School of Pathology (ETDs)
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Item Development of a multiplex HIV/TB point-of-care diagnostic assay based on the microarray(University of the Witwatersrand, Johannesburg, 2023) Malatji, Kanyane BridgettHIV/AIDS mortality is caused by opportunistic illnesses/infections that take advantage ofthe weakened immune system in infected individuals. In Africa, the most common of these opportunistic illnesses include infection by Mycobacterium tuberculosis (M.tb) responsible for tuberculosis (TB). HIV co-infection with M.tb has negative implications for disease management given that each pathogen accelerates the morbidity caused by the other. The effective management of patients infected with both pathogens is restricted by the fact that their diagnosis is done separately. The situation is more difficult in remote areas where patients must wait for much longer to obtain their TB diagnostic results. In addition, the current diagnostic tests for the detection of TB such as chest X-ray and bacterial culture have a long turnaround time, are expensive to perform, and require sophisticated equipment and trained personnel. It is in this context that this project sought to develop an HIV and TB multiplex microarray-based assay for the detection of the two diseases using one test. The project used a 2.5 x 7.6 cm epoxy-coated glass slide as well as high- binding 96 well plates to which HIV-1 p24 and M.tb CFP10, ESAT6 and pstS1 antigens, known to be markers of active TB, were immobilized. The immobilized antigens were then incubated with anti-p24, anti-CFP10, anti-ESAT6 and anti-pstS1 primary antibodies diluted in human serum to mimic physiological conditions where the antibodies would exist in the presence of other proteins. Detection of binding between the antigens and primary antibodies was achieved by means of secondary antibodies conjugated to either a fluorescence dye or horseradish peroxidase (HRP). In chapter two of the study, the immobilization of the HIV and TB antigens on the epoxy-coated glass slides as capture molecules of the HIV and TB antibodies diluted in human serum was performed. The antigen-antibody reactions detection were achieved by means of fluorescence dye conjugated secondary antibodies. This chapter also covered the sensitivity and specificity of the technology where the epoxy-coated glass slides were compared to the gold standard 96 well high-binding plates. Data showed that the HIV and TB antigen-antibody reactions were specific, and the slides were more sensitive relative to the 96 well high-binding plates with limits of detection many folds lower. To be specific, the limit of detection from the slides averaged 0.954 ng/ml compared to 4474.6 ng/ml for the plates. The detection limit concentrations of the slides were lower than the reported physiological concentrations of HIV and TB antibodies in infected individuals. Chapter two also focused on the evaluation of the antigens’ stability on the epoxy-coated glass slides by determining the optimal experimental pH buffer, temperature, storage condition (dry or wet), as well as the shelf-life. Data showed that the optimal pH and temperature for the HIV and TB antigens immobilized on the slides were pH 7.4 and 25 ˚C. Moreover, the antigens could be stored dry for at least 90 days without losing their function. Overall, this chapter showed that the epoxy-coated microarray slides performed better than the gold standard 96 well high-binding plates in terms of sensitivity; and that the immobilized antigens could remain stable for a long period, and do not require specialized storage conditions; thus, making the microarray technology a potential diagnostic tool for the multiplex detection of HIV and TB in the case of co-infection. Chapter three of the study focused on the proof-of-concept of the technology using human serum samples infected with HIV. The chapter showed that the technology could detect p24 antibodies in six out of seven samples infected with HIV, i.e., it detected p24 antibodies in 85.7% of samples known to be HIV positive. Furthermore, HIV negative samples also proved to be negative with this technology, thus no false positives were observed. Moreover, the technology was specific for HIV detection as no binding was observed on TB antigens. Therefore, these data support what was observed in the previous chapter when the HIV antibodies were spiked in normal human serum. Chapter four explored the application of the diagnostic technology for the point-of-care (POC) detection of HIV and TB antigen- antibody reaction, using HRP conjugated secondary antibodies, as well as the 2,2′-azino- bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and 3,3',5,5'-tetramethyl Benzidine (TMB) substrates for colour change based endpoint. This chapter also covered the sensitivity and specificity of the immunoassay in the high-binding 96 well plates and on epoxy-coated glass slides. Similar to what was observed in the previous chapter, the HIV and TB antigen-antibody interactions were specific, and the epoxy-coated microarray slides were more sensitive than the 96 well high-binding plates with limits of detection averaging 815-folds lower than the plates. Nevertheless, both platforms were found to be sensitive enough to be used for the POC detection of HIV and TB co infection using visual inspection. Furthermore, the stability of the antigens