Search for genetic determinants of Plasmodium falciparum malaria drug resistance in vitro and in vivo

Malaria remains the most deadly disease in the world with nearly 627 000 deaths and more than 200 million new clinical cases every year, the large majority occurring in sub Saharan Africa children aged < 5 years. This represents anyway a significant decrease as compared with the situation in the start of the Millennium. This is due in part to the worldwide adoption of artemisinin-based combination therapy (ACT). However these gains are being threatened. A pattern of progressive decreased susceptibility of the parasite to the ACT key drugs, the artemisinin derivatives is emerging. Another central drug is quinine, still the mainstay for the treatment of severe malaria in Africa.

The aim of this thesis was to contribute to the understanding of the genetic determinants of Plasmodium falciparum resistance to two key short half-life antimalarials, quinine and artesunate and to assess the parasite susceptibility to these drugs in Mali.

In a clinical study on the efficacy of quinine 100% of severe Plasmodium falciparum infected patients were cured. For the first time, the pfnhe1 microsatellite allele ms4760-1, previously proposed to be involved with parasite in vitro resistance to this drug was selected post treatment pointing for this marker as also involved in the in vivo sensitivity of the parasite to quinine. Conversely, t he ms4760 status of the initial infections was not predictive of the clinical outcome, leading to the conclusion that the ms4760-1 is likely a secondary factor of quinine resistance. The pfcrt K76T SNP was not shown to be under selection. In conclusion, albeit pfnhe1 has an undeniable contribution to the parasite response in vivo, other factors must be involved, supporting the view of quinine resistance as a complex multigenic trait.

P. falciparum decreased sensitivity to artemisinin and its derivatives have been recently reported in SE Asia, including Thailand. We therefore performed an explorative study based on the determination of the in vitro sensitivity (IC 50 ) of 47 culture adapted parasites from Mae Sot (Thai-Myanmar boarder) to a number of ACT drugs. These included artemisinin and dihydroartemisinin (DHA), the key metabolite of both artemether and artesunate. The open reading frames of the drug transporter genes pfcrt, pfmdr1, pfmrp1 and pfmrp2 were further studied. Correlation analyses revealed two novel candidate markers of multidrug resistance: the pfmdr1 F1226Y and pfmrp1 F1390I SNPs, which were associated with 2-3 fold, increases in the IC50 s of artemisinin and also the ACT partner drugs lumefantrine and mefloquine.

An artesunate monotherapy (7 days) efficacy trial was performed at Malian malaria setting with the objective of detecting possible delayed P. falciparum clearance phenotypes, an early sign of decreased drug susceptibility. The microscopic based assessment of the infections did not reveal any extended parasitaemia clearance times with a median clearance time of 32 hours. Nevertheless there were clear int er-individual differences in the clearance dynamics.

Recently, SNPs in the P.falciparum K13 propeller gene has been proposed to be markers of artemisinin resistance, i.e. of significantly increased clearance time in SE Asia. We therefore studied the polymorphisms in this gene in Mali and any possible association with the range of clearance times observed above. In addition, a set of samples from a previous cross section survey study, conducted prior to ACTs implementation, were analyzed in order to try to detect temporal changes in the sequence of the K13 propeller gene. The SE Asian mutations associated with artemisinin resistance were not found in Mali in any of the periods. Nevertheless, the K13 gene was found to be polymorphic in Mali even before the wide use of ACTs. No association was however found between polymorphism and parasite clearance rate. Interestingly, the SNPs found in the early cross-sectional study were different from those found in the later study . Further, the later study revealed mutations present near one of the key a.a. positions linked with resistance in Asia. These patterns merit further investigations.

Finally, a new qPCR based approach was used to revisit the artesunate monotherapy study. This had the aim of increasing the sensitivity of parasite detection, in order to obtain an improved phenotype of parasite clearance, and hence improved conditions to search for a correlation between the presence of K13 mutation and the trend of prolonged parasite clearance.

No clear association could be found even though the qPCR approach was able to find evidence of parasites 72 hours after artesunate treatment in more than 46% of infections previously considered as cleared by microscopy. Intriguingly no mutations in the K13 propeller gene were found among the parasites classified as fast clearers by this method (parasites cleared at 24 hours after treatment).

The result of the clinical trials showed high in vivo efficacies for both artesunate and quinine. However, this situation can rapidly change, as demonstrated by the recent emergence of artemisinin resistance in Asia. Molecular monitoring of any possible evolution and selection of antimalarial drug tolerance/resistance associated polymorphisms of genes such as Pfnhe-1 or K13 propeller are critical for optimal drug policies and sustained efficacy.

List of scientific papers

I. Kone A, Mu J, Maiga H, Beavogui AH, Yattara O, Sagara I, Tekete MM, Traore OB, Dara A, Dama S, Diallo N, Kodio A, Traoré A, Björkman A, Gil JP, Doumbo OK, Wellems TE, Djimde AA. Quinine treatment selects the pfnhe-1 ms4760-1 polymorphism in Malian patients with Falciparum malaria. J Infect Dis. 2013 Feb 1;207(3):520-7.
https://doi.org/10.1093/infdis/jis691

II. Veiga MI, Ferreira PE, Jörnhagen L, Malmberg M, Kone A, Schmidt BA, Petzold M, Björkman A, Nosten F, Gil JP.Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance. PLoS One. 2011;6(5):e20212.
https://doi.org/10.1371/journal.pone.0020212

III. Maiga AW, Fofana B, Sagara I, Dembele D, Dara A, Traore OB, Toure S, Sanogo K, Dama S, Sidibe B, Kone A, Thera MA, Plowe CV, Doumbo OK, Djimde AA. No evidence of delayed parasite clearance after oral artesunate treatment of uncomplicated falciparum malaria in Mali. Am J Trop Med Hyg. 2012 Jul;87(1):23-8.
https://doi.org/10.4269/ajtmh.2012.12-0058.

IV. Ouattara A, Kone A, Adams M, Fofana B, Walling A.M, Hampton S, Coulibaly D, Thera M.A, Diallo N, Dara A, Sagara I, Gil J.P, Bjorkman A, Takala S.H, Doumbo O.K, Plowe C.V and Djimde A.A. Polymorphisms in the K13-propeller gene in artemisinin susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali. Am J Trop Med Hyg [Accepted]
https://pubmed.ncbi.nlm.nih.gov/25918205

V. Kone A, Diallo N, Bechir B.K, Fofana B, MaigaA.W, Sissoko S, Dara A, Dama S, Bjorkman A, Gil J.P, Sutherland C, Doumbo O.K, and Djimde A.A. measuring the clearance of falciparum by the method of rt pcr relative quantification after treatment of uncomplicated malaria with artesunate in Mali. [Manuscript]