Puffer Fish Poisoning Research Articles

Sea temperature influences accumulation of tetrodotoxin in British bivalve shellfish

Tetrodotoxin (TTX), a potent neurotoxin mostly associated with pufferfish poisoning, is also found in bivalve shellfish. Recent studies into this emerging food safety threat reported TTX in a few, mainly estuarine, shellfish production areas in some European countries, including the United Kingdom. A pattern in occurrences has started to emerge, however the role of temperature on TTX has not been investigated in detail. Therefore, we conducted a large systematic TTX screening study, encompassing over 3500 bivalve samples collected throughout 2016 from 155 shellfish monitoring sites along the coast of Great Britain. Overall, we found that only 1.1 % of tested samples contained TTX above the reporting limit of 2 μg/kg whole shellfish flesh and these samples all originated from ten shellfish production sites in southern England. Subsequent continuous monitoring of selected areas over a five-year period showed a potential seasonal TTX accumulation in bivalves, starting in June when water temperatures reached around 15 °C. For the first time, satellite-derived data were also applied to investigate temperature differences between sites with and without confirmed presence of TTX in 2016. Although average annual temperatures were similar in both groups, daily mean values were higher in summer and lower in winter at sites where TTX was found. Here, temperature also increased significantly faster during late spring and early summer, the critical period for TTX. Our study supports the hypothesis that temperature is one of the key triggers of events leading to TTX accumulation in European bivalves. However, other factors are also likely to play an important role, including the presence or absence of a de novo biological source, which remains elusive.

Detection of Tetrodotoxin in Vomit and Urine from a Patient Suffering from Pufferfish Poisoning

We experienced a pufferfish poisoning case where no food residue was available to detect a causative agent. However, tetrodotoxin (TTX) was detected in vomit and urine samples from a patient using LC-MS/MS. Furthermore, we found a significant matrix effect in this analysis, indicating that the retention time of vomit and urine was not identical to the TTX standard solution and measured values multiplied by the dilution factors were not constant. Elimination of this matrix effect was attained by dilution of samples based on the retention time of the TTX standard solution, i.e., 10-time dilution of vomit test sample for LC-MS/MS analysis or 100-200-time dilution of urine one. Further research on urine analytical methods revealed that when TTX concentrations were too low to identify its peak on a chromatogram, TTX could be identified through a dilution procedure. It also showed that the application of the matrix-added TTX standard solution was effective for quantitative analysis under the influence of the matrix.

Rapid identification of Takifugu genus using visual loop-mediated isothermal amplification.

Some Takifugu species are commonly found in the coastal areas of China, Japan, Thailand, and Korea and cause pufferfish poisoning, which is toxic and even lethal to humans. From 2010 to 2015, there were 430 cases of pufferfish poisoning worldwide, resulting in 52 deaths. Identification of Takifugu species is imperative to reduce financial losses and ensure food safety. Here, visual loop-mediated isothermal amplification (LAMP) was applied to identify Takifugu species. Conserved regions within the mitochondrial DNA among different Takifugu species were selected to design LAMP primers. In 55min of amplification, sufficient DNA was obtained to observe the results with the naked eye, without the need for complicated instruments. The method was highly specific, with no cross-detection of 17 other fish species, namely, 7 Tetraodontiformes species and 10 commercially important fish. The method showed a detection limit of 0.1ng Takifugu DNA and was successfully validated to detect Takifugu in cooked fish and the vomitus of poisoned patients. This rapid and visual LAMP method is a useful tool to prevent false labeling, protect consumer rights, and reduce the risk of pufferfish poisoning. PRACTICAL APPLICATION: The loop-mediated isothermal amplification method established in this study can identify cooked or digested fish products containing 1% or more of Takifugu. Therefore, it can be used for the visual detection of Takifugu products and the medical diagnosis of Takifugu poisoning.

High Levels of Tetrodotoxin in the Flesh, Usually an Edible Part of the Pufferfish Takifugu flavipterus, Caused by Migration from the Skin and the Regional Characteristics of Toxin Accumulation

The consumption of a pufferfish, Takifugu flavipterus or komonfugu in Japanese, formerly known as Takifugu poecilonotus, is popular in Japan. However, T. flavipterus is frequently involved in cases of tetrodotoxin (TTX) poisoning in Japan. Although victims have usually consumed inedible parts, some cases are related to consumption of flesh. To improve the risk management of pufferfish poisoning, we studied TTX level in the flesh and skin of T. flavipterus. Ninety-seven specimens obtained from the Seto Inland Sea and landed in Fukuoka Prefecture were analyzed by liquid chromatography-tandem mass spectrometry. The flesh from six specimens was toxic (>10 MU/g = 2.2 mg/kg): one was in poor condition (not freeze–thawed); three were freeze–thawed before sample preparation; and two freshly prepared and in good condition (not freeze–thawed). The fillets were divided into outer and inner portions; the TTX levels in the outer portions were notably higher. The skin of the six specimens was moderately to extremely toxic: 165 MU/g (36.3 mg/kg) in the fresh specimen not in good condition, 600–950 MU/g (132–200 mg/kg) in freeze–thawed specimens, and 4500 and 6000 MU/g (990 and 1320 mg/kg) in the two fresh specimens. We concluded that TTX in the flesh migrated from the highly toxic skin. In addition, TTX levels in the skin appeared to be regionally specific. We recommend that toxic portions of T. flavipterus are removed as soon as possible after individuals are caught, and that fish from known highly toxic areas are not consumed.

Paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) of Korean pufferfish

Paralytic shellfish toxins (PSTs) and tetrodotoxin (TTX) are neurotoxins that display pharmacological activity that is similar to that of specific sodium channel blockers; they are the principle toxins involved in shellfish and puffer fish poisoning. In Korea, puffer fish is a very popular seafood, and several cases of accidental poisoning by TTX have been reported. Therefore, it is necessary to determine whether puffer fish poisoning incidents are caused by PSTs or by TTX. In this study, we used mouse bioassay (MBA) and liquid chromatograph-tandem mass spectrometry (LC-MS/MS) to determine the presence of PSTs and TTX in puffer fish from an area near Mireuk-do, Tong-Yeong on the southern coast of Korea from January through March, 2014. The toxicity of PSTs and TTX extracts prepared from three organs of each specimen was analyzed by MBA. Most of the extracts killed mice with typical signs of TTX and PSTs. The LC-MS/MS analysis of seven specimens of Takifugu pardalis and Takifugu niphobles, each divided into muscles, intestines, and liver, were examined for TTX. In T. pardalis, the TTX levels were within the range of 1.3–1.6 μg/g in the muscles, 18.8–49.8 μg/g in the intestines, and 23.3–96.8 μg/g in the liver. In T. niphobles, the TTX levels were within the range of 2.0–4.5 μg/g in the muscles, 23.9–71.5 μg/g in the intestines, and 28.1–114.8 μg/g in the liver. Additionally, the toxicity profile of the detected PSTs revealed that dcGTX3 was the major component in T. pardalis and T. niphobles. When PSTs were calculated as saxitoxin equivalents the levels were all less than 0.5 μg/g, which is below the permitted maximum standard of 0.8 μg/g. These findings indicate that the toxicity of T. pardalis and T. niphobles from the southern coast of Korea is due mainly to TTX and that PSTs do not exert an effect.

A fatal case of severe pufferfish poisoning

Abstract Introduction: Pufferfish poisoning is the main cause of death due to food poisoning in China. No effective treatment has been proposed so far. The artificial airway and early mechanical ventilation are crucial for the prognosis, but the optimal time for these interventions remains unclear. Here we present an unsuccessful use of mechanical respiratory support in a patient with pufferfish poisoning. Patient concerns: A 62-year-old male patient presented to the emergency department with complaints of numbness around the mouth. He gave a history of eating pufferfish 3 hours before the coming to the hospital. Diagnosis: He was diagnosed as pufferfish poisoning based on the history of consuming pufferfish and the symptom of numbness. Interventions: Gastric lavage was performed in time, but he did not receive protective endotracheal intubation. His condition worsened rapidly with respiratory failure and cardiac arrest. He received active treatment in the intensive care unit with mechanical ventilation. Outcomes: Spontaneous breathing was not restored during the intensive care unit stay. He underwent treatment with hyperbaric oxygen in another hospital and died of respiratory failure eventually. Conclusion: The establishment of artificial airway and early implementation of mechanical ventilation are crucial for good prognosis. Our case report provides a valuable experience for implementing early endotracheal intubation intervention in patients with pufferfish poisoning.

