The Potential Impact of Density Dependent Fecundity on the Use of the Faecal Egg Count Reduction Test for Detecting Drug Resistance in Human Hookworms

Current efforts to control human soil-transmitted helminth (STH) infections involve the periodic mass treatment of people, particularly children, in all endemic areas, using benzimidazole and imidothiazole drugs. Given the fact that high levels of resistance have developed to these same drugs in roundworms of livestock, there is a need to monitor drug efficacy in human STHs. The faecal egg count reduction test (FECRT), in which faecal egg output is measured pre- and post-drug treatment, is presently under examination by WHO as a means of detecting the emergence of resistance. We have examined the potential impact of density dependent fecundity on FECRT data. Recent evidence with the canine hookworm indicates that the density dependent egg production phenomenon shows dynamic properties in response to drug treatment. This will impact on measurements of drug efficacy, and hence drug resistance. It is likely that the female worms that survive a FECRT drug treatment in some human cases will respond to the relaxation of density dependent constraints on egg production by increasing their egg output significantly compared to their pre-treatment levels. These cases will therefore underestimate drug efficacy in the FECRT. The degree of underestimation will depend on the ability of the worms within particular hosts to increase their egg output, which will in turn depend on the extent to which their egg output is constrained prior to the drug treatment. As worms within different human cases will likely be present at quite different densities prior to a proposed FECRT, there is potential for the effects of this phenomenon on drug efficacy measurements to vary considerably within any group of potential FECRT candidates. Measurement of relative drug efficacy may be improved by attempting to ensure a consistent degree of underestimation in groups of people involved in separate FECRTs. This may be partly achieved by omission of cases with the heaviest infections from a FECRT, as these cases may have the greatest potential to increase their egg output upon removal of density dependent constraints. The potential impact of worm reproductive biology on the utility of the FECRT as a resistance detection tool highlights the need to develop new drug resistance monitoring methods which examine either direct drug effects on isolated worms with in vitro phenotypic assays, or changes in worm genotypes

Synergism between ivermectin and the tyrosine kinase/P-glycoprotein inhibitor crizotinib against Haemonchus contortus larvae in vitro

Anthelmintic resistance is a major problem in parasitic nematodes of livestock worldwide. One means to counter resistance is to use synergists that specifically inhibit resistance mechanisms in order to restore the toxicity, and hence preserve the usefulness, of currently available anthelmintics. P-glycoproteins (P-gps) eliminate a wide variety of structurally unrelated xenobiotics from cells, and have been implicated in anthelmintic resistance. Crizotinib is a tyrosine kinase inhibitor under development as a cancer therapeutic. The compound also inhibits P-gps, and has been shown to reverse multidrug resistance in cancer cells. We were therefore interested in determining if the compound was able to increase the sensitivity of Haemonchus contortus larvae to ivermectin, as measured by in vitro larval development and migration assays with a drug-resistant and a –susceptible isolate. In migration assays, co-administration of crizotinib increased the toxicity of ivermectin to resistant larvae (up to 5.7-fold decrease in ivermectin IC), and rendered the resistant larvae equally or more sensitive to ivermectin than the susceptible isolate. On the other hand, co-administration of crizotinib had no effect on ivermectin sensitivity in the susceptible isolate. In development assays, significant increases in the sensitivity of both the resistant (up to 1.9-fold) and susceptible (up to 1.6-fold) larvae to ivermectin were observed, although the magnitude of the observed synergism was less than seen in migration assays, and the resistant larvae retained significant levels of ivermectin resistance. By highlighting the ability of the P-gp inhibitor crizotinib to increase the sensitivity of H. contortus larvae to ivermectin, this study provides further evidence that P-gp inhibitors are potential tools for modulating the efficacy of anthelmintics. In addition, the differences in the outcomes of the two assays, with ‘resistance-breaking’ effects being much more marked in migration assays, suggest that some life-stage-specific aspects may exist in the interaction of ivermectin with P-gps in the two worm isolates

Drug-efflux and target-site gene expression patterns in Haemonchus contortus larvae able to survive increasing concentrations of levamisole in vitro

