Quantification of malaria parasite release from infected erythrocytes: inhibition by protein-free media

BACKGROUND: Intracellular malaria parasites leave their host erythrocytes to infect neighbouring cells after each cycle of asexual replication. No method is currently available for the direct quantification of parasite release. METHOD AND RESULTS: To quantify parasite release process, human erythrocytes infected with Plasmodium falciparum were injected into sealed chambers at optimal density, where they progressed through the end of the erythrocyte cycle. Each event of parasite release inside the chamber at the site of erythrocyte rupture leaves on the chamber wall a footprint, composed of 1) separated parasites, 2) a digestive vacuole with haemozoin, and 3) fragments of the ruptured membranes. These footprints are stable for hours, allowing precise identification using differential interference contrast (DIC) microscopy. The relative rate of parasite release is defined as the percent of such footprints out of all schizonts injected and incubated into chamber at 37°C for two hours. The method is highly reproducible, easy to perform, and does not require expensive equipment. Additionally, this method allows one to analyse cell and release site morphology, which adds information about the release process and the quality of the culture. The method is used here to show that swelling of schizonts caused by protein-free media inhibits parasite release. CONCLUSION: In this study, a novel method is described to count sites of parasite release by microscopy. Besides the direct estimation of parasite release from infected erythrocytes, this method provides a morphological evaluation of normal infected cells approaching the end of the plasmodial life cycle, or pathological forms accumulated as the result of experimental intervention in the parasite release process. One may now accurately estimate the relative parasite release rate at the time of cycle transition, without any obligatory coupling to parasite invasion.