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- ItemTools for analysis of Luminex immunoassay data: development of a robust pipeline and best practices recommendations(Stellenbosch : Stellenbosch University, 2023-02) Da Camara, Ncité Lima; Tromp, Gerard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Background and Scope: Immunoassays can be used to detect and measure the concentration of many antigens in a variety of specimens for the diagnosis of diseases, and for the detection of microbes and various illegal substances. In addition, they can be used to monitor and study processes, or differentiate infection, latent or active disease in a patient's immune response after infection with a pathogen by measuring the presence of specific antigens. Recent advances in the instrumentation include multiplex immuno-assays, e.g., Luminex 200 or Luminex MAGPIX®, powered by Luminex xMAP® technology. Analysis of data produced by the multiplex immunoassays is complex and current analytical approaches are highly subjective. Currently, there is no standard, robust approach for data pre-processing of multiplex immunoassay data. To overcome this knowledge gap, I developed a robust and standardized data pre-processing pipeline for multiplex immunoassay data to ensure reproducible data. In addition, provide recommendations for best practices of Luminex data generation and data pre-processing to ensure reproducible science by amending existing laboratory standard operating procedure (SOPs) and developed data pre-processing SOPs’ for the Stellenbosch University Bioinformatics Research Group. Design and Approach: I evaluated current laboratory processes, analysed existing Luminex data and drafted best practices recommendations that will ensure reliable input data for the pipeline. I implemented programmatic steps for data management, quality control, and pre-processing to provide high quality data for analyses. A variety of data preprocessing approaches were investigated, and a set of robust standardized options are provided to the user together with appropriate bioinformatics tools in the newly developed pipeline. Results: For robust and reproducible data generation and data preparation, standardization of manual laboratory best practices procedures are recommended and implemented to ensure standard file name convention, file format, file structure and standard plate layout. In addition, the development of a robust automated standardized data pre-processing pipeline using algorithms in R, freely available software will reduce variability and error introduced by humans. In addition, to ensure reliability and reproducibility of the results generated using this pipeline, the pipeline records metadata such as parameter settings, program, and package versions in the output. The methods were validated using existing de-identified Stellenbosch University https://scholar.sun.ac.za iv data sets from the Stellenbosch University Immunology Research Group. In the future the pipeline can be applied to newly generated data from a variety of immunological studies. Conclusions: This pipeline will standardize and speed up data pre-processing, as well as provide consistent and reproducible results with any complex analyses. Furthermore, provide the bioinformatician or statistician with a rapid means to pre-process Luminex data for subsequent analysis. The framework developed here can be easily applied to other data analysis projects from different biomedical fields.
- ItemAccuracy and impact of the MTBDRplus v2 and MTBDRsl v2 line probe assays for the detection of first-line and second-line drug resistant tuberculosis(2023-02) Pillay, Samantha; Theron, Grant; de Vos, Margaretha; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Combating drug-resistant tuberculosis (DR-TB) remains a challenge globally. Treatment success rates are often derailed by under-diagnosis and under-reporting of disease. Patients remain contagious for prolonged periods prior to initiation of appropriate treatment which is further exacerbated by the amplification of drug resistance and poor treatment outcomes. Using current and new diagnostic tools effectively is key to rapid diagnosis of tuberculosis and early detection of drug resistance. Firstly, (chapter 2) line probe assays (LPAs) frequent inability to generate a resistance call in paucibacillary specimens is problematic. We showed that while MTBDRplus and MTBDRsl tests work well on smear-negative specimens for detecting drug resistance, failure rates remained high. We demonstrated with the use of routine key programmatic data how time-to-reporting of results improved with the use of molecular assays and provided evidence on how standard-of-care can be improved in a programmatic context. Secondly, (chapter 3) LPA testing on smear-negative specimens is not always performed causing diagnostic delays and hindering their role as a direct front-line diagnostic tests. Thus, by using Xpertgenerated data we determined the ratio of actionable-to-non-actionable results and the number of missed resistant cases at varying thresholds. We demonstrated that Xpert semiquantitation category is superior to informing reflex LPA testing than smear status. In short, this method provides a framework by which laboratories that currently do not test smear-negative specimens to expand testing. Thirdly (chapter 4) current pathways using Xpert MTB/RIF or Xpert Ultra as frontline tests for diagnosing TB and rifampicin resistance lack further treatment guidance. We did a systematic review and assessed the performance of Xpert MTB/XDR for the detection of pulmonary tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and amikacin. Participants consisted of 1228 for pulmonary tuberculosis detection and 1141 for drug resistance. We found Xpert MTB/XDR is unlikely to test positive as a follow-up test for the detection of Mycobacterium tuberculosis in samples that test Xpert Ultra
