Development and implementation of molecular methods to genotype Plasmodium falciparum in uncomplicated malaria and severe malarial anemia in Gabon

Abstract

Understanding the population structure of malaria parasites and the host-parasite interaction is of fundamental interest and may help in the development of improved anti-malarial interventions. This thesis contributes to the improvement of procedures and methods for the characterization of the malaria parasite and reports current epidemiological characteristics of Plasmodium falciparum populations in Lambaréné, Gabon. The result of this thesis encourages the use of rapid diagnostic tests (RDT) not only as a diagnostic tool but also as a source of DNA that can be subsequently used for molecular assays to assess parasite diversity, species distribution, and genetic polymorphisms (e.g. mutations associated with drug resistance) as well as diagnostic performance of RDTs on large scale. In addition, performance and resolution of a genotyping method based on size polymorphisms of the msp1 gene, by using conventional polymerase chain reaction (PCR) followed by automated capillary electrophoresis (CE) (QIAxcel system, Qiagen) has been improved. Furthermore, in order to overcome the comparatively low sensitivity of conventional PCR, a barcoding assay with 9 single nucleotide polymorphisms (SNPs) for genotyping of low-density P. falciparum infection by Taqman-probe-based end-point PCR was adapted from a published method. The alternative use of these two approaches will help to improve the accuracy of parasite genotyping, covering different types of samples, applications, and lab settings. The improved techniques were applied in two case scenarios: i) an epidemiological survey and ii) to assess parasite population structure in different compartments of the body. It was observed that in Lambaréné and surroundings, the multiplicity of infection (MOI) and prevalence of chloroquine-resistance-associated mutations remain high; particularly, in the most rural areas and despite a change in malaria control recommendations, including withdrawal of chloroquine from the market. Decreasing MOIs in central areas of Lambaréné may be the result of urbanization and its effect on transmission intensity and spread of drug resistance, which results in less diverse malaria parasite populations. 55 Within its human host, malaria parasite population structure in both severely anemic and control patients was shown to be similar between bone marrow and peripheral blood. If the differences observed in some patients is of pathophysiological importance remains to be further investigated. My thesis highlights the crucial role of adequate genotyping approaches in the development of malaria eradication tools, identifies a commonly available source of DNA for retrospective studies and suggests an improvement in the guidelines for sample collection and molecular analyses for studying malaria in endemic regions.