Detection of Plasmodium falciparum: From Biomarker Identification and Characterisation to Application

Abstract

Malaria is one of the most deadly infectious diseases, responsible for 438 000 deaths and 214 million infections per year, mostly in developing countries. Ultra-sensitive detection of the asexual and sexual forms of Plasmodium spp., the causative agent of malaria, is crucial to reduce the staggering numbers of malaria infections with the ultimate goal of eradication, and it is important for research. Unfortunately all current detection methods and biomarkers for malaria have limitations. The performance of antigen-detection assays depends strongly on the biomarker detected, the antibodies used to target the biomarker and the detection method itself. The goal of this project was to improve the detection of Plasmodium falciparum by addressing these three main factors. First, novel biomarkers were identified in a bioinformatics approach, then experimentally characterized and evaluated for their suitability to use in diagnostic tests. KAHRP and PFD1170c were recognized as potential markers for asexual stage parasites. A particular focus was on biomarkers specific for gametocytes, as gametocytes are the only stage able to infect mosquitos, therefore crucial for the spread of the disease and prime targets for transmission blocking strategies. Potential gametocytes specific biomarkers were selected through a transcriptome study of field samples. The candidates were then GFP-tagged for characterization at the protein level. PFC0685w was identified as gametocyte specific biomarker. The detection limit of antigen-detection assays depends on the availability of high affinity antibodies targeting the biomarker. A limiting factor for the generation of high affinity antibodies is the difficulty of expressing Plasmodium antigens required for antibody generation. Dictyostelium discoideum was assessed as an alternative host for the recombinant antigen expression. KAHRP was successfully expressed in Dictyostelium discoideum using an inducible system. An immuno-qPCR, the so-called TT-lock immuno-qPCR was adapted for the quantitative detection of malaria parasites. In general, immuno-qPCR uses a DNA-conjugated antibody to detect a specific antigen by PCR amplification. Its advantage is an unprecedented sensitivity. However, one major challenge in immuno-qPCR is the synthesis of protein-DNA conjugates. The TT-lock immuno-qPCR uses the strong interaction between the DNA-binding protein Tus and a specific DNA sequence for the protein-DNA conjugation. The TT-lock immuno-qPCR has been optimized to detect PfLDH, a widely used biomarker in commercial available rapid diagnostic tests. The assay performed well in detecting recombinant PfLDH, but for the detection of parasite material it did not perform better than already existing methods. The findings of this study highlight the difficulties of improving malaria diagnosis using antigens. To find the right antibodies for a detection method is a challenging task, as each antibody needs to be evaluated experimentally and the generation of new antibodies does not guarantee a higher quality. In hand go the challenges to express recombinant antigen needed for antibody generation. Furthermore, the detection method optimized in this study needs further fine-tuning to push the limit of detection, even after extensive attempts of optimization. Show more