TOWARDS THE OPTIMSATION OF MALARIA RAPID DIAGNOSTIC TESTS

Nelson Lee (2010). TOWARDS THE OPTIMSATION OF MALARIA RAPID DIAGNOSTIC TESTS PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Nelson Lee
Thesis Title TOWARDS THE OPTIMSATION OF MALARIA RAPID DIAGNOSTIC TESTS
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2010-02
Thesis type PhD Thesis
Supervisor Dr. James McCarthy
Dr. Qin Cheng
Total pages 166
Total colour pages 20
Total black and white pages 146
Subjects 11 Medical and Health Sciences
Abstract/Summary ABSTRACT Malaria is a deadly parasitic disease affecting the tropics and subtropical regions of the world. Each year, there are more than 300 million cases of malaria in which more than one million people die. The ability to rapidly and reliably diagnose malaria infections is key to both the management of individual patients as well as public health efforts to control the disease. Malaria rapid diagnostic tests (RDTs) offer the potential for such rapid and reliable diagnosis, of which the histidine rich protein 2 (HRP2) based RDTs are most commonly used. However, field studies have reported variable sensitivities of these tests in different settings, to patients with varying levels parasitemia. It is therefore critical to identify the cause of this variation in sensitivity and suggest possible solutions. I hypothesised that the genetic variation of the HRP2 gene which determines the structure, epitope arrangement and epitope number of the protein, determine the affinity and avidity of the interaction between the HRP2 antigen and anti-HRP2 antibody, hence the sensitivity of the HRP2 detecting RDTs. I also hypothesised that the heat susceptibility of the anti-HRP2 antibodies are different and can have significant influence on the shelf life of the HRP2 RDTs. This study involved the characterization of a panel of 12 anti-HRP2 monoclonal antibodies to determine their suitability for use in RDTs and to find a combination of MABs that would give the highest sensitivity in the context of a RDT. Aims of this study are: 1. To determine the epitopes recognized by the anti-HRP2 monoclonal antibodies and investigate the impact of epitope diversity such as the number, structure and arrangement of epitopes in different strains on the antigen-antibody binding affinity and avidity. 2. To analyse the heat susceptibility and isotypes of the anti-HRP2 antibodies and to suggest a combination of MABs for the development of a new malaria RDT with the highest sensitivity and storage life. 3. To determine the diversity of the HRP2 epitopes in parasite isolates from geographically different regions and to determine specific motifs that will potentially be used for future antibody production. Key outcomes of the research included: a) Mapped the epitopes recognised by the MABs and correlated the epitope structure, length and number of times present in a HRP2 sequence with potential sensitivity. b) Determined and rank the MABs based on their binding avidity, rate of binding and the stability of binding, which are all important attributes for a RDT MAB. c) Heat susceptibility of the MABs were determined and recombinant HRP2 was produced for potential use as a positive control antigen. d) Identified the best combination of MABs to use to obtain a malaria RDT with the highest sensitivity and storage life. e) Identified a 10 amino acid HRP2 Motif for future MAB production that is present in all HRP2 strains sequenced and is present in the highest prevalence. This research has provided important knowledge on the potential causes of the variation in the sensitivity of the malaria RDTs. The approach used in this study has allowed for the characterisation of the interaction between the monoclonal antibodies and HRP2. The findings have allowed us to determine a possible combination of MABs that would result in a malaria diagnostic test with the highest potential sensitivity and stability. The study also suggested possibilities for the development of better anti-HRP2 MABs for use in future RDTs. Malaria RDTs is currently the best available option for the diagnosis of P. falciparum infections in most endemic regions of the globe. The outcomes of this research will potentially overcome the current difficulties in accurate and reliable malaria diagnosis and reduce the misdiagnosis of patients. The results obtained in this study will increase the pace of current malaria control and assist in global efforts to eradicate this deadly disease.
Keyword Rapid Diagnostic Tests (RDTs)
Malaria
Histidine Rich Protein (HRP)
Histidine Rich Protein 2 (HRP2)
Plasmodium falciparum
Rapid Diagnostic Tests (RDTs)
Malaria Diagnosis
Additional Notes Colour Pages: 21-22, 32, 69-71, 74, 85, 87, 90, 107-108, 111-117, 162

 
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Created: Tue, 23 Nov 2010, 16:09:22 EST by Mr Nelson Lee on behalf of Library - Information Access Service