Doctoral Degrees (Molecular Biology and Human Genetics)
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- ItemInvestigation of the antimycobacterial activity of fungal endophytes and their magnetic dendrimer stabilized nanoparticles against mycobacteria(Stellenbosch : Stellenbosch University, 2024-03) Hussan, Raeesa Hoosen; Mavumengwana, Vuyo; Malgas-Enus, Rehana; Loxton, Andre Gareth; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Tuberculosis (TB), is a disease caused by a single infectious agent Mtb, remains a global health threat that claimed the lives of 1.4 million people worldwide between 2020- 2021. Despite current anti tuberculosis treatment, TB remains one of the leading causes of infection highlighting the imminent need for drug discovery of alternative and novel treatments with distinct modes of action against Mycobacteria. In this context, the present study investigated the antimycobacterial activities of fungal endophyte metabolites functionalized on magnetic dendrimer stabilized nanoparticles (DSNPs) against mycobacterial models. To achieve the aim, fungal endophytes were isolated from four Fynbos families (Asteraceae, Lamiaceae, Ericaceae, and Droseraceae), sequenced and identified by phylogeny. Methanolic crude extracts obtained from cultivated fungi were tested for antimycobacterial activity against Mycobacterium tuberculosis H37Rv (Mtb H37Rv) and Mycobacterium smegmatis mc2155 (Msmeg mc2155). Metabolites within the bioactive fungal extracts were identified by untargeted liquid chromatography-mass spectrometry (LC-QTOF-MS/MS). The infection of THP-1 macrophage cells with Msmeg mc2155 was performed to determine the intracellular antimycobacterial activity of fungal crude extracts. The DSNPs were synthesized from modified magnetic iron oxide nanoparticles (MIONs) and G3-dendrimer micelles. Microscopic and spectral techniques (SEM, FTIR, UV-Vis, and ICP-AES) were used to characterize nanoparticles, followed by antimycobacterial activity assays. Twenty unique fungal isolates were identified with Penicillium being the abundant genera. Penicillium thomii, Diaporthe leucospermi, Penicillium rubens, Cadophora sp. and Penicillium sp. possess antimycobacterial activity. Cadophora sp. demonstrated the most significant inhibition of Msmeg mc2155 and the bioactivity was enhanced in the host-directed approach against intracellular Msmeg mc2155. The metabolites N-benzyl-1-tetradecanamine, N-benzyl-1-hexadecanamine, asperthecin, meleagrin, roquerfortine F, chlamydocin and neoxaline, (-)5-methylmellein, and 9-octadecenamide were identified with the latter two possessing antimycobacterial activity. The nanoparticles (G3- dendrimer micelles, MIONs and DSNPs) were successfully synthesized. G3-dendrimer micelles and DNSPs inhibited Msmeg mc2155 and the former Mtb H37Rv. MIONs possessed enhanced bioactivities against intracellular Msmeg mc2155. Functionalization of DSNPs with bioactive metabolites (nano metabolites) demonstrated no antimycobacterial activity against Msmeg mc2155 directly but antimycobacterial activity was observed intracellularly. Enhanced activity of nano-metabolites was observed directly against Mtb H37RV. Fungal extracts, nanoparticles and functionalized nanoparticles have distinct mechanisms of antimycobacterial activities against the strains of mycobacteria (Msmeg mc2155 and Mtb H37Rv). This study highlighted that the properties of nanoparticles greatly influence their antimycobacterial activities i.e., the ferrous content of a naked nanoparticle opposed to a modified nanoparticle. Functionalization of a nanoparticle contributes to its modification which has an impact on the antimycobacterial activity. Hence, this necessitates further investigations about nanoparticles, their properties and modifications against mycobacterial models. In addition, the nanoparticles have the potential to be modified to accommodate or scaffold existing anti-Tb drugs and to potentiate antimycobacterial effects through pathogen- and host directed approaches.
- ItemInfectiousness of TB patients and factors associated with the airborne survival of Mycobacterium tuberculosis: Tuberculosis Infectiousness and TrANsmission (TITAN) study(Stellenbosch : Stellenbosch University, 2024-03) Mahlobo, Zama; Theron, Grant; Venter, Rouxjeane; Mishra, Hridesh; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Tuberkulose is wêreldwyd die grootste aansteeklike oorsaak van sterftes en word van persoon tot persoon oorgedra via aërosols wat hoofsaaklik deur hoes geproduseer word. Alhoewel, dit is egter uitdagend om TB-aërosols te evalueer, beide van pasiënte en deur in vitro-modelle. Verder is daar 'n gebrek aan implementeerbare oplossings wat uit TB-aerobiologie navorsing stem. Ons het gepoog om kennisgapings onder hierdie tema aan te spreek. Eerstens (Hoofstuk 3), met behulp van ‘n “Human Aerosol Chamber”, het lugdraende Mycobacterium tuberculosis en druppels geëvalueer deur gebruik te maak van verskillende uitlesings (kultuur, DNS), en hoe dit verskil het op grond van verskillende respiratoriese oefeninge (gewoone-asemhaling, spontane hoes en gedwonge hoes). Ons het ook variante van verskillende gesigbedekkings ondersoek, wat moontlik minder stigmatiserende effek (gedoen voor COVID-19) sal he, asook hoe aansteeklikheid deur twee weke se effektiewe behandeling beïnvloed is. Ons het gewys dat 1) nie-chirurgiese vorms van gesigbedekking druppels wat deur mense met TB gegenereer word, kan verminder (die Stad Kaapstad oorweeg dit nou om hierdie alternatiewe gesigbedekkings aan te bied as deel van pogings om persoonlike asemhalingsbeskerming in gemeenskappe te bevorder), 2) hoesonderdrukking tydens gewoone-asemhaling kan steeds 'n infeksierisiko verteenwoordig, en 3) partikeltellings en “colony forming units” (CFU's) word nie met hoesfrekwensie geassosieer nie. Verder, gedurende 'n tydperk van studie-ontwrigting as gevolg van COVID-19, het ons 'n kommentaar geskryf hoe pandemie-mandaat persoonlike asemhalingsbeskerming TB-oordrag kan verminder (Hoofstuk 2). Na twee weke se behandeling was daar 'n beduidende toename in partikeltellings tydens spontane hoes en verminderde aërosolaansteeklikheid in alle respiratoriese oefeninge. Tweedens (Hoofstuk 4), maak pasiënte se heterogeniteit dit moeilik om spesifieke Mtb op ‘n beheerde wyse te bestudeer. Daarom het ons ‘n “Sampler for In Vitro Aerosols (SIVA) ontwerp. BCGbakteria was geaërosoliseer en gekwantifiseer deur gebruik te maak van kultuur- en DNS-uitlesings. Derdens (Hoofstuk 5), is fluoressensiediasetaat (FDA) kleuring gebruik as 'n vinnige kleuringsmetode om lewensvatbare Mtb bakteria in sputum te identifiseer en met aansteeklikheid in kontakte te korreleer. Die gebruik van FDA op sputum was nog noiit voorheen tesame met aërosoluitlesings geëvalueer nie. Die tegniek was geassesseer in vergelyking met Auramine O en kultuur en ons het gewys dat FDA sensitiwiteit nie beïnvloed word deur konsentrasie van Mtb teenwoordig in die monster nie. TITAN demonstreer die gebruik van 'n nuwe “Human Aerosol Chamber” om TB-aërosol aansteeklikheid te bestudeer (Hoofstuk 2 en 3), en die doeltreffendheid van gesigbedekking. Dit het 'n direkte impak op hoe die Stad Kaapstad strategieë implementeer om teen TB-oordrag te veg. Die opgedateerde SIVA-model voeg waarde toe in hoe ons Mtb-aerobiologie en aansteeklikheid bestudeer en het groot toepaslikheid vir toekomstige studies.
- ItemThe 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.
- ItemDiagnostic accuracy of novel sputum and non-sputum-based tuberculosis diagnostics in HIV-positive patients initiating anti-retroviral therapy(Stellenbosch : Stellenbosch University, 2023-12) Ndlangalavu, Gcobisa; Theron, Grant; Reeve, Byron; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Tuberculosis (TB) is a major health problem killing millions of people worldwide and is the leading cause of death among people living with HIV (PLHIV). PLHIV are at high risk of TB due to their weakened immune systems and 18 times more likely develop TB than people without HIV. Therefore, early detection of TB among PLHIV is fundamental and dependent on good TB diagnostics. The study addressed barriers and limitations of TB screening (triage) and diagnosis, with some limitations being facility-based active case finding, which is challenging, and delayed patient results. To highlight limitations and utility of tests in the pipeline, current and novel TB diagnostics were extensively reviewed (Chapter 2). Additionally, we tested selected PLHIV initiating antiretroviral therapy (ART), in whom TB detection is difficult due to their paucibacillary nature, are negative by the World Health Organization (WHO) recommended four-symptom screen (W4SS), and unable to produce sputum (Chapters 3 to 5). ART initiators were included irrespective of W4SS status, to challenge the existing TB testing algorithm in PLHIV. Formerly, W4SS was exclusively recommended for triage for further TB testing even in PLHIV. Due to W4SS’ suboptimal specificity and poor implementation, alternative triage tools are required, which we investigated. This includes C-reactive protein (CRP) (Chapter 3). CRP (≥10mg/l) had higher specificity than W4SS in ART initiators and reduces unnecessary downstream testing. Since CRP levels varied within groups of the same culture status, Chapter 4 investigated clinical and demographic factors associated with CRP levels, thereby informing which types of patients might be missed or falsely included by CRP-based triage algorithms. Low CD4 cell count, culture-confirmed TB, and increased TBScore II were strongly associated with elevated CRP. Sputum Xpert MTB/RIF Ultra (Ultra) is the frontline rapid pulmonary TB test in South Africa, however, supporting data PLHIV, among whom Ultra may offer sensitivity improvement over Xpert MTB/RIF (Xpert) is limited. Sputum expectoration is challenging in PLHIV, and easily accessible urine can mitigate this. Chapters 3 and 5 investigated the diagnostic accuracy of Ultra using urine versus Determine TB LAM Ag test (LF-LAM). Their performance was similar. We further investigated urine Fujifilm SILVAMP TB-LAM (FujiLAM) diagnostic accuracy versus that of LF-LAM (Chapter 5). FujiLAM had increased sensitivity and comparable specificity versus LF-LAM. However, FujiLAM’s diagnostic accuracy varied between different manufacturer lots. In summary, CRP (>10mg/l) would reduce unnecessary expensive downstream TB testing. CRP increases case detection especially in key and vulnerable populations. CRP data from this study contributed to a meta-analysis that informed the latest screening guidelines for TB, changing decades old triaging approaches. Sputum-Ultra is more sensitive than Xpert and detects ~1/2 W4SS-negative cases. Our sputum-Ultra data contributed to a systematic review evaluating the diagnostic accuracy of Xpert and Ultra, irrespective of W4SS status, and was the only study with Ultra data among PLHIV. TB detection in outpatient PLHIV can be improved with FujiLAM implementation if improved versions of the tests would be available. Non-sputum-based confirmatory tests with improved sensitivity are urgently needed to improve case detection especially in sputum-scarce and W4SS-negative people.
- ItemThe microbiome and inferred function in pulmonary and extrapulmonary tuberculosis.(Stellenbosch : Stellenbosch University,, 2023-12) Nyawo, Georgina Rumbidzai; Theron, Grant; Naidoo, Charissa; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Background (Chapter 1): Each year, up to 3–4% of all deaths worldwide are attributable to infection with the bacterial pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease, which amounts to almost 5,000 deaths each day1. The human microbiome produces metabolites that influence the immune system. Health, therefore, depends on the microbiome, however, the significance of the microbiome in tuberculosis (TB) is just beginning to be understood. Hypothesis: The primary hypothesis of this study is that by conducting microbiome analysis, it is possible to differentiate unique microbial communities found in different disease sites among individuals with pulmonary and extrapulmonary tuberculosis, and this can establish relationships between specific microbial taxa and clinical markers such as CRP levels. Aims and objectives: This study aimed to analyze the microbiome and predicted metagenome of individuals examined for pulmonary or extrapulmonary tuberculosis in an area with a high prevalence of HIV. The goal was to understand the microbial composition and its links to clinical characteristics in this population. Methods (Chapters 2-5): We leveraged ongoing diagnostic trials involving individuals with different forms of TB to characterise the microbiome before treatment. A range of samples were collected (i.e., cough aerosols, oral washes, induced sputum, bronchoalveolar lavage fluid, stool, site-of-disease fluids) from presumptive pulmonary TB or extrapulmonary TB (EPTB) people. Background DNA sampling controls (i.e., saline flush of medical apparatus) were additionally collected, where appropriate. Microbial DNA underwent 16S rRNA gene sequencing and was analysed, and functional metagenomes were inferred. The microbiomes of people with confirmed TB were compared to symptomatic, whilst accounting for clinical factors such as HIV status. Results (Chapters 2-5): In Chapter 2, we show (using a FDA-approved commercial device, PneumoniaCheck) that cough aerosols from people with pulmonary TB 1) contain detectable microbial readouts, including mycobacterial readouts, 2) Mycobacterium tuberculosis complex (MTBC) is detectable by Xpert MTB/RIF Ultra in cough aerosols, and 3) microbiota present in cough aerosols are distinct from those in the upper and lower airways. As TB lymphadenitis (TBL) is the most common form of EPTB, chapter 3 describes the site-of-disease lymph microbiome in people with presumptive TBL compared to symptomatic controls. Briefly, this includes the existence of distinct microbial community states termed ‘lymphotypes’ in definite TBs, and include: "Prevotella-Corynebacterium," "Prevotella-Streptococcus," and "Mycobacterium." The Mycobacterium lymphotype was associated with more severe disease, suggesting disease progression from Prevotella-Corynebacterium and Prevotella-Streptococcus states. The co-occurrence of Corynebacterium and Streptococcus species contributes to TBL pathology. Furthermore, definite TBs were enriched in microbial metabolic pathways related to fatty acids, amino acids, and short-chain fatty acids (SCFAs). These pathways have known proinflammatory immunomodulatory effects that inhibit TB control, and the Mycobacterium lymphotype showed the highest enrichment in these pathways. Chapter 4 describes the site-of-disease microbiome in people with presumptive tuberculous pericarditis (TBP) as it is the deadliest manifestation of EPTB, compared to symptomatic controls. The pericardial fluid (PF) of individuals with TBP differs from those without TBP in terms of both the microbial diversity and the relative abundance of specific taxa (not only Mycobacterium but also other genera like Lacticigenium, Kocuria, and Weeksella). We noted that HIV infection is associated with differences in the abundance of Mycobacterium in PF. Additionally, within individuals with both HIV and TBP (dTBs), the presence of Bifidobacterium is associated with more severe disease, indicated by HIV-positivity and lack of antiretroviral therapy (ART). PF microbial differences between people with definite TB (dTBs) and people without TB (nTBs) are accompanied by variations in functional capacities. Notably, dTBs show an enrichment of pathways involved in SCFA production. Moreover, PF microbiome is linked to distinct clinical and chest imaging characteristics. In Chapter 5, we evaluate the relationship between CRP, the microbiome, and TB by looking at how microbiome members in presumptive EPTB correlate with CRP, as a surrogate for inflammation. We, thus, sought to relate the microbial differences in EPTB to inflammation, represented by levels of the inflammatory marker C-reactive protein (CRP), using site-of-disease samples from our study. In people with presumptive tuberculous lymphadenitis (TBL), those with TB or HIV displayed higher CRP levels, suggesting an interplay between microbial diversity and inflammation. Mycobacterium relative abundance was found to positively correlate with CRP levels, especially in certain lymphotypes. These associations were not observed in individuals with presumptive tuberculous pericarditis (TBP), indicating distinct microbial influences between different TB manifestations. Discussion and conclusion (Chapter 7): In conclusion, this study aimed to improve our understanding of the microbiome's association with TB, especially in vulnerable subpopulations such as HIV-positive individuals. It sheds light on various aspects of the microbiome's association with TB, highlighting how the site-of-disease microbiomes are altered in TB and how specific microbial taxa and metabolic pathways may influence TB pathogenesis and severity. Elevated CRP levels correlated with certain lymphotypes in TBL, indicating distinct microbial influences between manifestations. This study establishes a basis for future research into the microbiome as a potential diagnostic or prognostic marker for TB, a disease that continues to pose a major global health threat.