A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility

Abstract

Background: Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods.

Methodology/principal findings: Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC(50) values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and -resistant isolates of H. contortus.

Conclusions/significance: Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.

Figure 1. RTCA unit differentiates between live and dead parasites from different developmental stages using the cell index readout.

Panel A: Micrograph of adult Ancylostoma caninum hookworms - females in the top two wells and magnified image on the left, and males in the bottom two wells. Note the gold circular electrodes covering the base of the E-Plate in the magnified image. Panel B: Cell index output generated by a single adult female A. caninum with and without exposure to thiabendazole (TBZ)*. Panel C: Haemonchus contortus L₃ cell index output in the presence of varying amounts of levamisole (LEVA)*. Panel D: H. contortus egg hatching in the presence of varying amounts of TBZ - curves show the average of duplicate experiments. Note that increasing drug concentrations result in less egg hatching and a corresponding lesser cell index output. * The cell index numerical value is not relevant to this analysis - the curves have been manually repositioned to assist with visualization of the data. The amplitude within each curve is the important feature of the data for this experiment.

Figure 2. Motility Index of Schistosoma mansoni paired adult worms generated from the cell index output.

The Motility Index is relative to the amplitude of the cell index curve. Panel A: Drug dilution series with praziquantel (PZQ); each curve is an average of minimum 3 experiments, error bars not shown to enhance clarity. Panel B: PZQ dose response curves used to generate IC₅₀ values generated from Motility Index analysis.

Figure 3. Real time IC₅₀ curves with 95% confidence interval error bars for a range of developmental stages of different helminths.

Panel A: Schistosoma mansoni paired adult worms with praziquantel (PZQ). Panel B: Adult female Ancylostoma caninum hookworms with TBZ magnified to aid visualisation; inset shows the entire data set. Panel C: Haemonchus contortus eggs with TBZ.

Figure 4. IC₅₀ values from RTCA unit can differentiate between LEVA-resistant and -sensitive lines of H. contortus L₃.

Panels A and B: Motility Index with selected LEVA concentrations, resistant and sensitive lines respectively. Curves are means of triplicate experiments. Error bars not shown for clarity of the figure. Panel C: Real time IC₅₀ curves of LEVA-resistant and -sensitive lines with 95% confidence interval error bars. Panel D: Real time IC₅₀ curves of IVM-resistant and -sensitive lines with 95% confidence interval error bars magnified to aid visualisation; inset shows the entire data set. * P<0.05, ** P<0.01, *** P<0.001.