A mechanistic model quantifies artemisinin-induced parasite growth retardation in blood-stage Plasmodium falciparum infection
Introduction
Plasmodium falciparum malaria is a major parasitic disease which causes severe morbidity and mortality in approximately half a million people annually (World Health Organization, 2015). Artemisinin (ART) and its derivatives (e.g. artesunate, dihydroartemisinin and artemether), used in combination with partner drugs, provide front-line protection, and have been responsible for dramatic reductions in disease burden over the past few decades (World Health Organization, 2015). Despite their clinical and public health effectiveness, the emergence of ART resistance and lack of alternative treatments places current control programs at risk (Ariey, Witkowski, Amaratunga, Beghain, Langlois, Khim, et al., 2014, Ashley, Dhorda, Fairhurst, Amaratunga, Lim, Suon, et al., 2014, Dondorp, Nosten, Yi, Das, Phyo, Tarning, et al., 2009, Phyo, Nkhoma, Stepniewska, Ashley, Nair, McGready, et al., 2012). Development of a comprehensive understanding of ART’s mechanism of action and associated effects on infected red blood cells (iRBCs) is therefore critical for development of optimised ART-based treatment regimens and maintenance of control program impact (Simpson et al., 2014).
Recent in vitro experiments (Dogovski, Xie, Burgio, Bridgford, Mok, McCaw, et al., 2015, Klonis, Xie, McCaw, Crespo-Ortiz, Zaloumis, Simpson, et al., 2013, Yang, Xie, Cao, Giannangelo, McCaw, Creek, et al., 2016), combined with advances in pharmacokinetic–pharmacodynamic (PK–PD) modelling (Cao et al., 2016), have established a platform to probe the parasite’s temporal response to antimalarial drugs. The key experimental advance underlying these in vitro studies was the application of short drug pulses, which enabled fine-scale measurement of the killing effect of drug (Cao et al., 2016). A normal life cycle of an iRBC for P. falciparum is approximately 48 h and is classified based on morphological appearance into three main stages: the ring stage (approximately 0–26 h post infection (h p.i.)), trophozoite stage (approximately 27–38 h p.i.) and schizont stage (approximately 39–48 h p.i.). Upon rupture at approximately 48 h p.i., iRBCs release merozoites, 8–12 of which successfully invade susceptible RBCs to initiate a new round of infection (Dietz, Raddatz, Molineaux, 2006, Simpson, Aarons, Collins, Jeffery, White, 2002, Zaloumis, Humberstone, Charman, Price, Moehrle, Gamo-Benito, et al., 2012). Dogovski et al. demonstrated that a short pulse of ART (or dihydroartemisinin (DHA)) can induce growth retardation, prolonging the 48 h life cycle (Dogovski et al., 2015). Importantly, they found that growth retardation did not stop parasite growth entirely and was thus considered to be distinct from parasite dormancy which “freezes” parasites for days to weeks (Codd, Teuscher, Kyle, Cheng, Gatton, 2011, Teuscher, Gatton, Chen, Peters, Kyle, Cheng, 2010).
Experimental identification of drug-induced growth retardation raises two questions: 1) By how much is the life cycle of a parasite prolonged in response to a short drug exposure pulse?; and 2) How influential is growth retardation when considering in vivo parasite killing using PK–PD models? These two questions are important because we expect any drug-mediated variation in the duration of one or more life stages to impact on the efficacy of the drug (given the well-established finding that drug can exert stage-specific killing effect to parasites (Cao, Klonis, Zaloumis, Dogovski, Xie, Saralamba, et al., Klonis, Xie, McCaw, Crespo-Ortiz, Zaloumis, Simpson, et al., 2013, Saralamba, Pan-Ngum, Maude, Lee, Tarning, Lindegardh, et al., 2011, Witkowski, Khim, Chim, Kim, Ke, Kloeung, et al., 2013). The alteration in drug efficacy may be significant if the prolonged stage(s) covered by the drug pulse exhibit very distinct killing effects. Quantification of growth retardation and assessment of its potential effect on in vivo parasite killing is therefore important in guiding further experimental investigations into drug activity and strategies for optimising ART-based combination therapies.
To the best of our knowledge, no model has yet been designed to quantify the growth retardation effect and predict its influence on in vivo parasite killing. Here we construct a mechanistic model of parasite growth to explain drug-induced growth retardation. We model the relationship between parasite age and the fluorescence intensity of a parasite’s RNA/DNA-binding dye. Through application to fluorescence data from in vitro experiments using the laboratory 3D7 strain by Dogovski et al. (2015), we provide the first quantification of growth retardation. We identify a concentration threshold above which the growth retardation is evident. Furthermore, the model accommodates two possible mechanisms of drug-induced growth retardation, which can be reliably identified in future experimental studies. Finally, by incorporating growth retardation into a PK–PD modelling framework, we simulate in vivo parasite killing upon exposure to a single dose of artesunate and demonstrate the importance of considering growth retardation in the design of optimal artemisinin-based dosing regimens.
Section snippets
Experiment and data
We first summarise the in vitro experiment in which parasite growth retardation was identified. We provide sufficient information for the purposes of model development and evaluation. For full details on the experimental implementation we refer the reader to the original publication (Dogovski et al., 2015).
Fig. 1 presents the experimental process. A culture containing tightly synchronised rings (3D7 strain; over 80% of the population within a one-hour age window) with an average age of 6 h p.i.
Quantifying the dependence of drug-induced parasite growth retardation on drug concentration
Fig. 5 presents the results of fitting the 5-parameter model to the SYTO-61 fluorescence data for ART (the best-fit parameter values are provided in Table S1 in the Supplementary material). The model correctly captures the ART-dependent change of shape in SYTO-61 fluorescence histograms — increasing ART concentration leads to an increase in the ring population (i.e. the mode with the lower fluorescence) at the expense of a decreased trophozoite population (i.e. the mode with the higher
Discussion
In this paper, we have studied ART-induced parasite growth retardation using a mechanistic model that considers the ring-to-trophozoite transition to be a two-stage process and exploits the differing rates of nucleic acid production (measured through SYTO-61 fluorescence intensity) in those two life stages. By fitting the model to fluorescence histogram data for the laboratory 3D7 strain, we have been able to identify the dependence of growth retardation on applied drug concentration Figs. 6A
Declaration of interest
We have no conflict of interest to declare.
Author contributions
PC, NK, MPD, LT, JAS and JMM conceived the study. NK and LT performed the experiment. PC, NK, SZ analysed the data. PC, SZ, DSK and DC developed the mathematical model. PC and JMM drafted the manuscript. All authors reviewed and edited the manuscript and gave final approval for publication.
Acknowledgements
The work was supported by the National Health and Medical Research Council of Australia [grant numbers 1100394, 1060357]; the Centre for Research Excellence ViCBiostat [grant number 1035261]; and the Centre for Research Excellence PRISM2 [grant number 1078068]. James M. McCaw was supported by an Australian Research Council Future Fellowship [grant number FT110100250]. Julie A. Simpson was supported by a National Health and Medical Research Council of Australia Senior Research Fellowship [grant
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