Abstract
The in vivo efficacy of potential antimalarials is usually evaluated by direct microscopic determination of the parasitaemia of Plasmodium-infected mice on Giemsa-stained blood smears. This process is time-consuming, requires experienced technicians and is not automatable. Therefore, we optimized a SYBR Green I (SYBRG I) fluorescence-based assay to fluorometers commonly available in many research laboratories. This technique was originally developed to assess parasitaemia in humans by cytometry. We defined optimal conditions with Plasmodium berghei-infected mice, standard lysis buffer (Tris, EDTA, saponin and Triton), whole blood cells and 2 h staining incubation with SYBRG I 2X. The fluorescence background generated by uninfected whole blood cells was low (around 4.6%), and the linearity high (r 2 = 0.96), with parasitaemia ranging from 1.4 to 60%. The Bland–Altman plot showed a strong correlation between SYBRG I and Giemsa gold standard method; Z′-factor was >0.5. These findings suggest that our fluorescence-based assay is suitable for in vivo antimalarial drug assessment in a malaria murine model. It can help to overcome the human bias found with microscopic techniques.
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Acknowledgements
We are grateful to Elizabeth Elliott for valuable editorial assistance. This study was funded by the Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias) and the French Ministère des Affaires étrangères et du Développement international (MAEE) through ECOSNORD 2013 program [HERMES-28693] and the Vicedecanatura de Investigación, Facultad de Ciencias, Universidad Nacional de Colombia.
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Arias, M.H., Deharo, E., Valentin, A. et al. Adaptation and optimization of a fluorescence-based assay for in vivo antimalarial drug screening. Parasitol Res 116, 1955–1962 (2017). https://doi.org/10.1007/s00436-017-5477-z
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DOI: https://doi.org/10.1007/s00436-017-5477-z