Performance and consistency of a fluorescence-based high-throughput screening assay for use in Babesia drug screening in mice

Mohamed Abdo Rizk,Ikuo Igarashi,Rasha Eltaysh,Mahmoud Aboulaila,Shimaa Abd El-Salam El-Sayed,Naoaki Yokoyama

doi:10.1038/s41598-017-13052-5

Abstract

In this study, we evaluated the validity of a fluorescence-based assay using SYBR Green I (SG I) stain for screening antibabesial compounds against B. microti in mice. Two different hematocrits (HCTs; 2.5% and 5%) were used. Correlating relative fluorescence units (RFUs) with parasitemia showed significant linear relationships with R2 values of 0.97 and 0.99 at HCTs of 2.5% and 5%, respectively. Meanwhile, the Z′ factors in a high-throughput screening (HTS) assay were within the permissible limit (≥0.5) at 2.5% HCT and lower than this value at 5% HCT. Taken together, the highest signal-to-noise (S/N) ratios were obtained at 2.5% HCT; therefore, we concluded that 2.5% was the best HCT for applying fluorescence assay in antibabesial drug screening in mice. Additionally, positive control mice and those treated with diminazene aceturate, pyronaridine tetraphosphate, and an allicin/diminazene aceturate combination showed peak parasitemia and fluorescence values on the same day post-inoculation. Moreover, using different concentrations of SG I revealed that the optimal concentration was 2x. In summary, considering that all experiments were applied under optimal laboratory conditions, fluorescence assay at 2.5% HCT using 2x SG I for B. microti parasite offers a novel approach for drug screening in mice.

Highlights

Babesiosis is a tick-borne disease of great economic importance in the animal industry
Significant linear relationships between relative fluorescence units (RFUs) and B. microti parasitemia were obtained with both HCTs (Fig. 1)
High-throughput screening (HTS) assay parameters inclusive of Z′ factor, S/N ratio, % CVmax, and % CVmin were calculated at both HCTs (2.5% and 5%) on days with peak of parasitemia to appraise of the assay quality

Summary

Introduction

Babesiosis is a tick-borne disease of great economic importance in the animal industry. The disease is caused by intraerythrocytic protozoan parasites of the genus Babesia, which affects animals and humans worldwide[1]. Our laboratory established a novel Babesia fluorescence assay (BFA) for large-scale drug screening against Babesia and Theileria parasites in vitro[9,10]. The assay depends on using SYBR Green I (SG I) stain, which binds to the double-stranded DNA of parasites[11] The validation of this assay depends on the statistical parameters of high-throughput screening assay (HTS), including Z′ factor (Z′), signal-to-noise (S/N) ratio, coefficient of variation at the maximum signal (% CVmax, positive control), and coefficient of variation at the minimum signal (% CVmin, negative control)[11]. In this study, we evaluated the validation of BFA assay for drug screening against the growth of B. microti in specific pathogen-free mice. Two antibabesial drugs, including diminazene aceturate (DA) and pyronaridine tetraphosphate (PYR), were used in this study

Methods

Results

Conclusion