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BACKGROUND AND AIMS: Amanita phalloides poisoning causes severe liver damage which may be potentially fatal. Several treatments are available, but their effectiveness has not been systematically evaluated. We performed a systematic review to investigate the effect of the most commonly used therapies: N-acetylcysteine (NAC), benzylpenicillin (PEN), and silibinin (SIL) on patient outcomes. In addition, other factors contributing to patient outcomes are identified. METHODS: We searched MEDLINE and Embase for case series and case reports that described patient outcomes after poisoning with amanitin-containing Amanita mushrooms. We extracted clinical characteristics, treatment details, and outcomes. We used the liver item from the Poisoning Severity Score (PSS) to categorize intoxication severity. RESULTS: We included 131 publications describing a total of 877 unique cases. The overall survival rate of all patients was 84%. Patients receiving only supportive care had a survival rate of 59%. The use of SIL or PEN was associated with a 90% (OR 6.40 [3.14-13.04]) and 89% (OR 5.24 [2.87-9.56]) survival rate, respectively. NAC/SIL combination therapy was associated with 85% survival rate (OR 3.85 [2.04, 7.25]). NAC/PEN/SIL treatment group had a survival rate of 76% (OR 2.11 [1.25, 3.57]). Due to the limited number of cases, the use of NAC alone could not be evaluated. Additional analyses in 'proven cases' (amanitin detected), 'probable cases' (mushroom identified by mycologist), and 'possible cases' (neither amanitin detected nor mushroom identified) showed comparable results, but the results did not reach statistical significance. Transplantation-free survivors had significantly lower peak values of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total serum bilirubin (TSB), and international normalized ratio (INR) compared to liver transplantation survivors and patients with fatal outcomes. Higher peak PSS was associated with increased mortality. CONCLUSION: Based on data available, no statistical differences could be observed for the effects of NAC, PEN or SIL in proven poisonings with amanitin-containing mushrooms. However, monotherapy with SIL or PEN and combination therapy with NAC/SIL appear to be associated with higher survival rates compared to supportive care alone. AST, ALT, TSB, and INR values are possible predictors of potentially fatal outcomes.

The deadly toxin α-amanitin is a bicyclic octapeptide biosynthesized on ribosomes. A phylogenetically disjunct group of mushrooms in Agaricales (Amanita, Lepiota, and Galerina) synthesizes α-amanitin. This distribution of the toxin biosynthetic pathway is possibly related to the horizontal transfer of metabolic gene clusters among taxonomically unrelated mushrooms with overlapping habitats. Here, our work confirms that two biosynthetic genes, P450-29 and FMO1, are oxygenases important for amanitin biosynthesis. Phylogenetic and genetic analyses of these genes strongly support their origin through horizontal transfer, as is the case for the previously characterized biosynthetic genes MSDIN and POPB. Our analysis of multiple genomes showed that the evolution of the α-amanitin biosynthetic pathways in the poisonous agarics in the Amanita, Lepiota, and Galerina clades entailed distinct evolutionary pathways including gene family expansion, biosynthetic genes, and genomic rearrangements. Unrelated poisonous fungi produce the same deadly amanitin toxins using variations of the same pathway. Furthermore, the evolution of the amanitin biosynthetic pathway(s) in Amanita species generates a much wider range of toxic cyclic peptides. The results reported here expand our understanding of the genetics, diversity, and evolution of the toxin biosynthetic pathway in fungi.

Amanita fuligineoides, a lethal mushroom discovered in China, contains abundant cyclic peptide toxins that can cause fatal poisoning. However, the MSDIN gene family encoding for these cyclic peptides in A. fuligineoides has not been systematically studied. In this research, the transcriptome sequencing of A. fuligineoides was performed and its MSDIN family members were analyzed. A total of 4.41 Gb data containing 30833 unigenes was obtained; sequence alignments throughout several databases were done to obtain their functional annotations. Based on these annotations, MSDIN genes were found and verified by RT-PCR. A total of 29 different core peptides were obtained: 3 toxin genes, encoding β-amanitin (β-AMA), phalloidin (PHD), and phallacidin (PCD), and 26 genes encoding unknown cyclic peptides, 20 of which are reported for the first time and may encode for novel cyclic peptides. Analysis of the predicted precursor peptides indicated that octocyclic peptides were the main MSDIN peptides synthesized by A. fuligineoides, accounting for the 45%. A phylogenetic analysis suggested that studied precursor peptides could be clustered into 7 clades, which might represent different functionalities. Results suggested that A. fuligineoides might have a strong capacity to synthesize cyclopeptides, laying the foundation for their excavation and utilization.

The paper presents results of AI diagnostics and treatment across the period of 2004-2020 pointing to the efficacy of two particular protocols.Quantitative determination of amanitins in blood (ATOs) and urine (ATOu) performed by the original ELISA kit, indicated upon mycological history and clinical symptoms of poisoning. ATOu positive cases were recommended our protocol; ATOu negative results excluded amanitin poisoning.out of 2876 fungal poisonings registered in Slovakia during the subjected period, were 698 AI suspected cases. In 557 of them, was AI reliably excluded, in 141 confirmed. Urinary ATOu correlated with the severity of poisoning in the range of 6-47 h after mushroom ingestion, without false negativity. Serum ATOs had no diagnostic value. 129 patients with confirmed AI received full treatment protocol with antidotes of penicillin plus silibinin. In this group died two patients of acute kidney injury in the early stages of poisoning and 127 patients were recovered. Silibinin without penicillin was used in 12 patients. One of them undergone liver transplantation and four patients died of fulminant liver failure, respectively intracranial hemorrhage. Treatment failure in the PNC + silibinin protocol was 1.5 % (2 of 127 patients), silibinin alone being 41.7 % (5 of 12 patients, p = 0.00058).Early diagnostics of amanitin intoxication based on mycological and clinical history and subsequent determination of urinary amanitin levels (ATOu) allows early initiation of treatment. The use of treatment protocol with antidotes of PNC and silibinin is of high therapeutic efficacy. The omission of PNC from the treatment protocol significantly worsens patients' prognosis.

