Infant Botulism: Checklist for Timely Clinical Diagnosis and New Possible Risk Factors Originated from a Case Report and Literature Review.

Infant botulism is a rare and underdiagnosed disease caused by BoNT-producing clostridia that can temporarily colonize the intestinal lumen of infants less than one year of age. The diagnosis may be challenging because of its rareness, especially in patients showing atypical presentations or concomitant coinfections. In this paper, we report the first infant botulism case associated with Cytomegalovirus coinfection and transient hypogammaglobulinemia and discuss the meaning of these associations in terms of risk factors. Intending to help physicians perform the diagnosis, we also propose a practical clinical and diagnostic criteria checklist based on the revision of the literature.

Botulism during SARS-CoV-2 pandemic: The importance of differential diagnoses.

Botulism is a neuroparalytic syndrome caused by a neurotoxin produced by Clostridium botulinum. We describe a patient with neurological symptoms associated with intoxication by Clostridium botulinum and infection by SARSCoV2. This report underlines that it is mandatory, even in case of SARS-CoV-2 positivity, to investigate all the causes of a clinical pattern.

Rapid Detection of Clostridium botulinum in Food Using Loop-Mediated Isothermal Amplification (LAMP).

Botulinum neurotoxins are considered as one of the most potent toxins and are produced by Clostridium botulinum. It is crucial to have a rapid and sensitive method to detect the bacterium Clostridium botulinum in food. In this study, a rapid detection assay of C. botulinum in food using loop-mediated isothermal amplification (LAMP) technology was developed. The optimal primers were identified among three sets of primers designed specifically based on the partial ntnh gene encoding nontoxic-nonhaemagglutinin (NTNH) for rapid detection of the target DNA in plasmids. The optimal temperature and reaction time of the LAMP assay were determined to be 64 °C and 60 min, respectively. The chemical kit could be assembled based on these optimized reaction conditions for quick, initial high-throughput screening of C. botulinum in food samples. The established LAMP assay showed high specificity and sensitivity in detecting the target DNA with a limit of 0.0001 pg/ul (i.e., ten times more sensitive than that of the PCR method) and an accuracy rate of 100%. This study demonstrated a potentially rapid, cost-effective, and easy-operating method to detect C. botulinum in food and clinical samples based on LAMP technology.

First confirmed case of infant botulism in Africa, caused by a dual-toxin-producing Clostridium botulinum strain.

Botulism, a rare life-threatening toxemia, is probably underdiagnosed in all of its forms in Africa. This study reports the first laboratory-supported case of infant botulism on the African continent. A 10-week-old, previously well infant presented with progressive global weakness, feeding difficulty, and aspiration pneumonia. During a lengthy hospitalization, a rare bivalent Clostridium botulinum strain, producing subtype B3 and F8 toxins and with a new multilocus sequence type, was isolated from stool. The infant was successfully treated with a heptavalent botulinum antitoxin infusion and pyridostigmine. Despite the relative rarity of infant botulism, this case illustrates the importance of maintaining a high level of clinical suspicion when assessing hypotonic infants. The value of modern diagnostic modalities in identifying and characterizing this under-recognized condition is also demonstrated.

Pathogenesis, clinical characteristics, diagnostics and treatment of bacterial foodborne diseases / Élelmiszer eredetu bakteriális megbetegedések patogenezise, klinikai jellegzetességei, diagnosztikája és kezelése.

Összefoglaló. Az élelmiszer-eredetu megbetegedések igen gyakoriak, bár pontos adatok nem állnak rendelkezésre, mivel az enyhe, gyorsan múló gastrointestinalis tünetekkel a betegek nem fordulnak orvoshoz, vagy nem történik diagnosztikus vizsgálat. Az amerikai Járványügyi és Betegségmegelozési Központ (CDC) adatai szerint az USA-ban évente 6 lakosból 1 esik át élelmiszer okozta tüneteken. Az ételintoxikációk során a baktérium által termelt toxinok okozzák a tüneteket, közülük a leggyakoribb a Clostridium perfringens, a Staphylococcus aureus és a Bacillus cereus okozta, élelmiszer-eredetu intoxikáció. A nem megfeleloen tárolt vagy hokezelt élelmiszerekben - beleértve a S. aureus által szennyezett anyatejet - ezen baktériumok életképesek maradnak, elszaporodnak, és toxint termelhetnek, illetve toxinjaik megorzik megbetegítoképességüket. Az étel elfogyasztása után 3-12 órával hányást, hasmenést okoznak. A tünetek többnyire 24 órán belül megszunnek. A Clostridium botulinum súlyos neurológiai tünetei miatt emelkedik ki a többi toxikoinfekció sorából. C. botulinum okozta tünetekre felnotteknél házi készítésu konzervek és húskészítmények elfogyasztása után jelentkezo gastrointestinalis vagy neurológiai tünetek esetén kell gondolnunk. A Clostridioides difficile szintén a toxinjai révén okoz súlyos, életveszélyes megbetegedést, továbbá az esetek 20-30%-ában számolnunk kell az infekció relapsusával. Növekvo gyakorisága miatt ismernünk érdemes a laboratóriumi és klinikai diagnosztika részleteit és a legmodernebb kezelési lehetoségeket, úgymint megfelelo mintavétel, mintatárolás és -szállítás, tenyésztés, toxinkimutatás, helyes tüneti kezelés, antibiotikumkombinációk, széklettranszplantáció és monoklonálisantitest-kezelés. Orv Hetil. 2020; 161(48): 2019-2028. Summary. Foodborne diseases are quite common, however, accurate data are not available because patients do not visit doctors with mild, rapidly resolving symptoms and diagnostic tests are not performed. The Centers of Disease Control and Prevention (CDC) estimates that, in the USA, 1 in 6 citizens gets food poisoning yearly. Symptoms of intoxication are due to the toxins produced by bacteria, mostly by Clostridium perfringens, Staphylococcus aureus and Bacillus cereus. These bacteria can survive in not properly stored or heated food, including S. aureus contaminated breastmilk. They can multiply and produce toxins causing intoxications. The gastrointestinal symptoms start 3-12 hours after consumption of the contaminated food and resolve in 24 hours. Clostridium botulinum causes severe neurological symptoms that should be suspected after consumption of home-made cans, smoked hams and sausages. The disease caused by Clostridioides difficile is not a foodborne one, but C. difficile causes severe infection via its toxins. Another problem is that C. difficile infection recurs in 20-30% of cases. Due to the increasing incidence of foodborne diseases, it is worth to learn the precise clinical and laboratory diagnostic algorithms including sampling, storage and transportation of samples, cultivation of bacteria and differential diagnosis of these diseases, furthermore the most up-to-date symptomatic and causative treatment options like antibiotic combinations, stool transplantation and monoclonal antibodies. Orv Hetil. 2020; 161(48): 2019-2028.

Emergency response to a cluster of suspected food-borne botulism in Abuja, Nigeria: challenges with diagnosis and treatment in a resource-poor setting.

Food-borne botulism is a rare, acute and potentially fatal neurologic disorder that results from ingestion of food contaminated by botulinum toxin released from the anaerobic, spore-forming, gram-positive bacterium Clostridium botulinum. We reported an unusual cluster of botulism outbreak with high case fatality affecting a family following ingestion of home-made fish. A suspected outbreak of botulism affecting three patients in a family of six was reported to the Nigeria Centre for Disease Control. A rapid response team investigated by line-listing all the family members, interviewed extended family members, caregivers, clinicians, and nurses to collect socio-demographic and clinico epidemiological information using a semi-structured questionnaires. We collected blood from patients and food samples and locally made drink from the family home for laboratory testing. All family members ingested the same home-made food within the 48hrs before onset of symptoms in the index case. The clinical presentation of the three affected cases (AR=50.0%) was consistent with botulinum poisoning. Two of the affected cases died (CFR=66.7%) within 48hrs of admission, before antitoxin was made available. The third case had a milder presentation and survived, after administration of appropriate antitoxin. The remaining three children developed no symptoms. None of the samples cultured Clostridium botulinum. The blood samples were negative for mouse lethality test. Our report describes the challenges of diagnosis and management of rare emerging infectious disease outbreaks in resource-constrained settings.

Outbreak of botulism type A in dairy cows detected by MALDI-TOF mass spectrometry.

Twenty-eight lactating dairy cattle in New York State were exposed to botulism toxin; 12 died and 16 recovered but never returned to full productivity. Pieces of a raccoon carcass were found in the total mixed ration on the first day of the outbreak. Clinical signs included anorexia, decreased milk production, decreased tongue tone, profound weakness, and recumbency. Clostridium botulinum type A (BoNT/A) was detected in rumen contents from 2 deceased cows via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, C. botulinum type C was cultured from the liver of a third cow, and C. botulinum neurotoxin-producing type C gene (bont/C) was detected via real-time PCR. On postmortem examination, 4 cows had findings suggestive of toxic myopathy, but the cause and significance of these lesions is unknown given that botulism is typically not associated with gross or histologic lesions. This outbreak of BoNT/A in cattle in North America was diagnosed via MALDI-TOF MS, a rapid and sensitive modality for detection of botulinum preformed neurotoxin.

Manure contamination with Clostridium botulinum after avian botulism outbreaks: management and potential risk of dissemination.

BACKGROUND: Persistence of Clostridium botulinum in the environment is well known. Getting rid of it after animal botulism outbreaks is so tricky, especially as far as manure concerns. This study aimed at 1. describing manure management on 10 poultry farms affected by botulism and 2. assessing the persistence of C botulinum in poultry manure after the outbreak. METHODS: Each farm was visited twice at two different manure storage times (two weeks after manure removal and two months later). Fifteen samples of manure were collected on each visit and C botulinum was detected using real-time PCR. RESULTS: Management of manure varied among poultry farms (classical storage, addition of quicklime, bacterial flora or incineration). C botulinum was detected in the manure of all 10 farms, 56.5per cent of samples being positive. C botulinum was detected significantly more frequently at the second visit (65.8per cent vs 49.7per cent, P<0.01) and on the surface of the pile (63.1per cent vs 50per cent, P=0.025). CONCLUSION: This study shows the persistence of C botulinum in poultry manure over time after a botulism outbreak and highlights manure management as a key health issue in preventing spore dissemination in the environment and recurrence of the disease.

Assessment of the risk of botulism from chilled, vacuum/modified atmosphere packed fresh beef, lamb and pork held at 3 °C-8 °C.

The safety of current UK industry practice (including shelf-life) for chilled, vacuum/modified atmosphere-packed fresh red meat (beef, lamb and pork) held at 3°C-8°C has been evaluated with respect to non-proteolytic Clostridium botulinum. UK industry typically applies a retail pack shelf-life at 3°C-8°C to 13 days for fresh red meat, with a maximum of 23 days for beef, 27 days for lamb, and 18 days for pork. An exposure assessment established that current commercial practice for fresh red meat provided strong protection with more than 1010 person servings marketed in the UK without association with foodborne botulism. A challenge test demonstrated that spores of non-proteolytic C. botulinum inoculated on chilled vacuum-packed fresh red meat did not lead to detectable neurotoxin at day 50 for beef, day 35 for lamb, or day 25 for pork (i.e. <40 pg type B toxin and type E toxin g-1 of meat). The products were visually spoiled many days before these end points. The exposure assessment and challenge test demonstrated the safety of current UK industry practices for the shelf-life of fresh, vacuum-packed beef, lamb and pork held at 3°C-8°C with respect to C. botulinum, and that botulinum neurotoxin was not detected within their organoleptic shelf-life.

High prevalence of Clostridium botulinum in vegetarian sausages.

Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.

Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment.

The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.

First record of mass wild waterfowl mortality due to Clostridium botulinum in Brazilian semiarid.

In 2008, 270 wild birds from aquatic environments were found dead or debilitated on the banks of smaller lakes that had been formed due to the decrease in the level of the holding lake of the Sobradinho Dam located on the São Francisco River in the Caatinga of Bahia, Brazil. The outbreak occurred months after the dam's partial drainage, with the formation of puddles that accumulated decomposing organic material. Amongst the 270 individuals examined and/or found dead, the majority (50%) of the birds found belonged to the Anatidae family. The debilitated birds presented neurological clinical signs including lack of motor coordination, weakness, grave flaccid paralysis in the legs, wings, neck and eyelids, diarrhea, and dyspnea. Tissue samples of the birds were collected, as were water samples and samples of the substrate of the lakes. Zoonotic arboviroses or heavy metals were not detected. Analyses of liver and digestive tract content samples through bioassay and serum neutralization in mice revealed the presence of type C botulinic toxin in the viscerae samples, and type D in sediment samples. According to our knowledge, this is the first record of an outbreak of botulism in wild birds in natural conditions in Brazil.

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks.

Avian botulism is a serious neuroparalytic disease mainly caused by a type C/D botulinum neurotoxin produced by Clostridium botulinum group III, one of the entwined bacterial species from the Clostridiumnovyisensulato genospecies. Its isolation is very challenging due to the absence of selective media and the instability of the phage carrying the gene encoding for the neurotoxin. The present study describes the development of an original method for isolating C. botulinum group III strains. Briefly, this method consists of streaking the InstaGene matrix extraction pellet on Egg Yolk Agar plates and then collecting the colonies with lipase and lecithinase activities. Using this approach, it was possible to isolate 21 C. novyi sensu lato strains from 22 enrichment broths of avian livers, including 14 toxic strains. This method was successfully used to re-isolate type C, D, C/D, and D/C strains from liver samples spiked with five spores per gram. This method is cheap, user-friendly, and reliable. It can be used to quickly isolate toxic strains involved in avian botulism with a 64% success rate and C. novyi sensu lato with a 95% rate. This opens up new perspectives for C. botulinum genomic research, which will shed light on the epidemiology of avian botulism.

Clostridium botulinum and Clostridium perfringens Occurrence in Kazakh Honey Samples.

The aim of this study was to assess occurrence of Clostridium botulinum and Clostridium perfringens in honey samples from Kazakhstan. Analyses were carried out using a set of PCR methods for identification of anaerobic bacteria, and detection of toxin genes of C. botulinum and C. perfringens. Among 197 samples, C. botulinum was noticed in only one (0.5%). The isolated strain of this pathogen showed the presence of the bont/A and ntnh genes. C. perfringens strains were isolated from 18 (9%) samples, and mPCR (multiplex PCR) analysis led to them all being classified as toxin type A with the ability to produce α toxin. Sequence analysis of 16S rDNA genes showed occurrence in 4 samples of other anaerobes related to C. botulinum, which were C. sporogenes and C. beijerinckii strains. C. botulinum prevalence in honey samples from Kazakhstan in comparison to the prevalence in samples collected from the other regions seems to be less. The highest prevalence of Clostridium sp. was noticed in the East Kazakhstan province. Our study is the first survey on BoNT-producing clostridia and C. perfringens prevalence in Kazakh honey.

Functional detection of botulinum neurotoxin serotypes A to F by monoclonal neoepitope-specific antibodies and suspension array technology.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and cause the life threatening disease botulism. Sensitive and broad detection is extremely challenging due to the toxins' high potency and molecular heterogeneity with several serotypes and more than 40 subtypes. The toxicity of BoNT is mediated by enzymatic cleavage of different synaptic proteins involved in neurotransmitter release at serotype-specific cleavage sites. Hence, active BoNTs can be monitored and distinguished in vitro by detecting their substrate cleavage products. In this work, we developed a comprehensive panel of monoclonal neoepitope antibodies (Neo-mAbs) highly specific for the newly generated N- and/or C-termini of the substrate cleavage products of BoNT serotypes A to F. The Neo-mAbs were implemented in a set of three enzymatic assays for the simultaneous detection of two BoNT serotypes each by monitoring substrate cleavage on colour-coded magnetic Luminex-beads. For the first time, all relevant serotypes could be detected in parallel by a routine in vitro activity assay in spiked serum and food samples yielding excellent detection limits in the range of the mouse bioassay or better (0.3-80 pg/mL). Therefore, this work represents a major step towards the replacement of the mouse bioassay for botulism diagnostics.

A severe outbreak of botulism in cattle in Central Italy.

Botulism in cattle is rarely reported in Italy. This study describes an outbreak of botulism in a dairy herd in Central Italy in September 2012, and the notably high mortality rate it caused. Differential diagnoses involving toxicology and bacteriology, and electrolyte imbalances, all proved negative. A multiplex polymerase chain reaction (PCR) for detecting the BoNT gene led to the identification of the causative agent as Clostridium botulinum type DC. The presence of the toxin was confirmed subsequently via mouse bioassay. Initially, the peracute deaths and ambiguous clinical signs delayed the diagnosis and, as a result, impeded identification of the source of the infection on the farm. The severity of the outbreak demonstrates that screening for animal botulism should always form part of the diagnostic protocols used to investigate sudden peracute deaths without apparent cause in livestock.

Identification and characterization of Clostridium botulinum strains associated with an infant botulism case in China.

Infant botulism was rarely reported in China. The second reported event of the disease including three cases occurred in 2015. In the present study, one (the third case) of the three cases was identified and investigated to trace the sources of transmission. Samples from feces and foodstuffs were used to isolate Clostridium botulinum strains. Each isolate was obtained from the baby's feces and opened powdered infant rice cereal, respectively. In this case, the C. botulinum strains were identified and characterized by combined mouse bioassay, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-throughput sequencing including single nucleotide polymorphisms (SNP). Results showed that the disease was caused by a type B strain of C. botulinum. Strains associated with this case as well as isolates from stored and historical samples were phylogenetically analyzed and compared. C. botulinum type B isolates from the infant feces and from an opened container of infant rice cereal were indistinguishable, suggesting that opened container of infant rice cereal is likely to be the source of transmission of spores to the infant. It is not clear that how the opened container was contaminated and the child was exposed since environmental testing was not performed. This study provides detailed information about usage of the three methods and references for dealing with other associated cases.

Botulism Outbreak in a Regional Community Hospital: Lessons Learned in Transfer and Transport Considerations.

Botulism is a potentially lethal disease caused by a toxin released by Clostridium botulinum. Outbreaks of botulism from food sources can lead to a Mass Casualty Incident (MCI) involving sometimes hundreds of individuals. We report on a recent outbreak of botulism treated at a regional community hospital with a focus on emergency medical services (EMS) response and transport considerations. Case Presentation: There were 53 patient evaluated for botulism at the sending facility. In total, 11 botulism exposures required intubation at the sending facility. Twenty-four patients were ultimately transported by critical care capable ALS crews with the majority (16) of these transports occurred in the first 24 hours. There was one fatality in the first days of the outbreak and a second death that occurred in a patient who died after long-term acute care (LTAC) placement several months after hospital discharge. Conclusion: Local EMS providers and public safety officers have a critical role in identifying and following up on potentially exposed botulism cases. The organization of transporting agencies and the logistics of transfer turned out to be 2 opportunities for improvement in response to this mass casualty incident.

Effect of Different Storage Conditions on Analytical and Sensory Quality of Thermally Processed, Milk-Based Germinated Foxtail Millet Porridge.

Foxtail millet porridge was prepared using germinated grains and milk and was evaluated for its storage stability after thermal processing at ultra-high temperatures (UHT) of 142 °C for 5 s and retort processing temperatures of 121.5 °C for 15 min. Various physical, chemical, and microbial changes of the porridge were studied for a storage period of 180 days at 25 ± 1 °C. Using consumer perception and survival analysis, the predicted shelf life of the UHT treated and retort processed foxtail millet porridge samples stored at 25 ± 1 °C was found to be 186 ± 9 days and 245 ± 15 days, respectively. Also, data from consumer liking, profiling, physical, chemical, and microbial parameters showed significant changes (P < 0.05) in the thermally treated packaged porridge samples over time. As the consumer overall acceptability decreased, the detection of positive attributes (thick and uniformly colored texture and appearance; grainy mouth texture; caramel taste and aroma) in the porridge decreased, while the detection of negative attributes (uneven, decolored, and curdled texture and appearance; sticky mouth texture; cooked, sour and off smell; cooked, sour and off taste) increased. The present study could establish a significant difference (P < 0.05) in the storage induced properties of UHT and retort processed porridge samples. The analytical evaluation of foxtail millet porridge found that UHT treated porridge was better in quality, but consumers preferred retort processed porridge. PRACTICAL APPLICATION: The quality and sensory attributes, evaluated for UHT treated and retort processed porridge samples during the storage period of 180 days, were found to be contradictory. Based on the results of CATA sensory analysis, the shelf life of UHT treated and retort processed porridge samples was predicted to be more than 6 months. Therefore, both UHT treatment and retort processing can be effectively applied to prepare a ready to eat milk based porridge using germinated foxtail millet grains.