Browsing by Author "Dickens, Caroline"

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    Influence of vitamin D receptor polymorphisms on biochemical markers of mineral bone disorders in South African patients with chronic kidney disease
    (2018-02) Waziri, Bala; Dix-Peek, Therese; Dickens, Caroline; Duarte, Raquel; Naicker, Saraladevi
    Background: It remains unclear whether genetic factors may explain the reported variation in the levels of biochemical markers of chronic kidney disease mineral and bone disorders (CKD- MBD) across ethnic groups. Therefore, the aim of this study was to examine the influence of vitamin D receptor (VDR) polymorphisms on secondary hyperparathyroidism and its association with vitamin D levels in black and white South African study participants. Methods: This was a cross sectional study involving 272 CKD stage 3- 5D patients and 90 healthy controls. The four major VDR polymorphisms (Bsm 1, Fok 1, Taq 1, and Apa1) were genotyped using the polymerase chain reaction- restriction fragment length polymorphism (PCR –RFLP) method. In addition, biochemical markers of CKD-MBD were measured to determine their associations with the four VDR polymorphisms. Results: With the exception of Taq I polymorphism, the distribution of the VDR polymorphisms differed significantly between blacks and whites. In hemodialysis patients, the Bb genotype was significantly associated with moderate secondary hyperparathyroidism (OR, 3.88; 95 CI 1.13–13.25, p = 0.03) and severe hyperparathyroidism (OR, 2.54; 95 CI 1.08–5.96, p = 0.03). This was consistent with the observed higher levels of median parathyroid hormone, fibroblast growth factor 23 and mean phosphate in patients with Bb genotype. This candidate risk genotype (Bb) was over represented in blacks compared to whites (71.0% versus 55.6%, p < 0.0001). In an unadjusted regression model, FokFf genotype was found to be significantly associated with the risk of developing severe vitamin D deficiency < 15 ng/ml (OR, 1.89; 95 CI 1.17–3.07, p = 0.01). Conclusion: The VDR Bb genotype is an independent predictor of developing secondary hyperparathyroidism in patients with end stage kidney disease. In addition, study participants with FokFf genotype are at increased of developing severe 25 -hydroxyvitamin D [25(OH)D] deficiency.
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    Occult hepatits B virus (HBV) infection in the Chacma Baboon (Papio ursinus orientalis)
    (2011-11-23) Dickens, Caroline
    Members of the family Hepadnaviridae have been detected in both avian and mammalian species. They have a very limited host range, and among the nonhuman primates, have been found to occur naturally in chimpanzees, gorillas, gibbons, orang-utans and woolly monkeys. The human hepatitis B virus (HBV) has been shown to infect chimpanzees, Barbary macaques and tree shrews. During the course of a previous study, to determine the susceptibility of baboons (Papio ursinus orientalis) to HBV infection, HBV DNA was detected in the serum of 2 baboons prior to their inoculation with HBV-positive human serum, raising the possibility that baboons are naturally infected with a hepadnavirus. Therefore the aim of this study was to determine the prevalence of HBV in wildcaught baboons and to molecularly and functionally characterise the virus isolated from these baboons. DNA was extracted from the sera of wild-caught baboons and four separate regions of the HBV genome amplified by nested polymerase chain reaction (PCR). Samples were only considered to be positive for HBV if at least three of these regions amplified. DNA was extracted from the liver tissue of one of the HBV DNA-positive baboons using a proteinase K digestion followed by a phenolchloroform extraction and ethanol precipitation. From this extract, the complete HBV genome was amplified by nested PCR of eight overlapping subgenomic fragments, and sequenced. This sequence was analysed phylogenetically using both the PHYLIP and Simmonic software packages. A selective real time PCR using SYBR®-green detection was used to detect covalently closed circular (ccc) DNA. RNA was extracted from the baboon liver tissue using a guanidinium-acidphenol extraction method, reverse transcribed and portions of the HBV genome amplified by nested PCR. Transmissibility of the virus was tested by injecting four experimentally naïve baboons individually with serum from four HBV DNApositive baboons and followed for 26 weeks. HBV was detected in the serum of 5/69 (7,2%) wild-caught baboons by Southern hybridization and in 11/49 (22,4%) adult and 4/20 (20,0%) juvenile wild caught baboons. This gave an overall prevalence of 21,7% in the baboon population surveyed. Serologically, the baboon sera were negative for all markers of HBV infection and alanine aminotransferse (ALT) levels were normal. In the one baboon liver tissue available, HBcAg was detected by immunohistochemical staining in some of the hepatocyte nuclei, but HBsAg was not detected. Phylogenetic analysis of the complete genome of the HBV isolate found it to cluster with subgenotype A2, a surprising result considering that subgenotype A1 predominates in South Africa. However, unlike other subgenotype A2 isolates, the basic core promoter had the G1809T / C1812T double mutation characteristic of subgenotypes A1 and A3 and the precore region had the G1888A mutation unique to subgenotype A1. These mutations in the basic core promoter and precore regions have previously been shown to reduce the expression of the precore and core proteins. Four additional mutations in the polymerase, surface, X and core open reading frames (ORFs) further differentiated the baboon HBV strain form the majority of previously sequenced subgenotype A2 isolates. cccDNA was detected at low levels in the baboon liver tissue. Regions of the precore/core and surface ORFs were amplified off reverse transcribed cDNA. These results demonstrate HBV replication in the baboon liver. Transmission of the virus was shown by the detection of HBV DNA in the sera of the four inoculated baboons at various times throughout the 26 week follow-up period. These baboons also showed transient seroconversion for HBsAg and HBeAg during this period with intermittent fluctuations in ALT levels. Moreover, using DNA extracted from liver tissue obtained at necropsy from one of the injected baboons, the sequence of the HBV surface gene amplified was found to be identical to the sequence of the isolate from inoculum. The finding of subgenotype A2 in the baboon is paradoxical because subgenotypes A1 and A3 as well as genotypes D and E predominate in Africa. The possibility exists that subgenotype A2 is an older strain that has been overtaken by these other strains. There is however a scarcity of subgenotype A2 sequencing data from Africa and a higher circulation of this subgenotype could be uncovered with more extensive molecular epidemiological studies in more remote areas. Alternatively, a recent discovery of alternative compartmentalization of subgenotype A2 infections in the peripheral blood lymphocyte population of individuals from India, where subgenotype A1 also predominates, could explain the lack of detection of this subgenotype in Africa. Occult hepatitis B infection is defined as the presence of HBV DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for HBsAg by currently available assays. The detection of HBV DNA in the baboon liver and serum in the absence of serological markers therefore classifies this infection as occult. To our knowledge, this is the first study to demonstrate a naturally occurring occult HBV infection in non-human primates.