Linkage disequilibrium between two distinct loci in chromosomes 5 and 7 of Plasmodium falciparum and in vivo chloroquine resistance in Southwest Nigeria
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Chloroquine (CQ) resistance in Plasmodium falciparum is associated with polymorphisms in loci on pfcrt and pfmdr1 genes. In this study, we determined the association and linkage disequilibrium between in vivo CQ resistance and P. falciparum polymorphisms in pfcrt gene at codon 76 and pfmdr1 gene at codon 86 in isolates obtained from 111 children with acute uncomplicated falciparum malaria in Nigeria. Patients were treated with standard dosage of CQ and followed up for 28 days. Filter paper samples were collected at enrollment and during follow-up for parasites genotypes and identification of pfcrt and pfmdr1 mutations. Association and linkage disequilibrium between mutant pfcrtT76 and pfmdr1Y86 alleles in pretreatment isolates of P. falciparum was determined. Fifty-five out of the 111 patients (49.5%) failed treatment. Single mutant pfcrtT76 or pfmdr1Y86 alleles were found in 55 out of 111 P. falciparum isolates screened at enrollment. Of these 55 isolates, the mutant pfcrtT76 and pfmdr1Y86 alleles were found in 84%. Both mutant pfcrtT76 (p=0.0196) and pfmdr1Y86 (p=0.000042) alleles were associated with in vivo CQ resistance. In addition, the mutant pfcrtT76 (p=0.047) and pfmdr1Y86 (p=0.006) alleles were significantly selected by CQ in patients who failed treatment. Association analysis between paired single alleles at pfcrt and pfmdr1 loci showed a significant association (p=0.0349 and χ 2=4.45) between the pfcrt T76 allele on chromosome 7 and the pfmdr1Y86 allele on chromosome 5 and that these two mutant alleles were in linkage disequilibrium (p=0.000, D′=0.64, and r 2=0.28). Considering the high level of CQ resistance and drug use in the study area, the observed linkage disequilibrium between the mutant pfcrtT76 and pfmdr1Y86 alleles is maintained epistatically through directional CQ selective pressure.
KeywordsLinkage Disequilibrium Malaria Pfmdr1 Gene Pfcrt Gene Intense Malaria Transmission
The authors thank all the patients and their parents or guardians for volunteering to participate in the study. We thank MR4 for providing all genomic DNA used as controls for PCR and RFLP experiments. We also Thank Dr. Dan Milner at the Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA for providing helpful comments on the manuscript. This study was supported by grants from the Fogarty International Centre, the Multilateral Initiative for Malaria in Africa (MIM)/Training in Tropical Diseases (TDR), the UNICEF/United Nations Development Programme/World Bank/WHO/TDR, and the Harvard Malaria Initiative and the International Atomic Energy Agency (IAEA) project RAF/0625. C. T. Happi is supported by a Fogarty International Research Collaboration Award no. NIH RO3TW006298-01A1, the IAEA project RAF/0625, and the WHO/TDR/PAG/South–South Initiative project ID A50337. G. O. G. is supported by the MIM/TDR project ID A20239.
- Djimde AA, Doumbo OK, Traore O, Guindo AB, Kayentao K, Diourte Y, Niare-Doumbo S, Coulibaly D, Kone AK, Cissoko Y, Tekete M, Fofana B, Dicko A, Diallo DA, Wellems TE, Kwiatkowski D, Plowe CV (2003) Clearance of drug-resistant parasites as a model for protective immunity in Plasmodium falciparum malaria. Am J Trop Med Hyg 69:558–563PubMedGoogle Scholar
- Fidock DA, Takashi N, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, Ursos LMB, Sidhu AS, Naude B, Kirk WD, Su X-Z, Wootton JC, Roepe PD, Wellems TE (2000) Mutation in the P. falciparum digestive vacuole transmembrane protein Pfcrt and evidence for their role in chloroquine resistance. Mol Cell 861:861–871CrossRefGoogle Scholar
- Happi TC, Thomas SM, Gbotosho GO, Falade CO, Akinboye DO, Gerena L, Hudson T, Sowunmi A, Kyle DE, Milhous W, Wirth DF, Oduola AMJ (2003) Point mutations in the pfcrt and pfmdr-1 genes of Plasmodium falciparum and clinical response to chloroquine, among malaria patients from Nigeria. Ann Trop Med Parasitol 97:439–451PubMedCrossRefGoogle Scholar
- Happi CT, Gbotosho GO, Folarin OA, Akinboye DO, Yusuf BO, Ebong OO, Sowunmi A, Kyle DE, Milhous W, Wirth DF, Oduola AMJ (2005) Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine-pyrimethamine resistance in malaria-infected patients from Nigeria. Acta Trop 95:183–193PubMedCrossRefGoogle Scholar
- Looareswan S, Viravan C, Webster HK, Kyle DE, Hutchinson DB, Candfield CJ (1996) Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand. Am J Trop Med Hyg 54:62–66Google Scholar
- Mayor AG, Gomez-Olive X, Aponte JJ, Casimiro S, Mabunda S, Dgedge M, Barreto A, Alonso PL (2001) Prevalence of the K76T mutation in the putative Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene and its relation to chloroquine resistance in Mozambique. J Infect Dis 183:1413–1416PubMedCrossRefGoogle Scholar
- Ochong EO, Van Den Broek IVF, Keus K, Nzila A (2003) Association between chloroquine and amodiaquine resistance and allelic variation in the Plasmodium falciparum multiple drug resistance 1 gene and the chloroquine resistance transporter gene in isolates from the upper Nile in Southern Sudan. Am J Trop Med Hyg 69:184–187PubMedGoogle Scholar
- Ranford-Cartwright LC, Taylor J, Umasunthar T, Taylor LH, Babiker HA, Lell B, Schmidt-Ott JR, Lehman LG, Walliker D, Kremsner PG (1997) Molecular analysis of recrudescent parasites in a Plasmodium falciparum drug efficacy trial in Gabon. Trans R Soc Trop Med Hyg 91:719–724PubMedCrossRefGoogle Scholar
- Sutherland CJ, Alloueche A, Curtis J, Drakeley CI, Ord R, Duraisignh M, Greenwood BM, Pinder M, Warhurst D, Target GAT (2002) Gambian children successfully treated with CQ can harbor and transmit Plasmodium falciparum gametocytes carrying resistant genes. Am J Trop Med Hyg 67:568–585Google Scholar
- Tinto H, Sanou B, Dujardin JC, Ouedraogo JB, Van Overmeir C, Erhart A, Van Marck E, Guiguemde TR, D’Alessandro U (2005) Usefulness of the Plasmodium falciparum chloroquine resistance transporter T76 genotype failure index for the estimation of in vivo chloroquine resistance in Burkina Faso. Am J Trop Med Hyg 73:171–173PubMedGoogle Scholar
- WHO (1996) Assessment of therapeutic efficacy of antimalarial drugs for uncomplicated malaria in areas with intense transmission. WHO/MAL/96-1077. GenevaGoogle Scholar