title: In vitro amodiaquine resistance and its association with mutations in pfcrt and pfmdr1 genes of Plasmodium falciparum isolates from Nigeria.
abstract: Amodiaquine (AQ) is currently being used as a partner drug in combination with artesunate for treatment of uncomplicated malaria in most endemic countries of Africa. In the absence of molecular markers of artemisinin resistance, molecular markers of resistance to AQ may be useful for monitoring the development and spread of parasites resistance to Artesunate-Amodiaquine combination. This study was designed to assess the potential role of polymorphisms on pfcrt and pfmdr1 genes and parasite in vitro susceptibility for epidemiological surveillance of amodiaquine resistance in Plasmodium falciparum. The modified schizont inhibition assay was used to determine in vitro susceptibility profiles of 98 patients' isolates of P. falciparum to amodiaquine. Polymorphisms on parasites pfcrt and pfmdr1 genes were determined with nested PCR followed by sequencing. The geometric mean (GM) of AQ 50% inhibitory concentration (IC-50) in the 97 P. falciparum isolates was 20.48 nM (95% CI 16.53-25.36 nM). Based on the cut-off value for AQ in vitro susceptibility, 87% (84) of the P. falciparum isolates were sensitive to AQ (GM IC-50=16.32 nM; 95%CI 13.3-20.04 nM) while 13% were resistant to AQ in vitro (GM IC-50=88.73nM; 95%CI 69.67-113.0nM). Molecular analysis showed presence of mutant CVIET pfcrt haplotype, mutant pfmdr1Tyr86 allele and the double mutant CVIET pfcrt haplotype+pfmdr1Tyr86 in 72%, 49% and 35%, respectively. The GM IC-50 of isolates harboring the wild-type pfcrt CVMNK haplotype+pfmdr1Asn86 allele (3.93nM; 95%CI 1.82-8.46 nM) was significantly lower (p=0.001) than those isolates harboring the double mutant pfcrt CVIET haplotype+pfmdr1Tyr86 allele (50.40 nM; 95%CI 40.17-63.24 nM). Results from this study suggest that polymorphisms in pfcrt and pfmdr1 genes are important for AQ resistance and therefore may be useful for epidemiological surveillance of P. falciparum resistance to AQ.
authors.affiliation: Malaria Research Laboratories, IMRAT, College of Medicine, University of Ibadan, Ibadan, Nigeria.
authors.forename: O A
authors.initials: OA
authors.lastname: Folarin
authors.forename: C
authors.initials: C
authors.lastname: Bustamante
authors.forename: G O
authors.initials: GO
authors.lastname: Gbotosho
authors.forename: A
authors.initials: A
authors.lastname: Sowunmi
authors.forename: M G
authors.initials: MG
authors.lastname: Zalis
authors.forename: A M J
authors.initials: AM
authors.lastname: Oduola
authors.forename: C T
authors.initials: CT
authors.lastname: Happi
mesh_terms: D000483:Alleles; D000655:Amodiaquine / Q000494:pharmacology*; D000962:Antimalarials / Q000494:pharmacology*; D002648:Child; D002675:Child, Preschool; D016054:DNA, Protozoan / Q000235:genetics; D005260:Female; D006239:Haplotypes; D006801:Humans; D007223:Infant; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D020125:Mutation, Missense; D009549:Nigeria; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D011110:Polymorphism, Genetic; D015800:Protozoan Proteins / Q000235:genetics*
chemical_list: D000655:Amodiaquine; D000962:Antimalarials; D016054:DNA, Protozoan; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; C513498:Mdr1 protein, Plasmodium falciparum; C416369:PfCRT protein, Plasmodium falciparum