Doctoral Degrees (Molecular Biology and Human Genetics)

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2020-12-11 Global transcriptomic investigation of the human macrophage response towards pathogenic/non-pathogenic mycobacteria
(Stellenbosch : Stellenbosch University, 2019-12) Mishra, Abhilasha Madhvi; Baker, Bienyameen; Leisching, Gina; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
Background:Tuberculosis (TB) is a major cause of infection-related mortalityworldwide. In 2017 an estimated 1.3 million people who were HIV-negative died of TB. An estimated 5-10% of infected individual develop active TB during their lifetime, while the remaining90% (of infected population) successfully control the bacteria. Also, some of the close household contacts of TB patients remain uninfected and healthy. Studying host immune response towards Mycobacterium tuberculosis(M. tb) can unfold the reason behind this enigma. Methods:We conducted a detailed investigation of in vitrohost response from human monocyte derived macrophages(hMDMs)towards different strains of mycobacteria(grown in detergent-freemedia), i.e. pathogenic (M. tbR179) andnon-pathogenic (M. smegmatisand M. bovisBCG). The host response was measured post-infection (at mRNA and protein levels) using AmpliSeq, quantitative real time polymerase chain reaction (qRT-PCR), multiplex ELISA (Luminex), intracellular mycobacterial survivaland cytotoxicity assay. Biological network analysis (ingenuity pathway analysis IPA) was performed to understand the gene regulatory networkinvolved in the pathophysiology associated with the host-immune system.Based on false discovery rate (FDR) and biological functions, we selected an inter-related gene family of interferon induced protein with tetratricopeptides (IFIT1, IFIT2 andIFIT3) from the list of 19 potential differentially expressed genes(DEGs)for knock-up (vector-based over-expression)/down experiments. This gene family is known to form a protein complex during viral infection to act against the antigen. Studyencompassing their role against bacteria is not well established.Therefore, we performed knocking-up of IFITsvia vector-based transfection and knocking-down via small interferingRNA (siRNA) approach to investigate their effect upon mycobacteria inside the host macrophages. Results:AmpliSeqanalysis found 19 DEGs at 12 hours post-infection across all three strains. We observed lower number of mycobacterial CFUs and higher host response (at both RNA and protein level) in hMDMs infected with M. smegmatisas compared to other two strains. Biological network analysis revealed interferon-interleukin associated signalling pathways as most prominent among the 19 differentially expressed genes.We found a differed host response towardsall three strains, which mayattributeto their pathogenicity. Messenger RNA and protein level comparisons at different time points, depicted strong role of interferon and interleukin associated gene network. This network was able to successfully counter M. smegmatisbut succumb to M. bovisBCG andM. tbR179. Most importantly, across all three strains, intra-cellular bacterial growth and survival measured through colony forming units (CFUs)decreased significantly upon knocking up of IFITs(IFIT1, IFIT2 andIFIT3),while we recordedan increase in CFUs upon knocking down ofIFITsin the host macrophages. Using multiplex ELISA, we found higher expression of key pro-inflammatory cytokines (i.e. IDO1, IFN-γ, IL-6, and IL-23) during knock-up (vector-based over-expression)of IFITsresulting in reduction of mycobacteria. Conclusion:Differentially expressed IFITs showed a strong effect against mycobacteria, which can be used as a promising therapeutic targetadjunct to anti-TB therapy. This knowledge will broaden the scope of host drug targets for resistance free bacteriostatic immuno-therapy.
Accuracy and impact of the MTBDRplus v2 and MTBDRsl v2 line probe assays for the detection of first-line and second-line drug resistant tuberculosis
(2023-02) Pillay, Samantha; Theron, Grant; de Vos, Margaretha; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Combating drug-resistant tuberculosis (DR-TB) remains a challenge globally. Treatment success rates are often derailed by under-diagnosis and under-reporting of disease. Patients remain contagious for prolonged periods prior to initiation of appropriate treatment which is further exacerbated by the amplification of drug resistance and poor treatment outcomes. Using current and new diagnostic tools effectively is key to rapid diagnosis of tuberculosis and early detection of drug resistance. Firstly, (chapter 2) line probe assays (LPAs) frequent inability to generate a resistance call in paucibacillary specimens is problematic. We showed that while MTBDRplus and MTBDRsl tests work well on smear-negative specimens for detecting drug resistance, failure rates remained high. We demonstrated with the use of routine key programmatic data how time-to-reporting of results improved with the use of molecular assays and provided evidence on how standard-of-care can be improved in a programmatic context. Secondly, (chapter 3) LPA testing on smear-negative specimens is not always performed causing diagnostic delays and hindering their role as a direct front-line diagnostic tests. Thus, by using Xpertgenerated data we determined the ratio of actionable-to-non-actionable results and the number of missed resistant cases at varying thresholds. We demonstrated that Xpert semiquantitation category is superior to informing reflex LPA testing than smear status. In short, this method provides a framework by which laboratories that currently do not test smear-negative specimens to expand testing. Thirdly (chapter 4) current pathways using Xpert MTB/RIF or Xpert Ultra as frontline tests for diagnosing TB and rifampicin resistance lack further treatment guidance. We did a systematic review and assessed the performance of Xpert MTB/XDR for the detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin. Participants consisted of 1228 for pulmonary tuberculosis detection and 1141 for drug resistance. We found Xpert MTB/XDR is unlikely to test positive as a follow-up test for the detection of Mycobacterium tuberculosis in samples that test Xpert Ultra
Analysis and application of evolutionary markers in the epidemiology of Mycobacterium tuberculosis
(Stellenbosch : Stellenbosch University, 2008-12) Van der Spuy, Gian Dreyer; Warren, R. M.; Van Helden, P. D.; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
This series of studies includes both methodological analyses, aimed at furthering our understanding of, and improving the tools used in molecular epidemiology, and investigative projects which have used these tools to add to our knowledge of the M. tuberculosis epidemic. Using serial isolates from tuberculosis patients, we have investigated the evolutionary rate of the IS6110 RFLP pattern. In accordance with other studies, we determined a ½-life for this epidemiological marker of 10.69 years, confirming its appropriateness for this purpose. We also identified an initial, much higher apparent rate which we proposed was the result of pre-diagnostic evolution. In support of this, our investigations in the context of household transmission of M. tuberculosis revealed that IS6110-based evolution is closely associated with transmission of the organism, resulting in a strain population rate of change of 2.9% per annum. To accommodate evolution within estimates of transmission, we proposed that calculations incorporate the concept of Nearest Genetic Distance (cases most similar in RFLP pattern and most closely associated in time). We used this to create transmission chains which allowed for limited evolution of the IS6110 marker. As a result, in our study community, the estimated level of disease attributable to ongoing transmission was increased to between 73 and 88% depending on the Genetic Distance allowed. We identified the duration of a study as a further source of under-estimation of transmission. This results from the artefactual abridgement of transmission chains caused by the loss of cases at the temporal boundaries of a study. Using both real and simulated data, we showed that viewing a 12-year study through shorter window periods dramatically lowered estimates of transmission. This effect was negatively correlated with the size of a cluster. Various combinations of MIRU-VNTR loci have been proposed as an alternative epidemiological marker. Our investigations showed that, while this method yielded estimates of transmission similar to those of IS6110, there was discordance between the two markers in the epidemiological linking of cases as a result of their independent evolution. Attempting to compensate for this by allowing for evolution during transmission improved the performance of IS6110, but generally had a deleterious effect of that of MIRU-VNTR. However, this marker remains a valuable tool for higher phylogenetic analysis and we used it to demonstrate a correlation between sublineages of the Beijing clade and the regions in which they are found. We proposed that, either the host population had selected for a particular sublineage, or that specific sublineages had adapted to be more successful in particular human populations. We further explored the dynamics of the epidemic over a 12-year period in terms of the five predominant M. tuberculosis clades. We found that, while four of these clades remained relatively stable, the incidence of cases from the Beijing clade increased exponentially. This growth was attributed to drug-sensitive cases although drug-resistant Beijing cases also appeared to be more successful than their non-Beijing counterparts. Possible factors contributing to this clade’s success were a greater proportion of positive sputum smears and a lower rate of successful treatment.
Analysis of copy number variation and disease mechanisms underlying Parkinson’s disease
(Stellenbosch : Stellenbosch University, 2016-03) Van der Merwe, Celia; Bardien, Soraya; Loos, Ben; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics
ENGLISH ABSTRACT : Parkinson’s disease (PD) is a neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental and genetic factors. To date, a number of PD-causing genes have been found. The PINK1 gene is of particular interest for this study, and mutations in this gene result in autosomal recessive inheritance of early onset PD. PINK1 plays a vital role in mitochondrial quality control and homeostasis, and in its absence it is thought to result in an accumulation of dysfunctional mitochondria in neurons, culminating in neuronal cell death. Whilst pharmacological and surgical interventions are available for PD, the current options exhibit adverse side effects with long term treatment. There is a great need to develop new treatments with i. less side effects and ii. that can simultaneously target the multiple pathways associated with this disorder. One molecule is curcumin, the core component of the curry spice turmeric, which is well known for its antioxidant and anti-inflammatory properties and has already been studied for its possible neuroprotective role in Alzheimer’s disease. The aim of the present study was to create a cellular model of PD by decreasing the expression of PINK1 in SH-SY5Y neuroblastoma cells. Thereafter, we aimed to test the protective effects of curcumin on this model in the presence and absence of a known stressor, paraquat. This study also aimed to detect possible copy number variation (CNV) in PINK1 (and other PD-causing genes) in a cohort of South African patients with PD, in order to obtain patient-derived fibroblasts to verify the results obtained from the original cellular model. PINK1 was knocked down using siRNA (Qiagen, USA) in SH-SY5Y neuroblastoma cells, and the knock down was verified by quantitative real time PCR (qRTPCR) and western blotting. Thereafter, PINK1 siRNA cells and control cells were separated into four treatment groups: i. untreated, ii. treated with 25μM paraquat for 24hours, iii. pre-treated with 2μM curcumin for 1hour then treated with 25μM paraquat for 24hours, or iv. treated with 2μM curcumin for 1hour, and various parameters of cellular and mitochondrial function were measured. Cell viability was measured by an MTT assay. Western blot analysis was performed using cleaved PARP and full-length caspase 3 markers to detect levels of apoptosis, and LC3-II and p62 markers to detect autophagic flux. Mitochondrial respiration experiments were completed on the Seahorse XF Analyser using the Mito Stress Test Kit and the Glycolysis Stress Test. Flow cytometry was utilised to measure mitochondrial membrane potential (MMP) using the JC- 1 fluorochrome, and mitochondrial network was analysed by fluorescent microscopy. For CNV detection, MLPA was performed on 210 South African PD patients and putative mutations were verified by qRTPCR on the Lightcycler 96. PINK1 was successfully knocked down at a gene and protein expression level. The PINK1 siRNA cells exhibited a significant decrease in cell viability (p=0.0036), and an increase in apoptosis (p=0.0144). A decrease in PINK1 expression also resulted in significantly decreased MMP (p=0.0008), mitochondrial respiration (p=0.0015), ATP production (p=0.002) and glycolytic capacity (p=0.0445). No significant changes were observed in the connectivity of the mitochondrial network, but autophagic flux was significantly increased in the PINK1 siRNA cells, as detected by increased LC3-II levels (p=0.0152). As expected, paraquat-treated cells exhibited decreased cell viability, increased apoptosis, decreased MMP, autophagic flux, and a more fragmented mitochondrial network. Paraquat treatment therefore successfully acted as a stressor on the cells. Curcumin pre-treatment followed by paraquat treatment rescued cell viability in control cells (p=0.003), and significantly decreased apoptosis in PINK1 siRNA cells (p=0.0018). Curcumin protected mitochondrial dysfunction in PINK1 siRNA cells by increasing MMP (p=0.0472) and maximal respiration (p=0.0014), as well as significantly increasing MMP (p=0.0307) and maximal respiration (p=0.032) in control cells. Additionally, curcumin treatment resulted in increased autophagic flux (p=0.0017) in stressed control cells. These results highlight a protective effect of curcumin against paraquat and against the damaging effects on the mitochondria in cells with decreased PINK1 expression. Lastly, MLPA analysis did not reveal any PINK1 CNV mutations in a total of 210 South African PD patients, and fibroblasts were therefore not obtained. A number of false positive mutations were identified that were not verified by qRTPCR. A common polymorphism M192L resulting in a false positive PARK2 exon 5 deletion was found in a number of patients, all of whom were of Black or Mixed Ancestry ethnic groups. One patient was shown to harbour a heterozygous deletion in PARK2 exon 4. In conclusion, PINK1 siRNA-mediated knock down in SH-SY5Y neuroblastoma cells can be used as a model of PD to study aspects of mitochondrial dysfunction. Furthermore, curcumin should be considered as a possible therapeutic target for PD, as it exhibits protective effects against paraquat at a mitochondrial level. Given the low toxicity of curcumin, and the fact that it is already part of a dietary regimen in most populations worldwide, further studies on elucidating its biochemical and cellular properties are therefore warranted. The use of natural compounds such as curcumin as therapeutic agents is currently a topical and fast-growing area of research, and holds much promise for clinical application in various diseases including neurodegenerative disorders such as Alzheimer’s disease and PD.
Analysis of host determining factors in susceptibility to tuberculosis in the South African coloured population
(Stellenbosch : University of Stellenbosch, 2009-12) De Wit, Erika; Hoal, Eileen; Van Helden, Paul; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: The infectious disease tuberculosis (TB) still represents a global threat due to its devastating effect on health and the subsequent high mortality rate. Previous studies have indicated that host genetic factors are implicated in host susceptibility to TB. Since TB is a complex disease, it can be assumed that susceptibility to M. tuberculosis has multiple genetic causative factors (as well as environmental causes). The current study focussed on a number of South African Coloured (SAC) individuals, some of whom were TB cases and others controls. Population substructure was tested in the admixed SAC population as it can be a strong confounding factor for association studies. Our results using the programme STRUCTURE indicated no population substructure in the SAC population. We further investigated the population structure of the SAC group using Affymetrix 500k SNP chip data which showed that the SAC population group has 4 major ancestral components: the Khoesan, European, African and Asian (Indian). A number of candidate polymorphisms in eight genes, previously indicated to play an important role in TB susceptibility, were tested in case-control associations studies. We found statistically significant associations between IFNGR1, IL-8, IL-1Ra and NRAMP1 polymorphisms and TB susceptibility in the SAC population. It has become increasingly evident that gene-gene interactions play a far more important part in an individual’s susceptibility to a complex disease than single polymorphisms would on their own. The importance of epistasis was clearly identifiable in this study with only four associations found between the individual variants and TB susceptibility, but eight instances of statistically significant gene-gene interactions. A combined data set consisting of 106 variants constructed from our database and also used for gene-gene interaction analysis yielded numerous statistically significant interactions. The interaction between the genotype of the human host and the bacterial strain genotype was also investigated and yielded interesting results. Owing to various polymorphisms in several cytokine genes, the protein levels of the main modulators of the immune system, cytokines and chemokines, are changed in several diseases such as infectious diseases and may affect susceptibility or resistance to TB. The functional polymorphisms or haplotype patterns in some of these cytokine genes might be vital for protective immune responses and may serve as biomarkers of protection or susceptibility to TB. The present study investigated 18 cytokines including pro-inflammatory, anti-inflammatory and chemokine factors in healthy (mantoux positive or negative) children using the Linco-plex immunoassay, and investigated potential interactions. The basic research will one day contribute to personalised genetics which may benefit infectious diseases such as TB. If individuals can be identified as potentially more vulnerable, they may require different vaccination strategies, a higher index of suspicion if exposed to TB, and prophylactic treatment.
Antimycobacterial activity of ascidian fungal symbionts
(Stellenbosch : Stellenbosch University, 2022-12) Tapfuma, Kudzanai Ian; Mavumengwana, Vuyo; Malgas-Enus, Rehana; Loxton, Andre Gerhard; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Tuberculosis (TB) is an infectious disease which primarily affects the lungs. Treatment of TB is complicated because the causative agent, Mycobacterium tuberculosis, is an intracellular pathogen which infects and kills cells of the innate immune system, while exhibiting intrinsic and extrinsic resistance to many of the currently available antimicrobial agents. A sizeable percentage of TB patients in the world-population are infected by M. tuberculosis strains which are resistant to currently utilized first- and second-line anti-TB drugs. Drug-discovery studies bioprospecting for compounds with novel anti-TB activities are therefore essential in order to control the spread of TB and to prevent a catastrophic pandemic. In this study, extracts from marine fungi were considered for antimycobacterial activity bioprospecting as they are largely underexplored. A total of 46 cultivable fungi were isolated from ascidians and 32 of these fungal isolates were sequenced and consequently identified. Among these fungi, the methanol crude extract from Clonostachys rogersoniana MGK33 was found to possess the highest antimycobacterial activity with minimum inhibitory concentrations of 0.125 and 0.200 µg/mL against Mycobacterium smegmatis mc2 155 and M. tuberculosis H37Rv, respectively. Untargeted metabolite profiling of the crude extract from C. rogersoniana MGK33 revealed the presence of bionectin F (among other compounds) which has previously been shown to possess antimicrobial activity in other studies. In silico molecular docking and simulation experiments in this study showed that bionectin F is a potential inhibitor of M. tuberculosis β-ketoacyl-ACP reductase (MabA). An attempt was then made to generate novel agents that would be composed of nanoparticles surface functionalized with the bioactive fungal extract from C. rogersoniana MGK33. In particular, mono-metallic SPIONs were synthesized using the co-precipitation method and then surface modified to produce bi-metallic superparamagnetic iron oxide nanoparticles (SPIONs) using nickel, zinc, gold, copper and silver, to produce Ni-SPIONs, Zn-SPIONs, Au-SPIONs, Cu-SPIONs and Ag-SPIONs. Functionalization was then performed using the MGK33 extract to produce Ni-SPIONs@MGK33, Zn-SPIONs@MGK33, Au-SPIONs@MGK33, Cu SPIONs@MGK33 and Ag-SPIONs@MGK33. Among these agents, Cu-SPIONs and Ag SPIONs were found to exhibit the strongest antimycobacterial activity, comparatively stronger than that of the counterparts, Cu-SPIONs@MGK33 and Ag-SPIONs@MGK33. In an experiment involving the treatment of RAW 264.7 macrophage cells infected with M. smegmatis mc2 155, the MGK33 extract exhibited the highest early apoptosis activity (9.61%), followed by Cu-SPIONs@MGK33 (3.34%), both agents tested at 1.96 µg/mL for 24 hours. The MGK33 extract further showed strong antimycobacterial activity against intracellular M. smegmatis mc2 155, compared with the nanoparticles synthesized in this study. Results in this study led to the conclusion that the marine fungus, C. rogersoniana MGK33 is a prolific source of compounds with antimycobacterial and immunomodulatory activity, and that further studies should be done to develop Cu-SPIONs and Ag-SPIONs into lead agents for anti-TB drug development.
