title: A deep sequencing tool for partitioning clearance rates following antimalarial treatment in polyclonal infections.
abstract: OBJECTIVE
  Current tools struggle to detect drug-resistant malaria parasites when infections contain multiple parasite clones, which is the norm in high transmission settings in Africa. Our aim was to develop and apply an approach for detecting resistance that overcomes the challenges of polyclonal infections without requiring a genetic marker for resistance.
  
  
  METHODS
  Clinical samples from patients treated with artemisinin combination therapy were collected from Tanzania and Cambodia. By deeply sequencing a hypervariable locus, we quantified the relative abundance of parasite subpopulations (defined by haplotypes of that locus) within infections and revealed evolutionary dynamics during treatment. Slow clearance is a phenotypic, clinical marker of artemisinin resistance; we analyzed variation in clearance rates within infections by fitting parasite clearance curves to subpopulation data.
  
  
  RESULTS
  In Tanzania, we found substantial variation in clearance rates within individual patients. Some parasite subpopulations cleared as slowly as resistant parasites observed in Cambodia. We evaluated possible explanations for these data, including resistance to drugs. Assuming slow clearance was a stable phenotype of subpopulations, simulations predicted that modest increases in their frequency could substantially increase time to cure.
  
  
  CONCLUSIONS
  By characterizing parasite subpopulations within patients, our method can detect rare, slow clearing parasites in vivo whose phenotypic effects would otherwise be masked. Since our approach can be applied to polyclonal infections even when the genetics underlying resistance are unknown, it could aid in monitoring the emergence of artemisinin resistance. Our application to Tanzanian samples uncovers rare subpopulations with worrying phenotypes for closer examination.
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON, Canada; nicole.mideo@utoronto.ca.
authors.forename: Nicole
authors.initials: N
authors.lastname: Mideo
authors.affiliation: Division of Transfusion Medicine, Department of Medicine, University of Massachusetts, Worcester, MA, USA; Program in Bioinformatics and Integrative Biology, University of Massachusetts, Worcester, MA, USA;
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Bailey
authors.affiliation: Program in Bioinformatics and Integrative Biology, University of Massachusetts, Worcester, MA, USA;
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Hathaway
authors.affiliation: Department of Parasitology, Muhimbili University of Health and Allied Sciences, Dar Es Salaam, Tanzania;
authors.forename: Billy
authors.initials: B
authors.lastname: Ngasala
authors.affiliation: Division of Immunology and Medicine, USAMC Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand;
authors.forename: David L
authors.initials: DL
authors.lastname: Saunders
authors.affiliation: US Army Medical Component, Armed Forces Research Institute of Medical Sciences, Phnom Penh, Cambodia;
authors.forename: Chanthap
authors.initials: C
authors.lastname: Lon
authors.affiliation: Division of Infectious Diseases, University of North Carolina School of Medicine, Chapel Hill, NC, USA;
authors.forename: Oksana
authors.initials: O
authors.lastname: Kharabora
authors.affiliation: Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON, Canada;
authors.forename: Andrew
authors.initials: A
authors.lastname: Jamnik
authors.affiliation: Division of Infectious Diseases, University of North Carolina School of Medicine, Chapel Hill, NC, USA;
authors.forename: Sujata
authors.initials: S
authors.lastname: Balasubramanian
authors.affiliation: Malaria Research, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden;
authors.forename: Anders
authors.initials: A
authors.lastname: Björkman
authors.affiliation: Malaria Research, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; Centre for Clinical Research Sörmland, Uppsala University, Sweden; Department of Women's and Children's Health, International Maternal and Child Health (IMCH), Uppsala University, Sweden;
authors.forename: Andreas
authors.initials: A
authors.lastname: Mårtensson
authors.affiliation: Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, USA;
authors.forename: Steven R
authors.initials: SR
authors.lastname: Meshnick
authors.affiliation: Center for Infectious Disease Dynamics, Department of Biology and Entomology, the Pennsylvania State University, University Park, PA, USA and.
authors.forename: Andrew F
authors.initials: AF
authors.lastname: Read
authors.affiliation: Division of Infectious Diseases, University of North Carolina School of Medicine, Chapel Hill, NC, USA; Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, NC, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Juliano
keywords: amplicon sequencing; artemisinin; drug resistance; ecology; malaria; within-host selection
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