Molecular markers of anti-malarial drug resistance in southwest Ethiopia over time: regional surveillance from 2006 to 2013.

Background: Drug resistance is one of the main reasons of anti-malarial treatment failures and impedes malaria containment strategies. As single nucleotide polymorphisms (SNPs) have been found to correlate with anti-malarial drug resistance, the surveillance strategy includes continuous monitoring of known molecular markers and detection of new mutation patterns. With the introduction of artemisinin-based combination therapy, selection of specific patterns has been observed worldwide.
Methods: From March to June 2013, whole blood was collected on filter paper from microscopically malaria positive patients in Jimma zone (District), southwestern Ethiopia. Plasmodium falciparum, Plasmodium vivax and mixed infections were included. SNPs were investigated by conventional or real-time PCR, restriction fragment length pattern analysis or sequencing. Results were compared to molecular patterns from Ethiopian isolates in 2004, 2006 and 2008/9.
Results: Plasmodium falciparum, P. vivax, and mixed infections were molecularly confirmed in 177, 80, and 14 samples, respectively. In P. falciparum, mutations in the pfcrt, pfmdr 1and pfATP 6 (SERCA) gene were investigated. Whereas the mutation in the pfcrt gene at codon 76 K was still found in 95.6% of all samples, the pfmdr 1 86 T mutation fell to 1.2% (2/163) in 2013 compared to 9% in 2008/9 and 86% in 2006 (P<0.001). The pfmdr 1 184 F mutation dominated with 100.0% (172/172) in 2013. Sequencing of the recently reported PF3D7&#95;1343700 kelch propeller domain showed no mutation at codon 476. First sequencing data of the pvmdr 1 gene from Jimma region revealed a prevalence of the mutations 976 F and 1076 L in 72.7% (16/23) and 100.0% (19/19) of the isolates, respectively.
Conclusion: Since the introduction of artemether-lumefantrine (AL) in Jimma, Ethiopia, in 2006, the prevalence of certain SNPs associated with AL use has increased. Markers for chloroquine resistance in P. vivax were highly frequent. Continuous molecular and clinical surveillance are of paramount importance.