Adaptation and optimization of a fluorescence-based assay for in vivo antimalarial drug screening.

Arias, Maria; Deharo, Eric; Valentin, Alexis; Garavito, Giovanny

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 = 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.

FLUORESCENCE; ANTIMALARIALS; DRUG use testing; PLASMODIUM; ANIMAL models in research; DRUG efficacy

Parasitology Research, 2017, Vol 116, Issue 7, p1955

0932-0113

Academic Journal

10.1007/s00436-017-5477-z