Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

Abstract Background: High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. It is essential to establish sensitive and reliable markers of PPQ resistance which can be adopted for monitoring the prevalence of drug resistance and guide drug policy change. Several genetic markers have been proposed such as copy numbers of P. falciparum multidrug resistance 1 ( pfmdr1 ) and plasmepsin 2 (PM2), and mutations in exonuclease (exo-E415G) and P. falciparum chloroquine resistance transporter (PfCRT) genes.Methods: To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia and the presence of exo-E415G and PfCRT mutations as well as PM2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. The efficacy of several drug combinations between standard clones and newly cloned P. falciparum Cambodian isolates was also measured.Results: The characterization of culture-adapted isolates revealed that exo-E415G and novel PfCRT mutations can confer PPQ-resistance, in the absence of PM2 amplification. In vitro testing of PPQ resistant parasites showed bimodal dose-response phenotype as previously reported in both Cambodian isolates and genome-edited Dd2 strains. Our finding is the first PPQ resistant clinical isolate with documented swollen digestive vacuole phenotype. From the field isolates, we were able to clone a new parasite line, 14-B5, which is sensitive to arteminsinin and piperaquine but resistant to chloroquine. Most of the drug combinations tested revealed antagonistic interaction against the 14-B5 line, indicating its different genetic background from other P. falciparum laboratory reference lines.Conclusions: Surveillance for PPQ resistance in regions that rely on DHA-PPQ as the first line treatment should depend on the monitoring of the reflective molecular markers. Bimodal dose response curves and swelling of the food vacuole can be used as PPQ resistant phenotype. The use of currently circulating parasite isolates is necessary for relevant drug combination development against current P. falciparum resistance profiles.