Paralytic Shellfish Poisoning Research Articles

Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry.

Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD).

Spatial distribution of toxic Alexandrium tamiyavanichii (Dinophyceae) in the southeastern South China Sea-Sulu Sea: A molecular-based assessment using real-time quantitative PCR (qPCR) assay

In this study, a quantitative real-time PCR (qPCR) assay targeting the second internal transcribed spacer (ITS2) of the nuclear-encoded ribosomal RNA gene (rDNA) was developed for Alexandrium tamiyavanichii, a harmful tropical marine dinoflagellate. This species is of concern because it produces toxins that cause paralytic shellfish poisoning (PSP). The qPCR assay employed hydrolysis probe technology and showed high specificity, with a detection limit of 102 gene copies (less than one cell equivalent). Using this assay, the spatial distribution of A. tamiyavanichii was assessed, for the first time, in the southeastern South China Sea and the Sulu Sea. Plankton samples were collected from 71 stations during a scientific cruise from the Research Vessel Sonne as part of the joint EU project on Stratosphere ozone: Halogens in a Varying Atmosphere (SHIVA), conducted in November 2011. The highest cell densities were detected offshore of Kuching, southern Borneo (150cellsl−1) and exceeded the threshold level of 20–40cellsl−1 where the bioaccumulation of PSP toxins by shellfish is of concern. The distribution of A. tamiyavanichii was patchy horizontally with the highest cell concentrations found mainly offshore of southern Borneo, and a heterogeneous vertical distribution was observed above the pycnocline. The A. tamiyavanichii qPCR assay proved its applicability, specificity and sensitivity, and provides an alternative implementation tool for harmful microalgae monitoring programs.

The death mechanism of the harmful algal bloom species Alexandrium tamarense induced by algicidal bacterium Deinococcus sp. Y35.

Harmful algal blooms (HABs) cause a variety of deleterious effects on aquatic ecosystems, especially the toxic dinoflagellate Alexandrium tamarense, which poses a serious threat to marine economic and human health based on releasing paralytic shellfish poison into the environment. The algicidal bacterium Deinococcus sp. Y35 which can induce growth inhibition on A. tamarense was used to investigate the functional mechanism. The growth status, reactive oxygen species (ROS) content, photosynthetic system and the nuclear system of algal cells were determined under algicidal activity. A culture of strain Y35 not only induced overproduction of ROS in algal cells within only 0.5 h of treatment, also decrease the total protein content as well as the response of the antioxidant enzyme. Meanwhile, lipid peroxidation was induced and cell membrane integrity was lost. Photosynthetic pigments including chlorophyll a and carotenoid decreased along with the photosynthetic efficiency being significantly inhibited. At the same time, photosynthesis-related gene expression showed down-regulation. More than, the destruction of cell nuclear structure and inhibition of proliferating cell nuclear antigen (PCNA) related gene expression were confirmed. The potential functional mechanism of the algicidal bacterium on A. tamarense was investigated and provided a novel viewpoint which could be used in HABs control.

Use of a Liquid Chromatographic Method for Assessment of Paralytic Shellfish Poisoning Toxin Profiles in Mussels and Clams from Uruguay

ABSTRACT For over 30 y, a mouse bioassay has been used to monitor paralytic shellfish poisoning (PSP) toxicity in shellfish harvested along the coast of Uruguay. Although providing a good controlmechanism for the safety of shellfish consumers, the assay is known to have several disadvantages. To assess the potential for use of liquid chromatography with fluorescence detection, a study was conducted to analyze shellfish samples harvested over a 20-y period using this alternative method. Mussels and clams were analyzed using the AOAC Official Method 2005.06 with results compared with those obtained from the original mouse bioassay and used to determine the presence of specific toxin congeners. The chromatographic results from nontoxic samples indicated no specificity issues resulting fromthe potential presence of naturally fluorescent coextractives but with complex and variable toxin distributions in samples designated PSP-positive by the bioassay. Although some species-related differences in toxin profile ...

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Scientific questionParalytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in dinoflagellates is extremely limited owing to their unique features. The origin and evolution of PST biosynthesis in these two kingdoms are still controversial. Technical significanceHigh-throughput omics technologies, such as genomics, transcriptomics and proteomics provide powerful tools for the study of PST biosynthesis in cyanobacteria and dinoflagellates, and have shown their powerful potential with regard to revealing genes and proteins involved in PST biosynthesis in two kingdoms. Scientific significanceThis review summarizes the recent progress in PST biosynthesis in cyanobacteria and dinoflagellates with focusing on the novel insights from omics technologies, and discusses the evolutionary relationship of toxin biosynthesis genes between these two kingdoms.

Gymnodinium catenatum Graham isolated from the Portuguese coast: Toxin content and genetic characterization

The bloom forming marine dinoflagellate Gymnodinium catenatum Graham has been linked to paralytic shellfish poisoning (PSP) outbreaks in humans. Along the Portuguese coast (NE Atlantic), G. catenatum shows a complex bloom pattern, raising questions about the origin and affinities of each bloom population. In this work, the variability within six cultured strains of G. catenatum isolated from Portuguese coastal waters (S coast, W coast and NW coast), between 1999 and 2011, was investigated. The strains were analyzed for toxin profiling and intra-specific genetic diversity. Regarding the toxin profile, differences recorded between strains could not be assigned to the time of isolation or geographical origin. The parameter that most influenced the toxin profile was the life-cycle stage that originated the culture: vegetative cell versus hypnozygote (resting cyst). At the genetic level, all strains showed similar sequences for the D1–D2 region of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA) and shared complete identity with strains from Spain, Algeria, China and Australia. Conversely, we did not find a total identity match for the ITS-5.8S nuclear rDNA fragment. After sequence analysis, two guanine/adenine (R) single nucleotide polymorphisms (SNP 1 and 2) were detected for all strains, in the ITS1 region. This species has been reported to present very conservative LSU and ITS-5.8S rDNA regions, though with few SNP, including SNP1 of this study, already attributed to strains from certain locations. The SNP here described characterize G. catenatum populations from Portuguese waters and may represent valuable genetic markers for studies on the phylogeography of this species.

Association between the Hygiene Index Values of Live Fresh Aquatic Products and Food-Borne Diarrhea in the Population of the Ningbo Area in China.

To investigate the association of the hygiene index values of live fresh aquatic products and food-borne diarrhea in the population of the Ningbo area in China. Volatile basic nitrogen (VBN), histamine (HIS), indole, tetrodotoxin (TTX), and paralytic, neurotoxic, amnesic and diarrhetic shellfish poisons (PSP, NSP, ASP, and DSP, respectively) in the samples of live fresh aquatic products and food-borne diarrhea cases in six studied districts were analyzed. Results indicate that the incidence rate of food-borne diarrhea is related to the hygiene index values. Aside from VBN, the main risk factors related to food-borne diarrhea in edible aquatic products include DSP (in marine fish, shrimp, and other shellfishes), NSP, and ASP (in marine shrimp and crab). Hygiene index values among different species were significantly different. No significant difference in the monitoring index values was found among the six different studied districts. The reported cases of food-borne diarrhea were positively associated with VBN and DSP in aquatic products in Haishu, Jiangbei, Zhenhai, and Beilun, as well as VBN and NSP in aquatic products in Jiangdong and Yinzhou. In conclusion, VBN, DSP, NSP, and ASP are important risk factors for the occurring of food-borne diarrhea in the population of the Ningbo area in China.

Optimization of a rapid QuEChERS sample treatment method for HILIC-MS2 analysis of paralytic shellfish poisoning (PSP) toxins in mussels

A rapid and simple QuEChERS sample treatment was proposed for the development of a selective hydrophilic interaction liquid chromatography-ESI-MS2-based method for the determination of saxitoxins (STXs) in mussel samples. Among different sorbents, ABS Elut-NEXUS phase, composed of polystyrene cross-linked with 50% divinyl benzene and poly(methyl methacrylate), provided the best results. The effects of experimental parameters, including sorbent amount, vortexing time and centrifugation time were investigated and optimized by experimental design. In particular, regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction response. The method was validated under the optimized conditions; detection and quantification limits in the 3–159 μg/kg and 7–436 μg/kg ranges respectively were obtained, except for C2 for which highest values were calculated due to its low ESI ionization efficiency. Finally, the analysis of twenty-eight mussel samples permitted to detect and quantify some of the investigated STXs, proving the applicability of the devised method.

Saxitoxin Modulates Immunological Parameters and Gene Transcription in Mytilus chilensis Hemocytes.

Saxitoxin (STX) is a neurotoxin produced by dinoflagellates in diverse species, such as Alexandrium spp., and it causes paralytic shellfish poisoning (PSP) in humans after the ingestion of contaminated shellfish. Recent studies have suggested that the immune functions of bivalves could be affected by harmful algae and/or by their toxins. Herein, hemocytes are the main effector cells of the immune cellular response. In this study, we evaluated the response of hemocytes from the mussel Mytilus chilensis to STX exposure in a primary culture. Cell cultures were characterized according to size and complexity, while reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein diacetate (DCFH-DA) assay. Finally, phagocytic activity was measured using both flow cytometry and fluorescence microscopy assays. Additionally, gene transcription of candidate genes was evaluated by qPCR assays. The results evidenced that exposures to different concentrations of STX (1–100 nM) for 24 h did not affect cell viability, as determined by an MTT assay. However, when hemocytes were exposed for 4 or 16 h to STX (1–100 nM), there was a modulation of phagocytic activity and ROS production. Moreover, hemocytes exposed to 100 nM of STX for 4 or 16 h showed a significant increase in transcript levels of genes encoding for antioxidant enzymes (SOD, CAT), mitochondrial enzymes (COI, COIII, CYTB, ATP6, ND1) and ion channels (K+, Ca2+). Meanwhile, C-type lectin and toll-like receptor genes revealed a bi-phase transcriptional response after 16 and 24–48 h of exposure to STX. These results suggest that STX can negatively affect the immunocompetence of M. chilensis hemocytes, which were capable of responding to STX exposure in vitro by increasing the mRNA levels of antioxidant enzymes.

Differences in the toxin profiles of Alexandrium ostenfeldii (Dinophyceae) strains isolated from different geographic origins: Evidence of paralytic toxin, spirolide, and gymnodimine.

Among toxin-producing dinoflagellates of the genus Alexandrium, Alexandrium ostenfeldii is the only species able to produce paralytic shellfish poisoning (PSP) toxins, spirolides (SPXs) and gymnodimines (GYMs). In this study we characterized and compared three A. ostenfeldii strains isolated from the Baltic, Mediterranean, and southern Chile Seas with respect to their toxin profiles, morphology, and phylogeny. Toxin analyses by HPLC-FD and LC-HRMS revealed differences in the toxin profiles of the three strains. The PSP toxin profiles of the southern Chile and Baltic strains were largely the same and included gonyautoxin (GTX)-3, GTX-2, and saxitoxin (STX), although the total PSP toxin content of the Chilean strain (105.83 ± 72.15 pg cell(-1)) was much higher than that of the Baltic strain (4.04 ± 1.93 pg cell(-1)). However, the Baltic strain was the only strain that expressed detectable amounts of analogues of GYM-A and GYM-B/-C (48.27 ± 26.12 pg GYM-A equivalents cell(-1)). The only toxin expressed by the Mediterranean strain was 13-desmethyl SPX-C (13dMeC; 2.85 ± 4.76 pg cell(-1)). Phylogenetic analysis based on the LSU rRNA showed that the studied strains belonged to distinct molecular clades. The toxin profiles determined in this study provide further evidence of the taxonomic complexity of this species.

片側性末梢性顔面神経麻痺を伴う麻痺性貝中毒の1例(Paralytic shellfish poisoning associated with unilateral peripheral facial nerve palsy: a case report)

