Browsing by Department "Department of Clinical Laboratory Sciences"

Now showing 1 - 20 of 151
  • Thumbnail Image
    Item
    Open Access
    A citywide, clonal outbreak of Pseudomonas aeruginosa during a drought
    (2021) Opperman, Christoffel Johannes; Centner, Chad M; Nicol , Mark P; Moodley, Clinton
    Background Outbreaks of community-acquired Pseudomonas aeruginosa are typically small and localized. We investigated an increase in P. aeruginosa clinical isolates in Cape Town, South Africa during a severe drought. Methods Cases were defined as P. aeruginosa isolated from any clinical sample, and “wild-type” as susceptibility to all antibiotics tested. Residential addresses of community-acquired wild-type cases were mapped. Whole genome sequencing and multi-locus sequence typing were used to determine clonality and identify virulence genes. A modified case-control study in a subset of patients with bloodstream infection compared demographic and clinical characteristics between sequence types. Results The outbreak lasted 10 months from December, 2016 to September, 2017 with 3,321 documented cases. At the peak, cases reached 2.3-fold baseline and the city's dams reached a nadir of 19% capacity. Cases were distributed widely across the city. Multi-locus sequence type (ST) 303 was found in 27 of 42 (64%) blood culture isolates of P. aeruginosa during the outbreak, one of 19 (5%) before, and none of 11 after. ST303 infection was independently associated with younger age, but not with co-morbidities nor increased mortality. Fifty-one virulence genes were differentially present in ST303 compared with other sequence types, including genes involved in biofilm formation, iron uptake, and gut penetration. Conclusion The investigation confirmed a citywide outbreak of P. aeruginosa coinciding with and potentially related to a severe drought. We identified a predominant outbreak-associated clone, ST303, which harboured genes that could contribute to virulence and survival in drought-related conditions. Enhanced surveillance for P. aeruginosa during periods of drought is recommended.
  • No Thumbnail Available
    Item
    Open Access
    A molecular approach to precision medicine in South African children with Epilepsy: towards a genetics-based diagnostic service for Epilepsy
    (2024) McIntosh, Caitlin; Esterhuizen, Alina; Fieggen Karen
    Background: Epilepsy is a neurological disorder characterised by unprovoked, recurring seizures. SubSaharan Africa carries the highest burden of epilepsy in the world, owing mainly to the increased risk factors such as infectious and parasitic disease, and traumatic brain injury. A proportion of this burden is genetic, however, genetic testing for epilepsy in South Africa is limited, currently only accessible in the private healthcare sector via international referral. Genetic testing in epilepsy is an internationally recognised diagnostic tool which can inform the diagnosis and patient management, with options for precision treatment, frequently resulting in improved outcomes. The main aim of this research project, therefore, was to design and validate a next-generation sequencing (NGS) gene panel for South African paediatric patients with drug-resistant epilepsy. The panel design was aimed primarily at the developmental and epileptic encephalopathies, where the yield of informative findings is highest, and the results often carry implications for treatment. The secondary aim of the project was to perform preliminary pharmacogenetic testing, to determine if variants previously associated with antiseizure drug metabolism in other populations are present in this study group. Materials and Methods: Forty probands with clinically complex, drug-resistant epilepsy with no identified, acquired cause were recruited from the neurology epilepsy service at the Red Cross War Memorial Children's Hospital in Cape Town. All 40 probands were tested with a panel of 78 genes selected on the basis of association with disease and clinical actionability. NGS data files were subject to variant prioritisation, followed by variant confirmation by cycle sequencing, segregation analysis, and final classification according to ACMG criteria. All 40 probands were tested with two pharmacogenomic arrays, one generalised array, the Veridose® Core Panel produced by Agena Bioscience (San Diego, USA), and one custom-designed anti-seizure medication-specific SNV array targeting eight SNVs across six different genes. Results: Three pathogenic variants were identified; two in SCN1A and one in GRIN2A, with a pickup rate of 7.5% (3/40). Two variants of uncertain significance were identified in GABRG2 and GRIN2B. The findings were aligned with the electroclinical features in each patient. The pharmacogenomic analysis revealed one variant, EPHX1 rs1051740, which appeared to be statistically significantly differently distributed in the study group to the general African population (P = 0.02). Three other variants, EPHX1 rs2234922, SCN1A rs3812718, and CYP2d6 s59421388 appear to be trending towards significance, with P-values of 0.08, 0.05, and 0.05, respectively. Conclusion: The main outcome of this project was the successful development of an NGS-based diagnostic protocol for paediatric epilepsy using the gene panel approach, now ready for implementation in the local diagnostic testing laboratory. Moreover, the genetic cause of epilepsy was identified in three study participants, with treatment implications in two (variants in SCN1A). The pharmacogenomic analysis in this project did not reveal specific insights owing to the small study group, but provided exposure to pharmacogenomic analysis, and may be used as a basis for further research into the pharmacogenomics of epilepsy in larger African cohorts.
