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.