麻痺性貝中毒は，口唇・舌・顔面のしびれから始まり，次第に四肢の麻痺へ広がり，重症の場合は呼吸筋麻痺で死亡することもある。現在，わが国では行政によって貝毒の調査が行われ，貝類の毒性が規制値を超えた場合は出荷停止の措置が取られるため，貝類摂食による中毒症例は極めて少ない。今回我々は，牡蠣の摂食による片側性末梢性顔面神経麻痺を伴う麻痺性貝中毒の症例を経験した。患者は55歳，女性。車で旅行中，高速道路のサービスエリアで牡蠣鍋を食べた。牡蠣を食べたとたんに口唇にやけどをしたようなしびれを感じた。食後5分後に舌の感覚が鈍く感じられ，食後10分後に飲み物が口角からこぼれ，呂律が回らなくなった。食後3時間半後に当院を受診した。来院時，構音障害は改善しており，四肢麻痺はなく，呼吸・循環に異常はなかった。口唇・舌・顔面のしびれを訴え，右末梢性顔面神経麻痺を認めた。右耳介周囲の皮疹や疼痛などは認めず，頭部CT・MRI検査で異常を認めなかった。麻痺性貝中毒と診断され，入院したが，呼吸筋麻痺の出現は認めず，第3病日に退院し，近医へ通院となった。顔面神経麻痺は発症12日目に消失し，以後再発はない。これまでに片側性末梢性顔面神経麻痺を伴う麻痺性貝中毒の報告はなく，本症例は極めて稀と考えられた。同様な症例に遭遇した場合，ベル麻痺と速断せず，貝類摂食の有無を含めた詳細な病歴聴取と経過観察入院が必要である。 Paralytic shellfish poisoning (PSP) begins with tingling and numbness of the lips, tongue, and face, and could be lethal due to respiratory muscle paralysis. PSP is quite rare in Japan today because it has been effectively prevented by regular surveys of shellfish poison by the government. A 55–year–old woman visited a restaurant in a rest area on a highway and ate a dish of oysters and vegetables. As soon as she ate an oyster, she felt tingling in her lips. In 10 minutes after the meal, coffee spilt from her mouth on drinking and she had dysarthria. There were no abnormalities of her respiration or circulation and no limb paralysis. Brain CT and MRI also revealed no abnormalities. She was admitted to our hospital because PSP associated with right unilateral peripheral facial nerve palsy (UPFNP) was diagnosed. Respiratory muscle paralysis did not develop, and she was discharged on the third day. The facial palsy disappeared on the twelfth day. There has been no reported case of PSP associated with UPFNP. When encountering a similar case, detailed medical history–taking and follow–up hospitalization are necessary, avoiding a premature diagnosis of Bell's palsy. 麻痺性貝中毒（paralytic shellfish poisoning，以下PSP）とは，毒のある貝を食べて発症する中毒の一つで，その症状は口唇・舌・顔面のしびれから始まり，次第に四肢の麻痺へ広がり，重症の場合は呼吸筋麻痺を来して死亡する恐れがある 1, 2ため，適切な対応が必要である。現在，わが国では行政によって貝類の毒性調査が行われ，貝類の毒性が規制値を超えた場合は出荷停止の措置が取られる 2, 3ので，PSP症例は極めて少なく，本症に遭遇したことがない救急医は多いと考えられる。今回我々は，片側性末梢性顔面神経麻痺を伴うPSPという極めて稀な症例を経験したので報告する。 患 者：55歳，女性 既往歴：34歳時に耳硬化症に対して右あぶみ骨手術 アレルギー：なし 現病歴：車で旅行中，高速道路のサービスエリアで牡蠣鍋を食べた。同行者は牡蠣鍋を食べていない。5個の牡蠣のうち3個目を右側の前歯で噛んだとたんに右口唇，右頬粘膜，舌の右側に灼熱感やピリピリしたしびれを感じ，やけどをしたかと思ったという。その後，食事を終えてサービスエリアを出発した。食後5分後に口唇や右顔面の腫れと舌の感覚の鈍さを自覚し，さらに食後10分後に飲み物が右口角からこぼれ，呂律が回らなくなった。呂律障害は30分ほどで改善したが，食後2時間後に腹痛・嘔気が出現した。食後3時間半後に当院救急外来を受診した。 来院時身体所見：意識清明，歩行可能。血圧135/75mmHg，脈拍66/min，呼吸は平静で呼吸数18/min，SpO2：96%（room air），体温36.3°C。胸部：呼吸音異常なし，心雑音なし。腹部：腸雑音正常，心窩部に軽度圧痛あり。反跳痛なし，筋性防御なし。神経学的所見としては，瞳孔両側3.0mm，対光反射両側正常，眼球運動両側正常，眼振なし。耳鳴，難聴，めまい症状なし。顔面は右末梢性顔面神経麻痺を認めた（柳原法で4/40点）（Fig. 1）。右口唇から右頬粘膜，右下顎の歯槽および舌の右側にかけて知覚鈍麻を訴えた。構音障害はごく軽度認めた。舌偏位，四肢麻痺，四肢の知覚異常は認めなかった。他の局所所見としては，右耳介周辺や右外耳に発疹や水疱は認めず，鼓膜所見にも異常は認めなかった。口唇には軽度腫脹があったが，発赤や熱傷は認めなかった。口腔内は右頬粘膜に軽度腫脹があったが，熱傷，水疱，舌の腫脹は認めなかった。全身皮膚の発赤・潮紅，蕁麻疹や眼瞼浮腫は認めず，頸部のstridorは聴取されなかった。 Photographs show right unilateral peripheral facial palsy. a: When smiling, loss of the right nasolabial fold and deviation of the mouth angle to the left were shown on the second day. b: When looking upward, the wrinkle didn't appear on her right forehead on the third day. 入院時検査所見：採血では特に異常を認めなかった（Table 1）。心電図では右軸偏位を認めたが，不整脈やST変化，T波異常などは認めなかった。胸部X線検査，頭部CT検査で異常を認めなかった。以上の結果よりPSPと診断した。食後4時間経過しており，構音障害は改善していることから，胃洗浄や活性炭投与は行わなかった。しかし，これから呼吸筋麻痺が出現する恐れがあるため，末梢ラインを確保し，経過観察入院とした。 入院後経過：来院2時間後，書類記入時に複視を訴えたが，6時間後には消失していた。脳梗塞を疑って頭部MRI検査を行ったが，特に異常を認めなかった。当初上腹部に強い腹痛を認め，第2病日には下痢を伴ったが，その後症状は軽快した。第2病日から食事を開始したが，舌の右側の味覚異常を訴えた。また右眼の乾燥を訴えた。第3病日には口唇の腫脹は軽減した。顔面神経麻痺は他覚的には変化はないが，患者は少し麻痺の改善を自覚するとのことであった。牡蠣摂食後36時間後までに呼吸筋麻痺の出現はなく，近医耳鼻科受診を指示して自宅退院とした。 退院後経過：第4病日，自宅近くの耳鼻科を受診し，右顔面神経完全麻痺（柳原法6/40点）に対して，アデノシン三リン酸二ナトリウム，メコバラミンを処方された。