Context: Amanita phalloides related toxicity from amatoxins can result in acute liver and multi-organ failure and is responsible for 90% of all mushroom poisoning death. However, more evidence is needed in regards to different management strategies. Case details: We present two cases of amanita mushroom ingestion who were treated with intravenous rifampicin. Discussion: Further study is needed to establish the efficacy and role of rifampicin in amatoxin related mushroom poisoning.

[Abstract Not Available] WOS:000636853400007 2-s2.0-85099128737 PubMed: 33444994

A 34-year-old woman from Thai origin developed acute liver failure after ingestion of a soup which contained the death cap (Amanita phalloides).In patients with poisoning due to amatoxin-containing mushrooms, gastro-intestinal complaints usually develop several hours after ingestion, followed by acute hepatic failure which occasionally leads to death. The incidence of reported mushroom poisonings in the Netherlands has increased in 2019, which is possibly associated with migration of asylum seekers who regularly pick and eat mushrooms.In the Netherlands mushroom intoxication is rare. Therefore, there is a lack of knowledge among health care personnel and foragers. The present case report highlights the importance of awareness of the poisonous death cap to prevent intoxications and optimize treatment decisions.

Background Amanitin-producing mushrooms, mainly distributed in the genera Amanita, Galerina and Lepiota, possess MSDIN gene family for the biosynthesis of many cyclopeptides catalysed by prolyl oligopeptidase (POP). Recently, transcriptome sequencing has proven to be an efficient way to mine MSDIN and POP genes in these lethal mushrooms. Thus far, only A. palloides and A. bisporigera from North America and A. exitialis and A. rimosa from Asia have been studied based on transcriptome analysis. However, the MSDIN and POP genes of many amanitin-producing mushrooms in China remain unstudied; hence, the transcriptomes of these speices deserve to be analysed. Results In this study, the MSDIN and POP genes from ten Amanita species, two Galerina species and Lepiota venenata were studied and the phylogenetic relationships of their MSDIN and POP genes were analysed. Through transcriptome sequencing and PCR cloning, 19 POP genes and 151 MSDIN genes predicted to encode 98 non-duplicated cyclopeptides, including α-amanitin, β-amanitin, phallacidin, phalloidin and 94 unknown peptides, were found in these species. Phylogenetic analysis showed that (1) MSDIN genes generally clustered depending on the taxonomy of the genus, while Amanita MSDIN genes clustered depending on the chemical substance; and (2) the POPA genes of Amanita, Galerina and Lepiota clustered and were separated into three different groups, but the POPB genes of the three distinct genera were clustered in a highly supported monophyletic group. Conclusions These results indicate that lethal Amanita species have the genetic capacity to produce numerous cyclopeptides, most of which are unknown, while lethal Galerina and Lepiota species seem to only have the genetic capacity to produce α-amanitin. Additionally, the POPB phylogeny of Amanita, Galerina and Lepiota conflicts with the taxonomic status of the three genera, suggesting that underlying horizontal gene transfer has occurred among these three genera.

The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.

Amatoxins (AMAs) are lethal toxins found in a variety of mushroom species. Detection methods are needed to determine the occurrence of AMAs in mushroom species suspected in mushroom poisonings. In this manuscript, we report the generation of novel monoclonal antibodies (mAbs, AMA9G3 and AMA9C12) and the development of a competitive, enzyme-linked immunosorbent assay (cELISA) that is sensitive at 1 ng mL&minus, 1 and shows selectivity for &alpha, amanitin (&alpha, AMA) and &gamma, amanitin (&gamma, AMA), and less for &beta, amanitin (&beta, AMA). In order to decrease the overall time needed for analysis, the extraction procedure for mushrooms was also simplified. A rapid (1 min) extraction procedure of AMAs using solvents as simple as water alone was successfully demonstrated using Amanita mushrooms. Together, the extraction method and the mAb-based ELISA represent a simple and rapid method that readily detects AMAs extracted from mushroom samples.

Amanita phalloides poisoning with a high mortality is a serious health problem in the world. The typical clinical manifestations are usually characterized by the absence of any symptoms followed by severe gastrointestinal disorders and acute liver failure. Inhibition of RNA polymeraseII (RNAP II) activity, apoptosis, and oxidative stress are considered as the major mechanism of amatoxins intoxication. The current treatment measures mainly include prevention of amatoxins absorption, elimination of absorbed amatoxins, potential antidotes therapy, and liver transplantation. Nevertheless, there are no widely accepted treatment criteria for Amanita phalloides poisoning. This paper will focus on the treatment measures based on the previous studies and provide the currently available information for clinicians.

Amanita subpallidorosea is a recently discovered lethal Amanita sect. Phalloideae species found in China that is clustered with A. virosa in the same clade based on molecular phylogenetic analysis. However, the cyclopeptide toxin contents of these lethal mushrooms remain poorly studied. In this study, the cyclopeptide toxins in A. subpallidorosea were reported for the first time and the cyclopeptide compositions of A. subpallidorosea and A. virosa species were systematically analyzed. Thirteen cyclopeptides and two unknown compounds were identified or observed from these two lethal mushrooms by high-performance liquid chromatography coupled with high-resolution mass spectrometry. Of the known cyclopeptides, the virotoxins alaviroidin, viroisin, and viroidin, which were previously thought to be restricted to A. virosa, were identified in A. subpallidorosea. The cyclopeptide compositions showed that there are diversities in the kinds and levels of amatoxins, phallotoxins, and virotoxins between A. subpallidorosea and A. virosa species, and that the amount of total toxins in the tested A. subpallidorosea is significantly higher than that in the tested A. virosa. Furthermore, consistency of the cyclopeptide toxins with the molecular phylogenetic relationships was demonstrated.

