Browsing by Subject "TB"

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    A systematic review: the role of neuroinflammation as a pathway to injury in traumatic brain injury
    (2025) Vermeulen, Marcia; Schutz, Charlotte; Meintjes, Graeme
    Background: HIV-associated tuberculosis has a high mortality. Chest x-rays are an adjunct diagnostic tool for tuberculosis but has high inter-reader variability, which may be reduced with chest x-ray scoring systems. We analysed and scored chest x-rays of hospitalised patients with HIV-associated tuberculosis and assessed the relationship of these chest x-ray scores with 12-week mortality and biomarkers of tuberculosis dissemination. Methods: In this cohort study, the chest x-rays of adult patients, admitted with a new diagnosis of microbiologically confirmed HIV-associated tuberculosis were scored using the Timika scoring system. We excluded patients without a valid test result for the 3 biomarkers of tuberculosis dissemination (urine lipoarabinomannan, TB blood culture and urine Xpert); valid chest x-ray; or who were lost to follow up. Results: Amongst 364 included participants, 73 (20%) died and 291 (80%) survived. Median age was 36 years and median CD4 count 57cells/mm3. 25% of participants had normal chest x-rays. No association was found between chest x-ray score and dissemination score. Higher chest x-ray score was associated with higher hazards of death using a multivariate analysis: every 10-point increase in chest x-ray score resulted in 9% increased hazards of death. Conclusion: In this cohort, a higher Timika chest x-ray score was associated with higher hazards of death at 12-weeks.
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    Aetiology of pleural effusions diagnosed by routine culture and molecular techniques in children living in a high tuberculosis-endemic setting
    (2025) Wordui, Seyram; Gray, Diane; Zampoli, Marco
    BACKGROUND: Confirming aetiology of pleural effusion in children may be difficult in tuberculosis (TB)-endemic settings. We investigated the role of polymerase chain reaction (PCR) and routine biochemical tests in discriminating pleural effusion caused by bacteria from other aetiologies. METHODS: This is a post-hoc analysis of a cross-sectional study among children with pleural effusion in a tertiary hospital in South Africa, incorporating data from PCR testing of stored pleural fluid. Aetiological classification was defined by microbiological confirmation. RESULTS: Ninety-one children were enrolled, median age 31 months (IQR 12-102). Aetiology of pleural effusion was 40 % (n=36/91) bacteria, 11% (n=10/91) TB, 3% (n=3/91) viruses, 11% (n=10/91) polymicrobial and 35% (n=32/91) had no pathogen identified. The commonest pathogen was Staphylococcus aureus (n=27/91; 30%) with similar yields on culture and PCR, followed by Streptococcus pneumoniae (n=12/91; 13%), detected more commonly by PCR. PCR reduced the number of children with unconfirmed aetiologies from 48 to 32. Characteristics of children with no pathogen most resembled those with TB. Pleural fluid lactate dehydrogenase ≥1716 U/L best discriminated bacterial pleural effusion from other aetiologies (sensitivity of 86%; specificity 95%). CONCLUSION: PCR improved detection of pathogens and reduced number of children with unconfirmed aetiologies in presumed exudative pleural effusion.
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    Catching a glimpse: the visualization of Mycobacterium tuberculosis from TB patient bioaerosols
    (2023) Dinkele, Ryan; Warner, Digby; Gessner, Sophia
    Transmission between hosts is crucial for the success and survival of the obligate human pathogen and aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb). Despite this, little is known about how and when Mtb is aerosolized nor the key metabolic and morphological determinants driving successful transmission. To address these knowledge gaps, my doctoral research sought to develop a microscopic method for the detection of aerosolized Mtb following liquidcapture within the respiratory aerosol sampling chamber (RASC). This was achieved through the combination of the mycobacterial cell wall probe, 4-N,Ndimethylamino-1,8-naphthalimide-trehalose (DMN-tre), with the arraying of bioaerosol samples on bespoke nanowell devices amenable to fluorescence microscopy. With this method, a median of 14 live Mtb bacilli (range 0-36) were detected in 90% of confirmed TB patients following 60 minutes of bioaerosol sampling. Three distinct DMN-tre staining patterns were identified among aerosolized Mtb, strongly suggestive of metabolic heterogeneity. Moreover, a low proportion of patients produced Mtb in small clumps. These observations highlight the advantages of using microscopy over conventional culture- or molecular-based techniques for probing the metabolic and morphological characteristics of aerosolized Mtb. Applying this method in a second study, we sought to understand how and when Mtb is aerosolized. To this end, we aimed to compare the aerosolization of Mtb and total particulate matter from patients with TB during three respiratory manoeuvres: tidal breathing (TiBr), forced vital capacity (FVC), and cough. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three manoeuvres returned similar rates of positivity for Mtb. No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling manoeuvres. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic TB patients. Assuming the number of viable Mtb organisms detected provides a proxy measure of patient infectiousness, this method suggests that TiBr is a significant contributor to TB transmission. In developing a novel platform for the detection of aerosolized Mtb, this work has suggested the need to re-examine old assumptions about Mtb transmission.
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    Correlates of tuberculosis and non-tuberculosis morbidity and immunity in sub-Saharan African HIV-exposed, uninfected infants
    (2024) Iwase, Saori Christina; Jaspan, Heather; Happel, Anna-Ursula
    Background: Perinatal HIV transmission has been considerably reduced due to successful intervention programs. Consequently, there is a growing population of infants who are HIV-exposed but uninfected (iHEU), particularly in sub-Saharan Africa. These infants experience an increased risk of morbidity compared to infants who are HIV-unexposed and uninfected (iHUU), predominantly due to infectious diseases. Although the mechanisms underlying this increased vulnerability remain unclear, it may be associated with their altered immunity and/or gut microbiota. Bacillus Calmette-Guérin (BCG) vaccination is an effective intervention to prevent severe tuberculosis (TB) disease in children. BCG vaccination also enhances heterologous protective immunity against infections through epigenetic reprogramming of innate immune cells (known as “trained innate immunity”). However, whether iHEU receive comparable protection from BCG induced immunity against TB and non-TB infection as iHUU remains elusive. Gut microbiota plays a critical role in immune development during infancy. A close relationship between gut microbiota and vaccine responses has been reported in iHUU, including tetanus toxoid (TT) vaccination. However, a limited number of studies longitudinally investigated the effect of in utero HIV exposure on the gut microbiota, and results are often conflicting. In addition, not many studies have compared the trajectory of gut microbiota between iHEU and iHUU across multiple countries. While several studies have indicated reduced immune responses against TT vaccination in iHEU compared to iHUU, the interplay between HIV exposure, gut microbiota, and vaccine response is largely unexplored. Aims: In this dissertation, we examined three potential contributing factors that may underlie the higher risk of morbidity observed among iHEU in sub-Saharan Africa. The specific aims were to examine whether BCG affords the same protection against TB infection (TBI) and disease in iHEU (corresponds to Aim 1), effect of HIV exposure on longitudinal gut microbiota composition and its association with TT vaccine response (corresponds to Aim 2), and optimization of epigenetic assay protocol, intended for future investigation of BCG-induced histone modifications in iHEU (corresponds to Aim 3). Methods and results: To assess TBI prevalence among iHEU and iHUU, a total of 418 mother-infant pairs from South Africa and Botswana were included. All infants received BCG vaccination at birth as per standard of care. T-SPOT.TB (ELISpot-based interferon-gamma release assay) was performed using cryopreserved peripheral blood mononuclear cells (PBMCs) from infants aged 9-18 months. The prevalence of TBI did not differ by the infant HIV exposure status, with 10 cases (3.4%) among iHEU and four cases (3.2%) among iHUU, none with symptoms of active TB disease. This trend was the same across two different African countries where the burden of HIV and TB is high. However, because of the lower T-SPOT.TB positivity than initially anticipated, we were under powered to conclude the effect. To assess whether gut microbial succession alters immunity in iHEU, we profiled longitudinal gut microbiota composition and associated this with TT vaccine responses in 354 mother-infant pairs from South Africa and Nigeria. Stool samples were collected at 1 and 15 weeks of life, and 16S ribosomal ribonucleic acid (rRNA) gene sequencing was performed. Plasma IgG anti-tetanus antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). The effect of HIV exposure on infant gut microbiota composition was relatively modest compared to the impact of age and geographical factors. However, HIV exposure and specific gut microbes were independently associated with the TT vaccine response at 15 weeks of age. Results for South Africa and Nigeria differed, possibly due to higher maternal anti-tetanus IgG titers and hence infant baseline titers in Nigeria. To optimize an epigenetic assay that can be applied to infant samples, monocytes and natural killer (NK) cells were isolated from cryopreserved PBMCs using fluorescence-activated cell sorting (FACS). Cleavage Under Targets and Tagmentation (CUT&Tag) was optimized for assessing the histone modifications, acetylation of histone H3 at lysine 27 (H3K27Ac), trimethylation of histone H3 at lysine 4 (H3K4me3), and trimethylation of histone H3 at lysine 27 (H3K27me3; also used as a positive control). The optimized protocol was then applied to a subset of infant samples (n = 14; aged between six and seven weeks). Optimal input cell number, polymerase chain reaction (PCR) cycles, and sequencing depth were carefully determined for the CUT&Tag assay. These adjustments were necessary to achieve the assay's feasibility and data quality. The optimized CUT&Tag protocol and fine-tuned data analysis strategy successfully exhibited its capability to analyze multiple histone modifications using only 5,000 infant monocytes or NK cells as an input sample. Conclusions: Prenatal HIV exposure and gut microbiota may independently influence infant TT vaccine response. This supports the existing notion that iHEU exhibit altered immunity. Although previous studies have indicated that iHEU experience a higher risk of infection than iHUU, our data suggested that BCG vaccination was equally protective against TBI, irrespective of HIV exposure status. The optimized CUT&Tag protocol will offer a useful tool for investigating histone modifications using ultra-low input samples. This will be employed in the future study to explore whether iHEU exhibit comparable epigenetic modifications induced by BCG vaccination as for iHUU, providing valuable insight into whether iHEU receive similar non-specific protection from BCG vaccination compared to iHUU.
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    Diagnostic outcomes of CT-guided lung biopsy in an HIV and TB co-infection prevalent region
    (2024) van Rensburg, Juan Janse; Said-Hartley, Mariam Qonita
    Background: Tuberculosis (TB) and the Human Immunodeficiency Virus (HIV) are a major burden on the health of the people in the Western Cape with approximately 56 000 new cases of TB diagnosed in 2018 and 16 287 incident cases of HIV during the same period. CT-guided lung biopsy is a limited resource primarily used for diagnosing suspected cancer. TB is occasionally diagnosed on samples obtained by CT-guided lung biopsy. Objectives: To quantify the burden of TB on the CT-guided lung biopsy (CTLB) service at Groote Schuur Hospital (GSH). Secondary aims include summarising the diagnoses observed in an HIV-positive subset and comparison of diagnostic performance of different methods for TB diagnosis on biopsy samples. Method: A retrospective review of results from 406 consecutive CTLB performed at a tertiary hospital in the Western Cape during 2018 and 2019. Results: The mean age of subjects was 61 years, and the male to female ratio 1.1:1. A total of 40 specimens (9.9%) had confirmed tuberculosis, 264 (65%) had malignancy, and 3 had concurrent TB and malignancy. Conclusion: This study confirmed a high yield of TB among CTLB specimens as expected in a TB-prevalent region. There was also a higher proportion of TB and of non-TB non-malignant causes of lung nodules and masses in the HIV-positive subset as compared to HIV negative subjects.
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    Does the inclusion of the cost and burden of adverse drug reactions associated with drug-resistant TB treatment affect the incremental cost-effectiveness of new treatment regimens? A case study from the introduction of bedaquiline in South Africa National TB Programme
    (2018) Bistline, Kathryn Lou; Sinanovic, Edina; Firnhaber, Cynthia
    South Africa has one of the world’s highest burdens of TB, HIV/TB co-infection, and drug-resistant TB. Second-line TB treatment is less effective, more expensive, and more toxic than treatment for drug-sensitive TB. Nearly 1 in every 5 persons who starts treatment for drug-resistant TB in South Africa will die; 1 in every 3 persons who survives treatments experiences permanent, profound hearing loss. For decades there was little progress in TB research, however, and so treatment with old regimens continued despite safety concerns. In 2012 the US and European regulatory authorities approved a new drug, bedaquiline, but only for treatment in cases with no other options. In 2015, the South African Medicines Control Council approved bedaquiline for drug-resistant TB, but only a limited number of doses were approved in the 2016/2017 annual budget and the focus, again, was only for the patients who had no other options. In order to inform policy makers in planning and budgeting for drug-resistant TB treatment, the aim of this thesis was to determine whether the simple calculation that bedaquiline was too expensive relative to standard regimens using kanamycin was too simple. Particularly, given the high burden of adverse drug reactions (ADR) associated with kanamycin, would the inclusion of the cost and burden of ADR affect the incremental cost effectiveness ratio of a new treatment regimen where bedaquiline replaces kanamycin? Analysis of the national drug-resistant TB case register showed that mortality during second-line treatment was early, primarily in the first 6 months of treatment, even when patients do not have extensive drug resistance. HIV-positive patients not on anti-retroviral therapy (ART) at initiation of drug-resistant TB treatment have the highest risk of mortality. The high early mortality is a real risk that clinicians have to balance when deciding to initiate ART and effective second-line TB treatment both as quickly as possible. Daily injections coupled with taking more than 10 pills each day are a heavy burden for patient compliance, but also pose concerns in terms of overlapping and compounding toxicities; this burden was confirmed through a meta-analysis of the pooled frequency of adverse events among cohorts with at least 25% of the patients HIV-positive. A competing risk analysis of a cohort of drug-resistant TB patients from Johannesburg – addressing the reality that patients may not have experienced an ADR because they died rather than because they were at lower risk – indicated that HIV-infected patients who are not yet stable on ART and second-line TB treatment are at the highest risk of ADR. A Markov model built and parameterized using the data from the South African national TB programme indicates that bedaquiline for all drug-resistant TB led to a small gain in effectiveness at a cost that was under the costs of the drug itself, due to savings from daily injection visits. While cost-effective, it was not clear that South African policy makers needed to move beyond the offer of bedaquiline for patients with extensive drug resistance. However, the calculation, and the decision point, were different once the costs and disability associated with ADRs was included in the analysis. Bedaquiline-based regimens offer a cost-saving and more effective alternative to an injection-based regimen for drug-resistant TB patients treated in the public sector in South Africa.
