Identification Of Toxins Research Articles

Amanitins in Wild Mushrooms: The Development of HPLC-UV-EC and HPLC-DAD-MS Methods for Food Safety Purposes

Mushroom poisoning remains a serious food safety and health concern in some parts of the world due to its morbidity and mortality. Identification of mushroom toxins at an early stage of suspected intoxication is crucial for a rapid therapeutic decision. In this study, a new extraction method was developed to determine α- and β-amanitin in mushroom samples collected from central Portugal. High-performance liquid chromatography with in-line ultraviolet and electrochemical detection was implemented to improve the specificity of the method. The method was fully validated for linearity (0.5-20.0 µg·mL-1), sensitivity, recovery, and precision based on a matrix-matched calibration method. The limit of detection was 55 µg mL-1 (UV) and 62 µg mL-1 (EC) for α-amanitin and 64 µg mL-1 (UV) and 24 µg mL-1 (EC) for β-amanitin. Intra- and inter-day precision differences were less than 13%, and the recovery ratios ranged from 89% to 117%. The developed method was successfully applied to fourteen Amanita species (A. sp.) and compared with five edible mushroom samples after extraction with Oasis® PRIME HLB cartridges without the conditioning and equilibration step. The results revealed that the A. phalloides mushrooms present the highest content of α- and β-amanitin, which is in line with the HPLC-DAD-MS. In sum, the developed analytical method could benefit food safety assessment and contribute to food-health security, as it is rapid, simple, sensitive, accurate, and selectively detects α- and β-amanitin in any mushroom samples.

Identification and Characterization of Clostridium perfringens Atypical CPB2 Toxin in Cell Cultures and Field Samples Using Monoclonal Antibodies.

A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical β2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin.

PAT: a comprehensive database of prokaryotic antimicrobial toxins.

Antimicrobial toxins help prokaryotes win competitive advantages in intraspecific or interspecific conflicts and are also a critical factor affecting the pathogenicity of many pathogens that threaten human health. Although many studies have revealed that antagonism based on antimicrobial toxins plays a central role in prokaryotic life, a database on antimicrobial toxins remains lacking. Here, we present the prokaryotic antimicrobial toxin database (PAT, http://bioinfo.qd.sdu.edu.cn/PAT/), a comprehensive data resource collection on experimentally validated antimicrobial toxins. PAT has organized information, derived from the reported literature, on antimicrobial toxins, as well as the corresponding immunity proteins, delivery mechanisms, toxin activities, structural characteristics, sequences, etc. Moreover, we also predict potential antimicrobial toxins in prokaryotic reference genomes and show the taxonomic information and environmental distribution of typical antimicrobial toxins. These details have been fully incorporated into the PAT database, where users can browse, search, download, analyse and view informative statistics and detailed information. PAT resources have already been used in our prediction and identification of prokaryotic antimicrobial toxins and may contribute to promoting the efficient investigation of antimicrobial toxin functions, the discovery of novel antimicrobial toxins, and an improved understanding of the biological roles and significance of these toxins.

Combined Molecular and Biochemical Identification of Alpha Toxin in Local Isolated Clostridium novyi from the Sheep Liver.

Clostridium novyi (C. novyi) causes deadly Black disease in sheep and rarely in other animals. Alpha toxin (α-toxin), the most apparent pathogen of this disease, is produced by C. novyi type B. Economic damages of C. novyi include sheep mortality costs, depreciation of affected farms, and health problems with infected carcasses. The identification of C. novyi and isolation of its pathogens by conventional methods is a time-consuming process, necessitating a simple and rapid method for isolating and detecting pathogenic C. novyi. Therefore, this study aimed to molecularly identify α-toxin in local C. novyi isolates from the sheep livers. In this study, 75 livers suspected of Black disease were sampled. The samples of the liver were cultured under anaerobic conditions. Some of the cultured colonies were used in biochemical tests. For molecular confirmation, the DNA of isolates was extracted, and the isolates were confirmed by the polymerase chain reaction (PCR) on the liver tissue and cultured samples using specific α-toxin primers. The PCR on α-toxin produced a band in the range of 609 bp, indicating that the samples belonged to C. novyi. According to the results, of 75 isolates, 18 isolates were confirmed as C. novyi. C. novyitype B was isolated from the liver and confirmed by biochemical and molecular characterization. The PCR assay ensured a sensitive and specific tool for the detection of C. novyi in the samples.

