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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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Abstract

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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Malaria Research Laboratories, Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria

    C. T. Happi, G. O. Gbotosho, O. A. Folarin, A. Sowunmi, O. M. Bolaji & B. A. Fateye

  2. Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Springs, MD, USA

    D. E. Kyle & W. Milhous

  3. Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA, USA

    C. T. Happi & D. F. Wirth

  4. Special Programme for Research and Training in Tropical Diseases (WHO/TDR), Geneva, Switzerland

    A. M. J. Oduola

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  1. C. T. Happi
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  2. G. O. Gbotosho
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  3. O. A. Folarin
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  4. A. Sowunmi
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Correspondence to C. T. Happi.

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Happi, C.T., Gbotosho, G.O., Folarin, O.A. et al. Linkage disequilibrium between two distinct loci in chromosomes 5 and 7 of Plasmodium falciparum and in vivo chloroquine resistance in Southwest Nigeria. Parasitol Res 100, 141–148 (2006). https://doi.org/10.1007/s00436-006-0246-4

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