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    Gastric lavage procedure animation
    (2010) Daya, Rupesh; Kibel, Maurice; Stent, Stacey
    This resource can be used to illustrate the gastric lavage procedure. Gastric lavage is the standard method of obtaining specimens for Tuberculosis (TB) diagnosis in young children. This animation can be used to demonstrate how this sensitive procedure is performed to medical students.
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    Generation of Liposomes to Study the Effect of Mycobacterium Tuberculosis Lipids on HIV-1 cis- and trans-Infections
    (2021-02-16) Pouget, Marion; Coussens, Anna K.; Ruggiero, Alessandra; Koch, Anastasia; Thomas, Jordan; Besra, Gurdyal S.; Wilkinson, Robert J.; Bhatt, Apoorva; Pollakis, Georgios; Paxton, William A.
    Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.
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    Hearing loss in the developing world: Evaluating the iPhone mobile device as a screening tool
    (2014) Peer, Shazia; Fagan, Johannes J
    BACKGROUND: Developing countries have the world's highest prevalence of hearing loss, and hearing screening programmes are scarce. Mobile devices such as smartphones have potential for audiometric testing. OBJECTIVES: To evaluate the uHear app using an Apple iPhone as a possible hearing screening tool in the developing world, and to determine accuracy of certain hearing thresholds that could prove useful in early detection of hearing loss for high-risk populations in resource-poor communities. METHODS: This was a quasi-experimental study design. Participants recruited from the Otolaryngology Clinic, Groote Schuur Hospital, Cape Town, South Africa, completed a uHear test in three settings - waiting room (WR), quiet room (QR) and soundproof room (SR). Thresholds were compared with formal audiograms. RESULTS: Twenty-five patients were tested (50 ears). The uHear test detected moderate or worse hearing loss (pure-tone average (PTA) >40 dB) accurately with a sensitivity of 100% in all three environments. Specificity was 88% (SR), 73% (QR) and 68% (WR). It was highly accurate in detecting high-frequency hearing loss (2 000, 4 000, 6 000 Hz) in the QR and SR with 'good' and 'very good' kappa values, showing statistical significance (p40 dB). It is highly sensitive for detecting threshold changes at high frequencies, making it reasonably well suited to detect presbycusis and ototoxic hearing loss from HIV, tuberculosis therapy and chemotherapy. Portability and ease of use make it appropriate to use in developing world communities that lack screening programmes.
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    Impact of immune reconstitution inflammatory syndrome on antiretroviral therapy adherence
    (2012) Nachega, Jean B; Morroni, Chelsea; Chaisson, Richard E; Goliath, Rene; Efron, Anne; Ram, Malathi; Maartens, Gary
    ObjectiveWe determined the impact of immune reconstitution inflammatory syndrome (IRIS) on antiretroviral therapy (ART) adherence in a cohort of 274 human immunodeficiency virus (HIV)-infected South African adults initiating ART.MethodsWe carried out a secondary analysis of data from a randomized controlled trial of partially supervised ART in Cape Town, South Africa. Monthly pill count adherence, viral suppression (HIV viral load < 50 c/mL), and IRIS events were documented. Poisson regression was used to identify variables associated with ART adherence below the median in the first 6 months of ART.ResultsWe enrolled 274 patients: 58% women, median age 34 years, median CD4 count 98 cells/μL, 46% World Health Organization clinical stage IV, and 40% on treatment for tuberculosis (TB). IRIS and TB-IRIS developed in 8.4% and 6.6% of patients, respectively. The median cumulative adherence at 6 months for those with an IRIS event vs no IRIS was 95.5% vs 98.2% (P = 0.04). Although not statistically significant, patients developing IRIS had a lower 6-month viral load suppression than those without IRIS (68% vs 80%, P = 0.32). ART adherence below the median of 98% was independently associated with alcohol abuse (relative risk [RR] 1.5; 95% confidence interval [CI] 1.2–1.9; P = 0.003) and IRIS events (RR 1.7; 95% CI 1.2–2.2; P = 0.001).ConclusionAlthough IRIS events were associated with slightly lower adherence rates, overall adherence to ART remained high in this study population. Concerns about IRIS should not deter clinicians from early ART initiation.
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    Investigating permeation of anti-mycobacterial agents in Mycobacterium tuberculosis and M. tuberculosis-infected macrophages in vitro as a model for early stage tuberculosis drug discovery
    (2021) Mabhula, Amanda N; Chibale, Kelly; Warner, Digby F
    Tuberculosis (TB) is the leading cause of death due to a single infectious disease and remains a major threat to global public health. The increasing emergence of multi-drug resistance to current anti-TB drugs, exacerbated by the long treatment duration, highlights the need for new effective treatments or strategies to shorten the treatment duration, improve patient adherence and curb the alarming rates of resistance. A key challenge to current strategies employed in the development of anti-TB drugs is the complexity in TB disease pathology which presents as a wide spectrum of lesions in patients presenting with the disease. These lesions occur at different anatomical loci in the same individual and at different stages as the disease progresses. In addition, the interaction between the causative agent, Mycobacterium tuberculosis, and its obligate human host induces physiologic and metabolic changes in the infecting bacillus that are specific to each lesion compartment, and dynamic. This is likely to influence M. tuberculosis susceptibility to antibiotic treatment and, consequently, affect treatment duration and possibly the development of drug resistance. A major limitation in current strategies to address this problem is translation of in vitro compound potency to in vivo efficacy. To reach the target site, a drug must first distribute and accumulate in the lesion microenvironments where bacteria reside: the macrophage host cell and the caseum. This thesis focused on the development of an in vitro infection model that could be used to predict drug penetration into M. tuberculosis-infected macrophages. Of particular interest was the extent to which host intracellular drug concentrations translate into effective antimycobacterial activity. To this end, the thesis comprised three key aspects: (i) characterization of physicochemical properties, antimycobacterial activities and M. tuberculosis-mediated metabolism of selected antiTB compounds; (ii) determination of intracellular drug permeation in resting, activated and foamy macrophages; and (iii) determination of the correlation (or not) between intracellular drug concentration and effective M. tuberculosis growth inhibition. The highly lipophilic natural product, fusidic acid (FA), its known human metabolite, 3-ketofusidic (3-ketoFA or GKFA37), and two C-3 alkyl esters (GKFA16 and GKFA17) as FA prodrugs were utilized in the study. In addition, another chemical class, the less lipophilic benzoxazole-based oxime derivatives were also investigated. Moxifloxacin (MXF), levofloxacin (LVF), bedaquiline (BDQ), rifampicin (RIF) and clofazimine (CFZ) were included for reference as known anti-TB drugs with varying lipophilicities. In chapter 2, FA and derivatives showed potent antimycobacterial activity (~1 µM) with selectivity indices (SI) >20 against the THP-1 macrophage cell line. Predicted