Crystal Structures of Botulinum Neurotoxin Subtypes A4 and A5 Cell Binding Domains in Complex with Receptor Ganglioside.

Botulinum neurotoxins (BoNT) cause the potentially fatal neuroparalytic disease botulism that arises due to proteolysis of a SNARE protein. Each BoNT is comprised of three domains: a cell binding domain (HC), a translocation domain (HN), and a catalytic (Zn2+ endopeptidase) domain (LC). The HC is responsible for neuronal specificity by targeting both a protein and ganglioside receptor at the neuromuscular junction. Although highly toxic, some BoNTs are commercially available as therapeutics for the treatment of a range of neuromuscular conditions. Here we present the crystal structures of two BoNT cell binding domains, HC/A4 and HC/A5, in a complex with the oligosaccharide of ganglioside, GD1a and GM1b, respectively. These structures, along with a detailed comparison with the previously reported apo-structures, reveal the conformational changes that occur upon ganglioside binding and the interactions involved.

Neurophysiological patterns of acute and post-acute foodborne botulism.

INTRODUCTION/AIMS: Neurophysiological patterns in patients with foodborne botulism are rarely described after the acute phase. We report data from a large Italian outbreak of botulism, with patients evaluated at different timepoints after poisoning. METHODS: Eighteen male patients (mean age 47 ± 8.4 y) underwent 22 clinical and neurophysiological evaluations (4 patients were re-evaluated). The resting compound muscle action potential (CMAP) amplitude, postexercise CMAP amplitude, CMAP change after high-frequency (50 Hz) repetitive nerve stimulation (HFRNS), and motor unit action potentials (MUAPs) were assessed in the acute (4-8 days after poisoning; 5 patients), early post-acute (32-39 days after poisoning; 5 patients), and late post-acute (66-80 days after poisoning; 12 patients) phases. RESULTS: In the acute, early post-acute, and late post-acute phases, respectively, reduced CMAP amplitudes were found in 100%, 20%, and 17% of patients; abnormal postexercise CMAP facilitation was observed in 100%, 40%, and 0% of patients; and pathological incremental responses to HFRNS were found in 80%, 50%, and 8% of patients. Baseline CMAP amplitudes, postexercise CMAP facilitation, and CMAP increases in response to HFRNS differed significantly between the acute and post-acute phases. Small MUAPs were found in 100% of patients in the acute and early post-acute phases and in 50% of patients in the late post-acute phase. DISCUSSION: The neurophysiological findings of foodborne botulism vary considerably according to the evaluation time point. In the post-acute phase, different neurophysiological techniques must be applied to support a diagnosis of botulism.

The First Reported Case of Infant Botulism in Korea: Treatable Infantile Neuromuscular Disease.

Botulism is a rare neuromuscular disorder caused by neurotoxins produced by Clostridium botulinum. The diagnosis of infant botulism may be obscured or delayed, as its presentation is similar to that of infantile neuromuscular disorders. We report the first Korean case of infant botulism in an acute progressive floppy infant with poor sucking and a weak cry. No abnormalities were found in all blood, cerebrospinal fluid, genetic test, nerve conduction study, and imaging studies. A stool-toxin test was finally performed under suspicion of infant botulism, and the result was positive. The patient was immediately treated with heptavalent botulism antitoxin. Follow-up after 3 months showed normal development with a complete resolution of all symptoms. Therefore, clinical suspicion of infant botulism, which is a treatable infantile neuromuscular disease, is essential for early diagnosis and prompt treatment in the differential diagnosis of a floppy infant.

Lambert-Eaton Myasthenic Syndrome, Botulism, and Immune Checkpoint Inhibitor-Related Myasthenia Gravis.

PURPOSE OF REVIEW: This article reviews the pathophysiology, epidemiology, clinical presentation, diagnosis, and treatment of Lambert-Eaton myasthenic syndrome (LEMS) and of botulism, and immune-related myasthenia gravis (MG) occurring in the context of immune checkpoint inhibitor therapy for cancer. RECENT FINDINGS: The suspicion that LEMS is rare but also likely underdiagnosed is supported by recent epidemiologic data. A validated, LEMS-specific scale now exists to assess and monitor disease, and symptomatic and immunomodulatory treatments are available. As presynaptic disorders of neuromuscular transmission, LEMS and botulism share electrodiagnostic abnormalities but have important distinguishing features. Knowledge of the clinical features of botulism is needed, particularly with continued cases of infant botulism, the opioid epidemic increasing the incidence of wound botulism, and medical use of botulinum toxin, which may cause iatrogenic botulism. Foodborne botulism remains rare. Prompt recognition of botulism and administration of antitoxin can improve outcomes. MG may be exacerbated or may present de novo in the context of immune activation from immune checkpoint inhibitor therapies for cancer. Immune-related MG commonly overlaps with myositis and myocarditis. Corticosteroids typically result in improvement. However, immune-related MG can be more fulminant than its idiopathic counterpart and may cause permanent disability or death. SUMMARY: The diagnosis of LEMS, botulism, or immune-related MG can generally be made from the patient's history, supplemented with directed questions, a physical examination designed to demonstrate abnormalities, and laboratory and electrodiagnostic testing. Early diagnosis and carefully selected treatment not only improve outcomes of the neuromuscular disease but can affect the prognosis of underlying malignancy, when present.

The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs.

A large number of bacterial toxins consist of active and cell binding protomers linked by an interchain disulfide bridge. The largest family of such disulfide-bridged exotoxins is that of the clostridial neurotoxins that consist of two chains and comprise the tetanus neurotoxins causing tetanus and the botulinum neurotoxins causing botulism. Reduction of the interchain disulfide abolishes toxicity, and we discuss the experiments that revealed the role of this structural element in neuronal intoxication. The redox couple thioredoxin reductase-thioredoxin (TrxR-Trx) was identified as the responsible for reduction of this disulfide occurring on the cytosolic surface of synaptic vesicles. We then discuss the very relevant finding that drugs that inhibit TrxR-Trx also prevent botulism. On this basis, we propose that ebselen and PX-12, two TrxR-Trx specific drugs previously used in clinical trials in humans, satisfy all the requirements for clinical tests aiming at evaluating their capacity to effectively counteract human and animal botulism arising from intestinal toxaemias such as infant botulism.

Botulism in pregnancy - a clinical approach to diagnosis and management.

Botulism is a life-threatening toxin mediated disease that often presents with a sudden rapid onset of paralysis of their skeletal muscles with subsequent respiratory compromise and sudden death. Given the natural physiological changes during pregnancy, pregnant women with botulism may experience a more exaggerated course and have worse outcomes compared to nonpregnant women. Medical providers caring for such patients should not only maintain a high level of suspicion for botulism, but administration of an antitoxin early in their care can help reduce morbidity and mortality, while awaiting confirmatory laboratory results. It is not uncommon for a medical provider caring for these women to mistakenly associate the signs and symptoms associated with botulism with pregnancy-related findings; therefore, this article illustrates a clinical algorithmic approach to caring for these women, including a systematic approach to establishing a diagnosis and management plan for pregnant women with botulism.

Electrophysiological abnormalities in iatrogenic botulism: Two case reports and review of the literature.

Therapeutic use of botulinum neurotoxin type A (BoNT/A) is effective, and generally safe. Nevertheless, iatrogenic botulism (IB) is rarely reported as a result of systemic spread of the BoNT/A, causing general weakness, bulbar symptoms and dysautonomia. Suggestive clinical feature are decisive to raise the diagnostic suspicion, which however needs a confirmation in the electrodiagnostic (EDX) study, above all to exclude other treatable diseases. In this study, we report 2 patients who developed IB after receiving therapeutic doses of BoNT/A, assessing the EDX changes, and reviewing the literature on EDX in IB. Although there is not enough data to draw solid conclusions we propose that, in a subject with suggestive clinical features and recent exposure to BoNT/A, the absence of a decremental or incremental response to repetitive nerve stimulation in muscles showing acute denervation changes, is a suggestive finding for the diagnosis of IB.

Brain functional changes in patients with botulism after illegal cosmetic injections of botulinum toxin: A resting-state fMRI study.

BACKGROUND: Botulinum toxin type A (BoNT-A) is generally considered safe and is widely used to treat a variety of clinical conditions involving muscle hyperactivity and for cosmetic purposes. However, the effects of BoNT-A poisoning (botulism) on brain function are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we investigated brain functions in 9 patients who received illegal cosmetic injections of botulinum and 18 matched controls by combining the analysis methods of regional homogeneity (ReHo) and amplitude of low-frequency fluctuation (ALFF) based on resting-state fMRI. Compared with the controls, the patients with botulism exhibited significantly reduced ReHo values in the left posterior lobe of the cerebellum extending to the right anterior lobe of the cerebellum, as well as in the right anterior lobe of the cerebellum extending to the parahippocampal gyrus and right posterior lobe of the cerebellum. The patients with botulism also showed weakened ALFF values in the right anterior lobe of the cerebellum extending to the left anterior lobe of the cerebellum and right posterior lobe of the cerebellum, as well as in the right anterior lobe of the cerebellum. CONCLUSIONS/SIGNIFICANCE: The results indicate that BoNT-A may modulate cerebral activation in specific areas, which may play roles in both the adverse effects of botulism and the mechanism underlying clinical treatment with BoNT-A.

Guillain-Barré syndrome mimicking botulism in early disease course.

A 42-year-old man, returning to Europe after a 2-month stay in China, reported cough and runny nose. Five days later, he developed neck discomfort and rapidly evolving weakness, spreading from his arms to the facial, bulbar and neck muscles, and then the legs. He developed dysphagia and breathlessness, and was intubated in the emergency department. Cerebrospinal fluid showed mildly elevated protein. On day 2 he had fixed dilated pupils, with absent oculocephalic reflexes, and a flaccid upper limb paralysis. MR scans of the brain and spinal cord were normal. The early features of descending weakness, bulbar involvement and fixed dilated pupils made it difficult to distinguish between Guillain-Barré syndrome and botulism, and early investigations were inconclusive. We initially gave both botulinum antitoxin and intravenous immunoglobulin, but initially delayed plasma exchange as this would have removed the botulinum antitoxin. Diagnostic testing for botulism has low sensitivity, so we urge pre-emptive treatment if it is clinically suspected.

Foodborne botulism due to ingestion of home-canned green beans: two case reports.

BACKGROUND: Foodborne botulism is a life-threatening, rapidly progressive disease. It has an incidence of less than 10 cases per year in Germany and mostly affects several previously healthy people at the same time. The only specific treatment is the administration of botulism antitoxin. According to the German guidelines administration of antitoxin is recommended only in the first 24 hours after oral ingestion of the toxin. CASE PRESENTATION: A 47-year-old white woman and her 51-year-old white husband presented with paralysis of multiple cranial nerves and rapidly descending paralysis approximately 72 hours after ingestion of home-canned beans. The disease was complicated by autonomic changes like hypertension, febrile temperatures, and a paralytic ileus. The diagnosis was confirmed by identification of botulinum neurotoxin type A in the serum of the woman. In accordance with the German guidelines, antitoxin was not given due to the prolonged time interval at diagnosis. Both patients had a long intensive care unit course requiring ventilation for approximately 5 months. Finally they recovered completely. CONCLUSIONS: A full recovery from foodborne botulism is possible even in patients with intensive care lasting several months. There are only case reports indicating that administration of antitoxin may shorten the course of the disease, even if given later than 24 hours after intoxication. Due to the rarity of the disease and its rapid course there are no randomized controlled trials. Thus, evidence of the superiority of this treatment is lacking. However, the prevailing view according to the German guidelines to administer antitoxin only within 24 hours after ingestion of the toxin should be questioned in the case of progression of the disease with proof of remaining toxin in the blood.

Black Tar Heroin Skin Popping as a Cause of Wound Botulism.

BACKGROUND: Botulism is a rare potentially fatal and treatable disorder caused by a bacteria-produced toxin that affects the presynaptic synaptic membrane resulting in a characteristic neuromuscular dysfunction. It is caused by either the ingestion of the toxin or the bacteria, inhalation, or wound infection. We present our observations with a descriptive case series of wound botulism secondary to black tar heroin (BTH) injection. METHODS: We report a retrospective single-center case series of 15 consecutive cases of wound botulism presenting to University Medical Center of El Paso. Medical records where reviewed to obtain demographic information, clinical presentation, treatment, and outcome. RESULTS: We identified fifteen patients with mean age of 47 years: twelve men, and three women. All had administered BTH through skin popping and had abscesses in the administration areas. By history, the most common symptoms were dysphagia (66%), proximal muscle weakness of upper and lower extremity (60%), neck flexor muscle weakness (33%), ophthalmoplegia (53%), bilateral ptosis (46%), dysarthria (53%), double vision (40%), blurred vision (33%), and dry mouth (20%). During the examination, the most common features noted were: proximal muscle weakness of upper and lower extremities (73%), ophthalmoplegia (53%), ptosis (46%). In patients with documented wound botulism, the pupils were reactive in 46%. All patients required mechanical ventilation and were treated with the trivalent antitoxin. Eleven patients (73.3%) were discharged home, two were transferred to a skill nursing facility, and two were transferred to long-term acute care facility. CONCLUSION: In our patients, BTH injection, involving the action of injecting under the skin acetylated morphine derivatives (mostly 6-monoacetylmorphine and 3-monoacetylmorphine), was associated with the development of botulism. The availability of BTH at the US-Mexican border is not surprising since it is frequently produced in Latin America. Its association with the development of botulism should be recognized early to allow a prompt diagnosis and treatment with the antitoxin. A clinical feature worth noting is the presence of normal pupillary light reflex in nearly half of patients. Therefore, the presence of a normal pupillary response does not exclude the presence of wound botulism.

Wound botulism, its neurological manifestations, treatment and outcomes: A case series from the Glasgow outbreak, 2015.

Background and aims We examined the neurological manifestations, treatment and outcomes of a subset of 25 patients within the largest ever outbreak of wound botulism in Europe. Methods and results All 25 cases were intravenous drug users. The most common presenting symptom was dysarthria in 19/25 (76%), followed by dysphagia in 12/25 (48%), blurred vision in 10/25 (40%) and double vision in 8/25 (32%). Microbiological analysis confirmed the diagnosis in nine cases (36%). Duration of admission positively correlated with time to antitoxin, time to wound debridement and female sex. Conclusion As the outbreak continued, hospital stays shortened, reflecting growing awareness of the outbreak and quicker treatment initiation.

Clinical Serum Therapy: Benefits, Cautions, and Potential Applications.

Blood serum from immunized humans or animals (e.g., horses) contains relevant antibodies and has been used as serum therapy to treat many diseases or envenomation events. The effectiveness of blood serum was initially discovered in 1890 when Kitasato and von Behring observed the effectiveness of this type of therapy against diphtheria and tetanus. Serum therapies played an important role in the advancement of modern medicine prior to the development of penicillin and steroids. At present, several types of serum therapy remain in clinical use. However, some physicians have a limited understanding of the nature and the benefits of serum therapy and the factors that require particular attention. In this review, we set out to clarify the benefits, cautions, and potential applications of serum therapy in the context of conditions such as gas gangrene, diphtheria, botulism, and tetanus and bites from three snake species (mamushi, habu, and yamakagashi) and the redback spider. It is hoped that this review will help clinicians to learn about clinical serum therapies and become familiar with their applications.

Retrospective survey to evaluate the safety and efficacy of Japanese botulinum antitoxin therapy in Japan.

Japanese botulinum antitoxins have been used for more than 50 years; however, their safety and therapeutic efficacy are not clear. In order to analyze the available data on botulinum antitoxin therapy in Japan, we surveyed published reports about botulism cases in which botulinum antitoxins were used, and retrospectively analyzed the safety and efficacy of the therapy. A total of 134 patients administered botulinum antitoxins were identified from published reports. Two cases of side effects (1.5%) were detected after antitoxin administration, both not fatal. The fatality rate was 9.4%, and more than 70% of the patients showed improvement in their symptoms and better clinical conditions than those not treated with antitoxins. These data suggest that the therapy with Japanese antitoxins is safe and highly effective.

Evidence for a natural humoral response in dairy cattle affected by persistent botulism sustained by non-chimeric type C strains.

Bovine botulism is a sporadic acute disease that usually causes catastrophic losses in the herds. The unusual clinical evolution of a persistent mild outbreak in a dairy herd, prompted us to characterize the neurotoxin gene profile of the strain involved and to evaluate whether seroconversion had occurred. Diagnosis was based on mild classical symptoms and was supported by PCR and bacteriological findings, which revealed the involvement of a non-mosaic type C strain. An in-house ELISA was developed to detect antibodies to botulinum neurotoxin type C and its performance was evaluated in a vaccination study. Fifty days after the index case, fecal and serum samples were collected from the 14 animals of the herd and screened for Clostridium botulinum and anti-botulinum neurotoxin antibodies type C, respectively. The in-house developed ELISA was also used to test 100 sera samples randomly collected from 20 herds. Strong ELISA reactions were observed in 3 convalescent and 5 asymptomatic animals involved in the studied outbreak. The ELISA-positive cows all tested positive for non-mosaic C. botulinum type C in the feces and the same strain was also detected in the alfalfa hay, suspected to be the carrier source. Ten out of the 100 randomly collected sera tested positive for anti-botulinum neurotoxin type C antibodies: 7 had borderline values and 3 from the same herd showed titers three times higher than the cut-off. We concluded that type C botulism in cattle may occur with variable severity and that prolonged exposure to sublethal doses of botulinum neurotoxin C may occur, resulting in detectable antibodies.

Outbreak of Botulism After Consumption of Illicit Prison-Brewed Alcohol in a Maximum Security Prison--Arizona, 2012.

The authors investigated the second botulism outbreak to occur in a maximum security prison in Arizona within a 4-month period. Botulism was confirmed in eight men aged 20 to 35 years who reported sharing a single batch of pruno made with potatoes. Initial symptoms included blurred vision, slurred speech, muscle weakness, ptosis, and dysphagia. All patients received heptavalent botulinum antitoxin, seven required mechanical ventilation, and all survived. The median incubation period was 29 hours. Sera from all patients and leftover pruno tested positive for botulinum toxin type A. Botulism should be considered among prisoners with cranial nerve palsies and descending, symmetric flaccid paralysis. Prison-brewed alcohol, particularly when made with potatoes, can be a vehicle for botulism and is associated with outbreaks of botulism in prisons.

Alcohol Production, Prevention Strategies, and Inmate Knowledge About the Risk for Botulism From Pruno Consumption in a Correctional Facility--Arizona, 2013.

During July to November 2012, two botulism outbreaks (12 cases total) occurred in one all-male prison; both were associated with illicitly brewed alcohol (pruno) consumption. Inmate surveys were conducted to evaluate and develop prevention and education strategies. Qualitative surveys with open-ended questions were performed among inmates from rooms where outbreaks occurred to learn about pruno consumption. Quantitative surveys assessed knowledge gained after the outbreaks and preferred information sources. For the quantitative surveys, 250 inmates were randomly selected by bed from across the correctional facility and 164 inmates were interviewed. Only 24% of inmates reported any botulism knowledge before the outbreaks and education outreach, whereas 73% reported knowledge after the outbreaks (p < .01). Preferred information sources included handouts/fliers (52%) and the prison television channel (32%).