Validation of a clinical assay for botulinum neurotoxins through mass spectrometric detection.

Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.

High risk and low prevalence diseases: Botulism.

INTRODUCTION: Botulism is a serious condition that carries with it a high rate of morbidity and mortality. OBJECTIVE: This review highlights the pearls and pitfalls of botulism, including presentation, diagnosis, and management in the emergency department (ED) based on current evidence. DISCUSSION: Botulism is a neuromuscular disorder caused by toxin production of clostridium species bacteria and is a challenging diagnosis that mimics several other conditions. Children account for the majority of patients, with a foodborne source most common, followed by wound sources, typically from intravenous drug injection. Classically, patients with botulism develop bilateral cranial nerve palsies and symmetric, bilateral, descending paralysis. However, patients may initially present with vague symptomatology, such as weakness and dry mouth, which can make diagnosis challenging. A careful history elucidating exposures such as intravenous drug use or consumption of non-commercial canned products can help differentiate botulism from other disorders causing neuromuscular weakness. If suspected, the Centers for Disease Control should be notified to mobilize antitoxin for treatment as soon as the diagnosis is suspected even prior to confirmatory testing. Antibiotics should be avoided in these patients, as they can potentiate toxin release, unless there is a concomitant infection requiring antibiotic therapy. Patients with botulism can develop respiratory compromise requiring emergent airway management. Prolonged neuromuscular blockade from botulism will lead to a variety of symptoms that require comprehensive intensive care unit level care. CONCLUSION: An understanding of botulism and its many potential mimics can assist emergency clinicians in diagnosing and managing this deadly disease.

Delayed onset post-traumatic wound botulism.

A 41-year-old man developed rapidly progressive cranial neuropathies and muscle weakness followed by respiratory failure, requiring ventilation support. On examination, there was marked bilateral ptosis and ophthalmoplegia with bulbar, neck and proximal upper limb weakness. He had a recent open left humeral fracture that eventually required amputation. Despite immunoglobulin therapy, his progressive weakness continued. Multiple investigation results were inconclusive. Eventually, botulinum type A toxin was found positive, by which time the therapeutic window for antitoxin had passed. He continued on supportive management and was treated for concomitant infections and nosocomial illnesses. He was subsequently weaned from respiratory support and has made a good neurological recovery.

Iatrogenic botulism. / Iatrogen botulisme.

This case study describes severe iatrogenic botulism following treatment with a botulinum toxin injection at a private clinic abroad.

Foodborne Botulism, Canada, 2006-20211.

During 2006-2021, Canada had 55 laboratory-confirmed outbreaks of foodborne botulism, involving 67 cases. The mean annual incidence was 0.01 case/100,000 population. Foodborne botulism in Indigenous communities accounted for 46% of all cases, which is down from 85% of all cases during 1990-2005. Among all cases, 52% were caused by botulinum neurotoxin type E, but types A (24%), B (16%), F (3%), and AB (1%) also occurred; 3% were caused by undetermined serotypes. Four outbreaks resulted from commercial products, including a 2006 international outbreak caused by carrot juice. Hospital data indicated that 78% of patients were transferred to special care units and 70% required mechanical ventilation; 7 deaths were reported. Botulinum neurotoxin type A was associated with much longer hospital stays and more time spent in special care than types B or E. Foodborne botulism often is misdiagnosed. Increased clinician awareness can improve diagnosis, which can aid epidemiologic investigations and patient treatment.

Infant Botulism, Israel, 2007-2021.

Infant botulism (IB) is an intestinal toxemia that manifests as descending paralysis, constipation, and, in some cases, respiratory failure. Laboratory-confirmed IB cases are rare, and recent data in Israel are lacking. We conducted a national multicenter retrospective study of laboratory-confirmed IB cases reported in Israel during 2007-2021. A total of 8 cases were reported during the study period. During 2019-2021, incidence may have increased because of a cluster of 5 cases. Infant median age for diagnosis was 6.5 months, older than previously reported (3 months). Most cases occurred during March-July. Honey consumption was reported in 1 case, and possible environmental risk factors (living nearby rural or construction areas, dust exposure, and having a father who works as a farmer) were reported in 6 cases. Although IB is rare, its incidence in Israel may have increased over recent years, and its epidemiology and risk factors differ from cases reported previously in Israel.

Duration of Fecal Excretion of Clostridium Botulinum and Botulinum Neurotoxin in Patients Recovering from Infant Botulism.

This study sought to determine duration of fecal excretion of Clostridium botulinum organisms and neurotoxin after onset of infant botulism in 66 affected infants. Median excretion was longer for type A than type B patients (organisms: 5.9 vs 3.5 weeks, toxin: 4.8 vs 1.6 weeks, respectively). Toxin excretion always ceased before organism excretion. Antibiotic therapy did not affect duration of excretion.

