Investigation of Markers of Artemisinin Resistance at Selected Intervals during the 72 h Period after Artemisinin based Combination Therapy Dosing in Kisumu Western Kenya

Background: Microscopic parasite detection during the course of treatment or follow-up suggests that a proportion of ACT treated children in Kenya do not completely clear Plasmodium falciparum parasitemia. Plasmodium falciparum mutation in chloroquine resistance transporter gene (Pfcrt76), multidrug resistance gene1 (Pfmdr1), deubiquitinating enzyme gene (Pfcubp-1) and clathrin vesicle associated adapter 2, u subunit encoding gene (Pfap2mu) and multidrug resistant protein 1 gene (Pfmrp1) have been associated with subsequent patent recrudescence after ACT treatment. As there are no validated markers of ACT resistance in Africa to-date, surveillance of changes in these polymorphisms during the course of treatment is useful in establishing their role in ACT treatment outcome. Methods: 118 P. falciparum 2013 to 2015 samples from ACT clinical efficacy studies were genotyped for frequency of drug resistance polymorphisms using sequence analyzers. Each sample was screened at least three to four-time points namely; day zero before start of treatment then days 2 and 3 after initiation of treatment plus the day of subsequent parasitemia by microscopy prior to day 42 for some of the subjects. Sequence analyzers were used to genotype for frequency of drug resistance polymorphisms, Pfmdr1 gene copy number and genetic diversity typing of the 12 microsatellite loci. Genetic diversity of parasite populations across four time-points was determined by analysis of 12 microsatellite loci. Worldwide Antimalarial Resistance Network’s parasite clearance estimator (PCE) was used to determine parasite clearance rates. Results: The new genes Pfap2mu and Pfubp had S145C and E1528D being most polymorphic with prevalence’s of 18% and 19%, respectively Pfmdr1 86,184 and 1246 had significant increase in wild type alleles between day zero and time-points 3 and 4. Microsatellite profile analysis show that the mean number of alleles in all the loci across the 8 populations ranged from 9.250 to 1.000. Poly α was the most polymorphic with 35 alleles. The mean unbiased HE was 0.672 while Shannon diversity index for the 8 populations was ranging between 0.182-0.000, none of the parasite analyzed had matching haplotypes. The mean parasite clearance half-life was 2.63 h (95% confidence interval [CI]) and the median clearance half-life was 2.24 h. The parasite clearance half-life ranged from 1.14-5.05 h. Conclusion: Increased wild-type Pfmdr1 86,184 and 1246 as well as polymorphisms in Pfap2mu and Pfcubp-1 in post day zero suggest that these genes could be responding to ACT dosing and therefore require continued monitoring. Samples with multiple copies of the Pfmdr1 did not indicate show effect on parasite clearance rate. Though there was no correlation between these profiles and clearance rates, further evaluations are needed to determine the potential public health implications of these observations and the utility of these loci as markers of artemisinin sensitivity in populations of P. falciparum worldwide.