A barcode of multilocus nuclear DNA identifies genetic relatedness in pre- and post-Artemether/Lumefantrine treated Plasmodium falciparum in Nigeria

Abstract

Background: The decline in the efficacy of artemisinin-based combination treatment (ACT) in some endemic regions threatens the progress towards global elimination of malaria. Molecular surveillance of drug resistance in malaria-endemic regions is vital to detect the emergence and spread of mutant strains.

Methods: We observed 89 malaria patients for the efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum infections in Lagos, Nigeria and determined the prevalence of drug resistant strains in the population. Parasite clearance rates were determined by microscopy and the highly sensitive var gene acidic terminal sequence (varATS) polymerase chain reaction for 65 patients with samples on days 0, 1, 3, 7, 14, 21 and 28 after commencement of treatment. The genomic finger print of parasite DNA from pre- and post-treatment samples were determined using 24 nuclear single nucleotide polymorphisms (SNP) barcode for P. falciparum. Drug resistance associated alleles in chloroquine resistance transporter gene (crt-76), multidrug resistance genes (mdr1-86 and mdr1-184), dihydropteroate synthase (dhps-540), dihydrofolate reductase (dhfr-108) and kelch domain (K-13580) were genotyped by high resolution melt analysis of polymerase chain reaction (PCR) fragments.

Results: By varATS qPCR, 12 (18.5%) of the participants had detectable parasite DNA in their blood three days after treatment, while eight (12.3%) individuals presented with genotypable day 28 parasitaemia. Complexity of infection (CoI) was 1.30 on day 0 and 1.34 on day 28, the mean expected heterozygosity (H_E) values across all barcodes were 0.50 ± 0.05 and 0.56 ± 0.05 on days 0 and 28 respectively. Barcode (π) pairwise comparisons showed high genetic relatedness of day 0 and day 28 parasite isolates in three (37.5%) of the eight individuals who presented with re-appearing infections. Crt-76 mutant allele was present in 38 (58.5%) isolates. The mdr1-86 mutant allele was found in 56 (86.2%) isolates. No mutation in the K-13580 was observed.

Conclusions: Persistence of DNA-detectable parasitaemia in more than 18% of cases after treatment and indications of genetic relatedness between pre- and post-treatment infections warrants further investigation of a larger population for signs of reduced ACT efficacy in Nigeria.

Keywords: Artemisinin-based combination; Drug resistance; Genetic relatedness; Parasite clearance; Residual parasitaemia; SNP barcode.

Fig. 1

Study flow chart

Fig. 2

Parasite count/μl by microscopy before and after artemether/lumefantrine treatment. (After day 1, the mean concentration was the same up to day 14 with re-appearing infections on days 21 and 28)

Fig. 3

Kaplan-Meier estimates of parasite reduction following artemether-lumefantrine treatment (Group A represents % reduction of parasitaemia on day 1, (ii) Group B represents % reduction of parasitaemia on day 3, Group C represents % reduction of parasitaemia on day 7, and Group D represents % parasitaemia beyond day 7

Fig. 4

Log₁₀ Parasite count/μl by varATS qPCR before and after artemether/lumefantrine treatment (Low-grade residual infections persist on days 1, 3, 7, 4 and 28)

Fig. 5

Prevalence of polymorphisms in Plasmodium falciparum drug resistance genes in pre-treatment parasite population (K13: Kelch 13 gene; Dhps: Dihydropteroate synthase; Pfcrt: Plasmodium falciparum chloroquine resistance transporter gene; Pfmdr1: Plasmodium falciparum chloroquine resistance transporter gene; Dhfr: Dihydrofolate reductase)