While there is some evidence that changes in nicotinic acetylcholine receptor (nAChR) subunits confer resistance to levamisole in gastrointestinal helminth parasites, the exact nature of the resistance mechanism(s) is unclear. We utilised the presence of a resistant fraction within the Wallangra 2003 isolate of Haemonchus contortus larvae in order to subdivide the population into three subpopulations of larvae able to survive increasing concentrations of the drug. We then measured gene expression levels in the subpopulations and the larval population as a whole, focusing on genes encoding the subunit components of levamisole-sensitive receptors, genes encoding ancillary proteins involved in receptor assembly, and P-glycoprotein (P-gp) genes. The subpopulation surviving the lowest levamisole concentration showed increases of 1.5- to 3-fold in a number of P-gp genes (Hco-pgp-3, -4, -10, and -14) alongside unchanged receptor genes, compared to the whole Wallangra larval population. On the other hand, the subpopulation surviving the intermediate levamisole concentration showed an increase in only a single P-gp (Hco-pgp-14), alongside decreases in some receptor subunit (Hco-unc-63a) and ancillary protein genes (Hco-unc-50, Hco-ric-3.1 and 3.1). The subpopulation surviving the highest levamisole concentration showed further decreases in receptor subunit genes (Hco-unc-63a and Hco-unc-29 paralogs) as well as genes involved in receptor assembly (Hco-unc-74, Hco-unc-50, Hco-ric-3.1 and 3.1), alongside no increased P-gp gene levels. This suggests a biphasic pattern of drug resistance in the larvae of this worm isolate, in which a non-specific P-gp-mediated mechanism confers low levels of resistance, while higher level resistance is due to altered receptor subunit composition as a result of changes in both subunit composition and in the levels of proteins involved in receptor assembly

The Effect of Egg Embryonation on Field-Use of a Hookworm Benzimidazole-Sensitivity Egg Hatch Assay in Yunnan Province, People's Republic of China

With the implementation of mass drug administration programmes for the control of human soil transmitted helminths there is a need to develop drug sensitivity monitoring tools to detect the emergence of resistance. The present study aimed to use an egg hatch assay to measure benzimidazole sensitivity in human hookworms in a field setting in Yunnan province, People's Republic of China, in order to assess whether the assay offered a practical means of monitoring drug sensitivity in human hookworms in such a location. The assay proved able to generate dose response data, which allowed for the drug sensitivity of the hookworms in the local children to be described; the mean IC50 was 0.10 ug/ml thiabendazole. The study also found that practical issues associated with stool collection procedures, specifically the embryonation of some eggs during the time elapsing between stool deposition and egg recovery, can have an impact on the drug sensitivity data. We suggest means for data analysis that overcome the impact of egg embryonation on drug dose response data, which should allow for the use of such assays at different field sites worldwide

The mediating role of innovation in the relationship between market orientation and university performance in Pakistan

Universities globally are going through a paradigm shift with a need to become more innovatively market-oriented to handle the issue of growing competition for funding, as well as attracting/retaining the international/local competent students and academicians. However, there appears to be a dearth of research on how such state of affairs could be addressed, particularly in the emerging economies like Pakistan. In the light of resource-based theory (RBT), as well as organizational-learning theory (OLT), literature suggests that market-orientation (MO) and innovation are to be the desirable unique resources, as well as the guiding philosophies, to enable universities for a more competitive performance. Hence, this study investigated how resources like marketorientation (MO), and innovation, can influence university performance (UP). The study also tested empirically the potential mediating effect of innovation on the MOUP relationship. In addition, how the dimensions of MO influenced the innovation and university performance (UP) were also tested empirically in the universities of Pakistan. Results of the PLS path modelling (with 369 respondents from the target public-sector universities) firstly confirmed significant effect of the “universal construct of MO” and two of its dimensions “the advising and mentoring, as well as the intelligencegeneration and response” on UP. However, one dimension of MO, which is the administration-leadership, was not significantly supported to directly influence the UP. Secondly, the study confirmed that there were significant direct effects of the “universal construct of MO”, as well as all of its dimensions, on innovation. Thirdly, the study also found that there was a significant effect of innovation on UP. Furthermore, the bootstrapping results found significant mediation of innovation between the MO-UP relationship. Hence, the results show that UP can be directly enhanced through MO and innovation. Even the use of innovation as a mediator can further strengthen the MO-UP relationship. Based on the findings, the study offers theoretical and practical implications, followed by its limitations and directions, for future research

