Browsing by Subject "Pathology"

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    Antigenic and immunological determinants of acute allergic susceptibility to meat in a uniquely defined cohort in the Eastern Cape
    (2023) Murangi, Tatenda; Levin, Michael; Horsnell William
    Allergic sensitization can occur after allergen exposure through the oral-mucosal or cutaneous route. Allergic remission is associated with a decrease in total and specific IgE levels to allergens. Ascaris lumbricoides is a potent inducer of IgE through the establishment of a strong Th2 environment. IgE induction following A. lumbricoides infection is a risk for allergic sensitization. Tick disruption of host skin during feeding has a systemic effect resulting in the induction of a Th2 phenotype with elevated IgE production. Raised IgE can be driven by exposure to parasite proteins and lipids with complex glycosylation patterns. Our study demonstrates the presence of alpha-gal in both adult and larval developmental stages of A. lumbricoides, Amblyomma hebraeum and Rhipicephalus evertsi. Alpha-gal glycosylation was prominent on 100kDa and 130-250kDa protein bands. A. hebraeum and R. evertsi showed differential expression of alpha-gal glycosylated proteins during feeding with band intensity increasing proportionally to an increase in feeding time in the salivary glands. Immunolocalization of alpha-gal in A. lumbricoides adult worms showed staining in the lining of the gastrointestinal tract while in A. hebraeum and R. evertsi, staining was prominent in the salivary glands. Screening for IgE demonstrated elevated IgE to A. lumbricoides in human research participants with challenge-proven alpha-gal allergy which positively correlated to alpha-gal IgE. Furthermore, non-alpha gal glycosylated A. lumbricoides antigens caused significant activation of a humanized rat basophil RS-ATL8 IgE reporter cell system after incubation with sera from alpha-gal allergic individuals. Interestingly, serum IgG4 from alphagal allergic individuals showed surface labelling of A. lumbricoides larvae invitro. Alpha-gal positive participants also demonstrated raised IgE and IgG4 towards A. hebraeum proteins. Proteomic analysis suggests alpha-gal glycosylation to be present on alpha-2-macroglobulin found in lysates from both A. lumbricoides and A. hebraeum. These findings present A. lumbricoides, A. hebraeum and R. evertsi as potential sources of sensitization to alpha-gal and hypersensitivity reactions including anaphylaxis in humans after the consumption of red meat or use of pharmaceutical products from a mammalian source
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    Apolipoprotein E variants, plasma lipids, lipoproteins and dysApolipoprotein E variants, plasma lipids, lipoproteins and dysβLipoproteinaemia during pregnancy in Zimbabwean women.
    (2005) Tanyanyiwa, Donald Moshen; Marais, A D
    This study of pregnant women in Zimbabwe therefore set itself the following aims: To describe lipid and lipoproteins during and after pregnancy, To examine the prevalence of apoE variants, To evaluate dysβlipoproteinaemia in pregnancy, The correlation between dysβplipoproteinaemia and the apoE genotypes. This is the first study to systematically examine lipids and lipoproteins during pregnancy in black Africans.
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    Assessment of the suitability of blood samples collected for toxicology for subsequent genetic analysis: A follow-up study after five years
    (2023) Grobbelaar, Jana; Davies, Bronwen; Pearce Brendon
    Therapeutic and recreational drug use is a common occurrence across the world. However, substance use may sometimes result in adverse drug reactions and death even when typically non-fatal drug doses are administered. This phenomenon may be caused by variants in the genes encoding drug-metabolising enzymes, which leads to altered drug metabolism and at times, toxicity. Cause or manner of death may not be apparent in these cases, even after conducting a standard autopsy and ancillary toxicological and histological investigations. A molecular autopsy may then be performed to identify an underlying genetic cause. Genetic testing is however not routinely conducted in forensic mortuaries, and historic backlogs within the National Forensic Chemistry Laboratories may delay the processing of toxicology samples by months or even years. As such, specimens that have undergone toxicological testing and were stored long-term are sometimes the only samples available to conduct subsequent molecular autopsies, should it be necessary for the cause of death determination. This study therefore aimed to assess whether blood specimens that were used for toxicological analyses could provide suitable DNA for downstream genetic analyses after an extended storage period of five years. In 2017, DNA was analysed from blood samples collected into vials containing sodium fluoride/potassium oxalate preservatives or vials without preservatives (grey and red top tubes, respectively). A subset of these vials underwent preparation for toxicological analyses at the time, prior to DNA extraction, while the remaining tubes underwent DNA extraction immediately and were stored in a molecular laboratory as controls. DNA analysis was then repeated one year later in a separate study, as well as five years later as part of the current study. DNA quantity and quality scores were significantly lower in red top tubes compared to grey top tubes, and toxicological processing did not significantly influence results. DNA concentration and quality also significantly decreased over time for all sample types. PCR amplification and Sanger sequencing results were mostly poor for red top tubes, but grey top tubes showed overall improvements in sequence quality. However, all DNA analysis results generally improved when DNA was extracted using a modified salting out method. Based on these results, it is suggested that forensic laboratories that often experience delays in sample processing should perform molecular autopsies using blood stored in sodium fluoride/potassium oxalate preservative coupled with a salting out DNA extraction method.
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    A bacteriological and pathological investigation of tuberculosis meningitis in the Western Province of the Cape of Good Hope.
    (1952) Coetzee, Jack Nicol
    Although there have always been sporadic reports of spontaneous cures in tuberculous meningitis, up to a few years ago this diagnosis almost invariably meant death within a few weeks or months. The therapeutic use of Streptomycin in this disease has created new hope for these patients and has stimulated tremendous interest in all aspects of the disease.
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    Barriers to tuberculosis drug discovery: the mycobacterial cell wall
    (2023) Whittaker, Caitlin; Warner, Digby; Egan Timothy John
    The mycobacterial cell wall is a highly complex macromolecular structure that provides intrinsic resistance to several anti-tuberculosis drugs, making it critical in the success of Mycobacterium tuberculosis infection. Multiple layers encapsulate the cell membrane, including arabinogalactan chains and mycolic acids that are covalently bonded to the peptidoglycan layer, resulting in a highly selectively permeable barrier that is unique to this genus. The current treatment for tuberculosis (TB) utilizes antibiotics that weaken the structural integrity of the cell wall to allow easier access for drugs that have intracellular targets. Although this approach is theoretically effective, patient adherence is often poor owing to the lengthy treatment times and negative side effects associated with the multidrug combination regimen. As such, rational drug design to develop more potent, faster-acting anti-TB compounds requires a comprehensive understanding of the composition and functioning of the mycobacterial cell envelope to ensure effective penetration through this barrier. Bioinformatic approaches to compound validation provide a crucial foundation for drug development, but empirical validation of these molecules can present a serious bottleneck in the drug discovery pipeline. Here, we investigated fluorescent click chemistry as a rapid and inexpensive means of ascertaining molecular properties that impact compound permeation of the mycobacterial cell envelope. The variability in permeation of different click-reactive moieties could be rapidly determined using fluorescent read-outs; this, in combination with the availability of a wide array of click-reactive side chains, presents a potentially powerful platform for establishing the properties required by a compound to effectively cross the mycomembrane. Enzymatic degradation of cell wall components further revealed the resilience of mycobacteria as the resulting organisms, spheroplasts, were capable of surviving in the absence of this seemingly essential protective layer. This presents a potentially novel form of intrinsic resistance whereby stripping of the cell wall could allow for tolerance to cell wall active antibiotics, a previously under-appreciated strategy that has been reported in other pathogenic bacteria. Together, these findings highlight the highly dynamic nature of the mycobacterial cell envelope and the need for further investigation into the properties of this structure that allow for such efficient antibiotic evasion.
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    Characterisation of T cell specificity, functional, activation and memory profiles associated with QuantiFERON TB Gold conversion and reversion
    (2021) Mpande, Cheleka Anne-Marie; Nemes, Elisa; Scriba, Thomas; Rozot, Virginie
    Recent acquisition of Mycobacterium tuberculosis (M.tb) infection is associated with a higher risk of tuberculosis disease, compared with remote, asymptomatic infection. M.tb infection, defined by a positive tuberculin skin test (TST) and/or IFN--release assay [IGRA e.g. QuantiFERON TB GOLD (QFT)], is commonly thought to be a chronic state. However, longitudinal studies have demonstrated the dynamic nature of M.tb infection, whereby TSTs and IGRAs revert from a positive to a negative test in some individuals, possibly an indication of bacterial clearance. Despite the first observation of discordant serial TST results over 80 years ago and the wide use of TSTs/IGRAs, there is still a limited understanding of immunological features associated with different stages of M.tb infection and discordant serial TST/IGRA results. Most studies of M.tb-specific immune responses in humans are based on cross-sectional comparisons between M.tb infection and active disease, with very few large cohort studies enabling a longitudinal assessment of different phases of infection. Thus, the main objective of this thesis was to gain a better understanding of changes in M.tbspecific CD4 T cell functional, memory and activation profiles associated with QFT conversion (acquisition of M.tb infection) and reversion (potential M.tb clearance). Our first aim was to characterise the homing, cytotoxic and functional capacity of M.tbspecific memory CD4+ T cells during recent and remote M.tb infection, with a special focus on stem cell memory T (TSCM) cells. TSCM cells play a critical role in maintaining long-lasting immunity, demonstrated by their superior longevity, proliferation and differentiation capacity compared to central memory (TCM) and effector (TE) cells. Before this study, our knowledge of TSCM cells was primarily based on virus-specific CD8+ TSCM cells. We demonstrate that M.tb-specific CD4+ TSCM cells are induced upon recent M.tb infection and maintained at steady-state during established infection. Despite being the least differentiated M.tb-specific memory subset and representing 2 years) M.tb infection, we also aimed to define an M.tb- specific T cell biomarker that can distinguish between the two infection states as current diagnostics fail to do so. Our second major finding demonstrated that recently infected individuals have lower proportions of highly differentiated IFN-+TNF+KLRG-1+ CD4+ TE cells and higher proportions of early differentiated IFN--TNF+IL2+KLRG-1- CD4+ T cells than remotely infected individuals in response to M.tb lysate but not CFP-10/ESAT-6 stimulation. Akin to their recent M.tb exposure, recently infected individuals had higher levels of T cell activation, regardless of M.tb antigen specificity, than remotely infected individuals. The degree of M.tb-specific CD4 T cell activation was identified as the best candidate biomarker for recent infection. The very same biomarker could also distinguish between progressors and non-progressors and identify individuals with active tuberculosis disease among healthy individuals with remote M.tb infection. We propose that, upon large-scale clinical validation, the T cell activation biomarker could be used as a screening test in conjunction with current tuberculosis diagnostics to guide the provision of either preventive or full tuberculosis therapy. These results have very important implications for targeting provision of preventive treatment to M.tb infected individuals at high-risk of tuberculosis, which is one of the top 10 strategies required to achieve tuberculosis elimination targets. Based on data from observational studies conducted during the pre-antibiotic era and guinea pig tuberculosis models, TST/IGRA reversion in humans is hypothesised to be associated with spontaneous (natural) clearance of infection. Similarly, individuals recently exposed to patients with tuberculosis who did not convert TST/IGRA (termed resistors) nor develop disease had M.tb-specific T cell responses that did not include IFN- production. However, clearance of M.tb infection is virtually impossible to demonstrate in healthy individuals. We clearly illustrated that acquisition infection is associated with induction of CD4+ Th1 functional and memory T cell subsets associated with increased antigen burden. We thus hypothesised that if reversion represents natural M.tb infection clearance then immune responses post-QFT reversion, if detectable, would be predominantly TSCM/TCM cells that have an IFN- independent cytokine expression profile and low T cell activation levels. Interestingly, QFT reversion was not associated with a decrease in CFP-10/ESAT-6-specific IFN-+ CD4 T cell responses detected by flow cytometry. Overall, CD4 T cell responses to CFP-10/ESAT-6 in reverters were of intermediate magnitude between non-converters and remotely infected individuals. These responses were very low in most reverters (regardless of QFT status), which may explain fluctuations around the QFT assay cut-off resulting in reversion of the test. In the reverters who had low but robustly detectable responses, CFP-10/ESAT-6-specific CD4 T cells showed low levels of M.tb-specific T cell activation, maintenance of both IFN- dependent and independent Th1 cytokine co-expressing profiles and a predominantly TTM/TE phenotype. Memory and functional profiles detected in reverters in response to M.tb lysate shared more characteristics with non-converters than persistently infected (QFT+) individuals. Based on these results, we conclude that QFT reverters represent a heterogenous population in the tuberculosis spectrum who may experience very low or no in vivo antigen exposure. Altogether, these results indicate that not everyone with a QFT+ test likely experiences ongoing in vivo M.tb exposure, as suggested by much lower T cell activation observed during remote M.tb infection and QFT reversion compared to recent M.tb infection. Whether ongoing in vivo antigen exposure is required to maintain memory responses against M.tb remains to be determined. It is possible that key features of T cell responses against M.tb, including magnitude and differentiation, are shaped by the antigen load experienced during primary infection, regardless of whether infection is subsequently cleared. Answering these questions is critical to inform the interpretation of the current immunodiagnostic assays and to determine who could be spared from preventive tuberculosis therapy. On the other hand, here we defined a biomarker of recent infection and tuberculosis disease, which could enable the provision of targeted treatment to those who would benefit the most.
