International Journal for Parasitology: Drugs and Drug Resistance最新文献

Development of resistance to deployed antimalarial drugs is inevitable and needs prompt and continuous discovery of novel candidate drugs. Therefore, the antimalarial activity of 125 compounds from the Medicine for Malaria Ventures (MMV) pathogen box was determined. Combining standard IC₅₀ and normalised growth rate inhibition (GR₅₀) analyses, we found 16 and 22 compounds had higher potencies than CQ respectively. Seven compounds with relatively high potencies (low GR₅₀ and IC₅₀) against P. falciparum 3D7 were further analysed. Three of these were tested on 10 natural P. falciparum isolates from The Gambia using our newly developed parasite survival rate assay (PSRA).

According to the IC₅₀, GR₅₀ and PSRA analyses, compound MMV667494 was most potent and highly cytotoxic to parasites. MMV010576 was slow acting but more potent than dihydroartemisinin (DHA) 72 h after exposure. MMV634140 was potent against the laboratory-adapted 3D7 isolate, but 4 out of 10 natural Gambian isolates survived and replicated slowly despite 72 h of exposure to the compound, suggesting potential drug tolerance and risk of resistance development.

These results emphasise the usefulness of in vitro testing as a starting point for drug discovery. Improved approaches to data analyses and the use of natural isolates will facilitate the prioritisation of compounds for further clinical development.

Anoplocephalid tapeworms are commonly occurring in grazing horses around the world. Two currently available anthelmintics have documented high efficacy against Anoplocephala perfoliata; praziquantel in various dosages ranging from 1.0 to 2.5 mg/kg and pyrantel pamoate administered at 13.2 mg base/kg. Anthelmintic resistance has not been reported in A. perfoliata, but anecdotal reports made during 2022 have suggested a possible loss of efficacy for both actives. This paper reports fecal egg count data from a Thoroughbred operation in Central Kentucky in 2023. Fifty-six yearlings were first dewormed with a combination of ivermectin (200 μg/kg) and praziquantel (1.5 mg/kg) and subsequently treated with pyrantel pamoate (13.2 mg base/kg). Fecal egg counts were determined at the day of treatment and again 14 days post-treatment. Two groups of mares (n = 39 and 45) were also treated with ivermectin/praziquantel and examined pre- and post-treatment. Low efficacy of ivermectin and pyrantel pamoate was demonstrated against strongylid parasites in the yearlings with mean Fecal Egg Count Reductions (FECRs) at 75.6% or below and upper 95% credible interval (CI) limits below 90% in all cases. Overall anti-cestodal FECR levels in the yearlings were 23.5% (95% CI: 11.2–48.0) for praziquantel and 50.9% (20.5–72.0) for pyrantel pamoate. Praziquantel eliminated anoplocephalid eggs from three of 17 yearlings, but another 5 yearlings went from negative to positive status following treatment. Pyrantel pamoate failed to eliminate anoplocephalid eggs from any of 14 treated tapeworm-positive yearlings. Nine of 84 mares tested positive for anoplocephalid eggs, and seven of these were still positive post praziquantel treatment. These findings sharply contrast data from historic field efficacy studies conducted for both actives and raise concern about anthelmintic resistance having possibly developed. This emphasizes the need for developing and refining antemortem methodologies for evaluating anti-cestodal treatment efficacy and for searching for possible alternative treatment options.

Ancylostoma caninum is the most common and important gastrointestinal nematode of dogs in the United States. Despite recent reports of A. caninum isolates resistant to all classes of anthelmintics, little is known about the frequency and extent of this anthelmintic resistance. The study aim was to evaluate the efficacy of three commercial anthelmintic products in the treatment of foxhound dogs with a history of persistent A. caninum infections. In the first phase of this study, 35 foxhounds were randomly divided into three treatment groups: moxidectin/imidacloprid (MI), pyrantel pamoate/febantel/praziquantel (PFP), and emodepside/praziquantel (EP). Fecal samples were collected on day 0, 11, and 33 post-treatment (PT), and hookworm eggs were quantified using the mini-FLOTAC technique with a multiplication factor of 5 eggs per gram (EPG). The fecal egg count reduction (FECR) on day 11 PT was 65% (95% CI: 62%–68%) for MI, 69% (95% CI: 66%–72%) for PFP, and 96% (95% CI: 94%–97%) for EP. On day 33 PT, the FEC in the MI and PFP groups returned to almost the same values as on day 0, while in the EP group, the FEC remained low. Since MI and PFP proved ineffective, 32 animals were randomly divided into two groups in the second phase. They were treated either with a combination of MI/PFP or EP. The FECR at day 13 PT for the combination MI/PFP was 89% (95% CI: 87%–91%) and 99% (95% CI: 98%–99%) for EP. These results suggest that this A. caninum population is resistant to multiple anthelmintics. Although the combination of MI/PFP improved the anthelmintic efficacy, the FECR remained below 90%. Future studies are indicated to evaluate further the epidemiology of persistent hookworm infections in dogs in the US and to identify more effective treatment protocols as they pose a significant health risk to canine and human health.

