L copies in relation to CCL4 production in a cohort of 14 long-term nonprogressors (LTNPs). While no associations between copy number and CCL4 production were observed, the LTNP cohort had significantly lower levels of CCL4 production than the HIV-1-uninfected cohort, and this was maintained when Black and Caucasian individuals were examined individually. When the LTNP cohort individuals were divided based on viral loads, individuals with viral loads <400 RNA copies/ml had significantly lower CCL4 production than those with viral loads >400 RNA copies/ml. This finding suggests a role for the amount of CCL4 produced in the reduced pace of HIV-1 progression observed in LTNP individuals. Since genetic variation other than copy number can also influence CCL4 protein production, we then proceeded with a thorough genetic characterization of the CCL4 gene of uninfected individuals and investigated any possible relationships with select variants and CCL4 production. Sequencing the complete gene and flanking regions of 23 Black and 32 Caucasian revealed several intra as well as extragenic SNPs, with one newly identified SNP at position -1063. The Black population exhibited a higher degree of variability compared to the Caucasian population. We described four haplotypes in the Caucasian population, three haplotypes in the Black population, and a single haplotype shared between both population groups. Of the four indels that were identified, a three bp deletion (rs3216921) was the only indel present in the Caucasian population and was not identified in the Black population. Of all the indels, this indel had the highest allelic frequency (14%). Comparisons of haplotypes and prevalent SNPs with protein production in both population groups did not show any significant differences. Caucasian individuals harbouring the 3bp deletion however, had significantly higher levels of CCL4 production (p=0.024). These results form a good base from which to further investigate the impact of select genetic variants on CCL4 production and possibly HIV-1 control. This study has succeeded in optimising a ddPCR assay for the copy number determination of the CCL4 genes and has interrogated the relationship between CCL4L copy number and CCL4 production in both HIV-1-uninfected individuals as well as a subset of LTNPs. The results suggest a complex, intricate regulation of CCL4 that appears to play a role in HIV-1 control. In conclusion, this study forms the basis for future work to build on and to further explore the role of this CCR5 ligand in HIV-1 disease.Item Epidemiology of tuberculosis meningitis in an area with a high prevalence of HIV-infection(2015-04-17) Chaya, ShaakiraIntroduction Mycobacterium tuberculosis meningitis (TBM) is a severe manifestation of extra-pulmonary tuberculosis (EPTB) in children, particularly under 5 years of age. Children are vulnerable to EPTB as they are immunologically immature and unable to contain Mycobacterium tuberculosis (MTB) infection in the lung. Common neurological sequelae of TBM include focal motor deficits, vision loss and hydrocephalus. Early stage diagnosis and timeous anti-tuberculosis treatment decreases the case fatality rate of TBM. Objective To characterise the burden, clinical presentation, laboratory markers and short-term outcome of TBM in HIV-infected and HIV-uninfected children. Methods The electronic databases of admission of children at Chris Hani Baragwanath Academic Hospital (CHBAH), between January 2006 and December 2011 with a diagnosis of TBM were reviewed. Individual patient records were retrospectively reviewed for clinical and laboratory data. In addition, admissions from the neurosurgery wards were also reviewed. In patients whose medical records were unavailable, laboratory data was used. Results The overall incidence of TBM in 2006 was 6.96 per 100 000 (95% Confidence Interval [95%CI]: 4.46-10.36), peaked at 9.87 per 100 000 (95% CI: 6.91-13.67) in 2009 and subsequently declined to 3.18 per 100 000 by 2011 (95% CI: 1.64-5.56). There was a 38.6% (95% CI: 10.0-58.0; p=0.011) reduction in the overall incidence of TBM when comparing the period 2006-2009 with the period 2010-2011. This decline was particularly evident in HIV-infected children (49.6% reduction; 95%CI: 1.05-74.35; p=0.042). There were no differences in the clinical symptoms of TBM or tuberculosis between HIV-infected and -uninfected