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<jats:title>Abstract</jats:title><jats:p>Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of <jats:italic>Plasmodium falciparum</jats:italic> 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 <jats:italic>in vitro</jats:italic> 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 <jats:italic>plasmepsin 2/3</jats:italic> 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 <jats:italic>plasmepsin 2/3</jats:italic> gene duplications, as well as <jats:italic>pfmdr1</jats:italic>. 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 plasmepsin 2/3 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 <jats:italic>plasmepsin 2/3</jats:italic> in Pursat 2013 to three copies in 2014-15 (25% to 64%). <jats:italic>Pfmdr1</jats:italic> duplications are absent from all samples in 2014 from Preah Vihear and Ratanakiri and 2015 from Pursat. This study shows increasing levels of <jats:italic>plasmepsin 2/3</jats:italic> gene amplifications across Cambodia from 2012 – 2015 and a complete reversion of <jats:italic>pfmdr1</jats:italic> mutant parasites in all study locations. The multiplex TaqMan assay is a robust tool for monitoring both <jats:italic>plasmepsin</jats:italic> and <jats:italic>pfmdr1</jats:italic> copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring <jats:italic>plasmepsin 2/3</jats:italic> duplications.</jats:p>

Original publication

DOI

10.1101/655209

Type

Journal article

Publisher

Cold Spring Harbor Laboratory

Publication Date

31/05/2019