Dual-Toxin ("Bivalent") Infant Botulism in California, 1976-2020: Epidemiologic, Clinical, and Laboratory Aspects.

OBJECTIVE: To assess the consequences of infant botulism that result from Clostridium botulinum strains that produce 2 botulinum toxin serotypes, termed "bivalent." STUDY DESIGN: Epidemiologic investigations used a standard questionnaire. Clostridium botulinum strains were isolated by standard methods. Botulinum neurotoxin (BoNT) serotypes and the relative amounts of toxins produced were identified using the standard mouse bioassay. BoNT subtypes and genomic locations were identified by DNA nucleotide sequencing. RESULTS: Thirty bivalent cases of infant botulism occurred in the 45 years (1976-2020), representing 2.0% of all California infant botulism cases, in the 3 geographic regions of southern California, the southern Central Valley, and mid-northern California. Toxin serotype combinations were Ba (n = 22), Bf (n = 7), and Ab (n = 1). More patients with illness caused by bivalent C botulinum Ba and Bf strains needed endotracheal intubation at hospital admission, 60.0% (18/30), than did patients with illness caused by monovalent BoNT/B strains, 34.3% (152/443). The Cbotulinum Ba and Bf strains produced BoNT/B5 and either BoNT/A4 or /F2. The Ab strain produced BoNT/A2 and /B1. All toxin gene clusters were on plasmids. CONCLUSIONS: Infant botulism caused by bivalent Cbotulinum strains occurs sporadically and in diverse locations in California. Affected patients with bivalent Ba and Bf strains lacked distinguishing epidemiological features but appeared to be more severely paralyzed at hospital presentation than patients with illness caused by only BoNT/B. These bivalent strains produced BoNT subtypes A2, A4, B1, B5, and F2, and all toxin gene clusters were on plasmids.

Botulism mimicking Guillain-Barre syndrome: The question of plasma exchange in an unusual case of acute paralysis.

Guillain-Barré syndrome (GBS) is an immune-mediated polyradiculoneuropathy and the most common cause of acute flaccid paralysis worldwide. GBS classically presents with acute, progressive, ascending weakness, reduced to absent reflexes, and albuminocytological dissociation on cerebrospinal fluid (CSF) analysis. Botulism is a neurotoxin-mediated acute descending flaccid paralysis with cranial nerve palsies and dysautonomia. Botulism in adults is caused by ingestion/inhalation of botulinum toxin or wound infection with Clostridium botulinum. Both GBS and botulism can rapidly precipitate respiratory failure; thus, prompt diagnosis and treatment are crucial to mitigate poor outcomes. Herein, we describe a case of botulism initially diagnosed as GBS given classic laboratory features, and describe the importance of careful consideration of the most appropriate therapeutic modalities in cases of acute flaccid paralysis, particularly regarding empiric administration of botulinum antitoxin and use of intravenous immune globulin in lieu of plasma exchange for potential GBS to prevent removal of antitoxin.

Foodborne botulism outbreak involving different nationalities during the Rugby World Cup: critical role of credit card data and rapid international cooperation, France, September 2023.

In September 2023, a severe outbreak of type B botulism with fifteen cases was linked to consumption of canned sardines at a restaurant in Bordeaux, France, during the Rugby World Cup. The cases were from seven countries. One death was recorded. Outbreak investigation using credit card data, rapid communication between health authorities of the affected countries and broad media communication allowed identification of cases and exposed persons and prevented further severe outcomes.

A large travel-associated outbreak of iatrogenic botulism in four European countries following intragastric botulinum neurotoxin injections for weight reduction, Türkiye, February to March 2023.

In March 2023, 34 associated cases of iatrogenic botulism were detected in Germany (30 cases), Switzerland (two cases), Austria (one case), and France (one case). An alert was rapidly disseminated via European Union networks and communication platforms (Food- and Waterborne Diseases and Zoonoses Network, EpiPulse, Early Warning and Response System) and the International Health Regulation mechanism; the outbreak was investigated in a European collaboration. We traced sources of the botulism outbreak to treatment of weight loss in Türkiye, involving intragastric injections of botulinum neurotoxin. Cases were traced using a list of patients who had received this treatment. Laboratory investigations of the first 12 German cases confirmed nine cases. The application of innovative and highly sensitive endopeptidase assays was necessary to detect minute traces of botulinum neurotoxin in patient sera. The botulism notification requirement for physicians was essential to detect this outbreak in Germany. The surveillance case definition of botulism should be revisited and inclusion of cases of iatrogenic botulism should be considered as these cases might lack standard laboratory confirmation yet warrant public health action. Any potential risks associated with the use of botulinum neurotoxins in medical procedures need to be carefully balanced with the expected benefits of the procedure.

[A case of botulism in the Czech Republic and current possibilities for detecting the neurotoxin produced by Clostridium botulinum].

