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A Comparative study between the prevalence of MTHFR A1298C SNP and homocysteine metabolism in an elderly black South African population
(Vaal University of Technology, 2018-08) Dippenaar, Luzanne; Lebea, P. J., Dr.; Grobler, C. J., Dr.
Background: Cardiovascular diseases are one of the most common causes of death worldwide. This is not only a problem in developed countries, it is of major concern for public health in developing countries as well. Increased homocysteine is an independent risk factor for cardiovascular diseases. Nutritional deficiencies of folate, vitamin B6 and vitamin B12 are associated with hyperhomocysteinemia. MTHFR A1298C, a single nucleotide polymorphism, is similarly linked with higher concentrations of homocysteine. The aim of this study was to determine the prevalence of MTHFR A1298C in a black elderly population, along with folate, vitamin B6 and vitamin B12 and to evaluate the effect on homocysteine levels. Methodology: The research design was an observational cross-sectional study and was ethically approved. A total of 84 elderly who attend a day-care centre (also met inclusion criteria) were purposively selected. DNA was extracted and frozen on the day of blood collection. The MTHFR A1298C genotype was determined with real time PCR. Homocysteine, folate, vitamin B6 and vitamin B12 serum levels were detected with commercial assay kits. Results: Homocysteine was found to be elevated with a median of 17.78 µmol/L (interquartile range 13.98-21.03 µmol/L). Serum folate, vitamin B6 and vitamin B12 medians were in the normal range. Although, 5.95% and 22.62% of the population were deficient and possibly deficient for vitamin B12, respectively. MTHFR A1298C frequency was as follow: 89.29% (AA), 9.52% (AC) and 1.19% (CC), with no significant correlation (p>0.05) with homocysteine. Vitamin B12 correlated significantly with homocysteine levels. Conclusion: Vitamin B12 deficiency had an effect on homocysteine levels. Overall, nutritional deficiencies are not responsible for the hyperhomocysteinemia in this population. In conclusion from this study showed MTHFR A1298C frequency in black South Africans does not contribute to homocysteine as a risk factor for cardiovascular disease. Keywords: Cardiovascular disease, elderly, folate, homocysteine, MTHFR A1298C, vitamin B6, vitamin B12.
Correlating the prevalence of C174G polymorphism with IL-6, TNF-α and Hs-CRP in an elderly black South African population
(Vaal University of Technology, 2019-03) Valentine, Jessica; Lebea, J., Dr.; Grobler, C. J., Dr.
Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence thereof is on the rise in developing countries due to the demographic transition and urbanization. The inflammatory process, atherosclerosis, is at the root of the majority of CVDs and is caused by unresolved inflammation. Various cardiovascular risk factors such as hyperglycaemia, dyslipidaemia, hypertension, smoking and aging stimulate the development of atherosclerosis through triggering inflammation. Being in a state of chronic low-grade inflammation therefor places an individual at higher risk of developing CVD, with inflammation playing a cause and effect role. The aim of this study was to investigate the inflammatory status of an elderly black South African population by analysis of inflammatory markers HS-CRP, TNF-α and IL-6, as well as the genetic polymorphism C174G associated with increased serum levels of IL-6 in some populations. The research was conducted in the field of Biomedical Sciences as a quantitative, cross-sectional, analytical observational design. The study was ethically approved and involved collection of 84 blood samples from volunteers in a purposively selected population as part of a larger collaborative study. Serum was used to analyse HS-CRP, TNF-α and IL-6 and DNA was extracted from whole blood for analysis of the C174G polymorphism. The median serum HS-CRP of 6.44mg/L (IQR = 2.82 - 9.86mg/L) fell within the highest risk (>5mg/L) of CVD and 75% of participants were at high (3.01-5mg/L) or very high (>5mg/L) risk. The median TNF-α of 0.00pg/mL was within the normal range and only 2.6% of participants had high serum TNF-α levels. The median serum IL-6 level was 1.92pg/mL and was also within the normal range with only 2.6% of participants who had high serum IL-6 levels. For the C174G polymorphism analysis, 98.6% had the GG, 1.4% the GC genotype and no participants had the CC genotype. The median serum IL-6 level of the homozygous GG group was 6.51mg/L, higher than the 4.13mg/L serum IL-6 of the heterozygous GC group. The difference in IL-6 should be considered with caution as only one participant had the C allele. A highly significant (p=0.001) correlation was found between HS-CRP and IL-6, as well as between IL-6 and TNF-α (p = 0.048). The elderly black Sharpeville community is in an increased inflammatory state which puts them at risk of CVD. The prevalence of the C allele in the C174G polymorphism is low in this population. Further research could be conducted as intervention studies to decrease the inflammatory state of the population and influence health policy changes to improve prevention of CVD.
Correlation of saliva and serum antibody titre response to pneumococcal capsular polysaccharides
(Vaal University of Technology, 2017-10) Madimabe, Metsekae Richard; Adrian, P. V., Dr.; Grobler, C. J., Dr.
Background: Infants and young children under the age of 2 years are vulnerable to Streptococcus pneumoniae infections, especially those who are born in developing countries. Antibiotic therapy was an effective treatment until resistance to antibiotics emerged, and then vaccines were developed to assistant with the treatment. The first successful conjugate vaccine was the 7-valent pneumococcal conjugate which resulted in a decrease in vaccine serotype IPD in infants and children below the age of two years. Objectives: The main aim of this study was to assess the saliva and serum antibody concentration response to pneumococcal capsular polysaccharides in children vaccinated and unvaccinated. Design: This is a sub-study within a retrospective analysis of a prospective cohort study on the safety and immunogenicity of 7-valent pneumococcal polysaccharide-protein conjugate vaccine (PncCV) and the immunogenicity of a H. influenzae type b conjugate vaccine (HibCV). Setting and participation: Infants aged between ≥ 4 and ≤ 10 weeks were enrolled, who only received BCG and Polio vaccine following birth. Infants were enrolled according to HIV status during routine antenatal screening in the obstetrics wards of the two hospitals, in Johannesburg and Cape Town. Measurements: Saliva IgG and IgA concentrations against pneumococcal capsular polysaccharides serotype 4, 6B, 7F, 9V, 14, 18C, 19F and 23F were quantified a by multiplex bead-based assay using the Luminex technology. Serum IgG against polysaccharides serotype 4, 6B, 7F, 9V, 14, 18C, 19F and 23F were measured by a competitive Enzyme Linked Immuno-Sorbent Assay (ELISA). Results: Post three primary vaccine doses, both serum IgG and saliva IgG and IgA antibody concentrations to vaccine serotypes were protective in children who received the vaccine. The antibody concentration in children whom did not receive the vaccine was much lower in comparison to the vaccine group in both serum and saliva to vaccine serotypes (P = 0.0001). And also high positive correlation (>0.6) was observed of IgG in serum and in saliva following vaccination. Conclusion: Pneumococcal conjugate vaccines induce pneumococcal capsular polysaccharide specific antibodies in both serum and saliva. However, there are differences between the vaccines’ ability to induce mucosal immune response and there are also serotype specific differences in the antibody concentrations and in the proportion of positive samples after a series of vaccinations. The pneumococcal conjugate vaccine in this study was able to induce mucosal immune memory: the anti-pneumococcal IgA concentrations also increased with age in the saliva of unvaccinated children.
Development of a HPLC method for the detection of Levetiracetam in blood of patients with epilepsy
(Vaal University of Technology, 2016-05) Engelbrecht, Lynette; Rheeders, M.; Grobler, C. J., Dr.
Approximately 1% of the world’s population has epilepsy, the second most common neurological disorder after stroke. In South Africa almost 1 in every 100 people has epilepsy, affecting all ages. Levetiracetam (LEV), marketed as Keppra® is an anticonvulsant drug used in the treatment of epilepsy. The daily dosage is 500 mg twice daily with a maximum of 3000 mg. The therapeutic range of LEV is between 12-46 μg/ml. Therapeutic drug monitoring (TDM) should be considered for LEV in patients with poor seizure control or long term treatment. TDM depends on accurate drug concentration measurements. In order to provide an accurate measurement, the High performance liquid chromatography (HPLC) method was developed, compared with a commercially available kit, and the stability of the samples was investigated. Ethical approval was obtained from the Human Research Ethics Committee (Medical), VUT (Ethics reference number: 2015024.4). The study was conducted from January to October 2015. This study involved three groups of volunteers who gave written consent. The first group were fifteen healthy MTech students in the Biomedical Technology Department at the Vaal University of Technology (VUT). Their blood samples were used for the analytical validation of the method and for the stability studies over a 4 weeks period. The second group were six patients from Pathcare Laboratories in Potchefstroom, Klerksdorp and Vereeniging who used Levetiracetam. Their blood samples were used to investigate the influence of different collection tubes as well as the handling and storage of samples on the LEV concentration. The third group were forty four patients from Pathcare Laboratories, Cape Town. Their blood samples were transported to Clinical Pharmacokinetic Laboratory (CPL) for routine therapeutic drug monitoring analysis of LEV and used to compare the newly developed HPLC method and the Commercial kit. The HPLC method was successfully developed and validated to determine LEV in human plasma/serum samples. The calibration curves showed good linearity (r2 =0,999) over the concentration range of 1 – 60 μg/ml. Accuracy, mean extraction recovery, lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were 98-112%, 97,15% (±1,57), 0,5 and 1,0 μg/ml respectively, in plasma standards. The method was shown to be simple and fast, reproducible and effective for routine laboratory analyses in the future. The agreement between the newly developed method and the ClinRep® HPLC complete commercial kit was the same and there was a statistical significant correlation between the two methods (average r=0.999; p-value > 0.0001, F-test with a true value =0). The method was much cheaper than the commercial kit, used less sample (100 μl) and had a longer running time (15 minutes) to ensure no endogenous interference. The costs of the developed method was 71-82% lower than the three commercial kits available in South Africa. Stability experiments were performed to evaluate the stability of LEV in human plasma/serum, simulating the same conditions which occurred during study samples’ analyses. The % RSD was lower than 5% under all the conditions: freeze, fridge, room temperature and auto sampler over the 4 week period. The results showed that both LEV and the I.S (internal standard) were stable in human serum/plasma under all these conditions. The influence of five different collection tubes, Gold (SST Gel), Red, Purple (EDTA)Green (Heparin) and Blue (Sodium Citrate) was investigated. In two patients, decreased levels were observed in tubes containing blue (sodium citrate) and Green (Heparin). The decrease was not statistically significant. This is an important observation and is an indication that anticoagulants may cause some problems due to drug-protein binding and interference in the matrix effect. A cost effective and reliable HPLC-method with minimal sample preparation time for the routine determination of LEV in plasma/serum samples was developed. It was also shown that the plasma/serum samples were stable at different temperatures over a time period. The only collection tubes that may interfere with the concentrations were the Green (Heparin) and Blue (Sodium Citrate) tubes.
Establishing the correlation between R353Q polymorphism and haemostatic markers in a black elderly community of Sharpeville Gauteng Province South Africa.
(Vaal University of Technology, 2019) Rodrigue, Tagne Wambo Joseph; Lebea, P. J., Dr.; Grobler, C. J., Dr.
Background: In a group of the elderlies (older person) in Sharpeville, Gauteng, South Africa, the majority live in poverty with a poor nutritional status. This makes them susceptible to develop infectious diseases as well as the risk of Chronic Diseases of the Lifestyle (CDL) such as cancer, diabetes, heart attack, obesity and hypertension. One of the most constant features of aging is the progressively elevated levels of coagulation factors such as FVII, fibrinogen, and impairment of fibrinolysis might play a role in the ageing process. These are associated with increased susceptibility to Cardiovascular diseases (CVD) commonly found. An association between elevated levels of FVII and R353Q polymorphism has been established as a risk factor for CVD. This genetic polymorphism R353Q characterizes the substitution in the exon 8 of the FVII gene of guanine-to-adenine, which results in the replacement of arginine (R) by glutamine (Q) in codon 353 of the F7gene. Objectives: The aim of this study was to evaluate the prevalence of R353Q polymorphism in correlation with haemostatic markers within an urban elderly community in South Africa. Method: This study was ethically approved, and it is an experimental research design on the prevalence of R353Q polymorphism in correlation with homeostatic status (Factor VII, Fibrinogen and PAI-1). The study was done in a black elderly population living in the Vaal triangle region of Sharpeville, attending a day care center, who gave consent to participate in the study. A purposely selected sample of 102 subjects, who met the inclusion criteria were used. The homeostatic status was measured by factor VII and fibrinogen measuring coagulation and PAI-1 measuring fibrinolysis. Results: The prevalence of R353Q genetic polymorphism was established in 14.5% of the sampled population. The prevalence of the RQ (AG) genotype was determined in the sample population with 6.5 % of elevated factor VII levels, 7.8% of increased fibrinogen levels (coagulation) and 10.5 % of decreased levels of PAI-1. The R(A) allele, was detected in 1.3% of the sampled population of normal levels of FVII, fibrinogen and PAI-1. The dominant allele G(Q) was present in 76.3% of the sampled population. An imbalance haemostatic marker was established in the sampled population with 61% of elevated levels of factor VII, 70% of elevated levels of fibrinogen and 88% had a decreased level of PAI-1. Conclusion: The prevalence of R353Q polymorphism was established in this sample population, having an imbalanced haemostatic status of hypercoagulation (factor VII and fibrinogen) and imbalance fibrinolysis (PAI-1), which are strongly associated to CVDs.
