Authors

A Ibrahim²; F Mohring²; E Manko²; D Van Schalkwyk²; J Phelan²; D Nolder²; S Borrmann⁴; SM Di Santi¹; F Nosten³; CJ Sutherland²; RM Moon²; T Clark²; S Campino²;  ¹ University of Sao Paulo, UK;  ² London School of Hygiene and Tropical Medicine, UK;  ³ Shoklo Malaria Research Unit, Thailand;  ⁴ University of Tübingen, UK

Discussion

Malaria caused by Plasmodium malariae is a neglected tropical disease with a wide geographic distribution, contributing up to 40% of cases in regions of Africa and South America. This parasite species has unique biological traits, including a quartan life cycle, an ability to persist in the blood causing chronic infections, and associations with severe disease including nephropathologies and anaemia. These unique features may underly the observed reduced parasite drug response and drive malaria recurrence, representing a challenge for disease elimination. To provide insights into P. malariae biology, we explore the genomic diversity of this parasite across 4 endemic regions, spanning 25 countries. Through selective whole genome amplification, we obtain whole genome sequence data for 235 clinical isolates of P. malariae and identify 1,288,675 genome-wide SNPs, filtered to 143,201 high-quality SNPs within the core genome. We determine population structure, demonstrating a clear separation in parasites from Africa and Asia, and shared ancestry between South American and African parasites. We identify signals of selection in genes associated with the human immune response (pmmsp3) in addition to orthologs of genes associated with artemisinin-induced latency (pmeIF2a). We identify mutations within orthologs of genes associated with drug susceptibility, including amino acid substitutions in the DHFR-TS, CRT, MDR2, K13, CORONIN, PI4K, MRP1 and UBP1 proteins. We investigate the DHFR protein further and find mutations which align with known resistance mutations in the P. falciparum ortholog and use this as a candidate for further functional analysis. Due to the lack of a stable culture method for P. malariae, we use orthologue replacement in P. knowlesi parasites at the DHFR locus to investigate pyrimethamine susceptibility in the P. malariae genotypes described. We demonstrate a P. malariae genotype at the DHFR locus which reduces pyrimethamine susceptibility in comparison to the P. knowlesi genotype, in addition to known control phenotypes validating the approach for drug susceptibility assays. Our study provides the first evaluation of P. malariae population structure and creates a valuable resource for elucidating important insights into the biology and pathogenesis of this neglected pathogen.