Estudio de los niveles de toxinas marinas en moluscos comerciales a la venta en grandes superficies

Traballo Fin de Grao en Veterinaria. Curso 2018-2019La presencia de algas marinas tóxicas está aumentando en todo el mundo y, por lo tanto, la acumulación de toxinas marinas en la cadena alimentaria representa una amenaza para la industria de los moluscos. Galicia es el principal productor de moluscos bivalvos en la Unión Europea (UE) y en las últimas décadas, ha sido objeto de numerosos cierres de polígonos de mejillones debido a la presencia de biotoxinas, siendo las toxinas lipofílicas las más habituales en las costas gallegas. En este trabajo se ha estudiado la presencia de toxinas lipofílicas en mejillones comerciales de la especie Mytilus galloprovincialis procedentes de 3 Rías gallegas (Ares-Sada, Arousa y Pontevedra) con el objeto de establecer el riesgo potencial para el consumidor a través de la ingesta de los moluscos. Los mejillones se compraron en grandes superficies y mercados de Lugo y se analizaron mediante cromatografía líquida acoplada a espectrometría de masas en tándem (LC-MS/MS). El análisis se llevó a cabo tanto para toxinas lipofílicas reguladas en la UE como para las toxinas emergentes.A presenza de algas mariñas tóxicas está aumentando en todo o mundo e, polo tanto, a acumulación de toxinas mariñas na cadea alimentaria representa unha ameaza para a industria dos moluscos. Galicia é o principal produtor de moluscos bivalvos na Unión Europea (UE) e nas últimas décadas, foi obxecto de numerosos peches de polígonos de mexillóns debido á presenza de biotoxinas, sendo as toxinas lipofílicas as máis habituais nas costas galegas. Neste traballo estudouse a presenza de toxinas lipofílicas en mexillóns comerciais da especie Mytilus galloprovincialis procedentes de 3 rías galegas (Ares-Sada, Arousa e Pontevedra) co obxectivo de establecer o risco potencial para o consumidor a través da inxestión dos moluscos. Os mexillóns adquiríronse en grandes superficies e mercados de Lugo e analizáronse mediante cromatografía líquida acoplada a espectrometría de masas en tándem (LC-MS/MS). A análise levouse a cabo tanto para toxinas lipofílicas reguladas na UE como para as toxinas emerxentes.The occurrence of marine harmful algae is increasing worldwide and therefore, the accumulation of marine toxins in the food chain represents a food safety threat in the shellfish industry. Galicia, which is the main producer of edible bivalve mollusc in the European Union (EU), it was subjected to recurring cases of mussel farm closures in the last decades due to the presence of biotoxins, being the lipophilic marine toxins the most common in our coasts. In this work, the presence of lipophilic marine toxins in commercial mussels Mytilus galloprovincialis from 3 Galician Rías (Ares-Sada, Arousa and Pontevedra) were studied. The objective was to stablish a potential risk through the ingest. Mussels were purchased in shopping centres and local markets in Lugo and were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The analysis was performed in both regulated lipophilic marine toxins in the EU and emerging toxins

LC–MS/MS Analysis of the Emerging Toxin Pinnatoxin-G and High Levels of Esterified OA Group Toxins in Galician Commercial Mussels

The occurrence of marine harmful algae is increasing worldwide and, therefore, the accumulation of lipophilic marine toxins from harmful phytoplankton represents a food safety threat in the shellfish industry. Galicia, which is a commercially important EU producer of edible bivalve mollusk have been subjected to recurring cases of mussel farm closures, in the last decades. This work aimed to study the toxic profile of commercial mussels (Mytilus galloprovincialis) in order to establish a potential risk when ingested. For this, a total of 41 samples of mussels farmed in 3 Rías (Ares-Sada, Arousa, and Pontevedra) and purchased in 5 local markets were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS). Chromatograms showed the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), pectenotoxin-2 (PTX-2), azaspiracid-2 (AZA-2), and the emerging toxins 13-desmethyl spirolide C (SPX-13), and pinnatoxin-G (PnTX-G). Quantification of each toxin was determined using their own standard calibration in the range 0.1%–50 ng/mL (R2 > 0.99) and by considering the toxin recovery (62–110%) and the matrix correction (33–211%). Data showed that OA and DTX-2 (especially in the form of esters) are the main risk in Galician mollusks, which was detected in 38 samples (93%) and 3 of them exceeded the legal limit (160 µg/kg), followed by SPX-13 that was detected in 19 samples (46%) in quantities of up to 28.9 µg/kg. Analysis from PTX-2, AZA-2, and PnTX-G showed smaller amounts. Fifteen samples (37%) were positive for PTX-2 (0.7–2.9 µg/kg), 12 samples (29%) for AZA-2 (0.1–1.8 µg/kg), and PnTX-G was detected in 5 mussel samples (12%) (0.4 µg/kg–0.9 µg/kg). This is the first time Galician mollusk was contaminated with PnTX-G. Despite results indicating that this toxin was not a potential risk through the mussel ingestion, it should be considered in the shellfish safety monitoring programs through the LC–MS/MS methods.This research has received funding from the following FEDER co-funded grants. From Conselleria de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From CDTI and Technological Funds, supported by Ministerio de Economía, Industria y Competitividad, AGL2014-58210-R, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830, RTC-2016-5507-2, and ITC-20161072. From the European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX. This work was also supported by the program “Juan de la Cierva 2016” from the Spanish Government. Paz Otero is recipient of a Postdoctoral Funding (Ref. IJCI-2016-27774)S

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID‑19

Background. COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. Methods. A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. Results. The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183–12.968], 0.025), viral RNA load (N1) (1.962 [1.244–3.096], 0.004); viral RNA load (N2) (2.229 [1.382–3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). Conclusions. SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID-19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li-Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the “Subvenciones de concesión directa para proyectos y programas de investigación del virus SARS‐CoV2, causante del COVID‐19”, FONDO–COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT) and fnally by the “Convocatoria extraordinaria y urgente de la Gerencia Regional de Salud de Castilla y León, para la fnanciación de proyectos de investigación en enfermedad COVID-19” (GRS COVID 53/A/20) (CA). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Infammation. APT was funded by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co-fnanced by the European Development Regional Fund (A Way to Achieve Europe programme). The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript