Ex vivosusceptibility to antimalarial drugs and polymorphisms in drug resistance genes of AfricanPlasmodium falciparum, 2016-2023: a genotype-phenotype association study
Authors: Rosado Jason , Fola Abebe A. , Cojean Sandrine , Sarrasin Véronique , Coppée Romain , Zaffaroulah Rizwana , Bouzayene Azza , Cicéron Liliane , Houzé Ludivine , Crudale Rebecca , Musset Lise , Thellier Marc , Pradines Bruno , Clain Jérôme , Bailey Jeffrey A. , Houzé Sandrine ,   Investigation Study Group
Published: July 2024

Background

Given the altered responses to both artemisinins and lumefantrine in Eastern Africa, monitoring antimalarial drug resistance in all African countries is paramount.

Methods

We measured the susceptibility to six antimalarials usingex vivogrowth inhibition assays (IC50) for a total of 805Plasmodium falciparumisolates obtained from travelers returning to France (2016-2023), mainly from West and Central Africa. Isolates were sequenced using molecular inversion probes (MIPs) targeting fourteen drug resistance genes across the parasite genome.

Findings

Ex vivosusceptibility to several drugs has significantly decreased in 2019-2023 versus 2016-2018 parasite samples: lumefantrine (median IC50: 23·0 nM [IQR: 14·4-35·1] in 2019-2023 versus 13·9 nM [8·42-21·7] in 2016-2018, p<0·0001), monodesethylamodiaquine (35·4 [21·2-51·1] versus 20·3 nM [15·4-33·1], p<0·0001), and marginally piperaquine (20·5 [16·5-26·2] versus 18.0 [14·2-22·4] nM, p<0·0001). Only four isolates carried a validatedpfkelch13mutation. Multiple mutations inpfcrtand one inpfmdr1(N86Y) were significantly associated with altered susceptibility to multiple drugs. The susceptibility to lumefantrine was altered bypfcrtandpfmdr1mutations in an additive manner, with the wild-type haplotype (pfcrtK76-pfmdr1N86) exhibiting the least susceptibility.

Interpretation

Our study onP. falciparumisolates from West and Central Africa indicates a low prevalence of molecular markers of artemisinin resistance but a significant decrease in susceptibility to the partner drugs that have been the most widely used since a decade –lumefantrine and amodiaquine. These phenotypic changes likely mark parasite adaptation to sustained drug pressure and call for intensifying the monitoring of antimalarial drug resistance in Africa.

Funding

This work was supported by the French Ministry of Health (grant to the French National Malaria Reference Center) and by the Agence Nationale de la Recherche (ANR-17-CE15-0013-03 to JC). JAB was supported by NIH R01AI139520. JR postdoctoral fellowship was funded by Institut de Recherche pour le Développement.

Research in context

Evidence before this study

Artemisinin-based combination therapies (ACTs) have been introduced since the 2000s as the first-line curative treatment of malaria. ACTs combine an artemisinin derivative, which rapidly reduces parasite load, with another antimalarial drug –known as partner drug-which eliminates the remaining parasites thanks to its longer half-life. This approach reduces the likelihood of parasites developing resistance to both drugs, thereby increasing treatment efficacy and delaying the emergence of resistance. However, resistance to artemisinins and then to some partner drugs was identified in Southeast Asia more than a decade ago and has spread throughout the region. Artemisinin partial resistance is now emerging in the East and Horn of Africa. It manifests as delayed parasite clearance from the bloodstream after treatment, increasing the parasite load in contact with the partner drug only and the likelihood of selecting resistant parasites. It is, therefore, important to monitor antimalarial drug susceptibility and drug resistance mutations in contemporary African isolates, especially in the understudied West and Central African regions, to anticipate the spread of multidrug-resistant parasites.

We searched for articles on antimalarial drug resistance published between January 1, 2000, and July 1, 2024, using the PubMed search terms “antimalarial resistance”, “Africa”, and “ex vivo”. Of the 69 published studies, only six encompassing a total of 827 isolates across five West and Central African countries from 2016 to 2022 combinedex vivodrug assays with genotyping data. Parasites with an increased rate ofex vivosurvival to artemisinins were reported in one study from Ghana (7/90 isolates in 2018) and another from The Gambia (4/41 isolates in 2017). Only the Ghanaian study reported mutations in the non-propeller domain ofpfkelch13gene, whereas the Gambian study reported mutations associated with reduced susceptibility to lumefantrine (7%, 3/41). In Mali, Senegal and Burkina Faso, most isolates were susceptible to commonly used antimalarial drugs (chloroquine, amodiaquine, piperaquine, mefloquine, lumefantrine and dihydroartemisinin) using standard growth inhibition assays. In Ghana, reduced susceptibility to artemisinin, mefloquine and amodiaquine was observed. The relative lack of recent data on parasite susceptibility to antimalarial drugs in recent parasites from West and Central Africa prompted us to conduct this study.

Added value of this study

Ex vivosusceptibility to six antimalarial drugs (dihydroartemisinin, lumefantrine, mefloquine, chloroquine, monodesethylamodiaquine, and piperaquine) and mutations in fourteen drug resistance genes were evaluated in 805 isolates collected between January 2016 and February 2023 from 35 African countries, mainly from West and Central Africa.

Median IC50values were in the low nanomolar range, indicating good potency againstP. falciparum. However, worrying trends emerged from 2019 onwards, with median IC50values for lumefantrine that increased from 13·9 nM in 2016-18 to 23 nM in 2019-23 and for amodiaquine from 20·3 nM to 35·4 nM.

The high prevalence of resistance alleles inpfdhfr,pfdhps,pfmdr1andpfcrtgenes underscores the sustained pressure exerted by antimalarial drugs on parasite populations. Notably, although the triple mutantpfdhfrN51I-C59R-S108N was highly prevalent, thedhfr-dhpsquintuple mutant (with extrapfdhpsA437G-K540E), which is responsible for sulfadoxine-pyrimethamine treatment failure in adults and children, was rare. In addition, the analysis revealed some geographic and temporal variations in mutation prevalence.

The genotype-phenotype association analysis performed in this study elucidates the relationship between genetic variants andex vivodrug susceptibility, providing valuable information for understanding the molecular basis of resistance and informing future treatment strategies. For example, mutations in thepfcrtandpfmdr1genes, mainly K76T and N86Y, were associated with altered susceptibilities to most drugs. Haplotypic association analysis further indicated that the two genes have cumulative effects on the susceptibility to lumefantrine, with the wild-type haplotype (pfcrtK76-pfmdr1N86) exhibiting the least susceptibility.

Implication of all the available evidence

While the susceptibility to most antimalarials suggests continued efficacy, the observed decrease in susceptibilities to lumefantrine and amodiaquine in parasites from West and Central Africa from 2019 onwards suggests an ongoing adaptation of parasites, possibly related to the increasing use of ACT treatments in Sub-saharan Africa since a decade. These phenotypic changes over time were accompanied by small changes in the prevalence of resistance alleles inpfcrtandpfmdr1genes. Additional changes, potentially leading to larger decreases in drug susceptibilities, can be expected over time. The large-scale analysis presented here provides invaluable, contemporary insights into the current landscape of susceptibility to antimalarial drugs and molecular markers of resistance inP. falciparumisolates from West and Central Africa. While the data suggests that ACTs and sulfadoxine-pyrimethamine are likely to be effective in these regions, the phenotypic changes we observed call for intensifying the monitoring of antimalarial drug resistance in Africa.