The evaluation of whole blood cytokine assay for diagnosis of M.tuberculosis infection in South African children with household tuberculosis contact
(Vaal University of Technology, 2019-04) Masilo, J. M.; Adrian, P. V., Dr.; Grobler, C. J., Dr.
Background: There are critical unmet needs for improved strategies in the detection and diagnosis of M.tuberculosis infection in children, and for prevention of tuberculosis disease in children. Bacillus Calmette-Guérin (BCG) vaccination has limited the utility of tuberculin skin testing (TST) in areas with high vaccine coverage. Objectives: The aim of this study was to estimate the prevalence of M.tuberculosis infection in children with household tuberculosis contacts, using QFT-GIT testing in comparison with TST. Methods: This study was a cross-sectional design to assess the performance of a new T-cell based blood test, namely QuantiFERON-TB Gold In Tube (QFT-GIT), for diagnosis of tuberculosis infection in the children (n=182) of adults (n=124) with pulmonary tuberculosis, additionally to determine the prevalence of M.tuberculosis infection in children with household tuberculosis contacts, using QFT-GIT testing in comparison with TST. The study was carried out at Chris Hani Hospital. For children involved in the study, tuberculosis exposure information was obtained, together with TST, QFT-GIT, and HIV testing. Data obtained from both experiments was statistically analysed using SPSS version 24 to determine whether there was a significant agreement between QFT-GIT and TST on the detection of M.tuberculosis prevalence in children with house hold contacts with confirmed M.tuberculosis infection. Results: This study examined the sensitivity and specificity of the QFT-GIT tests compared with the standard TST for diagnosing latent tuberculosis disease in paediatric contacts. Because of the lack of a latent tuberculosis "gold standard”, the specificity and sensitivity of QFT-GIT was calculated with a two-by-two table method. The specificity of the QFT-GIT was 84% and the sensitivity was 85%. There was a good correlation between QFT-GIT and TST (Cohen’s kappa of 0.705). Seventeen percent (17%) of the 182 children tested by QFT-GIT yielded indeterminate results. Age was associated with indeterminate QFT-GIT results in paediatric tuberculosis contacts. Point prevalence for QFT-GIT was recorded as 31% at baseline and 39.5% after six months indicating variability between QFT-GIT results at baseline and after six months. Conclusion: It was concluded that the prevalence of tuberculosis infection was common among South African children who live with an adult with active tuberculosis. The agreement between QFT-GIT assay and TST for the diagnosis of latent tuberculosis in children was high. Although TST and QFT-GIT assays appeared comparable, QFT-GIT showed higher positivity rate amongst those contacts with reported household tuberculosis exposure compared to TST. The QFTGIT assay was a better indicator of the risk of M.tuberculosis infection than TST in a BCG-vaccinated population.
The prevalence of HBV, HTLV, HIV and concurrent infections in blood recipients of the South African National Blood Service (SANBS)
(Vaal University of Technology, 2020-12) Willemse, Reynier; Vermeulen, M.; Grobler, C. J., Dr.
Background: Currently, the South African National Blood Services are not testing for HTLV and HTLV screening is not mandated by the WHO or by regulatory standards in South Africa. Looking at the uniquely high prevalence of HIV and HIV / HBV co-infections in the South African population and taking into account the literature that suggests that most of these infected patients will be receiving blood, exposing these patients to an additional burden like HTLV can result in an increased disease progression of HIV to AIDS and a poor prognosis in these infected patients. Study design and methods: A blinded cross-sectional study was performed. 7015 specimens were collected from all blood transfusion laboratories across South Africa excluding the Western Cape Blood Transfusion Service laboratories. The specimens collected were tested using the ABBOTT Alinity S® Immunochemiluminescent autoanalyser. All test results were confirmed with the Roche Cobas® E801 and E411 auto analyser. Results: Over all prevalence for HIV was 39.39% (N=2763), HBV 7.57% (n=531) and HTLV 0.70% (N=49). Concurrent infection for HIV/HBV 4.92% (N=345), HIV/HTLV 0.36% (N=25), HBV/HTLV 0.09% (N=6) and HIV/HBV/HTLV 0.07% (N=5). Conclusion: This study confirmed an overall high prevalence of HIV and HBV infections among patients receiving blood products from the SANBS. Compared to the general population, the HIV prevalence in blood recipients was two-fold higher. Patients receiving a blood transfusion from the SANBS have high rates of HIV, HBV and HTLV which should be taken into consideration when determining donor screening strategies.
To establish the prevalence of MTHFR C677T polymorphism in correlation with homocysteine metabolic markers in a black elderly community, in Sharpeville, Gauteng province in South Africa
(Vaal University of Technology, 2021) Pule, Pule Bongani; Lebea, P. J., Dr.; Grobler, C. J., Dr.
Background: Increased serum homocysteine is well known as an independent cardiovascular risk factor. Hyperhomocysteinemia may be due to several factors such as nutritional deficiencies and genetics. The MTHFR C677T polymorphism is associated with increased serum homocysteine. Folate and vitamin B12 play essential roles in lowering homocysteine levels. Limitations have been identified using serum vitamin B12 as a marker for vitamin B12 status due to lack an efficient of test. Holotranscobalamin has been reported as a more accurate marker for vitamin B12 status. Cardiovascular risk due to hyperhomocysteinemia has been confirmed among the elderly in Sharpeville. Knowledge of the prevalence of MTHFR C677T polymorphism among Black elderlies in South Africa is limited. Objectives: The main aim of the study was to evaluate the prevalence of MTHFR C677T polymorphism as a cardiovascular risk in an elderly black population in Sharpeville. Correlations between the presence of MTHFR C677T polymorphism and homocysteine metabolic markers were evaluated. Holotranscobalamin as a diagnostic test for vitamin B12 status was also assessed in this study. Materials and methods: This study was ethically approved by the Vaal University of Technology ethics committee (20140827-1ms). It was an observational, experimental study conducted in 102 elderly (≥60 years) attending the day-care centre in Sharpeville. Real-Time PCR was used to determine MTHFR genotypes. Folate and vitamin B12 were measured with AIA-PACK. Homocysteine levels were determined with an automated Konelab™ 20i and holotranscobalamin by ELISA. STATA 12 software was used for analysis of descriptive and inferential statistics. Results: The prevalence of MTHFR C677T polymorphism in this sample population was 19%. Heterozygous CT single nucleotide polymorphism was 17% and mutant homozygous TT was 2%. The majority (81%) of the subjects had wild type homozygous CC genotypes. No associations were found between MTHFR C677T genotypes and homocysteine and folate levels. Hyperhomocysteinemia was high (54%) and low (5%) folate deficiency found. No vitamin B12 deficiency was found however 7% were on the category of likely to be deficient. Sensitivity and specificity of holotranscobalamin were 100% and 95% respectively. Conclusion: The conclusions drawn from the study is that the prevalence of MTHFR C677T polymorphism is low within elderly in Sharpeville. There is a high risk of cardiovascular disease as a result of high prevalence of hyperhomocysteinemia. An intervention to lower homocysteine concentration of elderlies residing in Sharpeville is needed. Other genetic predisposing factors of increased homocysteine levels should be investigated.

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