Amodiaquine resistance in Plasmodium berghei is associated with PbCRT His95Pro mutation, loss of chloroquine, artemisinin and primaquine sensitivity, and high transcript levels of key transporters

Wellcome Open Research

article

Loise Ndung'u¹  Benard Langat³  Esther Magiri⁴  Joseph Ng'ang'a⁴  Beatrice Irungu²  Alexis Nzila⁵  Daniel Kiboi⁴ 

[1] PAUSTI, Jomo Kenyatta University of Agriculture and Technology;KEMRI- Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute;Department of Nursing and Nutritional Sciences, University of Kabianga;Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology;Department of Life Sciences, King Fahd University of Petroleum and Minerals;West Africa Centre for Cell Biology and Infectious Pathogens, University of Ghana;Kenya Medical Research Institute ,(KEMRI)/Wellcome Trust, Collaborative Research Program

关键词: Malaria;    Resistance;    Plasmodium berghei;    Amodiaquine;    Cross-resistance;   

DOI  :  10.12688/wellcomeopenres.11768.2

学科分类：内科医学

来源: Wellcome

【 摘 要 】

Background: The human malaria parasitePlasmodium falciparum has evolved drug evasion mechanisms to all available antimalarials. 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 parasitePlasmodium berghei ANKA as a surrogate ofP. falciparum to investigate the mechanisms of amodiaquine resistance.Methods: We used the ramp up approach to select amodiaquine resistance. We then employed the 4-Day Suppressive Test to measure the resistance level 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.Results: Submission of the parasite to amodiaquine pressure yielded resistant line within thirty-six passages. The effective doses that reduced 90% of parasitaemia (ED90) of the sensitive and resistant lines were 4.29mg/kg and 19.13mg/kg respectively. The selected parasite retained resistance after ten passage cycles in the absence of the drug and freezing at -80ºC for one month with ED90 of 20.34mg/kg and 18.22mg/kg. The parasite lost susceptibility to chloroquine by (6-fold), artemether (10-fold), primaquine (5-fold), piperaquine (2-fold) and lumefantrine (3-fold). Sequence analysis ofPlasmodium berghei chloroquine-resistant transporter revealed His95Pro mutation. We found no variation in the nucleotide sequences of Plasmodium berghei multidrug resistance gene-1 (Pbmdr1), Plasmodium berghei deubiquitinating enzyme-1 orPlasmodium berghei Kelch13 domain. However, high mRNA transcripts of essential transporters;Pbmdr1, V-type/H+ pumping pyrophosphatase-2 and sodium hydrogen ion exchanger-1 and Ca2+/H+ antiporter accompanies amodiaquine resistance.Conclusions: The selection of amodiaquine resistance yielded stable “multidrug-resistant’’ parasites and thus may be used to study shared resistance mechanisms associated with other antimalarial drugs. Genome-wide analysis of the parasite may elucidate other functionally relevant genes controlling AQ resistance inP. berghei.

【 授权许可】

CC BY