artificial membrane permeability assay (PAMPA) results suggested that FA and derivatives would readily permeate the cell membrane. M. tuberculosis metabolized the C-3 alkyl-ester prodrug GKFA17 to form both FA and 3-ketoFA, with complete hydrolysis of the prodrug. FA was metabolized to 3-ketoFA, but the low levels of the metabolite suggested that another unidentified metabolite, presumed to be 3-epifusidic acid (3-epiFA), was formed. In vitro assays revealed that the potent benzoxazole-based oxime carbamates (PMN1-201, PMN1-136 and PMN2-09) were rapidly hydrolyzed by M. tuberculosis and were also susceptible to spontaneous degradation in media, forming the poorly active corresponding free oximes (PMN1-199, PMN1-135 and PMN1-157). In chapter 3, the in vitro macrophage drug uptake assay showed that FA C-3 alkyl prodrugs, GKFA16 and GKFA17, accumulated in significantly higher amounts in resting macrophages in comparison to FA and GKFA37. Accumulation of MXF was comparable to the least accumulated FA derivative, GKFA37, and showed steady state intracellular concentrations over a 6-day period. While GKFA16 and GKFA17 showed continued increasing accumulation, intracellular concentrations of FA and GKFA37 decreased after 48 hours, suggesting a likely susceptibility to macrophage efflux. In infected macrophages, the presence of intracellular bacteria or increasing bacterial burden did not affect the host cell ability to accumulate the drugs. FA and derivatives exhibited bacteriostatic inhibition of intracellular mycobacterial growth. MXF showed a potent bactericidal effect, reducing intracellular bacterial counts significantly at 10x MIC, with complete sterilization at 50x MIC even though MXF accumulation was significantly less than that of FA alkyl esters. These results suggested that both the inherent activity of a compound and ability to accumulate within host cells drive cellular efficacy. Given that the C-3 alkyl ester prodrugs accumulated at significantly higher concentrations than FA and GKFA37, this demonstrates the limitations of this assay in ascertaining the impact of intracellular concentration on drug efficacy for bacteriostatic drugs while highlighting its ability to correlate drug penetration and intracellular activity for cidal drugs. The prodrug GKFA17 was shown to undergo metabolism in resting host cells and during infection to form FA and then 3-ketoFA. Therefore, the prodrug strategy could be used to increase intracellular exposure of FA as GKFA17 showed superior macrophage accumulation. Benzoxazole-based oxime carbamates and their corresponding free oximes failed to accumulate in host macrophages and this was corroborated by their failure to control host cell bacterial growth despite the potent in vitro activity against M. tuberculosis of the carbamates, suggesting that they are poorly permeable. Chapter 4 investigated drug permeation in different macrophage phenotypes known to exist in the granuloma during TB disease, including foamy and activated macrophages. The activation state of the host cell did not affect the ability to accumulate anti-TB drugs such as RIF and BDQ. However, FA and its prodrug GKFA17 were significantly reduced in M1 activated macrophages. Despite the significantly reduced intracellular concentration, activated macrophages treated with FA and derivatives showed superior intracellular M. tuberculosis growth inhibition, suggesting that macrophage activation potentiates the activity of these compounds. In order to assess the effect of foamy macrophage lipid bodies (LBs) on drug uptake and intracellular localization, oleic acid-induced foamy macrophages were treated with selected antiTB drugs and experimental compounds. FA and derivatives showed early increased accumulation in foamy cells compared to resting macrophages, while MXF, BDQ and RIF levels were not significantly changed. Intracellular:extracellular (I/E) ratios increased with increase in lipophilicity, with FA C-3 alkyl prodrugs exhibiting the highest I/E ratios of >100. Despite exhibiting increased foamy macrophage concentrations, FA and derivatives exhibited a similar reduction (bacteriostatic) in bacterial counts in both resting and foamy macrophages. The intracellular activity of RIF was also not affected by presence of LBsin foamy macrophages. BDQ, LVF and MXF, however, showed reduced intracellular efficacy against M. tuberculosis in foamy macrophages compared to resting macrophages, suggesting a role for LBs to impact intracellular drug distribution. In conclusion, this thesis demonstrates the potential utility in combining advanced analytical methods and an in vitro infection model to determine cellular drug permeation profiles that might be applied to prioritize compounds and combinations optimized for distribution to target bacterial populations. This will facilitate well-informed decision-making processes in progression of lead compounds in pre-clinical development and, therefore, may offer the potential to reduce high rates of attrition of compounds which enter clinical phase of development.
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    Investigating the molecular pathogenesis of tuberculosis-associated immune reconstitution inflammatory syndrome
    (2025) Moseki, Raymond; Meintjes, Graeme; Wilkinson, Robert J.; Riou, Catherine; Lai, Rachel
    Thesis summary This thesis comprises a series of studies that adapted a reverse translational approach to investigate the mechanisms underlying the immunopathogenesis of paradoxical tuberculosis associated immune reconstitution inflammatory syndrome (TB-IRIS). Paradoxical TB-IRIS is an aberrant immune response targeted against Mycobacterium tuberculosis or its residual antigens in patients with advanced human immunodeficiency virus (HIV) infection shortly after commencement of antiretroviral therapy (ART). TB-IRIS is characterized by hyperinflammation and tissue damage. Pulmonary and lymph node involvement are the most common clinical manifestations. The incidence of TB-IRIS can reach up to 54% in settings where both HIV and TB are endemic, such as the sub-Saharan-Africa region of the African continent. Approximately 25% of patients with paradoxical TB-IRIS will require hospitalization for syndromic management. The management and prevention of paradoxical TB-IRIS rely on the use of anti-inflammatories such as immunosuppressive corticosteroid intervention, which is associated with an increased risk of Kaposi sarcoma. Paradoxical TB-IRIS is potentially fatal, with cases of central nervous system (CNS) involvement having the highest fatalities (up to 56%). The underlying cause of paradoxical TB-IRIS is thought to arise from a dysregulated hyperinflammatory immune response during ART-mediated immune recovery, although the causal mechanisms and associated molecular pathways are incompletely defined. This knowledge gap compromises efforts to develop new diagnostic and prognostic modalities as well as safer and more efficacious therapeutic interventions. This served as the rationale for undertaking the studies reported in this thesis. Using clinical samples from the PredART intervention trial and the TB-ART observational study, this thesis investigated the cellular and molecular determinants of paradoxical TB-IRIS. Conventional scientific hypothesis-based approaches as well as unbiased hypothesis generating approaches were used, with major experimental approaches including ribonucleic acid (RNA) sequencing, flow cytometry and soluble analyte analyses. RNA extracted from whole blood of consenting participants from the Pred-ART intervention trial was sequenced to profile global gene expression. Cross-sectional and longitudinal analyses were performed to compare transcriptomic profiles in participants who developed paradoxical TB-IRIS to those who did not, at week 0, 2 and 12 on ART. Using pathway analysis, samples collected prior to the initiation of ART (week 0) in patients who later developed paradoxical TB-IRIS (cases) were characterized by the upregulation of transcripts encoding neutrophil-derived antimicrobial peptides and defensins and associated with hypoxemia compared to the TB-non-IRIS controls. These largely reflected biological processes involved in neutrophilic responses and adaptation to metabolic stress and dysfunction. Furthermore, pathways that were significantly downregulated in patients who later developed paradoxical TB-IRIS compared to those who did not, included chondroitin sulfate biosynthesis and interleukin (IL)-6. These finding suggest that paradoxical TB-IRIS is preceded by increased expression of antimicrobial peptides and the impairment of immune cellular function controlled by the extracellular matrix. Pathway analysis at week 2 on ART, which coincided with the median time of clinical manifestations in patients that developed paradoxical TB-IRIS, revealed the significant enrichment of neutrophil degranulation, interleukin (IL)-1 signalling, as well as type I and II interferon (IFN). Moreover, biological pathways that were significantly downregulated in cases as opposed to controls at week 2 on ART were largely overrepresented by epigenetic biological processes. These data suggested that neutrophil degranulation preceded and characterized the onset of paradoxical TB-IRIS. To validate these findings, we evaluated the associations between absolute neutrophil counts and neutrophil soluble markers, with paradoxical TB-IRIS at week 0 and week 2 on ART. No significant differences in absolute neutrophil counts were reported in participants who later developed paradoxical TB-IRIS compared to those who did not at week 0. However, azurocidin measured in plasma samples by enzyme-linked immunosorbent assay (ELISA) was significantly lower in samples from participants who later developed paradoxical TB-IRIS at week 0. This suggests that participants who are likely to develop paradoxical TB-IRIS might have impaired innate immune responses at week 0 compared to those who did not develop the syndrome. Absolute neutrophil counts were higher in cases compared to controls at week 2 on ART. Soluble neutrophil markers including human neutrophil peptide 1-3, myeloperoxidase, and neutrophil elastase were significantly higher in cases compared to controls at week 2 on ART. These data suggest that patients who develop paradoxical TB-IRIS have heightened neutrophil degranulation at the time of symptoms onset. Longitudinal perturbation of gene expression was investigated between week 0 and week 2 in people who developed paradoxical TB-IRIS and TB-non-IRIS controls, respectively. Among the top significantly upregulated biological pathways in samples of participants that developed paradoxical TB-IRIS were neutrophil degranulation, type I and II interferon signaling, IL-1 cytokine signalling, and pyroptosis. Biological pathways that were significantly downregulated in these patients included heme biosynthesis and oxygen-carbon dioxide gaseous exchange signalling metabolic dysfunction. In TB-non-IRIS controls, biological pathways that were significantly upregulated included epigenetic modulation of gene expression, collagen biosynthesis and extracellular cytoskeleton remodelling indicating anabolic processes that restore or maintain proper cellular functionality such as cellular signalling. Type I interferon signalling and immune responses to viral pathogens were among biological pathways that were significantly downregulated in people who did not develop paradoxical TB-IRIS at week 2 on ART compared to week 0, likely indicating decrease in antiviral responses as HIV viral load declined. The management and prevention of paradoxical TB-IRIS rely on the use of corticosteroids which is associated with an increased risk of Kaposi Sarcoma and herpes reactivations. Thus, there is a need to explore other anti-inflammatory but non-immunosuppressive drugs that can be repurposed for the management of IRIS-related pathologies. Gene expression data at week 2 on ART and previous published data indicated that aberrant inflammasome activation represent one of the molecular mechanisms that underpins the pathogenesis of paradoxical TB IRIS. We proposed that chemical inhibition of the inflammasome would result in a targeted reduction of inflammation. Two candidate drugs (anakinra and parthenolide) were evaluated and benchmarked against the standard of care (prednisolone) in an ex vivo mycobacterial cell stimulation model using patient samples. Both anakinra and parthenolide significantly reduced several markers of inflammation compared to prednisolone. However, parthenolide was the more potent in reducing cytokine production including IL-1 among others. These findings provide experimental evidence of in vitro use of novel anti-inflammatory agents. Anakinra has been approved for clinical use in inflammatory conditions such as rheumatoid arthritis and could potentially be repurposed for use in the context of paradoxical TB-IRIS. A hallmark of paradoxical TB-IRIS includes the partial reconstitution of Mtb-specific CD4 T cell responses within 3 months of initiating ART while on effective antituberculosis therapy. The phenotype and transcription factors of antigen-specific interferon-gamma producing (IFN+) CD4+ T cells and their potential involvement in the pathogenesis of paradoxical TB IRIS were characterized in an experimental mouse model of IRIS (by collaborators) as well as in human peripheral blood mononuclear cells (PBMC). In mice, antigen-specific CD4 T cells knocked out for Eomes showed significantly higher proportion of survival compared to wildtype mice. No significant differences in Eomes expression were observed in Mtb-300 specific, IFN+ CD4 T cells in PBMC from paradoxical TB-IRIS cases compared to controls at the onset of clinical symptoms. However, Eomes expression was higher in cases compared to controls in bulk CD4 T cells at week 2 on ART. HLA-DR expression was higher in cases compared to controls in Mtb300-specific IFN+ CD4 T cells at week 2 on ART. Additionally, Granzyme B producing, Mtb300-specific IFN+ CD4 T cells co-expressing Eomes and Tbet were significantly higher in cases compared to controls at week 2 on ART. These data indicate that HLA-DR expression on Mtb300-specific CD4 T cells producing IFN+ can potentially be developed as a diagnostic tool to identify individuals with paradoxical TB-IRIS. Collectively, these findings highlight the key contribution of neutrophils and Mtb-specific CD4 lymphocytes in the pathogenesis of paradoxical TB-IRIS. The gene expression findings suggest that the underlying mechanism involves interferon stimulated genes which trigger the secretion of proinflammatory cytokines and the aberrant activation of the inflammasome. Members of the IL-1 family of cytokines are secreted as catalytically inactive proenzymes that require the action of mature inflammasome for activation. IL-1a is the only exception since its proform and mature form are both fully functional. Active IL-1 family of cytokines are potent modulators of inflammation which is a defining feature in people who develop paradoxical TB-IRIS at the time of clinical symptom onset. In addition, the pathogenesis of paradoxical TB-IRIS appears to be mediated by the heightened degranulation of neutrophils which may cause acute inflammation and tissue damage.
