Electronic Theses and Dissertations (Masters)

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The role of regulatory T cells in adults in South Africa with active tuberculosis
(2010-01-28T10:11:24Z) Mayne, Elizabeth Sarah
Introduction: Regulatory T cells (Tregs) are increasingly being recognized as key immunological players in immunosuppression and have been seen to be permissive for certain infections. Aim: This study aimed to elucidate the role that Tregs play in symptomatic infection with Mycobacterium tuberculosis (TB), both with and without co-infection with human immunodeficiency virus type 1 (HIV 1) by quantification of these cells at ex vivo. It was then attempted to characterise the behaviour of FoxP3 positive cells in culture with stimulation. Methods: Peripheral blood mononuclear cells were purified from uninfected controls, patients with active TB, patients with HIV infection and patients with HIV infection and active TB. The frequencies of Tregs were assessed by flow cytometry at ex vivo and again after four days of culture with stimulation with anti-CD3, Purified protein derivative, tetanus toxoid and HIV peptide superpools (gag and nef). These frequencies were compared between the four groups of patients. The ability of Tregs and effector T cells to proliferate was also assessed. Interferon-γ secretion was used as a measure of effector T cell response to stimulation. vi Results: Frequencies of Tregs were significantly reduced in patients with active TB as compared with HIV infected patients and uninfected controls. Co-infected individuals showed a broad range of frequencies which were not significantly different from controls. These frequencies remained stable in culture with the exception of those individuals infected with HIV who showed a decline in the frequency of those cells expressing FoxP3 over the period. Cells expressing FoxP3 were not anergic and responded to stimulation. HIV specific proteins, in addition, resulted in specific effects on the Tregs with a positive interferon response to gag correlating with increased Treg frequencies and FoxP3 expression in CD4+ T cells correlated with the proliferative response of CD4+ T cells to Nef in HIV infected individuals. Conclusions: This study shows significant differences of frequencies of FoxP3 positive producing cells in the peripheral blood at ex vivo in patients with active TB. The function of these cells in this population is uncertain and further functional data and long-term clinical follow-up is required. In addition, the frequencies of these cells remained constant over time and showed proliferative response to stimuli (most notably CD3) suggesting that these cells may be generated in the periphery.
Unmasking serial murder: a comparison of a South African murder series with characteristics from the Federal Bureau of Investigation Serial Murder Database
(2015) Holland, Shakeera
The term ‘serial killer’ brings to mind notorious criminals whose crimes are so heinous as to test the limits of the most vivid imagination and make us question their humanity. What is the reality of serial murder? In 2005, the Federal Bureau of Investigation (FBI) hosted a symposium on serial murder, which brought together international experts in the field of serial murder with the aim of clarifying and understanding this multifarious crime. On the 12th of March 2008, Gcinumzi Richman Makhwenkwe, ‘The Moffat Park Serial Murderer’ was convicted of 5 counts of murder, 3 counts of rape and 3 counts of robbery with aggravating circumstances. The Department of Forensic Medicine and Pathology of the University of the Witwatersrand, based at the Johannesburg Forensic Pathology Service (FPS) Medicolegal Mortuary Facility performed the medicolegal investigations of death in all the victims. This research report explores the characteristics of serial murder and serial murderers as documented in the literature; documents the features and characteristics of the Moffat Park murder series; compares the features of this South African murder series to those from the findings of the FBI serial murder symposium; explores the role of the forensic medical practitioner in the investigation of the Moffat Park series and serves to educate and inform forensic medical practitioners of the features of serial murder as awareness may potentially lead to earlier identification of a murder series. This could ultimately lead to earlier implementation of specialist investigative methods, earlier apprehension of the serial murderer and most importantly fewer victims.
Rationalising laboratory workflow to improve the efficiency of diagnostic service delivery: A critical review of haematological malignancies by flow cytometric immunophenotyping at the Charlotte Maxeke Johannesburg Academic Hospital
(University of the Witwatersrand, Johannesburg, 2023) Naidoo, Maynolia; Glencross, Debbie
The 2006 Bethesda medical indications guideline provides concise indications for flow cytometric immunophenotyping (FCI), to enable rationalising a decision for sample processing. The local practice of processing every sample received for FCI places an enormous burden on the resources of the laboratory, and leads to unnecessary expenditure for state health. A ‘triage’ process based on the current Bethesda medical indications guideline may be beneficial in developing countries. The aim of this study was to determine how the implementation of a triage process would impact on the diagnostic service delivery, and the ability to detect or miss disease. A retrospective review of 500 bone marrow aspirate (BMA) samples submitted for FCI analysis was performed from October to December 2019. The sensitivity, specificity, and predictive values of the BMA cytomorphology against the FCI outcomes (‘test-all’) were determined. Thereafter, the Bethesda medical indications guideline was retrospectively applied to the same data set (‘triage’), to compare the decision to process or not to process samples, against objective evidence of disease in various BMA investigations. After exclusion of inadequate quality samples that preclude comparison, 429 ‘test-all’ and 455 triage cases were evaluated. The ‘test-all’ analysis revealed a 97.1% sensitivity and 89.8% specificity, with a 64.1% positive predictive value (PPV) but striking 99.4% negative predictive value (NPV). The triage was largely effective in identifying cases with disease, revealing a 100% sensitivity and 83.3% specificity, with a PPV of 32.5% and very high NPV of 93.8%. Without impacting clinical outcomes, the implementation of a triage process can reduce the burden of FCI testing by 18%. Preliminary cytomorphological review of the accompanying BMA is strongly recommended as an additional step to improve the overall PPV of the triage, while safely reducing unnecessary FCI sample processing in a further 56% of cases. The implementation of a triage process with modifications for local use in flow cytometry laboratories, would enable the appropriate rationalisation of resources, improve the cost- effectiveness, and overall diagnostic service delivery in developing countries like Sub-Saharan Africa
Audit of Lysosomal Storage Diseases Testing at the National Health Laboratory Service in Johannesburg from 2011-2020
(2023) Novellie, Michael
Lysosomal Storage Diseases (LSDs) are a group of Inborn Errors of Metabolism (IEM), due to the lack of a lysosomal enzyme. This results in toxic accumulation of metabolic waste products in various organs leading to neurodevelopmental regression, organ failure and premature death in the absence of treatment. Treatments for LSDs are limited. This study audited LSD diagnostic test requests received by the Division of Human Genetics, National Health Laboratory Service (NHLS) in Johannesburg from 2011 to 2020 with the aim of understanding the demand, appropriateness, and patient management of suspected LSD cases. A quantitative survey of all samples (1861 tests) referred to NHLS Johannesburg during the study period was performed. A total of 198 (13.3%) samples were rejected for testing mainly because of faulty sample collection. Of the 1663 that were accepted for testing 1457 (87.6%) tested negative, 73 (4.4%) were inconclusive and 133 (8.0%) tested positive. Fifty-five (3.1%) patients with LSD test requests, all of which were positive, were known to a Clinical Genetics unit. The most frequently requested test was for Fabry disease: 620 (33.3% of all requests), even though this disease is not the most prevalent LSD. Of the 603 accepted test requests for Fabry disease, only 6 (1.0%) tested positive. This suggests that some referring clinicians had unrealistic expectations of encountering this disease. It should be noted, however, that testing for Fabry disease is part of a broad diagnostic workup that may be applied even if the indication for testing is not specific. Access to LSD testing was unequal: private facilities were proportionally over-represented compared to public facilities; certain provinces with large referral centres (in KZN and Gauteng) were over-represented compared to smaller centres. Feedback and education of referring clinicians regarding indications for testing and importance of patient follow up, especially by clinical genetics services, are recommended. Follow up of positive MPS screening tests with specific diagnostic tests is essential. A system should be implemented where a medical geneticist phones the referring clinician and discusses further sample requirements (blood for enzyme analysis) and referral to a genetics clinic for all positive LSD screening tests. Future consideration should be given to designing a more systematic testing process, with the introduction of molecular testing to supplement biochemical testing.
Laboratory Evaluation of Aspergillus Galactomannan Lateral Flow Assays
(University of the Witwatersrand, Johannesburg, 2023) Ubbink, Anja; Chibabhai, Vindana
Invasive aspergillosis diagnosis is based on a combination of clinical, radiological, and mycological factors, including the detection of Aspergillus galactomannan antigen in serum and bronchoalveolar lavage fluid (BALF). Lateral flow assays (LFA) introduced for rapid detection of galactomannan in serum and BALF include the IMMY sōna Aspergillus LFA (IMMY LFA) and the Dynamiker QuicGMTM Aspergillus Galactomannan Ag LFA (QuicGM LFA). Objective To evaluate the performance of the IMMY LFA and QuicGM LFA in South Africa. Methods Serum and BALF samples previously tested by Platelia BioRad Aspergillus GM-EIA were analysed using the two different LFAs. Percentage agreement and precision was assessed. Results Forty-six serum- and 13 BALF samples were tested using the IMMY GM LFA and 48 serum- and 6 BALF samples were tested using the QuicGM LFA. Using an optical density ≥0.5 as positive, results were compared to the BioRad Aspergillus GM-EIA. For the IMMY LFA, serum samples had a positive percent agreement (PPA) of 0% (0/1); negative percent agreement (NPA) of 91% (41/45) and overall percent agreement (OPA) of 89% (41/46). BALF samples had a PPA of 75% (3/4), NPA 50% (5/10) and OPA of 57% (8/14). For the QuicGM LFA, serum samples had a PPA 0% (0/3), NPA of 96% (43/45) and OPA of 90% (43/48). BALF samples had a PPA of 100% (1/1), NPA of 100% (5/5) and OPA of 100% (6/6). For the IMMY LFA, between-day reproducibility for 72% (13/18) and 63% (5/8) for serum and BALF samples, respectively. Between-batch reproducibility was 89% (16/18) and 50% (4/8), respectively for serum and BALF samples. For the QuicGM LFA, between-day reproducibility was 75% (9/12) and 75% (3/4) for the serum and BALF samples, respectively. The between-batch reproducibility was 100% (8/8) for serum and 100% (3/3) for BALF. Conclusion A follow-up evaluation with a larger sample size utilizing clinical, radiological, and laboratory data is warranted to determine the assays’ clinical utility. What this study adds Invasive aspergillosis is a life-threatening disease, where a prompt diagnosis improves outcome. Currently there is no Aspergillus galactomannan assay available in the South African state sector. This study evaluates two lateral flow assays for the detection of Galactomannan in South Africa
Mutation Profiling of Paediatric Solid Tumours in a Cohort of South African Patients
(University of the Witwatersrand, Johannesburg, 2023) Manolas, Erin; Krause, Amanda; Lamola, Lindie
Childhood cancers are an emerging global health burden, with the highest increase in incidence and mortality rates occurring in low-middle income countries, such as South Africa (SA). Adding to this burden is the contribution of cancer-predisposing genes (CPGs), whose germline variants increase the risk of cancer development in childhood. These genes are largely associated with a set of disorders, known as cancer-predisposing syndromes (CPSs), which are characterised by an increased likelihood of cancer development and/or additional phenotypic malignant/non- malignant features. Next-Generation Sequencing (NGS) technologies have been key in determining the occurrence and contribution of such germline variants to paediatric cancer development across international research and diagnostic settings. However, these technologies have not been applied to paediatric cohorts in SA and thus there is a paucity of data regarding the contribution of germline variants to childhood cancer development in this setting. Through the design and evaluation of an NGS targeted-CPG panel, this study aimed to generate germline variant profiles of SA paediatric cancer patients, thereby gaining insight into their potential role in the pathogenesis of childhood cancers. NGS was performed on the genomic DNA from 32 solid-tumour paediatric cancer patients using an Ion Ampliseq 50 CPG panel design and the Ion Torrent S5 sequencing instruments. Germline variants were called using the Ion Torrent Suite™ software (v.5.12.0) and annotated using the Ensembl Variant Effect Predictor. Variants were filtered using a bioinformatics pipeline assessing variant data from population and public databases, computational data, functional studies data, genetic pedigrees indicating family history and phenotypic data. Variant evidence was further interpreted for variant prioritisation and classification according to the ACMG-AMP guidelines. All putative pathogenic/likely-pathogenic (P/LP) variants identified were validated via Sanger Sequencing. Seven pathogenic and/or likely pathogenic germline variants were identified and validated in seven patients. Three of these variants, identified in the NF1, RET, and TP53 genes, were detected in patients who presented with phenotypes consistent with their genetic findings and are associated with well-known CPSs (diagnostic yield - 3/32, ~9.4%). The remaining four variants, identified in the BRCA1, ERCC3, FAH, and RB1 genes, have not been previously associated with the patient’s cancer phenotype and therefore require further investigation. At the time of this project’s data generation, this is the first global report of the novel heterozygous, likely pathogenic FAH p.R162H variant. Additionally, although reported elsewhere, the majority of the variants identified in this study (6/7, ~86.7%) have been reported for the first time within the SA paediatric population. To our knowledge, this is the first study to utilise NGS technologies in the germline variant profiling of paediatric solid-tumour patients in SA and therefore has greatly added to filling the current knowledge gap. In addition, these findings have contributed towards the foundation for the development of a CPG sequencing panel suitable for implementation in a SA diagnostic setting
Assessing the propensity of drug resistant tuberculosis to enter and exit the differentially culturable state
(2024) Nonkula, Bomikazi
Tuberculosis (TB), one of the oldest and most contagious infectious diseases, continues to be a global health concern. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC) which comprises of several species. These species are further subdivided into strains based on subtle genetic differences. The success of M. tuberculosis as a pathogen can be attributed to its ability to survive various stresses by adopting different growth states. Previous studies have shown that sputum from TB infected patients harbours a large proportion of drug-tolerant bacteria that are unable to form colonies on agar plates but can grow in liquid media. This population of organisms, termed differentially culturable tubercle bacilli (DCTB), could be resuscitated to grow by supplementing liquid media with cell free culture filtrates from axenic cultures of wild type M. tuberculosis H37Rv or mutant H37Rv lacking all five resuscitation promoting factors (Rpfs). Laboratory models that induce this differentially culturable state are critical for studying the physiology and metabolism of these bacteria in order to develop new TB diagnostic tests. In this study, five Beijing and five LAM drug resistant strains of M. tuberculosis were selected and used to robustly generate DCTB through an in vitro stress model using carbon starvation. The most probable number (MPN) assay and colony forming units were used to determine the amount of DCTB. Furthermore, the phenotype of these cells was studied using microscopy as well as metabolic probes that target the peptidoglycan (PG) component of the bacterial cell wall. Our findings demonstrated that applying the carbon starvation model to clinical M. tuberculosis strains (Beijing and LAM) resulted in robust levels of DCTB, as evidenced by limited growth on agar plates and enhanced growth in liquid media supplemented with culture filtrate from LAM and Beijing strains. Comparison of cell length between carbon starved cells to those grown in routine laboratory media suggested that DCTB appeared to be non-replicating and significantly shorter. The metabolic activity of the starved cultures was restored when they were supplemented with H37Rv, LAM and Beijing culture filtrate. Our results also demonstrated that Beijing strains had a higher propensity to produce DCTB compared to LAM strains and that the supplementation with Beijing culture filtrate resuscitated more DCTB. Collectively, our findings allow for the advancement of experimental systems that enable further investigation of DCTB and the properties of the Beijing strain that facilitate better adoption of the differentially culturable state.
Machine Learning on biochemical data for the prediction of mutation presence in suspected Familial Hypercholesterolaemia
(2024) Hesse, Reinhardt
Background Familial hypercholesterolemia (FH) is a common monogenic disorder and, if not diagnosed and treated early, results in premature atherosclerotic cardiovascular disease. Most individuals with FH are undiagnosed due to limitations in current screening and diagnostic approaches, but the advent of machine learning (ML) offers a new prospect to identify these individuals. Our objective was to create a ML model from basic lipid profile data with better screening performance than low-density lipoprotein cholesterol (LDL-C) cut-off levels and diagnostic performance comparable to the Dutch Lipid Clinic Network (DLCN) criteria. Methods The ML model was developed using a combination of logistic regression, deep learning and random forest classification and was trained on a 70% split of an internal dataset consisting of 555 individuals clinically suspected of having FH. The performance of the model, as well as that of the LDL-C cut-off and DLCN criteria, were assessed on both the internal 30% testing dataset and a high prevalence external dataset by comparing the area under the receiver operator characteristic (AUROC) curves. All three methodologies were measured against the gold standard of FH diagnosis by mutation identification. Furthermore, the ML model was also tested on two lower prevalence datasets derived from the same external dataset. Results The ML model achieved an AUROC curve of 0.711 on the high prevalence external dataset (n=1376; FH prevalence=64%), which was superior to that of the LDL-C cut off alone (AUROC=0.642) and comparable to that of the DLCN criteria (AUROC=0.705). The model performed even better when tested on the medium prevalence (n=2655; FH prevalence=20%) and low prevalence (n=1616; FH prevalence=1%) datasets, with AUROC curve values of 0.801 and 0.856 respectively. Conclusions Despite the absence of clinical information, the ML model was better at correctly identifying genetically confirmed FH in a cohort of individuals suspected of having FH than the LDL-C cut-off tool and comparable to the DLCN criteria. The same ML model performed even better when tested on two cohorts with lower FH prevalence. The application of ML is therefore a promising tool in both the screening for, and diagnosis of, individuals with FH.
Molecular epidemiology of M and E protein coding genes from South African SARS-CoV-2 strains, 2020 to 2021
(2024) Marsden, Fabian
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the current pathogen causing the COVID-19 pandemic across the world. While vaccines that elicit anti-SARS-CoV2 antibodies have been developed and licenced, there is a reduced protection against variants of concern (VOCs) such as Beta, Delta and Omicron. This is due to the mutations within the spike (S) protein which is the antigen targeted by most vaccines. Other potential vaccine targets include the structural proteins namely the membrane (M) and envelope (E) proteins of SARSCoV-2 which are more conserved. In this study we aimed to determine the extent of genetic diversity in the M and E protein genes from South African SARS-CoV-2 strains and its impact on predicted B and T cell epitopes. M and E gene sequences were extracted from South African SARS-CoV-2 genomes obtained from the Global Initiative on Sharing All Influenza Data (GISAID) database for the period 01March 2020 to 31 December 2021. Maximum-likelihood phylogenetic tree analysis shows that among South African E gene sequences only the Omicron VOC sequences form a distinct cluster. Similarly, the Omicron M gene sequences also form a distinct cluster compared to the Wuhan reference strain, Beta and Delta sequences. The predicted T cell and B cell epitopes of M and E proteins were identified with specific regions that have shown to have identical regions in both the variants and the reference strain, this shows the conserved nature of the M and E genes. SARS-CoV-2 are shown to have varying antigenic probabilities for M and E proteins from each of the variants considered as probable antigens. The allergenicity and toxicity of the M and E proteins was assessed in the context of potential vaccine development with certain peptides of each shown to have toxic properties. The predicted B and T cell epitopes show that despite the presence of mutations in the VOCs’ derived protein sequences, there is a common epitopic region that is shared between the reference and the variants. There is a strong 9-mer coverage by the natural sequences despite some non-coverage due to non-silent mutations. The results from the epitope predication and HLA typing shows the conserved nature of the M and E proteins which highlights the potential use for the development of vaccines.
