Analysis of a plasmid encoding botulinum neurotoxin type G gene in Clostridium argentinense.

Clostridium argentinense produces botulinum neurotoxin type G (BoNT/G). We sequenced and analyzed the plasmid harboring the bont/G gene, designated pCAG, in C. argentinense strain 2740. The pCAG consisted of 140,070 bp containing the bont/G gene cluster. Although this gene cluster showed high similarities in its DNA sequence and ORF arrangement to those of other bont gene clusters, the other regions of the plasmid did not. A phylogenetic study suggested that pCAG had a unique evolutionary history compared with other clostridial bont-harboring plasmids. This suggests that pCAG is possibly a novel type of plasmid expressing the bont/G gene in C. argentinense.

Jitter Evaluation in Distant and Adjacent Muscles after Botulinum Neurotoxin Type A Injection in 78 Cases.

To study the jitter parameters in the distant (DM) and the adjacent muscle (AM) after botulinum neurotoxin type A (BoNT/A) injection in 78 patients, jitter was measured by voluntary activation in DM (n = 43), and in AM (n = 35). Patients were receiving BoNT/A injections as a treatment for movement disorders. Mean age 65.1 years (DM) and 61.9 years (AM). The mean jitter was abnormal in 13.9% (maximum 41.4 µs) of DM, and 40% (maximum 43.7 µs) of AM. Impulse blocking was sparse. We found no correlation of the mean jitter to age, BoNT/A most recent injection (days/units), number of muscles injected, total BoNT/A units summated, number of total BoNT/A sessions, beta-blockers/calcium channel blockers use, and cases with local spread symptoms such as eyelid drop/difficulty swallowing. Maximum mean jitter (41.4/43.7 µs) for DM/AM occurred 61 and 131 days since the most recent BoNT/A, respectively. The far abnormal mean jitter (32.6/36.9 µs) occurred 229 and 313 days since the most recent BoNT/A. We suggested that jitter measurement can be done after BoNT/A in a given muscle other than the injected one, after 8 (DM) and 11 (AM) months, with reference >33 µs and >37 µs, respectively.

Genomic Characterization of Newly Completed Genomes of Botulinum Neurotoxin-Producing Species from Argentina, Australia, and Africa.

Botulinum neurotoxin-producing clostridia are diverse in the types of toxins they produce as well as in their overall genomic composition. They are globally distributed, with prevalent species and toxin types found within distinct geographic regions, but related strains containing the same toxin types may also be located on distinct continents. The mechanisms behind the spread of these bacteria and the independent movements of their bont genes may be understood through examination of their genetic backgrounds. The generation of 15 complete genomic sequences from bacteria isolated in Argentina, Australia, and Africa allows for a thorough examination of genome features, including overall relationships, bont gene cluster locations and arrangements, and plasmid comparisons, in bacteria isolated from various areas in the southern hemisphere. Insights gained from these examinations provide an understanding of the mechanisms behind the independent movements of these elements among distinct species.

Is botulism type C transmissible to human by consumption of contaminated poultry meat? Analysis of a suspect outbreak in French Guyana.

Botulism type C was suspected in a 46-year old man after consumption of sick poultry from a flock where botulism type C was confirmed. The patient developed characteristic signs of botulism, but investigation of biological samples did not confirm the presence of Clostridium botulinum or botulinum toxin. Despite having classical botulism symptoms, the man recovered very quickly. This raises the question of botulism transmission to humans by ingestion of contaminated poultry.

Content/Potency Assessment of Botulinum Neurotoxin Type-A by Validated Liquid Chromatography Methods and Bioassays.

Botulinum neurotoxin type-A (BoNTA) is one of the seven different serotypes (A to G) produced by Clostridium botulinum. A stability-indicating size-exclusion chromatography (SEC) method was developed and validated, and the specificity was confirmed by forced degradation study, interference of the excipients, and peaks purity. The method was applied to assess the content and high-molecular-weight (HMW) forms of BoNTA in biopharmaceutical products, and the results were compared with those of the LD50 mouse bioassay, the T-47D cell culture assay, and the reversed-phase chromatography (RPC) method, giving mean values of 0.71% higher, 0.36% lower, and 0.87% higher, respectively. Aggregated forms showed significant effects on cytotoxicity, as well as a decrease in the bioactivity (p < 0.05). The employment of the proposed method in conjunction with the optimized analytical technologies for the analysis of the intact and altered forms of the biotechnology-derived medicines, in the correlation studies, enabled the demonstration of the capability of each one of the methods and allowed for great improvements, thereby assuring their safe and effective use.

Immunogenicity of a Bivalent Non-Purified Recombinant Vaccine against Botulism in Cattle.

Botulism is a potentially fatal intoxication caused by botulinum neurotoxins (BoNTs) produced mainly by Clostridium botulinum. Vaccination against BoNT serotypes C and D is the main procedure to control cattle botulism. Current vaccines contain formaldehyde-inactivated native BoNTs, which have a time-consuming production process and pose safety risks. The development of non-toxic recombinant vaccines has helped to overcome these limitations. This study aims to evaluate the humoral immune response generated by cattle immunized with non-purified recombinant fragments of BoNTs C and D. Cattle were vaccinated in a two-dose scheme with 100, 200 and 400 µg of each antigen, with serum sampling on days 0, 56, 120, and 180 after vaccination. Animals who received either 200 or 400 µg of both antigens induced titers higher than the minimum required by the Brazilian ministry of Agriculture, Livestock and Food Supply and achieved 100% (8/8) seroconversion rate. Animals vaccinated with commercial toxoid vaccine had only a 75% (6/8) seroconversion rate for both toxins. Animals that received doses containing 400 µg of recombinant protein were the only ones to maintain titers above the required level up until day 120 post-vaccination, and to achieve 100% (8/8) seroconversion for both toxins. In conclusion, 400 µg the recombinant Escherichia coli cell lysates supernatant was demonstrated to be an affordable means of producing an effective and safe botulism vaccine for cattle.

More Clinical Mimics of Infant Botulism.

OBJECTIVE: To ascertain the actual diagnoses of 76 patients (2005-2015) whose clinical presentations so closely resembled infant botulism that the patients were treated with Human Botulism Immune Globulin Intravenous (BIG-IV; BabyBIG), but whose illnesses subsequently were not laboratory confirmed as infant botulism ("clinical mimics" of infant botulism). STUDY DESIGN: The California Department of Public Health produces BIG-IV and distributes it nationwide as a public service (ie, not-for-profit) orphan drug to treat patients hospitalized with suspected infant botulism. During the study period, admission records and discharge summaries for all patients treated with BIG-IV but who lacked a laboratory-confirmed diagnosis of infant botulism were collected and abstracted. The patients' discharge diagnoses were identified, categorized, and compared with previously reported clinical mimics categories for 32 patients (1992-2005). RESULTS: From 2005 to 2015, 76 clinical mimic illnesses were identified. These illnesses were distributed into the 5 categories previously reported of (1) probable infant botulism lacking confirmatory testing (26.3%); (2) spinal muscular atrophy (19.7%); (3) miscellaneous (15.8%); (4) metabolic disorders (11.8%); and (5) other infectious diseases (10.6%). Of the 76 clinical mimic illnesses, 15.8% had no alternate diagnosis established and were therefore categorized as undetermined. CONCLUSIONS: Over the 23 years 1992-2015, patients presenting with illnesses so clinically similar to infant botulism that they were treated with BIG-IV had actual diagnoses that were distributed into 5 main categories. These categories and their individual components constitute a working bedside differential diagnosis of infant botulism.

OnabotulinumtoxinA for trigeminal neuralgia: a review of the available data / Toxina Onabotulínica-A para neuralgia do trigêmeo: uma revisão dos dados disponíveis

Trigeminal neuralgia (TN) patients may develop side effects from centrally acting drugs, have contraindications for neurosurgical procedures, or experience relapse during conventional therapies. OnabotulinumtoxinA (BoNT/A) has been reported to be effective for TN, although this finding has been challenged. An overview of the available evidence based on a narrative/qualitative analysis of the literature is presented. About 90% of patients who receive BoNT/A show an improvement, a higher figure than that reported for the placebo effect of BoNT/A for other headaches. Tolerability of BoNT/A is good, and its few side-effects are transient. The articles reviewed were mainly case reports, case series and open-label trials; however, randomized controlled trials have endorsed the efficacy of BoNT/A for TN. This evidence, together with a better understanding of the analgesic mechanisms of BoNT/A and its proven efficacy in treating other pain syndromes, supports the use of this toxin as a therapeutic option for TN.

Pacientes com neuralgia do trigêmeo (NT) podem apresentar efeitos colaterais decorrentes do uso de drogas psicoativas, contra-indicações a procedimentos neurocirúrgicos ou perda da eficácia destas terapias. A neurotoxina botulínica do tipo A (NTB/A) tem demonstrado ser eficaz no alívio da NT, ainda que este achado tenha sido contestado. Uma análise narrativa/qualitativa da literatura disponível é apresentada. Cerca de 90% dos pacientes que receberam NTB/A melhoram, um número superior aos atribuíveis ao efeito placebo da NTB/A em outras cefaléias. Além disso, a NTB/A mostrou uma baixa incidência de efeitos colaterais, transitórios. Embora a maioria dos artigos consistam de relatos de caso, séries de casos e ensaios abertos, ensaios clínicos randomizados controlados recentes reafirmam a eficácia da NTB/A na NT. Estas evidências, associadas ao melhor entendimento dos mecanismos analgésicos da NTB/A e a sua eficácia em outras síndromes dolorosas, ratificam a NTB/A como uma opção terapêutica para a NT.

Agonist-dependent modulation of cell surface expression of the cold receptor TRPM8.

The spatial and temporal distribution of receptors constitutes an important mechanism for controlling the magnitude of cellular responses. Several members of the transient receptor potential (TRP) ion channel family can regulate their function by modulating their expression at the plasma membrane (PM) through rapid vesicular translocation and fusion. The mechanisms underlying this regulation are not completely understood, and the contribution of vesicular trafficking to physiological function is unknown. TRPM8 receptors are expressed in mammalian peripheral sensory neurons and are essential for the detection of cold temperatures. Previously, we showed that TRPM8-containing vesicles are segregated into three main pools, immobile at the PM, simple diffusive and corralled-hopping. Here, we show that channel expression at the PM is modulated by TRPM8 agonists in F11 and HEK293T cells. Our results support a model in which the activation of TRPM8 channels, located at the PM, induces a short-lived recruitment of a TRPM8-containing vesicular pool to the cell surface causing a transitory increase in the number of functional channels, affecting intrinsic properties of cold receptor responses. We further demonstrate the requirement of intact vesicular trafficking to support sustained cold responses in the skin of mice.

Quality of life in individuals with cervical dystonia before botulinum toxin injection in a Brazilian tertiary care hospital / Qualidade de vida em indivíduos com distonia cervical antes da aplicação de toxina botulínica em um hospital terciário brasileiro

OBJECTIVE: The purpose of this study was to evaluate quality of life (QoL) in a Brazilian population of individuals with cervical dystonia (CD) without effect of botulinum toxin (BTx) or with only residual effect of BTx, and identify possible physical and social aspects that affect their QoL. METHOD: Sixty five out of sixty seven consecutive patients with CD were assessed with two instruments: Short-form Health Survey with 36 questions (SF-36) and Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS). RESULTS: Severity of CD (TWSTRS) correlated moderately with two SF-36 subscale: role-physical (r= -0.42) and body pain (r= -0.43). Women also scored worse in two subscale of SF-36: vitality (p<0.05) and mental-health (p<0.005). CONCLUSION: Severity of CD and gender (female) were the main factors related to a worse QoL perception. These findings may help health professionals to predict which characteristics could lead to worse QoL, and therefore, better target their interventions to lessen the burden caused by CD.

OBJETIVO: O objetivo deste estudo foi avaliar a qualidade de vida (QV) em uma população brasileira de indivíduos com distonia cervical (DC), que estavam sem o efeito da toxina botulínica ou com efeito residual da mesma, e identificar os possíveis aspectos físicos e sociais que afetam sua QV. MÉTODO: Sessenta e cinco de sessenta e sete pacientes consecutivos com DC foram avaliados com dois instrumentos: Short-form Health Survey com 36 questões (SF-36) e Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS). RESULTADOS: A gravidade da DC (TWSTRS) correlacionou-se moderadamente com duas sub-escalas da SF-36: aspectos físicos (r= -0,42) e dor (r= -0,43). Mulheres apresentaram piores pontuações em duas sub-escalas da SF-36: vitalidade (p<0,05) e saúde mental (p<0,005). CONCLUSÃO: Gravidade da DC e gênero (feminino) foram os principais fatores relacionados à pior percepção de QV. Estes achados podem auxiliar profissionais da saúde a identificarem quais características poderiam levar a uma pior QV, e assim direcionar melhor suas intervenções, atenuando os danos causados pela DC.

Botulinum toxin type-A effect as a preemptive treatment in a model of acute trigeminal pain: a pre-clinical double-blind and placebo-controlled study / Toxina botulínica do tipo-A no tratamento preemptivo da dor trigeminal aguda: estudo pré-clínico duplo cego placebo controlado

The purpose of this study was to investigate if botulinum neurotoxin type-A (BoNT/A) had a preemptive antinociceptive effect in a formalin-induced orofacial pain model (FT). To test this hypothesis, male Rattus norvegicus were injected with isotonic saline solution 0.9 percent or BoNT/A administered as a 40 μl bolus, lateral to their nose, at 24 hours, 8, 15, 22, 29 or 36 days pre-FT. The procedures were repeated 42 days later. Influence on motor activity was assessed through the open-field test. Pain scores corresponded to the time spent rubbing and flicking the injected area. Animals pre-treated with BoNT/A at the first protocol (8 days subgroup) showed reduced inflammatory scores (p=0.011). For the other groups no significant results were observed at any phase. Motor activity was similar in both groups. BoNT/A showed to be effective preventing inflammatory pain up to eight days after the first treatment, an effect not reproduced on the second dose administration.

O objetivo deste estudo foi investigar o efeito preemptivo da neurotoxina botulínica do tipo/A (NTBo/A) através de um modelo de dor orofacial induzida pelo teste da formalina (TF). Rattus norvegicus machos foram injetados no lábio superior com solução salina isotônica 0,9 por cento (SSI) ou NTBo/A (subgrupos 24 horas, 8, 15, 22, 29 ou 36 dias) antes do TF, em dois tratamentos farmacológicos e respectivas avaliações intercalados por 42 dias. Os escores da dor corresponderam ao tempo de fricção da região injetada. Após o primeiro pré-tratamento com NTBo/A no subgrupo 8 dias os escores da fase inflamatória foram menores do que no grupo SSI (p=0,011). Todas as outras comparações não foram significativas. Nos testes de atividade motora não ocorreram diferenças entre SSI e NTBo/A. A NTBo/A pode ser considerada como tratamento preemptivo das dores orofaciais quando utilizada até oito dias antes do estímulo álgico, não havendo consistência terapêutica após um segundo tratamento.

Botulism in Brazil, 2000-2008: epidemiology, clinical findings and laboratorial diagnosis / Botulismo no Brasil, 2000-2008: epidemiologia, achados clínicos e diagnóstico laboratorial

Botulism is a rare and potentially lethal illness caused by Clostridium botulinum neurotoxin. We describe the findings of a laboratorial investigation of 117 suspected cases of botulism reported to the surveillance system in Brazil from January 2000 to October 2008. Data on the number and type of samples analyzed, type of toxins identified, reporting of the number of botulism cases and transmission sources are discussed. A total of 193 clinical samples and 81 food samples were analyzed for detection and identification of the botulism neurotoxin. Among the clinical samples, 22 (11.4 percent) presented the toxin (nine type A, five type AB and eight with an unidentified type); in food samples, eight (9.9 percent) were positive for the toxin (five type A, one type AB and two with an unidentified type). Of the 38 cases of suspected botulism in Brazil, 27 were confirmed by a mouse bioassay. Laboratorial botulism diagnosis is an important procedure to elucidate cases, especially food-borne botulism, to confirm clinical diagnosis and to identify toxins in food, helping sanitary control measures.

Botulismo é uma doença rara e potencialmente letal, resultante da ação de uma neurotoxina produzida pelo Clostridium botulinum. No presente estudo, estão descritos os resultados da investigação laboratorial de 117 casos suspeitos de botulismo notificados ao sistema de vigilância, ocorridos no Brasil no período de janeiro de 2000 a outubro de 2008. Os dados obtidos sobre as fontes de transmissão, os tipos de toxina identificados e de amostras analisadas serão discutidos. Foram analisadas 193 amostras clínicas e 81 amostras de alimentos para detecção e identificação de neurotoxina botulínica. Entre as amostras clínicas, 22 (11,4 por cento) amostras apresentaram resultado positivo para toxina (nove do tipo A, cinco do tipo AB e em oito o tipo não foi identificado) e entre as amostras de alimentos, oito (9,9 por cento) foram positivas (cinco do tipo A, uma do tipo AB e em duas o tipo não foi identificado). Dos 38 casos considerados positivos para botulismo, 27 foram confirmados pelo bioensaio em camundongo. O diagnóstico laboratorial de botulismo é importante para elucidação dos casos, principalmente de botulismo alimentar, para confirmação dos diagnósticos clínicos e identificação das toxinas nos alimentos, provendo subsídios para as medidas de controle sanitário.

Botulinum neurotoxin type-A for primary stabbing headache: an open study / Toxina botulínica do tipo-A para o tratamento da cefaléia primária em punhaladas: um estudo aberto

Primary stabbing headache is an ultra-short headache, associated with primary headaches, more prevalent in women and with a poor response to therapy. The effect of botulinum neurotoxin type-A (BoNTA) on primary stabbing headache was investigated in 24 patients. Three patients showed complete remission. Nineteen patients showed a decrease in their primary stabbing headaches that started in the second week, and that was sustained during approximately 63 days. In two patients BoNTA showed no therapeutic effect. The BoNTA seems to be an excellent therapeutic option for primary stabbing headache.

Cefaléia primária em punhaladas (CPP) é uma cefaléia ultra-rápida, associada a cefaléias primárias, mais frequente em mulheres e com discreta resposta terapêutica. O efeito da neurotoxina botulínica do tipo A (NTBo-A) sobre a CPP foi investigado em 24 pacientes. Três pacientes apresentaram completa remissão dos sintomas. Dezenove pacientes mostraram uma redução que começou na segunda semana e que manteve-se por um período de 63 dias. Em dois pacientes a NTBo-A não apresentou nenhum efeito terapêutico. A NTBo-A parece ser uma excelente opção terapêutica no tratamento da CPP.