Consistency between the ichthyotoxicity and allelopathy among strains and ribotypes of Margalefidinium polykrikoides suggests that its toxins are allelochemicals

Harmful algal blooms (HABs) of the ichthytoxic dinoflagellate Margalefidinium polykrikoides have caused mass mortality of marine life around the world. While its toxic effects can impact fish, bivalves, coral, zooplankton, and even other phytoplankton, the toxin(s) and allelochemical(s) eliciting these impacts have yet to be definitely identified, leaving open the question as to whether its toxicity and allelopathic effects are caused by the same chemical agents. In this study, we investigated the ability of 10 strains of M. polykrikoides with different geographic origins and ribotypes to cause mortality in two strains of the dinoflagellate, Akashiwo sanguinea (allelopathy), and the sheepshead minnow, Cyprinodon variegatus (toxicity). Results showed that the potency of allelopathy against both strains of A. sanguinea and toxicity to the fish were significantly correlated across strains of M. polykrikoides (p < 0.001 for all). These results strongly support the notion that the major allelochemicals and toxins of M. polykrikoides are identical chemicals, an ecological strategy that may be more energetically efficient than the separate synthesis of toxins and allelochemicals as has been reported in other HABs. Our results also highlight the vital significance of the definitive identification of allelochemicals and toxins of M. polykrikoides and of the quantitative characterization of these compounds in the field where HABs of M. polykrikoides occur during blooms.

The invasive red seaweed, Dasysiphonia japonica, forms harmful algal blooms: Mortality in early life stage fish and bivalves and identification of putative toxins

In recent decades, the rate of introduction of non-indigenous macroalgae has increased. While invasive seaweeds often outcompete native species for substrata, their direct effects on marine life are rarely described. Here, we describe ‘red water’ events caused by the decay of blooms of the invasive red seaweed, Dasysiphonia japonica, in Great South Bay, NY, USA, and the ability of water from such events to induce rapid and significant mortality in larval and juvenile fish (Menidia beryllina, Menidia menidia, and Cyprinodon variegatus) and larval bivalves (Mercenaria mercenaria and Crassostrea virginica). All species studied experienced significant (p<0.05) reductions in survival when exposed to macroalgae in a state of decay, seawater in which the alga was previously decayed, or both. Both bivalve species experienced 50–60% increases in mortality when exposed to decaying D. japonica for ∼ one week, despite normoxic conditions. Among fish, significant increases (40–80%) in mortality were observed after 24 h exposure to decayed D. japonica and one-week exposures caused, on average, 90% mortality in larval M. beryllina, 50% mortality in juvenile (∼3 cm) M. menidia, and 50% mortality in larval C. variegatus. All fish and bivalve mortality occurred under normoxic conditions (dissolved oxygen (DO) >7 mg L–1) and low ammonium levels (< 20 µM), with the exception of C. variegatus, which expired under conditions of decayed D. japonica coupled with reduced DO caused by the alga. Screening of water with decayed D. japonica using liquid chromatography-mass spectrometry revealed compounds with mass-to-charge ratios matching caulerpin, a known algal toxin that causes fish and shellfish mortality, and several other putative toxicants at elevated levels. Collectively, the high levels of mortality (50–90%) of larval and juvenile fish and bivalves exposed to decaying D. japonica under normoxic conditions coupled with the observation of ‘red water’ events in estuaries collectively indicate the red seaweed, D. japonica, can create harmful algal blooms (HABs).

Dried urine spot and dried blood spot sample collection for rapid and sensitive monitoring of exposure to ricin and abrin by LC–MS/MS analysis of ricinine and l-abrine

Ricin and abrin are protein toxins found in plants. Their extreme toxicity raises concern for their use as bioterrorism agents. Extraction from castor bean seeds yields 1–5% ricin and 0.3–0.8% ricinine. Abrin and l-abrine were extracted from Arbrus precatorius beans in mass ratios of 0.12% and 0.45%, respectively. Hence, ricinine and l-abrine (which are small molecules) may serve as biomarkers for assessing exposure to the corresponding proteins.A new approach was developed for high-throughput rapid identification of ricinine and l-abrine in microsamples of whole blood - dried blood spots (DBSs) and urine - dried urine spots (DUSs). These microsample collection methods are known for their advantages in field- sampling storage and shipping. The new method is based on a rapid sample preparation (extraction in a methanol/water mixture) followed by LC–MS/MS targeted analysis, the total lab time is less than one hour. Therefore, it may serve for preliminary confirmation before a labor-intensive and lengthy verification procedure for the identification of the protein toxin itself is carried out. The detection limit of ricinine was 50 pg/mL in whole blood and 100 pg/mL in urine, and for l-abrine, 100 pg/mL in whole blood and 300 pg/mL in urine. DUS and DBS samples were highly stable, even after storage for several months at room temperature. This method was successfully implemented in a real forensic case where a limited volume of urine was collected 72 h after self-injection of a castor bean extract in a suicide event.

Identification and Expression Analysis of an Interacting Protein (LvFABP) that Mediates Vibrio parahaemolyticus AHPND Toxin Action.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus causing AHPND (VPAHPND) is the most serious disease affecting shrimp farming. The PirAvp and PirBvp toxins of VPAHPND are known virulence factors. However, the corresponding target protein in shrimp that mediates their action has not been identified. By screening yeast two-hybrid cDNA libraries from intestine, stomach, and hepatopancreas of Litopenaeus vannamei, the protein with the largest increase in gene expression in shrimp hepatopancreas in response to VPAHPND challenge was identified and designated LvFABP. Analysis revealed high sequence homology of the LvFABP gene and a lipocalin/cytosolic fatty acid binding gene. Yeast two-hybrid pairwise analysis, GST-pull down assay, and far-western blot assay were performed to determine the interaction between LvFABP and PirBvp. LvFABP was able to directly bind to PirBvp. The expression of LvFABP in the hepatopancreas was significantly higher at P23 and P27 developmental stages of L. vannamei. RNA interference (RNAi) of LvFABP reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the intestine, stomach, and hepatopancreas after VPAHPND challenge. We concluded that the LvFABP was involved in AHPND pathogenesis and acted as a VPAHPND toxin interacting protein. This is the first identification of VPAHPND toxin interacting protein from the shrimp digestive system by yeast two-hybrid library screening and were confirmed by in vitro protein interaction verification and in vivo challenge experiments. This study provides novel insight into the contributions of LvFABP towards AHPND pathogenesis in shrimp. The findings could inform AHPND preventative measures in shrimp farming.

Raman Spectroscopy Enables Non-invasive and Confirmatory Diagnostics of Aluminum and Iron Toxicities in Rice.

Metal toxicities can be detrimental to a plant health, as well as to the health of animals and humans that consume such plants. Metal content of plants can be analyzed using colorimetric, atomic absorption- or mass spectroscopy-based methods. However, these techniques are destructive, costly and laborious. In the current study, we investigate the potential of Raman spectroscopy (RS), a modern spectroscopic technique, for detection and identification of metal toxicities in rice. We modeled medium and high levels of iron and aluminum toxicities in hydroponically grown plants. Spectroscopic analyses of their leaves showed that both iron and aluminum toxicities can be detected and identified with ∼100% accuracy as early as day 2 after the stress initiation. We also showed that diagnostics accuracy was very high not only on early, but also on middle (day 4–day 8) and late (day 10–day 14) stages of the stress development. Importantly this approach only requires an acquisition time of 1 s; it is non-invasive and non-destructive to plants. Our findings suggest that if implemented in farming, RS can enable pre-symptomatic detection and identification of metallic toxins that would lead to faster recovery of crops and prevent further damage.

Comparative Evaluation of Three Immunoassays for the Simultaneous Detection of Clostridioides difficile Glutamate Dehydrogenase and Toxin A/B.

