Phenotypic and Genotypic Analysis of In Vitro -Selected Artemisinin-Resistant Progeny of Plasmodium falciparum

Abstract

Emergence of artemisinin resistance in Cambodia highlights the importance of characterizing resistance to this class of drugs. Previously, intermediate levels of resistance in Plasmodium falciparum were generated in vitro for artelinic acid (AL) and artemisinin (QHS). Here we expanded on earlier selection efforts to produce levels of clinically relevant concentrations, and the resulting lines were characterized genotypically and phenotypically. Recrudescence assays determined the ability of resistant and parent lines to recover following exposure to clinically relevant levels of drugs. Interestingly, the parent clone (D6) tolerated up to 1,500 ng/ml QHS, but the resistant parasite, D6.QHS340؋3, recovered following exposure to 2,400 ng/ml QHS. Resistant D6, W2, and TM91c235 parasites all exhibited elevated 50% inhibitory concentrations (IC 50 s) to multiple artemisinin drugs, with >3-fold resistance to QHS and AL; however, the degree of resistance obtained with standard methods was remarkably less than expected for parasite lines that recovered from 2,400-ng/ml drug pressure. A novel assay format with radiolabeled hypoxanthine demonstrated a greater degree of resistance in vitro than the standard SYBR green method. Analysis of merozoite number in resistant parasites found D6 and TM91c235 resistant progeny had significantly fewer merozoites than parent strains, whereas W2 resistant progeny had significantly more. Amplification of pfmdr1 increased proportionately to the increased drug levels tolerated by W2 and TM91c235, but not in resistant D6. In summary, we define the artemisinin resistance phenotype as a decrease in susceptibility to artemisinins along with the ability to recover from drug-induced dormancy following supraclinical concentrations of the drug.

Altered temporal response of malaria parasites determines differential sensitivity to artemisinin

“…Indeed, recent studies suggest that resistance in P. falciparum is associated with an altered temporal pattern of transcription (28) and may be linked to a region on chromosome 13 (29). In vitro selection of resistance to artemisinins also points to the involvement of a range of different genes (23,30). We predict that different molecular changes will affect the responses of different parasite stages, but that in many cases the alterations will affect the length of the lag time of the response, for example by decreasing the reactive iron level or the level of a downstream target molecule.…”

“…It is important to note that the approaches we have used do not address the question of whether subpopulations of dormant or tolerant parasites (23)(24)(25) are involved in resistance to ARTs. The ability of a subpopulation of parasites to enter a quiescent phase could contribute to artemisinin resistance; however, there is no need to invoke the presence of such subpopulations to explain the sensitivity differences that we observe; the behavior is that of the population as a whole and represents the stochastic drug response of the parasites.…”

Apicomplexan Energy Metabolism: Carbon Source Promiscuity and the Quiescence Hyperbole

“…Combination therapies employing artemisinin still represent the primary treatment of malaria, although drug-resistance is ever-increasing (www.who.int/mediacentre/ factsheets/fs094). Perhaps central to this is the fact that sublethal exposures to artemisinin (and derivatives) in vitro lead to arrest in P. falciparum ring-stages and subsequent transition to a morphologically-distinct 'dormant' state that is able to persist under drug pressure and recover after removal of the treatment [97,98]. Artemisinin-induced 'dormant rings' display reduced susceptibility to artemisinin itself [99,100], but remain susceptible to other drugs targeting the ETC, suggesting that they are still metabolically active [97].…”

Assessment of the Induction of Dormant Ring Stages in Plasmodium falciparum Parasites by Artemisone and Artemisone Entrapped in Pheroid Vesicles In Vitro

“…A plausible explanation for recrudescence is drug-induced quiescence or dormancy that protects ring-stage parasites against artemisinin exposure (9,10). The artemisinin-treated ring stages of P. falciparum thereby enter a temporary growth arrest (11,12), wherein they survive drug treatment, resuming normal growth once drug pressure is removed (13)(14)(15).…”