Piperaquine resistant CambodianPlasmodium falciparumclinical isolates: in vitro genotypic and phenotypic characterization

Background High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicatedPlasmodium falciparumin Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy. Methods To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeenP. falciparumisolates from northern Cambodia, and the presence of E415G-Exo andpfcrtmutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well aspfpm2copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly clonedP. falciparumCambodian isolates was also determined. Results The characterization of culture-adapted isolates revealed that the presence of novelpfcrtmutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence ofpfpm2amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone-proguanil combinations revealed synergistic antimalarial activity. Conclusions Surveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance.P. falciparumharbouring novelpfcrtmutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence ofpfpm2amplification was not required to render parasites PPQ resistant suggesting that the increase inpfpm2copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations againstP. falciparumstrains and when down-selecting novel drug candidates.