Immunoglobulins in COVID-19 pneumonia: from the acute phase to the recovery phase.

BACKGROUND: COVID-19 pneumonia causes hyperinflammatory response that culminates in acute respiratory syndrome (ARDS) related to increased multiorgan dysfunction and mortality risk. Antiviral-neutralizing immunoglobulins production reflect the host humoral status and illness severity, and thus, immunoglobulin (Ig) circulating levels could be evidence of COVID-19 prognosis. METHODS: The relationship among circulating immunoglobulins (IgA, IgG, IgM) and COVID-19 pneumonia was evaluated using clinical information and blood samples in a COVID-19 cohort composed by 320 individuals recruited during the acute phase and followed up to 4 to 8 weeks (n = 252) from the Spanish first to fourth waves. RESULTS: COVID-19 pneumonia development depended on baseline Ig concentrations. Circulating IgA levels together with clinical features at acute phase was highly associated with COVID-19 pneumonia development. IgM was positively correlated with obesity (ρb = 0.156, P = 0.020), dyslipemia (ρb = 0.140, P = 0.029), COPD (ρb = 0.133, P = 0.037), cancer (ρb = 0.173, P = 0.007) and hypertension (ρb = 0.148, P = 0.020). Ig concentrations at recovery phase were related to COVID-19 treatments. CONCLUSIONS: Our results provide valuable information on the dynamics of immunoglobulins upon SARS-CoV-2 infection or other similar viruses.

Matrix Metalloproteinase-9 Expression Is Associated with the Absence of Response to Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer Patients.

Triple-negative breast cancer (TNBC) is particularly challenging due to the weak or absent response to therapeutics and its poor prognosis. The effectiveness of neoadjuvant chemotherapy (NAC) response is strongly influenced by changes in elements of the tumor microenvironment (TME). This work aimed to characterize the residual TME composition in 96 TNBC patients using immunohistochemistry and in situ hybridization techniques and evaluate its prognostic implications for partial responders vs. non-responders. Compared with non-responders, partial responders containing higher levels of CD83+ mature dendritic cells, FOXP3+ regulatory T cells, and IL-15 expression but lower CD138+ cell concentration exhibited better OS and RFS. However, along with tumor diameter and positive nodal status at diagnosis, matrix metalloproteinase-9 (MMP-9) expression in the residual TME was identified as an independent factor associated with the impaired response to NAC. This study yields new insights into the key components of the residual tumor bed, such as MMP-9, which is strictly associated with the lack of a pathological response to NAC. This knowledge might help early identification of TNBC patients less likely to respond to NAC and allow the establishment of new therapeutic targets.

Prognostic Implications of the Residual Tumor Microenvironment after Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer Patients without Pathological Complete Response.

With a high risk of relapse and death, and a poor or absent response to therapeutics, the triple-negative breast cancer (TNBC) subtype is particularly challenging, especially in patients who cannot achieve a pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Although the tumor microenvironment (TME) is known to influence disease progression and the effectiveness of therapeutics, its predictive and prognostic potential remains uncertain. This work aimed to define the residual TME profile after NAC of a retrospective cohort with 96 TNBC patients by immunohistochemical staining (cell markers) and chromogenic in situ hybridization (genetic markers). Kaplan-Meier curves were used to estimate the influence of the selected TME markers on five-year overall survival (OS) and relapse-free survival (RFS) probabilities. The risks of each variable being associated with relapse and death were determined through univariate and multivariate Cox analyses. We describe a unique tumor-infiltrating immune profile with high levels of lymphocytes (CD4, FOXP3) and dendritic cells (CD21, CD1a and CD83) that are valuable prognostic factors in post-NAC TNBC patients. Our study also demonstrates the value of considering not only cellular but also genetic TME markers such as MUC-1 and CXCL13 in routine clinical diagnosis to refine prognosis modelling.

Circulating pyruvate is a potent prognostic marker for critical COVID-19 outcomes.