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    Characterisation of the germ cell tumours seen at the Red Cross War Memorial Children's Hospital (1956-1995)
    (1998) Gordon, Alan Ian; Gordon, Alan; Kaschula, R O C
    The aim of the current study is the characterisation (primarily pathological, but with clinical correlation) of the germ cell tumours seen in the Pathology Department of the Red Cross Childrens Hospital since its inception in 1956 (through to the year 1995, date of commencement of the study). Study population: Infants and children from birth to 13 years of age (of all population groups, but predominantly those from the disadvantaged black and mixed race communities of the Greater Cape Town metropolitan area).
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    Cooling rates of dummies under various degrees of air humidity, wind speed and air temperature
    (2013) Mfolozi, Sipho; Martin, Lorna
    Henssge observed that even a slight but permanent air movement accelerates cooling of a naked body significantly. However in those experimental studies the rate of air movement was not quantified. Today Henssge’s Nomogram method of thermometric thanatochronometry (mathematic estimation of the post-mortem interval using body temperature measurements) is used the world over and utilises various corrective factors for naked and clothed bodies in still/moving air. The purpose of this research was to correlate measured air flow rates (wind speed) and measured relative air humidity levels (RH) with post-mortem cooling rate in order to formulate appropriate corrective factors to be used with Henssge’s Nomogram. The effect of air flow rates and air humidity on the post-mortem cooling curve was studied within a range of air temperatures using gel-based models (Cooling Dummies) as substitutes for human bodies.
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    Determining the impact of Heligmosomoides polygyrus infection on the development of colitis
    (2019) Katsandegwaza, Brunette; Smith, Katherine; Horsnell, Bill
    The ability of helminths to regulate inflammatory disorders and the mechanisms by which they carry this out are of great scientific interest. Currently, established literature emphasises the protective role of helminth infection in mouse models of inflammatory bowel disease (IBD). Utilising two well-established murine models of human disease, the oxazolone and dextran sulfate sodium (DSS) models, I found that induction of murine IBD is highly sensitive to diet change and mouse gender. Using the gastrointestinal helminth Heligmosomoides polygyrus (H.polygyrus), I demonstrate that helminth infection exacerbates IBD in both the oxazolone and DSS models of colitis. Underlying helminth infection results in increased inflammation locally in the colon and systemically in the spleen in both models of IBD, as measured by histology and flow cytometry. Exacerbation of DSS colitis is dependent on the dose of H.polygyrus but is independent of the phase of H.polygyrus infection, with both acute and chronic infections resulting in the same phenotype. Helminth exacerbated DSS colitis is characterised by significant bacterial translocation to the spleen, which is concluded to be due to loss of intestinal epithelial integrity. Helminth infection also resulted in a microbial shift of translocating bacteria following DSS administration, as evidenced by gram staining and bacterial sequencing. Administration of an 8- strain probiotic during acute helminth infection ameliorated helminth exacerbation of DSS colitis, restored epithelial integrity and abrogated splenomegaly. This work uncovers an unexpected and novel role for live helminth infection in exacerbating IBD and suggests that helminth-induced dysbiosis of the microbiota may drive disease. These studies reveal restoration of the microbiota through probiotics or helminth eradication as potential therapies for the treatment of gastrointestinal inflammatory disorders.
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    Development of a GC-MS method to determine toxic alcohols and their metabolites in postmortem blood
    (2024) Grevel, Carl; Vuko, Loyiso; Davies Bronwen
    The intentional and accidental ingestion of toxic alcohols represents a health care and potential public health concern within South Africa. Household and industrial antifreezes and brake fluids contain ethylene glycol (ETG) and diethylene glycol (DEG), which cause toxicity within humans. Other toxic alcohols such as 1,4-butanediol (1,4-BD) and propylene glycol (PGL) also show toxicity within the human body. Some of these analytes have previously been implicated in cases of fatal poisoning. However, the extent to which toxic alcohols contribute to death in South Africa is yet to be determined, as these are not routinely investigated in forensic toxicological analysis of biological samples. The purpose of this study was to modify and characterise a gas chromatography-mass spectroscopy (GC-MS) method for the quantitative determination of ETG, DEG, 1,4-BD, PGL, and the toxic metabolite of ETG, glycolic acid (GCA), in postmortem whole blood samples at the Forensic Toxicology Unit (FTU) in the Western Cape, South Africa. We describe the alteration of an existing method that examines most of these target analytes among others by Meyer, Weber and Maurer (2011), utilising a lower quantity of N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) and post-mortem whole blood rather than plasma. The method was characterised according to parameters of calibration model, limit of detection, limit of quantification, bias, precision, processed sample stability, and carryover. Linearity was observed for all analytes between 25– 100 µg/mL with preliminary limits of detection at 25 µg/mL. Preliminary limits of quantification were 25 µg/mL for 1,4-BD, and 50 µg/mL for ETG, PGL, GCA and DEG. Recovery was calculated at ~40% for all analytes, and processed sample stability was calculated to be acceptable for up to 72 hours. The developed method was applied to several post-mortem cases of toxic alcohol ingestion at the Observatory Forensic Pathology Institute (OFPI). In conclusion, the method for the determination of toxic alcohols in post-mortem samples was successfully developed and characterised for a government forensic toxicology laboratory in the Western Cape. Future work will include the validation of this method to streamline analytical determination of the morbidity and mortality related to toxic glycols in the Western Cape
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    Epidemiology and genetic risk factors of suicidal behaviour in South Africa
    (2023) Kootbodien, Tahira; Ramesar, Rajkumar; Martin Lorna
    Background: Suicide is an urgent public health problem. Fatal suicidal behaviour (individuals who died by suicide) and non-fatal suicidal behaviour (attempted suicide, self-harm, and suicidal ideation) comprise a complex interplay of individual, social, environmental, and biological factors, that are not fully understood. Given the considerable societal cost associated with suicide and health inequality in South Africa, there is a critical need to determine the burden of suicide and risk factors associated with suicide, to understand who is most at risk to inform effective prevention efforts. Despite clear evidence of multiple risk factors, suicidal behaviour remains difficult to predict and prevent. Prevention efforts in South Africa may be limited by the lack of a national suicide prevention plan and the low base rate of individuals who died by suicide may impede research comparing fatal and non-fatal suicidal behaviour in settings such as ours. In addition, existing suicide data are derived primarily from high-income countries rather than lowand middle-income countries (LMICs) where suicide and poverty levels are high, and the mental health treatment gap is large. The purpose of this study was to broaden our understanding of suicidal behaviour in South Africa by combining various data sources, each representing a unique perspective of the problem, to build on existing knowledge that may inform suicide prevention strategies in South Africa. This thesis is organised into four studies and aimed to investigate risk factors associated with suicidal behaviour and to identify opportunities for targeted suicide prevention. The specific objectives of each study component were as follows: • To describe trends and demographic risk factors in deaths from suicide as well as other conditions that may include suicide to identify populations at risk in South Africa (Study 1). • To investigate the association between environmental and occupational organophosphate pesticide (OP) exposure and attempted suicide in adults admitted to hospital in Cape Town, South Africa (Study 2). • To explore the genetic architecture underlying suicidal behaviour and psychiatric disorders to understand the genetic factors that increase the risk of suicidal behaviour (Study 3). • To describe healthcare utilisation 12 months before suicidal behaviour among individuals who attempted suicide and who died by suicide, to identify opportunities for prevention in Cape Town, South Africa (Study 4). Methods: This thesis included an ecological time-series study of national suicide mortality data from Statistics South Africa using joinpoint regression analysis (Study 1, N=10.3 million recorded deaths from 1997 to 2016; 8,573 deaths from suicide); a conditional logistic regression analysis of an attempted suicide hospital-based case-control study (Study 2, N=400; 200 cases and controls); a genome-wide genetic correlation study of suicidal ideation, self-harm, attempted and fatal suicide (samples [n] ranged from 62,648 to 125,844), and selected psychiatric disorders (n ranged from 9,954 to 386,533) using a genomic structural equation modelling approach (Study 3); and a retrospective cohort of linked electronic health records of individuals who attempted suicide and were admitted to hospital and a case series of fatal suicides on whom forensic autopsies were performed at a mortuary in Cape Town (Study 4, N=484). Results: The key findings in this study show that (i) suicide mortality rates were consistently higher in men than women between 1997 and 2016 (Study 1). Suicide rates increased by 7.7% among young people aged 15 to 29 years. Hanging, poisoning and firearms were the most frequent methods of suicide used. Subgroup analysis showed suicide by hanging and poisoning mortality rates increased by 2.9% and 3.7% across 20 years. Suicide deaths were underreported and may be included among deaths by accidental injuries and undetermined intent. However, these patterns varied by method of death (hanging, poisoning and firearm injury) over the study period. The largest proportion of suicide deaths may be potentially misclassified as accidental hanging and hanging by undetermined intent, and to a lesser extent, accidental poisoning and poisoning by undetermined intent. In contrast, firearm-related deaths were more likely to indicate a homicide than a suicide death. Missing data on select sociodemographic variables limited the accuracy and generalisability of our findings. (ii) Pesticide use in homes and gardens was common (85%); however, there was no association between attempted suicide and environmental (household, garden, and occupational) OP exposure (Study 2). Hazardous drinking and unemployment with no household income were significantly associated with an increased risk of attempted suicide while sharing the house with more than three persons was protective. (iii) We observed strong significant genetic correlations (rg) between suicidal ideation, attempted suicide, and self-harm (rg range, 0.71 to 1.09) and moderate-to-strong genetic correlations between suicidal behaviour traits and a range of psychiatric disorders (Study 3). The strongest genetic correlation was noted for major depressive disorder and suicidal ideation (Ever contemplated self-harm, rg=0.86±0.07, p=1.62x10-36). Multivariate genomic analysis revealed a single (common) factor structure for suicidal behaviour traits, major depressive disorder, attention-deficit hyperactivity disorder (ADHD), and alcohol use disorder. Approximately 2,951 genes and 98 sub-network hub genes were associated with the common factor, and shared biological pathways include involvement in developmental biology, signal transduction and RNA degradation. (iv) Approximately two-thirds of cases had at least one prior visit to a health care facility in the 12 months leading to suicidal behaviour (Study 4). The prevalence of psychiatric disorders was lower for individuals who died by suicide than attempted suicides but both groups interacted equally (approximately 65%) with the healthcare system during the 12 months leading to suicidal behaviour. Patients who used primary care services in the year before dying by suicide attended for the management of their chronic conditions (such as cardiovascular disease and diabetes) and emergency medical care for assault-related injuries. For attempted suicides, common reasons for a healthcare visit were for management of their chronic condition, HIV care, and a psychiatric diagnosis of depression, bipolar, or substance use disorders. Conclusions: This study expands on previous research and shows that while common risk factors are shared by individuals with fatal and non-fatal suicidal behaviour, the degree of risk varies across suicide groups, age, and sex. Findings of increased genetic risk of suicidal behaviour among individuals with psychiatric disorders suggest that identification and early treatment of co-morbid psychiatric disorders (major depression, alcohol use disorder, ADHD, and schizophrenia) should be included in suicide prevention strategies. Evidence of potential misclassification of suicide death within accidental injuries and undetermined intent categories may explain the underestimation of suicide mortality reported in this study. Combined with the high proportion of missing data in the national vital statistics and poor data quality of external causes of death, these findings suggest a critical need for ongoing training on the cause of death certification and further interventions to improve suicide data quality. Further, continued monitoring of suicide mortality data and linking electronic health records may provide opportunities for suicide surveillance that can help identify where prevention strategies should be allocated for maximum benefit, such as primary healthcare outpatient facilities, emergency treatment centres, and antiretroviral clinics.
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    Evaluating the use of capillary electrophoresis in the forensic DNA profiling of burnt teeth
    (2023) Geldenhuys, Adriaan; Mole, Calvin; Martin Donna-Lee
    Fires are a frequent cause of death, both globally and in South Africa, and often, individuals are burnt beyond the point of visual recognition. Teeth maintain their structure and can withstand high temperatures; making them a possible source of DNA from burnt human remains. DNA profiling is the current gold standard in forensic human identification, however, limited literature pertaining to DNA profiling of burnt teeth exists. The aim of this study was thus to evaluate the success of capillary electrophoresis in the forensic DNA profiling of teeth burnt at different temperatures, using an optimised DNA extraction protocol. Tooth samples from 25 donors (n = 100 [4 teeth per donor]) were subjected to three burning conditions, one tooth was left unburnt to act as a control and three teeth were each burnt in a muffle furnace at 100 ˚C, 200 ˚C, and 300 ˚C. The colour and weight of the teeth were recorded before and after burning. DNA was extracted using an optimised demineralisation step. Extracted DNA was quantified through real-time PCR and profiled using capillary electrophoresis with the Promega PowerPlex® ESX 16 system. Teeth burnt at 100 ˚C resulted in the most full profiles (96 % ; n = 24/25), followed by teeth burnt at 200 ˚C (84 %; n = 21/25), with 16 % partial profiles obtained (n = 4/25). Teeth burnt at 300 ˚C resulted in a large number of failed profiles (88 % ; n = 22/25), and had a significant decrease in profiling success (p = 0.001) and concentration (p = 0.001), and were significantly more degraded (p = 0.001), compared to control samples and samples burnt at lower temperatures. These results suggest that conventional DNA profiling methods and the DNA extraction method used herein are suitable for obtaining full DNA profiles from teeth exposed to temperatures as high as 200 ˚C, however, more sensitive methods such as targeted next generation sequencing (NGS) would be recommended to obtain more insight into highly degraded and fragmented samples, such as those burnt at 300 ˚C.
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    Factors affecting diagnostic and prognostic performance of a transcriptomic signature of risk of tuberculosis in HIV-uninfected South African adults
    (2022) Mulenga, Humphrey; Hatherill, Mark; Scriba, Thomas J
    Background Host blood transcriptomic signatures, such as RISK11, have potential as tests for diagnosing and predicting tuberculosis. This thesis aimed to review the literature, evaluate host and non-host factors associated with variability of the RISK11 signature and impact on discriminatory performance and evaluate RISK11 performance in combination with tests of Mycobacterium tuberculosis sensitization. Methods A systematic review of discriminatory performance of transcriptomic signatures for tuberculosis was conducted. RISK11, QuantiFERON-TB Gold-Plus and host factors were analysed in a prospective cohort, in which a cross-sectional study of upper respiratory organisms was nested. Effects on RISK11 were quantified using multivariable generalised regression. Discriminatory performance of RISK11, and RISK11/QuantiFERON combinations, were quantified by area under the curve and/or sensitivity and specificity. Results In the literature, one signature (90% sensitivity; 74% specificity) met the minimal criteria for a triage test; one signature (86% sensitivity; 84% specificity) met the minimal criteria for a predictive test. In the prospective cohort, RISK11 scores were higher among individuals with prevalent tuberculosis (+18.90%), night sweats (+14.65%) and incident tuberculosis (+7.29%). Cough was associated with 72.55% higher RISK11 score in prevalent tuberculosis cases. Stratification by cough improved diagnostic performance from area under curve of 0.74 overall, to 0.97 in cough-positive participants. Adjustment for host factors affecting controls did not change RISK11 discriminatory performance. In the cross-sectional study, RISK11 scores were higher by +16.7%, +67.8% and +13.5% in participants with coronavirus, influenza and rhinovirus, respectively, such that RISK11 could not differentiate prevalent tuberculosis from upper respiratory viruses. Compared to RISK11, the Either-Positive test combination decreased diagnostic negative likelihood ratio from 0.7 to 0.3, and prognostic negative likelihood ratio from 0.9 to 0.3, but did not improve upon QuantiFERON alone. Compared to QuantiFERON, the Both-Positive test combination increased diagnostic positive likelihood ratio from 1.3 to 4.7, and prognostic positive likelihood ratio from 1.4 to 2.8, but did not improve upon RISK11 alone. Conclusion RISK11 holds promise as a triage test for tuberculosis. Further optimisation, or development of new signatures is needed to improve discrimination of subclinical tuberculosis, without cough, and to mitigate the impact of viral co-infection. RISK11/QuantiFERON combination testing is not recommended.
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    Firearm fatalities examined at Salt River medico-legal laboratory in 1999 and their investigative outcome by 2004
    (2004) Liebenberg, Linda; Lourens, Denise
    The Republic of South Africa has OIle of the most liberal and human rights based constitutions wor1dWlde, with a very idealistic vision of individual freedom, alleviation of poverty access to education and health care and safety Many of the ideals are In direct redress of the previous regime, where only select strata of the populace had easy access to what are now constitutional rights for all.
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    Genomics of sickle cell disease and fetal hemoglobin in African populations
    (2024) Esoh, Kum Kevin; Wonkam, Ambroise
    Background More than 300,000 babies are born with sickle cell disease (SCD) each year. About 79% of these births occurs in sub-Saharan Africa where the sickle variant is known to have originated in the genetic background of the ancestors of Agriculturalist populations. Although the variant is highly lethal, the protection it confers against severe malaria in its heterozygous form has resulted in its persistence in sub-Saharan Africa where malaria is endemic. Without intervention, 50–90% of affected children in many sub-Saharan African countries die before their fifth birthday. The search for a definitive cure or effective disease-modifying therapy is therefore an imperative. Fetal hemoglobin (HbF) has long been recognized to ameliorate SCD severity whereby patients harboring natural genetic variations that lead to the persistence of high HbF levels in their blood tend to live longer with fewer complications. The HbF quantitative trait is highly heritable (89%), and is therefore the focus of enormous research for therapeutic purposes. Genetic polymorphisms influencing HbF level have been identified in three major loci; BCL11A, HBS1LMYB, and HBG2. However, these loci jointly explain less than 30% of HbF variability in African sickle cell anemia (SCA) patients as compared to ~50% in African Americans and non-anemic Europeans. Genome-wide association studies have been employed to replicate two of the three major loci (BCL11A and HBS1L-MYB) in African SCA patients, as well as to identify new loci including BACH2 involving a meta-analysis of African Americans and Tanzanians, SLC28A3, TICRR, and PIEZO2 in Nigerians, and FRMPD4 in Tanzanians. While BCL11A and HBS1L-MYB have been replicated in Cameroonian SCD patients through candidate genotyping studies, there has yet to be a genome-wide investigation. Moreover, the long survival of SCA patients in Africa despite a higher disease severity and mortality in the region hints at the enrichment of African genomes with ‘protective' polymorphisms potentially impacting the level of HbF, the strongest modifier of SCD severity. A better understanding of the genetic architecture of HbF in African SCA patients is therefore needed to foster research into potentially novel sickle cell disease therapeutic targets. Aims and Methods This thesis project aimed to: 1) present an in-depth description of the evolutionary history of the sickle cell mutation and its implication for global genetic medicine through a synthesis of publicly available data. This included updating and summarising the global georeferenced databases of the sickle gene and the HBB gene cluster haplotypes, systematically and critically evaluating the age and place of origin of the sickle cell mutation by incorporating information about malaria with which its evolution is highly intertwined, summarising bibliographic information on the genetic modifiers of SCD severity, and, importantly, examining other gene variants that co-occur with the sickle gene in sub-Saharan Africa that might hint at possible co-evolution and effect on SCD severity; 2) provide a comprehensive overview of SCD in Sub-Saharan Africa, bringing out transferable strategies and recommendations for prevention and care, through bibliographic searches; and 3) to investigate the missing heritability of HbF in SCA patients of African ancestry from Cameroon, Tanzanian, and the USA through a multiple imputation panel and genome-wide association approach, with fine-mapping and functional analysis. Briefly, the performance of different reference haplotype panels on genotype imputation accuracy for African SCA populations from Cameroon and Tanzania was assessed. Genome-wide association analyses for the two populations using all the imputation panels was performed, and then meta-analyses of the two populations, as well as with summary statistics from the USA-based cohorts. Statistical fine mapping and extensive in silico functional analyses were next performed to determine the functional relevance of significant associations, while extensive haplotype structure analysis was performed to illuminate the reason for substantial heterogeneity in association signals across different populations of the same ancestry. Finally, gene-based and gene set enrichment analyses were undertaken to identify additional significant loci and significantly enriched pathways, while heritability analysis was performed to further appreciate the observations of multiple significant signals and to better understand the genetic architecture of HbF in African SCA patients. Results Evolutionary history of sickle cell mutation In this first part, we successfully updated the global georeferenced databases of the sickle gene and the HBB gene cluster haplotypes. We showed that changes in population dynamics, contributed by migration and gene flow, might be introducing some HBB haplotypes in regions where they were previously absent, reflecting changes in regional SCD severity. Through our systematic and critical evaluation of the origin of the sickle cell mutation, we identified limitations to the models that have been used to estimate the age of the mutation, and we determined that the mutation is likely older than its currently held age of 22,000 years. Using data on the emergence of malaria, we determined that the mutation is most likely to have originated somewhere in West-Central Africa. Importantly, we showed overlapping distribution of the sickle gene and other gene variants that are under natural selection in sub-Saharan Africa. Data suggest that some of these gene variants impact SCD severity, while others have known modifying effects on the disease severity. Overview of sickle cell disease in sub-Saharan Africa We provided and overview of SCD in sub-Saharan Africa with transferable strategies for prevention and care as part of the Lancet Haematology's 2021 series on hematological care priorities in sub-Saharan Africa. We touched on aspects of SCD such as epidemiology, burden, mortality and life expectancy, hematological parameters, diagnosis, pathophysiology, biological and genetic modifiers of severity, management, environmental determinants, and psychosocial effects. We also presented challenges and proposed recommendations for SCD research in subSaharan Africa. Impact of reference haplotype panels on genotype imputation in African sickle cell anemia populations To use the genomics of HbF to search for other, perhaps more effective, targets of gene editing for better management and perhaps cure of SCD, we first assessed the impact of imputing missing genotypes into genome-wide single nucleotide polymorphism (SNP) data using different reference haplotype panels. We used six different panels including one which we created ourselves from whole genome sequencing data of fifty Cameroonians. The key observations included: i) different imputation performance for different African populations; ii) different imputation performance among different imputation panels within each African population, indicating that one variant can be imputed with vastly different accuracies across different panels, reflecting differences in haplotype structures across the panels resulting from different tagging schemes. This underscoresthe complementary use of the panels, with an expectation of different patterns of association (panel-specific significant signals). Genome-wide association analysis, statistical and functional fine mapping As expected from our assessment of imputation performance, we observed multiple panelspecific significant signals. We replicated the major known loci including BCL11A and HBS1L-MYB, and uncovered fourteen novel loci. The most significant of the novel loci, FLT1, which was observed in Cameroonians and replicated in a meta-analysis with Tanzanians, has known role in hematopoiesis. Fine-mapping and in silico functional analyses suggest an important role in HbF induction. Gene-based, gene set enrichment, and heritability analyses Gene-based analysis confirmed significant signals in the three major HbF-influencing loci, BCL11A, HBS1L-MYB, and HBG2. Gene set enrichment analysis revealed an overwhelming enrichment of hematopoiesis related pathways, as well as hemoglobin as the major enriched biological component, while blood traits were the most enriched phenotypes. Consistent with these, we estimated HbF heritability in a joint cohort of Cameroonian and Tanzanian SCA patients at 94% suggesting an enrichment of these populations with HbF-influencing loci in a way that has probably been underappreciated, and that might be better revealed through whole-genome sequencing. Conclusion and perspectives Our study presented data with overwhelmingly support for a single African origin of the sickle cell mutation, with opportunity for further research on determining the true age of the mutation. Tracking the movement of the mutation through the distribution of the HBB gene cluster haplotypes highlighted changing population dynamics that are important for public health. Calling attention to gene variants whose distribution in sub-Saharan Africa overlaps with the sickle gene is important because the co-occurrence could mean co-evolution which might suggest an impact on SCD severity. Therefore, further evolutionary genetic studies are warranted to understand the interactions of these gene variants and the sickle gene. We equally showed that progress in implementing newborn screening and comprehensive care in some sub-Saharan African countries has been encouraging. Early diagnosis and family education on management can reduce morbidity, while immunisation and hydroxyurea therapy initiated in affected children as young as 9 months old can greatly improve quality and quantity of life. Research in sub-Saharan Africa is urgently needed to establish the exact prevalence, mortality, and morbidity, environmental and genetic factors affecting clinical complications, and life expectancy of patients with sickle cell disease, to facilitate the design of future risk models and to investigate novel routes for therapeutic options with the ultimate aim of improving the clinical outcomes of patients with SCD in all parts of the world. Finally, our discovery of new genes that are associated with blood levels of HbF which is the strongest modifier of SCD severity and the target of gene editing means that our work has tremendous potential towards discovery of novel SCD therapeutic targets, but also in improving our understanding of the genetic architecture of HbF in understudied African populations.
