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
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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.
