Browsing by Author "Van der Westhuizen, Benjamin Jacobus Cillie"
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- ItemPolyester functionalisation for mycobacterial capture(2018-03) Van der Westhuizen, Benjamin Jacobus Cillie; Van Reenen, Albert; Cronje, Lizl; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.ENGLISH ABSTRACT: Tuberculosis (TB) is one of the deadliest diseases known to man and ranks among the top ten global causes of fatalities. This stems from the fact that Mycobacterium tuberculosis (Mtb), the causative pathogenic bacterial species responsible for TB, has developed extensive mechanisms to elude detection and eradication by the human immune system. While its diagnosis in adults with a negative human immunodeficiency virus (HIV) status is commonplace, paediatric diagnosis can be arduous even with the aid of sophisticated techniques. Therefore, paediatric cases often do not receive the priority they deserve. This problem is further amplified in developing countries, since they are most affected by the disease and also do not have access to sophisticated diagnostic techniques. Diagnosis of TB in developing countries is primarily based on sputum smear microscopy, a proficient but limited technique. Children often develop a paucibacillary form of TB, in which case the concentration of bacteria in their sputum falls below the lowest limit of detection for microscopic detection. If the TB bacilli can be concentrated within the specimen collected from patients, the detection limit can likely be met, allowing for rapid detection. Since the bacilli are prone to adhere to polymeric surfaces, a high surface to volume, fibrous matrix is of interest. In this study, various means of surface functionalising poly(ethylene terephthalate) (PET) microfibres with a biological entity was investigated. The entity of interest was a lectin based ahesin, namely Concanavalin A (Con A). Con A has an affinity for the mannose groups on the Mtb cell wall. By functionalising these fibres, a substrate was created which could potentially act as a concentrating and capturing platform for the Mtb bacilli. It follows that since these types of fibres are commonly used for specimen collection swabs, an Mtb specific buccal swab could be created. This would allow bacilli collection by means of their entrapment upon wiping the oral mucosa of a patient with the swab. Affinity studies between the modified PET microfibres and an attenuated strain of Mycobacterium bovis namely the bacillus of Calmette-Gu´ erin (BCG) were conducted. The strain of BCG used, possessed a fluorescent protein reporter gene, namely mCherry-BCG. These studies were also conducted as a function of serial dilutions, to evaluate the Mtb capture ability of the modified substrates. The successful capture of BCG by the substrates was confirmed by the aid of confocal fluorescence microscopy (CFM) and scanning electron microscopy (SEM). These studies revealed that all of the surface functionalised substrates were able to successfully capture the bacteria. This was possible down to culture concentrations which fell well below the detection threshold of sputum smear microscopy. The surface modification, which proved most successful at BCG capture, was a cross-linked protein aggregate (CLPA) derivative. CLPA was covalently bound to the surface of the fibres, by means of glutaraldehyde (GA).