title: Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia.
abstract: BACKGROUND
  Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed.
  
  
  METHODS
  Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections.
  
  
  RESULTS
  Considerable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases.
  
  
  CONCLUSIONS
  The parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance.
authors.forename: Panita
authors.initials: P
authors.lastname: Gosi
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Lanteri
authors.forename: Stuart D
authors.initials: SD
authors.lastname: Tyner
authors.forename: Youry
authors.initials: Y
authors.lastname: Se
authors.forename: Chanthap
authors.initials: C
authors.lastname: Lon
authors.forename: Michele
authors.initials: M
authors.lastname: Spring
authors.forename: Mengchuor
authors.initials: M
authors.lastname: Char
authors.forename: Darapiseth
authors.initials: D
authors.lastname: Sea
authors.forename: Sabaithip
authors.initials: S
authors.lastname: Sriwichai
authors.forename: Sittidech
authors.initials: S
authors.lastname: Surasri
authors.forename: Saowaluk
authors.initials: S
authors.lastname: Wongarunkochakorn
authors.forename: Kingkan
authors.initials: K
authors.lastname: Pidtana
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Walsh
authors.forename: Mark M
authors.initials: MM
authors.lastname: Fukuda
authors.forename: Jessica
authors.initials: J
authors.lastname: Manning
authors.forename: David L
authors.initials: DL
authors.lastname: Saunders
authors.affiliation: Department of Immunology and Medicine, Armed Forces Research Institute of Medical Science (AFRIMS), Bangkok, Thailand. delia.bethell@afrims.org.
authors.forename: Delia
authors.initials: D
authors.lastname: Bethell
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D000962:Antimalarials / Q000627:therapeutic use*; D037621:Artemisinins / Q000627:therapeutic use*; D000077332:Artesunate; D002160:Cambodia; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy* / Q000469:parasitology; D008297:Male; D020066:Merozoite Surface Protein 1 / Q000235:genetics*; D008875:Middle Aged; D010963:Plasmodium falciparum / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics*; D055815:Young Adult
chemical_list: D000953:Antigens, Protozoan; D000962:Antimalarials; D037621:Artemisinins; D020066:Merozoite Surface Protein 1; D015800:Protozoan Proteins; C065974:merozoite surface protein 2, Plasmodium; C072492:glutamate-rich protein, Plasmodium; D000077332:Artesunate