MOLECULAR MARKERS ASSOCIATED WITH AMODIAQUINE RESISTANCE IN RODENT MALARIA PARASITE Plasmodium berghei ANKA

Abstract

Due to the high genetic plasticity of Plasmodium falciparum to evolve resistance to single drug, W.H.O recommended the use of Artemisinin combination therapies (ACT) for treatment of uncomplicated P. falciparum malaria in Sub- Saharan Africa. To date, the combination of amodiaquine-artesunate is the first or second line of choice for treatment of uncomplicated malaria in Africa. Mode of action of amodiaquine (AQ) is predicted to be similar to that of chloroquine (CQ), thus they may share similar resistance mechanisms. The main objective of this study was to investigate the markers associated with AQ resistance in P.berghei. To this effect, resistant parasites had to be selected. The study used rodent malaria parasite Plasmodium berghei ANKA as a surrogate model to P.falciparum. The study interrogated mechanism of AQ resistance four ways: i) resubmission of parasite to AQ pressure for a further 16 passages, determined the resistance levels after every 4 passages and the stability of the resistant lines by storing at -80 degrees for a month. ii) determined cross resistance profiles of the resistant line against chemically and mechanistically related and unrelated antimalarial drugs iii) assess quinolone resistance suspect gene multi-drug resistance gene-1 (mdr1), chloroquine resistance transporter (crt), deubiquitinating protease 1 (ubp) and kelch13 by PCR and sequencing iv) evaluated the expression profiles of putative transporter V-type/H+ pyrophosphatase-2 (vp2), mdr1, Ca2+/H+antiporter (cvx1) and Na+/H+ exchanger (nhe1) by qRT – PCR. From the results obtained, the effective dosage that reduced 99% of parasitaemia (ED99) of sensitive line and resistant line were 5.05mg/kg and 20.73mg/kg respectively. After freezing at -80 degree for at least one month, the resistant parasite remained stable with an ED99 of 18.22. This study further shows that AQ resistant phenotypes are cross resistant to chloroquine (6 fold), artemether (10 fold), primaquine (5 fold), piperaquine (2 fold) and lumefantrine (3 fold), suggesting they are xv ‘’multidrug resistant phenotype’’. Sequence analysis for single nucleotide polymorphisms (SNPs) in P. berghei chloroquine resistant transporter (Pbcrt), P. berghei multidrug resistance gene 1 (Pbmdr1), P. berghei deubiquitinating enzyme 1 (Pbubp1) and P. berghei Kelch13 domain (Pbkelch13) however revealed no SNPs. Polymorphisms in these genes is associated with quinoline and artemisinin resistance suggesting AQ resistant phenotype is controlled by other unknown variants. This study further provides, evidence that AQ resistance is associated with high mRNA transcripts in resistance compensatory and modulatory genes; Pbmdr1, Ca2+/H+ antiporter (vcx1), V-type H+ pumping pyrophosphatase 2 (vp2) and sodium hydrogen ion exchanger1 (nhe1). In conclusion, this study was able to develop stable multidrug resistant P.berghei by continuous submission of parasites to AQ drug pressure for 36 passages. The study further, reveals that amodiaquine is associated with cross resistance in LM, CQ. PQ, PMQ and ATM. Increased transcript level of mdr1, vp2, cvx1 and nhe1 is associated with amodiaquine resistance in P.berghei ANKA. The study further reveals that mdr1, crt, ubp1 and kelch 13 domain are not associated with amodiaquine resistance in P. berghei. This study therefore recommends that drug selection of the parasite should be continued to acquire a highly stable resistant P.berghei. The study further recommends more cross resistant studies using different classes of antimalarial drugs to increase the knowledge of resistance mechanism among different antimalarial drugs. A whole genome sequencing and transcriptome profiling of AQ resistant P.berghei should be carried out. This will reveal any novel mutation in the unknown causal gene that may be associated with amodiaquine resistance. To further the understanding of amodiaquine resistance, the validation of the suspected genes should be done using PlasmoGEM resources as well as CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats).