Botulismo del lactante en Argentina. Trabajo multicéntrico / Infant botulism in Argentina. Multicentric work

Resumen El botulismo del lactante (BL), es la forma más frecuente del botulismo humano en la actualidad, es una enfermedad "rara" o "huérfana" ya que afecta a menos del 0,05 % de la población. El objetivo del presente trabajo es determinar la Incidencia del BL en la Argentina, evaluar el diagnóstico y tratamiento realizado, comparar la evolución y las secuelas al alta en pacientes con y sin tratamiento específico y, considerar las características climáticas (precipitaciones y vientos) y los estudios de muestras de suelos de las provincias con mayor cantidad de casos de BL. Presentamos un estudio multicéntrico, de cohorte (longitudinal) observacional, retrospectivo analizando las historias clínicas de los pacientes con BL, que ingresaron a Unidades de Cuidados Intensivos Pediátricos con asistencia respiratoria mecánica, desde el 1 de enero de 2010 hasta 31 de diciembre de 2013. Se consideró: edad, sexo, días previos al ingreso hasta diagnóstico por laboratorio, total internación en Unidades de Cuidados Intensivos Pediátricos con asistencia respiratoria mecánica, alimentación por sonda nasogástrica, tratamiento y secuelas. En Argentina entre 2010 al 2013 se registraron 216 casos de BL. En este trabajo se analizaron 79 pacientes provenientes de 11 provincias, que ingresaron a Unidades de Cuidados Intensivos Pediátricos. La edad promedio de los pacientes ingresados fue de 4 meses, de los cuales 90% recibía alimentación materna. Dieciocho pacientes de seis provincias recibieron antitoxina botulínica equina. El promedio de días de enfermedad previos al ingreso fue de 2 días en los pacientes que recibieron tratamiento con antitoxina botulínica equina y 4 días en los pacientes no tratados. Diagnóstico de laboratorio (Toxina A y Clostridium botulinum) a los 5 días en los tratados con antitoxina botulínica equina, y a los 11,5 en los no tratados. En los pacientes tratados con antitoxina botulínica equina, el promedio de días de internación fue de 30 versus 70 días en los no tratados (p=0,0001). El promedio días en las Unidades de Cuidados Intensivos Pediátricos de los pacientes tratados fue de 20 versus 54 días en los no tratados (p=0,0001). Los días de asistencia respiratoria mecánica en los tratados fue de 16 versus 43 días en los no tratados (p=0,0001) y los tratados requirieron 29 días de alimentación por sonda nasogástrica versus 70 días en los no tratados (p=0,0001). El 40% de los pacientes tratados presentaron neumonía asociada a respirador versus el 56% de los no tratados (p=0,0038), sepsis el 11% versus el 34% (p=0,005) y secuelas al alta 6% versus 64% (p=0,0001), respectivamente. En zonas con mayor número de casos, se observó una alta frecuencia de esporas en los suelos, asociado a clima seco y ventoso. Los resultados sugieren que el tratamiento precoz con antitoxina botulínica equina es una alternativa hasta disponer de inmuno-globulina botulínica humana. Los climas secos y ventosos favorecen la enfermedad.

