Operational application of a rapid antibody-based detection assay for first line screening of paralytic shellfish toxins in shellfish

Harmful Algae 9 (2010) 636–646

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Operational application of a rapid antibody-based detection assay for first line screening of paralytic shellfish toxins in shellfish Chun-Kwan Wong, Patricia Hung, Edward A.L. Ng, Kellie L.H. Lee, Grace T.C. Wong, Kai-Man Kam * Biotoxin Laboratory, Microbiology Division, Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, Shek Kip Mei, Kowloon, Hong Kong

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 March 2010 Received in revised form 13 May 2010 Accepted 15 May 2010

Paralytic shellfish poisoning toxins (PSP-toxins) are potent neurotoxins associated with marine dinoflagellates and may accumulate in filter-feeding shellfish to cause food intoxication in human. Monitoring programs for PSP in shellfish rely heavily on the use of traditional mouse bioassay (MBA). Considerable progress has been made in developing a reliable, rapid and relatively convenient assay for mass screening of possible PSP contaminated samples. In this study, we investigated the potential application of a commercially available antibody-based assay for routine first line screening of PSPtoxins in shellfish collected in Hong Kong. Preliminary study showed that the Jellett Rapid PSP Test (JRPT) performed acceptably in detecting regulatory limit (80 mg STXeq per 100 g of shellfish tissue) of each standard toxin under shellfish matrix mediums and human urine, except GTX1,4 and NEO. The results indicated its potential applicability in field and outbreak situations. Upon applying the kits in 938 field samples for over 2 years, no false JRPT-positive result was determined from MBA-positive samples. However, JRPT was able to detect MBA false negative sample that exceeded the regulatory limit as determined by High Performance Liquid Chromatography (HPLC). On the other hand, a number of JRPT false positive results were revealed, based on HPLC data, suggesting that the effective or actual limit of detection for JRPT was less than 40 mg STXeq per 100 g of shellfish tissue. Of all MBA-JRPT-negative samples, 21% showed HPLC-positive results. Nevertheless, the PSP toxicity levels were below the limit. No false JRPT-negative result was found during the study. This indicated its potential efficacy for use as screening assay from public health perspective. HPLC analysis showed that STX, NEO, GTX2,3 and GTX5 were the most common PSP-toxins found in shellfish. The PSP-toxins profile and their relative abundance in shellfish were demonstrated to be potentially good biochemical markers for investigating and tracing the samples’ origin. In addition, 11% of samples were recorded as JRPT-invalid results, in which 78% came from oysters. Treatment of these JRPT-invalid samples by a centrifugation step followed by a centrifugal filtration was very effective (95% validity with clearer indication lines after reanalysis by JRPT) in reducing invalid results through removing potential interfering substance(s) in shellfish matrix. Complementary to MBA and HPLC toxins analysis, JRPT was shown to be an appropriate tool for rapid and mass screening of potential PSP-toxins contaminated shellfish. When public health actions are considered to verify JRPT-positive, HPLC-positive and uncertain cases samples, confirmatory test should be performed by gold method, MBA. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Centrifugal filter device Microalgae Paralytic shellfish poisoning Rapid kit Shellfish toxins

1. Introduction Paralytic shellfish poisoning (PSP) is a common foodborne biotoxin-induced disease caused by the consumption of filterfeeding molluscan shellfish contaminated with hydrophilic neurotoxins, PSP-toxins. These toxins are produced by some

* Corresponding author at: Rm. 731, 7/F, Public Health Laboratory Services Branch, Centre for Health Protection, 382 Nam Cheong St., Shek Kip Mei, Kowloon, Hong Kong. Tel.: +852 2319 8303; fax: +852 2776 1446. E-mail addresses: [email protected] (K.-M. Kam), [email protected] (C.-K. Wong), [email protected] (P. Hung). 1568-9883/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2010.05.004

toxigenic marine microalgae genera such as dinoflagellates Alexandrium, Gymnodinium, and Pyrodinium (Hallegraeff, 1993; Taylor et al., 1995; FAO, 2004). Since PSP-toxins contaminations in marine organisms are common natural phenomena, there have been reports describing PSP-toxins bioaccumulation in mollusks and some crustaceans such as crabs and lobsters (Lawrence et al., 1994; Shumway, 1995; Lehane, 2000; Llewellyn et al., 2002; Oikawa et al., 2002). These organisms accumulate the majority of the PSP-toxins in their tissues and digestive organs, and the toxins may be passed through the food chain to predators of higher trophic level including human. PSP-toxins are among the most potent aquatic biotoxins known (Halstead, 2002) which possess a tetrahydropurine comprising

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Fig. 1. Structures of the major PSP-toxins.

two guanidinium groups (Oshima, 1995a,b; Lehane, 2000) (Fig. 1). In general, the toxins can be classified into three major categories according to their toxicity levels (carbamate > decarbamoyl > Nsulfocarbamoyl toxins). Carbamate toxins, including saxitoxin (STX), neosaxitoxin (NEO) and gonyautoxins 1–4 (GTX1–4), are the most potent group of PSP-toxins and are estimated to be about 10– 100 times more toxic than the N-sulfocarbamoyl group toxins (Cembella et al., 1993; Wright, 1995). The substitution at R4 position results in a prominent variation in toxicity among the toxins. To date, over 26 PSP-toxins analogues have been discovered in which the toxicities of the major toxins have been well defined from experimental mice (Table 1). These neurotoxins can inhibit the normal action potential propagation of nerve impulses by blocking voltage-gated sodium channels, causing neuromuscular

paralysis (Lehane, 2000). PSP-toxins have little or no effect in shellfish potentially due to their genetic adaption to the toxins (Bricelj and Shumway, 1998; Bricelj et al., 2005), whereas in human, PSP symptoms are mainly characterized by neurological disorders varying from slight tingling or numbness (e.g. lips, tongue, face and extremities) sensations to fatal respiratory paralysis. Gastrointestinal symptoms, nevertheless, are less common. In severe cases, the victims may suffer from respiratory arrests in 2–12 h following consumption of lethal dose of PSPtoxins contaminated shellfish. PSP poses significant public health threat worldwide due to its high mortality rate (Lagos, 1998). Fatal cases due to consumption of high-level toxicity of PSP-toxins contaminated shellfish have been reported (Fortuine, 1975; Rodrigue et al., 1990; Gessner et al., 1997; Wang and Li, 1998;

Table 1 Relative toxicities of PSP-toxins on mice (Oshima, 1995a,b; Rourke et al., 2008). PSP-toxins

Toxin category

Molecular weights (g/mol) [HCl salt]

