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Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon deep sequencing (TADS) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, TADS  Plasmodium falciparum  apical membrane antigen 1 ( ama1 ), and multidrug resistance gene 1 ( mdr1 ), were used to characterize the complexity of infection (COI) and drug-resistance genotypes, respectively.    P. falciparum  positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Of the 30 participants, 9 presented with recurrent parasitemia from day 26 (624h) onwards. The  ama1  and  mdr1  genes were amplified and sequenced, while  msp1, msp2 and glurp  data were obtained from the original clinical study.   The COI was comparable between  ama1  and  msp1, msp2 and glurp , however, overall  ama1  detected more haplotypes. Based on  ama1 , a stable number of haplotypes were detected throughout treatment up until day 3. Additionally, a recrudescent infection was identified with an  ama1  haplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of  ama1  haplotypes and parasitaemia, we identified a fast ( 5h) clearing haplotype. As expected, only two  mdr1  haplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184.   This study highlights TADS as a sensitive tool for tracking parasite haplotypes throughout treatment and can detect variation in haplotype clearance estimates. TADS can also identify slow clearing haplotypes, a potential early sign of selection during treatment. Consequently, TADS has the capability of improving the discriminatory power to accurately distinguish recrudescences from reinfections.

Targeted Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial