Polyester functionalisation for mycobacterial capture

Van der Westhuizen, Benjamin Jacobus Cillie (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

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).

AFRIKAANSE OPSOMMING: Tuberkulose (TB) is een van die dodelikste siektes bekend aan die mens, en word beskou as op die ranglys van die top tien oorsake van sterftes wˆereldwyd. Dit is vanwe¨e die feit dat Mikobakterium tuberkulose (Mtb), die patogene bakteriese spesie wat verantwoordelik is vir TB, omvattende meganismes ontwikkel het, wat sy opsporing en uitwissing deur die menslike immuunstelsel te fnuik. Terwyl TB allerdaag by volwassenes met ’n negatiewe menslike immuniteitsgebreksvirus (MIV)-status gediagnoseer word, kan pediatriese diagnose moeilik wees selfs met behulp van gesofistikeerde tegnieke. Pediatriese gevalle ontvang dus dikwels nie die prioriteit wat hulle toekom nie. Hierdie probleem word verder vergroot in ontwikkelende lande, aangesien hulle die meeste deur die siekte geraak word en nie toegang het tot gesofistikeerde diagnostiese tegnieke nie. Diagnose in ontwikkelende lande word hoofsaaklik gebaseer op sputum-smeer-mikroskopie, ’n toereikende, maar beperkte tegniek. Kinders ontwikkel dikwels TB met ’n geringe bacilli telling (paucibacillary TB), in welke geval die konsentrasie van bakterie ¨e in hul sputum onder die laagste opsporingsperk vir mikroskopiese opsporing val. As die TB-bacilli gekonsentreer kan word binne die monster wat by pasi¨ente versamel word, kan die opsporingsperke waarskynlik bereik word, wat vinnige diagnose moontlik maak. Aangesien die bacilli geneig is om aan polimeriese oppervlaktes te heg, is ’n ho¨e oppervlak tot volume, veselagtige, matriks van belang. In hierdie studie is verskeie metodes ondersoek om die oppervlak van politileentereftalaat (PET) mikrovesels met ’n biologiese entiteit doelgerig te maak, om so die konsentrasie van TB bacilli in ’n monster te konsentreer. Die entiteit van belang was ’n lektien gebaseerde bindingsgroep, naamlik Concanavalin A (Con A). Con A het ’n affiniteit vir die mannose groepe op die Mtb selwand. Deur hierdie funktionalisering van die vesels is ’n substraat geskep wat potensie¨ el kan dien as ’n konsentrasieen vasvang- platform vir die Mtb-bacilli. Dit volg dat, aangesien hierdie soort vesels algemeen gebruik word as deppers om monster te neem, ’n Mtb-spesifieke depper geskep kan word vir gebruik in die wangholte. Dit sal die bacilli-versameling bemiddel, wanneer ’n monster van die mondslymvlies van ’n pasi¨ent geneem word. Affiniteitstudies tussen die gemodifiseerde PET-mikrovesels en ’n verswakte stam van Mikobakterium bovis, naamlik die basil van Calmette-Gu´ erin (BCG) is uitgevoer. Die BCG stam wat gebruik is, het n fluoresserende prote¨ıen-indikator bevat, naamlik mCherry-BCG. Hierdie studies is ook uitgevoer as n funksie van reeks verdunnings, om die Mtb-onderskeppingsvermo¨e van die aangepaste substrate te evalueer. Die suksesvolle verstrengeling van BCG deur die substrate is bevestig met behulp van konfokale fluoressensiemikroskopie en skanderingelektronmikroskopie. Hierdie studies het bevestig dat al die oppervlak gefunksioneerde substrate daartoe in staat was om die bakterie ¨e suksesvol vas te vang. Dit was moontlik selfs tot en met kultuurkonsentrasies wat ver onder die opsporingsdrempel van sputum-smeermikroskopie val. Die oppervlak-modifikasie wat die beste resultate gelewer het vir BCGonderskepping was ’n kruisgekoppelde prote¨ıen-aggregaat afgeleide, wat kovalent gebind was aan die oppervlak van die vesel, deur middel van Pentaan-1,5-dialdehied.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103744
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