Objective To describe the clinical course, treatment, and outcome of 5 dogs following ingestion of toxic Amanita spp. mushrooms containing amatoxins using an adapted version of the Santa Cruz protocol developed for people. Case series summary Five dogs were presented with clinical signs compatible with amanitin toxicity with witnessed ingestion noted in 3 of 5 dogs. Clinical findings included acute onset vomiting and diarrhea, lethargy, and hepatopathy including signs of fulminant hepatic failure (increased liver enzyme activities, hyperbilirubinemia, prolonged clotting times, and hypoglycemia were noted among these cases). Urine toxicological screening confirmed the presence of Amanita toxins in 4 cases with expert mycologist speciation in the fifth. Core interventions included percutaneous biliary drainage, use of octreotide, and early nil per os orders. All dogs survived to discharge with this treatment strategy. New or unique information provided This case series describes the use of a modified version of the Santa Cruz protocol to address amatoxin-induced fulminant hepatic failure in dogs. The protocol was safe, well tolerated, and all patients made a full clinical recovery.

A non-target screening method of cyclopeptide toxins and their analogues in mushroom was developed, using ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) followed by mass spectrometry databases retrieval and software tools analysis for the candidate analogues. Three cyclopeptide toxins in the toxic mushroom Amanita rimosa were firstly screened without standard, and two of them were unknown analogues which were tentatively identified by the accurate masses, isotopic patterns and characteristic fragments. A validated quantitative method was performed to rapidly quantify three major cyclopeptide toxins in the Amanita rimosa sample including α-manitin, β-amanitin and phalloidin, and their contents were detected to be 4.52 mg/kg, 2.37 mg/kg and 2.53 mg/kg, respectively. The developed method has good selectivity and sensitivity for rapid and comprehensive screening the cyclopeptide toxins and their analogues in mushrooms at trace levels. Successful non-target screening of trace cyclopeptide toxin analogues will guarantee the food safety in mushrooms consumption.

BACKGROUND Fractionated plasma separation and absorption (FPSA) is an extracorporeal liver support method that detoxifies accumulated toxins. There are limited data of its use in the treatment of Amanita phalloides intoxication. The objective of this study was to investigate whether FPSA before liver transplantation improves patients' short-term post liver transplantation survival in Amanita phalloides poisoning. MATERIAL AND METHODS The study population consisted of ten patients who had liver transplantation (LT) due to acute liver failure (ALF) caused by Amanita phalloides poisoning. Six patients were treated with FPSA before liver transplantation. All the patients who were started on FPSA were also placed on the liver transplantation list according to emergent liver transplantation criteria. RESULTS Patients treated with FPSA were in a more severe clinical condition presenting in higher mean MELD, total bilirubin, INR and ammonia along with more frequent hypoglycemia and hepatic encephalopathy grade 3/4. FPSA group had longer mean waiting time on the recipient list (3.5 vs. 1.25 days) but inferior thirty-day survival rate (16.5% vs. 100%). CONCLUSIONS When conservative medical modalities are ineffective, the only treatment for Amanita phalloides poisoning is a liver transplant. Although FPSA treated patients had inferior post-LT survival, FPSA was found to prolong the pre surgical waiting time for critically ill patients, consequently giving a chance of life-saving procedure.

Hyperbranched rolling circle amplification (HRCA) with a padlock probe (PLP) targeting the α-amanitin (α-AMA) gene, as a screening tool for the universal identification of lethal amanitas, was established in this study. With the isothermal HRCA assay, all of the lethal Amanita species tested from Phalloideae (10) were positive, while the non-Phalloideae Amanita species (15) and three amanitin-containing Lepiota and Galerina species were negative. Furthermore, the PLP based on α-AMA sequences from lethal Amanita species was effective for Amanita α-AMA, but not Amanita β-AMA or non-Amanita α-AMA. HRCA sensitivity was 100-fold higher than conventional PCR with a detection limit of 100 copies (recombinant plasmid containing α-AMA), and 0.2% lethal amanitas could be detected in dry mushroom blends. The HRCA method presented provided a rapid, specific, sensitive and low-cost identification tool for lethal amanitas.

Rapid and accurate identification of multiple toxins for clinical diagnosis and treatment of mushroom poisoning cases is still a challenge, especially with the lack of authentic references. In this study, we developed an effective method for simultaneous identification of amanita peptide toxins by liquid chromatography coupled with photodiode array detection and ion trap time-of-flight mass spectrometry. The accuracy and selectivity of the methodology were validated through similar multiple fragmentation patterns and characteristic ions of standard α- and β-amanitin. The developed method could successfully separate and identify major toxic constituents in Amanita mushrooms. Two amatoxins and three phallotoxins were confirmed in a single run through their fragmentation patterns and characteristic ions, which can be used as diagnostic fragment ions to identify mushroom toxins in complex samples. Furthermore, the performance of the developed method was verified by using real biological samples, including plasma and urine samples collected from rats after intraperitoneal administration of toxins. Thus, the development methodology could be crucial for the accurate detection of mushroom toxins without standard references.

