Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether–lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya

Abstract

Artemether–lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995–2003) and 745 after (post-ACT; 2008–2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995–1996 to 93.2% in 2014 and 0.0% in 2002–2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya.

In Vitro and Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity

“…Previous studies demonstrated that LU may select for CQ-sensitive alleles such as the Pfmdr-1 N86 and Pfcrt K76 alleles, and these alleles have been associated with AL treatment failure (46,47). Our study and recent studies in western Kenya show a significant increase in the prevalence of the WT allele at codon K76 of Pfcrt and an upward trend in the prevalence of WT alleles in Pfmdr-1 codons N86 and N1246 in parasites collected after the introduction of ACT in Kenya (31). Therefore, the use of AL as the first line of malaria treatment in Kenya may be contributing to the selection of these wild-type alleles.…”

“…For example, 10 years after the discontinuation of CQ for malaria treatment in Malawi, several studies demonstrated the return of CQ-sensitive parasites and an increase in the prevalence of parasites harboring the wild-type (WT) Pfcrt K76 codon (22,23). Similar trends were later reported from studies conducted in other parts of Malawi (24,25), Tanzania (26,27), and Kenya (28)(29)(30)(31), raising the possibility of the selected reintroduction of CQ for malaria treatment. In contrast, countries that delayed the withdrawal of CQ for malaria treatment demonstrated no significant decreases in the prevalence of CQ resistance parasites (reviewed in reference 32), implying that drug pressure plays a major role in the maintenance of drug-resistant parasites in a population.…”

Plasmodium Falciparum Multidrug Resistance Gene-1 N86Y-Y184F-D1246Y Polymorphisms in Northern Nigeria: Implications for the Continued Use of Artemether-Lumefantrine in the Region

“…Variations in the IC 50 of antimalarial drugs between parasite lines expressing wild type pfmdr1-Y184 or mutant 184F were shown to be closely linked to either of pfmdr1 N86 or 86Y alleles and not the 184F allele [53]. However, several epidemiological studies on the prevalence of pfmdr1 Y184F polymorphisms have shown that the Y184 allele is predominantly con ned to East and Central Africa while the mutant 184F allele predominates in West Africa [1,32,36,48]. Indeed, reports of the high occurrence of the mutant pfmdr1 184F in West Africa were corroborated by the present ndings in which we show that the prevalence of pfmdr1 184F was high in all the states, and especially in Kano, and that its prevalence is higher in North-West compared to North-East Nigeria.…”

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

“…Previously published primers for Pfcrt K76T and Pfmdr1 SNPs for allele polymorphisms were used for PCR and sequencing analyses. Concentration of PCR reactants, purifying of the successfully amplified targets for eventual sequencing or PCR-based single-base extension assays followed by contig assembly ad analysis were done as previously described [12][13][14]. Polymorphisms in the Pfap2mu and Pfubp1genes were determined by direct sequencing of 3 fragments encompassing codons 1-174, 121-399 and 377-621 in the former and codons 1463-1563 for the later as described by Henriques et al [2].…”