Making Decisions to Identify Forage Shrub Species for Versatile Grazing Systems

Grazing systems in many parts of the world face large challenges, including a declining natural resource base (e.g. soil fertility), marked fluctuations in feed production across seasons and years, climate change (including the contribution of greenhouse gases from livestock), and market demands for sustainable and ethical production systems. The ‘Enrich’ project was established in Australia (Revell et al. 2008; Bennell et al. 2010) within this broad context of emerging challenges to explore the potential of using Australian native perennial shrub species as part of the feedbase for sheep and cattle in southern Australia. The underlying rationale was to: add perennial shrub species into the existing annual-based pasture feedbase so that the forage system could tolerate extended dry periods but provide green edible plant material during periods where a ‘feed gap’ would otherwise exist; be productive on marginal soils where other productive options are limited (Masters et al. 2010); and have a positive effect on gut function and health (Vercoe et al. 2007); i.e. a versatile grazing system. This paper outlines the research approach that was taken, and reports on a ‘decision tree’ to prioritise species from an initial large list, based on a wide range of plant characteristics and how they can be used in a grazing system

Insecticidal activities of histone deacetylase inhibitors against a dipteran parasite of sheep, Lucilia cuprina

Histone deacetylase inhibitors (HDACi) are being investigated for the control of various human parasites. Here we investigate their potential as insecticides for the control of a major ectoparasite of sheep, the Australian sheep blowfly, Lucilia cuprina. We assessed the ability of HDACi from various chemical classes to inhibit the development of blowfly larvae in vitro, and to inhibit HDAC activity in nuclear protein extracts prepared from blowfly eggs. The HDACi prodrug romidepsin, a cyclic depsipeptide that forms a thiolate, was the most potent inhibitor of larval growth, with equivalent or greater potency than three commercial blowfly insecticides. Other HDACi with potent activity were hydroxamic acids (trichostatin, CUDC 907, AR-42), a thioester (KD5170), a disulphide (Psammaplin A), and a cyclic tetrapeptide bearing a ketone (apicidin). On the other hand, no insecticidal activity was observed for certain other hydroxamic acids, fatty acids, and the sesquiterpene lactone parthenolide. The structural diversity of the 31 hydroxamic acids examined here revealed some structural requirements for insecticidal activity; for example, among compounds with flexible linear zinc binding extensions, greater potency was observed in the presence of branched capping groups that likely make multiple interactions with the blowfly HDAC enzymes. The insecticidal activity correlated with inhibition of HDAC activity in blowfly nuclear protein extracts, indicating that the toxicity was most likely due to inhibition of HDAC enzymes in the blowfly larvae. The inhibitor potencies against blowfly larvae are different from inhibition of human HDACs, suggesting some selectivity for human over blowfly HDACs, and a potential for developing compounds with the inverse selectivity. In summary, these novel findings support blowfly HDAC enzymes as new targets for blowfly control, and point to development of HDAC inhibitors as a promising new class of insecticides. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology

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

Parasitic worms cause untold morbidity and mortality on billions of people and livestock. Drugs are available but resistance is problematic in livestock parasites and is a looming threat for human helminths. Currently, new drug discovery and resistance monitoring is hindered as drug efficacy is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods evaluated by eye using microscopy. Here we describe a novel application for a cell monitoring device (xCELLigence) that can simply and objectively assess real time anti-parasite efficacy of drugs on eggs, larvae and adults in a fully automated, label-free, high-throughput fashion. This technique overcomes the current low-throughput bottleneck in anthelmintic drug development and resistance detection pipelines. The widespread use of this device to screen for new therapeutics or emerging drug resistance will be an invaluable asset in the fight against human, animal and plant parasitic helminths and other pathogens that plague our planet

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions

Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance