http://hdl.handle.net/10386/194 Sun, 19 May 2013 18:32:07 GMT 2013-05-19T18:32:07Z http://hdl.handle.net/10386/774 Title: Comparative study of vitamin B12 and holotranscobalamin or active B12 as a marker for vitamin B12 deficiency at Dr George Mukhari Hospital Authors: Murray, Louise M. Abstract: Abstract Aim: This study was undertaken to compare the diagnostic sensitivity and specificity of total vitamin B 12 analyses to active B 12 (holoTC) analyses in a population of patients attending the Dr George Mukhari Hospital in Pretoria. Methods: Routine serum folate, full blood count (FBC), thyroid function test, homocysteine, serum total vitamin B 12 and active B 12 analyses were performed on 30 samples. Results: Serum folate was determined in all patients and 96% of the patients had a normal folate value. When looking at the FBC results it is important to note that three times as many males as females presented with anemia (36% versus 16%). Thyroid function tests were normal in 90% of patients. When the total vitamin B 12 test was preformed only 10% of patients tested positive for vitamin B 12 deficiency, in contrast to the active B12 analyses where 16% of patients tested positive for vitamin B12 deficiency. Both tests had a diagnostic sensitivity of 50%. The diagnostic specificity for total vitamin B12 was 93% in comparison with the 86% obtained by the active B12 analyses; when homocysteine was used as the true marker for vitamin B12 deficiency. Conclusion: Diagnostic sensitivity was the same and the total vitamin B12 test's specificity was better in comparison to the active B 12 analyses. Thus the active B 12 assay cannot be recommended for routine use, since it has no benefit. Description: Thesis (M Med (Chemical Pathology)) -- University of Limpopo, 2012. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10386/774 2012-01-01T00:00:00Z http://hdl.handle.net/10386/673 Title: Full length genome characterisation of Hepatitis B virus isolates at Dr George Mukhari Hospital in Pretoria, South Africa Authors: Magobo, Rindidzani Edith Abstract: 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. Description: Thesis (MSc. (Medical Virology))-- University of Limpopo, 2011. Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10386/673 2011-01-01T00:00:00Z http://hdl.handle.net/10386/672 Title: Burden of rotavirus disease and molecular characterization rotaviruses at Dr George Mukhari Hospital from 2003-2005 Authors: Seheri, Luyanda Mapaseka Abstract: Background: Rotavirus infection remains a significant clinical problem throughout the world, infecting almost every child younger than 5 years of age, despite socio-economic status or environmental conditions. Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants and young children. Implementation of an effective vaccine programme could reduce the incidence and severity of rotavirus disease. Decisions about new candidate rotavirus vaccines require reliable data on disease impact in both developed and developing countries. The aim of this study was to assess the burden of rotavirus associated disease at the tertiary care Dr George Mukhari Hospital, Ga-Rankuwa and the secondary care hospital Brits Hospital, Madibeng and to describe the genetic diversity of rotavirus strains circulating in Ga-Rankuwa and Brits communities over a similar time period as the testing of Rotarix@ vaccine. The broad objectives included; to perform a hospital-based burden of rotavirus disease in two different hospitals in the North West of Pretoria area, to conduct molecular characterization of rotaviruses circulating in the Pretoria region and lastly to devise an alternative molecular typing method to detect rotavirus VP6 subgroups. Materials and Method: To investigate the hospital-based burden of rotavirus disease, diarrhoeal stool samples were collected at Dr George Mukhari and Brits Hospitals from children less than 5 years of age. Group A rotavirus antigen was detected from the samples using commercially available rotavirus enzyme immunoassay IDEIA TM Rotavirus test (DAKO, Dakocytomation, Denmark). Genetic analyses of rotavirus strains were determined by polyacrylamide gel electrophoresis (PAGE) to characterize the electrophoretic patterns followed by analysis of the P and G genotypes by RT -PCR and multiplex PCR amplification of specific sequences of VP7 and VP4 genes. To devise an alternative molecular typing method to detect rotavirus VP6 subgroups, with subgroup specificities determined by both VP6 monoclonal antibodies and restriction fragment length polymorphism using restriction endonuclease Acil, Odel and Rsa I. Selected PCR amplicons (VP7 and VP6 genes) were purified, cloned and sequenced. Consensus sequences of the VP7 and VP6 genes were aligned and analysed manually with Chromaslite and BioEdit software packages. Multiple sequence alignment was implemented by Mafft software packages. The nucleotide and deduced amino acid sequences of the VP7 and VP6 genes were compared with reference strains available from GenBank. Multiple methods were used to construct phylogenetic trees and included neighbor¬ joining, maximum parsimony analysis and maximum likelihood distance. Bootstrap values were computed using 1000 replicates with Phylip and the MEGA softwares. The graphic representation of each phylogenetic tree was displayed with the Treeview program. Results: Between 2003 and 2005, a total of 2 514 diarrhoeal stool samples were collected. Of these, 527 (21%) were positive for group A rotavirus and the majority of children hospitalized were less than 2 years of age. The annual peak prevalences of group A rotavirus were 56%, 59% and 56% for 2003, 2004 and 2005, respectively and were observed during the autumn and winter months. The estimated incidence of gastroenteritis associated with rotavirus indicates that one in every 50 to 70 children in the area is likely to be hospitalized with rotavirus diarrhoea between birth and 2 years of age. During the three-year study period, ten, six and seven different RNA electrophoretic patterns were identified in 2003, 2004 and 2005, respectively. The VP6 genes of the representative strains (G1, G2, G3, G9, G8 and G12) were ana lysed with restriction endonuclease Acil, Ode! and Rsa!. The restriction endonucleases produced 11 unique restriction profiles (A-K). The VP6 RFLP results correlated well with strains displaying long RNA electropherotypes and VP6 subgroup 1/ specificity and also with strains displaying short RNA electropherotypes and exhibiting VP6 subgroup I specificity as determined with VP6 monoclonal antibodies. The genotypic distribution varied remarkably and major rotavirus strains detected in circulation during the study period included G2P[4] in 2003, G1 P[8] in 2004 and G3P[8]/ G3P[6] in 2005. Rotavirus strains carrying G8P[8] specificities and unusual G 12P[6] strains were also detected at low frequency The consensus VP7 nucleotide sequences, exhibited the greatest homology and identity (97-99%), when compared against corresponding international reference strains. The nucleotide sequence datasets were closely related to strains from South Africa, Vietnam, Bangladesh, East India, Republic of Congo, China, Russia, Thailand and Japan. The phylogenetic tree revealed the South African strains (G1-G3, G8-G9 and G12) clustered with international strains whereas the G1 strains clustered into two different lineages. Phylogenetic analysis of the VP6 gene revealed four lineages with international reference strains. The VP6 gene showed 97-99% identity at the deduced amino acids level with strains from Taiwan, Bangladesh, the United States and Brazil. Conclusion: This is the first study to estimate the disease burden associated with rotavirus diarrhoea in South Africa. The overall results confirm that rotavirus is the most common cause of severe diarrhoea. The epidemiology of rotavirus diarrhoea in South Africa correlates well with what has been reported in other countries. The proportion of hospitalization of rotavirus infection in children less than 5 years was estimated to an annual prevalence of 22.8% (95%CI 21.2%, 24.5%) at Dr George Mukhari Hospital, while at Brits Hospital was estimated at 18.2% (95%CI 14.9%, 22.1 %). Rotavirus genotypes circulating at Dr George Mukhari Hospital showed a high degree of diversity and the emergence of uncommon rotavirus strains such as G12. The emergence of novel rotaviruses in the region needs to be taken into account where vaccine efficacy is concerned. It is, thus, important to continue with such studies to monitor the rotavirus strains associated with severe gastroenteritis in a hospital setting before and after the introduction of a rotavirus vaccine. Results also indicated that RFLP analysis of VP6 amplicons might be a simple and reliable, alternative to MAb subgrouping. The sequence analysis of the partial length VP6 gene confirmed the location and the recognition sites of the restriction enzymes The RFLP analysis proved to have more potential to accurately detect different rotavirus subgroups. Description: Thesis (PhD (Medical Virology))-- University of Limpopo, 2010. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10386/672 2013-01-01T00:00:00Z http://hdl.handle.net/10386/671 Title: The usefulness of the adenosine deaminase assay for diagnosing tuberculosis pleuritis in immunocompromised patients at Dr George Mukhari tertiary laboratory, Pretoria Authors: Molaudzi, Mulalo Abstract: Mycobacterium tuberculosis is the most common cause of death world-wide and its incidence has been steadily increasing, which is more evident when comparing the global tuberculosis (T8) incidence of 9.24 million in 2006 to 9.27 million cases in 2007. African countries are the second most affected by the epidemic and South Africa is among the 22 highest burden countries most affected by T8 with a very high number of cases relative to the total population. The early diagnosis of tuberculosis and screening of contacts is the cornerstone for controlling spread of active T8 infection. T8 diagnosis becomes even more challenging in patients with immunosuppression (for example in human immunodeficiency virus (HIV) infected), in the case of latent infection and extra pulmonary T8 such as pleural T8. The definitive diagnosis of pleural T8 depends on the demonstration of M. tuberculosis in sputum, pleural fluid and pleural biopsy. Although acid fast bacilli (AF8) microscopy is a rapid, inexpensive and relatively simple method, it has low sensitivity. The culture method is more sensitive than AF8 microscopy, detecting 25-37% of all pleural tuberculosis cases however it takes 4 to 8 weeks for a visible growth on a solid medium. Therefore it is important to find a rapid and reliable test for the diagnosis of pleural T8 particularly in developing countries such as South Africa where there is a high T8 incidence and HIV infection rate. Description: Thesis (MSc (Med)(Microbiology)) -- University of Limpopo, 2012. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10386/671 2012-01-01T00:00:00Z