Comparison of the susceptibility of Plasmodium knowlesi and Plasmodium falciparum to antimalarial agents

Abstract

Background: The simian malaria parasite Plasmodium knowlesi is now a well-recognized pathogen of humans in South-East Asia. Clinical infections appear adequately treated with existing drug regimens, but the evidence base for this practice remains weak. The availability of P. knowlesi cultures adapted to continuous propagation in human erythrocytes enables specific studies of in vitro susceptibility of the species to antimalarial agents, and could provide a surrogate system for testing investigational compounds against Plasmodium vivax and other non-Plasmodium falciparum infections that cannot currently be propagated in vitro.

Objectives: We sought to optimize protocols for in vitro susceptibility testing of P. knowlesi and to contrast outputs with those obtained for P. falciparum under comparable test conditions.

Methods: Growth monitoring of P. knowlesi in vitro was by DNA quantification using a SYBR Green fluorescent assay or by colorimetric detection of the lactate dehydrogenase enzyme. For comparison, P. falciparum was tested under conditions identical to those used for P. knowlesi.

Results: The SYBR Green I assay proved the most robust format over one (27 h) or two (54 h) P. knowlesi life cycles. Unexpectedly, P. knowlesi displays significantly greater susceptibility to the dihydrofolate reductase inhibitors pyrimethamine, cycloguanil and trimethoprim than does P. falciparum, but is less susceptible to the selective agents blasticidin and DSM1 used in parasite transfections. Inhibitors of dihydroorotate dehydrogenase also demonstrate lower activity against P. knowlesi.

Conclusions: The fluorescent assay system validated here identified species-specific P. knowlesi drug susceptibility profiles and can be used for testing investigational compounds for activity against non-P. falciparum malaria.

Figure 1.

Influence of starting parasitaemia of P. knowlesi (A1-H.1) and P. falciparum (3D7) on assay quality for both the fluorescent and colorimetric methods. Parasites set to 1% haematocrit and varying parasitaemia (0.1%–2%) were cultured in the presence or absence of a supralethal concentration of chloroquine for 27 h (circles), 54 h (squares) or 81 h (diamonds) for P. knowlesi, and 48 h (cirlces) or 96 h (squares) for P. falciparum. Upon termination of the assay, the plates were read using either the SYBR Green I fluorescence assay (a, c, e and g) or the LDH assay (b, d, f and h). The signal window and Z′ factor were calculated for each assay. The signal window was calculated by dividing the average reading for the drug-free control by the average reading for the high chloroquine concentration (background) control. The assay quality was assessed by determining the Z′ factor using the formula described in Zhang et al.

Figure 2.

Effect of synchrony on drug susceptibility measured across the life cycle using the SYBR Green I method after one or two life cycles. EC₅₀ values for chloroquine (squares), dihydroartemisinin (circles) or pyrimethamine (diamonds) were determined from experiments initiated at the times shown on the y-axis using synchronized parasites at 1% parasitemia and 1% haematocrit that were incubated for either 27 h (a) or 54 h (b) for P. knowlesi, and 48 h (c) or 96 h (d) for P. falciparum. Signal window (squares) and assay quality (Z′ factor, circles) (e–h) were determined as in Figure 1.