  • Thumbnail Image
    Item
    Open Access
    A numerical protocol for death-time estimation
    (2021) Mfolozi, Sipho; Malan, Arnaud George; Bello-Ochende, Tunde; Martin, Lorna Jean
    A body's axial temperature distribution at death was experimentally demonstrated by the author to predict the postmortem temperature plateau (PMTP), which is known to affect the measured core temperature value and hence death-time estimation. Yet today's methods of death-time estimation apply only a single-point approximation of a body's core temperature in life as well as a single-point measurement of a body's core temperature after death. Four studies were carried out to understand the relationship between a body's axial temperature distribution and the PMTP. The first study numerically approximated antemortem temperature distribution in an MRI-built, high-definition, anatomicallys egmented 3D computational human phantom consisting of several hundred tissues. Metabolic heat generation (QQmm) and blood perfusion (wwbb) parameters were applied to all thermogenic tissue using the Pennes BioHeat Model. The study demonstrated that the antemortem axial temperature distribution was nonlinear, that tissue temperature distribution was inhomogeneous, and that the position and size of the antemortem central isotherm was predicted by the size, shape and location of the most thermogenic internal organ in a given axial plane. Numerical approximation of a body’s antemortem axial temperature distribution using this study’s materials and methods was proposed for death-time estimation. The second study examined postmortem axial heat transfer. The approximated antemortem axial temperature distribution constituted the initial condition. QQmm and wwbb were set to zero to simulate death. Postmortem cooling was simulated in still air, on a cold concrete floor and on a heated floor. The antemortem central isotherm that single-point core thermometry detects was the PMTP. Its size at death, body radius, axial thermometry-depth and length of the postmortem interval (PMI) all predicted PMTP length. The cold concrete floor shifted the central isotherm away from the floor, while the heated floor shifted it towards the floor. Ground temperature and material properties, along with the aforementioned PMTP predictors, result in variation in measured single-point core thermometry values, yet today’s death-time estimation methods do not measure, approximate or standardise them. This is a source of uncertainty. This study demonstrated that a body’s postmortem axial thermal profile was very specific to the PMI at which it exists, including during the PMTP that single-point core thermometry detects. This study proposed a body’s measured postmortem axial thermal profile for death-time estimation to reduce PMTP uncertainties. The study also proposed numerical modelling of the ground, its temperature and material properties. The third study proposed a multipoint axial thermometry (MAT) device to measure a body’s postmortem axial thermal profile. The author designed the device prototype. Its fabrication was outsourced. Empiric and numerical MAT studies were conducted on a cooling dummy and 3D human phantom, respectively. MAT curves indicated a parabolic shape. The fourth study proposed a numerical protocol for death-time estimation that iteratively tested a MAT profile measured at an unknown PMI from a decedent using the proposed MAT device against MAT profiles predicted by numerical simulations of sequentially longer candidate PMIs. A candidate PMI whose MAT profile matched was considered the PMI estimated by the protocol. The proposed protocol applied the exact historical meteorological temperatures that existed during the final estimated PMI. Application of the protocol was demonstrated using a fictitious scenario in which a candidate PMI within 120s of the final estimated PMI was excluded. Potential sources of uncertainty of the proposed protocol were discussed and concluding remarks on future research were made.
  • Thumbnail Image
    Item
    Open Access
    A pilot study on stature estimation of the South African male population using the post mortem Lodox® Xmplar-dr imaging device at the Salt River Forensic Medico-Legal Laboratory
    (2022) Venketsamy, Yomika; Heyns, Marise; Mole, Calvin; Dinkele, Elizabeth
    Identification of deceased individuals is of paramount importance in the South African constitution, with victim identification noted as a human right. Stature has been used to assist identification of an individual when skeletal remains are recovered. The usefulness of stature estimation using conventional x-rays, magnetic resonance imaging (MRI) and computed tomography (CT) measurements of long bones in a modern population has been researched in a number of countries, however, there has been limited research conducted on Lodox® bone scans as an added tool for stature estimation in the South African population. Forty-nine deceased males aged 21 to 61 years were scanned with Lodox® within 24 hours of entering Salt River Mortuary for a scheduled autopsy. Total stature was initially measured on the autopsy table with an embedded ruler. The body underwent a full body digital x-ray using the Lodox® Xmplar DR device. To measure length of bones on the Lodox® scans, full body images were exported in DICOM (Digital Imaging and Communication in Medicine) format and five long bone maximum lengths i.e. humerus, radius, ulna, femur and tibia of the bodies were digitally measured using the integrated Lodox® software. Lodox® image scan measurements found that the humerus, femur and tibia were the most statistically significant correlators of stature, individually. The univariate linear regression showed strong statistical significance for the humerus, femur and tibia with estimating stature. Multiple linear regression with the combination of humerus and ulna; femur and tibia; humerus, femur and tibia were statistically significant in determining stature. However, a combination of ulna and radius and the combination of all five bones overall regression was not statistically significant. Univariate and multiple linear regression formulas were created for the South African male population using Lodox® image scan measurements. Correlation and paired t-tests showed significant correlation between manual stature measurement at the mortuary and Lodox® measurements for stature.
  • Thumbnail Image
    Item
    Open Access
    A retrospective investigation of sudden unexpected death in the young investigated at Salt River Mortuary, Cape Town
    (2020) Vandayar, Yuvika; Heathfield, Laura Jane
    Sudden unexpected death in the young (SUDY) is the tragic fatality of seemingly healthy individuals aged between one and 40 years. Little is known about the demographics and risk factors of these cases at Salt River Mortuary (SRM), Cape Town. Therefore, this project aimed to retrospectively investigate the burden and profile of SUDY cases admitted to SRM, between 1 January 2016 and 31 December 2018. Of the total 11 588 cases admitted over this period, 833 (7.2 %) were SUDY cases, wherein males comprised the majority (64.3 %). Individuals were a median age of 31 ± 10.3 years at death, and the main location of death was ‘residential' (43.5 %). There were also significantly more males than females in the age category of 31 - 40 years who were found outdoors compared to all other locations (p < 0.001). Risk factors included physical activity, substance abuse, and co-morbidities with concomitant use of chronic medication. More than a third of individuals experienced breathlessness prior to death (45.0 %). Of cases with a confirmed natural cause of death, the main organ systems involved were pulmonary, cardiovascular, central nervous system and gastrointestinal, which parallels international trends. Akin to local studies, in analogous amounts, TB and pneumonia were the leading causes of death. Additionally, 21.1 % of cases were identified as candidates for genetic testing which may resolve undetermined cases or elucidate underlying predisposing factors to sudden death. Fortunately, 81.8 % had biological samples available for these retrospective analyses. Cases often had missing documentation which advocates for training to ensure compliance to standardised procedures. This study shows that males aged 31 ± 10.3 years with pulmonary and cardiac-related co-morbidities are the most vulnerable for SUDY whilst sleeping. Awareness interventions targeted at this population are thus needed in an attempt to reduce these tragic fatalities.