第5病日頃，味覚異常や舌の鈍い感覚は消失した。第7病日に受診歴のある他の耳鼻科を受診したが，顔面神経麻痺は18/40点に改善していた。プレドニンを30mgから開始し，1週間かけて漸減するように処方された。第12病日に顔面神経麻痺は完全に消失したことを自覚し，第14病日に麻痺の消失（柳原法40/40点）が確認された。以後再発はない。 保健所への届出：PSPとして所轄保健所及び厚生労働省に連絡した。保健所からは，「被疑食材は冷凍加工国産牡蠣であったが，同一素材を調理した同じ料理を食べた1名と，他の料理を食べた13名に症状はなく，患者の残食および同じロット番号の商品に貝毒は認められなかったことから，麻痺性貝中毒と断定はできないと判断した」と後日連絡を受けた。 PSPは各種二枚貝（カキ，アサリ，ホタテガイ，ムラサキイガイなど）を摂食して発症する中毒で 2，原因毒としてはサキシトキシン，ネオサキシトキシン，ゴニオトキシ群など多数の同族体があり 2，総称して麻痺性貝毒と呼ぶ。これらの毒は有毒プランクトンである渦鞭毛藻によって産生され，有毒プランクトンを捕食する各種二枚貝の，主に中腸腺に蓄積される。麻痺性貝毒のヒトの致死量はサキシトキシン換算で1～2mgと推定され 2，毒力としてはテトロドトキシンに匹敵し，青酸ソーダの1,000倍に相当する猛毒である 4。作用機序はテトロドトキシンと同様で，骨格筋や神経の膜電位依存性ナトリウムイオンチャネルに結合し，チャネル内へのナトリウムイオンの流入を阻害して神経伝達を遮断する 2。麻痺性貝毒は，水溶性で，一般的な加熱調理では分解されない 2。 麻痺性貝毒は，経口摂取後，口腔や小腸の粘膜から速やかに吸収され 3，一般的には30分〜3時間で口唇・舌のぴりぴり感や知覚鈍麻が生じ，次第に顔面・頸部・四肢末端のしびれ感が生じる 5。その他に胃腸症状，頭痛，めまいが生じる 6。重症例では，その後嚥下障害，全身の異常感覚，運動失調，四肢の脱力を生じ，進行すると呼吸筋麻痺，呼吸停止が生じる 6。重症例ほど症状進行が速く，呼吸筋麻痺へ進行すれば12時間以内に死亡することがある 5。軽症〜中等症では2〜3日で症状は消失するが，重症例では1週間も持続することがある 5。治療は，貝類摂食後早期であれば胃洗浄，活性炭投与を行うが，その他は対症療法しかなく，呼吸筋麻痺に対しては適切な人工呼吸管理を行えば確実に救命できる 3。 わが国では，1948年に愛知県で初めてPSPが疑われる中毒が発生し 4，1975年に三重県でA.catenellaによる赤潮が初めて観察され，貝類から麻痺性貝毒が検出された 4, 7。我々の渉猟し得た限りでは，2013年までに推定や未届けを含め，全国で中毒事件が18件発生し，164人の患者と4人の死者が出ている 3, 4, 8, 9, 10（Table 2）。死亡例のうち，文献上死因が明らかなのは1例のみで，1979年に北海道で発症したPSPの患者が呼吸麻痺を伴う全身麻痺で死亡している 4。井上ら 10は，貝摂食後2時間後に呼吸停止を来したが，適切な人工呼吸管理によって障害を残さず救命された1例を報告している。現在わが国では，厚生労働省が麻痺性貝毒の毒性が規制値を超える貝類の販売を禁止している 3。また各都道府県によって，二枚貝生産海域区分ごとのプランクトンの調査と定期的な貝の毒性調査が行われ，毒性が規制値を超えた場合は出荷停止措置が行われる 3。1978年に上記措置が取られてから，市場に流通した二枚貝によるPSPの発生はないが，個人で採取した貝類で稀に発生している。1998年～2013年では，PSPは2008年，2010年，2013年にそれぞれ1件ずつ発生し，患者は計6人，死者0人と報告されている 3。 一方，世界的には，PSPは1年間に約2,000症例が発生し，死亡率は15%との報告 11がある。また，1年間に1,600症例が発生し，約300例は致死的（死亡率18.8％）との報告 12もあり，PSPはその死亡率の高さからフグ中毒以上に恐れられている。 本症例の鑑別診断としては，脳血管障害，アナフィラキシー，ベル麻痺，神経性貝中毒が挙げられる。脳血管障害は，頭部CT・MRI検査で否定された。アナフィラキシーについては，全身皮膚の発赤・潮紅，蕁麻疹や眼瞼浮腫は認めず，口頭浮腫・喘息・ショックなどの症状を認めていないことから，否定的と考える。ベル麻痺については，単純ヘルペスウイルスの抗体検査を行っていないので，その点で否定はできない。しかし，本症例は，第4病日に麻痺の改善が認められ，第12病日に完全回復している。この回復の速さは，完全麻痺のベル麻痺からの回復としては例外的に早い。Peitersenら 13のベル麻痺患者1,701例の調査では，不完全・完全麻痺を含めて，発症から2週間以内に治癒した例はわずか3%と報告している。加えて，牡蠣の口唇への接触を契機に発症したこと，口唇や頬粘膜のしびれ，構音障害，複視，腹痛，下痢などの症状を伴うことから，ベル麻痺は否定的と考えた。神経性貝中毒は，口内のしびれとひりひり感，運動失調，温度感覚異常などの神経障害を特徴とし，食後1～3時間で症状が現れ，嘔気，嘔吐，腹痛，下痢などの胃腸障害を伴うこともあり，アメリカやニュージーランドで発生する 14。胃腸障害を伴うところは本症例に合致するが，神経性貝中毒はこれまで日本では発生報告がないこと，本症例は国産の牡蠣による発症であることから，神経性貝中毒は可能性が低いと考えた。 Sobelら 1は，確定診断の検査手段がない臨床現場では，PSPを24時間以内の貝類摂食の事実と臨床症状から診断すると述べている。井上ら 10も，PSPの診断は病歴，症状から行うのが一般的で，患者の生体試料や残食の試料から毒成分を検出することは難しいと述べている。今回，保健所の調査では，本症例はPSPと断定できないと判断された。しかし我々は，牡蠣摂食後から発症したことは間違いなく，PSPに合致する症状があり，ほかに可能性の高い疾患を挙げられないことから，PSPとの診断は妥当と考える。今回は，患者の生体試料の分析は行っていない。米国では，PSP疑いの患者を診察した医師は，直ちに地域の保健機関に報告し，患者の尿を凍結保存して検査施設に送るようである 15。患者が単独で，残食もない場合，患者の生体試料は診断の有力な根拠となる。わが国でも，公的機関で患者の尿を緊急検査するシステムの必要性を感じる。 顔面神経麻痺については，PSPの症状を詳細に記載した報告が少なく 9, 10，一般的な症状の一つである顔面の麻痺が顔面神経麻痺を指しているかどうか，明らかにできなかった。しかし我々が，PubMedと医中誌Webで検索した限りでは，顔面神経麻痺を伴ったPSPの報告はなかったことから，本症例は極めて稀と考えられた。本症例の顔面神経麻痺は片側性であったが，これは貝毒が口腔粘膜や消化管から血中に吸収された結果の症状ではなく，接触した右側の口腔粘膜から速やかに貝毒が浸潤した結果による局所的症状ではないかと考える。 本症例の症状発現は極めて早い。これは貝毒が高濃度だったことに起因することも考えられる。しかしその場合は，消化管からも毒が吸収され，四肢のしびれ・呼吸筋麻痺が出現するなど，重症化するのではないかと思われた。また，高濃度の貝毒を含む貝を摂食し，摂食後1分以内に症状が発現した症例 15はあるが，同じ貝毒濃度の貝を摂食し，発症までに1時間かかった症例 15もあることから，症状発現の早さは単に高濃度ということでは説明がつかないと考える。PSPは，個々の患者によって摂食した毒量と症状の重症度に相当な違いがみられ，それは毒に対する患者ごとの感受性の違いとされている 16。本症例では，貝毒が，局所浸潤は速いが弱毒性のものであった可能性や，患者の貝毒に対する感受性が異常に高かった可能性などが考えられた。 本症例は発症が牡蠣摂食直後であり，患者が問診で牡蠣の摂食を言いそびれることがなかったためPSPを見過ごすことはなかった。しかし患者が牡蠣の摂食を話さなければ，ベル麻痺と速断して帰宅させてしまい，数時間後に呼吸筋麻痺を来した可能性があったと考える。ベル麻痺と思われても，貝類摂食の有無を含めた発症時の詳細な病歴聴取は必要である。 これまでPSPによる片側性末梢性顔面神経麻痺の報告例はなく，本症例は極めて稀と考えられた。顔面神経麻痺の予後は良好であった。貝摂食後に発生した顔面神経麻痺の診療の際は，安易にベル麻痺と速断せず，詳細な病歴聴取でPSPを鑑別診断し，呼吸筋麻痺の発生に留意した入院・経過観察が必要である。 利益相反はない。

