Evolution of Plasmodium falciparum antimalarial drug resistance markers post-adoption of artemisinin-based combination therapies in Yaounde, Cameroon

Abstract

Objectives: This study aimed to investigate the evolution of Plasmodium falciparum antimalarial drug resistance markers by comparing the pre- and post-adoption of artemisinin-based combination therapies (ACTs) in Yaounde, Cameroon.

Methods: The molecular characterization of known antimalarial drug resistance markers (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and Pfk13) in P. falciparum-positive samples collected in 2014 and 2019-2020 was achieved using nested polymerase chain reaction, followed by targeted amplicon deep sequencing on the Illumina MiSeq platform. Data derived were compared with those published during the pre-ACT adoption period from 2004 to 2006.

Results: A high prevalence of Pfmdr1 184F, Pfdhfr 51I/59R/108N, and Pfdhps 437G mutant alleles was observed during the post-ACT adoption period. The Pfcrt 76T and Pfmdr1 86Y mutant alleles significantly declined between 2004 and 2020 (P <0.0001). Conversely, the resistance markers to antifolates, Pfdhfr 51I/59R/108N and Pfdhps 437G, significantly increased during the same study period (P <0.0001). We identified nine mutations in the propeller domains of Pfk13; although they were all present in single parasite isolates, none of them are known to confer artemisinin resistance.

Conclusion: This study documented a near-complete reversion to sensitive parasites for markers conferring resistance to the 4-aminoquinolines and arylamino alcohols in Yaounde. In contrast, the Pfdhfr mutations associated with pyrimethamine resistance are moving toward saturation.

Keywords: Artemisinin-based combination therapies; Cameroon; Drug resistance; Evolution; Plasmodium falciparum; Targeted amplicon deep sequencing.

Figure 1

Study profile of enrolled participants. AL, artemether-lumefantrine; AQ, amodiaquine; AS-AQ, artesunate-amodiaquine; NGS, next generation sequencing; PCR, polymerase chain reaction; Pfcrt, Plasmodium falciparum chloroquine resistance transporter gene (mutations at position C72S, V73K, M74I, N75E, and K76T); Pfmdr1, P. falciparum multidrug resistance 1 gene (mutations at position N86Y, Y184F, S1034C, N1042D, and D1246Y); Pfdhfr, P. falciparum dihydrofolate reductase gene (mutations at position Pfdhfr A16V, C50R, N51I, C59R, S108N/T, and I164L); Pfdhps, P. falciparum dihydropteroate synthase gene; (mutations at position I431V, S436A/F, A437G, K540E/N, A581G, and A613S/T); Pfk13, P. falciparum kelch 13 gene (mutations in the nonpropeller and propeller regions); SP, sulfadoxine-pyrimethamine; SP-AQ, sulfadoxine-pyrimethamine-amodiaquine; SSOP, sequence specific oligonucleotide probe.

Figure 2

Evolution of antimalarial drug policy in Yaounde, Cameroon (2002-2014). AL, artemether-lumefantrine; AQ, amodiaquine; AS-AQ, artesunate-amodiaquine; CQ, chloroquine; MD, microscopy diagnosis; PD, presumptive diagnosis; RDT, rapid diagnostic test; SP, sulfadoxine-pyrimethamine.