Clinical isolates of uncomplicated falciparum malaria from high and low malaria transmission areas show distinct pfcrt and pfmdr1 polymorphisms in western Ethiopia
Authors: Tadele Geletta , Jawara Aminata , Oboh Mary , Oriero Eniyou , Dugassa Sisay , Amambua-Ngwa Alfred , Golassa Lemu 
Published: January 2023

Abstract
Background
Chloroquine which was the first-line drug for the treatment of uncomplicated P. falciparum malaria in Ethiopia was officially abandoned in 1998, and replaced by sulphadoxine-pyrimethamine which in turn was replaced by artemether-lumefantrine (AL) in 2004. Pfcrt gene has been associated with chloroquine resistance and pfmdr1 gene can alter malaria parasite susceptibility to most of the current antimalarial drugs, including lumefantrine, mefloquine, and chloroquine. In the absence of chloroquine (CQ) and extensive use of AL for eighteen years, we determined polymorphisms of pfcrt haplotype and pfmdr1 SNPs in two sites of West Ethiopia with different levels of malaria transmission.
Methods
Health-facility based cross-sectional study was conducted at Assosa and Anger Gute areas. Finger-prick blood samples were collected from a total of 225 microscopically confirmed Plasmodium falciparum patients and spotted onto Whatman filter papers. For molecular genotyping, parasite DNA was extracted using the Chelex extraction method. High-Resolution Melting Assay (HRM) was used to determine the prevalence of pfcrt haplotypes at positions 72–76 and pfmdr1 SNPs at codon N86Y, Y184F, N1042D and D1246Y. Furthermore, the pfmdr1 gene copy number (CNV) was determined using real-time PCR. A p-value of less or equal to 0.05 was considered significant.
Results
Of the 225 samples, 95.5%, 94.4%, 86.7%, 91.1% and 94.2% samples were successfully genotyped with HRM for pfcrt haplotype, pfmdr1-86, pfmdr1-184, pfmdr1-1042 and pfmdr1-1246, respectively. A total of 46.5%( 100/215) of the clinical isolates in west Ethiopia carry the mutant pfcrt genotype. The mutant haplotypes was detected among 33.5% (52/ 155) and 80% (48/60) of isolates collected from the Assosa and Anger Gute sites, respectively. P. falciparum with chloroquine-resistant haplotypes were more prevalent in the Anger Gute area (low transmission site) as compared with the Assosa area (high transmission area)(COR = 8.4, P = 0.00). Pfmdr1- N86Y wild type and 184F mutations were found in 79.8%( 166/208) and 73.4% (146 /199) samples, respectively. No single mutation was observed at pfmdr1-1042 locus; however, 89.6% (190/212) of parasites in West Ethiopia carry the wild-type D1246Y variants. Eight pfmdr1 haplotypes at codonsN86Y- Y184F-D1246Y were identified with the dominant NFD 61% (122/200) followed by the wild type NYD haplotype (17%( 34/200). The proportion of parasites with multiple pfmdr1 copies was 8.4%( 19/225). There was no difference in the distribution of pfmdr1 SNPs, haplotypes and CNV between the two study sites (P > 0.05).
Conclusion
There is a gradual regaining of chloroquine-sensitive haplotype in the study areas after cessation of CQ use for the treatment of uncomplicated falciparum malaria; however, the return to the wild-type is higher in high malaria transmission site (Assosa) than in low transmission area (Anger Gute). A high prevalence of the wild-type alleles N86, D1042 and D1246 and of the mutant-type allele 184F was detected from both study sites. The NFD haplotype was the predominant haplotype of the N86Y-Y184F-D1246Y and 8.4% of the parasites carry multiple copies of the pfmdr1 gene. Continuous surveillance is needed to closely monitor the changes in the pfmdr1 SNPs, which are associated with the selection of parasite populations by ACT.