Limited Polymorphisms in Plasmodium falciparum Lines Exposed to Pure Artemisinin and Artemisia Annua Extracts

Artemisinin-based combination therapy has been a vital tool in malaria control and elimination programmes. However, artemisinin resistant P. falciparum parasites have emerged in Southeast Asia, posing a major threat to the effectiveness of ACT. Resistance results in prolonged parasite clearance in vivo and enhanced survival of ring-stage parasites in vitro. Therefore, understanding the genetic basis of resistance would be critical to the success treatment and intervention strategies. This study aimed at identifying single nucleotide polymorphisms associated with artemisinin and Artemisia annua resistance. Genetic analysis was done on P. falciparum lines W2 and D6, previously selected under pure artemisinin and Artemisia annua extracts. Genomic DNA was extracted using QIAamp blood mini kit. Libraries were sequenced on Illumina Miseq platform using 151bp paired-end chemistry. Sequencing read data from each sample was mapped against P. falciparum reference sequence version 3.1. One non-synonymous (NS) mutation K189T was identified in K13 gene. The Pfmdr1 mutation N86Y was detected in W2 parasite exposed to pure artemisinin at IC50 equivalents and notably, the Pfcrt CQ sensitive CVMNK genotype was retained. The study also identified one background mutation in Pfcrt (I356T) in W2 parasites exposed to artemisinin at IC50 equivalents. In conclusion, K13 mutation described here has not been linked to reduced parasite clearance or in vitro artemisinin tolerance. Pfmdr1 gene may putatively play a role in artemisinin resistance.