Solid-phase extraction clean-up of ciguatoxin-contaminated coral fish extracts for use in the mouse bioassay.

Florisil solid-phase extraction (SPE) cartridges were used for purifying ciguatoxin (CTX)-contaminated coral fish extracts, with the aim of removing extracted lipid but retaining optimal level of CTXs in the purified fractions. The CTX-containing fraction (target fraction) in fish ether extract was isolated and purified by eluting through a commercially available Florisil cartridge with hexane-acetone-methanol solvent mixtures of increasing polarity (hexane-acetone (4:1, v/v) < acetone-methanol (7:3, v/v) < 100% methanol). Application of Florisil SPE using acetone-methanol (7:3, v/v) condition facilitated the separation of 4.2 +/- 0.4 mg (mean +/- standard error of the mean (SEM)) of purified target fraction from 20 mg ether extract with good retention of CTXs. The mouse bioassay was used to demonstrate that the average CTX recovery of the target fraction from CTX-spiked samples was 75.8% +/- 3.3%, which was significantly increased by 96.7% +/- 15% when compared with CTX recovery from ether extracts (44.8% +/- 5.2%) without performing SPE purification. Over 70% of non-target lipids were removed in which no CTX toxicity was found. Moreover, the target fractions of both CTX-spiked and naturally CTX-contaminated samples gave more prominent toxic responses of hypothermia and/or induced more rapid death of the mice. The use of acetone-methanol (7:3, v/v) condition in the elution could significantly improve overall recovery of CTXs, while minimizing the possible interferences of lipid matrix from co-extractants on mice.

Features of ciguatera fish poisoning cases in Hong Kong 2004-2007.

OBJECTIVE: To review the clinical features and laboratory investigations of ciguatera patients in Hong Kong between 2004 and 2007 in order to show the timely sampling of implicated fish from ciguatera victims and application of validated mouse bioassay for confirming suspected clinical cases of ciguatera. METHODS: Diagnosis of the ciguatera victims was based on history of coral fish consumption and clinical presentations stated in official guidelines for clinical diagnosis of ciguatera fish poisoning in Hong Kong. Food remnants of coral fish samples were collected swiftly from ciguatera victims between 2004 and 2007 for ciguatoxins (CTXs) analysis. RESULTS: Major clinical symptoms in ciguatera patients included gastrointestinal and neurological effects including limb numbness and diarrhoea, which developed at 0.5 to 15 hours after consumption of fish. In most cases, neurological symptoms were more common than gastrointestinal symptoms. A broad range of attack rate (10%-100%) was observed in each ciguatera outbreak. Validated mouse bioassay on ether extracts of the food remnant samples confirmed that all were CTXs-positive (<0.5 - 4.3 MU/20 mg ether extract) and directly linked to the corresponding ciguatera cases. CONCLUSION: Consistency between clinical and laboratory analysis for ciguatera poisoning illustrates the application of laboratory mouse bioassay in a timely fashion for confirming ciguatera poisoning cases and implementing effective public health measures. With further improvement in laboratory techniques, features of ciguatera fish poisoning cases can be better defined. Further studies are needed to determine the risk of each class of CTXs (Pacific-, Indian- and Caribbean-CTXs) in Hong Kong.

Study of an outbreak of ciguatera fish poisoning in Hong Kong.

Ciguatera fish poisoning (CFP) has been a significant and increasing public health problem in Hong Kong since 1980s. With growing demand for imported live coral fishes, the number of people who suffered from this disease has also been increasing. An outbreak of CFP in 2004 was the second most prominent in record as compared with the most significant one that occurred in 1998. In 2004, out of a total of 823 reported food poisoning outbreaks involving 3159 persons, 65 incidents (7.9%) affecting 247 people (7.8%) were attributed to CFP. Validated mouse bioassay analysis of surveillance samples revealed that seven samples (13%) were confirmed to be contaminated with ciguatoxins (CTXs). Typical symptoms of CTXs were found in mice injected with 20mg of fish extracts. The causative fishes included Cheilinus undulatus, Epinephelus coioides, Plectropomus areolatus, and Plectropomus leopardus. Most of these CTX-positive samples analyzed had only trace amounts of CTXs in their extract, except a C. undulatus sample which contained a mice lethal dose (2.5MU/20mg ether extract). This fish species was also the major origin of coral fish that caused clusters of CFP in the last quarter of 2004. Cigua-Check analysis of 20 flesh grains from seven CTX-positive fishes, previously confirmed as CTX-positive samples by mouse bioassay, showed that 50% of flesh grains were CTX contaminated.