HOW TO DETECT CTXs?
CTXs detection represents a significant technical challenge, due to their chemical nature, the multiplicity of congeners and the low levels of toxins present in contaminated organisms. However, even if several detection tests are now available, currently there is no standard test duly validated by the scientific community, that could enable public authorities to establish a seafood security regulation at an international level. To date, the only reliable methods to detect CTXs are laboratory tests based on toxins mode of action (functional tests), or chemical properties.
IN VIVO TESTS
Biological test on mouse or “Mouse bio-assay” (MBA) was the first CTXs detection test used. It is based on the symptoms and mice survival time observed after a 24h-48h period, following an intraperitoneal (ip) or intravenous (iv) injection of toxic fish extract. Several animal species other than mice have been used: cats, chicken or mongooses that have a better CTXs sensitivity but require large amount of extracts or, conversely, methods using invertebrates, such as mosquitoes, crayfish, fly larvae or shrimps, which require lesser amounts of the extract. These tests were gradually replaced by other methods following the 3R rule: “Reduce, Refine, Replace”, based on CTXs chemical, pharmacological and immunological properties.
The radioligand-receptor test or “Receptor binding-assay” (RBA) is a neuropharmacological test based on the specific affinity of CTXs and brevetoxins (PbTxs) to the site 5 of VSSC’s alpha subunits found on excitables cells membranes. Concerning CTXs detection, RBA measures the binding of a radiolabelled toxin, tritiated PbTx ([3H]PbTx-3), to this receptor, which compete with unradiolabled CTXs contained within the extract to analyse. Well adapted to CTXs detection in complex and varied biological matrices, RBA offers a high sensitivity (10-10M detection limit) and allows the use of untreated or partially purified extracts. It is economically more viable than the mouse bioassay, as it is easily automated to allow maximum processing capacity, making it an ideal tool for large-scale ciguatera risk monitoring programs. However, due to the regulatory constraints imposed by radioelements storing and handling, it appears difficult to generalize this test. But, recent labeling of brevetoxin with a fluorescent element gives hope for a greater applicability of this test.
Cell toxicity test or “Cell based-assay” (CBA) is another functional test allowing to measure the “overall toxicity” of a sample, by measuring the viability of a cultured cells line exposed to toxic extracts. This test is commonly used for the detection of a wide range of marine toxins: e.g. those active on VSSCs (saxitoxins, tetrodotoxins, brevetoxins and ciguatoxins), those active on Na+/K+ ATPases pumps (palytoxins), maitotoxins acting on voltage sensitive calcium channels or VSCC, okadaic acid which inhibits serine / threonine protein phosphatases, or pectenotoxins and dinophysistoxins…Besides its capacity to detect a wide range of marine biotoxins, CBA is also very sensitive (10-12M) and replicable, making it an excellent CTXs standard detection test candidate.
Various immunological assays have been developed for CTXs screening: the radioimmunoassay (RIA) or the “sandwich” test or enzyme-linked immunosorbent assay (ELISA). These tests are based on the principle of a highly specific recognition between an antibody (CTXs anti-antibody) and its antigen (CTXs). Theoretically, this approach seems to be the most promising one for the implementation of a fast, reliable, sensitive (up to 5×10-12M) and cheaper screening test. Its operating principle could also enable high-throughput screening of marine samples, and, most of all, its direct use on the field by individuals. Two trials of developing such a test have been tried: the CIGUATECT ™ and Cigua-Check ® (ToxiTec Inc. / Oceanit). But, these test kits have been withdrawn from the market, partly due to high false positives and false negatives reactions.
CTXs’ complexity and chemical diversity, their low natural immunogenicity related to their polycyclic polyether nature, as well as the limited availability of pure standards of CTXs, partially, explains the apparent difficulties to develop a reliable test.
Physicochemical tests (e.g. HPLC, LC-MS / MS) are based on high performance liquid chromatography techniques coupled with detection of each toxins families using Ultra-violet (UV), fluorescence, or tandem mass spectrometry. With a great sensitivity, these tests allow distinction and quantization of the different CTXs congeners within the same toxic family, but they require, as a prerequisite, to have the corresponding pure standards of the toxins. Therefore, the main limitations of this technique are that it does not detect new toxic families and, unlike the so-called functional tests, it doesn’t provide indication on the fish sample “whole toxicity”. Also, this type of methodology appears difficult to adapt to a CTXs high-throughput screening due to the several preliminary purification steps of the biological matrices. Therefore, these tests are most of the time used as confirmatory testing.
As the contaminated organisms cannot be identified by their appearance, odor, color or taste, island populations (which are highly exposed to CP risk), have gradually developed a wide range of traditional tests in attempt to detect ciguatoxic specimens. Across south pacific territories, several detection methods coming form popular beliefs or long ancestral practices are used. These traditional tests consist in 1/ giving a piece of flesh or liver of the suspicious fish to an animal or insect and observing its reaction; 2/ observing the oxydation of a silver coin or some matches in contact with the flesh; or 3/ observing the appearance of the whole fish or some of its organs.
A study has verified the effectiveness of two traditional detection tests used in French Polynesia (the rigor mortis method and hemorrhagic test). The ciguatoxic status of fisf samples, based on the use of the two tests by local fishermen, was compared to toxicity analysis, of the same samples, by Receptor Binding Assay (RBA). Despite a predictability rate not exceeding 70%, the use of these tests combined with the population knowledge on suspicious toxic species and fishing areas, may help to significantly reduce the risk of CP within fish dependent communities, at the condition that the test users are accustomed to these tests. The opportunity for island populations of remote archipelagos to use on site and cost-effective validated traditional tests may therefore represent a day to day valuable asset in ciguatera risk management.
Examples of traditional tests used by French Polynesia’s fishermen to differentiate toxic fish over healthy fish.