INVESTIGATION OF PYRONARIDINE RESISTANCE IN PLASMODIUM BERGHEI ANKA ISOLATES.

Abstract

Malaria is a major worldwide health problem and the control of the disease is compromised by acquisition and spread of Plasmodium falciparum resistance to multiple antimalarial drugs, which has caused an increase in malaria related morbidity and mortality. In response, Artemisinin based Combination Therapies (ACTs) have been implemented in almost all malaria endemic areas in an attempt to reduce emergence of resistance. For instance, a combination of Artesunate (ASN)-Pyronaridine (PRD) was recently prequalified by WHO drug as an alternative for treatment of malaria in African setting. However pyronaridine, a benzonaphthyridine derivative has a long half-life, consequently predisposed to high selection pressure, hence high chances of emergence of resistance. To counter this problem, the mechanisms of resistance of existing drugs need to be fully elucidated. As a first step towards understanding resistance, the study first selected pyronaridine resistance by submitting Plasmodium berghei ANKA line in vivo to increasing Pyronaridine concentration for 20 successive passages over a period of six months. The effective doses that reduced parasitaemia by 50% (ED50) and 90% (ED90) determined in the standard four-day suppressive test for the parent line were 1.83 and 4.79 mgkg-1, respectively. After 20 drug pressure passages, the ED50 and ED90 increased by 66 and 40 fold, respectively. To determine the stability of resistance, the parasites were first cloned by limiting dilution, then grown in absence of drug for five passages and cryo-preserved at -80⁰C for one month. The effective doses determined later found that the resistance phenotypes remained stable. Using PCR amplification and sequencing, the coding sequence of Plasmodium berghei multidrug resistance gene 1 (PbMDR-1) was analyzed, which is orthologous to the gene associated with quinoline resistance in Plasmodium falciparum. The results did not show any nucleotide polymorphism within the coding region. Further broader sequencing is therefore recommended in order to associate PbMDR-1 gene to PRD resistance in Plasmodium berghei. Nonetheless, additional possible candidate genes involved in pyronaridine drug resistance should be explored.