First record of the mermithid nematode worm Isomermis lairdi parasitizing black flies in Spain

Funding Information: The authors declare that they have no conflicts of interest. We acknowledge the bioinformatics infrastructure of the Global Health and Tropical Medicine Center, which is funded through Fundação para a Ciência e a Tecnologia contract UID/04413/2020. Publisher Copyright: © 2022 Elsevier B.V.Mermithid nematodes are considered a promising biological control agent to reduce the population density of different blood-feeding vectors, i.e. black flies (Diptera: Simuliidae), which are important pests of medical and veterinary interest worldwide. Immature larvae of black flies were collected in a rill from La Rioja (Northern Spain) in the summer of 2016. Isomermis lairdi Mondet, Poinar & Bernadou, 1977 (Nematoda: Mermithidae) was found parasitizing eleven specimens of Simulium cryophilum s.l. (Rubtsov, 1959) (prevalence of 52%), which represent the first record of this nematode for Spain and the second for Europe. The confirmation of the nematode and the black fly species was carried out by both morphological and molecular approaches using the 18S ribosomal RNA and the cytochrome c oxidase subunit I genes. Phylogenetic analyses indicated that the collected specimens were Isomermis lairdi (99.4–99.9% identity with homologues from Africa) with a sequence divergence of 0.2%. The role of Isomermis lairdi as an alternative tool in the biological control of black flies in Spain should be further explored.publishersversionpublishe

Plan de emerxencias. Fundación Pública Urxencias Sanitarias de Galicia-061

A Fundación Pública Urxencias Sanitarias de Galicia-061 é a encargada de proporcionar, desde o momento que ocorre a emerxencia, un control da situación, unha primeira avaliación e unha asistencia sanitaria que logre salvar o maior número de vidas e volver á normalidade o antes posible. Para isto, a actuación sanitaria debe seguir unha metodoloxía perfectamente establecida, xa que as actuacións organizadas son as mellores ferramentas de traballo. Así pois, é necesario posibilitar normas de actuación o máis protocolizadas posible, para poder traballar nas mellores condicións de seguridade e manter unhas directrices xerais, onde cada persoa coñeza tanto a súa función como a do resto dos componentes do equipo, procedendo, ademais, á súa identificación funcional mediante signos externos (uniformidade, carteis, identificación, etc.); para facilitar o entendemento e a coordinación de todos os implicados en resolver a situación acaecida. Con este fin, preséntase o Plan de emerxencias que a continuación se expón, nun afán de dar sempre a mellor e máis axeitada resposta; obxectivo primordial desde que a FPUS de Galicia–061 se instaura como responsable da medicina prehospitalaria na nosa comunidade autónoma.La Fundación Pública Urxencias Sanitarias de Galicia-061 es la encargada de proporcionar, desde el momento en que ocurre la emergencia, un control de la situación, una primera evaluación y una asistencia sanitaria que logre salvar el mayor número de vidas y volver a la normalidad lo antes posible. Para esto, la actuación sanitaria debe seguir una metodología perfectamente establecida, ya que las actuaciones organizadas son las mejores herramientas de trabajo. Así pues, es necesario posibilitar normas de actuación lo más protocolizadas posible, para poder trabajar en las mejores condiciones de seguridad y mantener unas directrices generales, donde cada persona conozca tanto su función como la del resto de los componentes del equipo, procediendo, además, a su identificación funcional mediante signos externos (uniformidad, carteles, identificación, etc.); para facilitar el entendimiento y la coordinación de todos los implicados en resolver la situación acaecidad. Con este fin, se presenta el Plan de emergencias que a continuación se expone, en un afán de dar siempre la respuesta mejor y más idónea; objetivo primordial desde que la FPUS de Galicia-061 se instaura como responsable de la medicina prehospitalaria en nuestra comunidad autónoma

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Quantitative proteomics comparison of total expressed proteomes of anisakis simplex sensu stricto, A. pegreffii, and their hybrid genotype

The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes’ biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomarkers among A. simplex, A. pegreffii and their hybrid genotype; 19 of these biomarkers, encoded by ten loci, are specific allergens of Anisakis (Ani s7, Ani s8, Ani s12, and Ani s14) and other (Ancylostoma secreted) is a common nematodes venom allergen. The rest of the markers comprise four unknown or non-characterized proteins; five different proteins (leucine) related to innate immunity, four proteolytic proteins (metalloendopeptidases), a lipase, a mitochondrial translocase protein, a neurotransmitter, a thyroxine transporter, and a structural collagen protein. The proposed methodology (proteomics and statistical) solidly characterize a set of proteins that are susceptible to take advantage of the new targeted proteomicsThis work was partially supported by Spanish National Project AGL2015-68248-C2-1-R and the National Natural Science Museum. The proteomic analysis was performed in the Proteomics Facility of the Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019.Peer reviewe

Immunoreactive proteins in the esophageal gland cells of anisakis simplex sensu stricto detected by MALDI-TOF/TOF analysis

In plant and animal nematode parasites, proteins derived from esophageal gland cells have been shown to be important in the host-nematodes relationship but little is known about the allergenic potential of these proteins in the genus Anisakis. Taking into account the increase of anisakiasis and allergies related to these nematodes, immunoreactive properties of gland cell proteins were investigated. Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s. to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining which revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp.-allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, with 82 spots revealed in 2D. The corresponding protein bands and spots were excised from the silver-stained gel and protein assignation was made by MALDI-TOF/TOF. A total of 13 (including proteoforms) were unambiguously identified. The majority of these proteins are known to be secreted by nematodes into the external environment, of which three are described as being major allergens in other organisms with different phylogenetic origin and one is an Anisakis simplex allergen.This work was partially supported by Spanish National project AGL2015-68248-C2-1-R and National Natural Science Museum. The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019.Peer reviewe

Modeling survival curves of Anisakis L3 after isothermal heat treatments at lethal temperatures

Even though anisakiasis is considered, nowadays, a significant threat to public health all over the world, no attempt has been made up to date to mathematically describe the thermal susceptibility of Anisakis larvae in the third stage (L3). To fill this gap, in this paper, more than 10,000 free (non-encysted) Anisakis L3 were individually heat treated in a thermal cycler at temperatures between 44 °C and 61 °C for different exposure times. After heat exposition, viability was assessed in each larva, survival curves at isothermal conditions were derived, and the effectiveness of four kinetic models (fundamental kinetic model, Mafart model, and probit and logit models) in describing these curves was tested. Evaluation of larvae viability after heat exposition revealed sigmoidal survival curves that increased their steepness with temperature. Of the four models tested, the Mafart model was the one that best fitted the data only differing from the observed survival ratios by 0.12 units on average. Validation experiments performed at temperatures different to those used to create the model corroborated its predictive capacity. Future efforts should be focused in predicting larvae viability at non-isothermal conditions as those occurring during fish cooking.The work has been financed with the project ‘Mapping the effect of heating regimes on Anisakis death and fate of their allergens in fish muscle. Development of a decision support tool for optimal processing (HEATSAKIS)’. Reference: PID 2020-119201RB-I00/AEI/10.13039/501100011033. T.Peer reviewe

Respiratory analysis of intact Anisakis L3 as physiological tool to identify subtle changes in larvae subjected to thermal and/or chemical stress and still considered viable

Trabajo presentado al 48th West European FishTechnologists Association Meeting (WEFTA), celebrado en Lisboa (Portugal) del 15 al 18 de octubre de 2018.Human infection due to eating fish parasitized by live Anisakis larvae in the third stage (L3) is considered an important health problem and the application of treatments to ensure their mortality is crucial to prevent the risk of infection. Mobility is used as the method to assess viability. However, mobile larvae may not always be infective, and there is recognised a need to establish other methods to assess whether these larvae are capable of infecting humans. We suggest that mitochondria may become dysfunctional owing to various physical or chemical treatments applied to inactivate Anisakis L3, even if larvae survive these treatments and the oxygen consumption rate (OCR) could give valuable information about the actual mitochondrial function. We aimed to establish whether respiratory analysis of Anisakis L3 could identify differences between larvae considered viable, but that had been subjected to stress (thermal and/or chemical). The modulators FCCP and azide were used to obtain the basal, maximum, spare and residual respiration rates. The respiration analysis of larvae subjected to a certain temperature or environmental stress, (i.e. storage at 37 °C or in gastric juice), showed that mitochondria were affected compared to the untreated controls. The maximum respiratory capacity of larvae subjected to freezing could initially decrease immediately after thawing, but after some acclimatization they were able to recover their respiratory capacity fully. However, when treated larvae were stored at refrigeration temperatures their mitochondria became dysfunctional faster than those of untreated larvae. To conclude, Anisakis L3 responds to mitochondrial respiration modulators, so respiration analysis can be incorporated for in vivo assessment of mitochondrial function in this nematode. These measurements can be used as a tool to characterize L3 subjected to different stresses which, together with other indicators, help to give a broader picture of Anisakis L3 characteristics and potential infectivity.This work was supported by the Project ANIRISK (AGL2015-68248-C2), funded by the Spanish Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (FEDER).Peer Reviewe

Thermal patterns of heat treated Anisakis L3-infected fishery products allow separation into low, intermediate and high risk groups of potential use in risk management

Anisakis third-stage larvae (L3) is moderately tolerant to heat and, to mitigate the risk of live L3 intake in cooked seafood, it is important to define with precision at which point after heat treatment the parasite is no longer infective. We aimed to find thermal patterns that allowed to classify fish sandwiches spiked with Anisakis L3 into “low” (100% probability of mortality), “intermediate” and “high” risk groups. For that, experiments with varying set temperatures and heating times have been performed in conditions of different external heating tempera-tures. Decision points to classify the samples into terminal nodes associated to different risk groups have been obtained with decision tree analyses and then confirmed with linear discriminant analysis. Separation into two (i. e. low vs high +intermediate risk) or three (i.e. low, intermediate, and high risk) distinct thermal patterns (98% and 95.9% correct classifications by cross-validation respectively) was achieved. These results refine heating conditions reported in the EU Regulation, since reaching 60 ◦C for 1 min in the thermal centre is not sufficient to kill all L3. However, when factors such as relative temperature of heating or time to reach the set temperature are taken into account, other thermal conditions are found that are equally safe in terms of Anisakis L3 inactivation. This, together with the description of “intermediate” and “high” risk groups can help in the risk identification and management, as well as in providing clearer recommendations to consumersThis work was financed by the European Union's Seventh Framework Programme for Research, Technological Development and Demonstration under Grant Agreement 312068 (EU PARASITE) and Spanish (AGL2015-68248-C2-1-R) MINECO/FEDER (ANIRISK)

Absorption of Anisakis spp. proteins using human Caco-2 cellular model

Trabajo presentado a la 49th West European Fish Technologists Association (WEFTA), celebrada en Tórshavn (Islas Feroe) del 15 al 17 de octubre de 2019.The risk of exposure Anisakis spp. allergens is mainly through the digestive tract, its pathogenic potential depends not only on its resistance to gastrointestinal digestion but also on its ability to cross the barrier of the intestinal epithelium. Previous studies have demonstrated the resistance to gastrointestinal digestion of allergens, mainly low molecular weight (unpublished data). The aim of this piece of work was to study the ability to cross the intestinal epithelial barrier of Anisakis spp. antigens/allergens from Anisakis crude extract (CE) using human Caco-2 cellular model. The integrity of the Caco-2 cells monolayer was characterised measuring the transepithelial electrical resistance (TEER) and paracellular permeability and the analysis of the antigens/allergens transported through the epithelium was performed by SDS-PAGE and immunoblotting. The analysis of TEER shows a significant decrease after two hours in samples with Anisakis CE added. Immunodetection assays (WB) show that the passage of the allergen Ani s 4 might combine paracellular and transcellular transport. Regarding the paracellular permeability evaluated by Lucifer Yellow, the results obtained after incubation of cells with CE, support that the Anisakis CE compromises the integrity of the Caco-2 monolayer. Viability assays show there is not any cytotoxic effect. Putative mechanisms underlying the decrease in TEER have been analysed (protease and phosphatase inhibition, reactive oxygen species determination). However, we have not currently identified the mechanism involved and further studies will be performed. Associated to the TEER changes, we have observed disorganization of occludin by confocal microscopy. Anisakis allergens are able to cross the intestinal barrier, which would justify the presence of symptoms in some patients sensitized to Anisakis ssp. after the consumption of seafood products contaminated Anisakis allergens.This work was financed by the Spanish Project ANIRISK (AGL201568248C1C2) MINECO/FEDER