Multiplication rate variation in the human malaria parasite Plasmodium falciparum

Abstract

It is important to understand intrinsic variation in asexual blood stage multiplication rates of the most virulent human malaria parasite, Plasmodium falciparum. Here, multiplication rates of long-term laboratory adapted parasite clones and new clinical isolates were measured, using a newly standardised assay of growth from low starting density in replicate parallel cultures with erythrocytes from multiple different donors, across multiple cycles. Multiplication rates of long-term established clones were between 7.6 and 10.5 fold per 48 hours, with clone Dd2 having a higher rate than others (clones 3D7, HB3 and D10). Parasite clone-specific growth was then analysed in co-culture assays with all possible heterologous pairwise combinations. This showed that co-culture of different parasites did not affect their replication rates, indicating that there were no suppressive interactions operating between parasites. Multiplication rates of eleven new clinical isolates were measured after a few weeks of culture, and showed a spectrum of replication rates between 2.3 and 6.0 fold per 48 hours, the entire range being lower than for the long-term laboratory adapted clones. Multiplication rate estimates remained stable over time for several isolates tested repeatedly up to three months after culture initiation, indicating considerable persistence of this important trait variation.

Figure 1

Measurement of exponential multiplication rates of laboratory adapted clones of P. falciparum. (A) 6-day growth assays for four laboratory clones, showing parasite density as numbers of genome copies per microlitre (on a log₁₀ scale). Six biological replicates were performed using different donor erythrocytes. Estimation of parasite copy numbers by quantitative PCR was confirmed to be highly correlated with counts of parasites by microscopy of Giemsa-stained slides (Supplementary Figure S1). ‘3D7’ here refers to the genetically modified clone 3D7-HT-GFP whereas the other clones were not genetically modified. (B) General linear model estimates of the replication rates per 48 hours over the 6-day assays for each of the four laboratory clones (point estimates with 95% confidence intervals).

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^, . (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-GFP. 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).

Figure 3

Parasite multiplication rates (with 95% confidence intervals) of new P. falciparum clinical isolates, sampled from patients in three West African countries (GUI, Guinea; SEN, Senegal, MAL, Mali). (A) Eleven isolates were analysed after growth in culture for at least two weeks (between 16 and 32 days) prior to assay. Exponential growth was measured over 4 days using a general linear model approach. All assays were conducted with biological triplicates in separate flasks with different donor erythrocytes, except for SEN232 which was assayed in duplicate. Six of the isolates (SEN232, GUI246, MAL247, GUI249, MAL250, SEN251) were assayed in medium supplemented by Albumax and serum, whereas the other five were assayed by medium with Albumax alone as noted in the Materials and Methods (results were similar for the two groups, Mann-Whitney test P = 0.90). As a control, the laboratory adapted clone 3D7-HT-GFP (here labelled ‘3D7′) was tested in parallel in each assay, yielding a consistent multiplication rate of approximately 8 fold per 48 hours. (B) Four of the clinical isolates cultured for longer periods were assayed at three different time points after culture initiation (up to 76 or 100 days for each isolate). These showed no significant changes in multiplication rate over time in culture. Assays were conducted with biological triplicates, except for the last cultured time point for which the assay was conducted in duplicate.