title: Real-time polymerase chain reaction assay for the rapid detection and characterization of chloroquine-resistant Plasmodium falciparum malaria in returned travelers.
abstract: BACKGROUND
  Imported drug-resistant malaria is a growing problem in industrialized countries. Rapid and accurate diagnosis is essential to prevent malaria-associated mortality in returned travelers. However, outside of a limited number of specialized centers, the microscopic diagnosis of malaria is slow, unreliable, and provides little information about drug resistance. Molecular diagnostics have the potential to overcome these limitations.
  
  
  OBJECTIVE
  We developed and evaluated a rapid, real-time polymerase chain reaction (PCR) assay to detect Plasmodium falciparum malaria and chloroquine (CQ)-resistance determinants in returned travelers who are febrile.
  
  
  METHODS
  A real-time PCR assay based on detection of the K76T mutation in PfCRT (K76T) of P. falciparum was developed on a LightCycler platform (Roche). The performance characteristics of the real-time assay were compared with those of the nested PCR-restriction fragment-length polymorphism (RFLP) and the sequence analyses of samples obtained from 200 febrile returned travelers, who included 125 infected with P. falciparum (48 of whom were infected CQ-susceptible [K76] and 77 of whom were CQ-resistant [T76] P. falciparum), 22 infected with Plasmodium vivax, 10 infected with Plasmodium ovale, 3 infected with Plasmodium malariae malaria, and 40 infected with other febrile syndromes. All patient samples were coded, and all analyses were performed blindly.
  
  
  RESULTS
  The real-time PCR assay detected multiple pfcrt haplotypes associated with CQ resistance in geographically diverse malaria isolates acquired by travelers. Compared with nested-PCR RFLP (the reference standard), the real-time assay was 100% sensitive and 96.2% specific for detection of the P. falciparum K76T mutation.
  
  
  CONCLUSIONS
  This assay is rapid, sensitive, and specific for the detection and characterization of CQ-resistant P. falciparum malaria in returned travelers. This assay is automated, standardized, and suitable for routine use in clinical diagnostic laboratories.
authors.affiliation: Tropical Disease Unit, University Health Network-Toronto General Hospital, Toronto, Ontario, Canada.
authors.forename: Gabriella A
authors.initials: GA
authors.lastname: Farcas
authors.forename: Rainer
authors.initials: R
authors.lastname: Soeller
authors.forename: Kathleen
authors.initials: K
authors.lastname: Zhong
authors.forename: Alireza
authors.initials: A
authors.lastname: Zahirieh
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Kain
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D002738:Chloroquine / Q000494:pharmacology*; D004351:Drug Resistance; D006801:Humans; D007194:India; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000188:drug therapy / Q000469:parasitology*; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins; D009154:Mutation; D010963:Plasmodium falciparum / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015800:Protozoan Proteins; D012680:Sensitivity and Specificity; D014195:Travel
chemical_list: D000962:Antimalarials; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D002738:Chloroquine