Amaç: Alfa amanitin (α-AMA) içeren Amanita phalloides türü mantarlar, ölümcül mantar zehirlenmelerinin çoğundan sorumludur ve karaciğer toksisitesi ve akut karaciğer yetmezliği ile sonlanan ciddi zehirlenmelere neden olur. Silibinin ve penisilin G gibi mevcut antidotlar, α-AMA ile oluşturulan karaciğer toksisitesinin önlenmesi ve/veya tedavisinde yeterince etkili değildir. Çalışmamızın amacı, resveratrolün α-AMA'e bağlı karaciğer toksisitesi üzerindeki etkilerini in vivo deneysel toksisite modelinde araştırmak ve tedavide antidot olarak kullanılan silibinin ile karşılaştırmaktı.Yöntem: Balb/c farelerde, AST ve ALT değerlerinde anlamlı oranda yüksekliğe neden olan ve histomorfolojik olarak da karaciğerde hasara neden olan 1.4 mg/kg α-AMA ile in vivo toksisite modeli oluşturuldu. Kontrol grubunda -AMA+%0.9. sodyum klorür (NaCl), Dimetil sülfoksit (DMSO) grubunda, α-AMA+ DMSO, resveratrol gruplarında, 30 mg/kg resveratrol, α-AMA ile eş zamanlı (α-AMA +ER), 12 saat (α-AMA +12R) ve 24 (α-AMA +24R) saat sonra olmak üzere 3 farklı tedavi protokolünde uygulandı. 5 mg/kg silibinin ise (α-AMA +Sil) α-AMA ile eş zamanlı olarak 6 saat arayla uygulandı. Kırk sekiz saatlik deney protokolünün sonunda fareler sakrifiye edildi. Alınan serum örneklerinden AST, ALT, Total bilirubin, Direk bilirubin düzeyleri, karaciğer dokularından antioksidan enzimler ve histomorfolojik incelemeler yapıldı. TUNNEL ve aktif kaspaz-3 incelemeleri ile apoptozis değerlendirildi.Bulgular: AST ve ALT değerleri, α-AMA+ER ve α-AMA+12R gruplarında, histomorfolojik hasar skorları ise α-AMA+ER, α-AMA+12R, α-AMA+24R ve gruplarında α-AMA+SF grubuna göre anlamlı düşüş gösterdi. Resveratrol, karaciğerde mononükleer hücre infiltrasyonunu, nekrozu ve aktif kaspaz-3 immünopozitifliğini azalttı. Sonuç: İn vivo deneysel toksisite modelimizde, resveratrol, α-AMA'e bağlı gelişen karaciğer toksisitesini önlemede etkili bulundu. Sonuç olarak; resveratrol, Amanita phalloides türü mantarlarla zehirlenmelerde kullanılabilme potansiyeli olan yeni bir tedavi alternatifidir. Objective: Amanita phalloides species mushrooms containing alpha amanitin (α-AMA) are responsible for the majority of fatal mushroom intoxications and can lead to severe poisonings resulting in liver toxicity and acute hepatic failure. Existing antidotes, such as silibinin and penicillin G, are not sufficiently effective in the prevention and/or resolution of α-AMA induced liver toxicity. We investigated the effects of resveratrol on α-AMA induced liver toxicity and compared with silibinin, a known antidote in the in vivo toxicity model.Method: The in vivo toxicity model was established with 1.4 mg/kg α-AMA, which results in significant elevation in AST and ALT values in Balb/c mice and also causes histomorphological damage in the liver. α-AMA+0.9% sodium chloride (NaCl) was administered in the control group, and -AMA+dimethyl sulfoxide (DMSO) in the DMSO group, while 30 mg/kg resveratrol was administered in the resveratrol groups, in three different treatment protocols, simultaneously with α-AMA (α-AMA +ER), after 12 h (α-AMA +12R) and after 24 h (α-AMA +24R). Silibinin at 5 mg/kg (α-AMA +Sil) was administered at 6-h intervals simultaneously with α-AMA. Mice were sacrificed at the end of the 48-h experimental protocol. AST, ALT, total bilirubin, and direct bilirubin levels were investigated from serum specimens collected, while antioxidant enzymes and histomorphological examinations were performed from liver tissue. TUNEL and active caspase-3 were examined and apoptosis was evaluated.Results: The liver transaminase levels in α-AMA+SR and α-AMA+12R groups and histomorphologic injury score in the α-AMA+SR, α-AMA+12R, α-AMA+24R and α-AMA+Sil groups were significantly lower than that of the α-AMA+NS group. Resveratrol, decreased mononuclear cell infiltration, necrosis and active caspase-3 immunopsitivity in liver. Conclusion: Resveratrol was found to be effective in α-AMA induced liver toxicity with its anti-inflamatory properties. It is a promising antidote with the potential for use in the treatment of liver toxicity associated with Amanita phalloides type mushroom poisonings. 115

To the Editor,Amanita mushroom poisonings remain a clinical challenge given their asymptomatic incubation periods and lack of consistently efficacious treatment modalities. As most of these exposur...

Amanita exitialis Zhu L. Yang & T. H. Li is the species responsible for the largest number of mushroom-associated human poisonings and fatalities in South China due to its lethal cyclic peptide toxins. Prolyl oligopeptidase B (POPB) is considered a key enzyme in the production of the highly toxic cyclic peptide α-amanitin. However, the POPB gene of A. exitialis has not been studied. In the present study we cloned and sequenced the full-length A. exitialis POPB (AePOPB) gene. The aim was to verify the gene structure and functions of AePOPB. The full-length sequence of AePOPB is 3144 bp, including 18 exons encoding 730 aa, and the advanced structure is very similar to that of the previously reported POPB in Galerina marginata (GmPOPB). The amino acid sequence of AePOPB is highly homologous with those from other amanitin-producing lethal mushrooms, implying that AePOPB may have a similar role in the biosynthesis of cyclic peptide toxins. Expression levels of AePOPB were detectable in all parts and developmental stages of the fruiting bodies, and AePOPB was expressed more strongly at early development stages (early and late elongation stages). At early and late elongation stages, the expression peaks occurred in the stipe, whereas at early and late mature stages, the expression peaks occurred in the pileus. The expression patterns of AePOPB in different stages and different parts of the fruiting bodies were highly consistent with those of Aeα-AMA, which is required for α-amanitin accumulation. These results indicate that AePOPB should be involved in the α-amanitin biosynthesis in A. exitialis.

