A Novel High Throughput Assay for Anthelmintic Drug Screening and Resistance Diagnosis by Real-Time Monitoring of Parasite Motility

Abstract

BackgroundHelminth 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 FindingsHere 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 IC50 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/SignificanceOur 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.

In vitro anthelmintic activity of the essential oils of Zanthoxylum zanthoxyloides and Newbouldia laevis against Strongyloides ratti

“…The use of this laboratory model (S. ratti) allowed for an easy source of nematodes (larvae and eggs) and limited the size of the animal facilities. The use of S. ratti as a model could serve as an approach for evaluating plant extracts that are potentially useful for circumventing human helminth infection (Smout et al 2010). In our model, the two EOs that were tested had efficiency close to that of the positive control molecules: thiabendazole and levamisole.…”

In Vitro Efficacy of Moxidectin versus Ivermectin against Sarcoptes scabiei

“…The LC 50 for ivermectin at 24 h (1.8 M) is similar to that for a Cooperia sp. (1.7 M), Haemonchus contortus, and Strongyloides ratti (ϳ1.14 M) (25)(26)(27), although in the last two nematodes, a high-throughput motility assay demonstrated lower IC 50 s (Ͻ0.34 M) (28). Where the filarial nematodes Brugia malayi and Dirofilaria immitis are concerned, the ivermectin IC 50 s ranged from 4.6 to 28.2 M, depending on life stage (27,29).…”

Comparison of novel and existing tools for studying drug sensitivity against the hookworm Ancylostoma ceylanicum in vitro

“…Death was determined in 3-7 wells for each treatment. The repartition of death time points is shown by the proportion and analysed using the Fisher's exact test ( (Smout et al 2010), which is a great disadvantage for medium-to high-throughput assays. The cost of the xCELLigence apparatus and the E-plates might also be a drawback.…”

“…An innovative application of the system for nematode worms was recently described, monitoring motilities of Haemonchus contortus, Strongyloides ratti, Ancylostoma caninum and Schistosoma mansoni (Smout et al 2010). Measurements were displayed in the RTCA controller software (Roche Inc.) The xCELLigence System was equilibrated either to room-temperature or to 37°C for 2 hours before the experiment was started.…”