title: Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar.
abstract: BACKGROUND
  Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant P. falciparum isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings.
  
  
  METHODS
  This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect pfcrt 76T-bearing minority variant P. falciparum. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples.
  
  
  RESULTS
  Thirty one clinical P. falciparum isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the pfcrt K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant P. falciparum strains after therapy as detected by HTA. RFLP analysis failed to detect any pfcrt K76T-bearing isolates.
  
  
  CONCLUSIONS
  These findings indicate that genetically CQ-resistant P. falciparum are more common than previously thought in Madagascar even though the fitness of the minority variant pfcrt 76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.
authors.affiliation: University of North Carolina, Chapel Hill, USA. jjuliano@med.unc.edu
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Juliano
authors.forename: Milijaona
authors.initials: M
authors.lastname: Randrianarivelojosia
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ramarosandratana
authors.forename: Frédéric
authors.initials: F
authors.lastname: Ariey
authors.forename: Victor
authors.initials: V
authors.lastname: Mwapasa
authors.forename: Steven R
authors.initials: SR
authors.lastname: Meshnick
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology* / Q000627:therapeutic use; D002648:Child; D002675:Child, Preschool; D002738:Chloroquine / Q000494:pharmacology* / Q000627:therapeutic use; D004351:Drug Resistance / Q000235:genetics*; D005784:Gene Amplification / Q000187:drug effects; D005838:Genotype; D020180:Heteroduplex Analysis; D006801:Humans; D008163:Luminescent Measurements; D008270:Madagascar; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000235:genetics* / Q000469:parasitology; D026901:Membrane Transport Proteins / Q000032:analysis*; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis / Q000235:genetics; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D017354:Point Mutation / Q000235:genetics; D015800:Protozoan Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D000962:Antimalarials; D026901:Membrane Transport Proteins; D009692:Nucleic Acid Heteroduplexes; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D002738:Chloroquine