In Vitro Susceptibility of Plasmodium falciparum Isolates from the China-Myanmar Border Area to Piperaquine and Association with Candidate Markers

Plasmodium falciparum from the Greater Mekong subregion has evolved resistance to the artemisinin-based combination therapy dihydroartemisinin and the partner drug piperaquine. To monitor the potential westward spread or independent evolution of piperaquine resistance, we evaluated the in vitro susceptibility of 120 P. falciparum isolates collected at the China-Myanmar border during 2007 to 2016. The parasite isolates displayed a relatively wide range of piperaquine susceptibility estimates. While 56.7% of the parasites showed bimodal drug response curves, all but five generated area-underthe-curve (AUC) estimates consistent with a susceptible phenotype. Using the piperaquine survival assay (PSA), 5.6% parasites showed reduced susceptibility. Of note, parasites from 2014 to 2016 showed the highest AUC value and the highest proportion with a bimodal curve, suggesting decreasing effectiveness in these later years. Unsupervised K-mean analysis of the combined data assigned parasites into three clusters and identified significant correlations between 50% inhibitory concentration (IC50), IC90, and AUC values. No parasites carried the E415G mutation in a putative exonuclease, new mutations in PfCRT, or amplification of the plasmepsin 2 and 3 genes, suggesting mechanisms of reduced piperaquine susceptibility that differ from those described in other countries of the region. The association of increased AUC, IC50, and IC90 values with major PfK13 mutations (F446I and G533S) suggests that piperaquine resistance may evolve in these PfK13 genetic backgrounds. In addition, the Pfmdr1 F1226Y mutation was associated with significantly higher PSA values. Further elucidation of piperaquine resistance mechanisms and continuous surveillance are warranted.