Development of Species-Specific PCR Primers for the Rapid and Simultaneous Identification of the Six Species of Genus Takifugu.

ABSTRACTPufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

미나리 추출물의 유리 아미노산 특성과 복어독에 중독된 마우스의 치사 시간에 미치는 영향

The purpose of this study is to investigate the characteristics of free amino acids in dropwort (Oenanthe javanica) and get the basic data about detoxication of the natural sources such as dropwort, soybean sprout, ginger, wood sorrel and 4-aminopyridine (4-AP) against the puffer fish poisoning. Dropwort was extracted with hot water and free amino acid in dropwort was analyzed. Asparagine (160.1 mg %), glutamine(119.8 mg %) and γ-aminobutylic acid (36.5 mg %) were major free amino acids (total 417.3 mg %). An aliquot of each extract of dropwort, soybean sprout, ginger, wood sorrel extracts and 4-AP combined with puffer fish toxin was injected to the mice respectively and we have checked the survival time. When only the puffer fish toxin was injected to the mice, the average survival time of mice was 662 second. And as dropwort extract was injected into the mice from 5 to 40 mg, the average survival time of mice was prolonged from 853 second to 1,493 second (Increase life span 232 %, T/C). With the 30 mg of injecting, the results suggested that certain content of free amino acids in dropwort was involved in water soluble antidotal. Another extracts and 4-AP increased survival time considerably depending on the amount injected but not so considerable compared to dropwort extracts.

Puffer fish poisoning: a case report

Puffer fish poisoning after consumption of this fish is one of the most common causes of poisoning among people in the coastal regions of Asia. Puffer fish poison (tetradotoxin) is one of the most toxic substances still found on earth; it is about 275 times more lethal poison than cyanide and fifty times more potent than strychnine or curare poison. Still no anti toxin or antidote is being discovered yet. On 6th January 2021, among five members of a single family, all of them ingested puffer fish after cooking, everyone developed toxic manifestations. Father died after three hours on the way to hospital, mother died after 17 hours during treatment and three daughters improved without any residual complication after getting supportive treatment in this hospital.
 CBMJ 2019 January: vol. 08 no. 01 P: 35-38

New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

Outbreak of Puffer Fish Poisoning in Dhaka City

Background: Puffer fish poisoning is quite uncommon but not rare in Bangladesh. This is not a very common fish in Bangladesh and consumed predominantly by poor class of people as it is cheap. Puffer fishes are considered toxic because they possess a potent neurotoxin, tetrodotoxin (TTX) and its analogs.Methods: In October 2014, total 11 patients from two families got admitted in Medicine unit of Dhaka Medical College on the same day with the complaints of tingling, numbness, dizziness, vertigo, postural instability and breathlessness. Both the families admitted, have ingested Puffer fish. A case control study was done where the control were taken from the vicinity with same socioeconomic background. The control group decline of any ingestion of portion of puffer fish. Hence the results and discussion are based on meticulous observation of cases. All the cases were clinically analyzed on different variables.Results: Total 11 patients were from two families, lives half kilometer apart, from Dholaipar, Jatrabari, Dhaka. Mean age of cases were 25.18. Among eleven cases, 7 were male and 4 were female. Mean of lag period between Puffer fish intake and development symptom was 99.44 minute (SD 97.28). Mean of lag period between development of symptom and hospitalization in puffer fish intake group was 308.57 minute (SD 198.78).There were 4 deaths those didn’t receive Neostigmine and respiratory support. Total 2 patient received Neostigmineand they recovered. Neostigmine is protective in Puffer fish poisoning (RR .889 <1) but the association is not significant (95% CI on both sides of 1).Conclusion: Cluster of Puffers fish poisoning presenting as outbreaks are associated with serious consequence. The early presentation to health facility and timely neostigmine application and or respiratory support can be lifesaving.J MEDICINE Jan 2018; 19 (1) : 30-34

