Table 3 In vitro drug sensitivity assays developed for the determination of anti-schistosomula activity of drug compounds
Methods | Principles | Advantages | Disadvantages |
|---|---|---|---|
Microscope readouts without staining | Parasites are manipulated in vitro and the effect is assessed by bright field examination of morphology. Schistosomula viability is assessed using different criteria: - intracellular granularity - schistosomula movement - schistosomula shape alterations This makes use of a viability scale from 0 to 3 | - Used to discriminate between live and dead schistosomula after incubation | - The personnel should be well trained to distinguish diverse schistosomula phenotypes - The bright-field, light microscopic detection of schistosomula viability is subjective due to lack of immunological and molecular evidence that death has actually occurred when a schistosomula is immobile - The technique is slow (time consuming) and tedious - Replication of results is not always possible, because of the absence of uniformity between laboratories |
Microscope readouts with Staining using a single fluorescent dye | Parasites are manipulated in vitro and the effect is assessed by bright field examination of viable or dead cells | - Used to discriminate between live and dead schistosomula after incubation - Does not require extensive training of personnel in parasite morphology - The fluorescent bioassay is objective - Fluorescent bioassay can be adapted for use with adult worm schistosomes as well as other life stages | - Cannot be used for earlier time-points - Cannot be used to measure dose response drug effects - It is sometime difficult to clearly differentiate live schistosomula from dead ones |
Microscope readouts with Staining using dual fluorescent viability assay | Combination of the use of DNA intercalating dyes (ethidium bromide (EB), Propidium Iodide (PI)) with Carboxyfluorescein (fluorescein diacetate), Resazurin to easily assess the percentage of viable schistosomula present in a sample | - This bioassay was developed for 96 or 384 well microtiter plate optically clear - Designed for medium throughput (96 well) and high throughput (384 well) applications - Increase by 10-fold the number of compounds screened per month over existing microscopy methodologies - Does not require extensive training of personnel in parasite morphology - The fluorescent bioassay objectively and rapidly quantify schistosomula viability in a high-throughput format - Fluorescent bioassay can be adapted for use with adult worm schistosomes as well as other life stages | - The ability of the dual fluorescent dye to provide significant phenotypic data is slightly limited |
Motility assay | The assay uses the xCELLigence system for monitoring cells in a real-time manner. This technique is based on the detection of changing electrical currents running through mini gold electrodes incorporated into the bottom of tissue culture plates | - Simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion - Help remove subjectivity in helminth phenotype characterization - Results can be compared directly from different laboratories | - The xCELLigence equipment used in this technology is costly and may restrict its applicability |
Fluorometric L-lactate assay | Consist of the measurement of lactate levels that reflect clearly the viability of schistosomula and this also correlate with schistosomula numbers | - Can be used as simple surrogate marker - Promising new approach to assess the viability of schistosomula in drug sensitivity assays | - This technique requires that the supernatant must be removed from the drug assay without aspirating the schistosomula and the drug assay should be diluted to an acceptable fluorescence range as needed. These make the fluorometric L-lactate assay less than high-throughput |
CellTiterGlo® (Commercial luminescence-based cell viability kit) | Detection of schistosomula viability through quantitation of ATP | - Suitable for a Medium-Throughput Assay semi-automated for drug screening - Fast, highly reliable, sensitive and automation friendly | - Require a precise multi-drop dispenser to ensure an exact number of NTS present in each well |