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

Parasitology Research
Maria H AriasGiovanny Garavito

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.

References

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Citations

Apr 5, 2020·Marine Drugs·María Álvarez-BardónRafael Balaña-Fouce

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Methods Mentioned

BETA
fluorescence assay
Fluorescence
light microscopy
Assay
ELISA
flow cytometry

Software Mentioned

Excel®

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