In the Czech Republic, botulism is a rare life-threatening disease. A total of 155 cases have been reported since 1960; according to the ISIN (formerly EPIDAT) database, there have been only three isolated cases since 2013, with the exception of a single occurrence of familial botulism in 2013. In our work, we present the occurrence of botulism after ingestion of pâté of untraceable origin by a couple who were hospitalized for botulotoxin food poisoning in July 2022. Their neurological symptoms were dominated by dysarthria. After administration of antibotulinum serum, their condition improved significantly. Patient samples were analyzed using affinity carriers and MALDI mass spectrometry, a modern highly sensitive technique for detecting the presence of botulinum neurotoxins. Unlike traditional detection by a difficult and costly biological experiment on mice, the above analysis does not require the killing of laboratory animals.

Foodborne botulism in Poland in 2018-2021.

Objectives: The aim of this study is to assess the epidemiological situation of foodborne botulism in Poland in 2018-2021, compared to previous years. Materials and methods: The assessment is based on an analysis of aggregated surveillance data from the annual bulletin "Infectious Diseases and Poisons in Poland" from 2013-2021, as well as botulism case reports collected by sanitary-epidemiological stations and sent to the Department of Epidemiology of Infectious Diseases and Surveillance of the NIPH NIH - NRI or provided by EpiBase. Results: A total of 54 foodborne botulism cases were registered between 2018 and 2021, including 31 (57.4%) cases of laboratory-confirmed botulism intoxication. Most cases (46 cases, 85.2%) were sporadic. The declining trend in the number of notifications continued throughout the time period, and the incidence recorded in 2021 (0.02 per 100,000 population) was the lowest since the introduction of mandatory reporting and registration of botulism in Poland. The highest median annual incidence was recorded in the Warmisko-Mazurskie (0.18), but many voivodeships reported only single notifications or none. The decreasing trend in rural areas relative to the level of incidence in urban areas continued. Men (incidence in men more than twice that of women) and people aged 50-59 predominated. All patients were hospitalized. According to the State Sanitary Inspection, there were 3 deaths (mortality of 5.6%). Conclusions: In 2018-2021, the downward trend in the number of foodborne botulism notifications in Poland continued. At the same time, the considerable proportion of cases reported by physicians without laboratory confirmation and not meeting the definition of botulism adopted in other EU countries is noteworthy. The incidence in rural areas decreases to the level of incidence in urban areas, and the previously observed geographical distribution of cases diminishes.

Botulinum Neurotoxin C Dual Detection through Immunological Recognition and Endopeptidase Activity Using Porous Silicon Interferometers.

Botulinum neurotoxins (BoNTs) are the most potent toxins known in nature produced by Clostridium botulinum strains, which can cause life-threatening diseases in both humans and animals. The latter is of serious environmental and economic concern, resulting in high mortality, production losses, and rejection of contaminated animal feed. The available in vivo mouse assay is inadequate for real-time and on-site assessment of outbreaks. Herein, we present a reflective-based approach for the detection of BoNT/C while estimating its activity. Two adjacent porous Si Fabry-Pérot interferometers are simultaneously utilized to quantify minute BoNT/C concentrations by a competitive immunoassay and to assess their endopeptidase activity. The reflectivity signals of each interferometer are amplified by biochemical reaction products infiltration into the scaffold or by peptide fragments detachment from the nanostructure. The optical assay is highly sensitive in compliance with the in vivo approach by presenting a detection limit of 4.24 pg mL-1. The specificity and selectivity of the designed platform are cross-validated against BoNT/B and BoNT/D, also relevant to animal health. Finally, the analytical performances of both interferometers for real-life scenarios are confirmed using actual toxins while depicting excellent compliance to complex media analysis. Overall, the presented sensing scheme offers an efficient, rapid, and label-free approach for potential biodiagnostic elucidation of botulism outbreaks.

Current Developments in Diagnostic Assays for Laboratory Confirmation and Investigation of Botulism.

Detection of botulinum neurotoxin or isolation of the toxin-producing organism is required for the laboratory confirmation of botulism in clinical specimens. In an effort to reduce animal testing required by the gold standard method of botulinum neurotoxin detection, the mouse bioassay, many technologies have been developed to detect and characterize the causative agent of botulism. Recent advancements in these technologies have led to improvements in technical performance of diagnostic assays; however, many emerging assays have not been validated for the detection of all serotypes in complex clinical and environmental matrices. Improvements to culture protocols, endopeptidase-based assays, and a variety of immunological and molecular methods have provided laboratories with a variety of testing options to evaluate and incorporate into their testing algorithms. While significant advances have been made to improve these assays, additional work is necessary to evaluate these methods in various clinical matrices and to establish standardized criteria for data analysis and interpretation.