Background: Coronavirus-19 (COVID-19) disease is driven by an unchecked immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus which alters host mitochondrial-associated mechanisms. Compromised mitochondrial health results in abnormal reprogramming of glucose metabolism, which can disrupt extracellular signalling. We hypothesized that examining mitochondrial energy-related signalling metabolites implicated in host immune response to SARS-CoV-2 infection would provide potential biomarkers for predicting the risk of severe COVID-19 illness. Methods: We used a semi-targeted serum metabolomics approach in 273 patients with different severity grades of COVID-19 recruited at the acute phase of the infection to determine the relative abundance of tricarboxylic acid (Krebs) cycle-related metabolites with known extracellular signaling properties (pyruvate, lactate, succinate and α-ketoglutarate). Abundance levels of energy-related metabolites were evaluated in a validation cohort (n=398) using quantitative fluorimetric assays. Results: Increased levels of four energy-related metabolites (pyruvate, lactate, a-ketoglutarate and succinate) were found in critically ill COVID-19 patients using semi-targeted and targeted approaches (p<0.05). The combined strategy proposed herein enabled us to establish that circulating pyruvate levels (p<0.001) together with body mass index (p=0.025), C-reactive protein (p=0.039), D-Dimer (p<0.001) and creatinine (p=0.043) levels, are independent predictors of critical COVID-19. Furthermore, classification and regression tree (CART) analysis provided a cut-off value of pyruvate in serum (24.54 µM; p<0.001) as an early criterion to accurately classify patients with critical outcomes. Conclusion: Our findings support the link between COVID-19 pathogenesis and immunometabolic dysregulation, and show that fluorometric quantification of circulating pyruvate is a cost-effective clinical decision support tool to improve patient stratification and prognosis prediction.

Identification of New CTX Analogues in Fish from the Madeira and Selvagens Archipelagos by Neuro-2a CBA and LC-HRMS.

Ciguatera Poisoning (CP) is caused by consumption of fish or invertebrates contaminated with ciguatoxins (CTXs). Presently CP is a public concern in some temperate regions, such as Macaronesia (North-Eastern Atlantic Ocean). Toxicity analysis was performed to characterize the fish species that can accumulate CTXs and improve understanding of the ciguatera risk in this area. For that, seventeen fish specimens comprising nine species were captured from coastal waters inMadeira and Selvagens Archipelagos. Toxicity was analysed by screening CTX-like toxicity with the neuroblastoma cell-based assay (neuro-2a CBA). Afterwards, the four most toxic samples were analysed with liquid chromatography-high resolution mass spectrometry (LC-HRMS). Thirteen fish specimens presented CTX-like toxicity in their liver, but only four of these in their muscle. The liver of one specimen of Muraena augusti presented the highest CTX-like toxicity (0.270 ± 0.121 µg of CTX1B equiv·kg-1). Moreover, CTX analogues were detected with LC-HRMS, for M. augusti and Gymnothorax unicolor. The presence of three CTX analogues was identified: C-CTX1, which had been previously described in the area; dihydro-CTX2, which is reported in the area for the first time; a putative new CTX m/z 1127.6023 ([M+NH4]+) named as putative C-CTX-1109, and gambieric acid A.

Resistin and IL-15 as Predictors of Invasive Mechanical Ventilation in COVID-19 Pneumonia Irrespective of the Presence of Obesity and Metabolic Syndrome.

The cytokine signature present in COVID-19 could provide information on the pathogenic mechanisms of the disease and could identify possible prognostic biomarkers and possible therapeutic targets. In this longitudinal work, we studied the clinical and biochemical parameters and circulating cytokine levels of 146 patients at the time of admission for COVID-19 and 4-6 weeks later. The main objective of this study was to determine whether basal cytokines could be early prognostic biomarkers of COVID-19, and also to analyze the impact of comorbidities, such as obesity or metabolic syndrome (MS), in the cytokine profile. The levels of most inflammatory cytokines were elevated on admission in relation to the level that was reached 4-6 weeks later, except for IL-1ß, which was lower on admission; these levels were irrespective of the presence of obesity or MS since the cytokine storm masks these inflammatory processes. Among the cytokines analyzed, those that correlated with a worse prognosis of COVID-19 were resistin, IL-6, IL-8, IL-15, MCP-1 and TNF-α. Specifically, resistin and IL-15 are the best early predictors of requiring invasive ventilation. Therefore, resistin and IL-15 should be included in the personalized treatment decision algorithm of patients with COVID-19.

Adipokines as New Biomarkers of Immune Recovery: Apelin Receptor, RBP4 and ZAG Are Related to CD4+ T-Cell Reconstitution in PLHIV on Suppressive Antiretroviral Therapy.

A significant proportion of people living with HIV (PLHIV) who successfully achieve virological suppression fail to recover CD4+ T-cell counts. Since adipose tissue has been discovered as a key immune organ, this study aimed to assess the role of adipokines in the HIV immunodiscordant response. This is a multicenter prospective study including 221 PLHIV starting the first antiretroviral therapy (ART) and classified according to baseline CD4+ T-cell counts/µL (controls > 200 cells/µL and cases ≤ 200 cells/µL). Immune failure recovery was considered when cases did not reach more than 250 CD4+ T cells/µL at 144 weeks (immunological nonresponders, INR). Circulating adipokine concentrations were longitudinally measured using enzyme-linked immunosorbent assays. At baseline, apelin receptor (APLNR) and zinc-alpha-2-glycoprotein (ZAG) concentrations were significantly lower in INRs than in immunological responders (p = 0.043 and p = 0.034), and they remained lower during all ART follow-up visits (p = 0.044 and p = 0.028 for APLNR, p = 0.038 and p = 0.010 for ZAG, at 48 and 144 weeks, respectively). ZAG levels positively correlated with retinol-binding protein 4 (RBP4) levels (p < 0.01), and low circulating RBP4 concentrations were related to a low CD4+ T-cell gain (p = 0.018 and p = 0.039 at 48 and 144 weeks, respectively). Multiple regression adjusted for clinical variables and adipokine concentrations confirmed both low APLNR and RBP4 as independent predictors for CD4+ T cells at 144 weeks (p < 0.001). In conclusion, low APLNR and RBP4 concentrations were associated with poor immune recovery in treated PLHIV and could be considered predictive biomarkers of a discordant immunological response.

Epigenome-wide association study of COVID-19 severity with respiratory failure.

BACKGROUND: Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the coronavirus disease 2019 (COVID-19), exhibit a wide spectrum of disease behaviour. Since DNA methylation has been implicated in the regulation of viral infections and the immune system, we performed an epigenome-wide association study (EWAS) to identify candidate loci regulated by this epigenetic mark that could be involved in the onset of COVID-19 in patients without comorbidities. METHODS: Peripheral blood samples were obtained from 407 confirmed COVID-19 patients ≤ 61 years of age and without comorbidities, 194 (47.7%) of whom had mild symptomatology that did not involve hospitalization and 213 (52.3%) had a severe clinical course that required respiratory support. The set of cases was divided into discovery (n = 207) and validation (n = 200) cohorts, balanced for age and sex of individuals. We analysed the DNA methylation status of 850,000 CpG sites in these patients. FINDINGS: The DNA methylation status of 44 CpG sites was associated with the clinical severity of COVID-19. Of these loci, 23 (52.3%) were located in 20 annotated coding genes. These genes, such as the inflammasome component Absent in Melanoma 2 (AIM2) and the Major Histocompatibility Complex, class I C (HLA-C) candidates, were mainly involved in the response of interferon to viral infection. We used the EWAS-identified sites to establish a DNA methylation signature (EPICOVID) that is associated with the severity of the disease. INTERPRETATION: We identified DNA methylation sites as epigenetic susceptibility loci for respiratory failure in COVID-19 patients. These candidate biomarkers, combined with other clinical, cellular and genetic factors, could be useful in the clinical stratification and management of patients infected with the SARS-CoV-2. FUNDING: The Unstoppable campaign of the Josep Carreras Leukaemia Foundation, the Cellex Foundation and the CERCA Programme/Generalitat de Catalunya.

Predictive Biomarkers of COVID-19 Severity in SARS-CoV-2 Infected Patients with Obesity and Metabolic Syndrome.

In SARS-CoV-2-infected patients, obesity, hypertension, and diabetes are dangerous factors that may result in death. Priority in detection and specific therapies for these patients are necessary. We wanted to investigate the impact of obesity and metabolic syndrome (MS) on the clinical course of COVID-19 and whether prognostic biomarkers described are useful to predict the evolution of COVID-19 in patients with obesity or MS. This prospective cohort study included 303 patients hospitalized for COVID-19. Participants were first classified according to the presence of obesity; then, they were classified according to the presence of MS. Clinical, radiologic, and analytical parameters were collected. We reported that patients with obesity presented moderate COVID-19 symptoms and pneumonia, bilateral pulmonary infiltrates, and needed tocilizumab more frequently. Meanwhile, patients with MS presented severe pneumonia and respiratory failure more frequently, they have a higher mortality rate, and they also showed higher creatinine and troponin levels. The main findings of this study are that IL-6 is a potential predictor of COVID-19 severity in patients with obesity, while troponin and LDH can be used as predictive biomarkers of COVID-19 severity in MS patients. Therefore, treatment for COVID-19 in patients with obesity or MS should probably be intensified and personalized.

High circulating SDF-1and MCP-1 levels and genetic variations in CXCL12, CCL2 and CCR5: Prognostic signature of immune recovery status in treated HIV-positive patients.

BACKGROUND: The underlying mechanisms of incomplete immune reconstitution in treated HIV-positive patients are very complex and may be multifactorial, but perturbation of chemokine secretion could play a key role in CD4+T-cell turnover. METHODS: We evaluated the circulating baseline and 48-week follow-up concentrations of SDF-1/CXCL12, fractalkine/CX3CL1, MCP-1/CCL2, MIP-α/CCL3, MIP-ß/CCL4 and RANTES/CCL5, and we estimated their association with CXCL12, CX3CR1, CCR2, CCL5 and CCR5 single nucleotide polymorphisms (SNPs) to investigate multiple chemokine-chemokine receptor signatures associated with immune dysregulation preceding poor immune recovery. FINDINGS: The circulating concentrations and gene expression patterns of SDF-1/CXCL12 (CXCL12 rs1801157) and MCP-1/CCL2 (CCR2 rs1799864_814) were associated with immune recovery status. CCR2 rs1799864_814 and CCR5 rs333_814 (Δ32) determine the baseline plasma RANTES and MIP-α concentrations, respectively, in participants with poor immune response. INTERPRETATION: SDF-1/CXCL12 and MCP-1/CCL2 could be considered prognostic markers of immune failure despite suppressive antiretroviral therapy. The strong linkage disequilibrium (LD) between CCR2 rs1799864_814 and CCR5 rs1800024 indicated that the alleles of each gene are inherited together more often than would be expected by chance. FUNDING: This work was supported by Fondo de Investigacion Sanitaria and SPANISH AIDS Research Network (ISCIII-FEDER); AGAUR and Gilead Fellowship. FV and YMP are supported by grants from the Programa de Intensificación (ISCIII) and Servicio Andaluz de Salud, respectively. JVG,EY and LR are supported by the Instituto de Salud Carlos III (ISCIII). AR is supported by Departament de Salut, Generalitat de Catalunya and by the Instituto de Salud Carlos III (ISCIII).

Bioaccessibility of lipophilic and hydrophilic marine biotoxins in seafood: An in vitro digestion approach.

