BACKGROUND:Benin remains highly burdened by malaria, and data on antimalarial resistance surveillance are limited. This study assesses the evolution of molecular markers associated with antimalarial resistance in southern Benin between 2017 and 2024. METHODS:We analysed 358 dried blood spot samples collected from children in southern Benin (Kétonou, Kpomè and Klouékanmè) in 2017 and 2024. Molecular markers in the PfKelch13, Pfmdr1, Pfcrt, Pfdhfr, and Pfdhps genes associated with antimalarial drug resistance were investigated. Pfcrt haplotypes were characterised using quantitative PCR, while the remaining markers were analysed by nested PCR followed by Sanger sequencing. RESULTS:None of the detected non-synonymous PfKelch13 mutations (7/281) was a WHO-validated, candidate or potential marker of artemisinin partial resistance. The Pfcrt CVIET haplotype declined significantly from 88.1% (59/67) in 2017 to 35.4% (101/285) in 2024 (p < 0.001). The Pfmdr1 N86Y decreased from 11.6% (8/69) to 6.0% (17/285), while the NFD haplotype increased slightly from 56.9% (37/65) to 61.9% (169/273). No mutations were observed at Pfdhfr I164L or Pfdhps K540E. The Pfdhfr triple-mutant IRNI haplotype remained nearly fixed over time at 97.0% (64/66) in 2017 vs 96.4% (268/278) in 2024. A similar trend was observed for Pfdhps A437G at 98% in 2017 and 2024. The Pfdhps I431V rose from 8.3% (5/60) in 2017 to 10.5% (29/275) in 2024, whereas Pfdhps A581G remained stable at 8%. The combined Pfdhfr/PfdhpsIRN_G haplotype remained highly prevalent (96.5% vs 94.6%). Aside from the significant decline in CVIET, none of the other changes was statistically significant. CONCLUSION:Our findings show no mutations compromising ACT efficacy, nor any immediate threat to the continued use of SP. In addition, the observed re-emergence of chloroquine-sensitive genotypes suggests a shift in parasite population dynamics over time. Nevertheless, continuous molecular surveillance remains essential for detecting emerging resistance and informing timely policy decisions.
Anthelmintic resistance constitutes a global threat to the control of parasitic nematodes. Current research has primarily focused on parasite-intrinsic genetic mechanisms, while the contribution of the symbiotic microbial community remains a key knowledge gap. Here, we report that ivermectin (IVM) resistance in the gastrointestinal nematode Haemonchus contortus is associated with the abundance of the bacterium Stenotrophomonas maltophilia. A representative strain, designated SM1, was isolated from resistant populations, and its abundance was associated with the resistant phenotype. Depletion of SM1 increased larval susceptibility to IVM, whereas reintroduction of the bacterium partially enhanced IVM tolerance. Metabolic analysis indicated that SM1 converts IVM into demethylated and oxo-derivatives (M1, M4, and M7). Using in silico analysis, the putative cytochrome P450 monooxygenase (Cmp08160) with a possible participation in IVM biotransformation was identified. Collectively, these findings suggest that symbiotic bacteria can influence IVM susceptibility in H. contortus and highlight the relevance of considering host-microbiota interactions in studies of anthelmintic resistance.
There are restricted treatment options for Chagas disease, a global health risk causing 12,000 fatalities annually. This situation is exacerbated by environmental changes and migration, which is altering patterns of disease transmission, and by expansion of the vector range. The disease is caused by the protozoan parasite Trypanosoma cruzi, which possesses a divergent translation apparatus, characterized by enlarged ribosomes and a unique mRNA cap appended to the 5' end of a 39-nucleotide spliced leader sequence at the 5' end of all its mRNAs. Given that many antimicrobial agents are protein synthesis inhibitors and that there are significant differences between the T. cruzi protein synthesis machinery and that of its mammalian host, it is likely that T. cruzi-specific protein synthesis inhibitors could be employed for the treatment of Chagas disease. Such inhibitors can be identified by screening chemical libraries in an in vitro translation assay. However, lysates of T. cruzi cannot reinitiate translation of exogenous mRNAs. Here, we demonstrate that T. cruzi extracts derived from a strain deficient for hemin accumulation efficiently translated a capped and polyadenylated reporter mRNA. We optimized assay conditions and adapted them to a 384-well format. We further miniaturized this assay to a 5 μL volume, demonstrating its suitability for ultra-high throughput screening. Further, in a proof-of-principle pilot study, we identified quinazoline compounds that inhibit translation of reporter mRNAs by T. cruzi extracts. These compounds inhibited protein synthesis and proliferation of insect-derived forms and wild-type parasites growing in mammalian cells at a low micromolar range and a selectivity index higher than 200. This first-of-its-kind T. cruzi in vitro translation system will enable high-throughput screening of very large chemical libraries, while first-in-class hit compounds identified in the pilot study can be developed into lead compounds by design and synthesis of focused libraries to develop therapeutics for Chagas disease.
Gastrointestinal nematodes, including Haemonchus contortus, represent a major constraint to livestock production globally, and the increasing prevalence of anthelmintic resistance necessitates the discovery of new chemotypes. Natural products provide a rich and underexplored source of bioactive scaffolds for anthelmintic development. However, there remains a need to systematically optimise such scaffolds to define structure-activity relationships in whole-organism systems. Here, we investigated neolignan compounds from the fruits of Styrax suberifolius and assessed the impact of halogenation on their anthelmintic activity. Two known neolignans, suberifolioside A and equiselignan B, were isolated and used to generate a series of 11 halogenated derivatives using N-halosuccinimide chemistry. In addition, a new glycoside, suberifolioside B, together with two known metabolites, 7R,8S-dihydrodehydrodiconiferyl alcohol and 3',4-O-dimethylcedrusin, were characterised by spectroscopic and spectrometric methods, and the absolute configuration of equiselignan B was confirmed by X-ray crystallography. All compounds were evaluated for activity against larval stages of H. contortus using an established in vitro phenotypic assay. While no compounds significantly affected exsheathed third-stage larvae, multiple derivatives displayed activity against fourth-stage larvae, indicating pronounced stage-specific susceptibility. Notably, halogenation of the neolignan scaffold modulated biological activity, with several derivatives inducing substantial reductions in larval motility and distinct abnormal phenotypes. This modulation was non-linear, with both mono- and poly-halogenated derivatives displaying activity. A monobrominated ether analogue exhibited maximal motility inhibition approaching that observed for moxidectin under the assay conditions, highlighting the potential of this scaffold for optimisation. These findings demonstrate that halogenated neolignans represent a tractable chemical class for anthelmintic discovery and reveal stage-specific vulnerabilities in H. contortus. This work provides a foundation for further optimisation and prioritisation of neolignan derivatives for downstream studies of mechanism of action and translational potential.
Trichomoniasis, caused by Trichomonas vaginalis, is the most prevalent nonviral sexually transmitted infection worldwide. The occurrence of treatment failure with metronidazole (MTZ) and the adverse effects of currently available nitroimidazoles underscore the necessity for novel therapeutic candidates. From a library of synthetic thienopyrimidine derivatives, G-18 was identified as a lead compound. G-18 exhibited potent anti-T. vaginalis activity against the tested T. vaginalis isolate, with a minimum inhibitory concentration (MIC) of 4 μg/mL and a half-maximal inhibitory concentration (IC50) of 1 μg/mL at 24 h, and it significantly reduced trophozoite viability within 2 h. Flow cytometry confirmed a higher proportion of propidium iodide (PI)-positive parasites following G-18 treatment compared to MTZ treatment (51.2% vs. 10.4%). G-18 demonstrated limited cytotoxicity towards mammalian cells under the tested conditions. Scanning and transmission electron microscopy revealed pronounced ultrastructural damage, including surface roughening, membrane disruption, and organelle disorganization. These findings support G-18 as a promising anti-trichomonal lead compound with rapid in vitro activity and a preliminary selectivity profile under the tested conditions.
The Plasmodium falciparum multidrug resistance-1 (pfmdr1) gene, which encodes the ABC P-glycoprotein homolog PgH1, accumulates mutations that result in amino acid changes, namely single-nucleotide polymorphisms at codons N86Y, Y184F, and D1246Y, associated with altered parasite susceptibility to multiple antimalarial drugs. Among these antimalarials are compounds used in artemisinin-based combination (ACT) therapies, which combine a fast-acting but rapidly clearing artemisinin with a longer-lasting partner drug. The in vitro susceptibility of each of these drugs varies with the pfmdr1 genotype of the treated parasites. This study characterized sequence diversity within the pfmdr1 hinge domain and the association with pfmdr1 point mutations at codons 86, 184 and 1246 in population samples from Cameroon, the Democratic Republic of Congo, and Benin, two decades after the introduction of ACTs. The data were evaluated and compared with similar data reported before or during the early implementation of ACT-based treatment strategies. Among 780 successfully sequenced isolates, a high diversity of Arginine-rich repeats in the hinge domain was observed. A significant correlation between the (Asn-Asp-Asn) 7-2-9 variant and both the 86Y and the 1246Y mutation was observed (p < 0.005, OR = 12.6, respectively). We also uncovered, when examining haplotypes across the N86Y, Y184F, and D1246Y codons, that an accumulation of mutations was inversely correlated with diversity in the hinge region. The data suggest patterns consistent with selective pressure and/or linkage with reduced diversity as a potential result of ACT implementation.
Naegleria fowleri is a free-living amoeba that causes a rare but almost always fatal brain infection known as primary amoebic meningoencephalitis (PAM). Although amphotericin B (AmB) is considered the primary treatment for PAM, it is often recommended in combination with several other drugs due to side effects associated with high concentrations. In this study, we investigated whether histone deacetylase inhibitors (HDACis) enhance the amoebicidal activity of AmB against N. fowleri. Anti-proliferative and cytotoxic activities were assessed using the cell counting kit and lactate dehydrogenase assays, respectively, and apoptosis induction was evaluated by flow cytometry. Class-specific HDACis, including class I inhibitors MS275 and MGCD0103 and a class II inhibitor MC1568, showed minimal effects on trophozoite viability. In contrast, the pan-HDACis SAHA and JNJ significantly reduced trophozoite viability in a dose- and time-dependent manner. Furthermore, 10 μM SAHA and JNJ significantly reduced the encystation ratio of N. fowleri. Combined treatment with low doses of HDACi (1 μM) and AmB (0.5 μM) produced stronger amoebicidal and pro-apoptotic effects than either agent alone. These combination treatments also exhibited low cytopathic effects on human brain tumor cells. These findings suggest that SAHA and JNJ, particularly in combination with AmB, hold promise as therapeutic candidates against N. fowleri infection while potentially mitigating the severe side effects associated with AmB.
Anthelmintic drug resistance is a concern for the sustained control of gastrointestinal nematodes (GINs) in ruminant livestock globally. Evolutionary-epidemiological modelling, which considers both parasite dynamics and resistance dynamics in response to interventions, can be useful in determining which anthelmintic resistance management (ARM) strategies may be effective without compromising parasite control. We address two key questions in ARM. First, how to improve the measurement of AR in populations. Second, identifying effective ARM strategies to slow the spread of AR while maintaining effective parasite control. We developed a simulation framework which tracks the weather-dependent epidemiology of GINs and AR evolution, providing a highly flexible methodology to evaluate multiple ARM strategy options in a single modelling framework, allowing for novel insights due to direct comparisons between strategies. Simulations to refine our understanding of anthelmintic resistance management evaluated the impact of key areas of uncertainty, including transmission intensity, resistance intensity, resistance frequency, drug decay and linking faecal egg count reduction tests (FECRT) to resistance allele frequency. Large-scale simulations present a methodologically thorough evaluation of how treatment choices simultaneously impact epidemiological and evolutionary outcomes. Phenotypic classifications of resistance status using FECRT failed to capture fine scale changes in resistance allele frequency. The pharmacokinetics of drug decay strongly influenced ARM outcomes, and trade-offs between ARM and effective parasite control depends on genetic factors underpinning resistance. Combination therapies appear to be the most effective resistance management strategy evaluated. Our findings suggest practical implementations to manage anthelmintic resistance must simultaneously consider parasite transmission, pharmacology and parasite genetics to be robust and sustainable. We provide a rigorous simulation framework to enable such discussions allowing for a refinement into our understanding of parasite control in the presence of resistance evolution.
Metronidazole (MTZ) remains the first-line therapy for trichomoniasis; however, increasing resistance in Trichomonas vaginalis threatens treatment efficacy. Because iron availability enhances MTZ sensitivity, we investigated whether MTZ-induced parasite death involves ferroptosis-related mechanisms and explored alternative pathways contributing to drug resistance. Exposure of metronidazole-sensitive and -resistant isolates to ferroptosis inducers reduced parasite viability, yet neither ferroptosis inhibition nor iron chelation restored survival, and lipid peroxidation was not detected. These findings indicate that MTZ-associated cytotoxicity does not proceed via canonical ferroptotic pathways in T. vaginalis. Instead, resistant parasites exhibited elevated expression of thioredoxin-associated transcripts, consistent with enhanced redox defense capacity. Functional suppression of thioredoxin reductase (TrxR) activity by mitomycin C (MMC) significantly enhanced MTZ-mediated killing and produced a synergistic antiparasitic effect in resistant isolates. Although selective TrxR inhibition alone was insufficient to fully reproduce this phenotype, these findings implicate thioredoxin-dependent redox regulation as a contributor to MTZ susceptibility. Combination treatment was accompanied by selective metabolic reprogramming, including upregulation of pentose phosphate pathway genes linked to NADPH generation. Collectively, our results demonstrate that MTZ induces a non-ferroptotic form of cell death and identify thioredoxin-dependent redox buffering as a resistance-associated vulnerability. Targeting redox homeostasis through rational combination therapy may represent a mechanistically guided strategy to overcome metronidazole resistance in trichomoniasis.
OBJECTIVES:Anthelmintic resistance (AR) in gastrointestinal nematode (GIN) parasites of ruminant livestock has been widely reported in Africa but analysis of variation between regions and over time is lacking. The objective of this paper was to collate data on AR and, through meta-analyses, identify patterns in its occurrence in order to guide future research, practice and policy. RESEARCH DESIGN AND METHODS:A systematic literature review following PRISMA guidelines retrieved publications on AR, from which comparable data on treatment effectiveness using the faecal egg count reduction (FECR) test were extracted for meta-analysis using logistic and linear regression. RESULTS:Data were skewed towards East Africa and small ruminants and spanned 1995-2025. Of 646 articles identified, 60 were retained for analysis. There was evidence of failure across all drug groups: percent FECR (FECR%) was below 95% in sheep and goats in 48% of tests using benzimidazoles (BZ), and 57% for imidazothiazoles (IMTZ) and macrocyclic lactones (ML); in cattle, similar levels of treatment failure were reported but sample size was very low. FECR<95% was more commonly reported in sheep in southern and North Africa than in other regions, and in goats failure rates were higher for BZ and IMTZ than other drug groups and increased over time. FECR% declined in small ruminants over the reporting period for all drug groups and frequently involved survival of Haemonchus after treatment. CONCLUSIONS:Treatment failure due to AR appears to be widespread in ruminant livestock in Africa. This first meta-analysis shows increasing levels of AR over time for the three major anthelmintic drug groups in small ruminants, and similar levels of AR in GIN in cattle. These findings reinforce the need to adapt parasite control strategies to preserve anthelmintic efficacy. They also provide a baseline for future studies, which would benefit from clear and comparable methods for measuring AR and new in vitro technologies.
Therapeutic failure in Acanthamoeba Keratitis (AK) is largely driven by the high resistance of cysts to antimicrobial agents. However, the development of effective cysticidal drugs is hindered by the lack of standardized in vitro encystation protocols. Inconsistencies in induction methods can produce immature cells that lack the structural defenses of mature clinical walls, potentially leading to false-positive results in drug screening. This study aims to evaluate the impact of cyst maturity on drug resistance assays by systematically comparing two common liquid media-glucose-supplemented PBS (PBSEB) and alkaline encystation buffer (EB2)-within Acanthamoeba Genotype T4 backgrounds. We assessed morphological transitions, transcriptional profiles (EMSP, ATG8, and CS-I), ultrastructure (TEM), and functional resistance to sodium dodecyl sulfate (SDS) and chlorhexidine gluconate (CHG) using environmental reference and clinical isolates, including the stress-tolerant NCKU_D strain. Although both media induced morphological rounding and upregulated the autophagy marker ATG8, functional and molecular assays revealed distinct developmental outcomes. PBSEB-treated cells failed to significantly upregulate the critical early serine proteinase EMSP and exhibited insufficient CS-I expression, remaining highly susceptible to SDS lysis and lacking intact walls under TEM. Conversely, EB2 triggered a robust 11.8-fold increase in EMSP, yielding structurally mature, SDS-resistant cysts. Importantly, functional assays demonstrated that EB2-induced cysts exhibited significantly higher chemical resistance to CHG than the PBSEB group at 48 h, with this resistance advantage further widening by 72 h of induction. Based on these distinct profiles, we suggest that while PBSEB serves as a useful model for investigating cell detachment kinetics and early autophagic pathways, the EB2 protocol is more suitable for evaluating downstream therapeutic efficacy and product safety where functional, double-walled resistance is required to avoid false-positive outcomes in Genotype T4 drug screening.
The emergence and spread of artemisinin-resistant Plasmodium falciparum threaten recent progress in malaria control and highlight the urgent need for antimalarial agents with new mechanisms of action. Here, we performed a high-throughput phenotypic screening of the Nagoya Chemical Library, comprising 36,160 compounds. This yielded 1391 (3.8%) primary hits, further reduced by cytotoxicity profiling. Differential screening against wild-type 3D7 and transgenic 3D7-yDHODH parasites expressing yeast dihydroorotate dehydrogenase (DHODH) was done to identify mitochondrial electron transport chain inhibitors. This approach identified a compound with modest activity against parasite DHODH, confirmed by enzymatic assays with recombinant PfDHODH. A subset of 441 potent compounds with half-maximal concentration (EC50) < 6.5 μM was selected and profiled against the Dd2-derived multidrug-resistant panel. This analysis identified compounds likely targeting key parasite pathways, including phosphatidylinositol 4-kinase (PfPI4K) and P-type ATPase 4 (PfATP4). Chemical clustering of a selected subset of 350 compounds (<10 μM) revealed novel scaffolds with antiplasmodial potency that bear no structural resemblance to known antimalarial compounds. Collectively, these findings highlight novel chemotypes associated with validated antiplasmodial targets (PfDHODH, PfPI4K, and PfATP4) and uncover previously uncharacterized scaffolds with unknown targets. These compounds provide a foundation for further investigation of their mechanisms of action and optimization as potential antimalarial agents.
Trichomonas gallinae is a protozoan parasite of major concern in avian medicine, particularly in domestic pigeons (Columba livia). This study investigated the nitroimidazole susceptibility and T. gallinae occurrence in domesticated pigeons from Eastern Spain, kept for different competitions. A total of 220 pigeons from 11 lofts were sampled and examined by microscopy and culture, revealing 63.6% of infected birds. Genotyping identified genotype C as predominant, with occasional detection of genotype A, mixed A/C infections, and one isolate of Lineage III. In vitro susceptibility testing of 42 isolates showed a high occurrence (45.2%) of elevated metronidazole minimum lethal concentrations (MLC) values (MLC ≥40 μg/mL) ranging from 5 to >100 μg/mL in 9/11 pigeon lofts examined. MLC values were not associated with sex, age, or reproductive status. In contrast, higher IC50 values were observed in isolates obtained from pigeons bred for Pica competitions, and metronidazole exposure was associated with higher MLC values compared with ronidazole. No significant associations were found between in vitro nitroimidazole susceptibility values and environmental or management parameters, although suboptimal hygiene and high bird density were common in lofts with strains with reduced susceptibility to metronidazole. The findings of this study underscore the urgent need to establish evidence-based therapeutic protocols, strengthen biosecurity measures, and promote the development of novel therapeutic alternatives for the control of avian trichomonosis, in order to mitigate the spread of T. gallinae strains resistant to currently available drugs in pigeon populations.
Anthelmintic resistance in gastrointestinal nematodes (GIN) of cattle is of increasing concern, but the number of studies monitoring anthelmintic efficacy is insufficient to provide a representative picture. In this study, resistance to macrocyclic lactones (ML) and benzimidazoles (BZ) in GINs from 14 Scottish dairy farms (four non-organic, 10 organic) was evaluated using multiple approaches. The faecal egg count reduction test (FECRT) remains the primary tool for evaluating anthelmintic resistance in the field and was used on the four non-organic farms. Differing methodologies and recent guideline updates complicate the interpretation of FECRT results across studies, as alternative statistical frameworks produce varying confidence intervals that can alter conclusions. Nonetheless, resistance to BZs and MLs in Ostertagia ostertagiwas consistently detected with all analytical approaches on 3/3 and 4/4 non-organic farms, respectively. The egg hatch test, combined with nematode differentiation, was used to assess BZ resistance across all 14 farms. The 95% effective concentration values observed at both the population and species levels, especially for O. ostertagi (EC95: 0.045-1.294 μg/ml), were consistent with BZ resistance on 13/14 farms. Mixed amplicon sequencing was applied to 10 populations from seven farms, including both pre- and post-treatment FECRT samples. Resistance-associated polymorphisms in the β-tubulin isotype-1 gene were detected on 5/7 farms and were present at over 25% frequency in O. ostertagi, Cooperia oncophora, and Trichostrongylus spp. populations.The presence of resistance to both BZs and MLs on 3/4 non-organic farms and higher than expected EC values for BZ on 13/14 farms, showing reduced efficacy of BZ across nearly all farms, including organic, underscoring that anthelmintic resistance is not confined to a single management system. This finding emphasizes the need for sustainable parasite control across all production systems to prevent further development and spread of resistance. Although these data originate from Scotland, the high level of animal movement across the UK suggests that the findings are likely relevant to grazing systems throughout much of the country.
Cryptosporidium parvum is a zoonotic intestinal protozoan that causes severe diarrhea in children and immunocompromised individuals. The lack of effective therapies underscores the urgent need for novel drug targets. As C. parvum primarily relies on glycolysis for energy, fructose-1,6-bisphosphate aldolase (FBA) represents a promising target. Here, we characterized CpFBA and evaluated its potential as a therapeutic target. Sequence analysis revealed that CpFBA contains a signature domain associated with the type I fructose-bisphosphate aldolase superfamily and adopts a tetrameric structure. CpFBA protein localizes to both the cytoplasm and membrane of C. parvum sporozoites and presents in C. parvum excretory-secretory products (ESPs). Recombinant CpFBA protein (rCpFBA) exhibits typical aldolase activity, with a higher affinity for fructose-1,6-bisphosphate (FBP) (Km = 81.23 μM). Subsequently, molecular docking computationally predicted that morin may potentially bind to CpFBA. Anti-CpFBA serum or morin treatment significantly reduces glucose uptake and ATP levels of C. parvum sporozoites, and decreases parasite number in vitro. In C. parvum-infected suckling mice, morin treatment (60 mg/kg/day) significantly reduces oocyst shedding by 61.8%, alleviates C. parvum-induced weight loss and intestinal damage, preserves the expression of tight junction proteins (ZO-1, occludin), and suppresses the production of pro-inflammatory cytokines. These findings suggest that CpFBA is a promising drug target against C. parvum, and the natural compound morin resists C. parvum infection.
Benzimidazole resistance in Haemonchus contortus represents a widespread challenge in small ruminant production worldwide. Benzimidazoles remain one of the most commonly used drug classes, due to their broad spectrum of activity, affordability, and ease of administration. However, extensive and often inappropriate use of these compounds has led to the rapid emergence and global spread of resistance, particularly in H. contortus, a highly pathogenic and genetically diverse gastrointestinal nematode. Resistance to benzimidazoles is primarily associated with point mutations in the isotype-1 β-tubulin gene, most notably at codons 167 (F167Y), 198 (E198A), and 200 (F200Y), which result in modifications to the β-tubulin that prevent drug binding and compromise treatment efficacy. This review summarises the molecular mechanisms underlying benzimidazole resistance and provides an overview of currently available diagnostic approaches, including in vivo and in vitro assays, as well as molecular techniques such as real-time PCR, digital PCR, pyrosequencing, deep amplicon sequencing, and isothermal amplification methods. Despite regional variation in the distribution of resistance-associated polymorphisms, there is a consistent global predominance of the F200Y mutation. Finally, this review highlights the importance of integrating molecular surveillance with sustainable parasite control strategies to slow the further spread of benzimidazole resistance and preserve the long-term efficacy of available anthelmintics.
Trichinellosis is a food-borne zoonosis caused by Trichinella spiralis, for which effective treatment against the muscle larvae stage remains limited. To identify new anti-T. spiralis agents from medicinal plants, 30 extracts with reported or presumed antiparasitic relevance were screened in vitro. Among them, the 80% ethanol extract of Anaphalis margaritacea (AMEE) showed the strongest larvicidal activity, causing complete muscle larvae mortality within 72 h at 100 μg/mL and exhibiting a clear dose-dependent effect. Preliminary chemical characterization by UPLC-Q-TOF-MS/MS led to the tentative identification of eight major constituents, mainly organic acids and flavonoids. In a murine model of T. spiralis infection, oral administration of AMEE reduced muscle larvae burden by up to 59.7% in a dose-dependent manner. AMEE treatment also alleviated muscle fibre damage and inflammatory infiltration in infected muscle, accompanied by reduced levels of IL-6, IL-13, and TGF-β and decreased mRNA expression of Nqo1 and Sod2. To explore the basis of its antiparasitic activity, integrated proteomics, mitochondrial enzyme activity assays, and transmission electron microscopy were performed. AMEE markedly suppressed the activities of mitochondrial respiratory complexes I, II, and IV, reduced complex V activity, and induced mitochondrial swelling and cristae disruption in muscle larvae. These findings show that AMEE exerts significant anti-T. spiralis activity in vitro and in vivo, with evidence consistent with mitochondrial dysfunction in muscle larvae and attenuation of infection-associated muscle pathology.
Chagas disease, a neglected tropical disease caused by Trypanosoma cruzi, urgently requires next-generation therapeutics due to the limitations of use of benznidazole and nifurtimox, including adverse effects, long treatment period and still unproven efficacy in chronic cases. With limited knowledge of T. cruzi biology regarding validated targets that could be addressed from a drug discovery perspective, most drug discovery efforts associated to the identification of new T. cruzi active chemotypes has been relying upon cell-based assays. The failure of repurposing the CYP51 acting antifungal drug posaconazole through clinical trials has highlighted the need for a more sophisticated characterization of phenotypically identified T. cruzi inhibitors via more stringent triage compounds before engaging with downstream drug development. In this study, we have evaluated 2422 compounds against the intracellular amastigotes of T. cruzi to identify novel starting points for drug development. The 30 identified inhibitors of T. cruzi growth including 5 serotonin-dopamine receptor antagonists and 7 TGF-β receptor inhibitors were then used as chemical tools to profile the drug action effects in terms of T. cruzi amastigote quantification measurements, kinetics of action, susceptibility in different strains as well as forms of the parasite. In terms of kinetics of action, fast-acting compounds inhibited the proliferation of intracellular amastigotes within 48 h of incubation, whereas slow-acting compounds required prolonged exposure to achieve comparable growth inhibition. Although most compounds were active against both Y and Dm28c strains, the correlation between their IC50 values was only moderate. Among the 30 identified inhibitors, 6 displayed potent inhibitory activity against all three parasite life-cycle stages-amastigotes, trypomastigotes, and epimastigotes. Lastly, the "parasite painting" methodology was applied to trypomastigotes of the parasite to classify compounds based on morphological perturbations. The series of assays applied in this study offer tools to characterize and prioritize inhibitors for the downstream discovery process.
Theileriosis and babesiosis remain major constraints to livestock production, and increasing drug resistance necessitates alternative therapeutic strategies. Here, we evaluated the metal ionophore PBT2 for anti-piroplasm activity. PBT2 inhibited proliferation of two Theileria annulata schizont infected cell lines with IC50 values of 450.9 nM and 407.8 nM and significantly reduced parasite burden. Colony formation was suppressed in both cell lines, consistent with parasite-associated growth inhibition. Cytotoxicity in BoMAC cells was limited, yielding a selectivity index exceeding 60-fold. ICP-MS analysis of purified schizonts showed dose-dependent Zn accumulation and concomitant Mn depletion following PBT2 treatment, whereas Cu and Fe levels were not significantly altered. In infected cells, PBT2 increased reactive oxygen species levels and reduced total superoxide dismutase activity. Partial restoration of viability by Mn supplementation supports a functional link between Mn depletion and growth inhibition. In a Babesia microti murine model, PBT2 reduced peak parasitemia in a dose-dependent manner and ameliorated infection-associated anemia. Our findings indicate PBT2 as a promising, safe, and broad-spectrum anti-piroplasm chemical compound that could be used to develop the drug formulation for controlling the infections of drug resistant piroplasm in future.
Isoxazoline derivatives have rapidly become cornerstone ectoparasiticides in veterinary medicine, providing high efficacy and long-lasting protection against fleas, ticks, and other arthropods in companion animals and, more recently, in food-producing species. This review summarizes current knowledge on the chemistry, pharmacology, and toxicology of the main veterinary isoxazolines - fluralaner, afoxolaner, sarolaner, and lotilaner - with particular emphasis on safety profiles and broader One Health considerations. Chemically, these compounds are highly lipophilic, fluorinated molecules with distinctive stereochemistry; the selective use of biologically active S-enantiomers has improved potency while limiting off-target effects. Isoxazolines act through non-competitive antagonism of γ-aminobutyric acid (GABA) - and L-glutamate-gated chloride channels in arthropods, resulting in sustained neuronal hyperexcitation and parasite death, with markedly higher affinity for arthropod than mammalian receptors. Pharmacokinetically, the compounds exhibit variable oral bioavailability but share extensive plasma protein binding, large volumes of distribution, low clearance, and long elimination half-lives, which underlie their prolonged clinical efficacy but also raise concerns regarding tissue accumulation and delayed adverse effects. Controlled studies generally demonstrate wide safety margins; however, post-marketing pharmacovigilance has identified rare but sometimes serious neurological adverse events, particularly in predisposed animals or in the context of impaired efflux transport, such as ABCB1/MDR1 mutations or P-glycoprotein inhibition. The authorization of fluralaner for use in laying hens and evidence of residues in eggs approaching established maximum residue limits emphasize the importance of continued residue monitoring and refinement of withdrawal periods. Moreover, the environmental persistence of isoxazolines and their pronounced toxicity to non-target invertebrates highlight emerging ecotoxicological concerns. Collectively, veterinary isoxazolines represent a highly effective yet complex class of antiparasitic agents whose safe and sustainable use requires integrated pharmacological, toxicological, residue, and environmental risk assessment.
