Evaluation of a fluorescence-based method for antibabesial drug screening

Area of Science:

• Parasitology
• Biochemistry
• Drug Discovery

Background:

• In vitro drug screening against Babesia and Theileria parasites relies on parasitemia measurement.
• Microscopy is the standard method for parasitemia assessment but has limitations.
• Developing alternative, efficient screening methods is crucial for antiparasitic drug discovery.

Purpose of the Study:

• To evaluate a fluorescence-based method using SYBR green I stain for in vitro antibabesial drug screening.
• To compare the accuracy and reliability of the fluorescence method against traditional microscopy.
• To assess the potential of this fluorescence assay for high-throughput screening (HTS) of antiprotozoan agents.

Main Methods:

• In vitro cultures of Babesia bovis were used to evaluate the fluorescence assay.
• SYBR green I stain (SG I) was employed to measure parasite load via relative fluorescence units (RFU).
• The linearity between RFU and parasitemia was determined, and IC50 values for three drugs were calculated and compared to microscopy.

Main Results:

• A strong linear correlation (r(2) = 0.9944) was observed between RFU and parasitemia.
• IC50 values for diminazene aceturate and nimbolide determined by fluorescence closely matched microscopy-based results.
• The fluorescence method demonstrated robust assay quality metrics (Z' factor, S/N ratio, %CV) comparable to malaria research assays.

Conclusions:

• The fluorescence-based method using SYBR green I is a reliable and accurate tool for in vitro antibabesial drug screening.
• This method provides a viable alternative to microscopy, with potential for adaptation into a high-throughput screening assay.
• The assay's performance suggests its utility in accelerating the discovery of new treatments for babesiosis.