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    Newer and novel sputum versus non-sputum-based tools for the diagnosis of active tuberculosis in different patient sub-populations
    (2025) Esmail, Aliasgar; Dheda, Keertan
    Background Pulmonary tuberculosis (PTB) has a spectrum of presentation ranging from sub clinical/minimally symptomatic disease on one end, usually in community-based patients, to overt symptomatic TB with hospitalisation on the severe end of the spectrum. This spectrum of sub-clinical and clinical presentation of TB along with the site of TB (pulmonary vs. Extra-pulmonary TB) generate a number of sub-populations. Current diagnostic tools are not a ‘one size fits all' and have important differential limitations in these patient sub-populations including those with pauci-bacillary disease (e.g., in HIV-infected patients, minimally symptomatic patients with low burden disease in the community, and in patients with extra-pulmonary TB). Thus, the overarching objective of this PhD was to evaluate the diagnostic performance of frontline tests, including the newer and more sensitive Xpert Ultra and urine LAM in key active TB patient sub-populations (wherein data are limited). The main themes and sub-populations included in my thesis are as follows: 1) Hospitalised HIV-Infected patients (patients in the severe end of PTB spectrum): To determine the optimal and most cost-efficient diagnostic strategy incorporating sputum and LAM (lipoarabinomannan) for the detection of TB (chapter 1). 2) Smear-negative TB: Independent confirmation on the performance of the more sensitive Xpert Ultra (Cepheid MTB/RIF Ultra) in sputum archived samples (chapter 2). 3) Community-based minimally symptomatic patients (patient in the non-severe end of the PTB spectrum): Evaluation of point-of-care (POC) sputum Xpert Ultra for detection of TB in minimally symptomatic/at-risk population for TB and its impact in detecting potentially infectious patients (chapter 3). 4) Extra-pulmonary TB (TB serositis): Evaluation on the performance of conventional diagnostic tools, including Xpert Ultra in tuberculous pericarditis (chapter 4) and pleuritis (chapter 5) compared to a novel immunodiagnostic tool (IRISA-TB). Methods (sub-populations are underlined) In Chapter 1, I describe an evaluation of an algorithm that describes the optimal & cost efficient strategy to combine sputum GeneXpert and urine LAM (tests that are recommended by the WHO) in a hospitalized HIV-infected sub-population: This comprised a post-hoc analysis of 561 HIV-infected sputum-expectorating patients that was part of a larger parent trial. 5 different diagnostic strategies using sputum culture as a reference standard were explored (Xpert alone, LAM alone, sequential Xpert followed by LAM and vice versa [LAM in Xpert-negative patients and Xpert in LAM-negative patients], and both tests concurrently [LAM + Xpert]). A cost-consequence analysis was also performed. In Chapter 2, I evaluated the performance of GeneXpert Ultra in 272 selected and well characterized archived sputum samples including 104 patients with smear negative TB and 102 non-TB with a history of previous TB to accentuate and evaluate the clinical significance of trace readouts (i.e., a readout that usually corresponds to the detection of a very small amount of TB DNA). Assay-specific limit-of-detection (LOD) experiments were conducted using serial dilutions of Mycobacterium tuberculosis H37Rv to confirm the assay's detection threshold. In In Chapter 3, I evaluated the diagnostic performance of point-of-care (POC) Xpert Ultra for the detection of TB in minimally symptomatic community-based sub-population: 5,274 participants were rapidly screened to enrol 584 patients with suspected pulmonary TB from peri-urban high burden communities of Cape Town, South Africa. The utility of POC-Xpert Ultra in detecting likely infectious patients was also specifically evaluated. In Chapter 4, I described the diagnostic performance of pericardial fluid unstimulated interferon gamma (measured using the novel IRISA-TBTM test) and compared it to other same-day tests including Xpert Ultra and adenosine deaminase (ADA) in 99 South African patients with suspected pericardial TB using a composite reference standard including pericardial fluid, pericardial tissue TB culture, pericardial tissue histopathology and response to TB treatment. Similarly, In Chapter 5, I described the diagnostic performance of pleural fluid unstimulated interferon gamma (measured using the novel IRISA-TBTM test) and compared to other same-day tests including Xpert Ultra and adenosine deaminase (ADA) in 207 individuals from Cape Town, South Africa and Vellore, India. A composite reference standard including pleural fluid, pleural tissue TB culture, pericardial tissue histopathology and response to TB treatment was used to define the composite reference standard for TB. Results: Chapter 1: In the HIV-infected hospitalised patient sub-population, the incremental yield of LAM over Xpert was 29.6% (45/152) and that of Xpert over LAM was 75% (84/11). The incremental yield of LAM increased with decreasing CD4 count. The costs per TB case diagnosed were similar for the sequential and concurrent strategies ($1,617 to $1,626). Chapter 2: Xpert Ultra had a lower sputum-specific LOD compared to that of the G4 version of Xpert MTB/RIF (9 vs. 184 cfu/mL). 94% in both forms of EPTB in chapters 4 & 5. Conclusions: Overall, the performance of currently available diagnostic tools varied in different subpopulations due to differential mycobacterial load, the compartment interrogated, patient phenotype (e.g. HIV-infected), testing strategy, and clinical context (e.g. hospitalized versus community-based). Main findings from each chapter as summarized as follows: Main conclusion in chapter 1: Xpert-Ultra had a considerably lower limit-of-detection compared to older Xpert-MTB/RIF assay, however, trace-readouts in the context of previous TB, reduced the specificity of the assay slight (<5%). A third of the trace results (3/9) were likely false positive. Main conclusion in chapter 2: In sputum-expectorating hospitalized patients with advanced HIV and access to both Xpert and LAM, concurrent testing with sputum Xpert and urine LAM may be the best and most cost-effective strategy for diagnosing TB in this sub-population. Main conclusion in chapter 3: POC-Xpert Ultra, when used as part of a community-based ACF, missed ~50% of patients with culture-positive TB. However, Xpert detected almost all likely infectious cases. Main conclusions in chapters 4 & 5: Conventional TB diagnostic tests performed poorly in pericardial and pleural TB. IRISA-TBTM, a novel same-day immunodiagnostic test, outperformed Xpert Ultra for the diagnosis of pericardial and pleural TB. These data have implications for the clinical utility of newer diagnostic tools in patient sub-populations from TB and HIV endemic settings.
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    Nonlinear mixed-effects modelling of drug-drug interactions between antiretroviral therapy and tuberculosis treatment
    (2025) Kengo, Allan; Denti, Paolo; Resendiz Galvan, Juan Eduardo
    Human immunodeficiency virus (HIV) remains a significant global health challenge that affected approximately 39 million individuals in 2022, with majority residing in Africa. Among people with HIV (PWH), co-infection with tuberculosis (TB) is a leading cause of death. However, the concurrent treatment of HIV and TB often results in drug-drug interactions (DDIs), mediated especially by rifampicin, a key component of the TB regimen and potent enzyme and transporter inducer. These DDIs may compromise treatment safety and efficacy, potentially leading to therapeutic failure and increased risk of drug resistance. In this thesis, we utilized non-linear mixed effects modelling and data from studies in PWH and healthy volunteers to characterize DDIs between first- and second-line antiretroviral (ARV) and anti-TB drugs. Additionally, we performed simulations to assess treatment target attainment following current dosing recommendations in PWH. Our pharmacokinetic model of standard- and high-dose rifampicin in PWH identified lower bioavailability of the top-up capsule formulation as the cause of lower-than-expected drug exposures in participants on high-dose rifampicin. Furthermore, the reduced dolutegravir exposures in participants on concurrent high-dose rifampicin, compared to those on the standard-dose, were attributed to reduced bioavailability rather than enhanced clearance. Notably, our simulations demonstrated that doubling the dosing frequency of dolutegravir effectively counteracted the DDI with both standard- and high-dose rifampicin. Secondly, we characterized the DDI between ritonavir-boosted atazanavir (ATV/r) and rifampicin, both in plasma and within peripheral blood mononuclear cells (PBMCs). Rifampicin increased the clearance of ATV/r by threefold, and doubling the dosing frequency of ATV/r was sufficient to counteract this interaction and restore treatment target attainment. Notably, rifampicin did not affect atazanavir equilibration or accumulation in PBMCs, suggesting that plasma studies can reliably reflect intracellular processes. We also applied our model to an external dataset, estimating a twofold decrease in atazanavir clearance, likely due to ritonavir co-administration. Lastly, we found clofazimine, a second-line drug resistant TB (DR-TB) drug, to increase the clearance of levofloxacin by 15% but not affect the pharmacokinetics of cycloserine, linezolid, or isoniazid. This confirmed that clofazimine can be safely co-administered with other DR-TB drugs, as it poses minimal risk of significant DDIs. In conclusion, non-linear mixed effects modelling can be used to evaluate DDIs, and we recommend its incorporation in routine dose optimization and therapeutic drug monitoring programs to enhance treatment outcomes.
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    Rapid urine-based screening for tuberculosis to reduce AIDS-related mortality in hospitalized patients in Africa (the STAMP trial): study protocol for a randomised controlled trial
    (2016) Gupta-Wright, Ankur; Fielding, Katherine L; van Oosterhout, Joep J; Wilson, Douglas K; Corbett, Elizabeth L; Flach, Clare; Reddy, Krishna P; Walensky, Rochelle P; Peters, Jurgens A; Alufandika-Moyo, Melanie; Lawn, Stephen D
    Abstract Background HIV-associated tuberculosis (TB) co-infection remains an enormous burden to international public health. Post-mortem studies have highlighted the high proportion of HIV-positive adults admitted to hospital with TB. Determine TB-LAM and Xpert MTB/RIF assays can substantially increase diagnostic yield of TB within one day of hospital admission. However, it remains unclear if this approach can impact clinical outcomes. The STAMP trial aims to test the hypothesis that the implementation a urine-based screening strategy for TB can reduce all cause-mortality among HIV-positive patients admitted to hospital when compared to current, sputum-based screening. Methods The trial is a pragmatic, individually randomised, multi-country (Malawi and South Africa) clinical trial with two study arms (1:1 recruitment). Unselected HIV-positive patients admitted to medical wards, irrespective of presentation, meeting the inclusion criteria and giving consent will be randomized to screening for TB using either: (i) ‘standard of care’- testing of sputum using the Xpert MTB/RIF assay (Xpert) or (ii) ‘intervention’- testing of sputum using Xpert and testing of urine using (a) Determine TB-LAM lateral-flow assay and (b) Xpert following concentration of urine by centrifugation. Patients will be excluded if they have received TB treatment in the previous 12 months, if they have received isoniazid preventive therapy in the last 6 months, if they are aged <18 years or they live outside the pre-specified geographical area. Results will be provided to the responsible medical team as soon as available to inform decisions regarding TB treatment. Both the study and routine medical team will be masked to study arm allocation. 1300 patients will be enrolled per arm (equal numbers at the two trial sites). The primary endpoint is all-cause mortality at 56 days. An economic analysis will be conducted to project long-term outcomes for shorter-term trial data, including cost-effectiveness. Discussion This pragmatic trial assesses an intervention to reduce the high mortality caused by HIV-associated TB, which could feasibly be scaled up in high-burden settings if shown to be efficacious and cost-effective. We discuss the challenges of designing a trial to assess the impact on mortality of laboratory-based TB screening interventions given frequent initiation of empirical treatment and a failure of several previous clinical trials to demonstrate an impact on clinical outcomes. We also elaborate on the practical and ethical issues of ‘testing a test’ in general. Trial registration ISRCTN Registry (ISRCTN71603869) prospectively registered 08 May 2015; the South African National Controlled Trials Registry (DOH-27-1015-5185) prospectively registered October 2015.
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    Retrospective review of urinary lipoarabinomannan (ULAM) in the diagnosis of disseminated tuberculosis (TB) in medical inpatients at a district level hospital in Cape Town
    (2025) Erwee, Christie; Dlamini, Sipho
    Background: Mycobacterium tuberculosis (MTB) and Human Immunodeficiency Virus (HIV) remain a major public health concern in South Africa (SA) with high mortality rates in patients with MTB and HIV co-infection. In 2016 Urinary Lipoarabinomannan (ULAM) became available in SA to expedite the diagnosis and treatment of Tuberculosis (TB) in this vulnerable population. Objectives To describe the experience at a district level hospital following the availability of ULAM testing for investigating HIV positive TB suspects. Firstly, to define the population and clinical outcomes in this cohort and secondly to assess if the hospital's standard operating procedure (SOP) regarding ULAM testing was followed and what additional MTB investigations were performed. Methods An observational, retrospective cohort study of patients who underwent ULAM testing at Mitchells Plain District Hospital (MPDH) was conducted from 2016 until 2018. The following data was collected: age, gender, CD4 count, viral load (VL), whether Antiretroviral Treatment (ART) naïve or interrupted and duration of ART if on treatment. Additionally, MTB diagnostic tests were performed and finally, patient clinical outcomes with regards to length of stay (LOS) and 30-day mortality and whether TB treatment was initiated. Descriptive statistics were used to analyse the data. Results 324 participants were included in the study. 54.6% of participants were female and the median age of the entire cohort was 36 (IQR 30-34). Median CD4 count was 41 (IQR 16-76) with high viral load of log 4.9 (IQR 3.9-5.4). 44.1% of participants were ART naïve, 36.1% of participants were on treatment and 19.8% had interrupted treatment. The median duration of ART was 0.6 years (IQR 0.1-2). 750 MTB specific tests were performed in the cohort, 48% of the participants initiated on TB treatment had positive microbiological results proving MTB infection. Overall 30-day mortality was 12.7%. Conclusions This study showed participants who were severely immunocompromised with little ART exposure and a mortality rate of 12.7%. Patients were frequently initiated on empiric MTB treatment with a proportion of participants not being adequately investigated, particularly in the LAM negative group. Sputum was the most frequent MTB specific test performed. Clinicians adhered to the SOP put in place to guide the use of ULAM.
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    Strategies for optimising TB-HIV co-treatment in children and adolescents with drug-susceptible tuberculosis on rifampicin-based standard regimens
    (2025) Chabala, Chishala; Mcilleron, Helen
    Children with HIV are highly susceptible to tuberculosis (TB) and often require concurrent antiretroviral treatment (ART) and antituberculosis therapy. However, rifamycins, the key drugs for TB treatment, significantly interact with many ART classes, necessitating adjustments to one or both therapies. In African and other low-resource settings, modifications typically involve ART regimens due to constrained drug options. This thesis presents four pharmacokinetics and one clinical study evaluating strategies for HIV/TB co-treatment in children and adolescents receiving rifampicin-based TB therapy, nested within two clinical trials across four African countries. First, we assessed the World Health Organization's (WHO) weight-band dosing recommendations for first-line TB drugs using fixed-dose combination tablets. Next, we examined two dose-adjustment strategies involving lopinavir/ritonavir, a widely available protease inhibitor for first- and second -line ART, using both liquid and tablet formulations. We then investigated a twice-daily dosing regimen of tenofovir alafenamide for children co-treated with rifampicin. Lastly, we evaluated clinical outcomes in children with HIV/TB and non-severe TB who received standard antituberculosis dosing. Our findings show that while WHO revised dosing guidelines improve TB drug exposure in children, target levels are not consistently achieved across weight bands; children in the lowest and highest weight categories experience lower exposures compared to adult reference targets. For dose modification, the use of modified 8-hourly lopinavir/ritonavir liquid in younger children was ineffective, whereas double-dosing with tablet formulations provided adequate exposures in older children and adolescents, countering rifampicin's effects. Twice-daily tenofovir alafenamide dosing counteracts rifampicin's inducing effects but requires caution when administered with adjusted protease inhibitor doses. Finally, we found that children with HIV/TB who were treated for non-severe TB had worse treatment outcomes compared to their HIV- uninfected counterparts, but they could also receive a shorter antituberculosis treatment as the duration of treatment did not impact their tuberculosis outcomes. In summary, optimizing HIV/TB co-treatment for children is a constantly evolving area that must adapt to advances in both HIV and TB care. This research will contribute to the understanding of strategies that can be used to optimise the treatment of children living with HIV/TB for improved clinical outcomes.
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    Targeted depletion of RibF, a putative bifunctional FAD synthetase/ flavokinase in Mycobacterium smegmatis using CRISPR interference
    (2020) Raphela, Mabule Lucas; Warner, Digby F; Chengalroyen, Melissa D
    Tuberculosis (TB) is the leading killer globally owing to an infectious disease. There is consequently an urgent need to develop novel TB drugs and shorter regimens to treat the causative agent, Mycobacterium tuberculosis, an imperative which demands the identification of new drug targets in essential mycobacterial pathways. To that end, the work presented in this dissertation aimed to functionally characterize ribF, an essential gene in the mycobacterial riboflavin (RF; vitamin B2) biosynthetic pathway. Given the role of RF as a core component of the essential flavin cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), it was hypothesized that silencing ribF would disrupt the biosynthesis of all flavoproteins, crippling numerous (essential) processes within the organism. Moreover, based on previous observations in Bacillus subtilis, it was predicted that the mycobacterial ribF homolog might play a role in regulating the rib operon (comprising a cluster of RF pathway genes) – either directly by binding to the FMN riboswitch, or indirectly through the production of FMN from RF, in turn enabling riboswitch-mediated repression of downstream genes. CRISPR interference (CRISPRi) technology was used to generate an anhydrotetracycline (ATc)-inducible ribF hypomorph of M. smegmatis, a widely exploited mycobacterial model. Consistent with other organisms, ribF was shown to be essential for in vitro growth of M. smegmatis: CRISPRi-mediated depletion of ribF was bacteriostatic, resulting in a 10-fold growth inhibition in liquid media and corresponding to no reduction (0 log-fold change) in colony forming units (CFU). Moreover, targeted metabolomic analyses revealed that ribF depletion was associated with accumulation of 6,7-Dimethylribityllumazine (DMRL), suggesting that the disruption of RibF function blocked conversion of RF to the essential cofactors, FMN and FAD, in turn inhibiting cell growth. Notably, the lethality of ribF depletion could not be complemented chemically by exogenous supplementation of growth media with RF, FMN or FAD. Downregulation of ribF also caused enhanced susceptibility to the known cell wall-targeting agent, vancomycin, but not to the putative RibF domain inhibitor, thonzonium bromide, suggesting an alternative mechanism of action or impaired bacillary permeation. In summary, these data support the inferred essentiality of ribF in mycobacteria, in turn supporting future work which aims to target this enzyme for new TB drug discovery.
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    The diagnostic pathway to a surgical lymph node excision biopsy service in a HIV and TB endemic region in a Western Cape Tertiary Institution
    (2025) Potelwa, Tennis Tomo Camagu; Verburgh, Estelle; Malherbe, Francois
    In the HIV/TB endemic public care setting of the Western Cape, diagnostic consideration of patients with persistent lymphadenopathy is focused towards extra- pulmonary tuberculosis (EPTB), more than other infectious or malignant causes of lymphadenopathy. We investigated patients consecutively referred for lymph node excision at Groote Schuur Hospital for selection of, and results of, laboratory tests performed during the diagnostic pathway and possible impact on diagnostic delay. Eighty-six patients were included, 61 patients (71%) had no previous diagnosis to explain the lymphadenopathy, while 25 patients had a previous diagnosis of a haematological malignancy, cancer or tuberculosis. In the new patient group, EPTB was the commonest diagnostic outcome (24.6%, 15/61), followed by lymphoma (21.3%, 13/61) and cancer (14.8%, 9/61). HIV positive patients constituted 41% (n=25). Median time from presentation with lymphadenopathy to first excision biopsy was 55 days (IQR 22-106). Fine needle aspiration (FNA) cytology of lymphadenopathy was performed in 30/61 (49%) of patients and repeated in a third of these, while smear for AFBs and culture for M. tuberculosis were infrequently performed and GeneXpert MTB/RIF assay on FNA never performed. Furthermore, in the seven patients with a final diagnosis of lymphoma in whom FNA cytology was performed, cytology was not diagnostic of lymphoma. In patients with persistent lymphadenopathy, this study demonstrates how poorly structured diagnostic pathways contribute to unnecessary health care utilisation and diagnostic delay in readily treatable conditions. We need to implement accurate diagnostic pathways for patients with lymphadenopathy in South Africa's healthcare system, thus improving early diagnosis of both EPTB and lymphoma, potentially improving patient outcomes.
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    The role of antibodies in tuberculosis
    (2024) Jacobs, Ashley; Wilkinson, Robert
    The role of antibodies in tuberculosis (TB) has been debated for over a century. Antibodies against Mycobacterium tuberculosis (M.tb) are detectable in persons who appear to resist M.tb infection, and yet humoral immune activation is a consistent biomarker of TB disease progression. Antibody responses to TB could therefore be a dual-edged sword, whereby antibodies promote TB pathogenesis, but some people produce antibodies that contribute to immunity against M.tb. In this thesis, I have therefore investigated the role of antibodies in TB in multiple clinical cohorts. Firstly, I generated fully human monoclonal antibodies (mAbs) against M.tb from patients with ATB. I identify the antigen of one mAb as the secreted antigen GlnA1, and then show that this mAb promotes in vitro production of TNF-α, IL-6, and IL-1β in the absence of mycobacterial killing. Secondly, I investigated antibody responses against M.tb in persons who live with HIV (PLWH). PLWH fail to mount a significant increase in IgG levels against M.tb in ATB. M.tb-specific IgG responses are also detectable in different cohorts of PLWH with negative interferon gamma release assay (IGRA) tests. However, greater levels of M.tb-specific IgG associates with IGRA conversion. Thirdly, I showed the development of flow cytometric assays to study opsonization and antibody dependant cellular phagocytosis (ADCP) of live mycobacteria in BCG vaccinated adults. These methods show that BCG vaccination induces ADCP 28 days post-vaccination. Fourthly, I tested whether unswitched memory B cells, and specifically the marginal zone B cells (MZB) subset, are impacted by ATB. MZB respond to capsular pathogens and could thus recognize the M.tb cell wall. MZB are found to be depleted in ATB but are not enriched at the site of disease in PLWH with pericardial TB. The depletion of unswitched memory B cells in TB observed in this study resembles that reported in autoimmune disease. Taken together, I provide support to the notion that at least some antibodies against M.tb could contribute to immunopathology in TB. The fact that PLWH are at greater risk of disseminated TB, and mount less of an antibody response, however, supports a role for antibodies in preventing disseminated disease that remains to be studied.