Prevalence of paralytic shellfish poison-producing Planktothrix agardhii and Cylindrospermopsis raciborskii in a Brazilian semi-arid reservoir

Aim: The present study aimed to investigate the composition and toxicity of the phytoplankton in Sítios Novos reservoir, used mainly as drinking water supply for approximately 30,000 people.MethodsSamples were collected between January 2010 and June 2011.ResultsDuring this period 19 taxa of cyanobacteria and 22 of algae were identified. Out of 45 samples collected, algae accounted for no more than 10% of the quantified organisms in 44 samples. Cyanobacteria accounted for 100% of the organisms quantified in three samples and for 99% in other 29 samples. Among the cyanobacteria group, Planktothrix agardhii (Gomont) Anagnostidis & Komárek and Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subbaraju prevailed and both strains were isolated and identified as paralytic shellfish poison (PSP) producers. C. raciborskii strain has shown to produce SXT and dcSXT while P. agardhii strain has shown to produce dcGTX2 or 3.ConclusionsTo the author’s knowledge, this is the first report of PSP-producer cyanobacteria species isolated in Northeastern Brazil and the first reported of a P. agardhii synthesizing dcGTX2/3.

Prediction of on-site depuration of paralytic shellfish poisoning toxins accumulated in the scallop Patinopecten yessoensis of Ofunato Bay, Japan

In this paper, we present a decay equation for paralytic shellfish poisoning (PSP) toxins using long-term field data spanning more than 10 years in order to predict the detoxification period for the scallop Patinopecten yessoensis in Ofunato Bay, Japan. From the data, we obtained the date of maximum toxicity in the digestive gland (DG) of the scallop and then the date of detoxification. Next, we performed linear regression analysis between loge-toxicities and days after the maximum toxicity level for each study year. Toxicity declined at a rate of 1.5 %/day, and a period of 3–9 months was required for the scallop to achieve a toxicity level of 20 mouse units (MU)/g DG tissue, which is a critical level for determining monitoring frequency and area of scallop toxicity. We then estimated the number of days needed to reach 20 MU/g DG tissue (t 20) using the equations obtained by the above-mentioned analysis, and we performed another linear regression analysis between the loge (maximum toxicity) and t 20 for each year. The difference between the actual and predicted detoxification time ranged from −16 to 18 days. We conclude that these equations can be used to predict the depuration of PSP toxins from scallops in Ofunato Bay.

Lethal paralytic shellfish poisoning from consumption of green mussel broth, Western Samar, Philippines, August 2013.

In July 2013, the Philippines' Event-Based Surveillance & Response Unit received a paralytic shellfish poisoning (PSP) report from Tarangnan, Western Samar. A team from the Department of Health conducted an outbreak investigation to identify the implicated source and risk factors in coastal villages known for green mussel production and exportation. A case was defined as a previously well individual from Tarangan, Western Samar who developed gastrointestinal symptoms and any motor and/or sensory symptoms after consumption of shellfish from 29 June to 4 July 2013 in the absence of any known cause. The team reviewed medical records, conducted active case finding and a case-control study. Relatives of cases who died were interviewed. Sera and urine specimens, green mussel and seawater samples were tested for saxitoxin levels using high performance liquid chromatography. Thirty-one cases and two deaths were identified. Consumption of > 1 cup of green mussel broth was associated with being a case. Seawater sample was positive for Pyrodinium bahamense var. compressum and green mussel samples were positive for saxitoxin. Inspection revealed villagers practice open defecation and improper garbage disposal. This PSP outbreak was caused by the consumption of the green mussel broth contaminated by saxitoxin. As a result of this outbreak, dinoflagellate and saxitoxin surveillance was established, and since the outbreak, there have been no harmful algal blooms event or PSP case reported since. A "Save Cambatutay Bay" movement, focusing on proper waste disposal practice and clean-up drives has been mobilized.

Performance Characteristics of AOAC Method 2005.06 for the Determination of Paralytic Shellfish Toxins in Manila Clams, European Otter Clams, Grooved Carpet Shell Clams, Surf Clams, and Processed King Scallops.

An approach was developed for the verification of method performance of the AOAC 2005.06 LC-fluorescence detector (FLD) method for determination of paralytic shellfish poisoning (PSP) toxins in bivalve shellfish. This was developed following advice published by the Analytical Laboratory Accreditation Criteria Committee and applied to shellfish species that had not been previously subjected to a full single-laboratory validation scheme. The refined approach was developed following the need to assess performance in a number of shellfish species infrequently monitored through the UK statutory monitoring program, while reducing the impact and cost of the studies, most notably in terms of the use of valuable reference standards. The species assessed were manila clams (Ruditapes philippinarum), European otter clams (Lutraria lutraria), grooved carpet shell clams (R. decussatus), surf clams (Spisula solida), and king scallops (Pecten maximus) presented as adductor only or adductor plus roe. The method was assessed for sensitivity in terms of LOD and LOQ, toxin recovery, and method precision in each species. It incorporated the PSP toxins deemed toxic and/or prevalent in UK samples and commercially available as certified reference standards. The toxins studied included GTX1-5, dcSTX, STX, C1&2, and NEO. The toxins dcGTX2&3 were included for surf clams due to the prevalence of these toxins in this species as a result of toxin decarbamoylation. Method performance targets were met for each of the characteristics investigated. Consequently, the method was deemed fit for purpose for the screening and quantification of these clam and scallop species for PSP toxins by AOAC Method 2005.06 LC-FLD.

HARMFUL MICROALGAE ASSEMBLAGE IN THE AQUACULTURE AREA OF AMAN ISLAND, NORTHEN STRAIT OF MALACCA

A field survey was carried out in Aman Island, the northern Strait of Malacca, to investigate the occurrence of harmful microalgae. The area is known for marine finfish cage-culture and blood cockle farming. Plankton samples were collected fortnightly between July 2011 and September 2013. A total of 58 phytoplankton genera were recorded; 37 belong to the diatoms, and 21 genera were dinoflagellates. Harmful microalgae species were identified microscopically, of which nine are bloom-forming species that responsible for fish kills. These included Akashiwo sanguinea, Chaetoceros affinis, Cha. curvisetus, Cha. laevis, Ceratium furca, C. fusus, Cochlodinium sp., Karlodinium australe and Noctiluca scintillans. Three Alexandrium species were identified from the samples, i.e. A. leei, A. affine and Alexandrium sp. Pulau Aman; some species in the genus have been known to associate with Paralytic Shellfish Poisoning (PSP). Diarrhetic Shellfish Poisoning (DSP)-toxin producers were also encountered in the water samples, viz. Dinophysis acuminata, D. caudata, Dinophysis sp. and Prorocentrum micans. The harmful diatom, Pseudo-nitzschia spp. that associated with Amnesic Shellfish Poisoning (ASP), was commonly found in the water samples. Cell abundance of each harmful microalga was enumerated. The results showed that cell densities of harmful microalgae were relatively low throughout the sampling period. The microalgae community was dominated by Ceratium furca in May 2012, with cell densities exceeding 70,000 cells L-1; no fish kill event was reported during the time. Even though no fish kill or shellfish poisoning was reported in the area, he presence of harmful microalgae albeit at low cell densities, may pose a potential threat to human health and seafood biosecurity. Therefore, regular phytoplankton monitoring is necessary to safeguard human health and seafood industries in the country. Keywords: Aman Island; blood cockle; fish-cage; Harmful Algal Bloom; Malaysia; Strait of Malacca

Toxin profile of Gymnodinium catenatum (Dinophyceae) from the Portuguese coast, as determined by liquid chromatography tandem mass spectrometry.

The marine dinoflagellate Gymnodinium catenatum has been associated with paralytic shellfish poisoning (PSP) outbreaks in Portuguese waters for many years. PSP syndrome is caused by consumption of seafood contaminated with paralytic shellfish toxins (PSTs), a suite of potent neurotoxins. Gymnodinium catenatum was frequently reported along the Portuguese coast throughout the late 1980s and early 1990s, but was absent between 1995 and 2005. Since this time, G. catenatum blooms have been recurrent, causing contamination of fishery resources along the Atlantic coast of Portugal. The aim of this study was to evaluate the toxin profile of G. catenatum isolated from the Portuguese coast before and after the 10-year hiatus to determine changes and potential impacts for the region. Hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was utilized to determine the presence of any known and emerging PSTs in sample extracts. Several PST derivatives were identified, including the N-sulfocarbamoyl analogues (C1–4), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), and decarbamoyl derivatives, decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNeo) and decarbamoyl gonyautoxin 3 (dcGTX3). In addition, three known hydroxy benzoate derivatives, G. catenatum toxin 1 (GC1), GC2 and GC3, were confirmed in cultured and wild strains of G. catenatum. Moreover, two presumed N-hydroxylated analogues of GC2 and GC3, designated GC5 and GC6, are reported. This work contributes to our understanding of the toxigenicity of G. catenatum in the coastal waters of Portugal and provides valuable information on emerging PST classes that may be relevant for routine monitoring programs tasked with the prevention and control of marine toxins in fish and shellfish.

Integrative Monitoring of Marine and Freshwater Harmful Algae in Washington State for Public Health Protection

The more frequent occurrence of both marine and freshwater toxic algal blooms and recent problems with new toxic events have increased the risk for illness and negatively impacted sustainable public access to safe shellfish and recreational waters in Washington State. Marine toxins that affect safe shellfish harvest in the state are the saxitoxins that cause paralytic shellfish poisoning (PSP), domoic acid that causes amnesic shellfish poisoning (ASP) and the first ever US closure in 2011 due to diarrhetic shellfish toxins that cause diarrhetic shellfish poisoning (DSP). Likewise, the freshwater toxins microcystins, anatoxin-a, cylindrospermopsins, and saxitoxins have been measured in state lakes, although cylindrospermopsins have not yet been measured above state regulatory guidance levels. This increased incidence of harmful algal blooms (HABs) has necessitated the partnering of state regulatory programs with citizen and user-fee sponsored monitoring efforts such as SoundToxins, the Olympic Region Harmful Algal Bloom (ORHAB) partnership and the state’s freshwater harmful algal bloom passive (opportunistic) surveillance program that allow citizens to share their observations with scientists. Through such integrated programs that provide an effective interface between formalized state and federal programs and observations by the general public, county staff and trained citizen volunteers, the best possible early warning systems can be instituted for surveillance of known HABs, as well as for the reporting and diagnosis of unusual events that may impact the future health of oceans, lakes, wildlife, and humans.

PUBLIC AWARENESS ON HARMFUL ALGAL BLOOM (HAB) IN LAMPUNG BAY

Harmful algal blooms (HABs) cause various problems, such as water quality degradation, fauna mass mortality and impairment of human health. Water quality monitoring in Lampung Bay has been conducted by Lampung Marine Aquaculture Office (BBL) of the Ministry of Marine Affairs and Fisheries since 1994. Occurrence of Pyrodinium bahamense var. compressum (Pbc), a causative organism of paralytic shellfish poisoning (PSP), has been recorded but threats caused by HABs have never been reported. A study aimed to know the level of public knowledge and awareness about HABs in Lampung Bay has been conducted. The components of group respondents consisted oflocal governments, academics and coastal communities. Awareness from each component was examined includinggeneral HAB knowledge, HAB impact to the community, HAB occurence and local knowledge of HAB. Data of HAB knowledge were collected through questionnaire and focus group discussion. The result showed that 40.5% of respondents had knowledge of HABs phenomena and 51.3% respondents only know signs or indicators of HAB occurrence, while only 1.4% respondents had knowledge of local HAB occurences. The direct impact of HABs to the community was not commonly known by the respondents. Only a few cases of poisoning after eating seafood were reported. It can be concluded that there was lack of public knowledge and awareness on HABs in LampungBay. Intensive public awareness programs about HABs should be conducted in order to reduce risk towards HABs in Lampung Bay.

Detection of human exposure to saxitoxin and neosaxitoxin in urine by online-solid phase extraction-liquid chromatography-tandem mass spectrometry.

Saxitoxin (STX) and neosaxitoxin (NEO) are potent neurotoxins that cause paralytic shellfish poisoning (PSP). PSP typically occurs through the ingestion of bivalve shellfish that have consumed toxin producing dinoflagellates. Due to initial presentation of symptoms being nonspecific, a clinical measurement is needed to confirm exposure to these toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of STX and NEO in human urine with tandem mass spectrometry. A unique feature of this online method is the incorporation of a new synthetic (15)N4-STX labeled internal standard used for quantitation. Manual sample preparation time was reduced by approximately 70% for 98 urine samples as compared to a previously reported method. The lowest reportable limit for STX was improved from 5.0ng/mL to 1.01ng/mL and from 10.0ng/mL to 2.62ng/mL for NEO. Three analysts validated the method with 20 calibration curves total over 30 days with precision and accuracy within ±15% for all QCs. This new online method rapidly identifies STX and NEO exposure with improved sensitivity, which can facilitate the work of public health authorities to confirm the cases of PSP, complementing the many shellfish monitoring programs worldwide.