Maternal vaccination in South Africa: timing and completeness
(2024) Bourne, Julia
Maternal immunisation is an invaluable public health measure that protects not only the mother, but also the foetus and new-born infant against a host of diseases; and is recommended by both the World Health Organisation (WHO) and South African national health authorities. Pregnancy induces a heightened state of immune system vulnerability, leaving women more susceptible to severe influenza outcomes, whilst neonatal tetanus has a fatality rate of between 80-100% in the absence of medical intervention. Maternal immunisation against influenza and tetanus has been successfully utilised as a public health strategy across the globe to uphold maternal and neonatal health. Maintaining coverage is imperative for both diseases as influenza strains change seasonally and tetanus cannot be eliminated, highlighting the importance of continued maternal immunisation. This study aimed to describe the uptake of both influenza and tetanus vaccinations during pregnancy, the completion of the tetanus vaccination schedule and the timing of both influenza and tetanus immunisation within South African antenatal care facilities. In addition, this study described clinical and demographic factors affecting maternal immunisation uptake. Clinical and demographic data were collected in a parent study and were retrospectively analysed in this study using the statistical software Stata. Influenza vaccination uptake within the sampled population was found to be 16.62% (806/4851). The odds of influenza vaccination were significantly higher in women aged 21-30 years, and women with six or more ANC visits. Metro East Cape Town site in the Western Cape outperformed Gauteng sites, with significantly increased odds of influenza vaccination amongst women frequenting that site. Appropriate influenza immunisation: defined as immunisation occurring during the period of either 01/04/2017-31/07/2017 or 01/04/2018-30/06/2018, occurred in 74.86% (530/708) of the cohort. Women who were alcohol users were significantly more likely to receive an influenza vaccine – yet this may be explained by the Metro East site which had the higher influenza coverage having the highest prevalence of alcohol use during pregnancy. Of 7105 women, 7031 (98.96%) received at least one dose of tetanus toxoid vaccine (TTV). Of these women, 39.24% (2759) received one dose; 51.06% (3590) received two doses and 9.70% (682) received the recommended three doses of TTV in their index pregnancy. Tetanus schedule completion was significantly more likely in women ≤20 years, and those who presented for their booking antenatal care (ANC) visit in the first trimester. In addition, women with more than three visits had an increased likelihood of TTV schedule completion. The odds of TTV schedule completion were decreased by negatively parity and gravidity, values over one and less than six, and greater than one respectively. Women with hypertension were significantly less likely to receive three TTV doses compared to women without hypertension. Julia Bourne MSc(Vaccinology) Research Report: Version 2.0 v Tetanus immunisation schedule adherence prevalence was 0.60% (4/670) in women with three doses and 90.34% (3209/3552) in women with two. Improvements may be made in South African maternal immunisation coverage, with this study supporting the idea of targeted educational campaigns and a revision of the maternal immunisation schedule to include the tetanus, diphtheria & acellular pertussis vaccine instead of the tetanus toxoid vaccine.
Evaluation of the genetic and metabolic determinants of postprandial glucose variability in Black South Africans
(2024) Masango, Bontle
This study aimed to determine the metabolic and genetic factors that account for the variation in postprandial glucose (PPG) in Black South Africans (SA). The study included 794 participants from the middle-aged Sowetan Cohort (MASC). PPG was calculated using the integrated area under the curve (iAUC) in response to an oral glucose tolerance test. Principal component analysis was applied to 31 metabolic factors to generate clusters represented by principal component variables. Polygenic risk scores (PRS) were computed for each participant using variants and weights from a validated African type 2 diabetes PRS. Linear regression models were used to evaluate the variance. The PRS did not contribute to the PPG variability in men or women. Central fat, serum lipids, and liver enzymes explained 10.8% of PPG variability in women. In men, peripheral fat, serum lipids, liver enzymes, and steroid hormones explained 10.6% of PPG variability. Our work has identified metabolic factors that predict PPG variability in Black SA.
The use of insecticide treated eave ribbons as a protection tool against pyrethroid-resistant populations of mosquitoes that transmit malaria and dengue fever
(2024) Shirima, Ruth Severin
Vector control methods such as insecticide treated nets (ITNs) and indoor residual spraying (IRS) have been successful in preventing mosquito-borne diseases like malaria and dengue. However, these methods face challenges including insecticide resistance, high costs, logistical difficulties, low adoption rates, and limited durability. Therefore, there is a need for simpler and more affordable interventions that can be used on a large scale in disease-endemic communities to supplement current approaches. This study evaluated the effectiveness of using insecticide-treated eave ribbons as a potential tool for complementing the current vector control methods. Eave ribbons are pieces of hessian fabric that can be placed around the eave spaces of poorly constructed houses to kill or repel mosquitoes. Laboratory cone bioassays were conducted to assess the efficacy of eave ribbons treated with the organophosphate, pirimiphos-methyl, for killing the malaria vectors, Anopheles funestus and Anopheles arabiensis, and the dengue vector, Aedes aegypti, under varying exposure durations and insecticide doses. In addition, a semi-field experiment was done to assess the efficacy of eave ribbons treated with pirimiphosmethyl against the malaria vectors. Indoor and outdoor biting was assessed by the number of mosquitoes captured indoors in window exit traps and outdoors by human landing catches, respectively. Mortality of recaptured mosquitoes was recorded after 24, 48, and 72 hours. The findings revealed that treated eave ribbons resulted in higher mosquito mortality than the untreated ribbons, but the impact increased with increased exposure duration or dose. The semi-field study indicated moderate levels of bite prevention and mortality of the mosquitoes. At the doses of 1 g a.i./m2 and 2 g a.i./m2 pirimiphos-methyl, there was no significant protection against An. arabiensis, but at the dose of 4 g a.i./m2 pirimiphos-methyl, there was only significant protection against outdoor biting An. arabiensis, but not An. funestus. In conclusion, while insecticide-treated eave ribbons may have potential for controlling malaria and dengue vectors, further research is needed to validate their efficacy in field settings and to identify suitable insecticides or insecticide combinations that are safe and effective.
Impact of donor CYP3A5 genotype on pharmacokinetics of tacrolimus in South African paediatric liver transplant patients
(2024) Wheeler, Caitlin
Tacrolimus is characterised by a narrow therapeutic target range and wide interpatient variability. Pharmacogenetic research attributes CYP3A5 single nucleotide polymorphisms to the variable inter-patient tacrolimus concentration dose ratios (CDR). In this study, we compared the mean tacrolimus CDR in paediatric liver transplant recipients and their living liver donors’ CYP3A5 rs776746 T>C genotypes(*1/*1, *1/*3 and *3/*3), accounting for donor and recipient characteristics. The graft-to-recipient weight ratio and the CYP3A5 donor genotypes were found to be independent factors significantly impacting the mean tacrolimus CDR. Donor CYP3A5 expressors (*1/*1 and *1/*3) were shown to have significantly lower recipient tacrolimus CDRs, therefore, higher dosages would be required in comparison to CYP3A5 non-expressors to reach the same therapeutic target range. The potential implementation of a stratified medicine dosage algorithm, combining living liver donor CYP3A5 genotyping with the calculation of the graft-to-recipient weight ratio, may predict the optimal tacrolimus dosage schedules for liver transplant recipients.
Characterisation of RSV fusion proteins from South African patients with RSV disease, 2019 to 2020
(2024) Mabilo, Prince
Respiratory syncytial virus (RSV) is classified into subtypes A (RSV-A) and B (RSV-B), which are classified into different genotypes based on genetic variability of the G surface glycoprotein gene. The F surface protein gene is more conserved however variability in signal peptide, transmembrane domain, and antigenic sites have been reported. The study was conducted in the Virology laboratory, Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), South Africa. Study participants included patients of all ages from whom respiratory samples were submitted for respiratory viruses diagnoses from 2019 to 2020. The complete RSV F genes were amplified, library prep was preformed using the Nextera DNA prep kits and sequenced using amplicon-based next generation sequencing on the Illumina MiSeq sequencing platform. The Genome Detective Virus tool v2.27 was used to assemble sequencing reads and MEGA X was used for phylogenetic analysis. N-linked glycosylation and amino acid sequence variation was assessed. The overall prevalence of RSV was 5.8% (101/1 734). Seventy (69.3%; 70/101) RSV-positive samples were available for genetic characterisation of the F protein gene and thirty-one (30.7%; 31/101) were excluded due to insufficient sample volume. Only RSV-A strains were identified (91.2%; 31/34). Twenty three of thirty-one (74.2%) of the RSV F gene sequences from 2019 to 2020 clustered together with bootstrap values ranging from 64% to 99% and were NA1-like. A N-glycosylation site at position 120 gained by South African strains from 2018 is retained in strains from this study. This N-glycosylation site is present in approximately 25.8% of RSV strains from this study. The diversity of RSV-A F proteins was low, with amino acid variations observed at 30/571 (5.3%) sites. Ten mutations were detected in 4/6 antigenic sites (I, II, IV and V), with frequencies ranging from 0.3 to 100%. Antigenic changes seen exclusively among South African strains are: Y33H (0.3%) and V384T (7.3%) at site I and S275F (0.3%) at site II. Seven mutations associated with escape of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocyte (CTL) were predicted in seven epitopes. Overall amino acid mutation frequency for 2019 to 2020 RSV-A F genes is similar to that reported for South African strains from 2018 (3.3% to 6.7%). For the first time in South Africa we detected the S275F mutation which causes palivizumab resistance.
Exploring the interaction of host genetics and the gut microbiome in obesity in an African population
(2024) Schnell, Samantha Susan
Obesity is a highly prevalent health concern that is on the rise in Sub-Saharan Africa. Even though genetic variation and gut microbiota have been implicated in the development of obesity independently, the interactions between these factors have not been previously explored in a South African cohort. This study aimed to identify possible associations between host genomes, body mass index as a measure of obesity, and gut microbiota composition (in the form of V3-V4 16S rRNA sequencing) in a female African cohort. Polygenic risk scores predictive of body mass index in this cohort were generated to categorise the participants into high- and low-risk groups. Subsequently, several statistical analyses were performed comparing gut microbiota between these groups. High-risk participants with high body mass index had associations identified with increased abundances of Prevotella_9 and VadinBE97. In contrast, the low polygenic risk and low body mass index sub-group was associated with greater Bacteroides levels. This study acknowledges the plausible interactions between these factors in an African cohort.
Evaluating the clinical utility of a SNP-based microarray platform: a comparative study
(2024) Mayisela, Minenhle Peniel
Developmental disorders make up a majority of cases seen in genetic clinics at the National Health Laboratory Service (NHLS). Chromosomal microarray analysis (CMA) is the first line testing for individuals with developmental disorders and congenital anomalies. This study compared the clinical utility of single nucleotide polymorphism (SNP) - based array (CytoScan® Optima array) and a comparative genomic hybridization (CGH) array (SurePrint G3 Unrestricted CGH ISCA v2, 8x60 kb microarray) in diagnosis of developmental disorders. This was done by looking at the differences in copy number variant calls, segment size differences and gene content between the two microarrays. The cost to run each type of microarray test for diagnosis was also compared. The copy number variant calls made by the two platforms were comparable. The SNP-based array did provide additional information that would be useful in molecular diagnosis. The final recommendation was for the CGH array and the SNP-based array to be used interchangeably based on clinical requests at the Division of Human Genetics
Off-label evaluation of alternative specimen types: Cobas® plasma separation card for HIV viral load and dried blood spots for COVID-19 serology testing
(2024) Mampa, Thabiso Mmammitsi
Plasma is the preferred specimen for HIV viral load (VL) monitoring and COVID-19 serology testing but poses a challenge in resource-limited settings due to the need for venous blood, skilled phlebotomy, and cold storage for specimen integrity. In this study dried blood spots and novel plasma separation devices (PSC, HSSE, and VLPlasma) versus plasma were investigated as alternative specimen types. The plasma separation devices (PSD) were compared to DBS to determine if eliminating cellassociated nucleic acids could improve HIV VL performance. Paired PSD (n=72), DBS (n=72) and plasma (n=72) were prepared from HIV positive residual whole blood. Similarly, paired PSC, DBS (n=91) and plasma (n=91) were prepared from HIV positive prospective whole blood to assess PSC as an alternative specimen for use on the Abbott m2000. The eluates were processed on the GeneXpert (residual blood), Abbott m2000 (residual and prospective blood) and Roche cobas® 68/8800 (prospective blood). Using plasma as reference, residual blood: DBS outperformed PSC, HSSE and VLPlasma in terms of accuracy 91.8%, compared to 87.8%, 79.1% and 75%. Prospective blood: PSC had improved performance over DBS in terms of sensitivity (92.2% and 87.1%), specificity (65% and 61.9%), and accuracy (86.9% and 80.7%). Additionally, the performance of DBS was evaluated for COVID-19 serology testing in 45 PCR-confirmed, COVID-19 positive individuals by preparing laboratory paired DBS-plasma samples. DBS were eluted using two diluents followed by manual ELISA and results compared to reference plasma testing. DBS-PBS and DBS-manufacturer’s diluent showed the same accuracy (93.6%). Kappa values (0.817 and 0.845) and sensitivity (100% and 91.4%) were similar, but DBS-PBS showed low specificity (75%) compared to DBS-diluent (100%). Off-Label use of the cobas® PSC for HIV VL and DBS for COVID-19 serology testing provides expanded options for testing in resource-limited settings. Further evaluation on capillary blood and automated laboratory workflow optimisation would still be required prior to scaled implementation.
Extended characterization of multi-drug resistant organisms colonising neonates at a tertiary hospital in Johannesburg, South Africa.
(2024) Mntla, Nonkululeko Marcia
Neonatal deaths remain high globally, particularly in sub-Saharan Africa. A third of deaths are due to infections, often secondary to multi-drug resistant (MDR) organisms. The purpose of this study was to investigate the prevalence of MDR ESKAPE+ C. auris colonisation amongst hospitalised neonates, to determine risk factors associated with MDR colonisation, and perform antimicrobial resistance characterization of these isolates. Two hundred and fifty-eight swabs were collected from 86 hospitalised neonates at a tertiary South African hospital between November and December 2020. A total of 135 ESKAPE+ C. auris isolates were identified; 68% were MDR. Majority of neonates (65%) were colonised with extended spectrum betalactamase (ESBL) producing Klebsiella pneumoniae, followed by extensivelydrug resistant (XDR) Acinetobacter baumannii. New Delhi metallo-beta-lactamase (NDM) producing A. baumannii were more prevalent than carbapenemase producing Enterobacterales (CPE). A prolonged hospital stay, median=14 days (p<.001) was identified as a risk factor for MDR organism colonisation. The high prevalence of MDR ESKAPE+ C. auris colonisation supports use of non-invasive samples to determine colonisation prevalence. More data are needed to develop improved surveillance systems which should incorporate colonisation swabs and clinical biomarkers in neonates with independently established risk factors.
