Electronic Theses and Dissertations (Masters)

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    The usefulness of monocyte fluorescence as a biomarker of Tuberculosis infection at Chris Hani Baragwanath Academic Hospital
    (University of the Witwatersrand, Johannesburg, 2023-11) Moosa, Aamina Yunus; Vaughan, Jenifer; Hodkinson, Katherine
    Introduction: South Africa has the 5th highest burden of Tuberculosis (TB) as well as coinfection with Human immunodeficiency virus (HIV) worldwide. Routine laboratory methods have varying sensitivity and specificity. The Xpert MTB/RIF (GXPU) (Cepheid, Sunnyvale, CA), has lower sensitivity in sputum smear negative cases and poor quality sputum samples. A robust, non-sputum based, inexpensive biomarker of TB would be of value in such cases. Monocytes are the major leucocyte involved in the immune response to TB. The Sysmex haematology analysers (Sysmex, Kobe, Japan) measure monocyte activation via monocyte fluorescence (MO-Y). This study aimed to evaluate the MO-Y and other Sysmex extended differential parameters (EDPs) as biomarkers of TB infection in the local setting. Methods: The MO-Y and EDPs were retrieved from the analyser for 121 adult cases (56 with TB, 65 controls). Further information was obtained from the laboratory information system, including patient demographics and other laboratory results; TB culture, SARS-CoV-2 results, C-reactive protein level, HIV status, bone marrow biopsies and the cycle threshold (CT) values on positive GXPU analysis. The MO-Y, EDPs and full blood count (FBC) values were compared among patients with and without TB (HIV positive and negative). Statistical significance was assessed (P-value of <0.05). Results: The MO-Y did not show utility in identifying patients with TB. A sub-population of patients living with HIV (PLWH) with a CD4 <100 cells/ul showed significantly higher MO-Y levels, due to other opportunistic infections affecting monocytes. Neutrophil surface fluorescence (a marker of neutrophil activation), was significantly higher in PLWH and with concomitant TB infection, possibly due to immune activation, worse illness, or increased bacterial infection. Among the PLWH, those with TB had significantly lower CD4 counts, absolute lymphocyte counts and mean cell volume (MCV) values. The MCV (cut-off value 87 fL) showed the strongest diagnostic utility for discriminating PLWH with and without TB (AUC 0.79). Conclusion: The MO-Y is not a useful biomarker of TB, but is significantly elevated in PLWH with low CD4 counts. The MCV showed adequate discriminatory power for differentiating patients with and without TB, at a cut-off level of 87fL.
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    Characterisation of the genetic variation in pharmacogenes involved in anti-tuberculosis drug metabolism across African populations
    (University of the Witwatersrand, Johannesburg, 2024) Malinga, Thandeka Vuyiswa Bongiwe; Twesigomwe, David; Othman, Houcemeddine
    Tuberculosis (TB) is a major health burden in Africa. Although TB is treatable, anti-TB drugs are associated with adverse drug reactions (ADRs) which are partly attributed to pharmacogenetic variation. The distribution of star alleles (haplotypes) influencing anti-TB drug metabolism, is unknown in many African populations. This presents challenges in implementing genotype-guided therapy in Africa to decrease the occurrence of ADRs and enhance the efficacy of anti-TB drugs. Therefore, this study aimed to characterise the distribution of star alleles in genes that are involved in anti-TB drug metabolism (mainly isoniazid), namely CYP2E1, NAT1, NAT2, GSTM1 and GSTT1, across diverse African populations. We used 794 high-depth whole genome sequence datasets representative of eight Sub-Saharan African (SSA) population groups. Data sources included the 1000 Genomes Project and H3Africa AWi-Gen. CYP2E1, NAT1, NAT2, GSTM1 and GSTT1 star alleles were called from the WGS data using StellarPGx. Subsequently, novel star alleles were analysed, and their allele defining variants were annotated using the Ensembl Variant Effect Predictor. We present the distribution of both common and rare star alleles influencing anti-TB drug metabolism across various SSA populations, in comparison to other global populations. Various key star alleles were identified in the SSA study populations at relatively high frequencies including NAT1*10, GSTT1*0 (>50%), GSTM1*0 (49%), and NAT2*5B (21%). Additionally, we predicted varying phenotypic proportions for NAT1 and NAT2 (acetylation) and the GST enzymes (detoxification activity) between SSA and other global populations. Fifty potentially novel haplotypes were identified computationally across the five genes. This study provides insight into the distribution of star alleles in genes relevant to isoniazid metabolism across various African populations. The high number of potentially novel star alleles exemplifies the need for pharmacogenomics studies in the African context. Overall, our analysis provides a foundation for implementing pharmacogenetic testing in Africa to reduce the risk of ADRs related to TB treatment.
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    Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker
    (University of the Witwatersrand, Johannesburg, 2023) Tsolo, Semakaleng Theresia; Ranchod, Heena
    The World Health Organization has prioritized the development of non-sputum-based assays that are capable of detecting active Tuberculosis (TB). Tryptophan (tryp) is converted to kynurenine (kyn) by the rate-limiting enzyme indoleamine 2, 3- dioxygenase (IDO). IDO activity may serve as a biomarker for active TB. Dried blood spots (DBS) can be collected outside of medical institutions and are simple to transport. We wanted to explore the use of DBS as an alternative sample type to measure the kyn/tryp ratio and IDO mRNA gene expression in healthy people. METHODS We optimised methods for elution of dried blood spots, exploring various elution buffers. Following method optimisation, we enrolled 40 healthy participants, and collected whole blood and DBS samples. Kyn and tryp concentrations were measured using ELISA (ImmuSmol, France). IDO mRNA gene expression was determined by real-time PCR using two housekeeping genes GAPDH and BACT. Statistical analysis was performed to determine the correlation and agreement between peripheral blood samples and DBS. RESULTS For IDO activity, tryp showed good agreement between plasma and DBS with a median percentage similarity of 91.1%. In contrast, no agreement was observed for kyn with a median percentage similarity of 56.6%. The kyn/tryp ratio performed poorly due to poor detection of kyn in DBS. Percentage similarity between whole blood and DBS IDO mRNA expression using GAPDH 87.1%, while using BACT was 84.6%. Using either traditional sample types or DBS, there was no correlation between IDO gene expression and kyn/tryp ratio. CONCLUSION We showed that tryp was measurable in DBS. Tryp in DBS was 91.1% similar to values in plasma. Despite method optimization, there was poor agreement between DBS and plasma for kyn. Although IDO mRNA gene expression was detectable in DBS, method agreement with whole blood was unsatisfactory. Alternative methods for the stabilization of kyn in DBS should be explored in future studies. IDO mRNA expression should be measured from whole blood in future studies
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    Assessing the propensity of drug resistant tuberculosis to enter and exit the differentially culturable state
    (2024) Nonkula, Bomikazi
    Tuberculosis (TB), one of the oldest and most contagious infectious diseases, continues to be a global health concern. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC) which comprises of several species. These species are further subdivided into strains based on subtle genetic differences. The success of M. tuberculosis as a pathogen can be attributed to its ability to survive various stresses by adopting different growth states. Previous studies have shown that sputum from TB infected patients harbours a large proportion of drug-tolerant bacteria that are unable to form colonies on agar plates but can grow in liquid media. This population of organisms, termed differentially culturable tubercle bacilli (DCTB), could be resuscitated to grow by supplementing liquid media with cell free culture filtrates from axenic cultures of wild type M. tuberculosis H37Rv or mutant H37Rv lacking all five resuscitation promoting factors (Rpfs). Laboratory models that induce this differentially culturable state are critical for studying the physiology and metabolism of these bacteria in order to develop new TB diagnostic tests. In this study, five Beijing and five LAM drug resistant strains of M. tuberculosis were selected and used to robustly generate DCTB through an in vitro stress model using carbon starvation. The most probable number (MPN) assay and colony forming units were used to determine the amount of DCTB. Furthermore, the phenotype of these cells was studied using microscopy as well as metabolic probes that target the peptidoglycan (PG) component of the bacterial cell wall. Our findings demonstrated that applying the carbon starvation model to clinical M. tuberculosis strains (Beijing and LAM) resulted in robust levels of DCTB, as evidenced by limited growth on agar plates and enhanced growth in liquid media supplemented with culture filtrate from LAM and Beijing strains. Comparison of cell length between carbon starved cells to those grown in routine laboratory media suggested that DCTB appeared to be non-replicating and significantly shorter. The metabolic activity of the starved cultures was restored when they were supplemented with H37Rv, LAM and Beijing culture filtrate. Our results also demonstrated that Beijing strains had a higher propensity to produce DCTB compared to LAM strains and that the supplementation with Beijing culture filtrate resuscitated more DCTB. Collectively, our findings allow for the advancement of experimental systems that enable further investigation of DCTB and the properties of the Beijing strain that facilitate better adoption of the differentially culturable state.