- ItemNew and improved methods for the diagnosis of susceptibility to tuberculosis drugs(Stellenbosch : Stellenbosch University, 2023-01) Derendinger, Brigitta; Theron, Grant; De Vos, Margaretha; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Drug-resistant tuberculosis (DR-TB) is a global threat. Diagnosing patients with DR-TB and initiating them onto appropriate treatment, in the shortest possible time, is of utmost importance. The optimisation of existing rapid molecular assays and the implementation of drug susceptibility testing (DST) for new drugs to monitor patients on treatment is crucial in curbing transmission. Firstly (chapter 2), showed that Mycobacterium tuberculosis (Mtb) DNA recoverable from used Xpert MTB/RIF (Xpert) cartridges ‒ cartridge extract (CE) – that would otherwise be discarded, can be used for downstream second-line molecular DST. No additional DNA extraction or sample purification was needed. We defined a threshold of Xpert semiquantitative category “low” to ensure that no MTBDRsl assays are wasted on CE likely to give invalid results. This alleviated the need for collection of a second sputum specimen, thereby reducing diagnostic delays, and enabling patients to be placed on treatment sooner. This approach is being developed into a cartridge extraction device and will be evaluated by TB programmes. Secondly and thirdly (chapter 3 and 4), MTBDRplus and MTBDRsl focussed on WHOendorsed rapid molecular assays that have reported suboptimal sensitivities and high indeterminate rates especially in smear-negative specimens. We hypothesised that ramp rate (speed of temperature change between PCR cycles) could impact assay performance. We showed that correcting thermocycler ramp rate (manufacturer-recommended ramp rate ≤2.2°C/s) likely improved the yield of rapid diagnoses for first-and second-line DST, done with MTBDRplus and MTBDRsl respectively, especially in paucibacillary specimens. Our survey showed that suboptimal ramp rate is a common problem but is easily fixable. This manuscript informed WHO course training material (https://openwho.org/courses/multi-drug-resistant-tb). Finally (chapter 5), bedaquiline (BDQ), a lifesaving TB drug, is undergoing rapid scale-up but largely in the absence of DST. In a group of programmatic patients still culture-positive after ≥4 months of BDQ-based treatment, more than half had isolates that gained BDQ resistance (mostly acquisition, some transmission). Several Rv0678 and pepQ variants were associated with phenotypic resistance, many previously undescribed. Patients with baseline fluoroquinolone-resistance, clofazimine exposure, and ≤4 effective drugs were more likely to be BDQ-resistant. This thesis has resulted in four first author manuscripts (three published, one submitted). Additionally, two 2nd author manuscripts and seven manuscripts as a middle co-author. These nine total are briefly discussed in chapter 6 and can be found in the appendices (and included as ancillary publications). The candidate presented three times at international and twice at national peer-reviewed conferences. In summary, this work shows that second-line DR time-to-treatment initiation could be reduced by doing second-line DST on CE from used cartridges. The number of smear-negative patients in whom DST is possible will improve substantially after ramp rate correction for MTBDRplus and MTBDRsl. Finally, we show the existence of a potentially infectious pool of BDQ resistant strains created under programmatic conditions, as well as the challenges and risks associated with starting patients with complex TB-treatment histories on a regimen containing a novel drug without routinely available DST. Our findings also inform on how and in whom new TB drugs are prioritised for use.
- ItemInvestigating host and pathogen biomarkers of mycobacterium bovis and nontuberculous mycobacterial infection in African buffaloes (Syncerus caffer)(Stellenbosch : Stellenbosch University, 2023-01) Clarke, Charlene; Miller, Michele Ann; Goosen, Wynand Johan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: African buffaloes (Syncerus caffer) are important maintenance hosts of bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis). Accurate and rapid diagnoses are essential for detection of M. bovis infected buffaloes. However, various factors may impede diagnosis, including suboptimal sensitivity of mycobacterial culture and cross-reactive immune responses to nontuberculous mycobacteria (NTM). In addition, there are challenges with transporting samples from remote locations to laboratories, as well as risk of zoonotic infection of humans handling samples. This study broadly aimed to improve bTB diagnosis in buffaloes by investigating host and pathogen biomarkers of M. bovis and NTM infections. This was achieved by 1) evaluating a safe, rapid test procedure to detect the presence of pathogenic mycobacterial DNA in buffalo post-mortem and ante-mortem samples, 2) characterising NTMs present in buffaloes, and 3) developing a high specificity test algorithm to improve screening of historically bTB-free buffalo herds. PrimeStore® Molecular Transport Media (PS-MTM) ensures a safer bTB testing platform by inactivation of pathogens. In this study, it effectively preserved mycobacterial DNA in M. bovis infected buffalo oronasal and tissue swabs for molecular testing. The novel