Assessment of FDA-approved drugs against Strongyloides ratti in vitro and in vivo to identify potentially active drugs against strongyloidiasis.

BACKGROUND: Infections with Strongyloides stercoralis belong to the most neglected helminth diseases, and research and development (R&D) efforts on novel drugs are inadequate. METHODS: A commercially available library containing 1600 FDA-approved drugs was tested in vitro against Strongyloides ratti larvae (L3) at 100 µM. Hits (activity > 70%) were then evaluated against S. ratti adult worms at 10 µM. Morantel, prasterone, and levamisole were tested in the S. ratti rat model using dosages of 1-100 mg/kg. RESULTS: Seventy-one of the 1600 compounds tested against S. ratti L3 revealed activity above 70%. Of 64 compounds which progressed into the adult screen, seven compounds achieved death of all worms (benzethonium chloride, cetylpyridinium chloride, Gentian violet, methylbenzethonium chloride, morantel citrate, ivermectin, coumaphos), and another eight compounds had activity > 70%. Excluding topical and toxic compounds, three drugs progressed into in vivo studies. Prasterone lacked activity in vivo, while treatment with 100 mg/kg morantel and levamisole cured all rats. The highest in vivo activity was observed with levamisole, yielding a median effective dose (ED50) of 1.1 mg/kg. CONCLUSIONS: Using a drug repurposing approach, our study identified levamisole as a potential backup drug for strongyloidiasis. Levamisole should be evaluated in exploratory clinical trials.

Anthelmintic Activity and Cytotoxic Effects of Compounds Isolated from the Fruits of Ozoroa insignis Del. (Anacardiaceae).

Ozoroa insignis Del. is an ethnobotanical plant widely used in traditional medicine for various ailments, including schistosomiasis, tapeworm, and hookworm infections. From the so far not investigated fruits of Ozoroa insignis, the anthelmintic principles could be isolated through bioassay-guided isolation using Caenorhabditis elegans and identified by NMR spectroscopic analysis and mass spectrometric studies. Isolated 6-[8(Z)-pentadecenyl] anacardic (1), 6-[10(Z)-heptadecenyl] anacardic acid (2), and 3-[7(Z)-pentadecenyl] phenol (3) were evaluated against the 5 parasitic organisms Schistosoma mansoni (adult and newly transformed schistosomula), Strongyloides ratti, Heligmosomoides polygyrus, Necator americanus, and Ancylostoma ceylanicum, which mainly infect humans and other mammals. Compounds 1-3 showed good activity against Schistosoma mansoni, with compound 1 showing the best activity against newly transformed schistosomula with 50% activity at 1µM. The isolated compounds were also evaluated for their cytotoxic properties against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cell lines, whereby compounds 2 and 3 showed antiproliferative activity in both cancer cell lines, while compound 1 exhibited antiproliferative activity only on PC-3 cells. With an IC50 value of 43.2 µM, compound 3 was found to be the most active of the 3 investigated compounds.

Repeated Ivermectin Treatment Induces Ivermectin Resistance in Strongyloides ratti by Upregulating the Expression of ATP-Binding Cassette Transporter Genes.

Ivermectin (IVM) is a widely used anthelmintic. However, with widespread use comes the risk of the emergence of IVM resistance, particularly in strongyloidiasis. Adenosine triphosphate (ATP)-binding cassette (ABC) transporter genes play an important role in the IVM-resistance mechanism. Here, we aimed to establish an animal experimental model of IVM resistance by frequent treatment of Strongyloides ratti with subtherapeutic doses of IVM, resistance being evaluated by the expression levels of ABC transporter genes. Rats infected with S. ratti were placed in experimental groups as follows: 1) untreated control (control); 2) treated with the mutagen ethyl methanesulfonate (EMS); 3) injected with 100 µg/kg body weight of IVM (IVM); 4) treated with a combination of EMS and IVM (IVM+EMS). Parasites were evaluated after four generations. Extent of IVM resistance was assessed using IVM sensitivity, larval development, and expression of ABC genes. By the F4 generation, S. ratti in the IVM group exhibited significantly higher levels of IVM resistance than did other groups according to in vitro drug-sensitivity tests and inhibition of larval development (IC50 = 36.60 ng/mL; 95% CI: 31.6, 42.01). Expression levels of ABC isoform genes (ABCA, ABCF, and ABCG) were statistically significantly higher in the IVM-resistant line compared with the susceptible line. In conclusion, IVM subtherapeutic doses induced IVM resistance in S. ratti by the F4 generation with corresponding upregulation of some ABC isoform genes. The study provides a model for inducing and assessing drug resistance in Strongyloides.

A protocol for chemical mutagenesis in Strongyloides ratti.

Genetic analysis using experimentally induced mutations has been a most valuable tool in the analysis of various organisms. However, genetic analysis of endoparasitic organisms tends to be difficult because of the limited accessibility of the sexually reproducing adults, which are normally located within the host. Nematodes of the genera Strogyloides and Parastrongyloides represent an exception to this because they can form facultative free-living sexually reproducing generations in between parasitic generations. Here we present a protocol for the chemical mutagenesis of Strongyloides ratti. Further we evaluate the feasibility of identifying the induced mutations by whole genome re-sequencing.

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

The need for new anthelmintic with no chemical residues is becoming urgent. In a program aiming at the evaluation of plant as sources of new active molecules, the anthelmintic activities of the essential oils (EOs) obtained from either Zanthoxylum zanthoxyloides seeds or Newbouldia laevis leaves were evaluated against Strongyloides ratti by analyzing the results of two in vitro bioassays. These two plants and their tested parts were retained after an ethnopharmacology survey that confirmed their use by small-scale farmers for treatment of small ruminants affected by digestive helminths. The plants were harvested in Benin, and their EO were obtained by hydrodistillation. The EO yield of extraction was 0.65% (w/w) of for Z. zanthoxyloides seeds and 0.05% (w/w) for N. laevis. The chemical compositions of the two EOs were analyzed by gas chromatography coupled with mass spectrometry. The major constituents of the EO from Z. zanthoxyloides consisted of the following compounds: γ-terpinene (18 %), undecane (15 %), valencene (8.3 %), decanal (8.3 %), and 3-carene (6.7 %). In contrast, the major constituents of the EO from N. laevis leaves consisted of the following compounds: ß-caryophyllene (36 %) and eugenol (5.8 %). An egg-hatching inhibition (EHI) assay was developed and a larval migration inhibition assay was used on S. ratti to examine the effects of the EOs and to evidence their inhibitory concentrations (IC(50) and IC(90)) values on this nematode. Furthermore, the toxicity of the two EOs on Vero cell line was evaluated. When tested on S. ratti egg hatching, the two EOs resulted in similar IC(50) values (19.5 and 18.2 µg/ml for Z. zanthoxyloides and N. laevis, respectively), which were about sevenfold higher than that of the control (thiabendazole, IC(50) = 2.5 µg/ml). Larval migration was inhibited at similar concentrations for: Z. zanthoxyloides (IC(50) = 46 µg/ml), N. laevis (IC(50) = 51 µg/ml), and the control [levamisole (IC(50) = 36 µg/ml)]. No cytotoxicity was found on Vero cells because both EOs had IC(50) values higher than 50 µg/ml. Therefore, we have concluded that the EOs from two plants, used in folk medicine, may contain compounds with anthelmintic activity and could be used as improved traditional medicines or, at least, as food additives in a combined treatment for the control of helminth infections.

Strongyloides ratti: transplantation of adults recovered from the small intestine at different days after infection into the colon of naive and infection-primed Wistar rats, and the effect of antioxidant treatment on large intestinal parasitism.

Strongyloides ratti (Nagoya strain) is unique in that a portion of adults parasitizing the small intestine withstands 'worm expulsion', which starts at around day 8 post-infection (p.i.) by host immunity, and establishes in the large intestine after day 19 p.i. To investigate the mechanism, adults obtained from the small intestine at day 7 or 19 p.i. were transplanted into the colon of infection-primed immune rats. Adults obtained at day 7 p.i. were rejected quickly, whereas those obtained at day 19 p.i. could establish infection. Moreover, the body length and the number of intrauterine eggs increased in the large intestine. In a separate experiment, large intestinal parasitism was abolished by the treatment of host rats with an anti-oxidant, butylated hydroxyanisole. These results indicate that small intestinal adults between days 7 and 19 p.i. acquired the ability to parasitize the large intestine of immune rats, and that free radicals produced by the host may have played a significant role in the process.

Strongyloides ratti: in vitro and in vivo activity of tribendimidine.

BACKGROUND: Strongyloidiasis is a truly neglected tropical disease, but its public health significance is far from being negligible. At present, only a few drugs are available for the treatment and control of strongyloidiasis. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the activity of tribendimidine against third-stage larvae (L(3)) of Strongyloides ratti in vitro and against juvenile and adult stages of the parasite in vivo. S. ratti larvae incubated in PBS buffer containing 10-100 microg/ml tribendimidine died within 24 hours. A single 50 mg/kg oral dose of tribendimidine administered to rats infected with 1-day-old S. ratti showed no effect. The same dose administered to rats harboring a 2-day-old infection showed a moderate reduction of the intestinal parasite load. Three days post-exposure a significant reduction of the immature worm burden was found. Administration of tribendimidine at doses of 50 mg/kg and above to rats harboring mature S. ratti resulted in a complete elimination of the larval and adult worm burden. For comparison, we also administered ivermectin at a single 0.5 mg/kg oral dose to rats infected with adult S. ratti and found a 90% reduction of larvae and a 100% reduction of adult worms. CONCLUSION/SIGNIFICANCE: Tribendimidine exhibits activity against S. ratti in vitro and in vivo. The effect of tribendimidine in humans infected with S. stercoralis should be assessed.

The beta-tubulin genes of two Strongyloides species.

The World Health Organization is sponsoring major treatment programs with the aim of controlling helminth infection throughout the tropical world. Prominent among the anthelmintics recommended for use in these programs are drugs in the benzimidazole (BZ) class. Resistance to these drugs has been associated with polymorphisms in the beta-tubulin gene. We have cloned and sequenced the beta-tubulin genes of Strongyloides stercoralis and Strongyloides ratti and have proceeded to develop a protocol for genotyping single worms for polymorphisms in beta-tubulin. Our findings indicate that S. ratti has a single beta-tubulin gene, making DNA sequence analysis of a single larva PCR product a feasible means of studying BZ resistance in these species. Our genotyping test allows the identification of polymorphisms at codons 167, 198, and 200 in the Strongyloides beta-tubulin gene, thus enabling survey for BZ resistant genotypes.

The effect of non-immune stresses on the development of Strongyloides ratti.

Strongyloides ratti is a parasitic nematode of rats. The host immune response against S. ratti affects the development of its free-living generation, favouring the development of free-living adult males and females at the expense of directly developing, infective 3rd-stage larvae. However, how the host immune response brings about these developmental effects is not clear. To begin to investigate this, we have determined the effect of non-immune stresses on the development of S. ratti. These non-immune stresses were subcurative doses of the anthelmintic drugs Ivermectin, Dithiazanine iodide and Thiabendazole, and infection of a non-natural host, the mouse. These treatments produced the opposite developmental outcome to that of the host immune response. Thus, in infections treated with subcurative doses of Ivermectin, Dithiazanine iodide and in infections of a non-natural host, the sex ratio of developing larvae became more female-biased and the proportion of female larvae that developed into free-living adult females decreased. This suggests that the mechanism by which the host immune response and these non-immune stresses affect S. ratti development differs.

Strongyloides ratti: chemokinesis of glycolytic enzyme- and lectin-treated third-stage infective larvae in vitro.

The infective third-stage larvae (L3s) of Strongyloides ratti, a parasitic nematode in rodents, showed two types of chemokinesis on a gradient of sodium chloride (NaCl) in an in vitro agarose tracking assay. The types were a consistent directional avoidance behavior under unfavorable environmental conditions and a reduced avoidance behavior under favorable conditions. We examined the effects of treatments with glycolytic enzymes and lectins by analyzing the avoidance behavior. L-Fucose dehydrogenase, hyaluronidase, beta-glucosidase, alpha-mannosidase, beta-galactosidase, concanavalin A, wheat germ agglutinin and soybean agglutinin exhibited inhibitory or enhancive effects on chemokinesis. We also confirmed the sites of the amphids of L3s aside from the mouth at the anterior end by scanning electron microscopy, and that concanavalin A-binding sites existed in the vicinity of the amphids using lectin-histochemistry. The carbohydrate moieties in the amphids of S. ratti L3s may play an important role as chemosensors in perceiving environmental cues.

Strongyloides ratti: thermokinesis of glycolytic enzyme- and lectin-treated third-stage infective larvae in vitro.

The infective third-stage larvae (L3s) of a parasitic nematode of rodents, Strongyloides ratti, showed three types of thermokinesis on a temperature gradient using an in vitro agarose tracking assay method. These depended both on the pattern of gradient temperature and the prior culture temperature. Most L3s (> or = 80%) isolated from rat feces cultured at 25 degrees C and placed on a gradient at temperatures between 30 degrees C and 37 degrees C showed no directional response, at 22-29 degrees C more than 50% of the L3s showed positive thermokinesis, at 21 degrees C L3s showed positive, negative and no directional responses in the same ratio, while at 18-20 degrees C, L3s showed negative thermokinesis (approx. 40%) or no directional response (approx. 60%) as in our previous study. The present study describes the effects of glycolytic enzyme- and lectin-treated positive thermokinesis of L3s. alpha-Glucosidase or concanavalin A significantly exhibited inhibitory effects on thermokinesis.

Chemical mutagenesis of the parasitic nematode Strongyloides ratti to isolate ivermectin resistant mutants.

We describe a strategy for the mutagenesis of the free-living adult generation of Strongyloides ratti and selection of worms carrying new mutations in the subsequent F2 generation of infective larvae. We demonstrate that this strategy is successful via the selection of infective larvae that are resistant to the anthelmintic ivermectin at a concentration of 10 ng/ml. The majority of these larvae were unable to give rise to patent infections when used to infect parasite naive rats, implying that the majority of the ivermectin resistance mutations confer pleiotropic defects on parasitic, but not on free-living, development.

Activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of nematodes in rodents and the influence on worm survival.

The present investigations deal with the activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of three rodent nematodes. While emodepside acts strongly against the adult stages of the rat nematodes Nippostrongylus brasiliensis and Strongyloides ratti, as well as against the mouse nematode Heligmosomoides polygyrus, its actions against the larval stages of these nematodes vary according to the species. Thus, emodepside is highly effective against the lung and intestine larval stages of N. brasiliensis and S. ratti. By contrast. the larval stages of H. polygyrus in the intestine are only partly affected by higher emodepside dosages.

Nematocidal activities of thiabendazole and ivermectin against the larvae of Strongyloides ratti and S. venezuelensis.

With the aim of developing therapeutic agents for strongyloidosis, the disease caused by infection with Strongyloides stercoralis, we established a novel assay technique using S. ratti and S. venezuelensis as models for S. stercoralis. The newly developed assay technique was found to more accurately represent treatment-induced larval paralysis than existing assays. Our method uses paper disks impregnated with the test solution, which even allows materials that are sparingly soluble in water to be tested. An inverted microscope was used to observe the larval states, and these states were recorded using a digital camera. We observed the activities of ivermectin and thiabendazole against larvae and calculated larval motility and velocity. These two factors were then combined to determine the overall viability of larvae at selected concentrations. The activities of the anthelmintics were compared by calculating the concentrations at which 50% viability was demonstrated, or in other words, the concentration at which paralysis was caused in 50% of the individuals (50% paralysis concentration; PC(50)). Evaluations after 24h of exposure yielded the following reproducible PC(50) values for ivermectin and thiabendazole, respectively: S. ratti, 2.4 and 140 microM; and S. venezuelensis, 2.3 and 190 microM. After treatment with ivermectin, there was a tendency for larval motility to be greater than that of the controls at low concentrations, a result that might be associated with its mechanism of action.

Murine strongyloidiasis: the effects of cyclosporin A and thiabendazole administered singly and in combination.

A single Cyclosporin A (CsA) dose of 30 mg kg-1 given orally at day 4 post-infection (p.i.) to Sprague-Dawley rats infected with Strongyloides ratti, reduced the faecal larval count by 46.8 +/- 1.2%. CsA was equally effective when the same dose rate was administered subcutaneously at day 4 p.i., reducing the faecal larval count by 41.6 +/- 8.6%. Thiabendazole (TBZ) given orally at 5 or 10 mg kg-1 (single dose at day 4 p.i.) reduced the faecal larval counts by 57.1 +/- 4.1% and 69.0 +/- 9.6%, respectively. Orally administered CsA was less effective than 5 mg TBZ kg-1 (at day 4 p.i.) Co-administration of 5 mg TBZ kg-1 and CsA did not elicit synergy or additive efficacy, indicating that CsA did not antagonise the anti-strongyloides activity of TBZ. The data suggests that for patients with current, historical or serological evidence of strongyloidiasis, CsA may be used where immunosuppressive therapy is required for other concurrent reasons or when TBZ is contraindicated.