Amodiaquine resistance in is associated Plasmodium berghei with His 95 Pro mutation , loss of chloroquine , artemisinin

The human malaria parasite has evolved Background: Plasmodium falciparum complex drug evasion mechanisms to all available antimalarials. To date, the combination of amodiaquine-artesunate is among the drug of choice for treatment of uncomplicated malaria. In this combination, a short acting, artesunate is partnered with long acting, amodiaquine for which resistance may emerge rapidly especially in high transmission settings. Here, we used a rodent malaria parasite ANKA as a surrogate of to Plasmodium berghei P. falciparum investigate the mechanisms of amodiaquine resistance. : We used serial technique to select amodiaquine resistance by Methodology submitting the parasites to continuous amodiaquine pressure. We then employed the 4-Day Suppressive Test to monitor emergence of resistance and determine the cross-resistance profiles. Finally, we genotyped the resistant parasite by PCR amplification, sequencing and relative quantitation of mRNA transcript of targeted genes. Submission of ANKA to amodiaquine pressure yielded Results: P. berghei resistant parasite within thirty-six passages. The effective dosage that reduced 90% of parasitaemia (ED ) of sensitive line and resistant line were 4.29mg/kg and 19.13mg/kg, respectively. After freezing at -80oC for one month, the resistant parasite remained stable with an ED of 18.22mg/kg. Amodiaquine resistant parasites are also resistant to chloroquine (6fold), artemether (10fold), primaquine (5fold), piperaquine (2fold) and lumefantrine (3fold). Sequence analysis of revealed Plasmodium berghei chloroquine resistant transporter His95Pro mutation. No variation was identified in Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating or nucleotide sequences. enzyme-1 Plasmodium berghei Kelch13 domain Amodiaquine resistance is also accompanied by high mRNA transcripts of key transporters; , and Pbmdr1 V-type/H+ pumping pyrophosphatase-2 sodium 1,2 3 4 4 2