Background: In the medical laboratory, a step-by-step workflow for Clostridioides difficile infection (CDI) detection using glutamate dehydrogenase (GDH) and toxin A/B assays for initial screening, along with a nucleic acid amplification test (NAAT), has been recommended recently. In this study, we evaluated these three immunoassays for the simultaneous detection of GDH and Clostridioides difficile (CD) toxin A/B. Methods: A total of 304 stool samples were tested for the presence of GDH antigen and CD toxin A/B using VIDAS C. difficile GDH and toxin A/B (CDAB), RIDASCREEN C. difficile GDH and toxin A/B (RIDA), and C. DIFF QUIK CHEK COMPLETE according to the manufacturers’ recommendations. As complementary reference methods for GDH and toxin A/B detection in the three immunoassays, CD cultures using ChromID C. difficile agar and the Xpert C. difficile assay, respectively, were tested. Results: All three GDH assays showed overall substantial agreement with the CD culture. All three toxin A/B assays showed overall moderate agreement with the Xpert C. difficile assay. In comparison with consensus results, VIDAS GDH and QCC GDH showed almost perfect agreement, whereas RIDA GDH showed inferior but substantial agreement. All three toxin A/B assays showed almost perfect agreement. Conclusions: Since the QCC GDH and toxin A/B assay is relatively more sensitive and specific than the other two immunoassays for discriminating toxigenic or non-toxigenic CDI, QCC is very helpful for the simultaneous identification of GDH and CD toxin A/B in the initial step of the two-round workflow for diagnosing CDI.

Modern methods of detection and identification of microbial toxins that inhibit protein synthesis in cells

Pathogenic microorganisms and products of their metabolism, namely, bacterial protein exotoxins, are considered one of the main sources of biological threat. Microbial toxins are highly active and extremely dangerous to humans. Determining trace amounts of such compounds remains relevant in healthcare and biological protection sector. Timely qualitative- and quantitative-specific indication of biotoxins is a key component in the diagnosis and implementation of therapeutic and preventive measures. Pathogenic microorganisms and products of their metabolism, bacterial protein exotoxins, are considered one of the main sources of biological threat. Microbial toxins are highly active and extremely dangerous to humans. Determining trace amounts of such compounds remains relevant in healthcare and biological protection sector. Timely qualitative- and quantitative-specific indication of biotoxins is a key component in the diagnosis and implementation of therapeutic and preventive measures. The current state and prospects of development in formulating specific indications of microbial toxins that disrupt protein synthesis in cells are analyzed. Modern ideas about the structure and mechanism of action of these toxins are briefly presented. Possibilities were considered, the advantages and disadvantages of classical traditional and modern innovative methods for identifying bacterial toxins that inhibit protein synthesis in cells were compared, and classifications were provided. Examples of the use of various approaches to identify the most significant representatives of this group in both clinical material and in environmental objects, including regulated ones, were given. The review also listed modern domestic and foreign developments in formulating specific indications of microbial toxins inhibiting protein synthesis. The review summarizes the results of studies to determine the current directions in the development of tools and methods for rapid specific indication of microbial toxins. The main trends in the creation of new methods of toxicological screening as part of an effective national system for monitoring biological threats were analyzed. Prospects for the development and introduction to the market of domestic test systems and automatic analysis platforms for the detection of bacterial toxins in environmental objects and biological material were determined.

Serotype Features of 17 Suspected Cases of Foodborne Botulism in China 2019-2022 Revealed by a Multiplex Immuno-Endopep-MS Method.

Diagnosis of botulism caused by multiple serotypes of botulinum neurotoxin (BoNT) is still a challenge due to the lack of a reliable detection method. The present study develops a feasible laboratorial method based on an isotope dilution Immuno-Endopep-MS to detect BoNTs and determine their serotypes and activities in clinical samples. Eleven positive foodborne botulism cases out of a total of 17 suspected cases in China, 2019–2022, were determined by the established method. Blood, urine, vomitus, gastric mucosa samples, and food samples were employed and evidenced to be suitable for the detection. Results showed that, although single type A-intoxication was still the first cause among these foodborne botulism cases, other causes involving type E, type B, and their mixed types were also determined, providing a glimpse to the serotype profile of botulism happened in recent years in China. Furthermore, in order to provide insights into in vivo profiles of toxin serotypes, a comprehensive analysis of clinical specimens collected from one family of four patients was performed during a clinically and therapeutically relevant time frame. Serotypes and concentrations of BoNT in specimens revealed a good correlation with symptoms and progresses of disease. Additionally, serum was proved to be more suitable for detection of BoNT/A with a detection window up to 12 days. A urine sample, although rarely reported for foodborne botulism diagnosis, was validated to be suitable for testing BoNTs, with a longer detection window up to 25 days. To the best of our knowledge, this is the first comprehensive analytical research on in vivo profiles of serotypes A, B, and E in different types of specimens from mixed botulism cases. Our method and findings facilitate the toxin detection and identification by clinical diagnostic laboratories.

Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish.

Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3–8 µg kg−1) were fulfilled. The matrix effect, ranging from −9 to 19%, allowed the use of a calibration curve in solvent (3–320 µg kg−1 in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories.

Outbreak of Type C Botulism in Backyard Poultry in Santa Catarina, Brazil

Background: Botulism is a disease caused by the ingestion of neurotoxin produced by Clostridium botulinum, characterized by flaccid paralysis, which can lead to high mortality. They have seven types of neurotoxins (A, B, C, D, E, F, and G) and, in birds, most cases are attributed to type C. They are considered sources of botulinum toxins where the decomposition of organic matter occurs, like stagnant water and rotting food. The main feature of the disease in birds is ascending symmetric flaccid paralysis. The present study aims to describe an outbreak of type C botulism in backyard poultry in the state of Santa Catarina, Southern Brazil.Case: A visit was made to the property with 160 backyard poultry with a history of high mortality in the municipality of Agrolândia, Santa Catarina. Clinical signs were characterized by paralysis of the pelvic limbs, neck and pendular wings, which progressed to death within 48 h. There was a mortality rate of 37.5% (60/160) between March and May 2019. These birds were kept in an overcrowded environment, with different species (chickens, ducks, teals, and turkeys) fed irregularly. The water supplied was provided from kitchen exhaust, accumulating in puddles on the floor that contained organic matter residues such as animal feces, food waste and bone fragments. The disposal of the carcasses of birds that died was in the same enclosure, buried superficially, facilitating the access of other birds to dig them up and consume them. Necropsy was performed on 2 chickens and one duck, no macroscopic or histopathological lesions were observed. Blood, liver, and gastrointestinal content samples were sent for research and identification of botulinum toxin through the serum neutralization test in mice. The presence of type C botulinum toxin was confirmed in the liver chicken of one sampled animals.Discussion: The identification of type C botulism toxin enabled the characterization of the outbreak, which is the toxin most associated with episodes of botulism in birds. It is not always possible to identify the origin of the infection, as intoxication can occur by ingestion of water contaminated with organic waste, however, in this outbreak, as sources of poisoning in birds, it was specified and occurred due to the ingestion of water with organic matter that was stagnant in the floor of the enclosure, and also by ingesting contaminated carcasses present in the area. In subsistence farming, botulism outbreaks are reported with greater frequency in the Northeast and Southeast of Brazil generally, cases in which sanitary conditions and incorrect carcass disposal favor the occurrence of the disease. As noted in the present study, high mortality is a common feature of botulism. The evaluated signs and developed evolution were similar to previous studies, which ranged from 14 to 72 h. The absence of macroscopic and histopathological changes is commonly reported in cases of botulism in domestic animals, since botulinum toxin only causes functional changes, with no tissue damage. The association of clinical signs, epidemiology, post mortem evaluation and detection of type C botulinum toxin concludes the diagnosis of botulism. Avian influenza and Newcastle disease are important diseases that have neurological conditions and high mortality that should be distinguished from botulism. This report confirms the need to associate history, clinical signs, absence of lesions with laboratory research for the diagnosis of botulism in birds. In addition, it reinforces the importance of disclosing basic management measures to prevent the occurrence of outbreaks such as presented in this report.Keywords: Clostridium botulinum, neurotoxin, bird disease, paralysis.Descritores: Clostridium botulinum, neurotoxina, doença de ave, paralisia.

Токсичні синдроми при гострих отруєннях умовно їстівними та отруйними грибами

Актуальність дослідження обумовлена великою кількістю сезонних грибних отруєнь дикорослими отруйними та умовно їстівними грибами на території Київської області, що супроводжується тяжким перебігом отруєння, особливо серед дітей, високими показниками стаціонарної летальності, надзвичайною вартістю та складністю лікування. Метою даного дослідження був обраний аналіз найбільш поширених токсичних синдромів і невідкладних станів, що виникають при отруєнні дикорослими отруйними та умовно їстівними грибами, поширеними на території Київської області. Матеріалом дослідження були обрані випадки гострих отруєнь дикорослими отруйними та умовно їстівними грибами серед дорослого населення м. Києва та області за 1995–2015 рр. (усього 1587 випадків). Усі пацієнти проходили стаціонарне лікування на базі токсикологічного відділення Київської міської клінічної лікарні швидкої медичної допомоги. Проведений ретроспективний аналіз результатів клініко-лабораторного обстеження та лікування пацієнтів. Використовувалися методи дослідження: аналітичний та порівняльного аналізу.За результатами проведеного дослідження встановлено, що залежно від виду грибного токсину, фази гострого отруєння та відповідно до клінічних проявів усі випадки гострих отруєнь дикорослими грибами були розподілені на три групи. Перша група об’єднала випадки отруєння грибами з гастроентеротропною дією; друга — з нейротропною дією; третя — з гепатонефротропною дією. За період дослідження отруєння грибами гепатонефротропної дії становили 15,2 % (242 випадки); гастроентеротропної дії — 81,3 % (1314 випадків); нейротропної дії — 1,9 % (31 випадок). Різноманітність і варіабельність симптомів гострих отруєнь дикорослими грибами обумовлюють необхідність проведення ретельної диференційної діагностики з широким спектром соматичних та інфекційних захворювань. Усі патологічні стани, що трактувались як отруєння грибами, були розподілені наступним чином: отруєння високотоксичними грибами (блідою поганкою та аманітальними мухоморами); токсикоінфекції і ботулізм; гострі отруєння пестицидами, металами, радіонуклідами та іншими токсикантами, що містилися у грибах у надмірно високих концентраціях; гострі та хронічні в стадії загострення захворювання органів черевної порожнини.З огляду на те, що в Україні не проводиться лабораторна ідентифікація грибних токсинів через високу вартість досліджень, відсутність обладнання, складність мікологічної ідентифікації, діагноз «отруєння грибами» повинен базуватися на токсикологічному та епідеміологічному анамнезі, клінічних проявах, клініко-біохімічних даних і результатах допоміжних методів дослідження. У складних для діагностики випадках слід залучати суміжних спеціалістів (неврологів, гастроентерологів, хірургів, інфекціоністів).

Establishing Cell Models to Understand Cellular Toxicity: Lessons Learned from an Unconventional Cell Type.

The syndrome of uremic toxicity comprises a complex toxic milieu in-vivo, as numerous uremic substances accumulate and harm the organ systems. Among these substances, toxic and non-toxic players differently interfere with human cells. However, results from animal experiments are not always compatible with the expected reactions in human patients and studies on one organ system are limited in capturing the complexity of the uremic situation. In this narrative review, we present aspects relevant for cellular toxicity research based on our previous establishment of a human spermatozoa-based cell model, as follows: (i) applicability to compare the effects of more than 100 uremic substances, (ii) detection of the protective effects of uremic substances by the cellular responses towards the uremic milieu, (iii) inclusion of the drug milieu for cellular function, and (iv) transferability for clinical application, e.g., hemodialysis. Our technique allows the estimation of cell viability, vitality, and physiological state, not only restricted to acute or chronic kidney toxicity but also for other conditions, such as intoxications of unknown substances. The cellular models can clarify molecular mechanisms of action of toxins related to human physiology and therapy. Identification of uremic toxins retained during acute and chronic kidney injury enables further research on the removal or degradation of such products.

Morphological, molecular, and biochemical study of cyanobacteria from a eutrophic Algerian reservoir (Cheffia).

