title: Prevalence of pfcrt mutations in Congolese and Malawian Plasmodium falciparum isolates as determined by a new Taqman assay.
abstract: A real-time PCR assay was developed to detect the K76T point mutation in the Plasmodium falciparum putative chloroquine resistance transporter gene. The assay was used with malaria positive clinical isolates from Rutshuru in the eastern part of the Democratic Republic of the Congo (DRC) and from Malawi. The K76T mutation was found in 52/56 (93%) clinical isolates from the DRC, where chloroquine resistance is high, but in none of the 12 isolates tested from Malawi where chloroquine is now rarely used. Sixteen percent of specimens from the DRC had detectable levels of both wild-type and mutant alleles. The real-time PCR results were compared to results from a nested allele-specific PCR assay and from direct DNA sequencing. Using allele-specific PCR as the reference method, the new assay is 100% sensitive and specific towards the mutant allele. In addition to its low per-test cost, the new assay is fast, easily automated, sensitive and well-suited to large-scale epidemiological studies.
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina School of Medicine, CB 7290, Chapel Hill, NC 27599, USA.
authors.forename: Paul E
authors.initials: PE
authors.lastname: Wilson
authors.forename: Walter
authors.initials: W
authors.lastname: Kazadi
authors.forename: Deborah Demster
authors.initials: DD
authors.lastname: Kamwendo
authors.forename: Victor
authors.initials: V
authors.lastname: Mwapasa
authors.forename: Anne
authors.initials: A
authors.lastname: Purfield
authors.forename: Steven R
authors.initials: SR
authors.lastname: Meshnick
mesh_terms: D000818:Animals; D002675:Child, Preschool; D002738:Chloroquine / Q000627:therapeutic use; D016054:DNA, Protozoan / Q000235:genetics; D015023:Democratic Republic of the Congo; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D006801:Humans; D007223:Infant; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000469:parasitology*; D008295:Malawi; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D015800:Protozoan Proteins; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D002738:Chloroquine