title: Prevalence of Plasmodium falciparum anti-malarial resistance-associated polymorphisms in pfcrt, pfmdr1 and pfnhe1 in Muheza, Tanzania, prior to introduction of artemisinin combination therapy.
abstract: BACKGROUND
  A report of the chloroquine and amodiaquine resistance pfcrt-SVMNT haplotype in Tanzania raises concern about high-level resistance to the artesunate-amodiaquine combination treatment widely employed in Africa. Mutations in the pfmdr1 multi-drug resistance gene may also be associated with resistance, and a highly polymorphic microsatellite (ms-4760) of the pfnhe1 gene involved in quinine susceptibility has not been surveyed in Tanzania.
  
  
  METHODS
  A total of 234 samples collected between 2003 - 2006 from an observational birth cohort of young children in Muheza, Tanzania were analysed. In these children, 141 cases of P. falciparum infections were treated with AQ and 93 episodes were treated with QN. Haplotypes of pfcrt and pfmdr1 were determined by a Taqman assay, and ms-4760 repeats in pfnhe1 were assessed by nested PCR amplification and direct sequencing. Parasite population diversity was evaluated using microsatellite markers on five different chromosomes.
  
  
  RESULTS
  The pfcrt-CVIET haplotype was present alone in 93.6% (219/234) of the samples over the study period; the wild-type chloroquine- and amodiaquine-sensitive haplotype pfcrt-CVMNK was present in 4.3% (10/234) of the samples; and both haplotypes were present in 2.1% (5/234) of the samples. No significant change in wild-type pfcrt-CVMNK prevalence was evident over the 4-year period of the study. The pfcrt-SVMNT haplotype associated with high-level amodiaquine resistance was not detected in this study. The pfmdr1 locus was genotyped in 178 of these samples. The pfmdr1-YYNY haplotype predominated in 67.4% (120/178) of infections and was significantly associated with the pfcrt-CVIET haplotype. All samples carried the wild-type pfmdr1-N1042 codon. The ms-4760 repeat on pfnhe1 locus displayed 12 distinct haplotypes with ms-4760-1 predominating in the population. Analysis of these haplotypes showed no association of a particular haplotype with quinine treatment outcome.
  
  
  CONCLUSIONS
  The pfcrt-CVIET chloroquine resistance haplotype dominated in the collection of P. falciparum samples from Muheza. The pfcrt-SVMNT haplotype, which threatens the efficacy of amodiaquine and was reported in the same time period from Korogwe, Tanzania, 40 Km from Muheza, was not detected. Relative low prevalence of pfcrt-SVMNT in Africa may result from genetic or other factors rendering P. falciparum less supportive of this haplotype than in South America or other regions.
  
  
  BACKGROUND
  Trial Protocol Number: 08-I-N064.
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA. nahla.gadalla@nih.gov.
authors.forename: Nahla B
authors.initials: NB
authors.lastname: Gadalla
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA. gloria.tavera@case.edu.|Case Western Reserve University School of Medicine, Cleveland, OH, 44106, USA. gloria.tavera@case.edu.
authors.forename: Gloria
authors.initials: G
authors.lastname: Tavera
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA. jmu@niaid.nih.gov.
authors.forename: Jianbing
authors.initials: J
authors.lastname: Mu
authors.affiliation: Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania. earkabyemela@yahoo.com.
authors.forename: Edward R
authors.initials: ER
authors.lastname: Kabyemela
authors.affiliation: Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA. michal.fried@nih.gov.
authors.forename: Michael
authors.initials: M
authors.lastname: Fried
authors.affiliation: Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA. patrick.duffy@nih.gov.
authors.forename: Patrick E
authors.initials: PE
authors.lastname: Duffy
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA. jsa@niaid.nih.gov.
authors.forename: Juliana M
authors.initials: JM
authors.lastname: Sá
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, NIH, Rockville, MD, USA. twellems@niaid.nih.gov.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wellems
mesh_terms: D000655:Amodiaquine / Q000494:pharmacology / Q000627:therapeutic use; D000962:Antimalarials / Q000494:pharmacology* / Q000627:therapeutic use; D037621:Artemisinins / Q000494:pharmacology / Q000627:therapeutic use; D002675:Child, Preschool; D015331:Cohort Studies; D004338:Drug Combinations; D004351:Drug Resistance; D006801:Humans; D007223:Infant; D016778:Malaria, Falciparum / Q000188:drug therapy / Q000469:parasitology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D011110:Polymorphism, Genetic; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism; D013636:Tanzania
chemical_list: D000655:Amodiaquine; D000962:Antimalarials; D037621:Artemisinins; D004338:Drug Combinations; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D015800:Protozoan Proteins; D017923:Sodium-Hydrogen Exchangers; C513498:Mdr1 protein, Plasmodium falciparum; C416369:PfCRT protein, Plasmodium falciparum; C515299:amodiaquine, artesunate drug combination