Histamine and Scombrotoxins.

Histamine intoxications result when histamine-metabolizing enzymes are compromised or overwhelmed by dietary histamine in the human body. This can occur either due to metabolic enzyme deficiencies, such as in histamine intolerance to wines, aged cheese and other foods or from high concentrations of histamine following ingestion of decomposed fish. The presence of histamine in decomposed fish and fish products results from bacterial decarboxylation of free L-histidine following product mishandling. Consequently, histamine intoxications from mishandled fish, commonly referred to as scombrotoxin fish poisoning (SFP) or scombroid poisoning, require high levels of free L-histidine only found in certain species of pelagic fish. Differential diagnosis is required of clinicians since dietary histamine intoxications produce the same symptoms typical of release of endogenous histamine due to IgE -mediated seafood allergies or anisakiasis. Although high levels of dietary histamine are responsible for SFP, histamine has important physiological functions and tends to exert toxic effects only at doses beyond the physiological range. Endogenous histamine is essential to local immune responses, regulation of gastric acid secretion in the gut, and neurotransmission in the central nervous system. Scombrotoxins, postulated to explain histamine's augmented toxicity in scombrotoxic fish, are a milieu of histamine and other bioactives. Since time-and-temperature abuse is required to produce high levels of histamine in fish, management consists of ensuring proper handling by identifying hazards and critical control points (HACCP) and maintaining a "cold chain" from catch to consumption. Reference methods for detecting histamine have received increased attention and the European Commission has validated a popular precolumn dansylation-based HPLC method through inter-laboratory collaboration and studied method equivalence with the AOAC fluorescence method 977.13 recognized by Codex Alimentarius. Much progress has been made during the last decade in the development and validation of rapid screening methods for detecting histamine in food and especially in fish products. These include many innovative sensors and several validated commercial test kits, many of them based on a recombinant form of the enzyme histamine dehydrogenase (HD).

A Rapid Method for the Detection of Diarrhetic Shellfish Toxins and Azaspiracid Shellfish Toxins in Washington State Shellfish by Liquid Chromatography Tandem Mass Spectrometry.

BACKGROUND: Diarrhetic shellfish toxins (DSTs) in domestic shellfish and azaspiracids (AZAs) in imported products are emerging seafood safety issues in the United States. In addition to causing gastrointestinal illnesses, some of these toxins are also carcinogenic and genotoxic. Efficient analytical strategies are needed for their monitoring in U.S. domestic and imported shellfish. OBJECTIVE: In the US, DSTs and AZAs are the only lipophilic shellfish toxins addressed in regulations. Streamlining of existing methods for several classes of lipophilic toxins, based on liquid chromatography coupled with triple quadrupole mass spectrometry, was pursued. METHOD: The resulting simplified LC-MS/MS method is focused on the separation and detection of just the AZAs and total DSTs using a C18 Hypersil gold column. Filter vials are used to expedite and simplify sample handling. RESULTS: The method has a run time of 7.25 min. LOQs for the AZAs and DSTs in shellfish were 0.3-0.4 µg/kg. Recoveries (AZAs and total DSTs) for three spiking levels in three matrixes ranged from 68 to 129%. Trueness was established using certified reference materials. Method equivalence was established using shellfish provided blind by the Washington State Department of Health Public Health Laboratory (WA DOH PHL). Data obtained from these samples agreed well with data from another LC-MS/MS method used in harvest control by WA DOH PHL (R = 0.999; P < 0.0001). CONCLUSIONS: The LC-MS/MS method described offers more rapid sample handling and has excellent sensitivity, linearity, and repeatability.

Ultrahigh-Performance Hydrophilic Interaction Liquid Chromatography with Tandem Mass Spectrometry Method for the Determination of Paralytic Shellfish Toxins and Tetrodotoxin in Mussels, Oysters, Clams, Cockles, and Scallops: Collaborative Study.

BACKGROUND: An ultrahigh-performance LC (UHPLC)-tandem MS (MS/MS) method for determination of paralytic shellfish poisoning toxins and tetrodotoxin (TTX) in bivalve molluscs was developed. To be used for regulatory testing, it needed to be validated through collaborative study. OBJECTIVE: The aim was to conduct a collaborative study with 21 laboratories, using results to assess method performance. METHODS: Study materials incorporated shellfish species mussels, oysters, cockles, scallops, and clams and were assessed to demonstrate stability and homogeneity. Mean concentrations determined by participants for blind duplicate samples were used to assess reproducibility, repeatability, and trueness. RESULTS: Method performance characteristics were excellent following statistical assessment of participant data, with method trueness showing excellent method accuracy against expected values. No significant difference was found in the trueness results determined by different chromatographic column types. Acceptability of the between-laboratory reproducibility for individual analytes was evidenced by >99% of valid Horwitz ratio values being less than the 2.0 limit of acceptability. With excellent linearity and sensitivity fit-for-purpose over a range of mass spectrometer instruments, the UHPLC-MS/MS method compared well against other detection methods. It includes additional paralytic shellfish toxin (PST) analogues as well as TTX, which, to date, have not been incorporated into any other hydrophilic marine toxin official method of analysis. CONCLUSIONS: The results from this study demonstrate that the method is suitable for the analysis of PST analogues and TTX in shellfish tissues and is recommended as an official alternative method of analysis for regulatory control. HIGHLIGHTS: A new mass spectrometric method for PST and TTX has been validated successfully through collaborative study.

Remifentanil for Sedation of Children With Traumatic Brain Injury.

OBJECTIVE: To determine whether remifentanil would provide adequate sedation while allowing frequent and reproducible neurologic assessments in children admitted to the pediatric intensive care unit (PICU) with traumatic brain injury (TBI) during mechanical ventilation. DESIGN: Retrospective review. SETTING: Tertiary care PICU. PATIENTS: Thirty-eight patients over a 30-month period. MEASUREMENTS AND MAIN RESULTS: Median age was 9 years (interquartile range [IQR] 2.25-12 years). The median Glasgow Coma Scale (GCS) was 9 (IQR: 8-10). All patients were tracheally intubated and receiving mechanical ventilation. A continuous infusion of remifentanil was started at 0.1 µg/kg/min, and bolus doses of 0.25 to 1 µg/kg were administered every 3 to 5 minutes as needed to reach the desired sedation level. Infusions were stopped at least hourly to perform neurologic examinations. The median remifentanil dose was 0.25 µg/kg/min with an IQR of 0.1 and 0.6 µg/kg/min. The maximum dose for any patient in the cohort was 2 µg/kg/min. Median duration of therapy with remifentanil was 20 hours (IQR: 8-44 hours). Adequate sedation was achieved with sedation scores (State Behavioral Scale) meeting target levels with a median value of 100% of the time (IQR: 79%-100%). Neurologic examinations were able to be performed within a median of 9 minutes (IQR: 5-14 minutes) of pausing the infusion. No serious safety events occurred. In 68% of the patients, neurologic examinations remained reassuring during remifentanil infusion, and patients were extubated. The remaining patients were transitioned to traditional sedative agents for long-term management of their traumatic injuries once the neurologic status was deemed stable. CONCLUSION: This data suggest that remifentanil is a suitable sedative agent for use in children with TBI. It provides a rapid onset of sedation with recovery that permits reliable and reproducible clinical examination.

Validation Study of MaxSignal® Histamine Enzymatic Assay for the Detection of Histamine in Fish/Seafood.

Bioo Scientific Corp. has developed a rapid enzymatic quantitative assay for the determination of histamine in seafood. Fresh/frozen tuna, canned tuna, pouched tuna, and frozen mahi mahi samples were used for the validation study under the specific guidelines of the AOAC Research Institute Performance Tested MethodsSM program. Recoveries ranged from 82 to 107% at concentrations ranging from 6 to 72 ppm, with RSDr values between 0.8 and 6.5% (6-72 ppm). The linearity of the assay ranged from 0 to 108 ppm, with R2 values exceeding 0.99. The LOD was 0.9 ppm and the LOQ was 2.6 ppm for frozen tuna, which gave the lowest background level of contaminant. Cross-reactivity of the assay was tested against 14 other biogenic amines and was found to be minimal for all (<0.5%), except for agmatine (4.1%) and putrescine (0.9%). There was no observable interference from any tested biogenic amines. Product consistency was verified by validating lot-to-lot variations and variations within the same lot. Overall recoveries for all tested matrixes were within the acceptable range (80-120%). A 1-year claimed shelf life of the kit at 4°C was verified by accelerated stability study data collected on days 1, 15, and 32 at 25°C and by real-time stability testing at 1-month, 6-month, and 1-year at 4°C. No difference in histamine detection was observed in ruggedness testing, in which minor changes were introduced to the assay protocol. Good agreement was observed between AOAC Official MethodSM 977.13 and the MaxSignal® Histamine Enzymatic Assay method. Independent laboratory testing demonstrated that the MaxSignal method works with the same precision in the hands of minimally trained technicians as with the expert method developers. This study validates the performance of Bioo Scientific's rapid enzymatic method.

Dexmedetomidine for Sedation During Noninvasive Ventilation in Pediatric Patients.

OBJECTIVES: To describe the use of dexmedetomidine for sedation in a large cohort of nonintubated children with acute respiratory insufficiency receiving noninvasive ventilatory support. DESIGN: Single-center, retrospective, observational cohort study. SETTING: A large quaternary-care PICU. PATIENTS: The study cohort included 202 children receiving noninvasive ventilatory and a dexmedetomidine infusion within 48 hours of PICU admission over a 6-month period. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary respiratory diagnoses in the cohort (median age, 2 yr) included status asthmaticus (60%) and bronchiolitis (29%). Dexmedetomidine was infused for a median of 35 hours with a median hourly dose across the patient cohort of 0.61 µg/kg/hr (range, 0.4-0.8 µg/kg/hr). The target sedation level was achieved in 168 patients (83%) in the cohort for greater than or equal to 80% of the recorded values over the entire noninvasive ventilatory course, with dexmedetomidine as the only continuously administered sedative agent. While vital signs were frequently abnormal relative to age-based norms, clinical interventions were needed rarely to treat bradycardia (13%), hypotension (20%), and hypopnea (5%). The most frequently used of these interventions was a dexmedetomidine dose reduction, fluid bolus, and titration of noninvasive ventilatory support. Five patients (2.5%) required endotracheal intubation: three due to progression of their respiratory illness, one with septic shock, and one with apnea requiring resuscitation. In 194 of 202 patients (96%), the outcome of the noninvasive ventilatory course was successful with the patient being weaned from noninvasive respiratory support to nasal cannula or room air. CONCLUSIONS: Dexmedetomidine was often effective as a single continuous sedative infusion during pediatric noninvasive ventilatory. Cardiorespiratory events associated with its use were typically mild and/or reversible with dose reduction, fluid administration, and/or noninvasive ventilatory titration. Prospective studies comparing dexmedetomidine with other agents in this setting are warranted.

Validation of the Biofish-300 HIS Enzymatic Biosensor for the Detection of Histamine in Fishery Products.

The Biofish-300 HIS method is a simple, reliable, and specific enzymatic biosensor for the detection of histamine. This technology is highly specific and selective and allows quantification of histamine in fishery products (fresh/frozen and processed) in a short time frame (2-3 min). Histamine in raw tuna, raw mackerel, raw sardine, raw anchovy, boiled tuna, canned tuna in water, canned tuna in oil, canned mackerel in tomato sauce, canned pickled sardine, and canned salted anchovy was analyzed using a water-based extract. Matrix-specific assay procedures and calibration curves were used to enable analyses to be carried out across multiple sample types. The performance of this assay was examined using samples that were naturally contaminated (reference materials and interlaboratory studies) and spiked with histamine. All data were judged against previously established acceptance criteria. Performance measures were evaluated for linearity, selectivity, matrix, lot consistency, and robustness. Results produced in all performance measures, except robustness, were within acceptable ranges. Out-of-range robustness results reflected deviation in sample volume compared to the standard assay procedures. Positive interferences from the presence of agmatine were shown.

Potential Threats Posed by Tetrodotoxins in UK Waters: Examination of Detection Methodology Used in Their Control.

Tetrodotoxin is a neurotoxin responsible for many human fatalities, most commonly following the consumption of pufferfish. Whilst the source of the toxin has not been conclusively proven, it is thought to be associated with various species of marine bacteria. Whilst the toxins are well studied in fish and gastropods, in recent years, there have been a number of reports of tetrodotoxin occurring in bivalve shellfish, including those harvested from the UK and other parts of Europe. This paper reviews evidence concerning the prevalence of tetrodotoxins in the UK together with methodologies currently available for testing. Biological, biomolecular and chemical methods are reviewed, including recommendations for further work. With the recent development of quantitative chromatographic methods for these and other hydrophilic toxins, as well as the commercial availability of rapid testing kits, there are a number of options available to ensure consumers are protected against this threat.

Potential threats posed by new or emerging marine biotoxins in UK waters and examination of detection methodology used in their control: brevetoxins.

Regular occurrence of brevetoxin-producing toxic phytoplankton in commercial shellfishery areas poses a significant risk to shellfish consumer health. Brevetoxins and their causative toxic phytoplankton are more limited in their global distribution than most marine toxins impacting commercial shellfisheries. On the other hand, trends in climate change could conceivably lead to increased risk posed by these toxins in UK waters. A request was made by UK food safety authorities to examine these toxins more closely to aid possible management strategies, should they pose a threat in the future. At the time of writing, brevetoxins have been detected in the Gulf of Mexico, the Southeast US coast and in New Zealand waters, where regulatory levels for brevetoxins in shellfish have existed for some time. This paper reviews evidence concerning the prevalence of brevetoxins and brevetoxin-producing phytoplankton in the UK, together with testing methodologies. Chemical, biological and biomolecular methods are reviewed, including recommendations for further work to enable effective testing. Although the focus here is on the UK, from a strategic standpoint many of the topics discussed will also be of interest in other parts of the world since new and emerging marine biotoxins are of global concern.

Flow cytometric-membrane potential detection of sodium channel active marine toxins: application to ciguatoxins in fish muscle and feasibility of automating saxitoxin detection.

Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.

Scombroid poisoning: a review.

Scombroid poisoning, also called histamine fish poisoning, is an allergy-like form of food poisoning that continues to be a major problem in seafood safety. The exact role of histamine in scombroid poisoning is not straightforward. Deviations from the expected dose-response have led to the advancement of various possible mechanisms of toxicity, none of them proven. Histamine action levels are used in regulation until more is known about the mechanism of scombroid poisoning. Scombroid poisoning and histamine are correlated but complicated. Victims of scombroid poisoning respond well to antihistamines, and chemical analyses of fish implicated in scombroid poisoning generally reveal elevated levels of histamine. Scombroid poisoning is unique among the seafood toxins since it results from product mishandling rather than contamination from other trophic levels. Inadequate cooling following harvest promotes bacterial histamine production, and can result in outbreaks of scombroid poisoning. Fish with high levels of free histidine, the enzyme substrate converted to histamine by bacterial histidine decarboxylase, are those most often implicated in scombroid poisoning. Laboratory methods and screening methods for detecting histamine are available in abundance, but need to be compared and validated to harmonize testing. Successful field testing, including dockside or on-board testing needed to augment HACCP efforts will have to integrate rapid and simplified detection methods with simplified and rapid sampling and extraction. Otherwise, time-consuming sample preparation reduces the impact of gains in detection speed on the overall analysis time.

Flow cytometric detection of saxitoxins using fluorescent voltage-sensitive dyes.

By virtue of their ability to block depolarization of nerve cells, the saxitoxins exert the toxic effects associated with paralytic shellfish poisoning and allow for their detection through various methodologies. When veratridine-induced depolarization is followed using voltage-sensitive fluorescent dyes, the presence of these toxic blocking agents can be observed as a decrease in fluorescence of dye-treated nerve cells. Detection using flow cytometry provides for selection of the most responsive population of cultured mouse neuroblastoma (Neuro 2a) cells thereby enhancing assay sensitivity and this approach can be accomplished in real time. The method is demonstrated in preliminary studies using saxitoxin and crude shellfish extracts.

Marine and freshwater toxins.

In a very busy and exciting year, 2005 included First Action approval of a much needed official method for paralytic shellfish toxins and multiple international toxin symposia highlighted by groundbreaking research. These are the first-year milestones and activities of the Marine and Freshwater Toxins Task Force and Analytical Community. Inaugurated in 2004 and described in detail in last year's General Referee Report (1) this international toxins group has grown to 150 members from many regions and countries. Perhaps most important they are now making important and global contributions to food safety and to providing alternatives to animal-based assays. Official Method 2005.06 was first approved in late 2004 by the Task Force and subsequently Official First Action in 2005 (2) by the Methods Committee on Natural Toxins and Food Allergens and the Official Methods Board. This nonproprietary method (3) is a precolumn oxidation, liquid chromatographic method that makes good use of fluorescence detection to provide high sensitivity detection of the saxitoxins. It has also proven to be rugged enough for regulatory use and the highest level of validation. As pointed out in the report of method principle investigator and Study Director James Lawrence, approval of 2005.06 now provides the first official alternative to the mouse bioassay after many decades of shellfish monitoring. This past year in April 2005 the group also held their first international conference, "Marine and Freshwater Toxins Analysis: Ist Joint Symposium and AOAC Task Force Meeting," in Baiona, Spain. The 4-day conference consisted of research and stakeholder presentations and symposium-integrated subgroup sessions on ciguatoxins, saxitoxin assays and liquid chromatography (LC) methods for saxitoxins and domoic acids, okadaiates and azaspiracids, and yessotoxins. Many of these subgroups were recently formed in 2005 and are working towards their goals of producing officially validated analytical methods. (Abstracts from the Baiona 2005 meeting cited in this report can be found in the online version of the conference abstract book in the Files and Folders section of the Marine and Freshwater Toxins online community at www.aoac.org.) An active topic for discussion in Baiona and subsequent Task Force activities was the expert consultation for Codex which met in Oslo, Norway in 2004 (previously described and cited in last year's GR report, ref 1). The consultation group's executive summary report (http://www.fao.org/es/ESN/food/risk_biotoxin en.stm) describes suggested changes in action levels as well as methods, method validation, and other issues. September 2005 saw the AOAC Task Force efforts further supported by another symposium, "Marine and Freshwater Toxins: Quality Methods for Food Safety and International Trade," at the AOAC INTERNATIONAL Annual Conference in Orlando, Florida. The multidisciplinary talks at this full day symposium ranged from ciguatoxins to cyanobacterial toxins, and spanned toxicology, biochemistry, molecular biology and analytical chemistry. Again, the symposium preceded Task Force meetings. Toxin subgroups, including a new group on cyanobacterial toxins, met for engaging and productive subgroup discussions. All of these activities were preceded by a Wiley Award symposium for Task Force member Mike Quilliam of NRC Canada. These talks, presented at a half-day symposium on the first day of the Annual Meeting, focused on Quilliam's work with LC tandem mass spectrometry (LC/MS/MS) and certified reference standards and materials, and included related presentations by some of his many research collaborators. To maintain flow and continuity between symposia and between Task Force meetings, the group now uses new electronic discussion forums. Individual subgroup areas, under the Marine and Freshwater Toxins Task Force, comprise this online community. First introduced by AOAC INTERNATIONAL in early 2005, these new resources are being used to distribute information and to supplement the in-person subgroup meetings and electronic mail in the group's validation efforts.

Insulin dynamically regulates calmodulin gene expression by sequential o-glycosylation and phosphorylation of sp1 and its subcellular compartmentalization in liver cells.

O-glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (approximately 30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression.

Committee on Natural Toxins and Food Allergens. Marine and freshwater toxins.

There have been major developments this past year in the Marine and Freshwater Toxins topic area (formerly Phycotoxins). These include AOAC approval and inauguration of a new AOAC Presidential Task Force on Marine and Freshwater Toxins to accelerate methods validation, and the appointment of several new Topic Advisors. A joint FAO/IOC/WHO group addressing biotoxins in molluscan bivalves is also relevant to this report and to the new Task Force. The AOAC Presidential Task Force on Marine and Freshwater Toxins is an international group that, in late November 2004, consisted of 90 world experts and stakeholders. Chaired by this General Referee, the group establishes methods priorities based on analytical methods criteria, determines fitness for purpose, identifies and reviews available methodologies, recommends methodologies for validation, and identifies complementary analytical tools. Once appropriate analytical methodology has been identified or developed, the Task Force is able to identify financial and technical resources necessary to validate the methods. The first two formal meetings of the Task Force were held in Bethesda, MD, on May 19, 2004 and in St. Louis, MO, on September 22, 2004. These meetings were held in conjunction with the XI International IUPAC Symposium on Mycotoxins and Phycotoxins and the 118th AOAC INTERNATIONAL Annual Meeting and Exposition, respectively. The Bethesda meeting served to introduce members of the group to the AOAC Community/Task Force model and to discuss objectives, concerns, general workings, and communications. The meeting concluded on an encouraging note, with a commitment from AOAC to help provide financial resources for the review of nonproprietary methods deemed high priority by the Task Force. This development was seen as an important step toward reaching methods validation objectives. The terms of reference for the Task Force were approved by the AOAC Board of Directors in late June, 2004. They described the Task Force membership as composed of voting and nonvoting members, with the voting members consisting of 13 members (12 plus the Chair). Voting members comprise of a balance of government regulators, academics, and industry members. No single agency has more than 2 voting members. Task Force members serve as experts in the field and agree to identify other experts; recommend individuals who can serve on the Task Force and as Chair; develop and prioritize a list of marine and freshwater toxins that need validated methods; assist in identifying existing methods for validation through AOAC validation programs; and recommend to the AOAC INTERNATIONAL Board of Directors policies and procedures necessary to accomplish the mission of the Task Force. They endeavor to actively support the work of the Task Force through garnering of sources of funding (except where prohibited by employer); identifying potential participating laboratories, sample identification and acquisition; and increasing program awareness among stakeholders. They assist AOAC in the identification of study directors and in the development of quality measurement tools by participating in the validation of methods and by identifying venues for members of the Task Group or the community to gather and assist with meeting content. Prior to the September 2004, AOAC Annual Meeting, the Task Force approved a set of Analytical Methods Selection Criteria, which are critical to the mission of the Task Force. They can be found, along with the Terms of Reference, roster of members, and other information, on the Task Force Web site at http://www.aoac.org/marine toxins/task_force.htm. The September 22, 2004 Task Force meeting in St. Louis included discussion of 2 interlaboratory studies, a proprietary kit for domoic acid by enzyme-linked immunosorbent assay (ELISA; Biosense Labs AS, Bergen, Norway) and also a nonproprietary liquid chromatography (LC) method for paralytic shellfish poisoning (PSP) toxins by precolumn oxidation (James F. Lawrence, Health Canada). These 2 methods were recommended by the Task Force for review by AOAC in September 2004. The group also discussed future priority directions, aspects of interlaboratory studies and official methods of analysis, other methods validation issues, future meetings, and funding. In addition to the Task Force meeting, 2 subgroup meetings were held. One subgroup addressed strategies to replace the mouse bioassay for brevetoxins with alternative modern methods based on ELISA or LC/mass spectrometry (MS). Brevetoxin metabolites, toxicity issues, and extraction conditions as well as future field studies were addressed in detail. The receptor binding assay (RBA)/saxitoxins subgroup addressed several aspects of the methodology, radiolabeled saxitoxin, and comparisons of mouse bioassay and RBA response. Both subgroups were productive and were seen as very useful by the participants. Task Force attendees generally agreed that subgroups are the most effective means of progressing towards validation of new methods and of ensuring thorough discussions of methods under consideration. By the time of their next meeting (April 2005) at the "Marine and Freshwater Toxins Analysis: 1st Joint Symposium and AOAC Task Force Meeting" in Baiona, Spain, the Task Force will have several well developed new subgroups in the areas of okadaic acid and dinophysis toxins, yessotoxins, domoic acids, and ciguatoxins. Some of the subgroups will hold face-to-face meetings in Spain and others will meet at future symposia or joint meetings. It is likely that training sessions will be associated with multiple Task Force meetings planned for 2005. Details on these meetings can be found on the Task Force Web site. Although the Task Force has experienced rapid growth, the addition of new members to the group, especially industry and government stakeholders, is encouraged. Task Force member Michael Quilliam, NRC Canada, provided the information given below on a joint CODEX group of special relevance to the new Task Force. This group met in late September 2004. For more information, see http://www.who.int/foodsafety/chem/meetings/biotoxin/en/.

Detection of paralytic shellfish poison by rapid cell bioassay: antagonism of voltage-gated sodium channel active toxins in vitro.

Although cytotoxicity assays provide several advantages over mouse bioassays, sodium channel-blocking marine toxins, such as those associated with paralytic shellfish poison (PSP), require prolonged incubation periods of 24-48 h. This is in marked contrast to in vitro detection of sodium channel-enhancing marine toxins such as ciguatoxins or brevetoxins which can be accomplished in as few as 4-6 h. We developed a modified PSP cell bioassay that is as rapid as in vitro methods for sodium channel-enhancing toxins. The cell bioassay is based on a saxitoxin-dependent antagonism of the rapid in vitro effects of brevetoxin or ciguatoxin. Comparative analysis of naturally incurred PSP residues by both antagonism cell bioassay and the mouse bioassay demonstrated significant correlation. The simplicity, sensitivity, and enhanced kinetics of the new antagonism cell bioassay format provide the basis for development of a practical alternative to conventional mouse testing for PSP.