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

Tritten, Lucienne; Braissant, Olivier; Keiser, Jennifer

doi:10.1017/s0031182011001934

Cited by 27 publications

(36 citation statements)

References 43 publications

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“…The feeding inhibition assay has been used to analyze the feeding behavior of Ancylostoma spp. larvae [15-17]. Viable parasites might ingest medium containing the fluorescent molecule (bovine albumin conjugated to fluorescein), which allows the discrimination from dead parasites by microscopical observation of the stained intestinal tract of the worms [12,17].…”

Section: Introductionmentioning

confidence: 99%

Development of an in vitro drug sensitivity assay for Trichuris muris first-stage larvae

2013

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BackgroundTrichuriasis represents a major public health problem in the developing world and is regarded as a neglected disease. Albendazole and mebendazole, the two drugs of choice against trichuriasis display only moderate cure rates, hence alternative drugs are needed. To identify candidate compounds, in vitro drug sensitivity testing currently relies on the adult Trichuris muris motility assay. The objective of the present study was to develop a simple and cost-effective drug sensitivity assay using Trichuris muris first-stage larvae (L1).MethodsSeveral potential triggers that induce hatching of T. muris were studied, including gastrointestinal enzymes, acidic environment and intestinal microflora. Next, optimal culture conditions for T. muris L1 were determined assessing a wide range of culture media. T. muris L1 were incubated in the presence of mebendazole, ivermectin, nitazoxanide, levamisole or oxantel pamoate at 37°C. The viability of the parasites was evaluated microscopically after 24 hours. The usefulness of fluorescent markers (resazurin, calcein AM, ethidium homodimer-1 or fluorescein-conjugated albumin) in drug sensitivity testing was also assessed.ResultsThe established L1 motility assay provided accurate and reproducible drug effect data in vitro. IC50 values for oxantel pamoate, levamisole and nitazoxanide were 0.05, 1.75 and 4.43 μg/mL, respectively. Mebendazole and ivermectin failed to show any trichuricidal effect on L1. No correlation was found between data from the four fluorescent markers and the comparative motility assay.ConclusionsThe motility assay based on L1 was found suitable for drug sensitivity screening. It is rather simple, cost-effective, time-saving and sustains medium-throughput testing. Furthermore, it greatly reduces the need for the animal host and is therefore more ethical. None of the viability markers assessed in this study were found to be satisfactory.

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Section: Introductionmentioning

confidence: 99%

Development of an in vitro drug sensitivity assay for Trichuris muris first-stage larvae

2013

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“…[10][11][12][13][14][15][16] Therefore, the correlation between the anthelmintic activity of compounds against laboratory and field isolates of human hookworms remains largely unknown.…”

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“…[6][7][8][9] However, novel compounds identified using small animal models of hookworm infection are rarely screened for comparable activity against field isolates of human hookworms. [10][11][12][13][14][15][16] Therefore, the correlation between the anthelmintic activity of compounds against laboratory and field isolates of human hookworms remains largely unknown.…”

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confidence: 99%

In vitro Screening of Compounds against Laboratory and Field Isolates of Human Hookworm Reveals Quantitative Differences in Anthelmintic Susceptibility

Treger

Otchere

Keil

et al. 2014

The American Society of Tropical Medicine and Hygiene

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Abstract. A panel of 80 compounds was screened for anthelmintic activity against a laboratory strain of Ancylostoma ceylanicum and field isolates of hookworm obtained from school children in the Kintampo North District of the Brong Ahafo Region of Ghana. Although the laboratory strain of A. ceylanicum was more susceptible to the compounds tested than the field isolates of hookworm, a twofold increase in compound concentration resulted in comparable egg hatch percent inhibition for select compounds. These data provide evidence that the efficacy of anthelmintic compounds may be species-dependent and that field and laboratory strains of hookworm differ in their sensitivities to the anthelmintics tested. These data also suggest that both compound concentration and hookworm species must be considered when screening to identify novel anthelmintic compounds.Human hookworm disease results from infection by two genera of hookworms, Ancylostoma spp. and Necator americanus. Albendazole (ABZ) and mebendazole (MBZ) are the primary drugs used to treat hookworm infection. A recent meta-analysis and review of field-based studies have shown that single-dose treatment cure rates with ABZ and MBZ are low (72% and 15%, respectively).1,2 Compounding these treatment failure rates in humans is the well-documented emergence of drug resistance to ABZ and MBZ among parasitic nematodes of agricultural and veterinary importance. [3][4][5] In light of this information, the development of novel anthelmintic chemotherapeutics is necessary.Currently, the identification and development of novel anthelmintic compounds to cure hookworm infection rely on laboratory-based screening for inhibitors of egg hatching, larval motility and morphology, and/or adult worm survival.6-9 However, novel compounds identified using small animal models of hookworm infection are rarely screened for comparable activity against field isolates of human hookworms. [10][11][12][13][14][15][16] Therefore, the correlation between the anthelmintic activity of compounds against laboratory and field isolates of human hookworms remains largely unknown.Moreover, for those cases in which anthelmintic activity is investigated, the larvicidal, rather than ovicidal, properties of a compound are usually analyzed. [17][18][19][20] Ovicidal activity is more frequently and most readily assayed in resource-limited field settings, where larval and adult hookworm-based assays are impractical tools for assessing anthelmintic activity. [21][22][23]

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“…After entering a suitable host, iL3s shed their enveloping cuticle (‘exsheathment’) and commence feeding. Exposure to suitable conditions in vitro (e.g., serum components, glutathione analogs, elevated temperature and CO 2 ) can ‘activate’ iL3s to transform into host-stage L3s and initiate feeding (e.g., Hawdon and Schad, 1990, Tritten et al., 2012). Activated L3s are henceforth termed aL3s.…”

Section: Resultsmentioning

confidence: 99%

Microfluidic platform for electrophysiological recordings from host-stage hookworm and Ascaris suum larvae: A new tool for anthelmintic research

Weeks

Roberts

Robinson

et al. 2016

International Journal for Parasitology: Drugs and Drug Resistan

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The screening of candidate compounds and natural products for anthelmintic activity is important for discovering new drugs against human and animal parasites. We previously validated in Caenorhabditis elegans a microfluidic device (‘chip’) that records non-invasively the tiny electrophysiological signals generated by rhythmic contraction (pumping) of the worm's pharynx. These electropharyngeograms (EPGs) are recorded simultaneously from multiple worms per chip, providing a medium-throughput readout of muscular and neural activity that is especially useful for compounds targeting neurotransmitter receptors and ion channels. Microfluidic technologies have transformed C. elegans research and the goal of the current study was to validate hookworm and Ascaris suum host-stage larvae in the microfluidic EPG platform. Ancylostoma ceylanicum and A. caninum infective L3s (iL3s) that had been activated in vitro generally produced erratic EPG activity under the conditions tested. In contrast, A. ceylanicum L4s recovered from hamsters exhibited robust, sustained EPG activity, consisting of three waveforms: (1) conventional pumps as seen in other nematodes; (2) rapid voltage deflections, associated with irregular contractions of the esophagus and openings of the esophogeal-intestinal valve (termed a ‘flutter’); and (3) hybrid waveforms, which we classified as pumps. For data analysis, pumps and flutters were combined and termed EPG ‘events.’ EPG waveform identification and analysis were performed semi-automatically using custom-designed software. The neuromodulator serotonin (5-hydroxytryptamine; 5HT) increased EPG event frequency in A. ceylanicum L4s at an optimal concentration of 0.5 mM. The anthelmintic drug ivermectin (IVM) inhibited EPG activity in a concentration-dependent manner. EPGs from A. suum L3s recovered from pig lungs exhibited robust pharyngeal pumping in 1 mM 5HT, which was inhibited by IVM. These experiments validate the use of A. ceylanicum L4s and A. suum L3s with the microfluidic EPG platform, providing a new tool for screening anthelmintic candidates or investigating parasitic nematode feeding behavior.

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

Cited by 27 publications

References 43 publications

Development of an in vitro drug sensitivity assay for Trichuris muris first-stage larvae

Development of an in vitro drug sensitivity assay for Trichuris muris first-stage larvae

In vitro Screening of Compounds against Laboratory and Field Isolates of Human Hookworm Reveals Quantitative Differences in Anthelmintic Susceptibility

Microfluidic platform for electrophysiological recordings from host-stage hookworm and Ascaris suum larvae: A new tool for anthelmintic research

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