The nematode genome exhibits a vast array of Cys-loop receptors that are activated by a diverse set of neurotransmitters and anthelmintic drugs such as ivermectin and levamisole. While many Cys-loop receptors have been functionally and pharmacologically characterized, there remains a large subset of orphan receptors where the agonist remains unknown. We have identified an orphan Cys-loop receptor, LGC-39, from the parasitic nematode Haemonchus contortus that is a novel type of cholinergic-sensitive ligand-gated chloride channel. This receptor groups outside of the acetylcholine-gated chloride channel family, in the previously named GGR-1 (GABA/Glycine Receptor-1) group of Cys-loop receptors. We found that LGC-39 forms a functional homomeric receptor when expressed in Xenopus laevis oocytes and is activated by several cholinergic ligands including acetylcholine, methacholine and surprisingly, atropine with an EC₅₀ for atropine on the low μM range. A homology model was generated which revealed some key features of the LGC-39 ligand-binding pocket that may explain some of the elements important for atropine recognition of the LGC-39 receptor. Overall these results suggest that the GGR-1 family (now called LGC-57) of Cys-loop receptors includes novel acetylcholine-gated chloride channel subtypes and may represent important future drug targets.

Benzimidazole-2-carbamates (BZ, e.g., albendazole; ALB), which bind β-tubulin to disrupt microtubule polymerization, are one of two primary compound classes used to treat giardiasis. In most parasitic nematodes and fungi, BZ-resistance is caused by β-tubulin mutations and its molecular mode of action (MOA) is well studied. In contrast, in Giardia duodenalis BZ MOA or resistance is less well understood, may involve target-specific and broader impacts including cellular damage and oxidative stress, and its underlying cause is not clearly determined. Previously, we identified acquisition of a single nucleotide polymorphism, E198K, in β-tubulin in ALB-resistant (ALB-R) G. duodenalis WB-1B relative to ALB-sensitive (ALB-S) parental controls. E198K is linked to BZ-resistance in fungi and its allelic frequency correlated with the magnitude of BZ-resistance in G. duodenalis WB-1B. Here, we undertook detailed transcriptomic comparisons of these ALB-S and ALB-R G. duodenalis WB-1B cultures. The primary transcriptional changes with ALB-R in G. duodenalis WB-1B indicated increased protein degradation and turnover, and up-regulation of tubulin, and related genes, associated with the adhesive disc and basal bodies. These findings are consistent with previous observations noting focused disintegration of the disc and associated structures in Giardia duodenalis upon ALB exposure. We also saw transcriptional changes with ALB-R in G. duodenalis WB-1B consistent with prior observations of a shift from glycolysis to arginine metabolism for ATP production and possible changes to aspects of the vesicular trafficking system that require further investigation. Finally, we saw mixed transcriptional changes associated with DNA repair and oxidative stress responses in the G. duodenalis WB-1B line. These changes may be indicative of a role for H₂O₂ degradation in ALB-R, as has been observed in other G. duodenalis cell cultures. However, they were below the transcriptional fold-change threshold (log₂FC > 1) typically employed in transcriptomic analyses and appear to be contradicted in ALB-R G. duodenalis WB-1B by down-regulation of the NAD scavenging and conversion pathways required to support these stress pathways and up-regulation of many highly oxidation sensitive iron-sulphur (FeS) cluster based metabolic enzymes.

Naegleria fowleri is an opportunistic protozoan, belonging to the free-living amoeba group, that can be found in warm water bodies. It is causative agent the primary amoebic meningoencephalitis, a fulminant disease with a rapid progression that affects the central nervous system. However, no 100% effective treatments are available and those that are currently used involve the appearance of severe side effects, therefore, there is an urgent need to find novel antiamoebic compounds with low toxicity. In this study, the in vitro activity of six oxasqualenoids obtained from the red algae Laurencia viridis was evaluated against two different strains of N. fowleri (ATCC® 30808 and ATCC® 30215) as well as their cytotoxicity against murine macrophages. Yucatecone was the molecule with the highest selectivity index (>2.98 and 5.23 respectively) and it was selected to continue with the cell death type determination assays. Results showed that yucatone induced programmed cell death like responses in treated amoebae causing DNA condensation and cellular membrane damage among others. In this family of oxasqualenoids, it seems that the most significative structural feature to induce activity against N. fowleri is the presence of a ketone at C-18. This punctual oxidation transforms an inactive compound into a lead compound as the yucatecone and 18-ketodehydrotyrsiferol with IC₅₀ values of 16.25 and 12.70 μM, respectively. The assessment of in silico ADME/Tox analysis revealed that the active compounds showed good Human Oral Absorption and demonstrate that are found to be within the limit of approved drug parameter range. Hence, the study highlights promising potential of yucatone to be tested for therapeutic use against primary amoebic meningoencephalitis.

A large-scale Fecal Egg Count Reduction Test (FECRT) was integrated with ITS-2 rDNA nemabiome metabarcoding to investigate anthelmintic resistance in gastrointestinal nematode (GIN) parasites in western Canadian beef cattle. The study was designed to detect anthelmintic resistance with the low fecal egg counts that typically occur in cattle in northern temperate regions. Two hundred and thirty-four auction market-derived, fall-weaned steer calves coming off pasture were randomized into three groups in feedlot pens: an untreated control group, an injectable ivermectin treatment group, and an injectable ivermectin/oral fenbendazole combination treatment group. Each group was divided into six replicate pens with 13 calves per pen. Individual fecal samples were taken pre-treatment, day 14 post-treatment, and at monthly intervals for six months for strongyle egg counting and metabarcoding. Ivermectin treatment resulted in an 82.4% mean strongyle-type fecal egg count reduction (95% CI 67.8–90.4) at 14 days post-treatment, while the combination treatment was 100% effective, confirming the existence of ivermectin-resistant GIN. Nemabiome metabarcoding of third-stage larvae from coprocultures revealed an increase in the relative abundance of Cooperia oncophora, Cooperia punctata, and Haemonchus placei at 14 days post-ivermectin treatment indicating ivermectin resistance in adult worms. In contrast, Ostertagia ostertagi third-stage larvae were almost completely absent from day 14 coprocultures, indicating that adult worms of this species were not ivermectin resistant. However, there was a recrudescence of O. ostertagi third stage larvae in coprocultures at three to six months post-ivermectin treatment, which indicated ivermectin resistance in hypobiotic larvae. The calves were recruited from the auction market and, therefore, derived from multiple sources in western Canada, suggesting that ivermectin-resistant parasites, including hypobiotic O. ostertagi larvae, are likely widespread in western Canadian beef herds. This work demonstrates the value of integrating ITS-2 rDNA metabarcoding with the FECRT to enhance anthelmintic resistance detection and provide GIN species- and stage-specific information.

Worldwide distributed coccidiosis is caused by infection of both Eimeria species and Cystoisospora in the host intestine and causes huge economic losses to the livestock industry, especially the poultry industry. The control of such diseases relies mainly on chemoprophylaxis with anticoccidials, which has led to a very common drug resistance in this field. However, the genetic mechanisms underlying resistance to many anticoccidial drugs remain unknown. In this study, strains of E. tenella resistant to 250 mg/kg monensin were generated and characterized. Forward genetic approaches based on pooled genome sequencing, including experimental evolution and linkage group selection, were used to locate candidate targets responsible for resistance to monensin and diclazuril in E. tenella. A total of 16 nonsynonymous mutants in protein-coding genes were identified in monensin-resistant strains, and two genomic regions with strong selection signals were also detected in diclazuril-resistant strains. Our study reveals the genetic characterization of the experimental evolution and linkage group selection in Eimeria species, and also provides important information that contributes to the understanding of the molecular mechanism of drug resistance in coccidia.

Chagas disease causes a problematic pathology that can lead to megacolon and heart disease, and can even cause the death of the patient. Current therapies for this disease are the same as they were 50 years ago, are not fully effective and have strong side effects. The lack of a safe and effective therapy makes it necessary to search for new, less toxic and totally effective compounds against this parasite. In this work, the antichagasic activity of 46 novel cyanomethyl vinyl ether derivatives was studied. In addition, to elucidate the type of cell death that these compounds produce in parasites, several events related to programmed cell death were studied. The results highlight four more selective compounds, E63, E64, E74 and E83, which also appear to trigger programmed cell death, and are therefore postulated as good candidates to use in future therapeutics for Chagas disease.

Haemonchus contortus is the most pathogenic nematode in small ruminants and anthelmintic resistance (AR) hampers its efficient control. Early detection of AR status is required to reduce selection for AR and cannot be achieved using phenotypic tests. For benzimidazoles (BZs), the detection of AR-associated alleles characterised by single nucleotide polymorphisms (SNPs) in the isotype 1 β-tubulin gene allows early AR detection in strongyles. The F200Y, F167Y, E198A and E198L polymorphisms have been described in BZ-resistant populations with a clear variation in frequencies between regions. A novel digital PCR (dPCR) enables the detection of all of the above-described polymorphisms in H. contortus. Assays were validated using synthetic DNA fragments containing these SNPs. Then, larvae obtained and pooled at farm level from 26 Austrian and 10 Italian sheep farms were analysed. For all assays a detection limit of 15 copies/μl of resistance alleles and a high level of accuracy were demonstrated, allowing to detect allele frequencies of 1% in most samples. In Austrian samples, elevated frequencies of F200Y resistance alleles were detected on all farms. Polymorphisms in codon 167 and codon 198 were identified in H. contortus from Austria for the first time. In Italian samples, the frequency of resistance alleles was still comparatively low, but F200Y resistance alleles were traceable. In conclusion we developed for the first time dPCR assays that target all SNPs of relevance associated with BZ-resistance in H. contortus. Future research on AR development could benefit from an early onset of SNP-based surveillance that would include the developed assays for all SNPs of relevance. Improved surveillance in the long term will include other important, though less pathogenic, nematode genera in the analyses.