The application of immunological biomarkers and enhanced pathogen detection for the epidemiological characterisation of bovine tuberculosis in African Rhinoceros
(Stellenbosch : Stellenbosch University, 2024-01) Dwyer-Leonard, Rebecca Ann; Miller, Michele Ann; Goosen, Wynand Johan; Witte, Carmel; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: African rhinoceros, specifically the black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros, are iconic species that are under threat due to poaching for their horns, range/habitat loss, unbalanced genetic/demographic structure, climate change, and infectious diseases, including tuberculosis (TB). Mycobacterium bovis (M. bovis) infection, a cause of TB, has been identified in African rhinoceros populations in Kruger National Park (KNP), South Africa. An interferon-gamma release assay (IGRA) is routinely applied for testing of individuals earmarked for translocation out of the park, and for general surveillance purposes. However, relatively little is understood about the overall susceptibility and pathogenesis of TB in these species, and its impact on affected populations. This study had four broad aims: i.) to collate information on the epidemiology of Mycobacterium tuberculosis complex (MTBC) infections in African rhinoceros, ii.) to determine prevalence and risk factors for M. bovis infection in KNP rhinoceros, iii.) to assess the impact of refrigeration and delayed stimulation of rhinoceros whole blood on mitogen stimulated interferon-gamma (IFN-γ) production, to increase flexibility in implementation of testing, and iv.) to determine whether MTBC can be detected in nasal swabs from rhinoceros with immunological evidence of infection, as an indication of potential infectiousness. Drawing from existing literature on MTBC infections in other species and contexts, a foundational understanding of TB epidemiology in rhinoceros species was developed. In other species, demographic risk factors include sex and age, with males and adults generally being at higher risk than females and younger individuals. Review of limited historical information reflected similar age- and sex-associated patterns for TB in captive African rhinoceros, with more reports of TB disease in black rhinoceros than white rhinoceros. Intra-species transmission of MTBC in rhinoceros was also considered to be a potential source of infection. Free-ranging rhinoceros in bovine TB (bTB) endemic areas may be exposed to MTBC, likely shed by maintenance hosts in KNP such as African buffaloes (Syncerus caffer), greater kudus (Tragelaphus strepsiceros), or warthogs (Phacochoerus africanus), through shared environmental niches, and resources. Based on previous reports, hypotheses were generated then investigated in a population-based study of M. bovis infection in 437 African rhinoceros in KNP. We determined an estimated overall infection prevalence of 15.4% (95% CI: 10.4-21.0%) based on mycobacterial culture and IGRA results for animals sampled between 2016-2020. Notably, a significant spatial clustering of cases was detected near the southwestern park border, although infection was widely distributed. Multivariable logistic regression models, including demographic and spatiotemporal variables, showed a significant, increasing probability of M. bovis infection in white rhinoceros based on increased numbers of African buffalo herds in the vicinity of the rhinoceros sampling location. Spillover of infection from African buffaloes to white rhinoceros sharing the environment was suspected. There was also a significantly higher proportion of M. bovis infection in black rhinoceros in the early years of the study (2016-2018) than in 2019 and 2020, which coincided with periods of intense drought, although other temporal factors could be implicated. Species of rhinoceros, age, and sex were not identified as risk factors for M. bovis infection. Ante-mortem surveillance for M. bovis infection in the Kruger National Park (KNP) rhinoceros population currently relies on results from (QFT)-Mabtech equine interferon-gamma (IFN-γ) release assay (IGRA). However, the requirement for same-day processing of rhinoceros blood samples for the IGRA is a logistical challenge to performing this test, particularly in remote locations. A pilot study showed that relative concentrations of IFN-γ (based on optical density values) in mitogen stimulated whole blood plasma decreased significantly with increased time blood was stored post-collection and prior to QFT stimulation. These findings support a need for same-day processing of rhinoceros blood samples for QFT-IGRA testing, as per the current practice to ensure optimal test performance. It was previously unknown whether M. bovis-infected rhinoceros could shed mycobacteria in respiratory secretions. Previous studies suggested that subclinically M. bovis-infected rhinoceros may pose minimal transmission risk. However, recent advances that have improved detection of MTBC members in paucibacillary samples prompted further investigation of respiratory secretions from rhinoceros with immunological evidence of infection, to elucidate the potential for mycobacterial shedding. A pilot study detected M. bovis in 14/64 (22%; 95% CI: 13-33%) of the IGRA positive rhinoceros, and none in the IGRA negative rhinoceros (n = 11) studied, suggesting that M. bovis-infected rhinoceros may be a source of infection for other susceptible animals sharing the environment. Overall, these studies address important knowledge gaps related to surveillance and epidemiology of TB in African rhinoceros, specifically, the free-ranging populations in KNP. This has created awareness of the potential threat of this pathogen to the conservation of these species and highlighted important areas for future research that will contribute to understanding the multi-host TB ecosystem in KNP and other complex systems.
Application of spoligotyping in the understanding of the dynamics of Mycobacterium tuberculosis strains in high incidence communities
(University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences., 2007-03) Streicher, Elizabeth Maria; Victor, T. C.; Warren, R. M.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
Tuberculosis (TB) is a global health problem and demands rigorous control management efforts. A dramatic increase in the acquisition and spread of drug resistant TB globally has been observed in recent years. A grim picture has emerged for the control program with the discovery of extreme drug-resistant TB, which is virtually untreatable and is of immense concern for the future of TB control. In the last decade strain-specific genetic markers have been identified to examine the molecular epidemiology and spread of TB, including IS6110 DNA-fingerprinting and spoligotyping. Although spoligotyping has less discriminatory power than the gold standard, IS6110 DNA-fingerprinting, it is simpler, faster and less expensive, as it is PCR-based. Spoligotyping has been applied to enhance our understanding of the dynamics of drug susceptible and drug resistant strains of Mycobacterium tuberculosis in high incidence communities, by studying 3 aspects of the TB epidemic: molecular epidemiology of drug resistant TB, recurrent TB and the evolution of M. tuberculosis. By using spoligotyping and other genotypic and phenotypic analysis of drug-resistant M. tuberculosis isolates from the Western Cape Province of South Africa showed that drug resistance is widespread and recently transmitted. An emerging drug resistant M. tuberculosis outbreak has been identified, termed DRF150, which has specific genotypic characteristics and is resistant to 5 first-line drugs in 45% of the cases. Inappropriate chemotherapy; poor adherence to treatment and prolonged periods of infectiousness due to the delay in susceptibility testing has led to the development and spread of this drug resistant genotype. The study demonstrates the ability of the spoligotyping technique to accurately determine the pathogenic mechanism of recurrent disease by spoligotyping, making it useful in large-scale intervention studies. Application of spoligotyping and a newly developed PCR-method showed that the occurrence of multiple infections was higher than what was previously assumed and also more frequent in retreatment cases than new cases. These findings have important implications for the understanding of protective immunity, and the development and testing of new vaccines and drugs. Various different molecular markers including spoligotyping has been used to reconstruct the evolutionary history of isolates with less than 6 copies of IS6110 element (termed Low Copy Clade (LCC)), which were previously poor defined. It was also shown that LCC is widely disseminated and play an important role in the global tuberculosis epidemic. Reconstruction of the evolutionary relationship of M. tuberculosis Principal Genetic Group 2 strains, identified previously unknown genetic relationships between strain families and laid the foundation to establish correlations between genotype and phenotype. Spoligotyping signatures, created by evolution of the Direct Repeat region in M. tuberculosis, were identified, which will enable the analysis of the strain population structure in different settings and will also enable the rapid identification of strain families that acquire drug-resistance or escape protective immunity in drug and vaccine trials. This study contributed to our understanding of the molecular epidemiology of drug resistant TB, recurrent TB and the evolution of M. tuberculosis in high incidence communities.
Aspects on advanced procedures during endoscopic retrograde cholangiopancreatography for complex hepatobiliary disorders
(Stellenbosch : Stellenbosch University, 2021-03) Lubbe, Jeanne Adele; Moore, Samuel; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Biomedical Sciences: Molecular biology and human genetics.
Background: The rapid development in endoscopic technology and associated skills has led to an increase in more advanced procedures being performed during endoscopic retrograde cholangiopancreatography (ERCP). Knowledge is limited regarding clinical value, integration, and outcomes for single operator cholangiopancreatoscopy (SOCP) and endoscopic intervention in the different Bismuth-Corlette (B-C) locations in the hepatic hilum. Objectives: To determine the clinical value of SOCP in the diagnosis and treatment of complex hepatobiliary and pancreatic disease. To describe the nationwide integration of SOCP and the extent to which adverse events are influenced when SOCP is added to ERCP. To compare adverse events and reintervention rates after endoscopic stenting for malignant obstruction in the distal and hilar locations of the biliary tree. To compare outcomes after endoscopic transpapillary (ETP) and percutaneous transhepatic (PTH) stenting in the palliation of malignant hilar obstruction (MHO). Methods: In study I all SOCP procedures performed between March 2007-December 2014 at a tertiary highvolume endoscopy unit were separately graded according to a predefined 4-graded scale estimating therapeutic value and diagnostic yield. Study II was a nationwide case-control study nested within the cohort of ERCP procedures, with- or without SOCP, and registered in the Swedish Registry for Gallstone Surgery and ERCP (GallRiks) between 2007-2012. To assess risk factors for adverse events, multivariate logistic regression was performed, and odds ratios (OR) calculated. The GallRiks registry was also utilised in study III where all patients undergoing endoscopic stenting for malignant biliary obstruction between 2010-2017 (based on International Classification of Diseases (ICD) coding), were included. Kaplan-Meier analysis was employed to calculate stent patency and Cox proportional hazard models to calculate the risk for recurrent biliary obstruction after single metal stent placement. To compare ETP and PTH drainage approaches, a retrospective deconstructed analysis of palliative stenting procedures for MHO at two specialised referral centres over a 5-year period was performed. Within-group analyses were performed to explore outcomes for different B-C types and Kaplan-Meier and restricted mean survival time analyses were performed to calculate and compare duration of therapeutic success. Results: In 365 SOCP procedures, SOCP was found be of pivotal importance in 19% of patients, of great clinical significance in 44%, and did not affect clinical decision-making or alter clinical course in 37% of patients. In study II a learning curve was observed after first introduction of 408 SOCP procedures, and postprocedural adverse events (19.1% vs. 14.0%), pancreatitis (7.4% vs. 3.9%) and cholangitis (4.4% vs. 2.7%) were more prevalent when SOCP was added to ERCP. After multivariate analysis, the risk for postprocedural adverse events remained (OR 1.35, 95% CI [1.04 - 1.74]). In 4623 ERCP procedures performed for stenting of malignant strictures (1364 hilar), adverse events and 6-month reintervention rates were increased after hilar stenting compared to distal stenting (17.2% vs. 12.0%, 73.4% vs. 55.9%). On multivariate analysis the risk for reintervention was three times higher after single metal stent placement in the hilum compared to the distal biliary tree (HR 3.47, 95% CI [2.01-6.00], p<0.001). In 293 patients undergoing palliative stenting for MHO (52.2% ETP, 47.8% PTH), access and bridging success in the ETP and PTH groups were 83.5% vs. 97.2% and 90.2% vs. 84.5%, respectively. Technical and therapeutic success were equivalent between the two groups, but duration of therapeutic success was longer after ETP drainage, with a 3-month gain in duration of therapeutic success after adjustment for B-C type (95% CI [26-160], p=0.006). Cholangitis rates were equivalent (21.4% vs. 24.7%), while pancreatitis was more common in the ETP group and deaths more common in the PTH group. Conclusions: When added to ERCP, SOCP contributes significant clinical value in 64% of cases. However, there is an increased risk of intra- and postprocedural adverse events which, together with a learning curve, suggests that it should likely be performed in specialised high-volume centres. Regarding endoscopic intervention for MHO, stenting in the hepatic hilum compared to the distal biliary tree is associated with more adverse events and decreased stent patency. When comparing palliative ETP with PTH stenting for MHO, both approaches have similar technical and therapeutic success, with ETP drainage being more durable. Future studies should explore the complimentary role of both approaches in specific B-C types.
A candidate and novel gene search to identify the PFHBII-causative gene
(Stellenbosch : University of Stellenbosch, 2004-12) Fernandez, Pedro (Pedro Wallace); Corfield, Valerie A.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biochemical Sciences.
ENGLISH ABSTRACT: Heart failure due to cardiomyopathy or cardiac conduction disease is a major cause of mortality and morbidity in both developed and developing countries. Although defined as separate clinical entities, inherited forms of cardiomyopathies and cardiac conduction disorders have been identified that present with overlapping clinical features and/or have common molecular aetiologies. The objective of the present study was to identify the molecular cause of progressive familial heart block type II (PFHBII), an inherited cardiac conduction disorder that segregates in a South African Caucasian Afrikaner family (Brink and Torrington, 1977). The availability of family data tracing the segregation of PFHBII meant that linkage analysis could be employed to identify the chromosomal location of the disease-causative gene. Human Genome Project (HGP) databases have provided additional resources to facilitate the identification of positional candidate genes. Clinical examinations were performed on individuals of the PFHBII-affected family, and, where available, clinical records of subjects examined in a previous study by Brink and Torrington (1977) were re-assessed. Retrospective data suggested redefining the classification of PFHBII. Subsequently, linkage analysis was used to test described dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and cardiac conduction-causative loci on chromosomes 1, 2, 3, 6, 7, 9, 11, 14, 15 and 19 for their involvement in the development of PFHBII. Once a locus was mapped, bioinformatics tools were applied to identify and prioritise positional candidate genes for mutation screening. The retrospective and prospective clinical study redefined PFHBII as a cardiac conduction and DCM-associated disorder and simultaneously allowed more family members to be traced.Fortuitously, candidate loci linkage analysis mapped the PFHBII locus to chromosome 1q32, to a region that overlapped a previously described DCM-associated disorder (CMD1D), by the generation of a maximum pairwise lod score of 3.13 at D1S3753 (theta [θ]=0.0) and a maximum multipoint lod score of 3.7 between D1S3753 and D1S414. However, genetic fine mapping and haplotype analysis placed the PFHBII-causative locus distal to the CMD1D locus, within a 3.9 centimorgan (cM) interval on chromosome 1q32.2-q32.3, telomeric of D1S70 and centromeric of D1S505. Bioinformatics analyses prioritised seven candidate genes for mutation analysis, namely, a gene encoding a potassium channel (KCNH1), an extracellular matrix protein (LAMB3), a protein phosphatase (PPP2R5A), an adapter protein that interacts with a cytoskeletal protein (T3JAM), a putative acyltransferase (KIAA0205) and two genes encoding proteins possibly involved in energy homeostasis (RAMP and VWS59). The PFHBII-causative mutation was not identified, although single sequence variations were identified in four of the seven candidate genes that were screened. Although the molecular aetiology was not established, the present study defined the underlying involvement of DCM in the pathogenesis of PFHBII. The new clinical classification of PFHBII has been published (Fernandez et al., 2004) and should lead to tracing more affected individuals in South Africa or elsewhere. The identification of a novel disease-causative locus may point toward the future identification of a new DCM-associated aetiology, which, in turn, might provide insights towards understanding the associated molecular pathophysiologies of heart failure.
