Molecular methods for the detection of infectious diseases: bringing diagnostics to the point-of-care
File(s)
Author(s)
Malpartida Cardenas, Kenny Shannen
Type
Thesis
Abstract
Human infectious diseases represent a leading cause of morbidity and mortality globally, caused by human-infective pathogens such as bacteria, viruses, parasites or fungi. Point-of-care (POC) diagnostics allow accessible, simple, and rapid identification of the organism causing the infection which is crucial for successful prognostic outcomes, clinical management, surveillance and isolation. The research conducted in this thesis aims to investigate novel methods for molecular-based diagnostics. This multidisciplinary project is divided into three main sections: (i) molecular methods for enhanced nucleic acid amplification, (ii) POC technologies, and (iii) sample preparation. The application, design and optimisation of loop-mediated isothermal amplification (LAMP) is investigated from a molecular perspective for the diagnostics of emerging infectious pathogens and antimicrobial resistance. LAMP assays were designed to target pathogens responsible for parasitic (malaria), bacterial and viral (COVID-19) infections, as well as antimicrobial resistance. A novel LAMP-based method for the detection of single nucleotide polymorphisms was developed and applied for diagnostics of antimicrobial resistance, emerging variants and genetic disorders. The method was validated for the detection of artemisinin-resistant malaria. Furthermore, this thesis reports the optimisation of LAMP from a biochemical perspective through the evaluation of its core reagents and the incorporation of enhancing agents to improve its specificity and sensitivity. In order to remove cold-chain storage from the diagnostic workflow, the optimised LAMP protocol was designed to be compatible with lyophilisation. Translation of LAMP to the POC demands the development of detection technologies that are compatible with the advantages offered by isothermal amplification. The use of simple, accessible and portable technologies is investigated in this thesis through the development of: (i) a novel colorimetricLAMP detection method for end-point and low cost detection, and (ii) the combination of LAMP with an electrochemical biosensing platform based on ion-sensitive field effect transistors (ISFETs) fabricated in unmodified complementary metal-oxide semiconductor (CMOS) technology for real-time detection. Lastly, current nucleic acid extraction methods are not transferable to be used outside the laboratory. Research of novel methods for low-cost and electricity-free sample preparation was carried out using cellulose matrices. A novel, rapid (under 10 min) and efficient nucleic acid extraction method from dried blood spots was developed. A sample-to-result POC test requires the implementation and integration of molecular biology, cutting-edge technology and data-driven approaches. The work presented in this thesis aims to set new benchmarks for the detection of infectious diseases at the POC by leveraging on developments in molecular biology and digital technologies.
Version
Open Access
Date Issued
2021-11
Date Awarded
2022-03
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Georgiou, Pantelakis
Rodriguez Manzano, Jesus
Sponsor
Engineering and Physical Sciences Research Council
Grant Number
EP/L016796/1
Publisher Department
Electrical and Electronic Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)