Mouse units (MU) per mmole

Relative toxicity (Reference to STX)

STX GTX1 NEO GTX4 GTX3 dcSTX dcGTX3 GTX2 dcGTX2 C2 (GTX8) GTX5 (B1)a C4 C3 C1 (epiGTX8)

Carbamate Carbamate Carbamate Carbamate Carbamate Decarbamoyl Decarbamoyl Carbamate Decarbamoyl N-sulfocarbamoyl N-sulfocarbamoyl N-sulfocarbamoyl N-sulfocarbamoyl N-sulfocarbamoyl

372.2 447.8 388.2 447.8 431.8 329.2 388.8 431.8 388.8 – 415.8 – – –

2483 2468 2295 1803 1584 1274 935 892 382 239 160 143 33 15

1.0000 0.9940 0.9243 0.7361 0.6379 0.5131 0.3766 0.3592 0.1538 0.0963 0.0644 0.0576 0.0133 0.0060

a

Same toxicity to GTX6 (B2).

638

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Lehane, 2000; Garcı´a et al., 2004, 2005). In addition to posing potential health hazard to seafood consumers, incidents of PSP can also elicit dramatic economic impacts (Conte, 1984) to the shellfish markets and local tourism. In Hong Kong, a comprehensive biotoxin surveillance program is in place to prevent PSP illnesses by screening potential PSPtoxins contaminated shellfish in fish markets. For operational analysis of PSP-toxins, mouse bioassay (MBA) is a reliable and widely accepted method used by public health authorities of many countries for years since its first application on PSP test in 1930s (Sommer and Meyer, 1937; Lehane, 2000, 2001; Toyofuku, 2006). In addition, it is presently an official method of the AOAC International (AOAC, 2000), approved method by the United States Food and Drug Administration (USFDA) and reference method prescribed in European Union (EU) legislation (Commission regulation EC 2042/2005) for the determination of PSP-toxins in shellfish. MBA provides fast detection (1 h turn around time) of total PSP toxicity (mg STXeq/100 g shellfish tissue) in a sample material with detection limit at about 40 mg STXeq per 100 g of shellfish tissue (Ferna´ndez and Cembella, 1995; Bricelj and Shumway, 1998; Park et al., 1999), which is about 2-fold less than the regulatory level of 80 mg STXeq per 100 g of shellfish tissue. The survival time of the mice after injection of sample extract is the direct means for toxicity quantification (AOAC, 2000). However, there are several shortcomings including limited selectivity and specificity as well as problems with animal testing (Park et al., 1986; Salter et al., 1989; McNabb et al., 2005). In addition, the accuracy of the assay may be affected by salt effects, metals contaminations (McFarren, 1959; Park et al., 1986; McGulloch et al., 1989; LeDoux and Hall, 2000) as well as extraction pH (Park et al., 1986; Vale et al., 2008), especially when toxins are present at very low levels. For confirmation and characterization of PSP-toxins in shellfish, analytical method based on ion-pairing high performance liquid chromatography (HPLC) with either pre-column or post-column derivatization of the PSP-toxins prior to fluorescence detection (FLD) is beneficial to reveal the toxins profiles and concentrations in PSP-toxins contaminated samples (Oshima, 1995a,b; Lawrence et al., 2005). Currently, these two derivatization techniques are widely used while the Lawrence HPLC-FLD method is now an AOAC official method (Lawrence et al., 2005). In general, Oshima method provides better quantitative information of PSP-toxin profile than Lawrence one, though the latter has major advantage of fast analysis. Detection limits are 1–2 pg/injection (Lawrence et al., 1995) and 5–30 pg/injection (Oshima, 1995a) for pre-column and post-column techniques, respectively, which are far below than that of MBA. Major drawbacks of these techniques include high cost HPLC machine, consumables (e.g. reagents, solvents and columns), and time-consuming, particularly in handling and running large number of surveillance samples for toxins analysis. Considerable effort, therefore, is needed to develop fast and costeffective alterative to the MBA and HPLC methods in providing first line mass screening of toxins contaminated shellfish samples in surveillance programs. A commercially available antibody-based immunoassay, Jellett Rapid PSP Test (JRPT), formerly known as the MIST AlertTM test kit (Jellett et al., 2002; Mackintosh et al., 2002), is designed to provide a simple and rapid PSP screening for shellfish samples in an economical way. It is a qualitative strip-format test working on the principle of lateral flow immunochromatography which adopts polyclonal PSP-toxin antibodies for the toxins detection. The average detection limit of the JRPT kit is 40 mg STXeq per 100 g of shellfish tissue as stated by the manufacturer. The use of this assay may be beneficial for screening out large portion of PSP negative and/or low PSP toxicity samples in the markets and may subsequently reduce the number of live experimental mice needed

for AOAC MBA (Oshiro et al., 2006; Turrell et al., 2007; Costa et al., 2009; Laycock et al., 2010). In the present study, shellfish samples collected from surveillance program in Hong Kong were used for investigating level of PSP-toxins contamination by three detection methods, JRPT, HPLC and official AOAC MBA (three-tiered perspective). This three-tiered approach was attempted to provide both qualitative and quantitative information of PSP-toxins contamination status in the samples. The results obtained from JRPT were scrutinized and compared with those of the HPLC toxins analysis and MBA. On the other hand, sensitivities of JRPT to different standard PSP-toxins near to-, above- and below-the regulatory limit were also evaluated for different matrices of shellfish extracts. The objective of this study was to assess the applicability and efficiency of using a rapid antibody-based assay as a first line screening alternative to the routine AOAC MBA for testing PSP-toxins contaminated shellfish under surveillance program conditions, and at the same time to identify and solve any practical problem encountered. The long-term approach is to reduce the use of experimental animals as first line screening for PSP test without affecting the current provision of public health protection from PSP. 2. Materials and methods 2.1. Sample collection Shellfish samples were collected biweekly from different districts in Hong Kong markets under surveillance program conditions between Jan-2007 and Jun-2009. Samples were kept frozen or at 4 8C upon collection and delivered to Biotoxin Laboratory for toxins extraction and analysis. All frozen samples were thawed at 4 8C prior to extraction. 2.2. Standard PSP-toxins and calibration Standard STX dihydrochloride (100 mg/ml) (courtesy of Dr. Sherwood Hall, U.S. Food and Drug Administration (USFDA)) was used for AOAC MBA calibration. Certified standard toxins, STX, NEO, GTX1,4, GTX2,3, dcSTX, dcGTX2,3 and GTX5, were obtained from the Certified Reference Materials Program of the Institute for Marine Biosciences, National Research Council (NRC), Halifax, Nova Scotia, Canada, for calibration of HPLC machine and for measuring the sensitivity of JRPT to different toxins concentrations under different matrices. Toxicity factors provided by NRC were used for PSP-toxins toxicity quantification as STX dihydrochloride equivalents. All standard solutions were stored at 4 8C before use. 2.3. Extraction of PSP-toxins Briefly, the outer shell of shellfish was cleaned thoroughly under tap water. Whole scallops were used for scallop samples; muscles, hepatopancreas, digestive glands and gonads were used for crustacean (lobster) samples. About 150 g of shellfish tissues was collected and placed on a sieve, rinsed under tap water to remove sand and any foreign material, drained for another 5 min at room temperature. 100 g of tissues was weighed and put into a 400-ml beaker together with 100 ml of diluted HCl (0.10–0.18N). The mixture was homogenized for 1–2 min using ULTRATURRAX1 T25 homogenizer until homogeneous slurry obtained. Homogenate made was adjusted to acidic medium (pH 2.0–3.5), and then boiled gently for 5 min with constant stirring. Boiled homogenates was cooled to room temperature on ice-bath and adjusted to original weights by adding 0.003N HCl. The cooled mixture was adjusted to acidic medium to between pH 3.0 and 3.5 followed by centrifugation at 3000 rpm for 5–10 min. All acidic supernatants collected were transferred to 20-ml plastic vial and

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kept at 4 8C prior to testing by JRPT, HPLC-FLD and AOAC MBA subsequently. The remaining extracts were kept at 4 8C refrigerator for further analysis within 2 days in order to verify the positive samples in case of any suspicious results. Due to the possible discrepancy between results arising from stability issues (Indrasena and Gill, 2000a,b), the data from further analysis was used as a qualitative reference. 2.4. Jellett Rapid PSP Test kit Jellett Rapid PSP Test kit (Jellett Rapid Testing Ltd., Nova Scotia, Canada), was used as a first line screening tool for PSP-toxins in the shellfish samples. All batches of test kits are provided with certificates of analysis to certify the quality of the kits with respect to STX and NEO. The manufacturer’s instructions of the kits were strictly followed. Briefly, 100 ml of the acidic supernatant of sample extract was mixed with 400 ml of running PSP-saline buffer by pipetting up and down for at least three times. 100 ml of the resulting mixture was inoculated into sample well ‘‘S’’ on a JRPT strip which was then read between 35 and 60 min. A positive control (0.4 mg/ml STX dihydrochloride in 0.003N HCl, the STX dihydrochloride standard solutions were provided by the USFDA or the NRC of Canada) was performed to ensure the sensitivity and efficacy of the kit in each run. All lot numbers of the kits were tracked and different production lots were also cross-compared using STX standards to confirm consistency in performance across lots. The interpretation of the results was based on the criteria stated on the laboratory instruction sheet of the kit by analyzing the color intensity of C- and T-line: For a JRPT-negative result ( ve) of sample, the intensity of the T-line is darker than the positive Tline on the Instruction Sheet. For a JRPT-positive result of sample, the intensity of the T-line is equal to or fainter than the positive Tline. For a JRPT-invalid result of sample, the intensity of C-line is equal to or fainter than that of invalid example. For this study, JRPT-positive results (+ve) were rated in three levels: Level (1) strongly +ve: no visible T-line was seen except C-line, Level (2) +ve: the T-line was approximately 25–50% of the intensity of the C-line (i.e. the ratio of color intensity between C-line and T-line was approximately equivalent to that of positive result demonstration on the lab instruction sheet provided by the manufacturer), and Level (3) slightly +ve: the T-line was approximately 50–75% of the intensity of the C-line. Because the interpretation of the color intensity of the lines can be subjective for each laboratory analyst, the results of the JRPT were finally reviewed and verified by another trained laboratory analyst to ensure concurrence in the interpretation before confirming the results. 2.5. Evaluation of JRPT sensitivity with spiked PSP-toxins standards under different matrix mediums The matrix effect of different shellfish extracts on JRPT performance were evaluated with PSP-toxins standards. Acidic extracts of various shellfish (abalone, clams, conches, mussels, oysters and scallops), previously determined as free from PSPtoxins contamination (i.e. below detection limit of an assay) by JRPT, HPLC and MBA, were used as matrix mediums. Certified standard PSP-toxins were spiked individually to different shellfish extracts at, above and below the regulatory limit (80 mg STXeq/ 100 g of shellfish tissue) to measure the sensitivity performance of the JPRT kit. For this test, 13 toxins concentrations (10, 20, 40, 80, 160, 240, 300, 320, 400, 480, 640, 1280 and 2560 mg STXeq/100 g of shellfish tissue) between 1/8 times (0.125) and 32 times (32) of regulatory limit (RL) were adopted for testing the sensitivity of JRPT under different matrix mediums. In addition, a human urine sample, donated by a healthy volunteer, was used to investigate its matrix effect to PSP-toxins detection on JRPT. PSP-toxin standard

