title: High prevalence of pfcrt-CVIET haplotype in isolates from asymptomatic and symptomatic patients in south-central Oromia, Ethiopia.
abstract: BACKGROUND
  As a result of extensive chloroquine resistance (CQR) in Plasmodium falciparum in late 1990s, Ethiopia replaced CQ with sulphadoxine-pyrimethamine (SP) as first-line drug, which in turn was replaced by artemisinin combination therapy in 2004. Plasmodium falciparum resistance to CQ is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene. Understanding diversity in the P. falciparum genome is crucial since it has the potential to influence important phenotypes of the parasite such as drug resistance. Limited data is available regarding the type of pfcrt mutant allelic type, the effect of CQ withdrawal and diversity of the parasite population in south-central Oromia, Ethiopia.
  
  
  METHODS
  Finger-pricked blood spotted on Whatman 3MM filter papers were collected from falciparum malaria patients. Parasite DNA was extracted from individual blood spots on the filter papers. The presence of K76T mutations was determined using nested PCR for all isolates. Complete sequencing of mutations in pfcrt 72-76 was done for a set of randomly selected resistant isolates. Four microsatellite (MS) markers were analysed to determine the heterozygosity.
  
  
  RESULTS
  Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the pfcrt K76T mutation. All isolates were mutant at the K76T polymorphism. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Heterozygosity (H(e)) for MS flanking the pfcrt chloroquine resistance allele ranged from 0.00 (mscrt -29, -29.268 kb) to 0.21 (mscrt -2, -2.814 kb). H(e) ranged from 0.00 (msint 3, 0 kb) to 0.19 (msint 2, 0 kb) for MS within the pfcrt gene. Both intronic and MS flanking the pfcrt gene showed low levels of diversity.
  
  
  CONCLUSIONS
  pfcrt CQR allele seems to be fixed in the study area. Of the different haplotypes associated with CQR, only the CVIET genotype was identified. No reversal to the wild-type has occurred in Ethiopia unlike in many Africa countries where CQR parasites declined after cessation of CQ use. Decreased diversity in CQR isolates surrounding pfcrt suggests CQ selection and homogenization among CQR parasite population. While mutation in msint 3 and mscrt -29 of the mutant pfcrt allele is being fixed, it seems that mutations in msint 2 and mscrt -2 are still evolving and may indicate the start of re-diversification of the population from a fixed 76 T population.
authors.affiliation: Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia. lgolassa@gmail.com.
authors.forename: Lemu
authors.initials: L
authors.lastname: Golassa
authors.forename: Nizar
authors.initials: N
authors.lastname: Enweji
authors.forename: Berhanu
authors.initials: B
authors.lastname: Erko
authors.forename: Abraham
authors.initials: A
authors.lastname: Aseffa
authors.forename: Göte
authors.initials: G
authors.lastname: Swedberg
mesh_terms: D058345:Asymptomatic Infections; D002738:Chloroquine / Q000494:pharmacology*; D059788:Dried Blood Spot Testing; D004351:Drug Resistance; D005002:Ethiopia / Q000453:epidemiology; D006239:Haplotypes; D006801:Humans; D008288:Malaria / Q000453:epidemiology* / Q000469:parasitology*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D015995:Prevalence; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013997:Time Factors
chemical_list: D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D002738:Chloroquine