Phenotypic assessment and genetic validation of Plasmodium falciparum molecular markers associated with malaria chemoprevention in Senegal

Drug resistance in Plasmodium falciparum threatens to undermine malaria control and elimination efforts. Senegal is a malaria-endemic country that has implemented successive antimalarial and chemopreventive drug-based strategies for two decades. Sulfadoxine-pyrimethamine (SP) is used for chemoprevention in Senegal for intermittent preventive treatment in pregnancy (since 2004) and SP plus amodiaquine (AQ) is used for seasonal malaria chemoprevention (SMC, since 2013). Using whole genome sequence (WGS) data from malaria patient samples from health facilities across Senegal (2006 - 2022), we observed near fixation of Pfdhfr triple mutant and fluctuation in Pfdhps and Pfcrt mutation frequencies over time. It is unclear how these mutations influence drug resistance and fitness phenotypes in natural isolates; therefore, we evaluated natural parasite isolates with different Pfcrt, Pfmdr1, Pfdhps, and Pfdhfr haplotypes. Parasites were culture-adapted and phenotyped for antimalarial drug susceptibility and competitive growth (fitness). Pfcrt CVIET + A220S + Q271E + N326S + R371I and Pfcrt CVIET + A220S + Q271E + I356T + R371I mutants were significantly more resistant to monodesethyl-amodiaquine (md-AQ) compared to Pfcrt wild-type (WT) and Pfcrt CVIET + A220S + Q271E + R371I mutants. Pfdhfr triple mutants were significantly more pyrimethamine (PYR) resistant than Pfdhfr WT and revealed a range of phenotypes, but this was not explained by Pfgch1 copy-number. Pfdhps A437G parasites were significantly more sulfadoxine (SDX) resistant compared to Pfdhps wild-type and Pfdhps S436A mutants, suggesting that A437G is a key mutation for SDX resistance. Competitive growth assays between Pfdhfr-Pfdhps mutants revealed that Pfdhps mutations do not always result in fitness costs. Ongoing phenotypic assessment and genetic validation of these mutations in a Senegalese background is necessary to assess the impact of drug pressure, identify evolving genetic determinants of drug resistance, and provide molecular markers for ongoing surveillance to monitor and guide the use of drug-based interventions. AUTHOR SUMMARYDrug resistance is a major concern for both preventing and treating malaria, especially in Africa where most malaria cases and deaths occur. Since 2013, Senegal has been giving children under 10 years old a combination of sulfadoxine-pyrimethamine plus amodiaquine to prevent malaria during the transmission season, called Seasonal Malaria Chemoprevention (SMC), and plans to continue expanding its use. However, there is evidence from genetic surveillance that drug resistance mutations are present in Senegal which could render this antimalarial drug combination ineffective. Here we use natural P. falciparum isolates obtained from Senegalese patients that represent the extant parasite population to evaluate the consequences of evolving mutations on antimalarial drug resistance and fitness phenotypes. This study is one of the first to use natural parasites to assess the impact of naturally derived mutations on drug resistance and fitness phenotypes. Our results provide evidence that certain combinations of drug resistance mutations impact both parasite drug resistance and fitness, and therefore need to be closely monitored and can inform optimal antimalarial combinations for the prevention or treatment of malaria. This work informs the ongoing evolution of resistance and fitness phenotypes in malaria endemic settings that are introducing new multi first line therapies (MFTs) and SMC interventions that have been used for decades in Senegal. Our approach creates a framework for using genetic surveillance data to form a hypothesis, which can then be phenotypically tested by measuring the resistance and fitness levels of genetically diverse natural parasite isolates.