Genetic Structure of Malaria Parasites Obtained Over a Ten Year Period from Patients Residing in the Diverse Ecosystem of Kenya

Human malaria is caused by five species of parasites of the genus Plasmodium: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi. Of the five species, P. falciparum malaria is the most lethal, causing more than half a million deaths annually. Globally, approximately 3.3 billion people are at risk of the disease, the majority living in Africa. Global efforts to reduce malaria have benefitted from the use of artemisinin combination therapies, insecticidetreated bed nets, and the use of rapid diagnostic tests (RDTs). Despite the reduction in malaria-related mortality and morbidity, the burden of disease on African children, pregnant mothers, and international travelers is still huge. P. falciparum is subject to strong selective forces from host immune responses and antimalarial drug therapies. Additionally, sustained use of RDTs that target specific parasite antigens such as histidine rich protein-2 (HRP2) and 3 have been linked to replacement of wild type parasites with strains with deleted or mutated HRP-2/3 genes. We, therefore, propose to evaluate the genetic structure of P. falciparum in blood samples collected over a ten-year period from patients residing in diverse ecosystems of Kenya. Understanding the genetic structure and diversity of P. falciparum populations, and how these are shaped by temporal and spatial dynamics is important to monitor interventions, guide control and treatment policies, and identify hot spots of transmission. Blood samples for this study will be obtained from an ongoing surveillance study (KEMRI SSC # 1282, WRAIR #1402) that recruits patients (>1 year) who present to hospitals with fever (>38oC). The surveillance sites are distributed across different ecological zones in Kenya. The samples will be screened for the presence of P. falciparum by real-time quantitative PCR (qRT-PCR) and only samples positive for P. falciparum will be included in the analysis. Samples will be analyzed retrospectively (2008-2022). For this analysis, over 1000 samples collected at selected time points in five different geographical locations will be utilized. Methods to elucidate genetic structure will include a novel highly-multiplexed and low-cost (compared to whole genome sequencing) deep sequencing assay for P. falciparum recently described by Kattenberg (Kattenberg et al., 2022). The assay uses amplicon sequencing that targets different markers (antimalarial drug resistance markers, SNP barcodes (192) that have been designed to capture the diversity of P. falciparum populations in Africa, KEL1 lineage markers, ama1 gene, and HRP2/3 and flanking region genes) in a two-step PCR. Following amplification, the amplicon libraries will be sequenced using the next generation sequencing platform (Illumina MiSeq), available at USAMRD-Kenya. Using the relevant bioinformatics pipelines and population genetic software and packages such as Chromopainter, finestructure in R, the resulting data will be analyzed for the multiplicity of infection, population structure, and population differentiation and gene flow. These measures will be used to evaluate the impact of the past interventions and other selective pressures on the parasite population structure in different ecoregions of Kenya.