The Development of Aptamer-Based Technology for Point-Of-Care Malaria Diagnosis

This dissertation, "The Development of Aptamer-based Technology for Point-of-care Malaria Diagnosis" by Roderick Marshall, Dirkzwager, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Malaria is an infectious disease caused by Plasmodium parasites which infects 200 million people a year and claims the lives of 600 000 despite a range of treatments being available. Effective diagnosis is key to malaria elimination but currently only 2/3 of patients receive a diagnostic test prior to administration of antimalarials. The overprescription of antimalarials carries the danger of exacerbating resistance. Antibody-based rapid diagnostic tests (RDTs) have greatly helped diagnosis in the field with 160 million units being sold in 2013 however unreliability in transport and storage temperatures can reduce the effectiveness of these technologies. Aptamers are single stranded oligonucleotide biorecognition agents which are developed in an in vitro selection technique and can serve as stable alternatives to antibodies in diagnostic applications. In a previous report, an aptamer (2008s) was developed to specifically bind to the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) and its crystal structure was elucidated. Therefore, the objective of this study was to adapt the 2008s aptamer into a sensitive, stable and cost effective malaria test system which is suitable for point-of-care diagnosis. This was achieved in the form of an aptamer-tethered enzyme capture (APTEC) assay. The APTEC assay functions by tethering PfLDH out of blood samples through aptamer binding, washing away any other biomolecules then adding a development reagent which, using a coupled reaction and the intrinsic enzymatic activity of PfLDH, produces a blue colourimetric response to signify malaria infection. In its 96-well plate format, APTEC had a limit of detection (LOD) of 4.9 ngmL-1 for recombinant PfLDH and 0.024% parasitemia for erythrocyte samples infected with cultured 3D7 P. falciparum parasites. When testing healthy and P. falciparum positive patient samples, specificity of 100% and sensitivity of 80% was observed. This was the same sensitivity detected when a commercially available antibody-based RDT was used. In addition, the APTEC assay which incorporates the 2008s aptamer was found to provide species specific diagnosis for P. falciparum infections and not P. vivax. This specificity was confirmed using P. vivax patient samples and biophysical analysis of recombinant PvLDH with 2008s. APTEC was additionally adapted to a point-of-care format by developing a paper-based syringe test using 3D printing to aid rapid prototyping. The syringe test, whose paper test zone turns blue for malaria positive samples, was found to have an equivalent sensitivity as the plate assay but does not require any equipment or specific expertise to be used and has a total test time of under an hour. Further stabilisation experiments confirmed that the APTEC syringe test could stay stable for long periods of time even at elevated temperatures and cost analysis found that per test, the raw reagents only cost a few USD cents. In conclusion, a novel aptamer-based diagnostic test for malaria was developed, clinically verified and adapted to a point-of-care format. It is hoped that the APTEC test system described here will be further optimised for the commercial market and provide a sensitive, stable and cost effective alternative to antibody-based RDT technologies. Subjects: OligonucleotidesMalaria - Diagnosis