Clinical Guidelines for Diagnosis and Treatment of Botulism, 2021.

Botulism is a rare, neurotoxin-mediated, life-threatening disease characterized by flaccid descending paralysis that begins with cranial nerve palsies and might progress to extremity weakness and respiratory failure. Botulinum neurotoxin, which inhibits acetylcholine release at the neuromuscular junction, is produced by the anaerobic, gram-positive bacterium Clostridium botulinum and, rarely, by related species (C. baratii and C. butyricum). Exposure to the neurotoxin occurs through ingestion of toxin (foodborne botulism), bacterial colonization of a wound (wound botulism) or the intestines (infant botulism and adult intestinal colonization botulism), and high-concentration cosmetic or therapeutic injections of toxin (iatrogenic botulism). In addition, concerns have been raised about the possibility of a bioterrorism event involving toxin exposure through intentional contamination of food or drink or through aerosolization. Neurologic symptoms are similar regardless of exposure route. Treatment involves supportive care, intubation and mechanical ventilation when necessary, and administration of botulinum antitoxin. Certain neurological diseases (e.g., myasthenia gravis and Guillain-Barré syndrome) have signs and symptoms that overlap with botulism. Before the publication of these guidelines, no comprehensive clinical care guidelines existed for treating botulism. These evidence-based guidelines provide health care providers with recommended best practices for diagnosing, monitoring, and treating single cases or outbreaks of foodborne, wound, and inhalational botulism and were developed after a multiyear process involving several systematic reviews and expert input.

Benign food-borne type B botulism presenting as unilateral internal ophthalmoplegia: a case report.

BACKGROUND: Food-borne botulism is a rare neuromuscular junction disorder due to the effect of toxins released from Clostridium botulinum ingested by eating improperly stored food. Its classic manifestation is a rapidly evolving descending symmetrical flaccid paralysis with dysautonomia. CASE PRESENTATION: We have described a case of type B food-borne botulism with a benign clinical course characterized by an initially unilateral tonic mydriatic pupil. An extensive neurophysiological evaluation inclusive of pilocarpine eye drop(s) test, facial and limbs nerve stimulation and sudomotor tests, was decisively leading the diagnostic process. CONCLUSIONS: The importance of what has been described here lies in underlining that it is always advisable to consider food-borne botulinum intoxication, even in the case of unilateral/asymmetrical internal ophthalmoplegia without generalized progressive involvement of the voluntary muscles.

Outbreak of Foodborne Botulism Associated with a Commercially Produced Multipack Potato Product, Colorado: September 2019.

During September 2019, public health authorities in El Paso County, Colorado, were notified of four patients who had presented to nearby hospitals with clinical features consistent with botulism, a paralytic illness caused by botulinum neurotoxin. One patient died soon after presentation; the other three patients required intensive care but recovered after receiving botulism antitoxin. Botulinum toxin type A was detected in serum from all patients. On further investigation, all four patients had shared a meal that included commercially prepared roasted potatoes from an individual package without refrigeration instructions that had been left unrefrigerated for 15 d. Storage of the product at ambient temperature likely allowed botulism spores to produce botulinum toxin, resulting in severe illness and death. The manufacturer improved labeling in response to this outbreak. Public health officials should consider unrefrigerated potato products as a potential source of botulism; clinicians should consider botulism as a possible cause of paralytic illness.

Investigation and Identification of Food Poisoning Caused by Clostridium botulinum Type B1 in Shenzhen, China.

Clostridium botulinum produces botulinum neurotoxins (BoNTs), which cause people who ingest them to become seriously ill and sometimes die. In recent years, sporadic food poisoning cases associated with C. botulinum have occurred across the world. In 2016, two men were admitted to our hospital in Shenzhen, China, with foodborne botulism. In this study, we report on these two typical C. botulinum-related food poisoning incidents and the steps taken to identify and characterize the causative pathogen. We characterized the bacterial pathogen isolated from the first patient using cooked meat medium and egg yolk agar bacterial cultures under anaerobic conditions, and morphologically identified the isolate using Gram staining. The in vivo bioassay results in mice showed that the minimum lethal dose of the BoNTs produced by our isolate was 0.001-0.0001 mg/mL (LD50 of the culture was estimated to be 1.5812 mg/kg). Whole genome sequencing (WGS) results showed that the isolate was identified as C. botulinum B1 Okra. The causative strain was successfully isolated from the intestinal lavage fluid collected from the initial patient.

First report of foodborne botulism due to Clostridium botulinum type A(B) from vegetarian home-canned pate in Hanoi, Vietnam.

Even one case of foodborne botulism constitutes a public health emergency. We report a series of cases with delayed treatment due to delayed diagnosis. Clostridium botulinum type A(B) was isolated from vegetarian home-canned pate, but not from stool samples. These are the first recorded cases of foodborne botulism in Hanoi.