children. Previous history of TB was significantly higher in HIV-infected children compared to HIV-uninfected children (OR 4.63; 1.40-15.22; p=0.011). Tuberculin skin test positive-reactivity (OR 0.09; 0.02-0.43; p=0.002) and sputum culture positivity (OR 0.29; 0.10-0.86; p=0.025) were less common in HIV-infected compared to -uninfected children. Cerebrospinal fluid cytology and biochemistry results were similar between HIV-infected compared to -uninfected children. Morbidity (22.7% in HIV-infected vs. 33.0% in -uninfected) and mortality (6.4% in HIV-infected vs. 6.9% in -uninfected) were similar between HIV-infected and -uninfected children. Conclusion The incidence of TBM has decreased over the study period 2006 to 2011.This decrease was temporally associated with an increase in the uptake of antiretroviral treatment in HIV-infected individuals.Item The effect of tuberculosis infection on the body composition of HIV positive adult patients on HAART in Johannesburg South Africa(2015-04-13) Govathson, CarolineBoth HIV and tuberculosis (TB) have been documented to have detrimental effects on the nutritional status of those infected and nutritional status is a strong predictor of disease progression and survival. Body composition measures can be used as a proxy for nutritional status and takes into account body fat, muscle and water. It constitutes Fat Mass(FM), Fat Free Mass (FFM), Total Body Water (TBW), Extracellular Water (ECW), Intracellular water (ICW), Daily Energy Expenditure (DEE), Basal Metabolic Rate (BMR), phase angle and BMI which can be analysed as separate outcomes. Its use in evaluation of nutritional status has been reported to give more accurate results than the use of weight alone. We compared body composition measures and changes over a 12 month period in patients with HIV alone to patients with HIV and TB.Item Molecular characterization of hepatitis B virus (HBV) from mono-infected and HBV/human immunodeficiency virus (HIV) co-infected individuals in Sudan(2014-09-09) Yousif, MukhlidHepatitis 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.Item Genetics of HIV-associated sensory neuropathy in black Southern Africans(2014-02-18) Hendry, Liesl MaryHIV-associated sensory neuropathy (HIV-SN) is a common complication associated with human immunodeficiency virus (HIV)-infection. A common symptom of HIV-SN is pain. Variation at specific loci within certain candidate genes has been suggested to alter susceptibility to developing HIV-SN as well as the susceptibility to developing pain and the intensity of the pain experienced. Few studies, however, have been conducted in individuals of black African ancestry. The aim of the current research was to conduct an in-depth study, in a black Southern African population, of genes previously associated with susceptibility to developing HIV-SN (TNFA and surrounding genes and IL12B) and variations in pain susceptibility and intensity (GCH1, KCNS1, IL1B, IL6, CCL2 and CCR2) in other neuropathic pain states. Single nucleotide polymorphisms (SNPs) identified in the literature were supplemented with population appropriate tagSNPs to improve assessment of the genes in an African population. Genotyping of previously collected deoxyribonucleic acid (DNA) samples was carried out using a GoldenGate assay with VeraCode microbeads and data were read on an Illumina BeadXpress Reader. Data were statistically analysed to assess the association of the genetic variants with susceptibility to developing HIV-SN and pain and variability of pain intensity in those patients with painful HIV-SN. Although some SNPs and haplotypes in the genes investigated associated with HIV-SN susceptibility (TNFA region), pain susceptibility (TNFA region, IL12B, KCNS1, IL6 and CCR2) or pain intensity (TNFA region, KCNS1, IL1B and IL6), none of the results were consistent with that which has been found in previous studies in non-African populations. Reasons for this may be that associations are population-specific or model-specific. Limitations of the study included the use of a relatively small sample, the method of sampling (convenience sampling), genotyping failure and tagging inefficiency in some instances, and the fact that there is no Southern African population dataset to use for tagSNP selection. My findings emphasise the importance of conducting genetic association studies in separate ethnic groups.