Plasmodium falciparum kelch13 polymorphisms identified after treatment failure with artemisinin-based combination therapy in Niger

Abstract

Background: Artemisinin-based combination therapy (ACT) is the most effective treatment for malaria, and has significantly reduced morbimortality. Polymorphisms associated with the Plasmodium falciparum Kelch gene (Pfkelch13) have been associated with delayed parasite clearance even with ACT treatment.

Methods: The Pfkelch13 gene was sequenced from P. falciparum infected patients (n = 159) with uncomplicated malaria in Niger. An adequate clinical and parasitological response (ACPR) was reported in 155 patients. Four (n = 4) patients had treatment failure (TF) that were not reinfections-two of which had late parasitological failures (LPF) and two had late clinical failures (LCF).

Results: Thirteen single nucleotide polymorphisms (SNPs) were identified of which seven were non-synonymous (C469R, T508S, R515T, A578S, I465V, I437V, F506L,), and three were synonymous (P443P, P715P, L514L). Three SNP (C469R, F506L, P715P) were present before ACT treatment, while seven mutations (C469R, T508S, R515T, L514L, P443P, I437V, I465V) were selected by artemether/lumefantrine (AL)-five of which were non-synonymous (C469R, T508S, R515T, I437V, I465V). Artesunate/amodiaquine (ASAQ) has selected any mutation. One sample presented three cumulatively non-synonymous SNPs-C469R, T508S, R515T.

Conclusions: This study demonstrates intra-host selection of Pfkelch13 gene by AL. The study highlights the importance of LCF and LPF parasites in the selection of resistance to ACT. Further studies using gene editing are required to confirm the potential implication of resistance to ACT with the most common R515T and T508S mutations. It would also be important to elucidate the role of cumulative mutations.

Keywords: Artemisinin; Niger; P. falciparum; Pfkelch13; Resistance.

Fig. 1

Agarose gel electrophoresis for Pfmsp2/FC27. Lane 1 = Molecular size ladder, Hyperladder IV (50-1013 kb, Bioline); Lanes 2, 4, 6, 8, 14 and 16 = Msp2 allelic family FC27 at day 0; Lanes 3, 5, 9, 11, 13, and 17 = Msp2 allelic family FC27 on day of treatment failure; Lanes 7, 10, 12, and 15 indicate negative samples; Lane 18 is a negative control without DNA

Fig. 2

Gel electrophoresis for Pfmsp1/K1. Lane 1 = Molecular size ladder, Hyperladder IV (50-1013 kb, Bioline); Lanes 2, 4, 6, 8, 10, 12, 14,16, and 18 = Msp1 allelic family K1 at day 0; Lanes 3, 5, 7, 9, 11, 13, 15, 17, and 19 = Msp1 allelic family K1 on day of treatment failure; Lane 20 is a negative control without DNA

Fig. 3

PCR2 agarose gel for amplication of Pfkelch13 fragment. Nested PCR: Lane 1 = Molecular size Marker (Hyper ladder IV, 50–1013 bp, Bioline), Lane 2 -14 = band size of 849 pb, corresponding to the helix domain of the Pfkelch13 gene, Lane 15 = Molecular Weigh Marker, Lane 16 is a negative control without DNA and Lane 17 = 3D7 control Plasmodium DNA

Fig. 4

Comparison of field isolate sequences with the control sequence Pf_3D7

Fig. 5

Graphical representation of the design of the study and the most critical findings. The number of samples collected at day 0 was 155. Only 14 samples among these 155 samples from day 0 showed a treatment failure (TF) during follow up. Ten (10) samples out the 14 TF were reinfections. All samples from day 0 as well as those with TF without reinfection were submitted for sequencing. LCFs (late clinical failures); LPFs (late parasitological failures); WT (Wildtype); NS (non-synonymous); S (synonymous); CNR (Centre National de Reference du Paludisme); ND (University Notre Dame)