Amplicon Deep Sequencing Reveals Multiple Genetic Events Lead to Treatment Failure with Atovaquone-Proguanil in Plasmodium falciparum.

Atovaquone-proguanil (AP) is used as treatment for uncomplicated malaria, and as a chemoprophylactic agent against Plasmodium falciparum. Imported malaria remains one of the top causes of fever in Canadian returning travelers. Twelve sequential whole-blood samples before and after AP treatment failure were obtained from a patient diagnosed with P. falciparum malaria upon their return from Uganda and Sudan. Ultradeep sequencing was performed on the and markers of treatment resistance before and during the episode of recrudescence. Haplotyping profiles were generated using three different approaches: agarose and capillary electrophoresis, and using amplicon deep sequencing (ADS). A complexity of infection (COI) analysis was conducted. Y268C mutants strains were observed during an episode of recrudescence 17 days and 16 h after the initial malaria diagnosis and AP treatment initiation. No Y268C mutant reads were observed in any of the samples prior to the recrudescence. SNPs in the and genes were observed upon initial presentation. The haplotyping profiles suggest multiple clones mutating under AP selection pressure (COI > 3). Significant differences in COI were observed by capillary electrophoresis and ADS compared to the agarose gel results. ADS using revealed the lowest haplotype variation across the longitudinal analysis. Our findings highlight the value of ultra-deep sequencing methods in the understanding of P. falciparum haplotype infection dynamics. Longitudinal samples should be analyzed in genotyping studies to increase the analytical sensitivity.