A fast and wide linear range method was established for the determination of mushroom toxins amanitins (α-amanitin,β-amanitin and γ-amanitin) and phallotoxins (phallacidin and phalloidin) in wild mushrooms by online liquid chromatography-diode array detector-tandem mass spectrometry (LC-DAD-MS/MS). The mushroom toxins were extracted with methanol, and diluted with water. The extracts were separated on an XBridgeTM BEH C18 column (150 mm×3.0 mm, 2.5 μm) under pH 10.7, measured by DAD and then analyzed by MS/MS. Basic mobile phase conditions were applied to improve the ionization efficiency of hydrogen ion adducts. The baseline separation of the analytes was obtained within 15 min. The limits of detection (LODs) of the sample matrix were 0.005-0.02 mg/kg. The toxins were quantified by the results measured by MS/MS when the toxin contents less than 2 mg/kg, and quantified by the results obtained from DAD when the contents more than 2 mg/kg. The linear range was 0.05-500 mg/kg for the whole method in one injection. The method was successfully applied to the analyses of amanitins and phallotoxins in Lepiota brunneoincarnata and white Amanita.

As the currently known diagnostic DNA targets amplified in the PCR assays for detection of poisonous mushrooms have their counterparts in edible species, there is a need to design PCR primers specific to the genes encoding amanitins and phallotoxins, which occur only in poisonous mushrooms.The aim of the study was testing of PCR-based method for detection of all genes encoding hepatotoxic cyclic peptides - amanitins and phallotoxins present in the most dangerous poisonous mushrooms.Degenerate primers in the PCR were designed on the basis of amanitins (n=13) and phallotoxins (n=5) genes in 18 species of poisonous mushrooms deposited to Genbank of the National Center for Biotechnology Information.The specificity of the PCR assays was confirmed against 9 species of edible mushrooms, death cap - Amanita phalloides and panther cap - Amanita pantherina.Designed two couples of PCR-primers specific to amanitins and phallotoxins genes can be recommended for detection of Amanita phalloides and other mushroom species producing hepatotoxic cyclic peptides - amanitins and phallotoxins.

In this study, the toxicology of A. exitialis, a lethal mushroom found in China, and the toxicokinetics of peptide toxins contained in it were evaluated. Beagles were fed A. exitialis powder (20 or 60 mg/kg) in starch capsules, after which they were assessed for signs of toxicity, as well as biochemical and pathological changes. Ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to assay the peptide toxins. The total peptide toxins in A. exitialis was 3482.6 ± 124.94 mg/kg. The beagles showed signs of toxicity, such as vomiting and diarrhea, at 12–48 h following ingestion of A. exitialis. Furthermore, alanine transaminase and aspartate transaminase levels in plasma, as well as prothrombin time and activated partial thromboplastin time peaked at 36 h post A. exitialis ingestion. Furthermore, total bilirubin and alkaline phosphatase levels peaked at 48 h after A. exitialis ingestion. Three dogs that were administered 60 mg/kg A. exitialis died at 24–72 h after ingesting the capsules. Additionally, liver histopathological examinations showed hemorrhagic necrosis of hepatocytes. α-Amanitin, β-amanitin, and phallacidin were rapidly absorbed and eliminated from plasma after A. exitialis was ingested. A long latency period (12–24 h post A. exitialis ingestion) was observed in the dogs before the onset of gastrointestinal symptoms. There was acute liver damage thereafter. Gastric lavage and enhanced plasma clearance methods such as hemodialysis, hemoperfusion, or plasma exchange may be ineffective in removing amatoxins from blood at 12 h post A. exitialis ingestion. Enhanced excretion of amatoxins in urine could be effective within 2 days after ingestion of A. exitialis because amatoxins in 0–2 d urine accounted for more than 90% of the total urine excretion.

Dünyada yaklaşık olarak 5000 dolayında mantar sınıflandırılmış olup, bunlardan 100 dolayında türün zehirli olduğu tespit edilmiştir. Bunlar arasında yaklaşık 10 türün ölümcül zehirlenmelere neden olduğu bilinmektedir. Zehirli mantarlar arasında en iyi bilineni Amanita phalloides türüdür. Bu mantar içinde birçok toksin tanımlanmıştır. Genel olarak bu toksinler amanitinler (amatoksinler, amanotoksinler) ve fallotoksinler olarak 2 sınıfa ayrılırlar.Ulusal Zehir Danışma Merkezi 2008 verilerine göre gıda kaynaklı intoksikasyonlar tüm intoksikasyonların %3,3‟ünü, mantar intoksikasyonları ise gıda kaynaklı intoksikasyonların %43,5‟ini oluşturmaktadır. Mantar zehirlenmeleri bitkisel kaynaklı zehirlenmelerin neden olduğu ölümlerin %50„sini oluşturmaktadır.Düzce ili bol yağış alması sebebiyle çok sayıda mantar türü yetişmekte ve acil servislere mantar yeme ile ilişkili olabilecek şikayetler nedeniyle başvurular da sıkça görülmektedir. Bu çalışmada mantar yedikten sonra rahatsızlanan ve acil servisimize başvuran 33 hastanın demografik, klinik ve laboratuar özelliklerini inceleyerek elde edilen veriler ışığında günlük acil tıp pratiğinde hekime tanı, tedavi, takip ve prognoz tahmininde yardımcı olabilmek amaçlanmıştır.Gereç ve Yöntem:Düzce Üniversitesi Araştırma ve Uygulama Hastanesi Acil Servisi‟ne 01.09.2015-30.06.2016 tarihleri arasında mantar yeme sonucu semptom ve bulgular gelişmesi nedeniyle başvuran 18 yaşından büyük toplam 33 olgu çalışmaya dahil edilmiştir. Mantar yeme öyküsü olmayan 33 olgu da kontrol grubu olarak çalışmaya dâhil edilmiştir. Çalışmamız prospektif bir olgu kontrol çalışması olarak tasarlanmıştır. Hastaların demografik özellikleri, acil servise başvuru semptomları,mantarın acil servise başvurudan ne kadar süre önce alındığı, acil serviste takip süreci ve aldığı tedaviler değerlendirilmiştir. Hastalardan alınan venöz kan örneklerinde temel biyokimyasal değerler incelenmiştir. Ayrıca hastalardan alınan serum ve idrarda alfa amanitin düzeyi de ELİSA ile kitlerde belirtilen standart yöntem ile çalışılmıştır. Veriler kontrol grubu ile kıyaslanarak istatistiksel değerlendirmeler, Statistical Package for the Social Science (SPSS v22) programında yapılmıştır.Bulgular:Mantar alan hastalarda ve kontrol grubundan alınan kan ve idrar örneklerinde alfa amanitin saptanmamıştır. Erkek kadın oranı 5/6 idi. Hastaların yaşları 20 ile 80 arasında değişmekte olup, ortanca yaş 50 olarak bulundu. En sık görülen semptomlar bulantı (%94) ve kusma (%85) idi. Mantar alımından sonra acil servise başvuru zamanı ortanca 11 (min:2 – max:25) saatti. Mantar zehirlenmesi sonucu başvuran bireylerin %67 (n= 22) ‟sine mide lavajı yapılmıştır ve %88 (n= 29) ‟ine aktif kömür tedavisi verilmiştir. Hastaların tamamına intravenöz sıvı tedavisi uygulanmıştır. Hastaların %75,8 (n= 25)‟i acil servisten taburcu olurken, %24,2 (n= 8)‟sinin servise yatışı yapılmıştır.Sonuç:Bu çalışma, tanısı konamadığında ciddi komplikasyonlar hatta ölüme yol açabilen mantar zehirlenmeleri hakkında bilim insanlarına katkı sağlayacaktır. Acil servise bulantı ve kusma ile başvuran hastalarda, acil hekimi tarafından mantar yeme öyküsü mutlaka sorgulanmalıdır. Batı Karadeniz Bölgesinde mantar tüketimine bağlı zehirlenmeler oldukça sık görülmektedir. Özellikle kırsal kesimde doğadan toplanan yabani mantarların tüketimine bağlı zehirlenme olgularının sıklığı nedeniyle bölge halkı eğitilmelidir. Pazarlarda kontrolsüz satılan yabani mantarlara karşı halk sağlığı birimleri koruyucu önlemler almalıdır. Mantar zehirlenmeleri konusunda sağlık çalışanlarının eğitimi hasta yönetimi konusunda faydalı olacaktır. Mantar zehirlenmesi önlenebilir bir sağlık sorunu ve ölüm nedenidir. Mantar zehirlenmesi nedeniyle sağlık kuruluşlarına başvuruların sayısını azaltarak sağlık sistemiüzerindeki yükü hafifletmek ve can kayıplarını da azaltmak için doğadan toplanan yabani mantarların yenmemesi konusunda toplum bilinçlendirilmelidir.Anahtar kelimeler: Mantar zehirlenmesi, alfa amanitin, Amanita phalloides. Background and Objectives:Approximately 5000 mushroom species have been defined in the world, of which about 100 have been found to be poisonous. Among these, approximately 10 species are known to cause lethal poisoning. Among the poisonous mushrooms, Amanita phalloides is the best known. Many toxins have been identified in this mushroom. In general, these toxins are divided into 2 classes as amanitines (amatoxins, amanotoxins) and fallotoxins.According to the National Poison Information Center 2008 data, food-borne intoxications account for 3.3% of all intoxications and fungal intoxications account for 43.5% of food-borne intoxications. Mushroom poisonings constitute 50% of the deaths caused by plant-based poisonings.Because of the abundant rainfall, lots of mushroom species grow in Duzce province, and it is commonly seen that people admit to emergency departments with complaints which may be related to mushroom intake. In this study, it is aimed to be able to help the emergency physician in the process of diagnosis, treatment, follow-up and prediction of prognosis in daily emergency medicine practice in the light of the data obtained by examining the demographic, clinical and laboratory characteristics of 33 patients who bacame ill and applied to our emergency department after eating mushrooms.Materials and Methods:In this study, a total number of 33 patients who are over 18 and applied to Düzce University Hospital Emergency Department due to the development of signs and symptoms after mushroom intake between the dates 01.09.2015 and 06.06.2016viwere included. 33 cases without any history of recent mushroom intake were included as the control group. Our study was designed as a prospective case control study. The demographic characteristics of the patients, presenting symptoms, how long ago they consumed mushrooms, the emergency follow-up process and the treatments given were evaluated. Baseline biochemical parameters were examined in venous blood samples taken from patients. In addition, the level of alpha amanitin in serum and urine taken from the patients was also studied with ELISA using the standard method specified by the producer of the kit. Statistical evaluations were performed in the Statistical Package for the Social Science (SPSS v22) program.Results:Alpha amanitin was not detected in blood and urine samples taken from neither the study nor the control group. The male to female ratio was 5/6. The median age of the patients was 50 years (range 20 to 80). The most common symptoms were nausea (94%) and vomiting (85%). Mean of the time period between mushroom intake and emergency department application was 11 (min: 2 - max: 25) hours. Sixty seven percent (n= 22) of the patients in the study group had gastric lavage and 88% (n= 29) of them were given active charcoal. All of the patients were given intravenous fluid therapy. Seventy five point eight percent (n= 25) of the patients were discharged from the emergency department and 24.2% (n= 8) were hospitalized.Conclusion:This study will provide scientists with some useful information about mushroom poisonings that can lead to serious complications or even death when they can not be diagnosed promptly. When a patient presented to the emergency department because of nausea and vomiting, any history of recent mushroom intake must be questioned by the emergency physician. Poisoning due to consumption of mushrooms is very common in Western Black Sea Region. Particularly in rural areas, the population of the region should be trained because of the frequency of poisoning incidents due to the consumption of wild mushrooms collected from nature. Public health units should take protective measures against wild mushrooms which are sold at markets without any control. Training of health professionals on mushroom poisoning will beviiuseful in patient management. Mushroom poisoning is a preventable health problem and a cause of death. Community awareness on avoiding comsumption of the mushrooms collected from nature should be raised in order to reduce the number of applications to health facilities due to mushroom poisoning and to alleviate the burden on the health system and to reduce mortality.Keywords: Mushroom poisoning, alpha amanitin, Amanita phalloides. 81