A candidate and novel gene search to identify the PFHBII-causative gene
(Stellenbosch : Stellenbosch University, 2004-12) Fernandez, Pedro; Corfield, Valerie A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: Heart failure due to cardiomyopathy or cardiac conduction disease is a major cause of mortality and morbidity in both developed and developing countries. Although defined as separate clinical entities, inherited forms of cardiomyopathies and cardiac conduction disorders have been identified that present with overlapping clinical features and/or have common molecular aetiologies. The objective of the present study was to identify the molecular cause of progressive familial heart block type II (PFHBII), an inherited cardiac conduction disorder that segregates in a South African Caucasian Afrikaner family (Brink and Torrington, 1977). The availability of family data tracing the segregation of PFHBII meant that linkage analysis could be employed to identify the chromosomal location of the disease-causative gene. Human Genome Project (HGP) databases have provided additional resources to facilitate the identification of positional candidate genes. Clinical examinations were performed on individuals of the PFHBII-affected family, and, where available, clinical records of subjects examined in a previous study by Brink and Torrington (1977) were re-assessed. Retrospective data suggested redefining the classification of PFHBII. Subsequently, linkage analysis was used to test described dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and cardiac conduction-causative loci on chromosomes 1, 2, 3, 6, 7, 9, 11, 14, 15 and 19 for their involvement in the development of PFHBII. Once a locus was mapped, bioinformatics tools were applied to identify and prioritise positional candidate genes for mutation screening. The retrospective and prospective clinical study redefined PFHBII as a cardiac conduction and DCM-associated disorder and simultaneously allowed more family members to be traced. Fortuitously, candidate loci linkage analysis mapped the PFHBII locus to chromosome 1q32, to a region that overlapped a previously described DCM-associated disorder (CMD1D), by the generation of a maximum pairwise lod score of 3.13 at D1S3753 (theta [θ]=0.0) and a maximum multipoint lod score of 3.7 between D1S3753 and D1S414. However, genetic fine mapping and haplotype analysis placed the PFHBII-causative locus distal to the CMD1D locus, within a 3.9 centimorgan (cM) interval on chromosome 1q32.2-q32.3, telomeric of D1S70 and centromeric of D1S505. Bioinformatics analyses prioritised seven candidate genes for mutation analysis, namely, a gene encoding a potassium channel (KCNH1), an extracellular matrix protein (LAMB3), a protein phosphatase (PPP2R5A), an adapter protein that interacts with a cytoskeletal protein (T3JAM), a putative acyltransferase (KIAA0205) and two genes encoding proteins possibly involved in energy homeostasis (RAMP and VWS59). The PFHBII-causative mutation was not identified, although single sequence variations were identified in four of the seven candidate genes that were screened. Although the molecular aetiology was not established, the present study defined the underlying involvement of DCM in the pathogenesis of PFHBII. The new clinical classification of PFHBII has been published (Fernandez et al., 2004) and should lead to tracing more affected individuals in South Africa or elsewhere. The identification of a novel disease-causative locus may point toward the future identification of a new DCM-associated aetiology, which, in turn, might provide insights towards understanding the associated molecular pathophysiologies of heart failure.
The characterisation of N-Acetyltransferase (NAT) in Mycobacterium tuberculosis
(Stellenbosch : University of Stellenbosch, 2005-03) Sholto-Douglas-Vernon, Carolyn; Van Helden, P.; Victor, T.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: A gene coding for Arylaminie N-acetyltransferase (NAT) has been found in Mycobacterium tuberculosis, the casual agent of tuberculosis (TB). N-acetyltransferase acetylates and inactivates isoniazid (INH), which is a front line drug used in TB therapy. A guanine to adenine SNP at basepair 619 (G619A) has previously been identified in this gene, which results in a glycine to arginine change at amino acid 207 (G207R) (Upton et al. 2001). In this study the nat gene was further characterised. The frequency of the G619A SNP was analysed in 37 M tuberculosis strain families found in the Western Cape Province of South Africa, and it was found that the G619A SNP is conserved in two strain families (strain family 3 and strain family 28). Further sequence analysis identified a new thymine to cytosine SNP at base-pair 529 (T529C) resulting in a tyrosine to histidine change at amino acid 177 (Yl77H). This SNP was found only in isolates from strain family 3. These results imply that these SNPs may be used in epidemiology studies to classify isolates into these strain families. Using Real Time PCR, the expression of nat in M bovis BCG and M tuberculosis (reference strain H37Rv) was determined over a 7 and 28 day growth cycle, respectively. Using 16S rRNA as an endogenous control, the nat gene was shown to be expressed early during the growth curve and reach its maximum expression level at approximately mid-log phase. The expression of nat was induced in drug susceptible M tuberculosis isolates (reference strain H37Rv and isolate 1430 containing both SNPs) exposed to INH at a concentration of O.Oll-lg/ml, but minimal change in expression was observed in resistant isolates (isolate 816) exposed to INH at the same concentration. Mycobacterium bovis BCG cultures exposed to INH, at a final concentration of 0.28I-lg/ml, showed an increase in protein production. The increase of nat mRNA and NAT protein in M tuberculosis and M bovis BCG, respectively, implies that INH affects the expression of NAT. The NAT protein was localised to all fractions of the cell in Mycobacterium smegmatis, M bovis BCG and M tuberculosis, using the Western blot technique. However, protein fractions from the cell envelope region showed a protein (detected with specific NAT antibodies) that ran at a higher molecular weight (MW). This implies that the cytosolic hydrophilic NAT undergoes some type of post-translational process that may make it hydrophobic, and enable it to pass into the cell envelope region. These results show for the first time how nat is expressed during the entire growth cycle of M tuberculosis and M. bovis BeG. It was shown that nat is expressed early during the growth cycle of the bacterium reaching maximum expression levels at mid-log phase. These results are in concordance with those obtained using M. smegmatis nat mutants, which taken together, show that early expression of nat is important for early growth and development of mycobacteria. The results in this study also showed that NAT appeared to be translocated into the cell envelope of the bacterium, implying that NAT may be involved in one of the pathways needed for complete formation of the cell envelope. These results suggest that NAT may be an important target for drug development, as inhibitors of NAT could result in hindered growth and hence spread of the bacterium within its host. Inhibitors may also result in the incomplete development of the cell wall, enabling the host to combat the disease using its own immune system.
Characterization of the innate and adaptive immune systems during active TB disease and during treatment
(Stellenbosch : Stellenbosch University, 2019-04) Kotze, Leigh Ann; Walzl, Gerhard; Du Plessis, Nelita; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Individuals presenting with symptoms of active tuberculosis (TB) disease currently undergo a lengthy diagnostic procedure, followed by an intense six-month treatment regimen. Delays in accurate diagnoses and severe treatment side effects contribute to low treatment adherence and drive the emergence of drug resistant Mycobacterium tuberculosis (M.tb) strains. Improvements in diagnostic technologies have allowed for same day diagnoses, however the roll-out of such devices are limited to settings with stable infrastructure. Additionally, many patients do not require the full treatment regimen when diagnosed early or when presenting with mild disease, however few reliable methods are available for identifying fast-responders. Immune biomarkers show great promise in addressing the need for improved diagnostic and treatment response prediction techniques. This study aimed to investigate multiple promising biomarkers, including (1) promising host diagnostic biomarkers for accurate discrimination of active TB disease from other diseases at point-of-care level; (2) promising host cell surface biomarkers for identifying treatment response shortly after treatment initiation; and also (3) functional host biomarkers for elucidating host-pathogen interaction responses for translation into diagnostic or treatment response biomarkers. Host diagnostic biomarkers were investigated (Chapter 2) in individuals presenting with symptoms suggestive of active TB disease, stratified according to an algorithm based on a combination of clinical, radiological and laboratory findings. Nine acute-phase proteins were investigated in participants' serum, and a biosignature comprising the biomarkers C-reactive protein (CRP) and serum amyloid A (SAA), accurately discriminated between participants with and without active TB disease with a sensitivity of 87.4% and specificity of 75.7%, irrespective of human immunodeficiency virus (HIV) co-infection. The validated biosignature performance in Ascaris lumbricoides sensitized participants remained relatively stable (Chapter 3; Sensitivity of 78%; Specificity of 75%). Validation of this biosignature in scenarios where both HIV- and Ascaris co-infection were considered, identified a robust and reliable biosignature suitable for use in high-burden settings. Individuals with confirmed active TB disease and healthy controls were recruited to investigate cell surface biomarkers of treatment response (Chapter 4). Markers of interest (CD126, CD120b, CD62L, CD197, and CD58) were investigated via flow cytometry on various immune cell subsets across three time points (diagnosis, month 1, end of treatment). No markers were differentially expressed at month 1, whereas CD120b and CD58 were upregulated the end of treatment on CD4+ and CD8+ T-cells, limiting their use as early treatment response biomarkers. Innate and adaptive response cytokines were then investigated from isolated cell subsets (neutrophils, monocytes, T-cells, combination) and compared to whole blood under unstimulated and antigen-stimulated conditions (Chapter 5). The downregulation of protective innate cytokines in the whole blood compared to the culture of monocytes with T-cells, suggested an active suppressive mechanism. A potential innate suppressive immune cell, myeloid-derived suppressor cells (MDSC), was then investigated in TB patients and healthy controls. MDSC were significantly upregulated in peripheral blood mononuclear cells (PBMC) from active TB patients, and cytokine production from MDSC co-cultured with T-cells displayed T-cell-specific downregulation of IFN- (Chapter 6). MDSC should be considered as a potential TB diagnostic biomarker, and future studies investigating frequency changes during treatment will inform on their value as treatment response biomarker.
