Figure 2

Quantitation of parasite multiplication rates for four laboratory clones across three intraerythrocytic cycles for all pairwise co-cultured combinations. (A) Allele-specificity of qPCR assays for clone-specific parasite density measurement. Three different allele-specific sets of assays were used to analyse all pairwise combinations of four P. falciparum laboratory clones. Each assay was first evaluated in a comparison of observed versus expected ratios for a pair of heterologous clones. The assay shown here is described in this paper, and discriminates msp6 alleles to accurately quantify Dd2 and D10 DNA mixed at different ratios from 1:99 to 99:1. Two different assays to discriminate msp1 alleles, used to quantify parasite DNA in other heterologous mixtures (Supplementary Figure S2), have been previously described^{21, 22}. (B) Comparison of ratios of different parasite clones in mixtures assayed by fluorescence microscopy and allele-specific qPCR. There is a high correlation between the independent measurements (r ² = 0.984) across a range of ratios from 1:20 to 20:1 for the Dd2-GFP and D10-mKate2 clonal mixtures. (C) P. falciparum clone-specific parasite multiplication rates (with 95% confidence intervals) are similar in monoculture and pairwise co-cultured combinations. ‘3D7′ here refers to the genetically modified clone 3D7-HT-GFP³⁵. The allele-specific qPCR assays of parasite densities (at days 0, 2, 4 and 6) yield estimates of clone-specific growth per 48 hour period by general linear modelling of exponential growth over six days (values are listed in Supplementary Table S1).