Multiplexed nanopore amplicon sequencing to distinguish recrudescence from new infection in antimalarial drug trials

Background The assessment of antimalarial drug efficacy against Plasmodium falciparum requires PCR correction to distinguish recrudescence from new infections by comparing parasite genotypes before treatment and in recurrent infections. Nanopore sequencing offers a low-cost, portable, scalable, and rapid alternative to traditional methods, supporting the expansion and decentralization of sequencing in endemic, resource-limited settings, potentially providing rapid PCR-corrected drug failure estimates. Methods We optimized a multiplexed AmpSeq panel targeting six microhaplotypes for high and uniform coverage. We assessed sensitivity and specificity for detecting minority clones in polyclonal infections and evaluated genetic diversity across the microhaplotype markers. We used mixtures of four P. falciparum laboratory strains at different ratios and 20 paired patient samples from a clinical trial. A custom bioinformatics workflow was used to infer haplotypes from polyclonal infections, including minority clones, with defined cut-off criteria for accurate haplotype calling. Findings The nanopore AmpSeq assay achieved uniform and high read coverage across all six microhaplotype markers (median coverage: 12,989x to 15,440x for laboratory strain mixtures and 7,011x to 11,600x for patients' samples, respectively). We found high sensitivity in detecting minority clones (up to 50:1:1:1 in the 3D7:K1:HB3:FCB1 laboratory strain mixtures) and high specificity with less than 0.01% of all reads being false-positive haplotypes. Genetic diversity in the markers used was high (H_E ≥ 0.98 and up to 31 unique haplotypes in 20 paired samples with cpmp), and concordant results in classifying new infections and recrudescence across all markers used were observed in 18 (90%) of 20 paired samples. Interpretation Our study demonstrates the feasibility of nanopore AmpSeq for distinguishing recrudescence from new infections in clinical trials.