Characterization of the interaction between acetylcholinesterase and laminin : a template for discovering redundancy
(Stellenbosch : Stellenbosch University, 2012-03) Swart, Chrisna; Johnson, Glynis; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine, acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be able to promote various non-cholinergic functions, including cell adhesion and neurite outgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouse laminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the site on AChE recognised by certain monoclonal antibodies (MAbs) might be critical for differentiation. These MAbs were found to inhibit both laminin binding and cell adhesion in neuroblastoma cells. In this study, the structure and characteristics of this site were investigated, using the AChE-laminin interaction as a template as well as a detailed epitope analysis of the MAbs. The interaction sites of AChE and laminin were investigated using phage display, modelling and docking, synthetic peptides, enzyme linked immunosorbent assays (ELISAs) and conformational interaction site mapping. Docking of AChE with the single-chain variable fragments (scFvs) produced from the phage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Asp motif, the 40Pro-Pro-Met-Gly sequence, and the 59Val-Val-Asp-Ala-Thr-Thr (human) motif. Mouse AChE was found to interact with the basic structures Val2718-Arg-Lys-Arg- Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793; and Val2817-Glu-Arg-Lys2820, on the 1 G4 domain of laminin. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps with laminin’s heparin-binding site. Docking showed the major component of the interaction site on AChE to be the acidic Arg90-Glu-Leu-Ser-Glu-Asp95 (omega loop), and also involving the Pro40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61 Ala-Thr-Thr-Phe-Gln66. Epitope analysis showed the MAb’s major recognition site to be the sequence Pro40-Pro- Met-Gly-Pro-Arg-Arg-Phe48 (human AChE). The MAbs also reacted with the prolinerich sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu- Asp95. These results define the interaction sites involved in the AChE-laminin interaction and suggest that the interaction plays a role in cell adhesion. Despite the in vitro demonstrations of the importance of AChE’s non-classical functions, the AChE knockout survives. Results from this study suggest the possibility of functional redundancy between AChE and other molecules in early development. Using these in vitro findings that AChE is able to bind laminin-111, information on the interaction sites, as well as results from the monoclonal antibody (MAb) epitope analysis, the idea of redundancy was investigated. Docking and bioinformatics techniques were used to investigate structurally similar molecules that have comparable spatiotemporal expression patterns in the embryonic nervous system. AChE has been shown to be involved in the pathogenesis of Alzheimer’s disease, thus molecules associated with brain function and neurodegeneration were also investigated. Molecules with which AChE could be possibly redundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoprotein receptors and their variants. AChE was observed to dock with growth arrest-specific protein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminin interaction. The AChE interaction site was shown to resemble the apolipoprotein-binding site on the low density lipoprotein receptor, and related molecules, including the low density lipoprotein receptor-related molecule (LRP) and the sortilin-related receptor (SORL1). These molecules, along with apoE, are associated with Alzheimer’s disease. Resemblances to the triggering receptor on myeloid cells (TREM1) were also suggested; this is interesting as AChE has been implicated in both haematopoiesis and haematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternative ligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, and also suggested the existence of other binding partners. In conclusion, characterisation of the AChE-laminin interaction sites and investigation of structurally similar sites in other molecules suggests a role for AChE in the stabilization of the basement membrane of developing neural cells and provides a feasible explanation for the survival of the knockout mouse. Furthermore, the demonstrated similarity of the AChE interaction site to sites on molecules, notably the low density lipoprotein receptor family and SORL1 and their apolipoprotein ligands that are implicated in the pathology of Alzheimer’s disease, as well as the possible link to haematopoietic differentiation and cancers, warrants further investigation.
Clinical and laboratory investigation of latex allergy in healthcare workers
(Stellenbosch : Stellenbosch University, 2004-12) De Beer, Corena; Walzl, Gerhard; Cilliers, Jacques; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Healthcare workers (HCWs) wear latex gloves to protect themselves and their patients against the transmission of microbial, viral and bloodborne diseases. These individuals are primarily exposed to latex via cutaneous (direct contact) and mucocutaneous (inhalation of airborne allergens on glove powder) routes. Repeated exposure leads to the formation of circulating latex-specific IgE and subsequent sensitisation with varying clinical expression. The airconditioning system of the Tygerberg Hospital (TBH) complex was investigated for the presence of aerosolised cornstarch glove powder and proteins. Dust samples were collected from 14 areas with different levels of latex glove usage. Dust samples were spectrophotometrically compared to a calibration graph of pure glove powder. The detection of starch and proteins in all the dust samples confirmed the presence of glove powder and possibly airborne latex allergens in the airconditioning ducts. As expected, the high exposure areas showed the highest concentrations of both starch and proteins. It is possible that other proteins than latex were involved, but the confirmed high level of protein contamination should be a cause for concern. Correlation between starch and protein levels was highly significant (p<0.01) in all instances. A total of 500 questionnaires were circulated for completion by HCWs from TBH. The response rate was 69.8%. After considering specific inclusion criteria, a study group of 152 individuals was compiled (28 males, 124 females). All subjects had current latex exposure and suffered from at least three pre-defined symptoms. Serum was collected from all subjects and dermal fluid from 31 subjects. Total IgE and latex specific IgE analysis were done on all serum and dermal fluid samples. Latex-specific IgE was positive (>0.35 IU/ℓ) in 23 serum and six dermal fluid samples. Skin prick tests (SPTs)for latex were done on 59 subjects with negative serum latex-specific IgE and 34 had positive results. Twelve subjects with negative latex-specific IgE and latex SPTs underwent patch tests with the European Standard Series, a piece of latex glove and glove powder in petrolatum. Three subjects had positive results to one or more of these allergens. Western blot analysis for latex was done on all positive sera and dermal fluid collected from these subjects. Western blot analysis for latex proved to be more sensitive than the capRAST, because it was able to identify specific bands in samples with negative capRAST results. All subjects showed a band for Hev b 1, which has been confirmed as a powder-bound airborne allergen. Hev b 6.01 is associated with HCWs with cutaneous symptoms and this band was recognised by 81% of the subjects. These findings confirmed that airborne and cutaneous routes are the major routes of exposure in HCWs. According to their laboratory results, subjects were divided into the following subgroups and compared statistically: Group A (serum positive, n=23), Group B (SPT positive, n=34) and Group C (negative, n=25). Group D (withdrawn, n=70) could not be used for statistical comparisons, due to incomplete results. An overall latex allergy prevalence of 38% was found. Group A differed significantly from Group B and Group C for most clinical and special investigations. Group A and B were also combined to represent all subjects with positive results (Cohort AB). The Allergy Score and Class were highly significant when Cohort AB was compared to Group C. The selection of clinical symptoms was confirmed to be relevant and work-related deterioration on any of the symptoms should bear a high index of suspicion in the evaluation of latex allergy. Numerical indices and specific symptoms showed high positive predictive values and the Allergy Score produced statistical significance in the positive subgroups when compared to the negative subgroup. Paired statistical significance was confirmed between the Allergy Score and occupational exposure (number of years, hours and pairs per week). The areas with the highest occupational latex exposure in HCWs are the face and hands. Different occupations also have different levels of exposure and two subgroups of HCWs (16 laboratory technologists and 13 theatre staff) were investigated for sebum content on different facial areas and the palms and dorsal areas of both hands. Baseline measurements were done before putting on gloves. In 21 subjects follow up measurements were done following three to four hours of occupational exposure, but before washing their hands. Baseline and follow up values were compared for all the different anatomical regions. Levels on the forehead and cheeks increased over time, while the level on the nose decreased. All hand regions decreased significantly during occupational exposure, suggesting that glove powder contributes to dryness of the skin. In conclusion, the problem posed by latex allergy will not be solved overnight and will probably remain a major occupational hazard for years to come. It is currently not possible to avoid exposure to latex, but it is imperative to institute safety measures to prevent further sensitisation in predisposed individuals and manage those already affected.
Clinically relevant mutations contributing to drug resistance in Mycobacterium tuberculosis
(Stellenbosch : Stellenbosch University, 2017-12) Du Plessis, Juanelle; Sampson, Samantha Leigh; Wigneshweraraj, Sivaramesh; Warren, Robin Mark; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Single nucleotide variants are the underlying driver of drug resistance, strain fitness and adaptation in Mycobacterium tuberculosis and investigating the mechanistic and physiological aspects of these mutations is key to our understanding of the biology of this pathogen. Rifampicin, one of the most powerful first line drugs used to treat tuberculosis, inhibits transcription in M. tuberculosis by binding to the β subunit of RNA polymerase (RNAP). However, mycobacteria are able to evade binding of this drug by acquiring mutations in the rpoB gene. These resistance-conferring mutations are essential for the survival of M. tuberculosis in the presence of rifampicin, however they impart a fitness cost to the bacterium due to a presumed reduction in transcription efficiency and subsequent changes in gene expression. One of the mechanisms M. tuberculosis uses to buffer the effects of this fitness cost is to acquire compensatory mutations in rpoC and rpoA. As RNAP is at the core of all mechanisms of gene regulation in M. tuberculosis, it is not surprising that mutations within this enzyme led to pleiotropic effects. As this remains a poorly understood area of mycobacterial physiology, the current work encompasses a triad of studies which aims to better understand functional aspects of M. tuberculosis RNAP, and the role of rpoB and rpoC mutations in drug resistance. First, the effect of a bacteriophage protein, Gp2, was investigated to determine whether it is able to inhibit RNAP in M. tuberculosis. As Gp2 is known to bind to the β’ subunit of RNAP in Escherichia coli, positive findings from this work would provide a framework for the identification of novel compounds that inhibit transcription in the presence of rpoB mutations, affecting the β subunit. By way of in vitro and in silico analysis, it was found that Gp2 binds to and inhibits RNAP in M. tuberculosis, however to a much lesser degree than it does in its de facto host, E. coli. Nonetheless, future studies can build on our findings as in silico modification of Gp2 could identify a structure which allows for stronger binding affinity of the protein. Secondly, the effect of rpoB and rpoC mutations on the function of mycobacterial RNAP was investigated using a series of in vitro transcription assays. Radioactivity-based assays were performed using purified wildtype and mutant versions of the RNAP complex, to assess enzyme activity and promoter affinity. Furthermore, the use of a fluorescence-based assay was trialled to develop a comparable method without the use of radiolabelled nucleotides. Lastly, we undertook a study to understand the effect of rpoC mutations on the transcriptome of M. tuberculosis. For this purpose, serial clinical isolates were selected where the acquisition of an rpoC mutation was observed. These samples were used for whole genome sequencing and gene expression analysis, which revealed a potential link between the rpoC V483G mutation and upregulation of Rv2416c (eis) and Rv1258c (tap). Serendipitously, genomic data also revealed that an ald mutation was acquired alongside the rpoC mutation. Recently, ald has been described as a novel gene linked to D-cycloserine resistance in M. tuberculosis, however, to date, the mechanism of drug resistance has not been determined. Given the unique opportunity to study the effect of ald mutations on gene expression in our study, we investigated two genes which were found to be differentially expressed in a clinical isolate with an ald mutation. A turbidity-based microdilution assay revealed that upregulation of Rv0577, a putative glyoxalase, led to an increase in the minimum inhibitory concentration of D-cycloserine, a finding which provides novel insight into the mechanism of D-cycloserine resistance in M. tuberculosis. In summary, this body of work has contributed to existing knowledge surrounding drug resistance and compensatory adaptation in M. tuberculosis.
Combining different types of highly parallelised technology datasets for bioinformatic analysis in the context of biomarker discovery for tuberculosis disease and treatment response
(Stellenbosch : Stellenbosch University, 2021-12) Maasdorp, Elizna; Tromp, G. C.; Walzl, G.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
Background: Monitoring of tuberculosis (TB) treatment response currently relies on month 2 sputum culture. It is a poor predictor of ultimate treatment failure and recurrent disease and has a long turnaround time of up to six weeks. A biomarker of treatment response to identify patients at high risk of poor outcomes will benefit both TB patient care and TB research. It has been shown that patients with negative end-of-treatment sputum culture can still have a highly inflammatory picture on Positron Emission Tomography and Computerised Tomography (PET-CT) scans at the same time point. This inflammation may be in response to the presence of viable Mycobacterium tuberculosis bacilli which were not sampled in sputum, or not culturable, or it may be due to ongoing immune dysregulation, which is a well-known phenomenon in TB. Currently, we are not able to distinguish between these potential scenarios, but despite this, it is clear that PET-CT provides complementary information to microbiology at the end of treatment. Since PET-CT is expensive and not widely available, it would be most practical to obtain similar information to what PET-CT provides, from a blood-based biomarker. Several whole blood gene expression signatures have been discovered for diagnosis of active TB and sub-clinical disease, but none have yet attained to the World Health Organisation’s target product profiles for new diagnostic tests for TB. Gene expression signatures have also been investigated as biomarkers of treatment response, but none has been validated, as treatment response studies require many participants and long follow-up times, leading to a scarcity of adequate treatment response data sets. Objective To utilise treatment outcome groups derived from end-of-treatment PET-CT scans, as an alternative dependent variable to microbiological treatment outcome groups in predictive modelling, and to discover whether PET-CT could be replaced by multiplexed immunoassay data, gene expression data, or both in combination. Methods Two existing treatment response data sets were utilised – one from a study with follow-up during TB treatment until 2 years after treatment completion, and one early bactericidal activity study with two- week follow-up. Both studies produced PET-CT and multiplexed immunoassay (Luminex) data, while the longer study, which was the main focus of this thesis, additionally included gene expression data. Unsupervised hierarchical clustering of selected quantitative PET-CT variables at end-of-treatment was performed to create two outcome groups, independent of microbiological results, for predictive modelling. RNA-sequencing data and Luminex data at three time points, for the same cohort of patients, were used to predict membership of the PET-CT outcome groups, both separately and in combined models. Immunoassay data were also used in predictive regression models to explain a proportion of the variance in quantitative PET-CT variables. Results Two clusters of patients were identified from the PET-CT variables – one consisting of 23 participants with a predominantly inflammatory ("hot") lung picture, including seven of eight participants who failed treatment, and the second consisting of 76 participants with a less inflammatory or even resolved lung picture ("cold"). Both gene expression and Luminex data models could predict cluster membership and achieved cross-validation classification areas-under-the-curve (AUCs) that ranged from 0.74 to 0.90 at end-of-treatment. The models also achieved similar AUCs at the diagnosis time point. Combining gene expression and Luminex data in classification models did not improve on the classification accuracy of the separate models. Luminex analyte regression models explained 55% of the variance of the total glycolytic activity index PET-CT variable in a test and a validation set. A Luminex analyte classification model could also identify presence of cavities 1 mm or larger, with AUCs of 0.83 and 0.75 in the test and validation sets, respectively. Differential gene expression, gene ontology, pathway and weighted gene co-expression analysis focusing on the two PET-CT clusters, highlighted known immunological and TB-related processes that differed between the clusters and provided justification for using treatment outcome groups based on PET-CT, as a complementary strategy to using microbiological treatment outcome groups. Conclusion At the end of TB treatment, PET-CT provides complementary information to microbiological treatment outcomes, that could be utilised in specific scenarios in future studies to monitor treatment response. As PET-CT is expensive and not widely available, it is highly desirable to replace it with a biomarker measured in peripheral blood. I showed that gene expression or protein measured in peripheral blood could potentially replace PET-CT, but discovery of such a biomarker will benefit from a study designed for that purpose, and the availability of independent data sets for validation.
Combining different types of highly parallelised technology datasets for bioinformatic analysis in the context of biomarker discovery for tuberculosis disease and treatment response
(Stellenbosch : Stellenbosch University, 2021-03) Maasdorp, Elizna; Tromp, G. C.; Walzl, G.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
Background: Monitoring of tuberculosis (TB) treatment response currently relies on month 2 sputum culture. It is a poor predictor of ultimate treatment failure and recurrent disease and has a long turnaround time of up to six weeks. A biomarker of treatment response to identify patients at high risk of poor outcomes will benefit both TB patient care and TB research. It has been shown that patients with negative end-of-treatment sputum culture can still have a highly inflammatory picture on Positron Emission Tomography and Computerised Tomography (PET-CT) scans at the same time point. This inflammation may be in response to the presence of viable Mycobacterium tuberculosis bacilli which were not sampled in sputum, or not culturable, or it may be due to ongoing immune dysregulation, which is a well-known phenomenon in TB. Currently, we are not able to distinguish between these potential scenarios, but despite this, it is clear that PET-CT provides complementary information to microbiology at the end of treatment. Since PET-CT is expensive and not widely available, it would be most practical to obtain similar information to what PET-CT provides, from a blood-based biomarker. Several whole blood gene expression signatures have been discovered for diagnosis of active TB and sub-clinical disease, but none have yet attained to the World Health Organisation’s target product profiles for new diagnostic tests for TB. Gene expression signatures have also been investigated as biomarkers of treatment response, but none has been validated, as treatment response studies require many participants and long follow-up times, leading to a scarcity of adequate treatment response data sets. Objective To utilise treatment outcome groups derived from end-of-treatment PET-CT scans, as an alternative dependent variable to microbiological treatment outcome groups in predictive modelling, and to discover whether PET-CT could be replaced by multiplexed immunoassay data, gene expression data, or both in combination. Methods Two existing treatment response data sets were utilised – one from a study with follow-up during TB treatment until 2 years after treatment completion, and one early bactericidal activity study with twoweek follow-up. Both studies produced PET-CT and multiplexed immunoassay (Luminex) data, while the longer study, which was the main focus of this thesis, additionally included gene expression data. Unsupervised hierarchical clustering of selected quantitative PET-CT variables at end-of-treatment was performed to create two outcome groups, independent of microbiological results, for predictive modelling. RNA-sequencing data and Luminex data at three time points, for the same cohort of patients, were used to predict membership of the PET-CT outcome groups, both separately and in combined models. Immunoassay data were also used in predictive regression models to explain a proportion of the variance in quantitative PET-CT variables. Results Two clusters of patients were identified from the PET-CT variables – one consisting of 23 participants with a predominantly inflammatory ("hot") lung picture, including seven of eight participants who failed treatment, and the second consisting of 76 participants with a less inflammatory or even resolved lung picture ("cold"). Both gene expression and Luminex data models could predict cluster membership and achieved cross-validation classification areas-under-the-curve (AUCs) that ranged from 0.74 to 0.90 at end-of-treatment. The models also achieved similar AUCs at the diagnosis time point. Combining gene expression and Luminex data in classification models did not improve on the classification accuracy of the separate models. Luminex analyte regression models explained 55% of the variance of the total glycolytic activity index PET-CT variable in a test and a validation set. A Luminex analyte classification model could also identify presence of cavities 1 mm or larger, with AUCs of 0.83 and 0.75 in the test and validation sets, respectively. Differential gene expression, gene ontology, pathway and weighted gene co-expression analysis focusing on the two PET-CT clusters, highlighted known immunological and TB-related processes that differed between the clusters and provided justification for using treatment outcome groups based on PET-CT, as a complementary strategy to using microbiological treatment outcome groups. Conclusion At the end of TB treatment, PET-CT provides complementary information to microbiological treatment outcomes, that could be utilised in specific scenarios in future studies to monitor treatment response. As PET-CT is expensive and not widely available, it is highly desirable to replace it with a biomarker measured in peripheral blood. I showed that gene expression or protein measured in peripheral blood could potentially replace PET-CT, but discovery of such a biomarker will benefit from a study designed for that purpose, and the availability of independent data sets for validation.
Deciphering the physiology of drug tolerant and resistant Mycobacterium tuberculosis
(Stellenbosch : Stellenbosch University, 2021-03) Pule, Caroline; Sampson, Samantha Leigh; Louw, Gail Erika; Warren, Robin Mark; Mouton, Jacoba Martina; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Poor adherence to treatment for tuberculosis (TB) disease and the rising incidents of drug resistant Mycobacterium tuberculosis strains are factors that negatively influence TB control. The current study was designed to explore some of the key knowledge gaps concerning M. tuberculosis physiology; looking at the effect of the diverse M. tuberculosis genetic backgrounds and the presence of ropB mutation on the transcriptome, looking at M. tuberculosis host response and the likelihood as to whether induced mycobacterial tolerance can provide a reservoir from which genetic resistance can arise. Exploring some of these key knowledge gaps was imperative, given the fact that, lengthy anti-TB drug treatment could be required to entirely eradicate some of M. tuberculosis strains, and non-compliance with completing treatment might lead to the emergence of multidrug (MDR)-TB. Firstly, we investigated the effect of M. tuberculosis strains with different genetic backgrounds on their total transcriptomic profiles (as a proxy for the physiological state). Secondly, we examined the influence of rpoB Ser531Leu mutation and the effect of isoniazid (INH) treatment (24h) at sub-lethal concentrations on the transcriptomic profiles of rifampicin (RIF)-resistant (K636RIF) and susceptible (K636WT and H37RVWT) M. tuberculosis strains, using RNA-sequencing (RNA-Seq) and Real-Time quantitative polymerase chain reaction (RT-qPCR) techniques. RNA-Seq analysis identified significantly differentially expressed genes in the transcriptomes of K636WT, H37RVWT and K636RIF M. tuberculosis strains. Our comparative transcriptomic data of K636WT relative to H37RvWT M. tuberculosis strains demonstrated that different genetic backgrounds influenced the total transcriptome. We demonstrated that rpoB Ser531Leu mutation has an impact on the transcriptional responses of K636WT relative to K636RIF M. tuberculosis strains. Our data did not demonstrate an effect of INH treatment on the transcriptomes of M. tuberculosis strains from different genetic backgrounds, making this one of our limitations. We then assessed the host immune response after infection with RIF-resistant (K636RIF and H37RvRIF) and susceptible (K636WT and H37RVWT) M. tuberculosis strains using the luminex x multi-analyte profiling (xMAP) technology and enzyme-linked immunosorbent assay (ELISA). Our host response data (Chapter 4) revealed no differences in host response to K636WT and H37RvWT M. tuberculosis strains from different genetic backgrounds. In contrast, there were differences in host response to K636WT and K636RIF M. tuberculosis strains in a RAW264.7 macrophage model of infection. This was confirmed by the observed varying secretion levels of cytokines and chemokines (IL-6, IL-12p40 and RANTES) required to mediate M. tuberculosis growth and survival after 24 - 48h of infection. We further investigated whether viable but non-replicating (VBNR) persisters Mycobacterium smegmatis sub-populations, when exposed to high INH concentrations, may provide a pool from which genetic resistant mutants can arise. We used a combination of a fluorescence dilution (FD) reporter system, flow cytometry and fluorescence-activated cell sorting (FACS) to detect, quantify and separate VBNR and actively replicating (AR) M. smegmatis bacterial populations following INH treatment. Subsequently, we performed PCR to amplify the katG and inhA promoter and Sanger sequencing to identify mutations in these genes that are commonly associated with INH resistance. Our flow cytometry results showed successful detection of VBNR and AR bacterial populations in M. smegmatis::pTiGc following INH pre-treatment at high concentration (30x MIC) for 72h. Mutation frequencies of different sorted populations were determined as 3.51% for M. smegmatisVBNR, 5.20% for M. smegmatisAR and 0.02% for M. smegmatis UNT. Sanger sequencing data demonstrated a high percentage of mutations in the inhA promoter (C-15T) (76% in VBNR; 64% in AR) compared to mutations in the katG gene (48% in VBNR; 44% in AR). However, the difference was not statistically significant (p > 0.1). This study addressed the following knowledge gaps: it advanced our understanding about the M. tuberculosis physiology. It confirmed that strain genetic background and the presence of rpoB Ser531Leu mutation may play a role in the physiological state of M. tuberculosis strains as reflected in their transcriptomes. It confirmed that host response in vitro is influenced by M. tuberculosis strain genotype and that infection with K636WT, H37RvWT and H37RvRIF M. tuberculosis strains will result in the secretion of pro-inflammatory cytokines and chemokines while infection with K636RIF M. tuberculosis strain (with rpoB Ser531Leu mutation) might induce secretion of anti-inflammatory cytokines (second line of host defense). This study was the first to successfully use a FACS analysis in combination with the FD reporter system to detect, isolate and quantify VBNR from AR M. smegmatis, following INH pre-treatment at high concentrations. We speculate that our results showed that the VBNR persisters‟ sub-population is likely to provide a reservoir from which genetic resistant mutants can arise, when treated with high INH concentrations as made evident by the observed INH resistant mutants in VBNR M. smegmatis. This work contributed further knowledge to finding better strategies to prevent the spread of emerging MDR, as well as extensively drug resistant M. tuberculosis.
Detection and Characterization of Mycobacterial Infections Occurring in Phacochoerus africanus (Gmelin, 1788) (Common Warthog)
(Stellenbosch : Stellenbosch University, 2018-12) Roos, Eduard Otto; Miller, Michele Ann; Parsons, Sven David Charles; Olea-Popelka, Francisco; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
ENGLISH ABSTRACT: Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex and the cause of bovine tuberculosis (bTB), has an extensive host range that includes livestock and wildlife. While warthogs are considered spill-over hosts for bTB, they could potentially become reservoir hosts, if conditions are favourable, i.e. increased population densities. With limited knowledge on the infection status of warthogs in South Africa and their epidemiological significance for other species, it is imperative to have readily available diagnostic tests for warthogs. Therefore, this study aimed to (i) establish reference cohorts of M. bovis-infected and uninfected warthogs; (ii) utilize these for the development and evaluation of diagnostic tools that can distinguish between infected and uninfected individuals; (iii) determine the seroprevalence of bTB in warthogs using the newly developed diagnostic tools; and (iv) characterize the genetic diversity of M. bovis isolates from warthogs. Three serological assays, i.e. the indirect PPD ELISA, the TB ELISA-VK® and the DPP® VetTB Assay, could distinguish between M. bovis-infected and uninfected warthogs with high sensitivity (75-88%) and specificity (79-89%). The overall seroprevalence from four M. bovis-endemic locations was high, i.e. 38%. Furthermore, three tests measuring the cellmediated immune responses of warthogs were developed. A cytokine release assay measuring interferon gamma induced protein 10 was able to distinguish between M. bovisinfected and uninfected warthog with a sensitivity of 68% and a specificity of 84%. The comparative intradermal tuberculin test classified 100% of culture-negative warthogs as test negative and 81% of culture-positive warthogs as test positive. Lastly, CXCL9, 10, 11, IFNG and TNFA gene expression were significantly increased in whole blood from M. bovis-infected warthogs in response to antigen stimulation, with CXCL10 showing the greatest mean fold increase. High genetic diversity of M. bovis isolates from warthogs was confirmed through spoligotyping and whole genome sequencing. Two distinct clades of M. bovis were identified by WGS, even though they shared the same spoligotype patterns This study has demonstrated that warthogs develop measurable and specific immune responses to M. bovis infection, which can be used to identify infected individuals ante-mortem. Furthermore, these tests will facilitate epidemiological studies of bTB in warthogs. With a high culture prevalence in warthogs from bTB endemic areas such as uMhkuze Nature Reserve and the Greater Kruger National Park, and high seroprevalence, warthogs seem to be highly susceptible to M. bovis infection. This suggests that warthogs may be an ideal sentinel species and strengthens the case that, under certain circumstances, they could be maintenance hosts. The genetic diversity of M. bovis isolates and the identification of two distinct clades challenges the current hypothesis that a single dominant strain circulates within a specific geographical location. Warthogs as a species should receive greater attention as potential disease maintenance hosts or as sentinels for bTB disease surveillance.

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