Disease Progression in Plasmodium knowlesi Malaria Is Linked to Variation in Invasion Gene Family Members

Abstract

Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥0.34 p = <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human population must give cause for concern to malaria elimination strategists in the Southeast Asian region.

Plasmodium knowlesi clinical isolates from Malaysia show extensive diversity and strong differential selection pressure at the merozoite surface protein 7D (MSP7D)

“…The Philippine, MR4 and the Hackeri Strains formed a separate group along with Malaysian Borneo isolates. The distinct sub-populations observed in other invasion gene of P. knowlesi like MSP1P, DBPRII, NBPXA and TRAP was not observed in MSP7D [23, 25–27, 31, 49]. (Fig.5).…”

“…Studies on mitochondrial and ssrRNA genes in P. knowlesi from patients and wild macaques identified two distinct sub-populations which clustered geographically to Peninsular Malaysia and Malaysian Borneo [23]. Additionally, genetic and genomic studies from Malaysia identified 3 distinct sub-populations; two originating from Sarawak and one from Peninsular Malaysia [24–27] thereby highlighting the complexity of infections in humans and the challenges for control as well as vaccine design.…”

Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi

“…However, even the humanadapted P. knowlesi line invades cynomolgus RBCs more efficiently than human RBCs, suggesting that invasion efficiency may remain a bottleneck in human infections. Interestingly, variation in NBPXb, which had been found not to bind to human RBCs, also has been linked to disease severity in humans (7,16). The role of NBPXb and variants of both P. knowlesi NBPs now can be investigated in vitro.…”

Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi

“…Particular alleles of two erythrocyte binding protein genes in P. knowlesi were found to be associated with specific disease progression profiles, such as differences in parasitaemia, and markers of disease severity such as hemoglobin levels, platelet levels, renal dysfunction, etc. in a population of knowlesi malaria patients (Ahmed et al , 2014). These two genes are also known to show differential binding between macaque and human erythrocytes (Semenya et al , 2012).…”