Genetic basis of resistance in Plasmodium falciparum parasites exposed to pure artemisinin and Artemisia annua extracts

Abstract

Artemisinin-based combination therapies are the current front-line antimalarials in majority of the countries where the disease is endemic. Their safety and tolerability profile is excellent. However, the emergence of artemisinin resistance in Southeast Asia is alarming. Resistance is characterized by prolonged parasite clearance rates (CR) following treatment and reduced ring-stage susceptibility to artemisinin. 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 Plasmodium falciparum lines W2 (CQ-resistant strain from Indochina) and D6 (CQ-sensitive strain from Sierra Leone), previously selected under pure artemisinin and Artemisia annua extract for 3 years. Genomic DNA was extracted using QIAamp blood mini kit and libraries were sequenced on Illumina Miseq platform using 151bp paired-end chemistry. D6 and W2 parasites cultured without drug exposure were used as controls. Paired-end short reads were mapped against P.falciparum reference genome sequence version 3.1. One non-synonymous mutation K189T (70%) was identified in the Plasmodium/Apicomplexa-specific domain of K13 gene. The Pfmdr1 mutation N86Y was detected in W2 parasites exposed to artemisinin drug at IC50 equivalents and notably, all the parasites showed a single Pfmdr1 copy number. The Pfcrt K76T mutation was detected in two samples, W2 parental line and W2 parasites exposed to ART drug. Also, one background mutation was identified in Pfcrt gene (I356T) in W2 parasites exposed to pure artemisinin at 1C50 equivalents. This study showed a very limited variability in K13 gene sequence and the K189T mutation described here has not been linked to reduced parasite clearance or in vitro artemisinin tolerance. The Pfmdr1 gene may putatively play a role in artemisinin resistance although additional studies are required to conclusively confirm this. Additionally, parasites exposed to Artemisia annua harbored wildtype alleles in all the gens analyzed here. Therefore, no mutation was associated with Artemisia annua resistance.