Most of our knowledge about the molecular and cellular biology of malaria parasites is based on “lab strains”, particularly a small number of Plasmodium falciparum isolates that have been cultured in vitro for years. Using the same clonal populations in laboratories all around the world helps with reproducibility and comparability of studies, for example when testing novel anti-malarial drugs. However, how lab strains differ from clinical isolates has been surprisingly poorly investigated, and there is a risk that new understanding and treatments or vaccines being developed may be skewed towards lab strains.
We previously showed that adaptation to in vitro culture may lead to acquisition of premature stop codons in transcription factors and other key genes1. The new and larger study we present here characterises the phenotypes of a larger set of clinical isolates that were cultured continuously for 5 months (Fig 1). At regular intervals, we performed whole genome sequencing and measured the parasite multiplication rate in an exponential growth assay2. The multiplication rates varied substantially among the isolates but most increased over time, indicating that strains adapt to their in vitro environment, although premature stop codon mutants only emerged in a minority of the isolates during culture. Very importantly, the multiplication rates at all times correlated with the initial parasitaemia levels in the patient on the day the blood sample was collected. In other words, an isolate derived from a patient with relatively high parasitaemia will multiply in culture on average faster than an isolate from lower parasitaemia, a trend still observed after 5 months of culture adaptation (Fig 2).
The parasite multiplication rate is theoretically an important determinant of virulence, and here we show that for P. falciparum this is at least partly intrinsic to each strain. Such initial variation occurring in natural infections persists in vitro even while culture adaptation proceeds. This discovery encourages different areas of research: What is the main cellular mechanism of the multiplication rate variation? Here we found only limited variation in the number of merozoites produced per multiplication cycle, but the invasion efficiency remains to be measured, and the multiplication cycle duration also needs to be investigated as potentially important. Larger studies on the multiplication rate phenotypes of clinical isolates will be needed to address to what extent the variation is encoded in parasite genomic differences, or due to epigenetic regulation.
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Figure 1. Parasite multiplication rates in a panel of 24 new Ghanaian P. falciparum clinical isolates over five months of continuous culture. Multiplication rates of the clinical isolates increased over time, with a mean of 5.1-fold after 77 days and 6.4-fold after 153 days (Mann-Whitney test).
Figure 2. Parasite multiplication rates correlate significantly with levels of parasitaemia measured in patients. Univariate significant correlation with patient parasitaemia levels for multiplication rates assayed after 153 days of culture.
- Claessens, A., Affara, M., Assefa, S. A., Kwiatkowski, D. P. & Conway, D. J. Culture adaptation of malaria parasites selects for convergent loss-of-function mutants. Sci. Rep. 7, 41303 (2017).
- Murray, L. et al. Multiplication rate variation in the human malaria parasite Plasmodium falciparum. Sci. Rep. 7, 6436 (2017).