Triclabendazole resistance in Fasciola hepatica: First report in sheep from the Santa Cruz province, Argentinian Patagonia.

In the fall of 2022, decreased triclabendazole (TCBZ) efficacy against F. hepatica was suspected in a sheep farm located in the Santa Cruz province, Argentinian Patagonia. Since TCBZ-resistance in F. hepatica has never been reported in this province, this study aimed to confirm potential TCBZ-resistance in F. hepatica and to evaluate the efficacy of closantel (CLO) and nitroxinil (NTX), through faecal egg count reduction test (FECRT), and the efficacy of albendazole (ABZ) through the in vitro egg hatch test (EHT) in sheep. Sixty-eight (68) animals were selected from a herd of eighty (80) female Merino naturally infected with F. hepatica based on eggs per gram of F. hepatica (EPGFh) counts and assigned into four (4) groups (n = 17 per group): Group Control, animals did not receive anthelmintic treatment; Group TCBZ, animals were orally treated with TCBZ (12 mg/kg); Group CLO, animals were orally treated with CLO (10 mg/kg); and Group NTX, animals were subcutaneously treated with NTX (10 mg/kg). The fluke egg output was monitored on days 0 and 21 post-treatment. For the EHT, liver fluke eggs were isolated from faecal samples (approx. 50 g) collected from animals of the control group. TCBZ efficacy against liver fluke was 53.4%, confirming the presence of TCBZ-resistant isolates on the farm. CLO and NTX were highly effective (100%) for the treatment of F. hepatica on this farm. The EHT was carried out in two different laboratories, in which was observed an ABZ efficacy of 95.8 (Bariloche) and 96.5% (Tandil). These results indicate the ABZ susceptibility of this F. hepatica isolate and the inter-laboratory precision of the test.

Identification and validation of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening.

Blastocystis sp. is among the most frequent intestinal protists identified in humans globally. However, characterization of Blastocystis subtype diversity in humans is ongoing. We report here the identification of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening involving colonoscopy and fecal testing (microscopy, culture, PCR). The full-length ssu rRNA gene sequence of the protist was generated using MinION long-read sequencing technology. The validity of the novel subtype was confirmed via phylogenetic and pairwise distance analyses of the full-length ST41 sequence and all other valid subtypes. The study provides reference material essential for conducting subsequent experimental studies.

Development and evaluation of an anatomically designed and 3D printed device to enhance orotracheal intubation success in rabbits by inexperienced veterinarians.

OBJECTIVE: To assess whether the use of a three-dimensional (3D) printed device enhances the success rate of orotracheal intubation in rabbits. STUDY DESIGN: Prospective, crossover randomized controlled trial. ANIMALS: A total of six mixed-breed rabbits. METHODS: A device to guide the endotracheal tube was designed based on computed tomography images and then manufactured using 3D printing. Rabbits were randomly assigned for intubation by two inexperienced veterinarians using the blind (BLI), borescope- (BOR) or device- (DEV) guided techniques. Success rate, number of attempts, time to success, injury scores and propofol dose were recorded and compared. Significance was considered when p < 0.05. RESULTS: Success rate was higher in DEV (58.3%) than in BLI (8.3%) (p < 0.023), but not different from that in BOR (41.6%). Total time until successful intubation was lower in DEV (45 ± 23 seconds) and BOR (85 ± 62 seconds) than in BLI (290 seconds; p < 0.006). Time for the successful attempt was lower for DEV (35 ± 10 seconds) and BOR (74 ± 43 seconds) than in BLI (290 seconds; p < 0.0001). The propofol dose required was lower for DEV (2.3 ± 1.2 mg kg-1) than for BLI (3.4 ± 1.6 mg kg-1) (p < 0.031), but not different from BOR (2.4 ± 0.9 mg kg-1). Number of attempts and oxygen desaturation events were not different among techniques (p < 0.051 and p < 0.326, respectively). Injury scores [median (range)] before and after attempts were different in BLI [0 versus 1 (0-3), p < 0.005] and BOR [0 (0-1) versus 1 (0-3), p < 0.002] but not in DEV [0 (0-2) versus 0 (0-3), p < 0.109]. CONCLUSIONS AND CLINICAL RELEVANCE: The device facilitated orotracheal intubation with a time similar to the borescope-guided technique but faster than the traditional blind technique.

Antinematodic effect of Enterococcus faecalis CECT7121 using Trichinella spiralis as a model of nematode infection in mice.

Nematode infections affect a significant percentage of the human population worldwide, especially in developing countries. There are a small number of drugs available to treat these infections, with variable outcomes. Therefore, the potential use of probiotics to help control parasitic infections has emerged as a suitable option. The main goal of this work was to assess the antinematodic effect of the probiotic Enterococcus faecalis CECT7121 (EFCECT7121) in vitro and in vivo, using Trichinella spiralis as a nematode model of infection. The in vitro assay showed a reduction in T. spiralis larvae viability of 31.6% when compared with the control group (6.3%) after 48 h incubation with EFCECT7121. Nevertheless, the isolated antimicrobial peptide AP7121 when inoculated at different concentrations did not reveal any larvicidal effect. Different EFCECT7121 treatment schemes in mice were evaluated, and the reduction of the enteral and parenteral burden of T. spiralis was determined. In addition, the protective effect of EFCECT7121 combined with the conventional anthelmintic albendazole (ABZ, 5 mg/kg) was also assessed. The oral administration of EFCECT7121 previous T. spiralis infection produced a reduction in the larvae per gram (LPG) of mice muscle tissue ranging from 32.8 to 47.9% on the 28th day post-infection. ABZ alone and the combination EFCECT7121 + ABZ produced a reduction of the LPG of muscle tissue of 62 and 60.7%, respectively. Results obtained in the current work support the hypothesis that probiotics such as EFCECT7121 have an antinematodic effect, and their combination with conventional anthelmintic drugs may result useful for improving clinical and parasitological outcomes.

Testing Albendazole Resistance in Fasciola hepatica.

The egg development test is a useful in vitro tool to detect albendazole (ABZ) resistance in Fasciola hepatica. ABZ is the only flukicidal compound with ovicidal activity. The described test is based on the ABZ capacity to affect parasite egg development and hatching in susceptible parasites, while this effect is lost in ABZ-resistant liver fluke isolates. Among many advantages, it is noted that the diagnostic test can be performed on eggs isolated from fecal samples (sheep and cattle), avoiding the sacrifice of animals necessary in controlled efficacy trials. The egg development test described here is a simple, inexpensive, and accessible method, previously employed for diagnosis of ABZ resistance in F. hepatica.

Albendazole treatment in laying hens: Egg residues and its effects on fertility and hatchability.

This work characterized the egg residual concentrations of albendazole (ABZ) and its sulphoxide (ABZSO) and sulphone (ABZSO2 ) metabolites and evaluated their effect on egg fertility and hatchability after ABZ treatments to laying hens. Seventy hens were allocated in groups: Group-1 was the control without treatment; Group-2 received a single ABZ oral dose (10 mg/kg); Group-3, -4 and -5 were treated with ABZ in medicated feed over 7 days at 10, 40, or 80 mg kg-1  day-1 , respectively. Eggs were analyzed to determine the ABZ/metabolite level by HPLC or subjected to incubation to evaluate the fertility and hatchability. Only ABZSO and ABZSO2 metabolites were quantified in egg after ABZ single oral administration with maximum concentrations of 0.47 ± 0.08 and 0.30 ± 0.07 µg/ml, respectively. ABZ and its metabolites were found in eggs after 7-day ABZ treatments. The egg residue exposure estimated as AUCs (areas under the concentration vs. time curve) were 100.5 (ABZ), 56.3 (ABZSO) and 141.3 µg hr g-1 (ABZSO2 ). ABZ administration did not affect the egg fertility at any dosages. Egg hatchability was not affected by ABZ treatment at 10 mg/kg in medicated feed, but it decreased when the dose was 4-8 times higher. These results should be considered when ABZ is used for deworming laying hens.

Assessment of serum pharmacokinetics and urinary excretion of albendazole and its metabolites in human volunteers.

BACKGROUND: Soil Transmitted Helminth (STH) infections negatively impact physical and mental development in human populations. Current WHO guidelines recommend morbidity control of these infections through mass drug administration (MDA) using albendazole (ABZ) or mebendazole. Despite major reductions in STH associated morbidity globally, not all programs have demonstrated the expected impact on prevalence of parasite infections. These therapeutic failures may be related to poor programmatic coverage, suboptimal adherence or the exposure of parasites to sub-therapeutic drug concentrations. As part of the DeWorm3 project, we sought to characterize the serum disposition kinetics and pattern of urinary excretion of ABZ and its main metabolites ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) in humans, and the assessment of the duration and optimal time point where ABZ and/or its metabolites can be measured in urine as an indirect assessment of an individual's adherence to treatment. METHODOLOGY/PRINCIPAL FINDINGS: Consecutive venous blood and urine samples were collected from eight (8) human volunteers up to 72 h post-ABZ oral administration. ABZ/metabolites were quantified by HPLC. The ABZSO metabolite was the main analyte recovered both in serum and urine. ABZSO Cmax in serum was 1.20 ± 0.44 µg/mL, reached at 4.75 h post-treatment. In urine, ABZSO Cmax was 3.24 ± 1.51 µg/mL reached at 6.50 h post-ABZ administration. CONCLUSION/SIGNIFICANCE: Pharmacokinetic data obtained for ABZ metabolites in serum and urine, including the recovery of the ABZ sulphoxide derivative up to 72 h in both matrixes and the recovery of the amino-ABZ sulphone metabolite in urine samples, are suggesting the possibility of developing a urine based method to assess compliance to ABZ treatment. Such an assay may be useful to optimize ABZ use in human patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT03192449.

Cystic echinococcosis therapy: Albendazole-loaded lipid nanocapsules enhance the oral bioavailability and efficacy in experimentally infected mice.

Therapeutic failures attributed to medical management of cystic echinococcosis (CE) with albendazole (ABZ) have been primarily linked to the poor drug absorption rate resulting in low drug level in plasma and hydatid cysts. Lipid nanocapsules (LNCs) represent nanocarriers designed to encapsulate lipophilic drugs, such as ABZ. The goals of the current work were: (i) to characterize the plasma and cyst drug exposure after the administration of ABZ as ABZ-LNCs or ABZ suspension (ABZ-SUSP) in mice infected with Echinococcus granulosus, and ii) to compare the clinical efficacies of both ABZ formulations. Enhanced ABZ sulphoxide (ABZ-SO) concentration profiles were obtained in plasma and cysts from ABZ-LNC treated animals. ABZSO exposure (AUC0-LOQ) was significantly higher in plasma and cyst after the ABZ-LNC treatments, both orally and subcutaneously, compared to that observed after oral administration of ABZ-SUSP. Additionally, ABZSO concentrations measured in cysts from ABZ-LNC treated mice were 1.7-fold higher than those detected in plasma. This enhanced drug availability correlated with an increased efficacy against secondary CE in mice observed for the ABZ-LNCs, while ABZ-SUSP did not reach differences with the untreated control group. This new pharmacotechnically-based strategy could be a potential alternative to improve the treatment of human CE.

Enhancement of the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica using the metabolic inhibitor ketoconazole.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered by using the metabolic inhibitor, ketoconazole (KTZ) to inhibit the cytochrome P450 (CYP 450) system within Fasciola hepatica. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP 450 enzyme system was inhibited by a 2 h pre-incubation in KTZ (40 microM). Flukes were then incubated for a further 22 h in NCTC medium containing either KTZ; KTZ + nicotinamide adenine dinucleotide phosphate (NADPH; 1 nM); KTZ + NADPH + TCBZ (15 microg/ml); or KTZ + NADPH + triclabendazole sulphoxide (TCBZ.SO;15 microg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible isolate than the TCBZ-resistant isolate. However, co-incubation with KTZ and TCBZ/TCBZ.SO led to more severe surface changes to the TCBZ-resistant isolate than with each drug on its own, with greater swelling and blebbing of the tegument and even the loss of the apical plasma membrane in places. With the Cullompton isolate, there was limited potentiation of drug action in combination with KTZ, and only with TCBZ.SO. The results support the concept of altered drug metabolism within TCBZ-resistant isolates and indicate that this process may play a role in the development of drug resistance.

Potentiation of triclabendazole sulphoxide-induced tegumental disruption by methimazole in a triclabendazole-resistant isolate of Fasciola hepatica.

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 microM), then incubated for a further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nM); MTZ+NADPH+TCBZ (15 microg/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 microg/ml). Changes to fluke ultrastructure following drug treatment and metabolic inhibition were assessed using transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than triclabedazole-resistant isolate. However, co-incubation with MTZ+TCBZ, but more particularly MTZ+TCBZ.SO, led to more severe changes to the TCBZ-resistant isolate than with each drug on its own, with severe swelling of the basal infolds and mucopolysaccharide masses in the syncytium, accompanied by a reduction in numbers of secretory bodies. The synthesis and production of secretory bodies in the tegumental cells was severely affected as well. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action. The results support the concept of altered drug metabolism in TCBZ-resistant flukes, and this process may play a role in the development of drug resistance.

Drug transfer into target helminth parasites.

The pharmacokinetics of an anthelmintic drug includes the time course of drug absorption, distribution, metabolism and elimination from the host and determines the concentration of the active drug that reaches the location of the parasite. However, the action of the anthelmintic also depends on the ability of the active drug to reach its specific receptor within the target parasite. Thus, drug entry and accumulation in target helminths are important issues when considering how best to achieve optimal efficacy. Passive drug transfer through the external helminth surface is the predominant entry mechanism for most widely used anthelmintics and is discussed in this article. Despite the structural differences between the external surface of nematodes (the cuticle) and the external surface of cestodes and trematodes (the tegument), the mechanism of drug entrance into both types of helminth depends on the lipophilicity of the anthelmintic and this is the major physicochemical determinant for the drug to reach a therapeutic concentration in the target parasite. Understanding the processes that regulate drug transfer into helminth parasites is an important aspect in improving the control of parasites in human and veterinary medicine.