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    HIV alters the expression of miRNA hsa-miR-200c-3p in B-cells, leading to enhanced migration of lymphoma cells
    (2018) Ramorola, Beatrice Relebogile; Mowla, Shaheen; Shires, Karen
    Background: The sub-Saharan African region is one that is affected most by the HIV/AIDS pandemic, with South Africa being the country with the highest number of infected individuals at 7.06 million. Infection with HIV is often associated with co-morbidities, including HIV-associated Non-Hodgkin’s Lymphomas (HIV-NHLs). Burkitt’s lymphoma (BL), a highly aggressive cancer, is one of the most common NHLs associated with HIV infection. Despite receiving highly active anti-retroviral therapy, the prognosis for this HIV-associated lymphoma remains poor and the incidence keeps on increasing in this group of patients. Recent studies have shown that microRNA (miRNA) dysregulation play essential roles in the pathogenesis of many cancers, including NHLs. While several human pathogenic viruses have been shown to deregulate cellular miRNAs, to date, no comprehensive studies have been carried out to determine whether HIV infection can lead to miRNA dysregulation in B-cells, which may contribute to the development of HIV-associated lymphomas. Objective: This research project aimed to validate the differential expression of selected miRNAs which were identified as potentially important in a PCR array, and characterise their roles in Burkitt’s lymphoma cells exposed to an attenuated strain of HIV-1, compared to control cells. Methods: Single-tube TaqMan miRNA assays were used to validate the previously observed differential expression of four selected miRNAs in Burkitt’s lymphoma cell lines (Ramos and BL41) exposed to HIV-1 compared to matched-microvesicle treated (control) cells. Following validation, the role of miRNA hsa-miR-200c-3p in the development of HIV-associated BL was investigated. This was done by using online bioinformatic prediction tools, as well as literature searches, to identify gene targets. Thereafter, the differential expression of a selected gene target was investigated by qPCR and western blotting. The functional significance of the observed changes in miRNA and gene expression was investigated by performing cell viability and migration assays. Results: Three upregulated (hsa-miR-575, hsa-miR-363-3p and hsa-miR-222-3p) and one downregulated (hsa-miR-200c-3p) miRNAs that were significantly deregulated by 2-fold or more (p< 0.05) in the PCR array were selected for validation. Thereafter, the miRNA hsa-miR200c-3p was selected for further analysis. Upon exposure to attenuated HIV-1, hsa-miR-200c3p was downregulated in the BL cell line Ramos, and this was reproducible in a second BL cell line BL41. The transcription factors ZEB1 and ZEB2, which are involved in cancer cell migration, were identified as targets of hsa-miR-200c-3p. Contrary to what is expected, the mRNA expression of both genes was found to be significantly downregulated in Ramos and BL41 exposed to attenuated HIV-1. At the protein level, in the Ramos cells, ZEB1 and ZEB2 matched what was observed for the mRNA. In contrast, both ZEB1 and ZEB2 protein were upregulated in BL41 cells under the same treatment conditions. At the functional level, the migration of both cell lines was enhanced when exposed to attenuated HIV-1, compared to control cells. Conclusions: The present study has demonstrated that HIV-1 has the ability to modulate cellular miRNA expression in Burkitt’s lymphoma cells. Of these miRNAs, hsa-miR-200c-3p is consistently downregulated when two BL cell lines were exposed to HIV. The ZEB transcription factors ZEB1 and ZEB2, which promote Epithelial-to-Mesenchymal Transition (EMT) through enhancing cellular migration, were investigated as hsa-miR-200c-3p targets. The mRNA levels of ZEB1 and ZEB2 were downregulated in both cell lines under the same experimental conditions. This is contrary to what is expected, since miRNAs lead to the attenuation of transcription or translation of their target genes and a downregulation of a miRNA should lead to an upregulation of its target. However, protein expression rather than mRNA expression has been described as a more accurate indication of target validation for miRNAs. The protein expression levels for ZEB1 and ZEB2 correlated with the mRNA expression results observed in the Ramos cells. In the BL41 cells, ZEB1 and ZEB2 protein levels were upregulated. Furthermore, in both cell lines, an increase in migratory ability was observed when cells were exposed to attenuated HIV-1. These results demonstrate that exposure to HIV enhances the cancer phenotype and that this is potentially due to changes in cellular miRNA expression brought about by the virus or viral components. Future studies should focus on gain-offunction and loss-of-function studies to determine whether the increase in cell migration is specifically due to a decrease in hsa-miR-200c-3p.
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    HIV-1 strain-specific neutralizing antibody responses and the dynamics of viral evolution
    (2016) Majara, Lerato Charlotte; Williamson, Carolyn; Anthony, Colin Scott
    It is widely held that for an HIV-1 vaccine to provide sterilizing immunity, it would need to elicit broadly neutralizing antibodies (bnAbs). However, factors underlying the development of these antibodies are not clear. There is evidence to suggest that in some individuals who develop bnAbs, the development of breadth is influenced by the co-evolution of the transmitted/founder (t/f) virus and earlier strain-specific neutralizing antibody (ssnAb) responses. Here we characterized the viral evolution, ssnAb and bnAbs responses in CAP292, an HIV-1 infected woman who developed bnAb responses from one year post infection. We used single genome amplification (SGA) to characterize viral evolution at four time points: at acute infection; after the development of strain-specific neutralizing responses; at the first detection of the broadly neutralizing antibody response; and lastly, at the peak of the broad response. We identified the t/f virus, and generated chimeric viruses from this to determine the targets of the ssnAb responses. A panel of site-directed mutant viruses were used to map the specificity of the bnAb responses. Our data indicated that infection was most likely founded by a single virus and that the first wave of ssnAbs emerged at 14 weeks post infection (w.p.i), targeting the V1V2 loop of Envelope (Env). A second wave of ssnAbs, possibly targeting the C3V4 region, emerged by 30 w.p.i. Two distinct viral clusters were detected by the time the bnAb response peaked, suggesting the presence of distinct escape pathways. Mapping of the bnAb specificities indicated that CAP292 produced PGT128-like bnAb responses targeted toward the 332 glycan.
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    Human Immunodeficiency Virus/Human Papillomavirus co-infection and host molecular genetics of cervical carcinoma
    (2019) Chambuso, Ramadhani Salum; Ramesar, Raj; Gray, Clive; Williamson, Anna-Lise
    A subgroup of women who are co-infected with human immunodeficiency virus type 1 (HIV1) and human papillomavirus (HPV) progress relatively rapidly to cervical disease regardless of the number of absolute CD4 count. During infection, viral peptides are recognized by the host immune system. It is reasonable to propose that the development of viral-associated cancers, like cervical cancer, requires interference with specific immune-response genes. This thesis investigates this proposition with consideration of host molecular genetic alterations and variations of the human leukocyte antigen class II (HLA II) genes as one of the groups of immune-response genes that are involved in directing CD4 T-cell responses during infection, in the instance of cervical cancer progression in HIV-1/HPV co-infected women. Study I, reviewed the available literature on host molecular genetics and HIV-1/HPV coinfection on cervical cancer progression. This study suggests that the dual pro-oncogenic effects of HPV oncoproteins E6/E7 and the HIV-1 oncoprotein Tat, may exacerbate and accelerate the rate of cervical disease progression in a subgroup of HIV-1-positive women. Additionally, HIV-1-positive cervical cancer has three important carcinogenesis steps: firstly, HPV integration into the host genome, secondly, dual pro-oncogenic effects of HPV oncoproteins E6/E7, and the HIV-1 Tat oncoprotein in the host genome and, thirdly, the accumulation of repeated, unrepaired genetic mutations and genetic alterations within the host chromosomal DNA. Genetic variations or mutations that affect the following host gene categories were suggested to be responsible for cervical cancer susceptibility and disease progression; (i) genes for the immune-response against oncogenic HPV infection, (ii) oncogenes, (iii) tumour-suppressor genes, (iv) apoptosis-related genes, (v) DNA damagerepair genes, and (vi) cell cycle-regulatory genes. However, studies II, III and IV are linked together and listed according to the specific objectives of this thesis. Study II, characterized the distribution of HPV genotypes within cervical tumour biopsies from a cohort of 181 HPV-unvaccinated South African women and studied the relationships with HIV-1 infection, age of patients, absolute CD4 count, CD4 percentage and the stage of cervical disease, and identified the predictive power of these variables for cervical disease stage. Distribution of HPV genotypes was related to the stage of cervical disease in HIV-1-positive women. Older age was a significant predictor for invasive cervical cancer (ICC) in both HIV-1-seronegative (p<0.0001) ) and HIV1-positive women (p=0.0003, q=0.0003). Sixty-eight percent (59/87) of HIV-1-positive women with different stages of cervical disease presented with CD4 percentage below or equal to 28 and a median absolute CD4 count of 400 cells/µl (IQR 300-500 cells/µl). Of the HIV-1-positive women, 75% (30/40) with ICC, possessed ≤28% CD4 cells versus 25% (10/40) who possessed >28% CD4 cells (both p< 0.001, q<0.001). Furthermore, 70% (28/40) of women with ICC possessed absolute CD4 count >350 cells/µl compared to 30% (12/40) who possessed absolute CD4 count ≤ 350 cells/µl (both p< 0.001, q< 0.001). Study III, was the first case-control study to investigate the association of HIV-1/HPV coinfection with specific host HLA II-DRB1 and -DQB1 alleles in cervical cancer. Two hundred and fifty-six (256) women of the same ethnicity were recruited, comprising 56 cases and 200 age-matched controls. A total of 624 HLA-DRB1 and -DQB1 class II genotypes were studied. HLA II-DQB1*03:01 and -DQB1*06:02 alleles were associated with cervical cancer in HIV-1/HPV co-infected women (p=0.001 and p< 0.0001, respectively) while HLA II-DRB1*13:01 and -DQB1*03:19 were rare or absent in women with cervical disease when compared to the control population (p=0.012 and 0.011, respectively). Study IV, aimed to investigate the host genetic alterations that may be involved in rapid tumour progression in HIV-1/HPV co-infected women. The frequency of loss of heterozygosity (LOH) and microsatellite instability (MSI) at the HLA II locus on chromosome 6p was analysed in cervical tumour biopsy DNA, with regard to HIV-1/HPV co-infection in 164 women. Seventy-four women were HIV-1-positive and ninety women were HIV-1-seronegative. Tumour DNA from HIV-1/HPV co-infected women demonstrated a higher frequency of LOH/MSI at the HLA II locus at 6p21.21 than tumour DNA from HIV1-seronegative women (D6S2447, 74.2% versus 42.6%; p=0.001, q=0.003), D6S2881 at 6p21.31 (78.3% versus 42.9%; p=0.002, q=0.004), D6S1666 at 6p21.32 (79% versus 57.1%; p=0.035, q=0.052), and D6S2746, at 6p21.33 (64.3% versus 29.4%; p< 0.001, q< 0.001), respectively. This thesis provides novel insights and adds to the existing knowledge on the relationships between HIV-1/HPV co-infection, CD4 immune status, host HLA II allele variations and genetic alterations at chromosome 6p in association or likely protection to cervical disease in the studied cohort of South African women. Identification of host molecular genetic susceptibility to disease with regard to viral infection is important for individualized molecular targeted prevention of cervical cancer.
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    Improving point-of-care diagnosis of tuberculosis: development and evaluation of novel technologies
    (2017) Moodley, Vineshree Mischka; Nicol, Mark P; Dorman, Susan E
    With an estimated third of all tuberculosis (TB) cases being missed, the need to develop rapid, simple and accurate diagnostic tests is critical. The last five years has seen an unprecedented activity in the development of a range of new tests. However, a major concern is that not all marketed TB tests have been assessed rigorously, particularly in terms of diagnostic accuracy, robustness under operational conditions in the field, and practical usefulness. This dissertation comprises a compilation of diagnostic clinical studies of novel point-of-care tests, namely a chemiresistive "TB breath-analyser"; a lipoarabinomannan (LAM) urine dipstick, and an adaptation of the Xpert®MTB/RIF assay for use on blood. Lastly, there is a modification of the sputum collection device (SCD) to enable specimen processing without the requirement of a biosafety cabinet. The chemiresistive sensor, which detects volatile organic compounds released by Mycobacterium tuberculosis in a patient's breath, demonstrated a high sensitivity (100%) and specificity (92%) for distinguishing patients with active TB from healthy controls. However, sensitivity (74%) and specificity (63%) were lower when the culture-negative participant group was compared to the culture-positive participants. The test shows potential as a useful screening test for TB with further refinement of the sensor technology. The LAM dipstick was shown to be useful in hospitalised HIV-infected patients with CD4 T-cell counts <200 cells/μL reinforcing the data from other studies. Although the blood Xpert®MTB/RIF assay showed some utility in diagnosis of TB in hospitalised patients with very advanced HIV, given the poor sensitivity and specificity, and the requirement for specialised equipment as well as a large volume of blood for testing, it is unlikely that Xpert®MTB/RIF testing on blood will contribute much over other existing diagnostics in resource-limited settings. Finally, the redesigned SCD offers a solution to biosafety concerns with minimal impact on patient acceptability and clinical care.
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    Investigating the current scope and potential of forensic entomology in decomposition cases at Salt River Mortuary, Cape Town, South Africa
    (2023) Laubscher, Tyrian; Mole, Calvin
    Decomposition cases form a fraction of the medico-legal cases conducted at Salt River Mortuary (SRM). With prolonged time since death and increased decomposition, entomological evidence becomes increasingly important in the estimation of minimum post-mortem interval. Currently no standard protocol exists for the handling of entomological evidence by SRM personnel and there is a lack of information about the issues that may impact the handling of these death scenes and associated entomological evidence. Therefore, this study aimed to investigate the current scope of cases involving entomological evidence at SRM. This was achieved by performing a six-year retrospective review of medico-legal cases performed at SRM from 2015 to 2020, and interviewing SRM personnel to gather data regarding attended scenes, methods and processes used on the scenes, and issues they may have faced. A total of 264 decomposition cases were examined at SRM in the six-year period, with 109 (41.3%) presenting with insect activity. Data about variables such as scene type, weather season, decomposition stage, burial or covering of remains, and open wounds were extracted from the case files. As expected, a greater proportion of cases presented with entomological evidence in the warmer summer and spring seasons compared to the cooler seasons, with no significant difference in the distribution between years (p=0.62). Insect activity was predominantly found in indoor cases, but this is not statistically significant (p=0.50). Most cases presenting with entomology activity were associated with early-stage decomposition. No association was observed between the presence of open wounds and insect activity. The interviews provided data that could not be extracted in the reviews, due to personal experience being provided by personnel. The primary themes emerging from the interviews were related to the insufficient training on the handling of entomological evidence, poor availability of resources for the handling of the entomological evidence, and scene dependent variables that differ between scenes and impacts how a scene is handled. This study identified areas that need improvement and provides a better understanding of entomological activity associated with decomposition cases. There is potential for the greater utility of forensic entomologists in medico-legal cases, and the implementation of a standardised entomological protocol along with proper training of personnel may improve medico-legal investigations of decomposition cases.