The emergence and spread of CQR Plasmodium falciparum in malaria-endemic countries has led to alterations in the anti-malarial treatment policy and the introduction of artemisinin-based combination therapies (ACT) for treatment of uncomplicated malaria . The failure of CQ to clear P. falciparum asexual parasites in Ethiopia was first reported in 1986 in areas bordering Somalia, Kenya and Sudan. A steady increase in the number of refractory isolates was observed in all malarious areas in Ethiopia and as a result CQR P. falciparum became a major public health threat in 1990s . In response to high levels of P. falciparum resistance to CQ and then sulphadoxine-pyrimethamine (SP), Ethiopia adopted artemether-lumefantrine (AL or Coartem©) as first-line therapy in 2004 [3–5]. According to the current national malaria diagnosis and treatment guidelines, AL and CQ are first-line treatment for uncomplicated P. falciparum and Plasmodium vivax infections, respectively. For all clinical infections without laboratory confirmation, AL, which is effective against both P. falciparum and P. vivax, is the first-line treatment. The reduced susceptibility of parasite strains to artemisinin necessitates assessment of the current status of CQR in Ethiopia. CQ is a drug of interest for study because of its efficacy, affordability, easy administration, prophylaxis potential, low toxicity and relatively few side effects .
It has been recognized that CQR falciparum malaria is caused by mutations in two genes, the P. falciparum CQ resistance transporter (pfcrt) and multidrug resistance transporter-1 (pfmdr1) however the former is a stronger predictor of CQR than pfmdr1. Polymorphisms in the pfcrt gene segregate precisely with two distinct drug response classes, considered either CQS or CQR. All P. falciparum clinical samples that are resistant to CQ contain the K76T mutation . Amino acid change from lysine to threonine at position 76 (K76T) appears necessary for the resistance phenotype and is the most reliable molecular marker of resistance of the various pfcrt mutations. It is unfortunate to notice that strains of P. falciparum resistant to artemisinin have appeared in the Cambodia–Thailand border region . While CQS strains are characterized by the CVMNK haplotype, irrespective of geographic origin, two major haplotypes defined by specific mutations at amino acid positions 72-76 of pfcrt, CVIET and SVMNT, are associated with the geographic origin of CQR . The former haplotype is predominantly found in Southeast Asia and Africa, whereas the SVMNT haplotype is characteristic of South America, Papua New Guinea , and the Philippines .
A decrease in drug resistant pfcrt alleles has been reported following discontinuation of drug pressure  in Africa although this process can vary greatly between countries. A long-term decline in the use of CQ can lead to resurgence of drug-sensitive populations of P. falciparum. This raises the possibility that, in time, the drug could be re-introduced . Understanding the influence of CQ withdrawal on local malaria parasite populations has great significance. In Malawi, for instance, the frequency of the mutation declined from 85% in 1992 to 13% in 2000 in one study  and from 17% in 1998 to 2% in 2000 in another study  after CQ was withdrawn as the first- line drug. Studies in Kenya  and China  have reported complete and partial recovery of CQS parasites after the use of CQ was abandoned. This recovery was thought to be due to the re-introduction of susceptible parasites harbouring a CQS pfcrt[17, 18], although the possibilities of back mutation in pfcrt at position 76 may not be underestimated. Continued surveillance of markers of resistance to withdrawn drugs has been indicated with the prospect that decreases in resistance may make it possible to reuse this safe and cheap drug [4, 17]. The overall trend in the reversal of the resistance could be related to a country’s drug policies although data regarding the impact of CQ withdrawal on circulating parasite population is not well studied in Ethiopia.
When advantageous mutations such as drug resistance mutations spread through a population, unrelated markers that flank these loci are carried along through linkage disequilibrium by a “hitch-hiking effect”, thereby removing genetic variation from chromosomal regions surrounding the locus . Unlike in CQS alleles, there is a marked drop in variability and an increase in linkage disequilibrium with CQR associated alleles in ~40 kb region flanking the pfcrt gene . MS repeats, occurring on the average approximately every 1 kb [20, 21], are common in the P. falciparum genome and have been recently used to analyse both intra- and inter-population relationships among drug-resistant P. falciparum strains.
Drug treatment has not only resulted in positive selection at codons conferring resistance but also alleles at loci closely linked to these genes, due to limited recombination in the chromosome regions containing these genes since drug selection was imposed . If a particular mutation in the target molecule is fixed in the parasite population due to the drug pressure, its flanking MS markers will start showing minimum variation . Thus, selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite . MS markers associated with drug resistance enabled identification of drug resistance origin as well as tracing spread of these mutations . Drug- resistant genotypes are characterized by reduced diversity around the major resistant alleles . Thus, the expected heterozygosity (He) values of the flanking MSs will be reduced among mutant parasites. Diversity of haplotypes existed in the parasite population before CQR but after the selection, parasites bearing the pfcrt76K allele are eliminated and those with pfcrt76T survive and expand in the population. Thus, in areas where CQ has been widely used, there will be homogeneity of the loci in this chromosome region of the resistant parasites around the pfcrt whereas diversity of MS alleles will still be found in sensitive parasites. Despite high level of CQR in Ethiopia, pertinent data regarding the type of mutant pfcrt genotype are lacking.
Applications of MS markers flanking the genes have revealed that drug resistance appears to have resulted from the migration of limited resistant lineages to many endemic regions however the resistance haplotypes present in Ethiopia is not known. Despite the migration of resistant lineages, multiple resistant lineages independently evolved in several endemic regions. Indeed, the geographic origin and spread of resistance can be estimated by the genotyping of pfcrt at positions 72-76 and MS haplotyping flanking this locus. Although Ethiopia is highly endemic for P. falciparum malaria and CQR P. falciparum isolates occur widely, there is scarcity of data regarding the pfcrt genotypes and diversity among the parasite population. Authors hypothesize that although CQ was replaced by ACT for treatment of P. falciparum malaria in the country, unlike in many African countries, the withdrawal CQ may not result in the reduction of CQR genotype for the fact that it is still the first-line treatment for P. vivax and co-infections are inevitable. The purpose of this study was, therefore, to determine the CQR haplotypes, to unveil the magnitude of P. falciparum CQR genotypes 14 years after its withdrawal and to determine the genetic diversity in CQR alleles in south-central Oromia, Ethiopia.