use of Xpert MTB/RIF Ultra with PS-MTM stored swab samples demonstrated that this commercial qPCR assay has promise for rapid, sensitive, and potentially in-field application, for detecting M. bovis shedding ante-mortem and infection post-mortem. A high diversity of NTM species was identified in a large percentage of buffalo oronasal secretion and tissue sample cultures by hsp65 or rpoB PCR amplicon Sanger sequencing, with M. avium complex members the most frequently detected. Genes encoding virulence factors, ESAT-6 and CFP-10, were present in more than half of the samples, which may be indicative of potential crossreactivity with bTB immunoassays. A commercial line-probe assay detected NTMs from DNA directly extracted from oronasal swabs, and the Ultra showed high specificity amidst the high presence of NTMs following direct application on oronasal swabs. Serial bTB test algorithms, with QuantiFERON® -TB Gold Plus (QFT) interferon-gamma (IFN-γ) release assays (IGRA) and QFT IFN-γ inducible protein-10 release assays (IPRA), showed the greatest specificity in a historically bTB-free buffalo herd, compared to parallel testing or individual tests. Presence of NTMs in oronasal swabs did not appear to impede assay specificity. In an unrelated historically bTB-free herd, serial testing accurately identified truly infected buffaloes (positive on IGRA and IPRA), which were confirmed M. bovis infected. In summary, buffaloes have a high diversity of NTMs present that may impede bTB diagnostic tests. However, assay specificity did not appear to be affected by NTM presence. High specificity was achieved when IGRA and IPRA were interpreted in series, and results demonstrated the value of this test algorithm to screen buffalo herds with no history of bTB. The Ultra assay maintained specificity in the presence of NTMs and accurately and rapidly identified M. bovis infected buffaloes post-mortem and ante-mortem from swabs stored in a pathogen inactivation media, PSMTM. These results demonstrate that rapid accurate differentiation of M. bovis infected and uninfected buffaloes can be achieved using methods described in this thesis.
- ItemAntimycobacterial activity of ascidian fungal symbionts(Stellenbosch : Stellenbosch University, 2022-12) Tapfuma, Kudzanai Ian; Mavumengwana, Vuyo; Malgas-Enus, Rehana; Loxton, Andre Gerhard; Allie, Nasiema; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Tuberculosis (TB) is an infectious disease which primarily affects the lungs. Treatment of TB is complicated because the causative agent, Mycobacterium tuberculosis, is an intracellular pathogen which infects and kills cells of the innate immune system, while exhibiting intrinsic and extrinsic resistance to many of the currently available antimicrobial agents. A sizeable percentage of TB patients in the world-population are infected by M. tuberculosis strains which are resistant to currently utilized first- and second-line anti-TB drugs. Drug-discovery studies bioprospecting for compounds with novel anti-TB activities are therefore essential in order to control the spread of TB and to prevent a catastrophic pandemic. In this study, extracts from marine fungi were considered for antimycobacterial activity bioprospecting as they are largely underexplored. A total of 46 cultivable fungi were isolated from ascidians and 32 of these fungal isolates were sequenced and consequently identified. Among these fungi, the methanol crude extract from Clonostachys rogersoniana MGK33 was found to possess the highest antimycobacterial activity with minimum inhibitory concentrations of 0.125 and 0.200 µg/mL against Mycobacterium smegmatis mc2 155 and M. tuberculosis H37Rv, respectively. Untargeted metabolite profiling of the crude extract from C. rogersoniana MGK33 revealed the presence of bionectin F (among other compounds) which has previously been shown to possess antimicrobial activity in other studies. In silico molecular docking and simulation experiments in this study showed that bionectin F is a potential inhibitor of M. tuberculosis β-ketoacyl-ACP reductase (MabA). An attempt was then made to generate novel agents that would be composed of nanoparticles surface functionalized with the bioactive fungal extract from C. rogersoniana MGK33. In particular, mono-metallic SPIONs were synthesized using the co-precipitation method and then surface modified to produce bi-metallic superparamagnetic iron oxide nanoparticles (SPIONs) using nickel, zinc, gold, copper and silver, to produce Ni-SPIONs, Zn-SPIONs, Au-SPIONs, Cu-SPIONs and Ag-SPIONs. Functionalization was then performed using the MGK33 extract to produce Ni-SPIONs@MGK33, Zn-SPIONs@MGK33, Au-SPIONs@MGK33, Cu SPIONs@MGK33 and Ag-SPIONs@MGK33. Among these agents, Cu-SPIONs and Ag SPIONs were found to exhibit the strongest antimycobacterial activity, comparatively stronger than that of the counterparts, Cu-SPIONs@MGK33 and Ag-SPIONs@MGK33. In an experiment involving the treatment of RAW 264.7 macrophage cells infected with M. smegmatis mc2 155, the MGK33 extract exhibited the highest early apoptosis activity (9.61%), followed by Cu-SPIONs@MGK33 (3.34%), both agents tested at 1.96 µg/mL for 24 hours. The MGK33 extract further showed strong antimycobacterial activity against intracellular M. smegmatis mc2 155, compared with the nanoparticles synthesized in this study. Results in this study led to the conclusion that the marine fungus, C. rogersoniana MGK33 is a prolific source of compounds with antimycobacterial and immunomodulatory activity, and that further studies should be done to develop Cu-SPIONs and Ag-SPIONs into lead agents for anti-TB drug development.