This study aimed to assess the bioaccessibility of different marine biotoxins in naturally contaminated shellfish and fish gonads using an in vitro digestion methodology. In general, hydrophilic toxins (domoic acid, paralytic shellfish poisoning toxins and tetrodotoxins) showed higher bioaccessibility than lipophilic ones (okadaic acid and azaspiracids). The bioaccessibility of toxins from the okadaic acid group ranged from 69% (raw European razor clams) to 74% (raw donax clams). Regarding azaspiracids, 47% of the initial content was bioaccessible in steamed blue mussel. As for hydrophilic toxins, 100% of the initial content was bioaccessible after digestion in raw shellfish and puffer fish gonads. The total tetrodotoxin bioaccessibility in puffer fish gonads decreased significantly after steaming. The profile of tetrodotoxins changed during the digestion process: TTX and 11-norTTX-6S-ol analogues decreased significantly after digestion, but the 5,6,11-trideoxy TTX analogue increased in both raw and steamed puffer fish gonads. These preliminary findings confirm the need to consider bioaccessibility data in future seafood risk assessment, as such information enables a more accurate and realistic estimation of potential seafood hazards, particularly in what concerns lipophilic toxins, therefore, constituting a crucial tool in the refinement of regulatory limits for the presence of biotoxins in seafood.

Development and validation of a maleimide-based enzyme-linked immunosorbent assay for the detection of tetrodotoxin in oysters and mussels.

The recent detection of tetrodotoxins (TTXs) in puffer fish and shellfish in Europe highlights the necessity to monitor the levels of TTXs in seafood by rapid, specific, sensitive and reliable methods in order to protect human consumers. A previous immunoassay for TTX detection in puffer fish, based on the use of self-assembled monolayers (SAMs) for the immobilization of TTX on maleimide plates (mELISA), has been modified and adapted to the analysis of oyster and mussel samples. Changing dithiol for cysteamine-based SAMs enabled reductions in the assay time and cost, while maintaining the sensitivity of the assay. The mELISA showed high selectivity for TTX since the antibody did not cross-react with co-occurring paralytic shellfish poisoning (PSP) toxins and no interferences were observed from arginine (Arg). Moreover, TTX-coated maleimide plates stored for 3 months at -20°C and 4°C were stable, thus when pre-prepared, the time to perform the assay is reduced. When analyzing shellfish samples, matrix effects and toxin recovery values strongly depended on the shellfish type and the sample treatment. Blank oyster extracts could be directly analyzed without solid-phase extraction (SPE) clean-up, whereas blank mussel extracts showed strong matrix effects and SPE and subsequent solvent evaporation were required for removal. However, the SPE clean-up and evaporation resulted in toxin loss. Toxin recovery values were taken as correction factors (CFs) and were applied to the quantification of TTX contents in the analysis of naturally-contaminated shellfish samples by mELISA. The lowest effective limits of detection (eLODs) were about 20 and 50µg/kg for oyster extracts without and with SPE clean-up, respectively, and about 30µg/kg for mussel extracts with both protocols, all of them substantially below the eLOD attained in the previous mELISA for puffer fish (230µg/kg). Analysis of naturally-contaminated samples by mELISA and comparison with LC-MS/MS quantifications demonstrated the viability of the approach. This mELISA is a selective and sensitive tool for the rapid detection of TTX in oyster and mussel samples showing promise to be implemented in routine monitoring programs to protect human health.

Immunosensor array platforms based on self-assembled dithiols for the electrochemical detection of tetrodotoxins in puffer fish.

The recent detection of tetrodotoxins (TTXs) in European fish and shellfish has emphasized the urgent need to develop specific, selective, rapid and easy-to-use methods for their detection to assess the potential risk posed to human health. For this purpose, a dithiol self-assembled monolayer (SAM)-based immunoassay previously performed on maleimide plates (mELISA) has been adapted to gold electrode arrays for the development of an electrochemical immunosensor for TTX. The electrochemical SAM-based immunosensor designed herein, provided an oriented, stable and spaced sensing platform for the determination of TTX, attaining a limit of detection of 2.6 ng mL-1. The applicability of the biosensor array was demonstrated by the accurate quantifications obtained in the analysis of different tissues of several puffer fish species (Lagocephalus lagocephalus, L. sceleratus and Sphoeroides pachygaster) caught along the Mediterranean coast of Spain. The good agreements found between the TTX concentrations determined by the immunosensor array platforms and those determined by mELISA, surface Plasmon resonance (SPR) immunosensor and liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis, proved the feasibility of the approach. The electrochemical immunosensor enables the determination of TTXs at levels as low as 0.07 mg TTX equiv. kg-1 tissue, thus, well below the Japanese value of 2 mg TTX equiv. kg-1 tissue used as a criterion to consider puffer fish safe for consumption. Compared to the colorimetric SAM-based approach, the immunosensor array described herein shows promise towards the development of disposable, portable and compact analysis tools applicable in monitoring programs for the surveillance of fishery products.

Identification of ciguatoxins in a shark involved in a fatal food poisoning in the Indian Ocean.

Severe food poisoning events after the consumption of sharks have been reported since the 1940s; however, there has been no clear understanding of their cause. Herein, we report for the first time the presence of ciguatoxins (CTXs) in sharks. The identification by mass spectrometry of CTXs, including two new analogues, in a bull shark (Carcharhinus leucas) that was consumed by humans, causing the poisoning and death of 11 people in Madagascar in 2013 is described. Typical neurotoxic ciguatera symptoms were recorded in patients, and toxicological assays on extracts of the shark demonstrated CTX-like activity. These results confirm this episode as a ciguatera poisoning event and expand the range of pelagic fish species that are involved in ciguatera in the Indian Ocean. Additionally, gambieric acid D, a molecule originally described in CTX-producing microalgae, was identified for the first time in fish. This finding can contribute to a better understanding of trophic relations within food webs. The present work confirms that consumption of sharks from the Indian Ocean should be considered a ciguatera risk, and actions should be taken to evaluate its magnitude and risk in order to manage shark fisheries.

Evaluation of tetrodotoxins in puffer fish caught along the Mediterranean coast of Spain. Toxin profile of Lagocephalus sceleratus.

Although consumption of Tetraodontidae species is prohibited in the EU, intoxications are still reported. The evaluation of tetrodotoxins (TTXs) by mass spectrometry (LC-MS/MS and LC-HRMS) and a screening immunoassay (mELISA) in tetraodontid fishes caught along the Western Mediterranean Sea revealed high concentrations of TTXs in Lagocephalus sceleratus while no TTXs were identified in L. lagocephalus and Sphoeroides pachygaster individuals. The high TTXs content found in the L. sceleratus analysed herein demonstrate the occurrence of highly toxic puffer fish in the Western Mediterranean Sea. Being L. sceleratus a recent invasive species in the Mediterranean, surveillance, risk assessment and risk management measures are necessary. The strategy used within this research work could be a valuable tool for future food safety monitoring.

New advances in electrochemical biosensors for the detection of toxins: Nanomaterials, magnetic beads and microfluidics systems. A review.

The use of nanotechnology in bioanalytical devices has special advantages in the detection of toxins of interest in food safety and environmental applications. The low levels to be detected and the small size of toxins justify the increasing number of publications dealing with electrochemical biosensors, due to their high sensitivity and design versatility. The incorporation of nanomaterials in their development has been exploited to further increase their sensitivity, providing simple and fast devices, with multiplexed capabilities. This paper gives an overview of the electrochemical biosensors that have incorporated carbon and metal nanomaterials in their configurations for the detection of toxins. Biosensing systems based on magnetic beads or integrated into microfluidics systems have also been considered because of their contribution to the development of compact analytical devices. The roles of these materials, the methods used for their incorporation in the biosensor configurations as well as the advantages they provide to the analyses are summarised.

Detection of Tetrodotoxins in Puffer Fish by a Self-Assembled Monolayer-Based Immunoassay and Comparison with Surface Plasmon Resonance, LC-MS/MS, and Mouse Bioassay.

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

Alternative methods for the detection of emerging marine toxins: biosensors, biochemical assays and cell-based assays.

The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs.