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.

Botulismo infantil: Comunicación de un caso clínico y revisión de la literatura / Infant botulism: Case report and review

Botulism is a rare disease in Chile and of the known clinical presentation, infant botulism is the most common. We report the case of a previously healthy seven month oíd male infant with a two weeks history of rinorrea, cough, fatigue, constipation and progressive weakness after the consumption of honey. Stool cultures were positive for Clostridium botulinum group 1 type A and electromyography was compatible with the diagnosis. The patient evolved with arterial hypertension, interpreted as secondary to autonomic dysfunction, which responded to calcium channel blockers. Muscle tone improved progressively during the following four weeks. Infant botulism is a potentially fatal disease; diagnosis can be difficult given the broad clinical manifestations. Prevention should focus on education of parents of infants as well as medical personnell.

El botulismo es un trastorno poco frecuente en nuestro país. De las formas conocidas, el botulismo infantil da cuenta de la mayoría de los casos. Comunicamos el caso clínico de un paciente de siete meses, sexo masculino, sin antecedentes mórbidos. Historia de dos semanas de coriza, tos y decaimiento. Tras la ingesta de miel presentó exacerbación de la sinto-matología respiratoria, constipación y debilidad muscular progresiva. Se analizó muestra de heces resultando positiva para Clostridium botulinum grupo I tipo A. El estudio electromiográñco fue compatible con el diagnóstico. Presentó hipertensión arterial, atribuyéndose a disfunción autonómica, con buena respuesta a bloqueadores de los canales de calcio. Recuperó progresivamente el tono muscular. En un control ambulatorio se apreciaba importante regresión de la sinto-matología. El botulismo infantil es una enfermedad potencialmente letal de no tratarse oportunamente y de difícil diagnóstico, ya que su presentación es similar a otros cuadros clínicos. Es necesario educar a padres y personal médico sobre las medidas de prevención para los lactantes bajo doce meses de edad.

[Cloning, expression and purification of snap-25 proterin]

Clostridial neurotoxin inhibits neurotransmitter release by selective and specific intracellular proteolysis of synaptosomal associated protein of 25KDa [SNAP-25], synaptobrevin/VAMP-2 and syntaxin. SNAP-25 is one of the components that forms docking complex in synaptic ends. This protein is subtrate for botulinum neurotoxins types A,C, and E. Each of these toxin serotypes specifically cleaves SNAP-25 in a particular position and thereby block docking and synaptic vesicle membrane fusion and finally prevents neurotransmitter exocytosis and transition of neurotic signals. Recombinant production of SNAP-25 in the laboratory can be used as a subtrate for the detection of clostridium botulinum types A, and E neurotoxins. In order to use the protein as a subtrate for detection of different types of clostridium neurotoxins in-vitro the protein was produced by recombinant technique. The cDNA from SNAP-25 was synthesized from total RNA purified from frozen Rattus norvegicus brain. and amplified by RT-PCR The amplified fragment was cloned into pET32a expression vector. The identity of recombinant protein was confirmed by Western blot using specific antibody and finally the recombinant protein was purified through an affinity column chromatography [Ni-NTA]. The optimum conditions of expression of SNAP-25 were found to be IPTG[1mM] and incubation at 37°C for 5 hours. The recombinant protein was isolated and purified using Ni-NTA column with imidazole at a concentration of 25OmM. Using enterokinase to cut the fision at 37°C comparatively yielded better results than room temperature. The protein retained its structure during the purification process being suitable for cutting and further tests. The purified protein we obtained can be used as subtrate for detection of clostridium botulinum types A, and E toxins

High-level expression of the Hcc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli and its immunogenicity as an antigen / 生物工程学报

A completely synthetic gene encoding the He domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, -50 kD) was constructed with oligonucleotides. After expressed in Escherichia coli, soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%-53% of soluble total proteins) and the purified yield was more than 30 mg/L by one-step purification. Then, the purified AHc was used to vaccinate Balb/c mice, which developed a strong and specific immune response as expected following administration of AHe protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble, stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab, but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.

Modification and high level expression of protective antigen fragment of botulinum neurotoxin serotype A / 生物工程学报

Designed a pair of primers through modifying N-terminal bases (5bps) of gene after ATG but not changing amino acid, and amplified a smaller mutated gene sequnce (468bp) containing two protective antigenic determinants from pBlue-BoNTaHc, N-terminal codon of mutated gene fragment is changed from low to high frenqence in E. coli. Mutated gene was ligated into pGEM-T vector and sequenced, then, cloned into a expression plasmid pBV220. As a result, cloned gene was expressed in insoluble form by temperature inducing (from 30 degrees C to 42 degrees C) in E. coli. Expression product is 40% of total proteins and is of specific binding activity to antibody in ELISA. The successful modification and high level expression of protective fragment of botulinum neurotoxin serotype A(BoNTaHc468) gene is conducive to further study on antitoxin and vaccine.

The First Outbreak of Botulism in Korea / 대한임상미생물학회지

Botulism is a rare neuroparalytic disease caused by neurotoxins of Clostridium species. A ten-year-old girl and her mother were admitted to a hospital with symptoms of progressive dizziness, blurred vision, slurred speech, constipation and difficulty in swallowing. These characteristic manifestations and clinical course prompted an examination of the possibility of botulism. Mouse bioassay performed with mother's stool demonstrated type A botulinum toxin and culture of the mother's stool was positive for Clostridium botulinum type A. This is the first case of botulism in Korea.