Abstract Infant botulism (BL), the most common form of human botulism today, is a "rare" or "orphan" disease as it affects less than 0.05% of the population. The objective of this work is to determine the incidence of BL in Argentina. Evaluate the diagnosis and treatment performed. To compare evolution and sequelae at discharge in patients with and without specific treatment. Consider the climatic characteristics (precipitations and winds) and the studies of soil samples from the provinces with the highest number of BL cases. We present a retrospective, observational, multicenter, cohort (longitudinal) study analyzing the medical records of patients with BL, who were admitted to Pediatric Intensive Care Units with mechanical ventilation, from January 1,2010 to December 31,2013. The following were considered: age, sex, days prior to admission, until laboratory diagnosis, Pediatric Intensive Care Units, me-chanical respiratory assistance, average hospital days, nasogastric tube feeding, treatment and sequelae. In the country, 216 cases of BL were registered between 2010 and 2013. We analyzed 79 who were admitted to Pediatric Intensive Care Units from 11 provinces. Average age 4 months. Maternal nutrition 90%. Eighteen patients (6 provinces) received equine botulinum antitoxin .Mean days of illness prior to admission: 2 in those treated with equine botulinum antitoxin and 4 in those not treated. Laboratory diagnosis (Toxin A and Clostridium botulinum) at 5 days in treated with equine botulinum antitoxin, at 11.5 in untreated. Patients with equine botulinum antitoxin average hospital days 30 vs 70 in untreated patients (p=0.0001). Mean Pediatric Intensive Care Unit days 20 vs 54 (p=0.0001) of mechanical respiratory assistance 16 vs 43 (p=0.0001) and nasogastric tube feeding 29 vs 70 (p=0.0001). Those treated presented ventilator-associated pneumonia 40% vs 56% (p=0.0038) and sepsis 11% vs 34% (p=0.005). Sequelae at discharge 6% vs 64% (p=0.0001) in those not treated. In areas with a higher number of cases, high frequency of spores in soils, dry and windy weather. The results suggest that early treatment with equine botulinum antitoxin is an alternative until human botulinum immunoglobulin is available. The dry and windy climates favor the disease.

Botulismo del lactante grave por toxina tipo A y uso de antitoxina equina AB luego de 5 días de evolución, reporte de caso y revisión bibliográfica / Severe infant botulism due to type A toxin and use of equine AB antitoxin AB after 5 days of evolution, a case report and literature review

Resumen El botulismo del lactante (BL) es una enfermedad neuroparalítica potencialmente grave que afecta a niños menores de un año, ocasio nada por la ingesta y germinación de esporas de la bacteria del género Clostridium en tubo digestivo y la producción in situ de toxina botulínica (TB). Ésta se absorbe de manera intermitente y puede ser sostenida en el tiempo, condicionando una mayor exposición a la TB respecto a otras formas de botulismo. La TB representa el agente más letal conocido para el ser humano, con capacidad de producir parálisis flácida descendente, insuficiencia respiratoria y la muerte. Los lactantes representan la población más susceptible a esta toxiinfección. El eje central del manejo del BL radica en el diagnóstico precoz y tratamiento de sostén adecuado y oportuno. Si bien en la bibliografía consultada se describe que el tratamiento específico con antitoxina botulínica humana (BabyBIG® reduce el tiempo de hospitalización y estadía en Unidad de Cuidados Intensivos, la misma no se encuentra disponible en muchos países, incluida la Argentina. En nuestro país se encuentra disponible la antitoxina botulínica de origen equino (AtBE) bivalente A-B. La misma no posee indicación formal para el tratamiento del BL por la escasa experiencia en esta población, su corta vida media y los efectos adversos descritos, como son la sensibilización a antígenos equinos de por vida y posibles reacciones anafilácticas más graves en lactantes, basados en trabajos de la década de 1980 y opiniones de expertos. Se presenta el caso de una paciente de 5 meses asistida en el Hos pital de Niños "Superiora Sor María Ludovica" con BL severo, con requerimientos de asistencia ventilatoria mecánica y deterioro clínico durante la internación. Recibió AtBE a los 48 días de enfermedad, con respuesta favorable, a partir de una búsqueda bibliográfica sobre la eficacia y el perfil de seguridad de la AtBE en BL grave y la eficacia de su administración luego de 5 días de inicio del cuadro. A pesar de no haberse hallado bibliografía que avale la eficacia de la AtBE pasados 5 días de evolución, se plantea su uso en pacientes con BL grave e indicadores compatibles con presencia de TB en circulación, como la intensificación de la hipotonía muscular o la identificación de TB en materia fecal o suero. La búsqueda realizada arrojó datos sobre posibles beneficios de su uso, tanto antes como después de los 5 días de evolución del cuadro, y la ausencia de reportes de reacciones adversas severas en lactantes. Se concluye que el uso de la AtBE podría ser una opción terapéutica frente a la ausencia de BabyBIG® en pacientes con BL grave confirmado que requieran cuidados intensivos con soporte ventilatorio mecánico, frente a indicadores compatibles con TB circulante, independientemente del tiempo de evolución.

Abstract Infant botulism (BL) is a potentially serious neuroparalytic disease that affects children under one year old, caused by the ingestion and germination of spores of the Clostridium genus bacterium in the digestive tract and the in situ production of botulinum toxin (TB), which is absorbed intermittently and can be sustained over time, with longer exposure time to TB than other botulism forms. The TB represents the most lethal toxin known to humans and can cause descending flaccid paralysis, respiratory failure and death. Infants represent an especially susceptible population. Early diagnosis and supportive care are the cornerstone of BL management. Although specific treatment with human botulinum antitoxin (BabyBIG® has shown to reduce the hospitalization time and Intensive Care Unit stay in the consulted bibliography, it is not currently available in many countries, including Argentina. Botulinum antitoxin of equine origin (AtBE) bivalent A-B is available in our country. This antitoxin has not a formal indication in BL due to the limited experience of its use in this population, its short half-life and the adverse effects described, such as lifelong sensitization to equine antigens and possible more severe anaphylactic reactions in infants, based on studies from the 1980s and expert opinions. We present the case of a 5 month old patient assisted at the Children's Hospital "Superiora Sor María Ludovica" with severe BL, in need of mechanical ventilatory assistance and worsening of her clinical state during hospitalization, who received ATBE at 48 days of illness with a favorable response. A bibliographic search was carried out on the efficacy and safety profile of AtBE in severe BL and the efficacy of its administration after 5 days of illness onset. Even though bibliography on efficacy of ATBE after 5 days of evolution was not found, its use is proposed in patients with compatible indicators of circulating TB, such as worsening of muscular hypotonia or TB presence in feces or serum in severe ill patients. The carried out search has shown data of the possible benefits of its use, both before and after 5 days of disease onset, and the absence of severe adeverse reaction reports in infants. We concluded that the use of AtBE could be a therapeutic option in absence of BabyBIG® in patients with confirmed severe BL who require intensive care with mechanical ventilatory support and compatible indicators with circulating TB, regardless of the evolution time.

Wound botulism presenting as dysphagia to an ENT ward.

A 44-year-old man with a background of heroin injection drug use was referred to the ear, nose and throat team with a sore throat and dysphagia. He was treated with intravenous antibiotics and steroids for suspected uvulitis. He developed progressive bulbar weakness and symmetrical descending weakness of the upper extremities over a 12-hour period and was intubated prior to transfer to the intensive care unit.Botulinum heptavalent antitoxin was administered, and subsequent PCR assay confirmed Clostridium botulinum neurotoxin B from his most recent injection site. He was found unconscious on the ward 3 days following extubation. Postmortem confirmed he died from heroin intoxication.This case highlights the importance of considering wound botulism in injection drug users presenting with unexplained weakness, particularly of the lower cranial nerves. Botulism is not characteristically associated with signs of localised or systemic infection in contrary to other bacterial complications of injection drug use.

Esophagogastric junction outflow obstruction: Where are we now in diagnosis and management?

Esophagogastric junction outflow obstruction (EGJOO) is a major motility disorder based on the Chicago Classification of esophageal motility disorders. This entity involves a heterogenous group of underlying etiologies. The diagnosis is reached by performing high-resolution manometry. This reveals evidence of obstruction at the esophagogastric junction, manifested by an elevated integrated relaxation pressure (IRP) above a cutoff value (IRP threshold varies by the manometric technology and catheter used), with preserved peristalsis. Further tests like endoscopy, timed barium esophagram, and cross-sectional imaging can help further elucidate the underlying etiology and rule out mechanical causes. Treatment is tailored to the underlying cause. Similar to achalasia, treatment targeting lower esophageal sphincter disruption like pneumatic dilation, peroral endoscopic myotomy, and botulinum injection are used in patients with functional EGJOO and persistent symptoms.

Quality of horse F(ab')2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab′)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p < 0.05. Results Horse F(ab′)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates. Conclusions Anti-botulinum AB (bivalent), anti-diphtheric, antitetanic and anti-rabies horse F(ab′)2 immunoglobulins probably contain acceptable levels of aggregates, although other improvements on the preparations must be carried out. Protein profile analysis and in vitro anticomplementary activity of F(ab′)2 immunoglobulin preparations should be included as quality control steps, to ensure acceptable levels of aggregates, contaminants and whole IgG molecules on final products, reducing the chances of adverse reactions in patients.(AU)

Effective and rapid treatment of wound botulism, a case report.

BACKGROUND: The latest news shows several cases of contaminated heroin that is found in different parts all over Europe. This information can be helpful for the emergency doctors to find the correct diagnosis of wound botulism in patients who are intravenous drug users. CASE PRESENTATION: We describe a case of a 40-year-old man who presented to the emergency department in 2016. He suffered from mild dysarthria, diplopia, dysphagia and ptosis since two days. The CT-scan of the cerebrum and the liquor were without any pathological results. We found out that the patient is an intravenous drug user and the clinical examination showed an abscess in the left groin. So we treated him with the suspected diagnosis of wound botulism. In the emergency operation we split the abscess, made a radical debridement and complementary treated him with a high dose of penicillin g and two units of botulism antitoxin. The suspected diagnosis was confirmed a few days later by finding the Toxin B in the abscess and in the patient's serum. In the following days the neurological symptoms decreased and the wound healing was without any complications. The patient left the hospital after nine days; the antibiotic therapy with penicillin g was continued for several days. In a following examination, 14 days after the patient's discharge of the hospital, no further symptoms were found and the abscess was treated successfully without any problems. CONCLUSION: Because wound botulism is a very rare disease it can be challenging to the attending physician. This case shows a fast treatment with full recovery of the patient without any further disabilities, which can be used for the future.

mRNA mediates passive vaccination against infectious agents, toxins, and tumors.

The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA-lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA-mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.

Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins.

Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001). Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.

Evidências de revisões sistemáticas Cochrane sobre uso da toxina botulínica / Evidence from systematic reviews Cochrane on use of botulinum toxin

Contexto: A toxina botulínica tem sido utilizada no tratamento de várias situações clínicas (de modo regulamentado ou não). Assim, é importante mapear a efetividade e a segurança de cada indicação, fornecendo evidências para a tomada de decisão. Objetivo: Mapear evidências de revisões sistemáticas Cochrane sobre o uso da toxina botulínica. Métodos: Revisão narrativa com busca sistematizada da literatura (overview) que incluiu revisões sistemáticas Cochrane. Resultados: Entre as 82 revisões sistemáticas da busca inicial, 24 preencheram os critérios de inclusão. As revisões sistemáticas concluíram que: (a) a toxina botulínica é eficaz para: bexiga hiperativa, dor no ombro, distonia cervical,lombalgia e ciatalgia, sialorreia na doença do neurônio motor/esclerose lateral amiotrófica, espasmos hemifaciais, esclerose múltipla, espasticidade de membros superiores na paralisia cerebral, blefaroespasmo; (b) as evidências atuais são insuficientes para apoiar o uso da toxina botulínica para: hipertrofia do masseter, dor miofascial, sialorreia na paralisia cerebral, disfonia espasmódica, estrabismo, disfunção de esfíncter esofágico superior e desordens neurológicas de deglutição; (c) há evidências de que a toxina botulínica não é eficaz para cervicalgia subaguda e crônica, pé cavo, espasticidade de membros inferiores na paralisia cerebral, acalasia primária, síndrome do desfiladeiro torácico e fissura anal. Conclusão: Nas situações clínicas para as quais há ensaios clínicos randomizados, eles possuem qualidade metodológica limitada, aumentando o risco de viés e reduzindo a confiança nos resultados. São necessários ensaios clínicos de qualidade para embasar o uso da toxina botulínica em situações clínicas nas quais ela já é utilizada, mesmo sem aprovação por órgão regulamentador nacional.

Substrate-based inhibitors exhibiting excellent protective and therapeutic effects against Botulinum Neurotoxin A intoxication.

Potent inhibitors to reverse Botulinum neurotoxins (BoNTs) activity in neuronal cells are currently not available. A better understanding of the substrate recognition mechanism of BoNTs enabled us to design a novel class of peptide inhibitors which were derivatives of the BoNT/A substrate, SNAP25. Through a combination of in vitro, cellular based, and in vivo mouse assays, several potent inhibitors of approximately one nanomolar inhibitory strength both in vitro and in vivo have been identified. These compounds represent the first set of inhibitors that exhibited full protection against BoNT/A intoxication in mice model with undetectable toxicity. Our findings validated the hypothesis that a peptide inhibitor targeting the two BoNT structural regions which were responsible for substrate recognition and cleavage respectively could exhibit excellent inhibitory effect, thereby providing insight on future development of more potent inhibitors against BoNTs.

Recent developments with metalloprotease inhibitor class of drug candidates for botulinum neurotoxins.

Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, and these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.

Prolonged prophylactic protection from botulism with a single adenovirus treatment promoting serum expression of a VHH-based antitoxin protein.

Current therapies for most acute toxin exposures are limited to administration of polyclonal antitoxin serum. We have shown that VHH-based neutralizing agents (VNAs) consisting of two or more linked, toxin-neutralizing heavy-chain-only VH domains (VHHs), each binding distinct epitopes, can potently protect animals from lethality in several intoxication models including Botulinum neurotoxin serotype A1 (BoNT/A1). Appending a 14 amino acid albumin binding peptide (ABP) to an anti-BoNT/A1 heterodimeric VNA (H7/B5) substantially improved serum stability and resulted in an effective VNA serum half-life of 1 to 2 days. A recombinant, replication-incompetent, adenoviral vector (Ad/VNA-BoNTA) was engineered that induces secretion of biologically active VNA, H7/B5/ABP (VNA-BoNTA), from transduced cells. Mice administered a single dose of Ad/VNA-BoNTA, or a different Ad/VNA, via different administration routes led to a wide range of VNA serum levels measured four days later; generally intravenous > intraperitoneal > intramuscular > subcutaneous. Ad/VNA-BoNTA treated mice were 100% protected from 10 LD50 of BoNT/A1 for more than six weeks and protection positively correlated with serum levels of VNA-BoNTA exceeding about 5 ng/ml. Some mice developed antibodies that inhibited VNA binding to target but these mice displayed no evidence of kidney damage due to deposition of immune complexes. Mice were also successfully protected from 10 LD50 BoNT/A1 when Ad/VNA-BoNTA was administered up to 1.5 hours post-intoxication, demonstrating rapid appearance of the protective VNA in serum following treatment. Genetic delivery of VNAs promises to be an effective method of providing prophylactic protection and/or acute treatments for many toxin-mediated diseases.

Outbreak of wound botulism in people who inject drugs, Norway, October to November 2013.

In October and November 2013, four cases of wound botulism were confirmed in people who inject drugs (PWID) in Norway. Two additional cases are suspected. Because of the international distribution pathways for heroin ­ the likely source of the outbreak ­ healthcare workers and public health authorities in other countries should remain vigilant for wound botulism in PWID. This outbreak serves as a reminder that countries should ensure access to botulinum antitoxin in case of outbreak situations.

The life history of a botulinum toxin molecule.

There is an emerging literature describing the absorption, distribution, metabolism and elimination of botulinum toxin. This work reveals that the toxin can be absorbed by both the oral and inhalation routes. The primary mechanism for absorption is binding and transport across epithelial cells. Toxin that enters the body undergoes a distribution phase, which is quite short, and an elimination phase, which is comparatively long. During the distribution phase, botulinum toxin migrates to the peri-neuronal microcompartment in the vicinity of vulnerable cells, such as cholinergic nerve endings. Only these cells have the ability to selectively accumulate the molecule. When the toxin moves from the cell membrane to the cell interior, it undergoes programmed death. This is coincident with release of the catalytically active light chain that paralyzes transmission. Intraneuronal metabolism of light chain is via the ubiquitination-proteasome pathway. Systemic metabolism and elimination is assumed to be via the liver. The analysis of absorption, distribution, metabolism and elimination of the toxin helps to create a life history of the molecule in the body. This has many benefits, including: a) clarifying the mechanisms that underlie the disease botulism, b) providing insights for development of medical countermeasures against the toxin, and c) helping to explain the meaning of a lethal dose of toxin. It is likely that work intended to enhance understanding of the fate of botulinum toxin in the body will intensify. These efforts will include new and powerful analytic tools, such as single molecule-single cell analyses in vitro and real time, 3-dimensional pharmacokinetic studies in vivo.