  • Thumbnail Image
    Item
    Open Access
    A study of allergy in the Western Cape
    (1974) Orren, Angela
  • Thumbnail Image
    Item
    Open Access
    An investigation of Epstein-Barr Virus (EBV) latency type and MYC gene aberrations in plasmablastic lymphoma diagnosed at Groote Schuur Hospital, Cape Town, South Africa
    (2020) Kriel, Raymond Frank; Ramburan, Amsha; Govender, Dhirendra
    Introduction: Plasmablastic lymphoma (PBL) is a rare, aggressive, AIDS-associated non-Hodgkin lymphoma. The pathogenesis of PBL is incompletely understood, however association with the Epstein-Barr virus (EBV) and the MYC gene, have been identified as important pathogenic mechanisms. Aims and objectives: To characterise the EBV latency in a cohort of patients diagnosed with PBL at Groote Schuur Hospital (GSH), by means of immunohistochemistry. To determine MYC gene aberrations using fluorescent in situ hybridisation (FISH). Materials and methods: The cohort comprised PBL cases diagnosed from 2005-2017. EBER ISH was used to confirm EBV infection. Manual immunohistochemistry using three monoclonal antibodies for EBV latent proteins, (EBNA1, EBNA2 and LMP1) was used to determine the latency type. Manual MYC FISH was performed on all PBL cases using a dual colour break apart rearrangement probe. Results: Forty-nine cases of PBL were included in this study. Forty-one cases were positive for EBER ISH. Thirty-seven (78.7%) cases showed HIV/EBV coinfection. Latency 0 was observed in 29 (70.7%) cases, latency 1 in 8 (19.5%) and latency 2 in 4 (9.8%) cases. MYC FISH was performed on all 49 PBL cases, of which 30 (61.2%) yielded a result. MYC was intact in 11 (36.7%), translocated in 8 (26.7%) and 11 (36.7 %) cases showed copy number variations. Conclusion: Our research demonstrated 37 (90.2%) of the EBV positive PBL cases showed a restricted latency pattern of 0 or 1. Furthermore we found that MYC gene aberrations consisting of translocations and copy number variations occurred in 19 cases (63.3%) , with copy number variations being higher than cited in current literature. Our study is also the first to investigate PBL EBV latency in SA. An uncommon finding was the existence of MYC gene aberrations in HIV positive, EBV negative PBL cases.
  • Thumbnail Image
    Item
    Open Access
    The ancient evolutionary history of polyomaviruses
    (Public Library of Science, 2016) Buck, Christopher B; Van Doorslaer, Koenraad; Peretti, Alberto; Geoghegan, Eileen M; Tisza, Michael J; An, Ping; Katz, Joshua P; Pipas, James M; McBride, Alison A; Camus, Alvin C; McDermott, Alexa J; Dill, Jennifer A; Delwart, Eric; Ng, Terry F F; Farkas, Kata; Austin, Charlotte; Kraberger, Simona; Davison, William; Pastrana, Diana V; Varsani, Arvind
    Author Summary: Polyomaviruses are a family of DNA-based viruses that are known to infect various terrestrial vertebrates, including humans. In this report, we describe our discovery of highly divergent polyomaviruses associated with various marine fish. Searches of public deep sequencing databases unexpectedly revealed the existence of polyomavirus-like sequences in scorpion and spider datasets. Our analysis of these new sequences suggests that polyomaviruses have slowly co-evolved with individual host animal lineages through an established mechanism known as intrahost divergence. The proposed model is similar to the mechanisms through with other DNA viruses, such as papillomaviruses, are thought to have evolved. Our analysis also suggests that distantly related polyomaviruses sometimes recombine to produce new chimeric lineages. We propose a possible taxonomic scheme that can account for these inferred ancient recombination events.
  • Thumbnail Image
    Item
    Open Access
    Apoptosis in Haematopoietic progenitors
    (2009) Rossouw, Sophia Catherine; Greenberg, Jacquie
    Introduction: Intracranial pressure (ICP) monitoring is a cornerstone of care for patients with severe traumatic brain injury (TBI). The primary goal of ICP treatment is to preserve brain oxygenation, and since brain oxygenation is usually not measured, the control of ICP is used as a surrogate marker. However studies indicating that cerebral hypoxia/ischemia may occur in the face of adequate ICP and cerebral perfusion pressure (CPP) suggest that the interaction between ICP and brain oxygenation is poorly understood and warrants further investigation. This is of particular importance in the context of children in whom the interpretation of relationships between intracranial factors is even more complex due to changing physiological norms with age. To date little scientific data exists in children and treatment threshold values are often extrapolated from adult guidelines. This study aims to better understand the relationship between ICP and brain oxygenation measured as brain tissue oxygen tension (PbtO2) in a large paediatric cohort suffering from severe TBI. Specifically analysis 1) investigated ICP and PbtO2 profiles over time following TBI, 2) examined the relationship between ICP and PbtO2 from time-linked paired observations, 3) explored various critical thresholds for ICP and PbtO2, and 4) interrogated digital data trends depicting the relationship between ICP and PbtO2. The level of agreement between hourly recorded and high frequency electronic data for ICP and PbtO2 was also evaluated. Method: Paired ICP and PbtO2 data from 75 children with severe TBI were tested with correlation and regression. Additional analyses controlled for mean arterial pressure (MAP), arterial partial pressure of oxygen (PaO2), CPP, arterial partial pressure of carbon dioxide (PaCO2) and haemoglobin (Hb) using multivariate logistic regression analysis and general estimating equations. Various thresholds for ICP were examined; these included age-related thresholds to account for the potential influence of age. Receiver-operating curves (ROCs) were used to graphically demonstrate the relationships between various thresholds of ICP and various definitions of low PbtO2. These were constructed for pooled and individual patient data. Interrogation of electronically recorded data allowed for case illustrations examining the relationship between ICP and PbtO2 at selected time points. Hourly and electronic data were compared using Bland and Altman plots and by contrasting the frequency of ICP and PbtO2 perturbations recorded with each system. 5 Result: Analyses using over 8300 hours of paired observations revealed a weak relationship between ICP and PbtO2, with an initially positive but weak slope (r = 0.05) that trended downwards only at higher values of ICP. Controlling for inter-individual differences, as well as MAP, CPP, PaO2, PaCO2 and Hb did not strengthen this association. This poor relationship was further reflected in the examination of threshold ICP values with ROCs, no singular critical ICP threshold for compromised brain oxygenation was discernible. Using age-based thresholds did not improve this relationship and individual patient ROCs demonstrated inter-individual heterogeneity in the relationship between ICP and PbtO2. However, it was clear that in individual patients ICP did exhibit a strong negative relationship with PbtO2 at particular time points, but various different relationships between the 2 variables were also demonstrated. A high level of agreement was found between hourly and electronic data. Conclusion: These results suggest that the relationship between ICP and PbtO2 is highly complex. Although the relationship in individual children at specific time points may be strong, pooled data for the entire cohort of patients, and even for individual patients, suggest only a weak relationship. This is likely because several other factors affect PbtO2 outside of ICP, and some factors affect both independently of each other. These results suggest that more study should be directed at optimising ICP thresholds for treatment in children. The use of complimentary monitoring modalities may assist in this task. Depending on the adequacy of measures of brain perfusion, metabolism or oxygenation, it is possible that targeting a range of ICP values in individual patients may be appropriate; however this would require detailed investigation.
  • Thumbnail Image
    Item
    Open Access
    Applications of aspiration lung biopsy with special reference to the pathogenesis of the resolution of acute and chronic lobar pneumonia
    (1951) Woolf, Colin Rael; Forman, F; Landau, A
    Lung biopsy is neither widely known nor practiced and it was only in 1949 that i first came across a paper on this subject. The title was: "Cellular analysis of the aspiration lung biopsy from normal and some pathological conditions by Z. Godlowski" (1949). The very term "lung biopsy" conjures up the picture of a needle being introduced into an air filled, very vascular structure where the bleeding of an injured vessel cannot readily be stopped, where the stage is set for air embolisms and where tension pneumothorax may occur. it was with great surprise but also an apparently innocuous procedure. Unfortunately, at that time, there was no opportunity to use the method. In 1950 I became the University Assistant at the New Somerset Hospital in Cape Town. Many of the cases admitted to the wards presented with chest pathology. Patients with pneumococcal lobar pneumonia were not infrequent and occasional cases did not resolve as expected but went on to become so-called chronic pneumonia. What happened when an acute lobar pneumonia went on to the chronic stage and why did this occur? it was suggested that investigae this problem.
  • No Thumbnail Available
    Item
    Open Access
    Astrocyte-mediated immune modulation during mycobacterial infection
    (2023) Geyer, Sohair; Jacobs, Muazzam
