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.
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.
Role of novel biomarkers in predicting chronic kidney disease progression among black patients attending a tertiary hospital in Johannesburg, South Africa
(University of the Witwatersrand, Johannesburg, 2023) Meremo, Alfred Jackson; Naicker, Saraladevi; Duarte, Raquel; Paget, Graham; Dickens, Caroline
Background: Chronic kidney disease (CKD) is a leading health issue and its magnitude has been increasing globally; where the developing countries are the most affected and they are the least equipped to deal with its associated consequences. Chronic kidney disease can rapidly and quietly progress to late CKD stages in impoverished environments. Early recognition of patients who are likely to develop end-stage kidney disease (ESKD) is important. Methodology: A prospective longitudinal study was conducted on CKD patients of black ethnicity attending at the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) renal outpatient clinic in South Africa, as from September 2019 to March 2022. Patients provided blood and urine samples for investigations in the laboratory at study enrolment (0) and at the 24 months follow up. The concentrations of the transforming growth factor isoforms [(TGF)-β1, TGF-β2 and TGF-β3) were determined in serum and urine at baseline using the Human TGF-β duoset ELISA. Data were descriptively and inferentially processed by the REDcap and analyzed using STATA version 17 and multivariable logistic regression analysis was applied to find out the predictors of CKD progression. Results: A total of 312 patients were recruited into the study; the median age was 58 (IQR 46 -67) years and 162 (51.9 %) were male. Hypertension was present in majority (96.7 %) of the patients. Diabetes mellitus was present in 38.7 % of patients and 38.1 % of the study patients had both hypertension and diabetes mellitus. A total of 297 (95.2%) patients completed the study. Death was reported in 5 (1.6%) patients and 10 (3.2%) of patients were lost to follow up. The prevalence of CKD progression was 49.5%, 33% had CKD remission and 17.5% had CKD regression while the prevalence of CKD progression by change in uPCR > 30% was 51.9%. Almost half (47.8 %) had a sustained decline in eGFR of > 4 ml/min/1.73 m2 /year or more, 35.0% of the patients moved to a more severe stage of CKD and 19.9% had more than 30% 6 decline in eGFR in two years. For patients with CKD progression, 54.9% patients were men and at baseline, their median age was 59 (46 - 67) years, urine protein creatinine ratio (uPCR) increased at 0.039 (0.015-0.085) g/mmol, eGFR was 37 (32 -51) mL/min/1.73 m2; the median serum TGF-β1 was 21210 (15915 – 25745) ng/L and the median urine TGF-β3 was 17.5 (5.4 –76.2) ng/L. For those who had CKD progression, hypertension was present in the majority (95.2%) of the patients. Diabetes mellitus was present in 59 (40.1%) patients and 58 (39.5%) patients had both hypertension and diabetes mellitus; 48.3% had severely increased proteinuria, 45.6% patients had anaemia, 34.0% had hyperuricemia and 17.7% had hypocalcaemia at baseline. For those patients with CKD progression vs those without CKD progression, the baseline median serum TGF-β1 was 21210 (15915 – 25745) ng/L vs 24200 (17570 – 29560) ng/L, the baseline median urine TGF-β3 was 17.5 (5.4 – 76.2) ng/L vs 2.8 (1.8 – 15.3) ng/L; however, baseline serum and urine TGF-β isoforms did not predict progression of CKD on univariate and multivariable analyses. Regarding use of medications among patients with CKD progression, calcium channel blockers (amlodipine) were used by majority (85.2 %) of the patients. Diuretics were used by 63.4% of the patients and 31.7 % of the patients were using insulin. Variables associated with CKD progression after multivariable logistic regression analysis were moderately elevated proteinuria (OR 2.1, 95% CI (1.1 – 3.9), P= 0.019), severely elevated proteinuria (OR 6.1, 95 % CI (3.2 – 11.6), P = 0.001), hyponatraemia (OR 4.5, 95% CI 1.8 - 23.6, P= 0.042), hypocalcaemia (OR 3.8, 95 % CI 1.0 - 14.8, P = 0.047), anaemia (OR 2.1, 95% CI 1.0 - 4.3, P= 0.048), elevated HbA1c (OR 1.8, 95 % CI 1.2 - 2.8, P = 0.007), diabetes mellitus (OR 1.8, 95 % CI 1.9 - 3.6, P = 0.047), current smoking (OR 2.8, 95 % CI 1.9 - 8.6, P = 0.049), medications which were calcium channel blockers (OR 2.07, 95 % CI 1.04 – 4.12, P = 0.038), diuretics (OR 2.35, 95 % CI 1.37 – 4.00, P = 0.002), insulin (OR 1.96, 95 % CI 1.01 – 3.84, P = 0.048) and baseline serum calcium levels (OR 0.06, 95 % CI 0.01 -0.64, P = 0.019). An increase in uPCR > 30% at two years identified most patients with CKD progression; clinicians and nephrologists should utilize change in uPCR > 30% at two years to identify those patients with CKD who are likely to progress more rapidly, who require closer surveillance and monitoring with emphasis on slowing or stopping progression of the CKD. Conclusion: Our study has demonstrated a higher prevalence of CKD progression in a prospective longitudinal study among black patients than that reported in previous studies. CKD progression was associated with current smoking, hyponatremia, hypocalcemia, anaemia, elevated HbA1c, diabetes mellitus, and proteinuria. While patients with CKD progression had lower baseline concentrations of serum TGF-β1 and increased baseline urinary TGF-β3 concentrations, baseline serum and urine TGF-β isoforms did not predict progression of CKD. The roles of the various serum and urine TGF-β isoforms in CKD progression at baseline are still unclear and highlight the importance of further studies to determine their isoform specific effects.