Combination interaction of proton pump inhibitors with fluconazole against multidrug resistant Candida auris
(2024) Galane, Kamogelo
A significant challenge with controlling C. auris infections is its resistance to multiple antifungal agents, including azoles. Alternative strategies are required to combat the problem of antifungal resistance. Some of the suggested approaches include combination therapy and anti-virulence treatment strategies. There is growing interest in combining proton pump inhibitors (PPIs) with available antifungal drugs for better treatment outcomes. In this study, we investigated the antifungal activity of PPIs omeprazole and pantoprazole alone and in combination with fluconazole against resistant C. auris isolates. The anti-virulence activity of the synergistic combination against resistant C. auris isolates was determined. Lastly, the effect of the synergistic combination on C. auris energy-dependent efflux-activity was established. The CLSI broth micro-dilution method was used to determine the antifungal susceptibility of 25 C. auris isolates against antifungal agents (fluconazole and amphotericin B) and PPIs (pantoprazole and omeprazole). The combination interaction of fluconazole and PPIs was established by determining the fractional inhibitory concentration index (FICI) of each combination. Time-kill curves were used to determine the antifungal activity of the synergistic combination over time and to confirm the combination interaction. All 25 C. auris isolates were screened for their ability to adhere to epithelial cells and proteinase secretion using microscopy and culture technique respectively. The effect of the synergistic combination on isolates with positive pathogenicity markers was determined. Most of the C. auris isolates were resistant to fluconazole (92%) and all were sensitive to amphotericin B (100%). Omeprazole and pantoprazole had antifungal activity against C. auris at MIC values of 3125 µg/ml and 15625 µg/ml, respectively. The combination of fluconazole and pantoprazole reduced fluconazole MIC values by 4-fold, from 125 µg/ml to 31.2 µg/ml. The combination had synergistic effect against most C. auris isolates (56%), additive effect in 36% and indifference in 8%. There was no synergism in the fluconazole and omeprazole combination. However, the combination had antagonist effect against most C. auris isolates (56%). All C. auris isolates displayed some degree of adherence to epithelial cells and 96% produced proteinases. Fluconazole and pantoprazole combination significantly reduced adherence (p values < 0.05) and proteinase secretion (p values< 0.001) at inhibitory and subinhibitory concentrations. Pantoprazole inhibited fluconazole efflux at inhibitory and subinhibitory concentrations. The study showed that C. auris isolates express virulence factors such as adherence and proteinase production. The combination of pantoprazole and fluconazole has antifungal and anti-virulence activity against resistant C. auris isolates. In addition, it was shown that pantoprazole can attenuate fluconazole resistance by inhibiting the activity of energydependent efflux-pumps and it has synergistic effect with fluconazole. Therefore, pantoprazole has the potential to improve therapy outcomes when azoles are used.
Comparison between lupus nephritis in HIV positive patients and HIV associated immune complex glomerulonephritis with “lupus–like” features: a clinicopathologic study
(2024) Mathaba, Margaret Masala
Background: Systemic lupus erythematosus (SLE) is an autoimmune disease seen commonly in black females of childbearing age. More than half of the patients present with renal disease or lupus nephritis complications. Coinfection with HIV in patients with lupus nephritis is rare. Despite Africa having the highest rate of HIV infection in the world, and there is no available data on the coexistence of HIV and Lupus nephritis. HIV is associated with a wide spectrum of renal diseases, including “lupus-like” HIV-Associated Immune Complex Kidney Disease (HIVICK). The most prevalent renal lesions in “lupus-like” HIVICK is diffuse proliferative lupus nephritis Objectives: This study aimed to compare and correlate the demographics, epidemiology, pathological and clinical findings of HIV positive patients with lupus nephritis and those with “lupus-like” HIVICK. Methods: This retrospective chart review study was conducted at the Charlotte Maxeke Johannesburg Academic Hospital in 5 years (2014-2018). We reviewed case reports that met our criteria for cases with lupus nephritis and cases with “lupus-like” HIVICK and allocated a lupus class according to the report findings. Results: Out of 2174 renal reports, 25(1.14%) patients were diagnosed with lupus nephritis and nine (0.41%) with “lupus-like” HIVICK. There were significant differences in age, serology (urea and creatinine), clinical presentation and lupus class. Conclusion: The occurrence of both HIV associated lupus nephritis and ‘lupus like’ HIVICK is rare. In our setting, the former is more common than the latter. We observed clinical and pathological differences which may be used to diagnose these disease entities.

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