Directional selection at the pfmdr1, pfcrt, pfubp1, and pfap2mu loci of Plasmodium falciparum in Kenyan children treated with ACT

Abstract

Background: The efficacy of artemisinin-based combination therapy (ACT) for Plasmodium falciparum malaria may be threatened by parasites with reduced responsiveness to artemisinins. Among 298 ACT-treated children from Mbita, Kenya, submicroscopic persistence of P. falciparum on day 3 posttreatment was associated with subsequent microscopically detected parasitemia at days 28 or 42.

Methods: DNA sequences of resistance-associated parasite loci pfcrt, pfmdr1, pfubp1, and pfap2mu were determined in the Mbita cohort before treatment, on days 2 and 3 after initiation of treatment, and on the day of treatment failure.

Results: Parasites surviving ACT on day 2 or day 3 posttreatment were significantly more likely than the baseline population to carry the wild-type haplotypes of pfcrt (CVMNK at codons 72-76; P < .001) and pfmdr1 (NFD at codons 86, 184, 1246; P < .001). In contrast, variant alleles of the novel candidate resistance genes pfap2mu (S160N/T; P = .006) and pfubp-1 (E1528D; P < .001) were significantly more prevalent posttreatment. No genetic similarities were found to artemisinin-tolerant parasites recently described in Cambodia.

Conclusions: Among treated children in western Kenya, certain P. falciparum genotypes defined at pfcrt, pfmdr1, pfap2mu, and pfubp1 more often survive ACT at the submicroscopic level, and contribute to onward transmission and subsequent patent recrudescence.

Keywords: artemisinin; drug resistance; genetics; malaria.

Figure 1.

Haplotype frequencies at codons 1463–1563 of pfubp1 in 123 pretreatment isolates. A 303-nucleotide fragment was amplified from genomic DNA using primers described in the text, and sequenced directly. Sequences were ordered from the most frequent to the least, and aligned in Clustal W and formatted in Boxshade, with additional hand-editing for clarity. Codon 1258, as identified by Borrman et al [19] is arrowed. Haplotype 1 is identical to the sequence found in the 3D7 reference genome. *Haplotypes 7 and 8 carry a synonymous mutation at codon 1518, encoding Asn.

Figure 2.

Global sequence diversity in pfap2mu (left) and pfubp1 (right) across genome sequences from 631 P. falciparum isolates mapped to the 3D7 reference genome [23]. Colored bars at any chromosome position (horizontal axis) denote a nonreference substitution at that residue in the relevant isolate (vertical axis). At top is the regional origin of isolates in the analysis, comprising 8 laboratory isolates (L), 367 isolates from West Africa, 88 from East Africa, 151 from SE Asia, and 17 from Oceania (Oc). Codon S160N/T of pfap2mu and codon 1528 of pfupb1 are highlighted by red arrows at the bottom of the figure. For pfubp1, the position of 2 carboxy-terminal introns is also marked. Previous annotation of this region of chromosome 1 as 2 distinct genes (PFA0215w and PFA0220w) is shown above the right-hand figure.