Poisonings with Amanita phalloides toxins require fast diagnosis in order to avoid expensive and unnecessary therapies. Initial clinical assessment in combination with urinary amanitin analysis is necessary for a definite diagnosis. Therefore, a simple, fast, and robust method was developed for reliable detection of α- and β-amanitin as well as for fully validated quantification of α-amanitin in human urine. After simple dilution and centrifugation of the urine sample, a fast on-line extraction using a Transcend TLX-II system based on turbulent flow chromatography (TurboFlow) was established. A new TurboFlow mode was introduced, the pseudo quick elute mode (PQEM), which had more options for method optimization than the generic quick elute mode (QEM). It allowed running several modes in one valve arrangement. The PQEM showed better practicability in routine and emergency analysis than the previously used methods. After extraction, the fast 15min LC-high resolution (HR)-MS/MS analysis allowed reliable identification of α- and β-amanitin based on fragments identified using so-called HR pseudo MS(3) experiments. According to international recommendations, the requirements for full validation including the parameters selectivity, calibration, accuracy, precision, recovery, matrix effects, and stability were fulfilled for α-amanitin. The method was successfully applied to the analysis of authentic urine samples containing amatoxins. In conclusion, this method allowed the determination of amatoxins using the novel PQEM in a faster, robust, and more reliable way than existing methods, making it suitable for daily routine and especially emergency toxicological analysis.

Amatoxins are produced primarily by 3 species of mushrooms: Amanita, Lepiota, and Galerina. Because amatoxin poisonings are increasing, the objective of this review was to identify all amatoxin-containing mushroom species, present a toxidromic approach to earlier diagnoses, and compare the efficacies and outcomes of therapies. To meet these objectives, Internet search engines were queried with keywords to select peer-reviewed scientific articles on amatoxin-containing mushroom poisoning and management. Descriptive epidemiological analyses have documented that most mushroom poisonings are caused by unknown mushrooms, and most fatal mushroom poisonings are caused by amatoxin-containing mushrooms. Amanita species cause more fatal mushroom poisonings than other amatoxin-containing species, such as Galerina and Lepiota. Amanita phalloides is responsible for most fatalities, followed by Amanita virosa and Amanita verna. The most frequently reported fatal Lepiota ingestions are due to Lepiota brunneoincarnata, and the most frequently reported fatal Galerina species ingestions are due to Galerina marginata. With the exception of liver transplantation, the current treatment strategies for amatoxin poisoning are all supportive and have not been subjected to rigorous efficacy testing in randomized controlled trials. All patients with symptoms of late-appearing gastrointestinal toxicity with or without false recovery or quiescent periods preceding acute liver insufficiency should be referred to centers providing liver transplantation. Patients with amatoxin-induced acute liver insufficiency that does not progress to liver failure will have a more favorable survival profile with supportive care than patients with amatoxin-induced acute liver failure, about half of whom will require liver transplantation.

Eating mushrooms known to contain amatoxin is fraught with serious complications. The analysis of the relevant literature publications revealed no article with the description of the histological picture of the internal organs in the subjects intoxicated with amatoxin. It is known, however, that such poisoning is associated with the severe irreversible injuries to all intracellular protein structures the character of which depends on time. Specifically, acute amatoxin intoxication produces the well apparent clinical picture within 6 days after intake of the poison. It is characterized by acute renal and hepatic insufficiency in the combination with the injury to the conducting system of heart and the myocrardium itself. Thereafter, the disseminated intravascular coagulation (DIC) syndrome developed accompanied by the signs of progressive tissue hypoxia that ended in death on day 9. The histological study has demonstrated necrotic foci in the liver and oedematous hepatic stroma. Kidneys underwent multiple hemorrhages, necrosis of convoluted tubules and well apparent hydropic protein dystrophy of their epithelium. The adrenal glands showed up signs of necrosis and hemorrhage. It is concluded that poisoning with mushrooms (amatoxin) should be regarded as the most probable cause of the condition requiring differential diagnostics between acute gastroenteritis and renal insufficiency.Употребление в пищу грибов, содержащих аматоксин, опасно серьезными осложнениями. Анализ источников литературы не выявил ни одного сообщения, в котором бы приводилась гистологическая картина внутренних органов у лиц, отравившихся аматоксином. Известно, что при таких отравлениях необратимые изменения обусловлены повреждением всех клеточных белковых структур и имеют различную временнýю составляющую. В случае острого отравления аматоксином развернутую клиническую картину в виде острой почечной и печеночной недостаточности, а также поражения проводящей системы сердца и самого миокарда наблюдали на 6-е сутки. Смерть наступила на 9-е сутки на фоне массивного ДВС-синдрома и признаков прогрессирующей тканевой гипоксии. При гистологическом исследовании наблюдали очаги некроза и отек стромы печени, множественные кровоизлияния, некроз извитых канальцев почек, резко выраженную белковую гидропическую дистрофию эпителия извитых канальцев, некроз и кровоизлияния в ткани надпочечников. Отравление грибами (аматоксином) необходимо рассматривать в качестве наиболее вероятной причины при дифференциальной диагностике острого гастроэнтерита и почечной недостаточности.

