Detection of molecular markers of antimalarial resistance

We assessed polymorphisms in the PfKelch13 gene by nested PCR amplification covering the full length of the gene (total 2,181 bp) and sequenced the gene by ABI Sequencer (Macrogen, Seoul, Republic of Korea) as described previously. We monitored cross-contamination by adding negative control samples in every run. Sequencing results were aligned against PfKelch13 of reference strain 3D7 (putative 9PF13_0238 NCBI Reference Sequence [3D7]: {"type":"entrez-nucleotide","attrs":{"text":"XM_001350122.1","term_id":"124513603","term_text":"XM_001350122.1"}}XM_001350122.1), using Bioedit software (Abbott, Santa Clara, CA, US). Two study technicians assessed polymorphic patterns blinded to the origin of the sample.

We quantified Pfplasmepsin2/3 gene copy number using relative quantitative real-time PCR based on Taqman probe on a Corbett Rotor-Gene Q (Corbett Research, Mortlake, NSW, Australia). Primers and probes have been described previously [56]. We performed amplification in triplicate on a total volume of 25μl as multiplex PCR using a QuantiTect Multiplex PCR NoROX Kit (Qiagen, Hilden, Germany). Every amplification run contained 9 replicates of calibrators and triplicates without template as negative controls. Plasmodium-specific beta-tubulin served as an internal standard for the amount of sample DNA added to the reactions.