in the 96 well high-binding plates using colour change detection was also evaluated. The antigens were found to be stable in the high-binding plates at different pH and temperature conditions; however, pH 7.4 and 25 ˚C were optimal. In addition, the antigens were stable when stored dry in the plates for a period of three months. In addition, between the two HRP substrates used, TMB was faster and more sensitive to the HIV and TB antigen-antibody reactions than the ABTS substrate, and the difference was statistically significant (p<0.05). The importance of this chapter is that it eliminated the need for sophisticated equipment to detect the presence of HIV and TB antibodies, as the detection could be achieved by visual inspection. Overall, data in this chapter supported further development of the microarray technology for the POC HIV and TB co-infection diagnosis. Chapter five attempted to produce the CFP10, ESAT6, and pstS1 TB antigens in plants to reduce the cost associated with the current commercially available bacteria-produced antigens.Item 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, HoucemeddineTuberculosis (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.Item Evaluation of novel assay formats for indoleamine 2, 3 dioxygenase as a tb biomarker(University of the Witwatersrand, Johannesburg, 2023) Tsolo, Semakaleng Theresia; Ranchod, HeenaThe 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 studiesItem Assessing the propensity of drug resistant tuberculosis to enter and exit the differentially culturable state(2024) Nonkula, BomikaziTuberculosis (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.Item The characterization of Mycobacterium tuberculosis amidase-like proteins(2024) Matlhabe, OfentseTuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis, is a significant global threat to human health. The emergence of drug resistant M. tuberculosis necessitates the identification of new drug targets for the development of new, shorter regimens. The peptidoglycan (PG) core of the M. tuberculosis cell wall is a potential source of drug targets because it is unique to bacteria and plays a vital role in a multitude of cellular processes and host-mediated immune responses. PG is constantly remodelled by PG synthases and hydrolases in response to external stimuli. This research focuses on N-acetylmuramyl-Lalanine amidases (amidases), PG hydrolases that are implicated in bacterial growth, cell division, virulence and antibiotic tolerance. More specifically, this PhD aims to characterize the M. tuberculosis Ami1 (Rv3717) homologue and to highlight its potential as a drug target. Genotypic characterization of a previously generated M. tuberculosis mutant strain lacking ami1 (H37Δami1S) was conducted prior phenotypic assessments. The deletion of ami1 had no significant effect on growth rate and cell division in standard 7H9 media. In contrast, the growth rate of H37Δami1S was significantly reduced when grown in Sauton’s minimal media with (1%) or without glycerol as a carbon source. We then surmised that Ami1 possibly plays a role in intracellular survival, where host-derived carbon sources support bacterial growth. The survival of H37Δami1S was reduced in IFN-γ stimulated U937 macrophages. H37Δami1S displayed increased susceptibility to rifampicin when assessed by broth microdilution. This observation was credited to a weakened, more permeable cell wall. Consistent with this, H37Δami1S exhibited an increased ethidium bromide uptake. Subsequently, we hypothesized that H37Δami1S may display alterations in antibiotic tolerance/persistence. In a 7-day time-course experiment, H37Δami1S displayed increased susceptibility to vancomycin, ethionamide and isoniazid as evidenced by declining bacterial survival. We interrogated the isoniazid-associated phenotype further, by assessing the transcription of all three amidase-encoding genes. Only ami1 was induced following exposure to isoniazid whereas the expression of ami3 and ami4 remained at basal levels. The regulation of the ami1 gene was explored further through bioinformatics, which revealed two putative transcriptional regulators predicted to bind upstream of ami1, namely Rv1423 and Rv1776c. Protein homology modelling detected HTH DNA binding domains in both proteins. These proteins were then cloned for recombinant expression in the pET29a+ system for purification. Rv1776c was successfully expressed and purified. Electrophoretic mobility shift assays yielded preliminary data that suggested that Rv1776c binds the promoter region of the ami1 gene. Attempts to optimize binding were unsuccessful. To further evaluate the role of Rv1776c and Rv1423 in regulating ami1 gene expression, we over-expressed the regulators, using the tetracycline operator, and assessed effect on cell wall stability, via an ethidium bromide diffusion assay. Over-expression of Rv1776c was not achieved despite increasing concentrations of anhydrotetracycline, suggesting possible downstream regulation of Rv1776c; however, over-expression of Rv1423 was achieved. An increase in ethidium bromide uptake was observed in strains over-expressing Rv1776c and Rv1423. Increasing anhydrotetracycline concentrations in both strains resulted in marginal decreases in the transcription of ami1. Overall, this study has demonstrated that Ami1 plays a vital role in how M. tuberculosis utilizes a carbon source during normal growth and survival in vitro. Moreover, the transcription of ami1 is specifically and directly responsive to isoniazid exposure, possibly via two transcriptional repressors. This work therefore supports further characterization and development of Ami1 as novel drug target in M. tuberculosis.Item Biomarkers to predict Tuberculosis treatment response(2024) Boshielo, ItumelengTuberculosis (TB) is a chronic disease caused by Mycobacterium tuberculosis (Mtb). Despite the implementation of multifaceted TB prevention and control efforts, a significant number of people still die from TB. Consistent with this, an uptick in TB-related mortality was recently noted, which has been ascribed to the negative effects of Coronavirus disease-2019 (COVID-19) on TB programs. The complex life cycle of Mtb is largely due to the use of immune evasion mechanisms to establish initial infection, remain dormant in the host, and reactivate pathogenicity under favourable circumstances. The prolonged TB treatment regimen is necessitated by the slow response of bacterial populations to standard TB chemotherapy, a phenomenon that may be caused by persistent, drug-tolerant bacteria. Scientific literature has provided evidence for these types of bacterial populations in the form of Differentially Culturable Tubercle Bacilli (DCTB). It has been demonstrated that DCTB represent drug tolerant bacteria that appear to be cleared at slower rate than organisms detected by routine culture methods. However, it remains unclear if DCTB populations elicit different immune responses when compared to their conventionally culturable counterparts. Herein, we address this question by optimizing a laboratory model for the generation of DCTB in vitro and test the capacity of clinical isolates of Mtb from Lineage 2 (Beijing) and Lineage 4 (LAM) to adopt the DCTB state. Using the Most probable number (MPN) assay, in the presence of culture filtrate (CF) as a source of growth factors to resuscitate DCTB, and colony forming units, the amount of DCTB in our model was quantified. As demonstrated by the limited growth on agar plates and increased growth in liquid media supplemented with CF from an axenic culture of Mtb, our findings demonstrated that carbon starvation was able to generate DCTB from clinical Mtb strains. After generating these populations, we stimulated whole blood with DCTB and conventionally culturable populations and report on the stimulation of a select set of cytokines (IFN-γ, IL-4, IL-5, IL-6, IL-12p70 and TNF-α) using a Bead Array Multiplex Immunoassay. In comparison to H37Rv-DCTB and LAM-DCTB, Beijing-DCTB induced significantly reduced levels of IL-5 and TNF-α. When comparing cytokine production between culturable and DCTB populations, within a single strain, we noted that LAMDCTB was delayed in the production of IFN-γ whilst Beijing-DCTB was not able to induce production of this cytokine when compared to conventionally culturable counterparts. These data suggest that shifting to a non-replicating DCTB state does indeed affect the ability of clinical isolates to induce immune responses. Based on these observations, we next set out to determine if DCTB affects immune responses during treatment of Mtb infected individuals. In prior work, using a prospective observational cohort, we demonstrated a substantive heterogeneity in clearance of DCTB in individuals with drug susceptible TB. We were able to classify these response patterns into three broad groups including (I) participants who were able to clear DCTB within the first two weeks of treatment (treatment-responsive); (II) those with delayed ability to clear these organisms (delayed-responsive) and (III) a group of individuals where DCTB did not change substantively during treatment (non-responders). Given these stark differences in treatment response patterns, we hypothesized that the immune responses associated with these patterns would be substantively different. In the second component of this work, we set out identify immune biomarkers that predict an effective response of DCTB to TB treatment. To quantify cytokines, chemokines and growth factors in plasma from these groups, we used a 65-plex Luminex assay, with a broad selection of targets. Statistically significant differences between these groups were analysed using the Kruskal-Wallis test with Dunn’s multiple comparisons, with p<0.05 was considered as statistically significant. When compared to patients who had TB and HIV co-infection, the number of cytokines that may possibly be used to report on the effectiveness of TB treatment was significantly higher in Mtbonly infected patients. This suggests that HIV infection significantly reduces the number of cytokines that can be used to report on TB treatment response. The ROC analysis of I-TAC, G-CSF and VEGFA showed that these cytokines have a significant discriminatory power to distinguish treatmentresponsive and non-responsive patients from HCs using DCTB as the measure of treatment response. No unifying cytokine signature that predicted DCTB response in all groups was identified. Together, our results indicate that some inflammatory markers are elevated in individuals with TB that rapidly clear bacteria during treatment. Given that these responses are based on DCTB, which represent drug tolerant populations, these select cytokines may be useful in evaluating the effectiveness of novel shorter TB treatment regimens.