Development of copy number assays for detection and surveillance of piperaquine resistance-associated plasmepsin 2/3 copy number variation in Plasmodium falciparum

Long regarded as the epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified a single nucleotide polymorphism as well as a copy number variation molecular marker that associate with clinical and resistance. The copy number polymorphism is a duplication of a region containing members of the plasmepsin multi-gene family of proteases. To accurately and quickly determine the presence of copy number variation in the duplication in field isolates, we developed a quantitative PCR assay using TaqMan probes. We validated copy number estimates using a separate SYBR green-based quantitative PCR assay as well as a novel breakpoint assay to detect the hybrid gene product. Field samples from 2012 – 2015 across 3 sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of gene duplications, as well as . We found high concordance across all methods of copy number detection. For samples derived from dried blood spots we found a greater than 80% success rate in each assay, with more recent samples performing better. We found evidence of extensive copy number amplifications in Pursat (94%, 2015) and Preah Vihear (87%, 2014), and lower levels in Ratanakiri (16%, 2014) in eastern Cambodia. We also see evidence of a shift from two copies of in Pursat 2013 to three copies in 2014-15 (25% to 64%). duplications are absent from all samples in 2014 from Preah Vihear and Ratanakiri and 2015 from Pursat. This study shows increasing levels of gene amplifications across Cambodia from 2012 – 2015 and a complete reversion of mutant parasites in all study locations. The multiplex TaqMan assay is a robust tool for monitoring both and copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring duplications.