Dynamics of parasite growth in genetically diverse Plasmodium falciparum isolates.

Multiple parasite lineages with different proliferation rates or fitness may coexist within a clinical malaria isolate, resulting in complex growth interactions and variations in phenotype. To elucidate the dynamics of parasite growth in multiclonal isolates, we measured growth rates (GRs) 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 of each parasite line were maintained over four consecutive asexual cycles (192 h), with thin smears prepared at each 48-h cycle to estimate GR and fold change in parasitemia (FCP). Cell cycle time (CCT), the duration it takes for ring-stage parasites to develop into mature schizonts, was measured by monitoring the development of 0-3-h post-invasion rings for up to 52 h post-incubation. Laboratory lines 3D7 (MRA-102) and Dd2 (MRA-150) were used as controls. Significant differences in GR, FCP, and CCT were observed between parasite isolates and clonal lineages from each isolate. The parasite lines studied here have well-defined growth phenotypes and will facilitate basic malaria research and development of novel malaria interventions. These lines are available to malaria researchers through the MR4 collection of NIAID's BEI Resources Program.

Dissection of haplotype-specific drug response phenotypes in multiclonal malaria isolates.

Natural infections of Plasmodium falciparum, the parasite responsible for the deadliest form of human malaria, often comprise multiple parasite lineages (haplotypes). Multiclonal parasite isolates may exhibit variable phenotypes including different drug susceptibility profiles over time due to the presence of multiple haplotypes. To test this hypothesis, three P. falciparum Cambodian isolates IPC_3445 (MRA-1236), IPC_5202 (MRA-1240) and IPC_6403 (MRA-1285) suspected to be multiclonal were cloned by limiting dilution, and the resulting clones genotyped at 24 highly polymorphic single nucleotide polymorphisms (SNPs). Isolates harbored up to three constituent haplotypes, and exhibited significant variability (p < 0.05) in susceptibility to chloroquine, mefloquine, artemisinin and piperaquine as measured by half maximal drug inhibitory concentration (IC50) assays and parasite survival assays, which measure viability following exposure to pharmacologically relevant concentrations of antimalarial drugs. The IC50 of the most abundant haplotype frequently reflected that of the uncloned parental isolate, suggesting that a single haplotype dominates the antimalarial susceptibility profile and masks the effect of minor frequency haplotypes. These results indicate that phenotypic variability in parasite isolates is often due to the presence of multiple haplotypes. Depending on intended end-use, clinical isolates should be cloned to yield single parasite lineages with well-defined phenotypes and genotypes. The availability of such standardized clonal parasite lineages through NIAID's BEI Resources program will aid research directed towards the development of diagnostics and interventions including drugs against malaria.