Amanita exitialis is a lethal mushroom that was first discovered in Guangdong Province, China. The high content of amanitin in its basidiocarps makes it lethal to humans. To comprehensively characterize the A. exitialis transcriptome and analyze the Amanita toxins as well as their related gene family, transcriptome sequencing of A. exitialis was performed using Illumina HiSeq 2000 technology. A total of 25,563,688 clean reads were collected and assembled into 62,137 cDNA contigs with an average length of 481 bp and N50 length of 788 bp. A total of 27,826 proteins and 39,661 unigenes were identified among the assembled contigs. All of the unigenes were classified into 166 functional categories for understanding the gene functions and regulation pathways. The genes contributing to toxic peptide biosynthesis were analyzed. From this set, eleven gene sequences encoding the toxins or related cyclic peptides were discovered in the transcriptome. Three of these sequences matched the peptide toxins α-amanitin, β-amanitin, and phallacidin, while others matched amanexitide and seven matched unknown peptides. All of the genes encoding peptide toxins were confirmed by polymerase chain reaction (PCR) in A. exitialis, and the phylogenetic relationships among these proprotein sequences were discussed. The gene polymorphism and degeneracy of the toxin encoding sequences were found and analyzed. This study provides the first primary transcriptome of A. exitialis, which provided comprehensive gene expression information on the lethal amanitas at the transcriptional level, and could lay a strong foundation for functional genomics studies in those fungi.

Yaykasli, Kursat/0000-0001-7550-6370; Saritas, Ayhan/0000-0002-4302-1093; Saritas, Ayhan/0000-0002-4302-1093; Kaya, Ertugrul/0000-0003-0081-682X WOS: 000328658600028 PubMed: 24139877 Although rarely seen, Amanita phalloides var. alba, a variety of A. phalloides type mushrooms, causes mushroom poisoning resulting in death. Since it is frequently confused with some edible mushrooms due to its white colored cap and macroscopic appearance, it becomes important in toxicological terms. Knowledge of the toxin amount contained in this mushroom type is invaluable in the treatment of cases involving poisoning. In this study, we examined the toxin levels of various parts of the A. phalloides var. alba mushroom growing Duzce region of Turkey. Toxin analyses were carried out for A. phalloides var. alba, which were collected from the forests Duzce region of Turkey in 2011, as a whole and also separately in its spore, pileus, gills, stipe and volva parts. The alpha amanitin, beta amanitin, gamma amanitin, phalloidin and phallacidine analyses of the mushrooms were carried out using the RP-HPLC method. A genetic analysis of the mushroom showed that it had similar genetic characteristics as A. phalloides and was a variety of it. The lowest toxins quantity was detected in spores, volva and stipe among all parts of the mushroom. The maximum amount of amatoxins was measured in the gills. The pileus also contained a high amount of amatoxins. Generally, amatoxins and phallotoxin concentrations were lower as compared to A. phalloides, but interestingly all toxins other than gamma toxin were higher in the spores of A. phalloides var. alba. The amount of toxin in all of its parts had sufficient concentrations to cause death. With this study, the amatoxin and phallotoxin concentrations in A. phalloides var. alba mushroom and in its parts have been revealed in detail for the first time. (C) 2013 Elsevier Ltd. All rights reserved.

Saritas, Ayhan/0000-0002-4302-1093; Yaykasli, Kursat/0000-0001-7550-6370; Saritas, Ayhan/0000-0002-4302-1093; Kaya, Ertugrul/0000-0003-0081-682X WOS: 000365745600013 PubMed: 23719849 Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure -, -, and -amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Duzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. -, -, and -Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 x 4.6 mm; 5 mu m particle) was used. The least amount of -amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

Background Diagnosis and management of Amanita mushroom poisoning is a challenging problem for physicians across the United States. With 5902 mushroom exposures and two resultant deaths directly linked to Amanita ingestion in 2009, it is difficult for physicians to determine which patients are at risk for lethal toxicity. Identification of amatoxin poisoning can prove to be difficult due to delay in onset of symptoms and difficulty with identification of mushrooms. Consequently, it is difficult for the Emergency Physician to determine proper disposition. Further, treatment options are controversial. Objectives To review current data to help health care providers effectively identify and treat potentially deadly Amanita mushroom ingestions. Case Reports We present two cases of Amanita mushroom ingestion in the northeastern United States treated with N-acetylcysteine, high-dose penicillin, cimetidine, and silibinin, a semi-purified fraction of milk thistle-derived silymarin, as part of their treatment regimen. The mushroom species was identified by a consultant as Amanita Ocreata . Conclusions We present the successful treatment of 2 patients who ingested what we believe to be an Amanita species never before identified in the northeastern United States.