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In Vitro Growth Phenotypes of Single Parasite Lineages Cloned from Multiclonal Malaria Isolates

Closeup of the head of a brown mosquito with a blurred background.

ASTMH 2021 Annual Meeting

Virtual Event

November 18, 2021


Measurement of malaria parasite proliferation in cultured erythrocytes is critical for elucidating key determinants of phenotypes, including drug susceptibility, virulence, and fitness. Multiple parasite lineages with different proliferation rates or fitness may coexist within a clinical isolate, resulting in complex growth interactions and variations in phenotype. We measured proliferation rates of three Plasmodium falciparum Cambodian isolates, including IPC_3445 (MRA-1236), IPC_5202 (MRA-1240), IPC_6403 (MRA-1285), and parasite lineages previously cloned from each of these isolates by limiting dilution. Following synchronization, in vitro cultures were maintained over four consecutive asexual parasite life cycles, with parasite sampling at the end of every cycle to estimate parasitemia and growth rate. In parallel with clinical isolates and component parasite lineages, growth rates and relative changes in parasitemia were measured for laboratory parasite lines 3D7 (MRA-102) and DD2 (MRA-150) as controls. We observed significant differences in fold-change in parasitemia (FC), and parasite growth rate (GR) between parasite isolates and clonal lineages that make up each isolate. For example, while isolate MRA-1240 exhibits similar a proliferation rate to one of its constituent lineages, MRA1240-hap1 (GR: 1.03 ± 0.02 vs. 1.02 ± 0.05; FC: 67 ± 6 vs. 62 ± 2; p > 0.05), the other two component lineages (MRA1240-hap2 and MRA1240-hap3) exhibit markedly different growth profiles. We observed that the most abundant parasite haplotype often dominates the growth phenotype, masking the effect of minority haplotypes akin to recent observations from drug susceptibility testing. Our results also show diminished proliferation of isolate MRA-1236 (GR: 0.90 ± 0.02; FC: 39 ± 4) relative to the component lineages MRA1236-hap1 (GR: 1.11 ± 0.02; FC: 101 ± 3) and MRA1236-hap2 (GR: 1.05 ± 0.04; FC: 88 ± 6) suggestive of competitive suppression. All parasite lines are available through BEI Resources and have well-defined in vitro growth phenotypes useful for both research and development of interventions against malaria.

Download the poster to explore the growth phenotypes of single parasite lineages cloned from multiclonal malaria isolates



Standwell Nkhoma, headshot

Standwell C. Nkhoma, PhD

Scientist, BEI Resources

Standwell Nkhoma, PhD, is an experienced parasitologist with a range of research interests in infectious diseases including malaria, and a strong passion for developing new biomedical research tools. He works within ATCC Federal Solutions as a scientist on a NIAID-funded contract to deliver malaria products, services, and standards for the BEI Resources Repository and to our commercial clients. His research & development (R&D) work focuses on developing new and improved approaches to enhance the authentication of MR4-BEI Resources reagents and establishing CRISPR/Cas9 technologies for editing parasite genomes in-house to examine the functional impact of genetic variation on important biomedical phenotypes including drug resistance and pathogen replication. Recent outputs from this work include two published manuscripts demonstrating how complex interactions between parasite lineages within a single malaria isolate affect phenotypic variation and evolution (International Journal for Parasitology: Drugs and Drug Resistance 2021; 15:152–161 and Molecular and Biochemical Parasitology 2023; 254:111552). Data in these manuscripts underscore the need for cloning clinical isolates to yield single parasite lineages with well-defined genotypes and phenotypes. Such clonal lineages are useful for screening candidate antimalarials and as standards for conducting drug resistance surveillance. Dr. Nkhoma earned his PhD in Molecular Biology and Biochemistry from the University of Liverpool, UK.

Female, Aedes aegypti mosquito, from a left lateral perspective, while she was in the process of acquiring a blood meal from her human host

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