Authors

Discussion

Artemisinin remains effective as the first line drug; however, according to epidemiological studies in Southeast Asia, delay clearance of the parasitized erythrocytes has been documented, emphasizing artemisinin resistance surveillance. Under a light microscope, artemisinin-exposed Plasmodium falcipalum form a dead-like morphology but capable of reentering the intraerythrocytic growth, establishing a hypothesis of dormant parasites. However, the existence of dormant form of P. falciparum in erythrocytes remain controversial in part due to morphological similarity between dormant and pyknotic parasites. A lack of tools to discriminate both stages further limit our ability to monitor the dormancy of P. falciparum. Here, we demonstrated the efficacy of a new laboratory assay that is compatible with the artemisinin susceptibility test. This research brings a concept of cell permeability to develop a trio fluorochrome alternative to the ring-stage survival assay (RSA). As a result of the use of two different types of DNA-binding fluorophores, the parasite membrane loss selective activity after artemisinin exposure, allowing enter of cell impermeant fluorophore. By contrast, the membrane of the infected erythrocyte at the ring-stage retains a selective cell membrane regardless of the parasite survival status. Together with mild detergent, we could identify nonviable parasites would lose such selective membrane properties similar to the process of cell apoptosis or necrosis. This approach clearly confirmed that the dormant parasites retained an intact membrane and resumed growth within 6 days, whereas those that lost the parasite membrane failed to regrow, ensuring an indicator for truly dormant parasites. Therefore, the use of this proposed method effectively discriminated between dormant and dead parasites in an RSA. In conclusion, this assay is a simple and rapid technique which makes a significant contribution to advancing diagnosis and treatment of malaria.