Population genetics of Plasmodium falciparum parasites exposed to artemisinin treatment in patients taking part in an efficacy clinical trial in Kisumu county, western Kenya.

Abstract

Artemether-lumefantrine (AL), adopted in Kenya for malaria treatment, remains highly efficacious. However, there are heightened concerns because resistance to Artemisinin combination therapy (ACTs) is now well documented in Southeast Asia (SEA). This resistance is associated with slow parasite clearance rates (CRs). There is now need for more genetically determined artemisinin resistance data from malarious regions that use ACT for malaria treatment. Therefore, the main objective of this study was to determine the role that population genetics plays in Plasmodium falciparum parasites exposed to artemisinin treatment amongst patients taking part in an efficacy clinical trial in Kisumu county, western Kenya. To accomplish this, two sets of archived samples were used. The first set was collected before the introduction of AL (pre- ACTs n= 29) and the second set from a randomized open labeled trial (post-ACTs n= 92). A panel of 12 microsatellites (MS) and 91 single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome were used to genotype the parasites. The 12 MS were selected because they have previously been shown to effectively differentiate P. falciparum strains. Similarly, the 91 SNPs have previously been shown in a SEA study to target minor allele frequency greater than 5% and are highly polymorphic therefore important in identifying similar parasites. The SNPs were also used to determine genes that may be involved in influencing parasite CRs. The 12 MS were highly polymorphic in post-ACTs parasites and showed greater genetic diversity compared to pre-ACTs parasites (p < 0.0226 at 95% CI). Most post-ACTs parasites achieved clearance within 42 hours of treatment with AL having a median xvi clearance half-life of 2.55 hours (range 1.19-5.05). Of the 91 SNPs assessed, 78 (85%) generated allele calls in all parasites and showed positive correlation with parasite clearance half-life. Based on SNP analysis, only 15 of 92 (16.3%) post-ACTs parasites analyzed were single-clone infections and genetically related. Interestingly, no SNPs in the K13-propeller gene were observed in the Kenyan parasites. Among the 78 SNPs with positive correlation of parasites clearance, 3 SNPs on chromosome 12 and 14 were associated with delayed parasite CRs. These 3 SNPS encode Plasmodium proteins with unknown functions. Based on results from this study, it can be recommended that advanced technologies such as use of the CRISPR-Cas9 system should be applied to validate the role these 3 SNPs have in influencing delay in parasite CRs. In conclusion, these three SNPs may serve as genetic markers associated with ACTs resistance in Kenya.