Real-time PCR for chloroquine sensitivity assay and for pfmdr1-pfcrt single nucleotide polymorphisms in Plasmodium falciparum.
Plasmodium falciparum drug resistance is a major problem in malaria endemic areas. Molecular markers and in vitro tests have been developed to study and monitor drug resistance. However, none, used alone, can provide sufficient data concerning the level of drug resistance and to issue precise guidelines for drug use policies in endemic areas. We propose real-time PCR for the simultaneous detection of pfcrt and pfmdr1 genes mutations and to determine the half-maximal inhibitory response (IC(50)) of antimalarial drug. Using hybridization probes and SybrGreen technology on LightCycler instrument, point mutations of pfcrt and pfmdr1 genes have been successfully detected in 161 human blood samples and determination of IC values was applied to chloroquine-sensitive and chloroquine-resistant strains. Moreover, mixed infections caused by P. falciparum clones with wild-type or mutant alleles could be efficiency separated. The aim of this study was not to provide definitive data concerning the rate of mutations in an endemic area, but to describe a powerful method allowing the quantification of DNA for IC(50) determination and the detection of major pfmdr1 and pfcrt mutations.
de Monbrison
Frédérique
F
Laboratoire de Parasitologie, Mycologie Med.et Pathologie Exotique, Hospices Civils de Lyon, Faculté de Médecine, Université Claude Bernard, E.A. 3087, Lyon, France. fdmonb@rockefeller.univ-lyonl.fr
Raynaud
Delphine
D
Latour-Fondanaiche
Christine
C
Staal
Anne
A
Favre
Sébastien
S
Kaiser
Karine
K
Peyron
François
F
Picot
Stéphane
S
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D052160:Benzothiazoles; D002738:Chloroquine / Q000494:pharmacology*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D004351:Drug Resistance; D031541:Fluorescence Resonance Energy Transfer; D014644:Genetic Variation; D006801:Humans; D020128:Inhibitory Concentration 50; D016778:Malaria, Falciparum / Q000097:blood / Q000188:drug therapy; D009930:Organic Chemicals / Q000737:chemistry; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics; D011804:Quinolines
D018528:ATP-Binding Cassette Transporters; D000962:Antimalarials; D052160:Benzothiazoles; D016054:DNA, Protozoan; D003959:Diamines; D009930:Organic Chemicals; D015800:Protozoan Proteins; D011804:Quinolines; C059987:mdr gene protein, Plasmodium; C098022:SYBR Green I; D002738:Chloroquine