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostrea gigas), native oysters (Ostrea edulis) common mussels (Mytilus edulis) and hard clams (Mercenaria mercenaria), but not found in cockles (Cerastoderma edule), razors (Ensis species) or scallops (Pecten maximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrum cordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Puffer fish poisoning: summary of case reports from Thailand

Puffer fish poisoning: summary of case reports from Thailand

A rapid method for tetrodotoxin (TTX) determination by LC-MS/MS from small volumes of human serum, and confirmation of pufferfish poisoning by TTX monitoring

A simple and rapid detection method for tetrodotoxin (TTX), a powerful sodium channel blocker, in small volumes of the serum of patients with pufferfish poisoning, was developed using an ultrafiltration spin column. The separation and identification of TTX was performed by liquid chromatography (LC) with a multi-mode ODS column and tandem mass spectrometry. TTX and an internal standard (voglibose) were monitored and quantitated using ion transitions: the respective precursor-to-product ion combinations, m/z 320/162 for TTX and 268/92 in MRM mode. The recoveries of TTX and voglibose were 91.0–110.8% and 104.7–107.4%, respectively, and with high accuracy (intra-run, 4.35–5.29%; inter-run, 2.95–5.79%) and linearity (0.5–200 ng/ml serum: r = 0.9994). The lower limit of quantification was 0.5 ng/ml serum. In patients, maximum serum TTX concentrations were 30.2 ng/ml serum for patient 1 on day 0 and 56.1 ng/ml serum for patient 2 on day 1. These results are important for the treatment of patients and for the identification of poisoning as well as for the determination of the cause of the food poisoning.

A Rapid Sample Preparation Procedure Using MonoSpin CBA and Amide Columns for Tetrodotoxin Detection in Serum and Urine Using LC–MS/MS Analysis

Clinical diagnosis of tetrodotoxin (TTX) poisoning can be difficult because of the lack of characteristic morphological findings and a screening test, such as an immunoassay. Here, we present a fully validated method for the analysis of TTX in serum and urine. In this method, serum and urine samples were extracted using MonoSpin CBA or amide columns, followed by LC–MS/MS analysis. The TTX was eluted from the column by 0.1 mL of 10 % acetic acid solution, and was directly injected into LC–MS/MS. An Agilent 1200 HPLC system equipped with a HILIC separation column (Zorbax HILIC Plus 2.1 × 100 mm, 3.5 μm) was used for isocratic elution, with a mobile phase of 10 mM ammonium formate with formic acid (95:5, v/v), along with 5 mM trifluoroacetic acid and 2 % acetonitrile. TTX was detected with an Agilent 6410 mass spectrometer utilizing positive electrospray ionization and multiple reaction monitoring. Limits of quantification for serum and urine were established to be 1 and 0.5 ng mL−1, respectively. Limits of detection for serum and urine were 0.5 and 0.25 ng mL−1, respectively. Intra-day and inter-day precision varied from 1.5 to 8.5 %. The recovery was >86.5 % for both matrices. In this method, the sample preparation process prior to injection into the LC–MS/MS takes approximately 10–15 min, which reduces the extraction time to one-tenth of that of previous methods. The application of this method was further verified by analysis of biological materials from a patient suffering from puffer fish poisoning.

Rapid Detection of Chemical Hazards (Toxins, Dioxins, and PCBs) in Seafood

Among the various hazards occurring in fish and seafood chemical hazards and in particular toxins (ciguatera, scombroid fish poisoning, paralytic shellfish poisoning, neurotoxic (brevetoxic) shellfish poisoning, puffer fish poisoning, diarrhetic shellfish poisoning) have an important place in food poisoning cases. On the other hand, some of the chemical hazards are often due to the pollution of the environment (heavy metals, dioxins, polychlorinated biphenyls, and halogenated aromatic hydrocarbons) and their detection is neither rapid nor facile. As a result there was a great need for developing new rapid and effective methods toward the chemical hazards determination mainly because of their high toxicity. The aim of this review is to provide the information about the new up-to-date detection techniques (Immunological, Chemical and Biochemical, and Molecular assays) in conjunction with detection limits. The latter is made possible by means of inclusion of seven comprehensive and, in most case cases, very extended tables. A reference is also made on the risk characterization of toxins as regards their importance to food contamination or poisoning.

Marine Fish Toxins in Thailand: Report of 6 Suspected Ciguatera Cases

Food poisoning or gastroenteritis is a common diagnosis for individuals who have nausea, vomiting and diarrhea after ingestion of fish. When also manifesting neurological symptoms, these are blamed on puffer fish poisoning, but ciguatera fish poisoning (CFP) can also manifest only with gastroenteritis, without neurological complications and remain undiagnosed. We report patients who fulfilled criteria of CFP suffering from severe GI disturbances, neurological manifestations, compromised cardiovascular status and autonomic dysfunction. All recovered within 1 - 3 days with supportive treatment. There were two small outbreaks: one in Bangkok in 2007 (2 patients) and another (2009) in Phuket (4 patients). All patients consumed the same unidentified fish portion and had severe GI symptoms. One had acute ventilatory failure requiring intubation whereas the remaining had neurological disturbances consisting of paresthesia, severe vertigo and ataxia. Absence of reflex tachycardia was noted in all patients who had severe volume depletion and shock. The most severe patient could be extubated within 24 hours and was discharged in 48 hours. All of the remaining recovered completely within 48 hours. Severe abdominal pain, nausea and vomiting and autonomic dysfunction in the form of bradycardia in the presence of hypotension were seen. Dramatic recovery within 48 hours occurred in all cases. None of the fish considered to have been responsible was available for testing. Physicians should be aware of CFP intoxication in the differential diagnosis of gastroenteritis-like syndromes after eating seafood.

An Overview on the Marine Neurotoxin, Saxitoxin: Genetics, Molecular Targets, Methods of Detection and Ecological Functions

Marine neurotoxins are natural products produced by phytoplankton and select species of invertebrates and fish. These compounds interact with voltage-gated sodium, potassium and calcium channels and modulate the flux of these ions into various cell types. This review provides a summary of marine neurotoxins, including their structures, molecular targets and pharmacologies. Saxitoxin and its derivatives, collectively referred to as paralytic shellfish toxins (PSTs), are unique among neurotoxins in that they are found in both marine and freshwater environments by organisms inhabiting two kingdoms of life. Prokaryotic cyanobacteria are responsible for PST production in freshwater systems, while eukaryotic dinoflagellates are the main producers in marine waters. Bioaccumulation by filter-feeding bivalves and fish and subsequent transfer through the food web results in the potentially fatal human illnesses, paralytic shellfish poisoning and saxitoxin pufferfish poisoning. These illnesses are a result of saxitoxin’s ability to bind to the voltage-gated sodium channel, blocking the passage of nerve impulses and leading to death via respiratory paralysis. Recent advances in saxitoxin research are discussed, including the molecular biology of toxin synthesis, new protein targets, association with metal-binding motifs and methods of detection. The eco-evolutionary role(s) PSTs may serve for phytoplankton species that produce them are also discussed.

Consequences of the increasing prevalence of the poisonous Lagocephalus sceleratus in southern Turkey

BackgroundThe increasing Indo-Pacific migration has affected the biodiversity of the Mediterranean Sea, and the prevalence of the puffer fish (Lagocephalus sceleratus), a well-known poisonous migrant, is increasing. The fish, which...

A Case of Hemoperitoneum due to Spontaneous Rupture of Visceral Artery Aneurysm after Ingestion of Puffer Fish

Ingestion of puffer fish can cause intoxication, which produces a wide range of symptoms due to the presence of neurotoxins, such as tetrodotoxin, in puffer fish. Abdominal pain is just one of the symptoms that should be treated with symptomatic and supportive therapy. This study reports a case of a 56-year-old male patient with abdominal pain, who was admitted to the emergency room with a diagnosis of puffer fish poisoning. In this case, the abdominal pain did not improve, but rather, the symptoms worsened. Finally, the cause of the abdominal pain was found to be hemoperitoneum due to active bleeding at the greater omentum, as observed on abdominal computed tomography; the source of bleeding was the right colic artery branch. Embolization was performed successfully, and the post-intervention course was uneventful. The patient was discharged without any complications within 13 days after admission.