The cyanobacteria management in water bodies requires a deep knowledge of the community composition. Considering the reliable and thorough information provided by the polyphasic approach in cyanobacteria taxonomy, here we assess the cyanobacterial community structure of the Cheffia reservoir from Algeria. Cyanobacteria were identified on the basis of morphological traits and next-generation sequencing (NGS); toxins-related genes were localized in addition to the identification of toxins; temperature and nutrient level of water samples were also determined. The polyphasic approach was essential for cyanobacteria investigation; 28 genera were identified through 16S rRNA metabarcoding with the dominance of taxa from Microcystis (34.2%), Aphanizomenon (20.1%), and Planktothrix (20.0%), and morphological analysis revealed the association in this water body of five species within the genus Microcystis: M. aeruginosa, M. novacekii, M. panniformis, M. ichthyoblabe, and M. flos-aquae. The presence of mcyE genotypes was detected; moreover, HPLC-PDA and LC-ESI-MS/MS revealed the production of microcystin-LR. Results obtained in our study are very important since this ecosystem is used for water supply and irrigation; as a consequence, a good water management plan is essential.

Rapid, presumptive identification of seed-based toxins using direct analysis in real time mass spectrometry (DART-MS) and its variants

Detection of seed-based toxins is a need for forensic chemists when suspected poisonings occur. The evidence that is found is often physically unidentifiable, as the seeds are mashed to extract the toxin. This work investigates potential strategies for rapid detection of seed-based toxins and seed mashes containing these toxins using chemical signatures obtained by direct analysis in real time mass spectrometry (DART-MS). Seven toxins (digoxin, digitoxin, hypaconitine, hyoscyamine, lanatoside, oleandrin, and scopolamine) and six seeds containing these toxins were studied. While detection of four of the toxins was readily attainable, detection of digoxin, digitoxin, and lanatoside was hindered by the inability to thermally desorb these larger compounds under normal operating conditions. The use of DART-MS variants capable of higher desorption temperatures (thermal desorption (TD)-DART-MS and infrared thermal desorption (IRTD)-DART-MS) enabled detection of these compounds. Detection of toxins from direct analysis of seed mashes and methanolic seed mash extracts was found to be compound and technique dependent. Principal component analysis (PCA) of generated mass spectra enabled differentiation of seed species, even in cases where the toxins were undetectable.

Haff disease in the pandemic COVID-19 period in Brazil

Objective: This study describes and characterize the cases of Haff disease reported in Brazil during the COVID-19 pandemic period. Descriptive and cross-sectional study using public data from the National Center for Strategic Information and Response in Health Surveillance nacional. The Risk Communication number 17 of 09/15/2021 was accessed. The description of suspected cases was extended to all Brazilian states where notifications occurred, from January 1, 2020, to September 15, 2021. There was an increase in cases of Haff's disease in Brazil, from 2020 onwards, with 46 in the Northeast region of Brazil. Eighty-five cases and three deaths were reported between January and September 2021 in the North, Northeast, South and Midwest regions. Most cases occurred in regions of greater heat such as the coastal region of the Northeast and around regions of the Amazon River Basin. A critical period for coping with the SARS-CoV-2 virus pandemic in Brazil, cases of disease of Haff could be reported in various regions of country. Despite the etiological suspicions, studies, and follow-up of the cases, it is still not possible to affirm that the disease is caused by biomolecules of the Palytoxin-Like Toxins group. The possible identification of the responsible toxin(s) will be essential for a complete understanding of the etiopathogenic mechanism of the disease and adequate development of prevention, treatment, and cure measures.

The Laboratory Diagnosis of Clostridioides difficile Infection: An update of current laboratory practice.

Clostridioides difficile can cause colitis and is associated with hospital acquired infections. The C. difficile infection (CDI) is due to production of toxins A and B which bind to epithelial cell surface receptors and triggers signaling pathways, leading to loss of epithelial barrier function, apoptosis, and inflammation, culminating in diarrheal disease. In early days, laboratory diagnosis of CDI was based on cell culture, identification of toxins, and their cytopathic effects. These assays were replaced by enzyme immunoassays for the detection of C. difficile toxins and the GDH house-keeping gene for improved specificity. Later, molecular assays with higher sensitivity were introduced which are becoming easier to incorporate into the test algorithm. The diagnosis of CDI and significance of laboratory results can be challenging with asymptomatic colonization of C. difficile in some patients. Test result interpretation is even more challenging due to multiple guidelines, emerging resistant C. difficile ribotypes, as well as differences in disease prevalence. An accurate test result for diagnosis of CDI depends on selecting patients with high pre-test probability, collecting an acceptable stool specimen, and a thorough understanding of current test methods.