Targeted Genomic Surveillance Unveils Genetic Variations Linked to Regional Malaria Drug Resistance Dynamics in India

<h4>Background</h4>India has made substantial progress in reducing <i>Plasmodium falciparum</i> malaria cases and has set a target to eliminate malaria by 2030. Although artemisinin-based combination therapy (ACT) treatment remains effective, tracking regional differences in genetic variants associated with antimalarial resistance is required for effective drug policy implementation.<h4>Methods</h4>We analyzed 238 <i>P. falciparum</i> clinical samples from 6 Indian states by sequencing 15 parasite genes associated with reduced drug effectiveness. The method involved nanopore sequencing of target gene amplicons derived from dried blood spots using a highly-sensitive PfMDR15 surveillance panel.<h4>Results</h4>India's historical policy of artesunate-sulfadoxine-pyrimethamine in central India and artemether-lumefantrine in the Northeast has shaped contrasting resistance profiles. In the Northeast, chloroquine resistance persisted at high frequency (<i>Pfcrt</i> K76T and CVIET haplotype; <i>Pfaat1</i> S258L), alongside quintuple and sextuple <i>Pfdhfr-Pfdhps</i> haplotypes conferring complete sulfadoxine-pyrimethamine resistance. Central India showed variable chloroquine resistance (parasites largely retained wild-type <i>Pfcrt</i>) and emerging lumefantrine tolerance (<i>Pfmdr1</i> Y184F, <i>Pfaat1</i> S258L). Interestingly, Delhi (Central India) parasites resembled profiles from the distant Northeast, which borders South East Asia. The detection of <i>Pfaat1</i> S258L, previously reported only from Africa and associated with reduced lumefantrine susceptibility, suggests convergent evolution under ACT partner-drug pressure. No WHO-validated <i>Pfk13</i> artemisinin resistance mutations were detected, supporting continued efficacy of ACT.<h4>Conclusions</h4>India's resistance landscape is fragmented, with signals of expanding lumefantrine tolerance and importation or evolution of globally relevant mutations. These findings highlight the importance of integrating molecular genomic surveillance into malaria control policy to monitor and protect ACT effectiveness and advance malaria elimination.