Screening for antifolate and artemisinin resistance in Plasmodium falciparum dried-blood spots from three hospitals of Eritrea

Abstract

Background: Antimalarial drug resistance is a major challenge hampering malaria control and elimination. About three-quarters of Eritrea's population resides in the malaria-endemic western lowlands of the country. Plasmodium falciparum, the leading causative parasite species, has developed resistance to basically all antimalarials. Continued surveillance of drug resistance using genetic markers provides important molecular data for treatment policies which complements clinical studies, and strengthens control efforts. This study sought to genotype point mutations associated with P. falciparum resistance to sulfadoxine-pyrimethamine and artemisinin, in dried-blood spots from three hospitals in the western lowlands of Eritrea.

Methods: Dried-blood spot samples were collected from patients visiting Adi Quala, Keren and Gash Barka Hospitals, between July and October, 2014. The patients were followed up after treatment with first line artesunate-amodiaquine, and dried-blood spots were collected on day three after treatment. Nested polymerase chain reaction and Sanger sequencing techniques were employed to genotype point mutations in the Pfdhfr (PF3D7_0417200), Pfdhps (PF3D7_0810800) and PfK13 (PF3D7_1343700) partial gene regions.

Results: Sequence data analyses of PCR-positive isolates found wild-type artemisinin haplotypes associated with resistance (Y493Y, R539R, I543I) in three isolates, whereas four mutant antifolate haplotypes associated with resistance were observed in six isolates. These included the triple-mutant Pfdhfr (S108N, C59R, N51I) haplotype, the double-mutant Pfdhfr (N51I, S108N) haplotype, the single-mutant Pfdhfr (K540E) haplotype, and the mixed-mutant Pfdhfr-Pfdhps (S108N, N51I + K540E) haplotype. Other findings observed were, a rare non-synonymous Pfdhfr V45A mutation in four isolates, and a synonymous Pfdhps R449R in one isolate.

Conclusions: The mutant antifolate haplotypes observed indicate a likely existence of full SP resistance. Further studies can be carried out to estimate the prevalence of SP resistance. The wild-type artemisinin haplotypes observed suggest artemisinin is still an effective treatment. Continuous monitoring of point mutations associated with delayed parasite clearance in ART clinical studies is recommended.

Keywords: Eritrea; Plasmodium falciparum; antifolate; artemisinin; drug resistance; genetic markers.

Figure 1.. Flow diagram showing the sample characteristics.

Figure 2.

Jalview visualization of multiple sequence alignments depicting nsy- and sy-point mutations: Pfdhfr (N51I, S108N, V45A) occurred in all four isolates (KH013, GBH017, AQH010, AQH009), C59R was identified in one isolate (KH013); Pfdhps (K540E) occurred in all four isolates (KH013, GBH017, AQH010, AQH009), R449R was identified in one isolate (GBH015) and Pfdhps (K540E) occurred in all four isolates (KH012, GBH014, GBH015, GBH017) PfK-13, established no point mutations in all six isolates, wild type amino acids retained at c.554(S), c.566(V), c.569(A), c.578(A), c.580(C), c.493(Y), c.539(R), c.543 (I).