A comparison between two in vitro techniques to detect resistance to ivermectin in field populations of Cooperia spp. in cattle

Anthelmintic resistance in beef cattle production is a well-known worldwide problem, contributing to the economic losses caused by gastrointestinal nematodes. Resistance to ivermectin (IVM) is present in 93.5% of farms in Argentina, Cooperia spp. being the most prevalent genus (100%). Diagnosing AR under field conditions is currently done using the faecal egg count reduction test, which has been long used but lacks sensitivity to detect resistance in its early stages. In trying to improve this, in vitro techniques have been developed for different compounds and different parasites, and tested mainly in sheep parasites. As part of a large study on IVM-resistant populations of Cooperia spp. in beef farms, this assay was designed to evaluate two in vitro techniques, the micro-agar larval development test (MALDT) and the larval migration inhibition test (LMIT), on proven resistant (R) and susceptible (S) field populations. Both populations had been previously characterised by controlled-efficacy tests, showing that the efficacy of ivermectin against R and Se Cooperia was 66.3% and 99.5%, respectively. For the MALDT, eggs of both Cooperia isolates were exposed to twelve anthelmintic concentrations, from 4.7x10⁻¹⁰M to 2.18x10⁻¹¹M. The obtained EC50 values were: 6.93x10-9M (95%CI: 6.37x10⁻⁹M to 7.49x10⁻⁹M) for the R population and 8.33x10-10M (95%CI: 7.86x10⁻¹⁰M to 8.8x10⁻¹⁰M) for the S one, with correlation coefficients (R²) of 0.92 y 0.93, respectively; the resistance factor (RF) was 8.31. For the LMIT, ensheathed L3 were exposed to eight concentrations, from 10⁻⁵M a 5x10⁻⁹M. The EC50 values were 6.33x10-8M (95%CI: 5.30x10-8M to 7.49x10-8M) for the R population, and 8.03x10-8M (95%CI: 5.49x10-8M ? 1,19x10-7M) for the S population, with R² of 0.87 y 0.52, respectively; and a RF of 0.79. Based on these preliminary results, the MALDT would be a useful in vitro technique to detect field populations of IVM-resistant Cooperia nematodes.Fil: Fuentes, Mariana Elisabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Lloberas, Maria Mercedes. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Luque, Sonia Elisabet. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Bernat, Gisele. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Riva, Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Fiel, Cesar Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Fernández, Alicia Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina28th Conference of the World Association for the Advancement of Veterinary ParasitologyIrlandaUniversity College DublinTrinity College DublinQueen’s University Belfas

Development and validation of an Enzyme-Linked Immunosorbent Assay for the detection of antibodies against Trichinella spp. in domestic pigs in Argentina

Trichinellosis is endemic in Argentina, with human outbreaks having mainly domestic pigs as focalised sources of infection. Enzyme-Linked Immunosorbent Assay (ELISA), based on excretory/secretory T. spiralis muscle larvae antigens (E/S ML) is the most recommended technique for epidemiological surveillance to detect anti-Trichinella antibodies in domestic pigs. Available international ELISA kits are expensive and need to be validated in each country. Thus, the aim of the present study was to evaluate the performance of an in-house indirect ELISA based on E/S ML antigen. The artificial digestion (AD) was used as reference technique. A total of 343 swine serum samples were used to define sensitivity, specificity, accuracy and repeatability of the ELISA. Sera tested were from 91 outdoor pigs associated with local outbreaks, 12 of them diagnosed as positive by AD, and 242 serum samples from intensive-rearing farms with strict hygienic conditions and negative results by AD. Sera from 2 pigs experimentally infected with 10,000 T. spiralis ML (bled at five different days post infection) were also included. E/S ML antigen was diluted at 5 μg/ml in coating buffer, samples were diluted 1/100 in wash buffer and peroxidase conjugated rabbit anti-pig IgG was used at 1/2500 dilution (Sigma). Reaction was developed using ortho-phenylene-diamine diluted in a citric acid buffer 0.07% H2O2. After 15 minutes, reaction was stopped with H2SO4 2.5N. The cut-off point was calculated based on the OD values of 5 negative controls (NC) using the formula: Mean NC ± 3 SD. The diagnostic sensitivity and specificity of the ELISA were 95.5% and 98.4%, respectively. The accuracy was 98.2%. The inter-assay repeatability (4 plates) calculated for 1 positive and 4 negative controls was under 20%. The performance of this ELISA suggests that it could be used as a reliable and practical tool for seroepidemiological studies in Argentina.Fil: Riva, Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Steffan, Pedro Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Amanto, Andres Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Davila, Agustin. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Bernat, Gisele. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Fernández, Alicia Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Fuentes, Mariana Elisabet. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Estein, Silvia Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina28th International Conference of the World Association for the Advancement of Veterinary ParasitologyDublinReino UnidoUniversity College of Dubli

Weaning from mechanical ventilation in intensive care units across 50 countries (WEAN SAFE): a multicentre, prospective, observational cohort study

Background Current management practices and outcomes in weaning from invasive mechanical ventilation are poorly understood. We aimed to describe the epidemiology, management, timings, risk for failure, and outcomes of weaning in patients requiring at least 2 days of invasive mechanical ventilation. Methods WEAN SAFE was an international, multicentre, prospective, observational cohort study done in 481 intensive care units in 50 countries. Eligible participants were older than 16 years, admitted to a participating intensive care unit, and receiving mechanical ventilation for 2 calendar days or longer. We defined weaning initiation as the first attempt to separate a patient from the ventilator, successful weaning as no reintubation or death within 7 days of extubation, and weaning eligibility criteria based on positive end-expiratory pressure, fractional concentration of oxygen in inspired air, and vasopressors. The primary outcome was the proportion of patients successfully weaned at 90 days. Key secondary outcomes included weaning duration, timing of weaning events, factors associated with weaning delay and weaning failure, and hospital outcomes. This study is registered with ClinicalTrials.gov, NCT03255109. Findings Between Oct 4, 2017, and June 25, 2018, 10 232 patients were screened for eligibility, of whom 5869 were enrolled. 4523 (77·1%) patients underwent at least one separation attempt and 3817 (65·0%) patients were successfully weaned from ventilation at day 90. 237 (4·0%) patients were transferred before any separation attempt, 153 (2·6%) were transferred after at least one separation attempt and not successfully weaned, and 1662 (28·3%) died while invasively ventilated. The median time from fulfilling weaning eligibility criteria to first separation attempt was 1 day (IQR 0–4), and 1013 (22·4%) patients had a delay in initiating first separation of 5 or more days. Of the 4523 (77·1%) patients with separation attempts, 2927 (64·7%) had a short wean (≤1 day), 457 (10·1%) had intermediate weaning (2–6 days), 433 (9·6%) required prolonged weaning (≥7 days), and 706 (15·6%) had weaning failure. Higher sedation scores were independently associated with delayed initiation of weaning. Delayed initiation of weaning and higher sedation scores were independently associated with weaning failure. 1742 (31·8%) of 5479 patients died in the intensive care unit and 2095 (38·3%) of 5465 patients died in hospital. Interpretation In critically ill patients receiving at least 2 days of invasive mechanical ventilation, only 65% were weaned at 90 days. A better understanding of factors that delay the weaning process, such as delays in weaning initiation or excessive sedation levels, might improve weaning success rates

Weaning from mechanical ventilation in intensive care units across 50 countries (WEAN SAFE): a multicentre, prospective, observational cohort study

Background: Current management practices and outcomes in weaning from invasive mechanical ventilation are poorly understood. We aimed to describe the epidemiology, management, timings, risk for failure, and outcomes of weaning in patients requiring at least 2 days of invasive mechanical ventilation. Methods: WEAN SAFE was an international, multicentre, prospective, observational cohort study done in 481 intensive care units in 50 countries. Eligible participants were older than 16 years, admitted to a participating intensive care unit, and receiving mechanical ventilation for 2 calendar days or longer. We defined weaning initiation as the first attempt to separate a patient from the ventilator, successful weaning as no reintubation or death within 7 days of extubation, and weaning eligibility criteria based on positive end-expiratory pressure, fractional concentration of oxygen in inspired air, and vasopressors. The primary outcome was the proportion of patients successfully weaned at 90 days. Key secondary outcomes included weaning duration, timing of weaning events, factors associated with weaning delay and weaning failure, and hospital outcomes. This study is registered with ClinicalTrials.gov, NCT03255109. Findings: Between Oct 4, 2017, and June 25, 2018, 10 232 patients were screened for eligibility, of whom 5869 were enrolled. 4523 (77·1%) patients underwent at least one separation attempt and 3817 (65·0%) patients were successfully weaned from ventilation at day 90. 237 (4·0%) patients were transferred before any separation attempt, 153 (2·6%) were transferred after at least one separation attempt and not successfully weaned, and 1662 (28·3%) died while invasively ventilated. The median time from fulfilling weaning eligibility criteria to first separation attempt was 1 day (IQR 0-4), and 1013 (22·4%) patients had a delay in initiating first separation of 5 or more days. Of the 4523 (77·1%) patients with separation attempts, 2927 (64·7%) had a short wean (≤1 day), 457 (10·1%) had intermediate weaning (2-6 days), 433 (9·6%) required prolonged weaning (≥7 days), and 706 (15·6%) had weaning failure. Higher sedation scores were independently associated with delayed initiation of weaning. Delayed initiation of weaning and higher sedation scores were independently associated with weaning failure. 1742 (31·8%) of 5479 patients died in the intensive care unit and 2095 (38·3%) of 5465 patients died in hospital. Interpretation: In critically ill patients receiving at least 2 days of invasive mechanical ventilation, only 65% were weaned at 90 days. A better understanding of factors that delay the weaning process, such as delays in weaning initiation or excessive sedation levels, might improve weaning success rates. Funding: European Society of Intensive Care Medicine, European Respiratory Society

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field