The gymnodimines and spirolides are phycotoxins classified into a heterogeneous group of marine biocompounds called cyclic imines. Although there is no clear evidence of their toxicity to humans, gymnodimines and spirolides are highly toxic to rodents and constitute a source of false positives in lipophilic toxin detection by the mouse bioassay. Using nicotinic acetylcholine receptor-enriched membranes of Torpedo, and fluorescent alpha-bungarotoxin, we developed a fluorescence polarization assay to detect and quantify gymnodimine-A and 13-desmethyl C spirolide. The presence of these cyclic imines in solution inhibited the interaction of fluorescent-labeled alpha-bungarotoxin with nicotinic acetylcholine receptors in a concentration-dependent manner. The sensitivity of the assay is in the order of nanomolar concentrations of gymnodimine and 13-desmethyl C spirolide. Okadaic acid, yessotoxin, and brevetoxin-2, three lipophilic marine toxins, did not interfere with this assay. A suitable extraction method in shellfish was also developed. The gymnodimine-A and 13-desmethyl C spirolide recovery rates of mussel matrix extraction with acetone/chloroform were 63.6% +/- 3.5% and 87.4% +/- 5.3%, respectively. In summary, this inhibition assay is capable of gymnodimine-A and 13-desmethyl C spirolide detection in mussel extracts with enough sensitivity and specificity to quantify these toxins in the range of 50-2000 microg/kg and 70-700 microg/kg of shellfish meat, respectively.

The detection of paralytic shellfish poisoning (PSP) toxins in contaminated shellfish is essential for human health preservation. Ethical and technical reasons have prompted the search for new detection procedures as an alternative to the mouse bioassay. On the basis of the detection of molecular interactions by surface plasmon resonance (SPR) biosensors, an inhibition assay was developed using an anti-GTX2/3 antibody (GT13-A) and a saxitoxin-CM5 chip. This assay allowed for quantification of saxitoxin (STX), decarbamoyl saxitoxin (dcSTX), gonyautoxin 2,3 (GTX2/3), decarbamoyl gonyautoxin 2,3 (dcGTX2/3), gonyautoxin 5 (GTX5), and C 1,2 (C1/2) at concentrations from 2 to 50 ng/mL. The interference of five shellfish matrixes with the inhibition assay was analyzed. Mussels, clams, cockles, scallops, and oysters were extracted with five published methods. Ethanol extracts and acetic acid/heat extracts (AOAC Lawrence method) performed adequately in terms of surface regeneration and baseline interference, did not inhibit antibody binding to the chip surface significantly, and presented STX calibration curves similar to buffer controls in all matrixes tested. Hydrochloric acid/heat extracts (AOAC mouse bioassay method) presented surface regeneration problems, and although ethanol-acetic acid/dichloromethane extracts performed well, they were considered too laborious for routine sample testing. Overall the best results were obtained with the ethanol extraction method with calibration curves prepared in blank matrix extracts. STX recovery rate with the ethanol extraction method was 60.52+/-3.72%, with variations among species. The performance of this biosensor assay in natural samples, compared to two AOAC methods for PSP toxin quantification (mouse bioassay and HPLC), suggests that this technology can be useful as a PSP screening assay. In summary, the GT13-A-STX chip inhibition assay is capable of PSP toxin detection in ethanol shellfish extracts, with sufficient sensitivity to quantify the toxin in the range of the European regulatory limit of 80 microg/100 g of shellfish meat.

The number of new daily infections is one of the main parameters to understand the dynamics of an epidemic. During the COVID-19 pandemic in 2020, however, such information has been underestimated. Here, we propose a retrospective methodology to estimate daily infections from daily deaths, because those are usually more accurately documented. Given the incubation period, the time from illness onset to death, and the case fatality ratio, the date of death can be estimated from the date of infection. We apply this idea conversely to estimate infections from deaths. This methodology is applied to Spain and its 19 administrative regions. Our results showed that probable daily infections during the first wave were between 35 and 42 times more than those officially documented on 14 March, when the national government decreed a national lockdown and 9 times more than those documented by the updated version of the official data. The national lockdown had a strong effect on the growth rate of virus transmission, which began to decrease immediately. Finally, the first inferred infection in Spain is about 43 days before the official data were available during the first wave. The current official data show delays of 15–30 days in the first infection relative to the inferred infections in 63% of the regions. In summary, we propose a methodology that allows reinterpretation of official daily infections, improving data accuracy in infection magnitude and dates because it assimilates valuable information from the National Seroprevalence Studies.