639

solutions in diluted HCl (0.003N HCl) were adopted as positive control to directly evaluate the kit performance, without interference from matrices of shellfish extracts or human urine. 2.6. Treatment of sample extracts with JRPT-invalid results All sample extracts with invalid results from JRPT were treated with centrifugation followed by a centrifugal filtration process before being reanalyzed by JRPT kits. Briefly, 2 ml of extract from JRPT-invalid sample was transferred to a centrifugal plastic vial and centrifuged at 10,000  g for 10 min. 400 ml of supernatant was collected from central clear portion of supernatant layer (avoid extracting any residual suspension in the portion) and was then added into sample reservoir of a Microcon1 YM-10 centrifugal filter device (Millipore, Bedford, MA, USA). The membrane nominal molecular weight limit in Daltons (proteins) = 10,000; maximum initial sample volume is 500 ml. The whole device was centrifuged at 10,000  g for 30 min. Centrifugation at above 14,000  g may result in membrane breakage. 100 ml of filtrate collected from the device’s filtrate tube was reanalyzed by a JRPT kit. Any suspicion of the result was further verified by HPLC-FLD and MBA. 2.7. HPLC analysis of PSP-toxins The acidic shellfish extract (a sub-sample of the same extract used for the JRPT) was centrifuged at 10,000  g for 5–10 min. The supernatant (3 ml) collected was passed through a Sep-Pak C18 cartridge column (Waters Corporation, Milford, MA) pre-washed with 10 ml of methanol followed by 10 ml of distilled water for equilibrium. The first 1.5 ml was discarded and the next 0.5 ml was collected in the cartridge of an ultra-filtration kit (Amicon Ultrafree1 MC 10,000) followed by a centrifugation at 10,000  g for 40 min. 10 ml of the supernatant was injected into the HPLC system for toxins analysis. PSP-toxins profiles and characteristics were analyzed by using ion-pairing chromatography with post-column derivatization according to the method described by Oshima (1995a). PSP-toxins including STX, NEO, dcSTX, GTX1,4, GTX2,3, dcGTX2,3 and GTX5 were adopted for analysis. The HPLC system was set up in accordance with the methods and procedures described by Oshima (1995b). Concentrations of PSP-toxins were determined by comparing the peak areas of PSP-toxins with their corresponding standards. The overall toxicities of the samples were represented as STX equivalents (mg STXeq/100 g shellfish). For calibration of HPLC, at least five different concentrations of standard solutions were injected into the machine for analysis in each run. The mean of peak areas for each concentration was plotted to construct a linear regression curve. 2.8. Mouse bioassay The ICR (CD-1) female mice (20  2 g) were obtained from the Laboratory Animals Service Centre of the Chinese University of Hong Kong. They were acclimated for at least 24 h and housed in a controlled environment at 23  2 8C, 54–56% humidity, and a 12h:12-h light–dark cycle system. Standard rodent chows (PMI Nutrition International Inc., USA) and tap water were supplied ad libitum. Mouse bioassay for PSP was performed in accordance with the official AOAC procedures for paralytic shellfish poison (AOAC, 2000) except that the shellfish tissue was homogenized after addition of diluted HCl. Reliability of the assay was maintained by keeping regular training of analysts with appropriate conversion factor (CF) values. Three mice were weighed and injected intraperitoneally (i.p.) with 1 ml of shellfish extract (sub-sample of the same extract used for the JRPT). The treated mice were then closely observed for PSP

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symptoms for 60 min. The median survival time was adopted for toxicity (MU) calculation. Sample was regarded as negative (i.e. not detected or below the detection limit of the assay) when mice survived after 60 min. The toxicity was expressed as mg STXeq/100 g of shellfish tissues based on AOAC method. All mice used were sacrificed after PSP experiment in compliance with ethical standards and guidelines. 3. Results 3.1. Sensitivity of JRPT to individual PSP-toxins under different matrix mediums The performance of JRPT on different PSP-toxins under different matrix mediums is summarized in Table 2. In general, all the PSP standard toxins could be detected by JRPT kits at or approaching to half of the regulatory limit (i.e. 40 mg STXeq/100 g of shellfish tissue) as stated by the manufacturer, except GTX1,4 and NEO. For testing GTX1,4 under all matrix mediums, at least 4 times (abalone extract) or even up to 32 times (mussel extract) of regulatory limit concentration was required for obtaining Level (2) +ve result in JRPT. In the present study, the limit of detection (LOD) of the kit to GTX1,4 was significantly in excess of the regulatory limit. For NEO under both diluted HCl and mussel matrix mediums, +ve results could be exhibited only when 2 times regulatory limit concentration was applied. Although testing NEO at regulatory level under mussel matrix medium could clearly show that the color intensity of C-line was stronger than that of T-line, the color intensity of Tline did not match the positive result demonstration on the laboratory instruction sheet provided by the manufacturer. In general, the JRPT performed better in the presence of shellfish matrix and human urine because ve results appeared at lower toxins concentrations than those under the diluted acid medium. It should be noted, however, that JRPT performed particularly poor in the detection of GTX1,4 under mussel extracts medium, though JRPT was able to detect STX, GTX2,3, dcSTX, dcGTX2,3 and GTX5 at

concentrations well below the regulatory limit. Moreover, most of the toxins that could be detected by JRPT were at 2-fold less than the regulatory limit under shellfish matrix mediums. 3.2. Comparative analysis of PSP-toxins contamination in field samples using JRPT, HPLC and MBA During the 2-year study period, a total of 938 shellfish samples were collected from different markets in Hong Kong. These surveillance samples included 197 clams, 234 oysters, 274 scallops, 155 mussels, 74 gastropods (abalone, conches and whelk) and 4 crustaceans (lobsters). Of the 938 samples analyzed by JRPT, 86 (9.2%) were positive including 34 Level (1) strongly +ve, 36 Level (2) +ve and 16 Level (3) slightly +ve results. 721 (77%) were confirmed negative and JRPT could not be performed in 31 cases (3.3%) mainly due to sticky nature of the samples. There were, however, 100 cases (11%) that were determined as JRPT-invalid results because color intensity of C- and/or T-line(s) could not match with either +ve or ve criteria as shown on the laboratory instruction demonstration sheet. Details of comparative analysis between MBA (gold standard), JRPT and HPLC are summarized in Table 3. For the 19 MBA-positive cases, 17 JRPT-positive (all were Level (1) strongly +ve) results were confirmed which were revealed subsequently to have consistent results with HPLC data. The toxicity levels of the samples were between 20 and 1650 mg STXeq/100 g of shellfish tissue. Therefore, no false positive result was found in JRPT as compared with the corresponding HPLC and MBA tests. At the same time, JRPT-invalid results were found amongst two MBApositive samples, in which the toxicity levels determined by HPLC analysis were at 21.1 and 31.1 mg STXeq/100 g of shellfish tissue. For the 919 MBA-negative samples, 69 were JRPT-positive including 17 strongly +ve, 36 +ve and 16 slightly +ve cases. This showed the potential false negative determinations from MBA. Besides the three JRPT-positive samples which could not be used for HPLC analysis because of unavailability or sticky nature of the

Table 2 Summary of detection concentrations of different PSP-toxins applied on JRPT kit under different matrix mediums for getting at least Level (2) +ve result. Matrix mediums

JRPT result

Toxin level (mg STXeq/100 g of matrix) needed for getting at least a Level (2) +ve result GTX5

dcGTX2,3

dcSTX

GTX2,3

GTX1,4

NEO

STX

0.003N HCl

+ve ved

0.125 RL NA

0.5 RL 0.25 RL

0.5 RL 0.25 RL

0.25 RL 0.125 RL

16 RL 8 RL

2 RL 1 RL

0.5 RL a 0.25 RL

Conch extract

+ve ved

0.5 RLb NA

0.5 RL NA

0.5 RL NA

0.5 RL NA

b

5 RL 2 RL

1 RL 0.25 RL

0.5 RL NA

Scallop extract

+ve ved

0.5 RLb NA

0.5 RLe NA

0.5 RL NA

0.5 RL 0.25 RL

8 RL 2 RL

1 RL 0.25 RL

0.25 RL 0.125 RL

Clam extract

+ve ved

0.25 RLb NA

0.25 RLe NA

0.25 RL NA

0.25 RLb 0.125 RL

6 RL 4 RL

1 RL 0.5 RL

0.25 RL NA

Oyster extract

+ve ved

0.25 RLb NA

0.25 RL NA

0.5 RLe NA

0.125 RLb NA

8 RL 6 RL

0.5 RL 0.25 RL

0.25 RLb NA

Mussel extract

+ve ved

0.25 RL NA

0.5 RL 0.25 RL

0.5 RL 0.125 RL

0.25 RLa NA

32 RLe 16 RLe

2 RLe 1 RLc,

0.5 RLa 0.125 RL

Abalone extract

+ve ved

0.5 RL NA

1 RLe NA

0.5 RLb,e 0.25 RL

0.5 RLb,e NA

4 RLe 2 RLe

1 RL 0.5 RL

0.25 RLa,e NA

Human urine

+ve ved

0.25 RL NA

0.5 RL 0.25 RL

0.25 RL NA

0.125 RL NA

8 RL 2 RL

1 RL 0.5 RL

0.25 RL NA

a,e

e

b

RL: Regulatory Level (80 mg STXeq/100 g of shellfish tissue). NA: Not Applicable. Study was not performed here to investigate the toxin level required for a JRPT-negative ( ve) result to be observed. a Level (1) strongly +ve result was obtained at this level. b Level (1) strongly +ve result was obtained at this level with a remnant/shadow found at T-line of JRPT strip. c C-line color intensity was clearly sharper than T-line, but the color intensity of T-line did not match positive result demonstration on the lab instruction sheet provided by the manufacturer. d JRPT-negative ( ve) result began to occur at this toxin concentration. e 2 replicates using different sample extracts. The value reported was an average of the 2 replicates.

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Table 3 Comparative analysis of results between MBA, JRPT and HPLC. HPLC PSP toxicitya

MBA

JRPT

HPLC

(20–1650)

ve 919

+ve 69

+ve 66 ve 0 NA/Sticky 3

Invalid 98

+ve 35 ve 62 NA/Sticky 1

+ve 0 ve 0 NA/Sticky 0

NA/Sticky 31

+ve 1 ve 12 NA/Sticky 18

+ve 0 ve 0 NA/Sticky 0

ve 721

+ve 151 ve 513 NA/Sticky 57

MBA

JRPT

HPLC

+ve 19

+ve 17

+ve 17 ve 0 NA/Sticky 0

Invalid 2

+ve 2 ve 0 nil 0

NA/Sticky 0

ve 0

(21.3, 31.1)

HPLC PSP toxicitya (0.1–95.3)

(0.3–22.5)

(6.5)

(0.04–37.8)

A total of 59 JRPT positive controls were used in this study: all were Level (1) strongly +ve results. NA/Sticky: The samples used for the test were either (1) not available due to small volume of the sample extracts or (2) sticky nature of the sample extracts which were not applicable for JRPT inoculation or HPLC injection. a mg STXeq/100 g shellfish tissue.

sample extracts, HPLC results from the other 66 JRPT-positive cases showed presence of PSP-toxins at toxicity levels between 0.1 and 95.3 mg STXeq/100 g of shellfish tissue. Among these, 2 samples were determined to have toxicity levels above half of the regulatory limit (one was over 40, and another was over 80 mg STXeq/100 g of shellfish tissue). In terms of detection limit of JRPT (40 mg STXeq/100 g of shellfish tissue), 82% of ‘‘false JRPTpositive’’ (PSP-toxins toxicity levels below 40 mg STXeq/100 g of shellfish tissue) results were identified when compared with their corresponding HPLC data. For the cases of JRPT-invalid, NA/Sticky and ve, PSP-toxins were detected at toxicity levels between 0.04 and 22.5 mg STXeq/100 g of shellfish tissue. In particular, toxicity level of PSP-toxins exceeding the regulatory limit was not found in any of the JRPT-invalid cases. On the other hand, PSP toxicity levels

were found in JRPT-negative ( ve) cases as compared with their corresponding HPLC analysis results. Nevertheless, toxicity levels for those negative cases ranged between 0.04 and 37.8 mg STXeq/ 100 g of shellfish tissue, which were far below the regulatory limit of 80 mg STXeq/100 g of shellfish tissue. According to HPLC results, there were still 36% and 3% of HPLC-positive cases found from JRPT-invalid and JRPT NA/Sticky samples, respectively. Overall, the rates of positive results determined by MBA and HPLC analysis were about 2.0% and 29%, respectively. This indicated that the HPLC method was able to detect PSP-toxins at levels below the detection limit of MBA. The relative distribution (relative percentage: nmol%) of PSPtoxins under each category of PSP contaminated shellfish is shown in Fig. 2. Overall, STX, NEO, GTX2 and GTX5 were found to be the

Fig. 2. The relative percentage (nmol%) of PSP-toxins in different shellfish collected in Hong Kong. Data are represented by mean  S.E.M.

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Table 4 PSP-toxins composition and their relative percentages (nmol%) in MBA-positive shellfish samples.

Scallops 2–9 were collected from three different districts in Hong Kong at different dates after an occurrence of PSP incident. a mg STXeq/100 g of shellfish tissue; bold text: exceed the regulatory limit (80 mg STXeq/100 g of shellfish tissue).

most common PSP-toxins among the shellfish samples. In general, the PSP contaminated shellfish comprised a suite of PSP-toxins in their tissues except gastropod samples. PSP-toxins found in PSP contaminated gastropods were sparse and the most abundant PSPtoxins detected were STX and NEO. For scallop samples, each of the four toxins including STX, NEO, GTX2 and GTX5 contributed more than 15% abundance among the total PSP-toxins analyzed. When comparing with a study of PSP-toxins profile in samples belonging to the same batch of contaminated scallops (Wong et al., 2009), the most common PSP-toxins found were STX, GTX2,3 and GTX5. Similar relative proportion in toxins profile among scallops was also found. On the contrary, for clam samples, NEO was relatively less abundant than that in other samples. For mussel and oyster samples, more than 20% of each of NEO and GTX2 were found together with a suite of other PSP-toxins at different relative percentages. Table 4 shows the PSP-toxins composition among the MBApositive samples. STX and GTX2,3 were the predominant toxins found in samples at toxicity levels over 40 mg STXeq/100 g of shellfish tissue. For MBA-positive results at around or less than 40 mg STXeq/100 g of shellfish tissue, the common PSP-toxins were either STX or GTX2,3, or both. By analyzing the toxins pattern of JRPT-positive samples, STX and/or GTX2,3 were predominant PSP-toxins found in positive cases of Level (1) strongly +ve, Level (2) +ve and Level (3) slightly +ve. No undetected PSP toxicity, therefore, was found when compared with HPLC results. For JRPTnegative but HPLC-positive samples, the average PSP toxicity levels were less than 10 mg STXeq/100 g of shellfish tissue. Comparatively few PSP-toxins were identified in those samples with such low toxicity levels. Nevertheless, MBA analysis showed that there

was no false negative result indication in those JRPT-negative samples. By analyzing the distribution pattern and relative molar percentages (nmol%) of PSP-toxins in all MBA-positive samples (Table 4), different suites of PSP-toxins were also found with different relative distribution percentages in shellfish. JRPT examination of these samples demonstrated that all were Level (1) strongly +ve results. Of interest to note was that the relative distributions of PSP-toxins were comparable between Scallops 2 and Scallops 9. It was subsequently found that these samples were collected from enhanced surveillance of three different districts at different times after an occurrence of PSP incident, and analysis of the distribution patterns of PSP-toxins among those scallop samples suggested that they might come from the same source. 3.3. Treatment for the JRPT-invalid result samples Among the sample extracts tested by JRPT kits, a significant number of JRPT-invalid results were revealed. In the period of study, 100 out of 938 (11%) samples showed JRPT-invalid results in which majority (78%) of these samples were oysters (Fig. 3). There was no information on seasonality or possible reproductive state linkage for these invalid effects. Treatment of these JRPT-invalid result samples extracts with a 10,000  g centrifugation in 10 min followed by another 30 min of 10,000  g centrifugation in a centrifugal filter device (Microcon1) yielded 95% validity after reanalysis with JRPT kits (Table 5). Overall, the color intensity of Cand T-line became sharper than those without the treatment. Either centrifugation or centrifugal filtration alone would not give a valid JRPT result after retest by JRPT (data not shown). Among 59

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fere the color expression or movement of GTX1,4 on the absorption pad of JRPT kits. 4. Discussion

Fig. 3. Category of shellfish in JRPT-invalid result samples.

JRPT-invalid samples randomly selected for the treatment and analysis (Table 5), five samples were subsequently identified as JRPT-positive (3 strongly +ve and 2 +ve results) after the treatment, and the results were consistent with those determined by HPLC analysis. After treatment, therefore, no false positive result was identified when comparing JRPT-positive samples and their corresponding HPLC data. On the other hand, 86% of the JRPTinvalid samples were finally confirmed as JRPT-negative results after retest by JRPT, which were then consistent with the results obtained in MBA test. As a result, no false negative result was found in JRPT tests after the centrifugation/filtration treatment. Nevertheless, about 33% of these JRPT-negative samples were found to have trace toxicity levels (0.33 13.5 mg STXeq/100 g shellfish tissue) of PSP-toxins by HPLC analysis at levels far below the PSP regulatory limit. For sample extracts originally determined as positive (+ve) or negative ( ve) by JRPT kits, treatment with centrifugation/ centrifugal filtration did not affect the final validity of the results. No false positive or negative result was found after the treatment. As regards the three mussel extracts previously spiked with 16-fold higher of regulatory limit of GTX1,4, there was no indication of positivity when tested by JRPT kits before treatment (Table 2). However, on application of centrifugation/ centrifugal filtration step on these three originally JRPT-negative results mussel extracts, it eventually produced three valid JRPTpositive results including a Level (2) +ve result and two Level (3) slightly +ve results. These showed the potential effect of interfering substance(s) present in extracts which might inter-

Because of unpredictable and significant public health threat of PSP contamination in shellfish, constant surveillance and regulatory control on PSP-toxins contaminated shellfish in the market is an important strategy to prevent exposure to this life-threatening biotoxin. Although traditional monitoring (AOAC MBA) is a recommended detection tool and reference method for scrutinizing potential PSP contamination in shellfish, adopting both fast and reliable alternatives is still a constant urge. This can provide efficient assays for mass screening of PSP-toxins contaminated shellfish samples, and on the other hand, lead to reduction on the use of experimental animals in biotoxin test (Directive 86/609/ EEC). In addition to using chemical analysis of PSP-toxins by High Performance Liquid Chromatography-Fluorescence Detection (HPLC-FLD) (Luckas et al., 2003; Lawrence et al., 2005; Rourke et al., 2008) and Liquid chromatography-Mass Spectrometry (LCMS) (Turrell et al., 2008) for studying the PSP-toxins composition and characteristic, antibody-based method is considered highly desirable in view of its capability to provide a rapid and comparatively convenient means for detecting molecular structure(s) of the toxins, so as to provide qualitative as well as quantitative information for toxins determination. In order to incorporate a detection assay that can be used operationally in a surveillance system for first line screening of PSP-toxins, sensitivity and selectivity to each of the key component toxin is the crucial factor in assessing the efficiency and efficacy of the assay before implementation for regulatory monitoring program. In general, our study on JRPT sensitivity showed that it performed acceptably (within 2-fold of the regulatory limit without a large margin for error) in detecting regulatory level of each standard toxin individually under different shellfish matrix mediums, except GTX1,4 and NEO. Although the performance of JRPT to NEO was regarded as acceptable to respond at regulatory level under different matrices besides mussel extracts, the overall sensitivity was significantly lower than that of PSP-toxins GTX5, dcGTX2,3, dcSTX, GTX2,3 and STX. In addition, the limit of detection of JRPT kit to GTX1,4 was significantly above the regulatory level. These results were consistent with other studies that reported poor efficiency at detecting both GTX1,4 and NEO

Table 5 Analysis of JRPT-invalid result extracts after treatment of a centrifugation followed by another centrifugation in a centrifugal filtration device. PSP test results

No. of samples

JRPT-invalid MBA results MBA PSP toxicitya HPLC results HPLC PSP toxicitya

59

JRPT +ve MBA results MBA PSP toxicitya HPLC results HPLC PSP toxicitya

3

JRPT ve MBA results MBA PSP toxicitya HPLC results HPLC PSP toxicitya

16

a b

JRPT results after treatment of centrifugation plus centrifugal filtration Strongly +ve

+ve

3 2 +ve, 1 ve 44, 44, 0 3 +ve 21, 21, 9.4

2 1 +ve, 1 53, 0 2 +ve 31, 5.9

– – – – –

2 1 +ve, 1 95, 0 2 +ve 73, 10

– – – – –

1b NA – NA –

Slightly +ve ve

Invalid

Validity %

51 51 ve 0 17 +ve, 34 ve 0.33–13.5, 0

3 3 0 3 0

95

– – – – –

100

0 1 +ve 3.6

– – – – –

2b NA – NA –

13 13 0 13 0

– – – – –

100

– – – – – 1

ve

ve

ve

ve ve

ve ve

mg STXeq/100 g shellfish.

Three PSP-toxins free mussel extracts (i.e. MBA, JRPT and HPLC: negative results) spiked with 1280 mg STXeq/100 g shellfish of GTX1,4. The mussel extracts were adopted from the same extract used for testing the Lowest Limit of Detection (LOD) of GTX1,4 on JRPT kit under mussel matrix medium. NA: Not applicable.

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(Jellett et al., 2002; CEFAS, 2007; Laycock et al., 2010), in particular the GTX1,4 epimer. Recent work from Laycock et al. (2010) showed the same weakness on sensitivities of both GTX1,4 and NEO even though efforts had been placed to raise antibodies with better cross-reactivity to these two toxins. Because the antibodies may have diverse responses to PSP-toxin analogues and toxins mixtures (Usleber et al., 2001; CEFAS, 2007; Laycock et al., 2010), further studies on sensitivity and specificity of the JRPT assay are necessary to assess its performance under different field situations and in variety of toxins mixtures. As regards the sensitivity of detection of PSP-toxins in human urine, the results showed that the kit was effective to detect almost all PSP-toxins at or well below the regulatory level, except poor sensitivity was still found for GTX1,4. Previous reports showed removal of PSP-toxins from human body through excretion in urine (Llewellyn et al., 2002; Garcı´a et al., 2004, 2005, 2010). Metabolic transformations such as (1) enzymatic oxidation and glucuronidation of N1 group in the tetrahydropurine nucleus of STX and GTX2,3 to form the hydroxy (–OH) analogues of NEO and GTX1,4 epimers pair, respectively; and (2) hydrolysis of carbamoyl group of STX to form its decarbamoyl analogue dcSTX, are responsible for toxins conversion before urination. It was suggested that a longer period of time (48 h) was required for these metabolic oxidation and hydrolysis to take place (Garcı´a et al., 2004). The two metabolic products, GTX1,4 and NEO, had been confirmed to have low cross-reactivity with JRPT kits. A study describing PSP-toxins analysis in urine samples of four PSP patients (Garcı´a et al., 2005) revealed that there was no GTX2,3 biotransformation in patients for 8 h after ingestion of toxic shellfish that contained only GTX2,3. The half-life of STX in human was also reported as less than 10 h (Gessner et al., 1997). By adopting the JRPT for preliminary study at clinical diagnostic level, it is recommended to collect patients’ urine samples within 8 h after the onset of PSP symptoms. This can help alleviate the over dependence on detection of PSP-toxin analogues that have lower sensitivities (GTX1,4 and NEO) for PSP determination. Moreover, toxins analysis using HPLC is required for PSP-toxins confirmation and characterization. Presently, there are still few studies describing the detailed and complete metabolic mechanisms of PSP-toxins transformation in human. In our present study, information on matrix effect of PSP-toxins in urine medium is still at a preliminary stage. More data, therefore, should be gathered before applying the kit for future extensive clinical use. By using JRPT for surveillance on PSP-toxins contaminated shellfish in the market for over 2 years, no false positive result was obtained out of a total of 17 MBA-positive samples (Table 3). There was a good coherence with the corresponding HPLC toxins analysis. However, replicate analysis from JRPT showed false negative MBA results in a sample containing PSP toxicity level above the regulatory limit. With reference to a toxin profile generated for this sample, 8 PSP-toxins including GTX1, dcGTX3, GTX5, GTX2,3, NEO, dcSTX and STX were identified. In terms of the stated detection limit of the JRPT kit (40 mg STXeq/100 g of shellfish tissue), we obtained high percentage (82%) of ‘‘false positive’’ results in JRPT when compared to our HPLC reference data. However, all ‘‘false positive’’ results observed from JRPT could be explained by the characterization of toxin profiles obtained from HPLC analysis. This high ‘‘false positive’’ rate was shown to have significant difference from previous studies carried out by Jellett et al. (2002) and Costa et al. (2009), in which they reported their ‘‘false positive’’ rates at 14% and 58%, respectively. The reason for our high HPLC ‘‘false positive’’ rate might be due to the lack of toxicity information of other PSP-toxins such as C-toxins, which might contribute significant amount of toxicity to the total PSPtoxins toxicity. From our experience, we found peaks (significant peak height) at the region of those ‘‘false positive’’ chromatograms where the C-toxins might probably be located (data not shown).

Nevertheless, the detection of only six PSP-toxins, such as STX, NEO and GTX1,4, by HPLC can still provide a reasonable estimation of the total PSP toxicity in shellfish samples (Asp et al., 2004). Although in 21% of MBA-JRPT-negative samples were HPLCpositive, the toxicity levels (0.04–37.8 mg STXeq/100 g of shellfish tissue, Table 3) were well below the regulatory limit, indicating a low probability of getting false negative results from JRPT examination. From public health perspective, it was good to have no false JRPT-negative result found over two years of field trial period. This leads to the potential application and adoption of this assay for operational screening for PSP-toxins contaminated shellfish. Nevertheless, HPLC toxins analysis is highly recommended as follow-up means for PSP-toxins characterization and confirmation in order to complement the JRPT information, especially for those JRPT-invalid results and NA/sticky samples where uncertainties in PSP contamination still exist. The official regulatory gold method, MBA, would be used as an ultimate confirmatory test for JRPT-positive, HPLC-positive and any uncertain cases (e.g. JRPT-invalid samples). The other AOAC validated method for PSP-toxins, such as Lawrence’s HPLC-FLD method (Lawrence et al., 2005), may also be used as an additional reference. For PSP-toxins analysis, the most common and abundant PSPtoxins worldwide include STX, NEO and GTX2,3 (Garcı´a et al., 2010). In the present study, STX, NEO, GTX2 and GTX5 were the most abundant toxins quantified by HPLC analysis in shellfish samples collected in Hong Kong (Fig. 2). Moreover, STX and GTX2,3 were also the most predominant toxins found in MBA-positive samples above 40 mg STXeq/100 g of shellfish tissue. Toxins analysis from previous studies showed that shellfish samples collected from southern China and Aleutian Islands of Alaska comprised considerable abundance of GTX2,3 and C-toxins (Anderson et al., 1996; Costa et al., 2009). Except NEO, the other toxins STX, GTX2 and GTX5 had been confirmed to have good cross-reactivity with JRPT antibodies in accordance with the present and previous studies (Jellett et al., 2002; CEFAS, 2007; Laycock et al., 2010). This might indicate the potential suitability of applying JRPT kits for providing general screening test of PSPcontaminated shellfish in our situation. Concerning the MBApositive samples collected from a PSP incident, JRPT could unambiguously identify the scallop samples (scallops 2–9) that showed significant PSP toxicity level above regulatory limit (MBA and HPLC data). Those samples were then found to be collected on different dates from individual districts. According to the composition profile and relative percentages of PSP-toxins analyzed in those samples using HPLC analysis, these scallops might root in a same source and/or a same batch. As a result, this demonstrated that detailed PSP-toxins patterns and their relative abundance (i.e. toxin profiles) in shellfish samples would be good and potential biochemical markers for investigating the origin and traceability of shellfish samples in PSP cases. For public health purposes, creation of a library of profiles with their corresponding geographic origins would be a reasonable next step, as this can assist in tracing back to contamination sources through analysis of the toxin profiles of implicated samples. The rates of JRPT-invalid results ranged from 0.7 to 5% in accordance with Oshiro et al. (2006) and CEFAS (2007). For our cases, there were about 11% of JRPT-invalid results in which most of them (78%) were extracts of oyster samples. For these samples with invalid results, 37% contained detectable levels of PSP-toxins by HPLC toxins analysis. Moreover, levels exceeding the PSP regulatory limit were also confirmed in MBA examination, showing the potential false negativity of highly toxic samples with JRPT-invalid result samples. Therefore, cautious attention must be taken to handle these types of JRPT-invalid samples. It is important to note that presence of interfering substance(s) in

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oysters or other shellfish extract samples could affect mobilization or interaction of antibody color complex on the absorption pad. However, there was still a lack of adequate information to clarify this uncertainty at this stage. In our study, we found that the application of centrifugation plus a centrifugal filtration in a Microcon1 device could resolve this practical problem by reducing significantly the number of JRPT-invalid results. The rate of validity after this treatment could yield up to 95% upon re-analysis by JRPT kits. In addition, the color expressions on both C- and T-line were sharper than those without such treatment. It was likely that the treatment help remove the interfering substance(s) in the samples and facilitate the molecular movement and/or antigens–antibodies interaction on JRPT kits. This refinement step could further reduce the need for experimental mice in JRPT-invalid result samples for AOAC MBA toxicity test. In order to imitate certain possible conditions that the shellfish may encounter with, such as cooking in pan together with lemon juice or acidic sauce (e.g. vinegar), and reaching highly acidic medium in human stomach, the standard AOAC extraction procedure (hydrochloric acid extraction with boiling treatment) was adopted for extraction of aqueous PSP-toxin analogues from the shellfish. But, there were some limitations in our study as no Ctoxins (C1–4) was analyzed by HPLC. Information on toxicity contribution by C-toxins was in question. It is because standard AOAC extraction method may facilitate the inter-conversions (hydrolysis) of some low toxicity PSP-toxin analogues, N-sulfocarbamoyl C-toxins (C1,2 and C3,4) and GTX6, to their more potent analogues (GTX2,3 and GTX1,4) and NEO, respectively. In addition, GTX5 can also be converted to STX through this extraction procedure (Turrell et al., 2007; Earnshaw, 2003). This standard extraction may ensure the most conservative measurement of PSP toxicity for shellfish in the PSP monitoring programs, so as to provide maximum public health protection to people from PSP intoxication. Owing to the possibility of incomplete hydrolysis of this N-sulfocarbamoyl toxins group to their more potent analogues (Anderson et al., 1996; Turrell et al., 2007), there was still a risk of under detection for these toxins in the samples, in particular those shellfish ingesting C-toxins rich dinoflagellates (Oshima, 1995a; Anderson et al., 1996; Mak et al., 2003). Therefore, if the JRPT is sensitive to these toxins and they are also present at moderate level, a false positive response (relative to the MBA) could result. In order to maximize the interconversions of those toxins to their more potent analogues, it is quite possible that complete hydrolysis can take place upon standardization of extraction pH, time and temperature of the AOAC extraction procedure (Anderson et al., 1996). The JRPT assay was considered sensitive to Nsulfocarbamoyl analogues such as GTX5 and C1–2 (Mackintosh et al., 2002; Asp et al., 2004; Turrell et al., 2007), suggesting the potential capability of the assay in identifying the low PSP toxicity samples. However, actual cross-reactivity of JRPT to GTX5 and C1– 2 would be in doubt at this stage owing to recent findings from Laycock et al. (2010) showing that those low toxicity toxins, GTX5 and C1–2, might partially degrade to more potent analogues of STX and GTX2,3, respectively. Harmful algal blooms, that cause biotoxins illnesses including those responsible for PSP, are global public health threat, and have a trend of increasing frequency and intensity (Hallegraeff, 1993; Anderson, 1994; Van Dolah, 2000). Therefore, PSP would be a significant public health concern in the future, and subsequently increase the demand of laboratory PSP testing. Studies showed that the application of PSP rapid detection assays would reduce experimental mice used in AOAC MBA by 30–60% (Mackintosh et al., 2002; Oshiro et al., 2006; Turrell et al., 2007). Recently, the JRPT was also accepted by the Interstate Shellfish Sanitation Conference (ISSC), USA as a screening assay for PSP contaminated shellfish (Oshiro et al., 2006; Costa et al., 2009). Given that about

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98% of MBA tests in Hong Kong were determined as negative results based on this study, it is proposed that the application of JRPT for first line screening of PSP-toxins may be useful for minimizing the number of bioassays for PSP-toxins screening and toxicity confirmation. Although HPLC method can provide detailed PSP-toxins profiles and a more sensitive tool for PSP-toxins characterization and quantification, it can only offer a complementary assessment of PSP risk. In addition, its laborious procedures and tedious preparation steps would hinder its use for monitoring in surveillance program. Basically, toxicity-based AOAC bioassay is still preferable for final total PSP toxicity confirmation in order to support the results obtained from antibody-based and chemical assays. Conflict of interest The authors declare that there is no conflict of interest in this study. Acknowledgements The authors are indebted to the Director of Health, Dr. P.Y. Lam for the permission to publish this article and particularly grateful to the staff of Public Health Laboratory Centre for their support and help in this study, and staff of Food and Environmental Hygiene Department in collection of samples.[SS] Declaration: All commercial products, trade names or materials mentioned in this article do not represent any suggestion, approval or endorsement for use by authors’ department and division.

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