    Central nervous system tuberculosis (CNS-TB) is the severest clinical extra-pulmonary manifestation of tuberculosis (TB) disease and constitutes approximately 1% of global cases. Little is known about the cells that regulate immune responses during CNS-TB infection. Microglia are the prime resident immune-effector cells in the CNS; astrocytes however also regulate innate and adaptive immunity in CNS disease and injury. Astrocytes play a progressive role in maintaining the structural and functional integrity of the CNS while supporting neuronal function and participating in host protection during infection of the CNS. These cells exist as distinct populations with complex morphological identities and functional modifications suited to their micro-environment. The principal aim of this study was to elucidate the genomic profile and cellular immune responses of astrocytes to characterise their immunomodulatory potential during CNS-TB infection. The ability of astrocytes to internalise mycobacteria was evaluated by immunocytochemistry and immunohistochemistry. The immune regulatory role of astrocytes was assessed through transcriptomic profiling, flow cytometry and Luminex analysis. Outcomes were validated in mouse models, through flow cytometric analysis of infected brain cells. The novel findings presented here collectively demonstrate the sophistication and complexity of astrocyte activity and behaviour during host immunity. For the first time, astrocytes showed internalisation of M. bovis BCG and M. tuberculosis bacilli, demonstrating that astrocytes are target cells for non-virulent and virulent mycobacterial strains. Extensive transcriptomic analysis of astrocytes revealed elevated expression of multiple pathways in infected cells, particularly those involved in inflammation and immune regulation, and emphasised the innate immune component. Notably, various pro-inflammatory cytokines essential to host defence during CNS-TB infection, such as IL-1b and TNF, were upregulated by astrocytes following the mycobacterial challenge. The data suggests that astrocytes may modulate host immune responses by regulating blood-brain barrier permeability and facilitating immune cell recruitment and activation at the site of infection. This potential was demonstrated by their enhanced expression and production of well-described pro- and anti-inflammatory factors as well as chemotactic factors following mycobacterial infection. Furthermore, increased expression of neurotrophic factors by astrocytes was observed, which can assist in the recovery and repair of the CNS during CNS-TB. By supporting the survival and function of neurons and modulating immune cell activity, astrocyte-derived neurotrophic factors can help to limit infection-induced damage and promote the resolution of inflammation. The dichotomous behaviour of astrocytes during CNS-TB was demonstrated, as they can contribute to the maintenance and protection of CNS function and host immune responses while potentially enhancing pathology during infection. This study explored astrocyte contributions to host immune responses during CNS-TB infection by examining their regulation of CNS inflammation in the presence of mycobacterial challenges, and highlighted the intricate interplay of cytokines/chemokines that need careful modulation to achieve optimal outcomes. These findings demonstrated that astrocytes are crucial regulators of host immunity during mycobacterial infection and play a progressive role in maintaining the structural and functional integrity of the CNS
  • No Thumbnail Available
    Item
    Open Access
    Basil cell carcinoma: an immunohistochemical assessment of P53, BCL-2 and CD138 in low and high-risk histological subtypes
    (2023) De Stadler, Janet; Roberts, Riyaadh
    Basal cell carcinoma (BCC) is the leading cancer in males and the second commonest cancer in females in South Africa. The cost to the health sector is expected to rise given the increasing global incidence rates, particularly of aggressive BCCs. Improved understanding of BCC is paramount to enhance early detection and screening which could potentially offset these rising costs. Specific histological patterns of BCC have been defined as high-risk for recurrence by the World Health Organisation. The different BCC subtypes are not simply architectural patterns but may represent differences in aetiopathogenesis and protein expression, and impact future targeted therapies. Upregulation of the Hedgehog pathway and TP53 inactivation are the two most common events in the development of BCCs. High-risk BCC patterns have been observed to show increased expression of tumoural p53 and decreased expression of BCL-2 and CD138 compared with low-risk patterns. In addition, peritumoural expression of CD138 has been noted to increase in the stroma of high-risk BCCs. These observations have largely been made by light microscopy rather than with digital analysis.
  • Thumbnail Image
    Item
    Open Access
    Building evidence for improving childhood immunisation coverage in Africa.
    (2012) Wiysonge, Shey Umaru Charles; Hussey, Gregory D; Schoub, Barry D
    The Expanded Programme on Immunisation has the potential to substantially reduce child mortality and contribute to achieving the Millennium Development Goals. We assessed the programme’s performance in Africa, the reasons for poor performance, and effective interventions for improving its performance on the continent. We used a combination of methods including systematic reviews, bibliometric analyses, generalised linear models, and grading of the quality of evidence. We found that African countries have made extraordinary advances since childhood immunisation programmes began in 1974. However, there exist wide inter-country and intra-country differences, and the quality of immunisation data is poor. Besides, vaccines are administered well after the recommended ages in many countries; leaving children exposed to deadly vaccine-preventable diseases for long periods. In addition, Africa’s contribution to the global immunisation research output is minimal. There is no association between research productivity and immunisation coverage in Africa, which may signal lack of interactive communication between policymakers and researchers. Furthermore, individual and contextual factors (defined at community and country levels) are independently associated with low immunisation coverage; suggesting that immunisation system strengthening should address people and the communities and societies in which they live. Lastly, we found moderate-to-high quality evidence that interactive educational meetings, audit and feedback, supportive supervision; and use of community health workers, parent reminders, home visits, interactive communication, mass media, and material incentives have the potential to improve childhood immunisation coverage in Africa.
  • Thumbnail Image
    Item
    Open Access
    Characterization of a unique sulfoxide synthase found in pathogenic trypanosomes
    (2013) Mashabela, Gabriel Tshwahla Makgotloduwa; Steenkamp, D J; Gammon, David W
    Includes abstract. Includes bibliographical references.
  • Thumbnail Image
    Item
    Open Access
    Characterization of human foreskin Langerhans cells
    (2021) Qumbelo, Yamkela; Chigorimbo-Tsikiwa, Nyaradzo; Gray, Clive
    Background: It is known that medical male circumcision (MMC) decreases HIV acquisition by up to 60%. One hypothesis is that MMC removes a foreskin (FS) that harbors different immune cells that are HIV target cells such as CD4+ macrophages, T, Langerhans (LCs), and dendritic cells (DCs). However, there have been different reports on whether the inner FS or outer FS has more HIV target cells. While LCs have been implicated in HIV transmission, their role remains controversial. Studies have shown that LCs can transmit the virus to T cells, which increases infection. On the contrary, others have reported that LCs prevent infection by degrading the virus through a langerin-dependent pathway. One of the factors that plays a major role in HIV transmission is their state of maturity and activation, which can be influenced by co-infection and other immunological processes. The aim of this study was to isolate, quantify and characterize Langerhans cells in the inner FS and outer FS from men undergoing MMC and to evaluate the phenotype of matured and activated LCS. Differences in the proteome of the inner FS and outer FS tissues were further investigated. Methodology: FS were obtained from men undergoing voluntary MMC from clinics and hospitals in the Western Cape (Age 18 years or older). Epidermal FS cells were extracted using crawl (migratory) assay and liberase enzyme digestion. Langerhans cells were isolated by density gradient centrifugation, sorted, quantified and immune-profiled by flow cytometry. CD1a and CD207 were used to identify Langerhans cells while HLA-DR, CD80, CD86 and CD40 were used as markers of maturity and activation. The gene expression profile of sorted LCs was also examined by single-cell sequencing with seq-well. Lastly, the differences in the proteome of the inner FS and the outer FS migrated epidermal cells were assessed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results: Langerhans cells were an average of 85% pure post-sorting. The numbers of Langerhans cells between the inner FS vs. outer FS were not statistically different (mean: 0.56% vs. 0.68% (SD=0.37) from migratory cells and 0.28% vs. 0.45% (SD=0.18) from enzyme digest, p-value >0.05, n=9). Sequencing showed that the sorted cells pooled from 5 participants (inner and outer FS) had different gene expression profiles. Furthermore, two groups of cells were identified from the sorted LCs based on their gene expression profile. The identified cells were monocyte-like and melanocyte-like cells. The monocyte-like cells were identified as LCs based on their gene expression profile while the melanocyte-like cells were identified as the contaminating cells as the cell purity was not 100%. Upon activation with tumor necrosis factor alpha (TNF-α), activated LCs isolated by the migration assay had similar proportions of cells expressing surface maturity and activation markers (HLA-DR, CD40 and CD80/86) when compared to the unstimulated controls (inactivated) (mean: 73.53% vs. 75.66%, n=9, p-value >0.05, SD=4.4) However LCs that were isolated by the migration assay expressed markers of activation at a higher level compared to LCs isolated by Liberase enzyme digestion (mean: 79.4% vs. 40%, p-value < 0.05 n=9, SD=23). Proteomics showed that the inner FS had an over-abundance of proteins involved in the interleukin 7 response and mRNA catabolic processes, while the outer FS had more spindle zones and cornified envelope proteins that were over-abundant when comparing inner and outer FS from 5 participants. Discussion and Conclusion: The study successfully extracted, sorted and immunoprofiled Langerhans cells using different methods and from different FS compartments (inner FS versus outer FS). When LCs were spontaneously migrated and isolated using the “crawl method”, they showed a more mature and activated phenotype compared to non-migrating “skin resident” immune cells. No differences were found between cells that were stimulated with inflammatory cytokines relative to unstimulated migratory cells in proportion of cells expressing activation markers. However, it was observed that cells isolated by liberase enzyme digestion showed significantly lower proportions of activation markers relative to migratory cells. Using LC-MS/MS-based proteomics; the inner FS exhibited high expression of proteins involved in the interleukin 7 response while the outer FS exhibited high expression of structural proteins, which suggests that the inner FS might be more involved in immunity as interleukins can stimulate immune response while the outer FS has a more structural role than the inner FS.
  • Thumbnail Image
    Item
    Open Access
    Characterizing the cellular latent reservoir of HIV-1 and the effect of immune activation on characteristics of the reservoir
    (2022) Ismail, Sherazaan Dineo; Burgers, Wendy A; Williamson, Carolyn; Riou, Catherine; Abrahams, Melissa-Rose
    Since the advent of antiretroviral therapy (ART) and the resultant suppression of viraemia in the majority of people living with HIV-1 (PLWH) on ART, HIV-1 infection has become manageable and PLWH have similar life expectancies as uninfected persons. However, ART is not curative, is needed lifelong, and its cessation leads to the recrudescence of viraemia. This is due to the formation of a latent reservoir that is long-lived and stable over time, precluding HIV-1 cure. The factors affecting reservoir formation, establishment, and kinetics are not fully understood. Furthermore, differences exist at the population level in disease progression in PLWH depending on ethnicity, biological sex, and infecting viral subtype. Similarly, differences in the latent reservoir of HIV-1 have been described, although less extensively. Understanding what shapes the latent HIV-1 reservoir is critical for developing strategies for cure. Furthermore, it is imperative that cure research is undertaken in diverse populations to ensure coverage of knowledge across different demographics. The latter will ensure that a cure strategy can be developed that will be globally implementable. In the Introductory chapter of this thesis, I provide a detailed review of the current literature and address the need for cure research in low-and middle- income countries. If a global cure is to be achieved, the burden of HIV-1 will need to be addressed in many different populations, most notably African women, as women bear the burden of HIV-1 globally. In South Africa, the country in the sub-Saharan African region with the highest prevalence of HIV-1, women are roughly twice as likely to be living with HIV than men (aged 15 to 49), with a prevalence rate 6% higher than the national average of 19%. Since women are underrepresented in HIV-1 research in general and more specifically in cure studies due to the paucity of research in countries outside of the global North, reservoirs and cure strategies ii need to be characterized in this context. Furthermore, while early treatment is the WHO standard of care for people diagnosed with HIV, a large majority of PLWH only initiated treatment in chronic infection. Since early ART is known to restrict formation of the latent reservoir of HIV-1, research in both early and late ART initiators is necessary. This research focused on characterising the viral reservoir in South African women in a well-established cohort of women who were recruited during acute HIV infection and followed until treatment initiation (which occurred during chronic infection) and beyond. Overall, this thesis focuses on characterising immune activation and inflammation during the course of both untreated and treated HIV-1 infection in a cohort of South African women and subsequently determining whether clinical or immune measures influence characteristics of the latent reservoir of HIV-1. T cell activation and the levels of soluble inflammatory cytokines in plasma were determined in forty-six women in the CAPRISA 002 Acute infection cohort. Chapter 2 describes the cellular immune activation and inflammation profiles of these participants throughout the course of infection at the following timepoints: acute infection, oneyear post-infection, and within a year preceding ART initiation, and two- and four- years postART initiation. T cell activation peaked in chronic infection and reduced dramatically after ART initiation. CD4+ and CD8+ T cell activation reached a post-treatment nadir by two years after ART initiation. Cytokine measures were within the ranges reported in the literature for PLWH. Notably CXCL-10 levels in plasma decreased significantly between two- and four years post-ART, indicating that it may be a sensitive marker of ongoing systemic inflammation in people on ART. In short, the T cell activation and inflammation profiles of the women in this study reflected what has been observed in other cohorts. iii The size of the replication-competent HIV-1 reservoir, measured by quantitative viral outgrowth assay after 5 years of suppressive antiretroviral therapy (ART), was quantified in twenty women of the cohort. In Chapter 3, the clinical and immunological correlates of reservoir size were investigated. Predictive modelling showed that the size of the replicationcompetent reservoir is directly related to viral load and CD4+ T-cell counts over the course of infection, although these measures do not fully predict reservoir size. We found that, in addition to viral load and CD4+ T-cell count, CD8+ T-cell activation within the year preceding ART, nadir CD4+ T-cell count, and baseline as well as on-treatment CD4:CD8 ratio at the time of sizing was associated with replication-competent reservoir size. We provide evidence that the late CD8+ T-cell activation level before treatment, together with viral loads and CD4+ T-cell counts, are directly related to the size of the replication-competent reservoir of HIV-1. Our results are consistent with the hypothesis that the host immune milieu near the time of ART initiation plays an important role in shaping the durable reservoir of HIV infection that persists on ART. Another characteristic of the HIV-1 reservoir is persistence: the presence of all forms of HIV1 within cells and tissues that contribute to pathogenesis, including defective, non-induced, and non-integrated forms of HIV-1. In Chapter 4, total HIV-1 DNA levels were measured as a proxy for viral persistence in thirty-one participants, and the correlates thereof investigated. The HIV-1 DNA levels in this cohort were similar to those reported in the literature for other cohorts where participants initiated therapy in late chronic infection. HIV-1 DNA levels did not differ significantly between two- and four years post-ART, but there was a trend to lower HIV-1 DNA when measuring pol versus gag gene frequencies in peripheral blood mononuclear cells (PBMC). These findings indicate that HIV-1 DNA decay rates may differ depending on the gene being measured, even when using the same assay. A weak significant correlation was iv found between CD4+ T cell counts at ART initiation and the change in HIV-DNA levels between two-and four years on ART. There was a significant correlation between residual CD4+ T cell activation at four years post-ART initiation and gag copies per million PBMC. A trend towards a correlation was found between CD4+ T cell activation and pol copies per million PBMC at the same timepoint. Finally, we found significant correlations between several cytokines at one-year post-infection and within one year pre-ART. These findings further solidify the hypothesis that the immune milieu around the time of ART initiation and after may play a complex role in formation of the viral reservoir of HIV-1. Our studies show a significant link between chronic immune activation and replication competent reservoir size, and also ongoing immune activation and viral persistence on ART. Further studies into whether these immune measures affect the timing of establishment and clonality of the reservoir in this cohort are ongoing and will inform the field about whether differences in cure strategies will need to be explored for those PLWH who had high levels of chronic immune activation before treatment initiation and subsequent shaping for the long-lived viral reservoir.
  • Thumbnail Image
    Item
    Open Access
    Clinical-pathological characterisation of children with B-cell non-Hodgkin lymphoma over a ten year period at a tertiary centre in Cape Town
    (2020) Kriel, Magdalena; Phillips, Lee-Ann; Davidson, Alan; Pillay, K; Hendricks, M
    Background: We characterized B-cell non-Hodgkin lymphoma (NHL) cases over ten years at a tertiary children's hospital to contribute to the body of knowledge on pediatric lymphoma in developing countries with a high human immunodeficiency virus (HIV) burden. Methods: A retrospective cohort study using clinical and laboratory records of children newly diagnosed with B-cell NHL from January 2005 to December 2014. Results: Seventy-five children ≤ 15 years were included. The majority had Burkitt lymphoma (n = 61). Twenty-five percent (n = 19) were HIV positive and 16% (n = 12) had concurrent active tuberculosis. Bulky disease was present in 65.7% (n = 46) and 30.1% (n = 22) were classified as Lymphomes Malins B (LMB) risk group C. The five year survival estimates for HIV-negative and HIV-positive children were similar in our cohort: 81% vs. 79% for eventfree survival and 85% vs. 83.9% for overall survival. Of three children with Burkitt lymphoma, HIV and LMB group C, two died within one year. Conclusions: Irrespective of HIV status, the survival of children in our B-cell NHL cohort compares favorably with cure rates in developed nations, although advanced disease remains associated with a poor prognosis. Characterization of childhood NHL cases contributes to accurate risk stratification and tailored treatment.
  • Thumbnail Image
    Item
    Open Access
    Comparative population genetics of the German shepherd dog in South Africa
    (2009) Coutts, N J; Harley, E H
    Modern breeding practices strive to achieve distinctive phenotypic uniformity in breeds of dogs, but these strategies are associated with the inevitable loss of genetic diversity. Thus, in parallel with the morphological variation displayed by breeds, purebred dogs commonly express genetic defects as a result of the inbreeding associated with artificial selection and the reduction of selection against disease phenotypes. Microsatellite marker analyses of 15 polymorphic canine loci were used to investigate measures of genetic diversity and population differentiation within and between German-bred and South African-bred German shepherd dogs. These data were quantified by comparison with typically outbred mongrel or crossbred dogs. Both the imported and locally-bred German shepherd dogs exhibited similar levels of genetic diversity. The breed is characterised by only a moderate loss of genetic diversity relative to outbred dogs, despite originating from a single founding sire and experiencing extensive levels of inbreeding throughout the history of the breed. Non-significant population differentiation between the ancestral German and derived South African populations indicates sufficient contemporary gene flow between these populations, suggesting that migration resulting from the importation of breeding stock has mitigated the effects of random genetic drift and a population bottleneck caused by the original founder event in South Africa. Significant differentiation between the combined German shepherd dog population and the outbred dogs illustrates the effects of selection and genetic drift on the breed since its establishment just over 100 years ago.
  • Thumbnail Image
    Item
    Open Access
    Comparison of the two lumpy skin disease virus vaccines, Neethling and Herbivac, and construction of a recombinant Herbivac-Rift Valley fever virus vaccine
    (2015) Omar, Ruzaiq; Williamson, Anna-Lise; Douglass, Nicola
    There are two broad aims to this project. The first aim is to compare and characterise two lumpy skin disease virus (LSDV) vaccines namely the vaccine based on attenuated Neethling LSDV (nLSDV) and Herbivac®LS (Herbivac). The second aim is to construct a recombinant LSDV expressing Rift Valley fever virus (RVFV) genes. An LSDV vaccine is critical for sustainable control of lumpy skin disease (LSD). There are four commercially available live attenuated vaccines for LSDV, nLSDV, Herbivac, Lumpyvax and the Kenyan strain sheeppox virus (KS-1). In this study Herbivac was characterised by comparing it to its parent, nLSDV. Growth curves of the two viral strains were conducted in cell culture as well as in embryonated hens’ eggs. No notable difference in the growth rate of the two strains could be detected when the viruses were grown in cell culture, however a notable difference was detected when the viruses were grown on the chick allantoic membranes (CAMs) of embryonated hens’ eggs. When grown on CAMs a faster growth rate was observed for nLSDV compared to Herbivac. nLSDV also killed the embryos at 4 d.p.i where Herbivac did not. The two strains were then further characterised through histological analysis of CAMs after infection with each of the viruses. Overall, higher levels of hyperplasia and hypertrophy were observed in CAMs infected with either nLSDV or Herbivac compared to uninfected CAMs. Herbivac-infected CAMs resulted in thicker chorionic membranes and larger pocks compared to nLSDV. RVFV and LSDV both contribute to the disease burden among cattle in Africa and the Arabian Peninsula. The main aim of this study was to construct a recombinant Herbivac which expresses immunogenic proteins of Rift Valley fever virus (Herbivac-RVFV). Herbivac-RVFV was designed to express specific RVFV genes selected for their antigenic properties. The genes selected are also representative of the genes from recent viral outbreaks in the horn of Africa. The selection of outbreak relevant RVFV genes involved phylogenetic analysis of all full length M-segment and NC gene sequences available on Genbank. Phylogenetic trees were constructed for M-segments and NC genes and groups identified which were highly representative of sequences from recent outbreaks of the virus. Consensus sequences were derived from these groups and included in the transfer vector. The phylogenetic analysis also revealed that the sequences of current RVFV vaccines are phylogenetically distant from viruses isolated from current outbreaks, although high levels of sequence conservation was maintained across all viral strains. This is the first study in which the RVFV genes coding for proteins that will induce a protective immune response (Gn and Gc, as well as the nucleocapsid (NC) gene) were selected so as to be representative of current outbreak strains of the virus. These genes were inserted between LSDV ORFs 49 and 50, a novel insertion site. The transfer vector also contained an eGFP marker gene and an ECO-GPT selection gene, located outside of the LSDV flanking sequences. This meant a two-step isolation procedure, first to isolate the recombinant containing the entire transfer vector with eGFP and ECO-GPT, and then to isolate a recombinant with only the RVFV genes and not eGFP and ECO-GPT. Transient expression of RVFV proteins in cells infected with Herbivac and then transfected with the transfer vector was confirmed via western blotting and immunofluorescence. Here the proteins Gn, Gc and NC were shown to be expressed. In the present study, a single crossover Herbivac-RVFV recombinant was isolated through multiple passaging of cell lysates, originally obtained from Herbivac-infected FBT cells transfected with the transfer vector, in the presence of mycophenolic-acid selection medium.
  • Thumbnail Image
    Item
    Open Access
    Comprehensive proteomic profiling of clinically relevant strains of Mycobacterium tuberculosis
    (2014) Peters, Julian S; Blackburn, Jonathan
    Tuberculosis is an airborne infectious disease caused by the bacillus known as Mycobacterium tuberculosis. Despite limited genetic variability, Mycobacterium tuberculosis strains exhibit vast discrepancies in phenotypic presentation in terms of virulence, elicited immune response and transmissibility. This study aims to use Mass Spectrometry (MS) tools to quantitatively and qualitatively investigate the total proteome expressed by various epidemiologically significant strains within the Mycobacterium tuberculosis complex (MTBC) as well as a clinically relevant non-tuberculous Mycobacteria (NTM) strain when cultured in vitro. We aim to use the experimental data obtained using discovery mass spectrometry to identify candidate proteins to use in the design of multiple reaction monitoring (MRM) MS experiments for targeted biomarker validation in patient derived biological samples such as sputum. Liquid chromatography mass spectrometry (LC MS/MS) and data capture were carried out using the LTQ Orbitrap Velos. 1D LC was carried out on gel fractionated samples to increase proteome coverage. This allowed a significant increase in the number of protein identifications of up to 80% proteome coverage per strain. Comparative analysis of the datasets was carried out to identify and define the core-proteome expressed across all strains as well as to identify differentially expressed proteins amongst the strains.