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Functional characterisation of tetanus and botulinum neurotoxins binding domains.

Tetanus and botulinum neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial neurotoxins.

Animals↗

Mortality of tetanus at the Lagos University Teaching Hospital, Nigeria.

Records of 349 tetanus patients, aged 10 years and above, admitted to the Lagos University Teaching Hospital, Nigeria, between 1990 and 1999 were reviewed. The male:female ratio was 1.98:1, and the ages were between 10 and 88 years, with a mean age of 29.8 years. The overall case fatality rate (CFR) of tetanus was 36.96% (33.19% for men and 44.44% for women). The CFR is similar to that previously reported in the hospital, but higher than that reported from Europe and North America. The lowest CFR was in the 10-19-year age group, and there was a trend towards increasing CFR with increasing age. Tetanus patients in the intensive care unit (ICU) had a significantly higher CFR than those in the medical wards. Unlike in developed countries, where management of tetanus in ICU has resulted in a decrease in CFR, the CFR of tetanus at the Lagos University Teaching Hospital has not significantly reduced over the years.

Adolescent↗

Respiratory failure in tetanus: case report and review of a 25-year experience.

The objectives of the study were to describe a novel presentation of tetanus and to review the course of the respiratory component and the treatment and management of the disease. A case report is presented with a review of a 25-year experience at Mayo Clinic. We describe the case of a 65-year-old woman who presented with persistent hiccups, dyspnea, and pleurisy of 3 days duration that was caused by tetanus from inadequate secondary immunity. She required intubation for progressive trismus and laryngospasm-associated respiratory failure. Infusion of lorazepam did not control her spasms. Refractory spasms and hiccups resolved with fentanyl and cisatracurium therapy. After 3 weeks, the patient was weaned from the ventilator with complete recovery. In the past 25 years, nine additional patients have presented to Mayo Clinic with acute tetanus. Respiratory failure requiring intubation developed in seven patients, and six of the seven intubated patients survived with minimal deficits. The prognosis of tetanus is favorable if it is diagnosed promptly and if treatment and supportive measures are begun. To our knowledge, this is the first report of a patient with acute tetanus presenting with hiccups. This report also confirms the results of previous studies that suggested a need for improved immunity in the elderly population.

Acute Disease↗

Immunogenicity and safety of an inactivated hepatitis A vaccine administered concomitantly with diphtheria-tetanus-acellular pertussis and haemophilus influenzae type B vaccines to children less than 2 years of age.

OBJECTIVE: The availability of a hepatitis A virus vaccine for infant and early childhood immunization could reduce the transmission of hepatitis A virus in the United States. This study evaluated the immunogenicity and safety of a hepatitis A virus vaccine (Havrix, GlaxoSmithKline Biologicals, Rixensart, Belgium) administered concomitantly with diphtheria-tetanus-acellular pertussis and Haemophilus influenzae type b vaccines to children < 2 years. METHODS: In this open, comparative, multicenter study, 1084 healthy children aged 11 to 25 months were allocated (4:4:3:3:4 ratio) to 5 treatment groups based on age and previous vaccination history. Subjects 11 to 13 months of age received 2 doses of hepatitis A virus vaccine 6 months apart (N = 243). Subjects aged 15 to 18 months received 2 doses of hepatitis A virus vaccine 6 months apart (N = 241); or hepatitis A virus vaccine, diphtheria-tetanus-acellular pertussis, and H influenzae type b at month 0 and the second dose of hepatitis A virus vaccine 6 months later (N = 183); or diphtheria-tetanus-acellular pertussis and H influenzae type b at month 0 and hepatitis A virus vaccine at months 1 and 7 (N = 175). Subjects 23 to 25 months of age received hepatitis A virus vaccine at months 0 and 6 (N = 242). Immune responses were measured at baseline and 30 days after vaccine doses, and solicited and unsolicited adverse events were collected. RESULTS: After 2 doses of hepatitis A virus vaccine, all of the subjects in all of the groups were seropositive. Coadministration of hepatitis A virus vaccine with diphtheria-tetanus-acellular pertussis and H influenzae type b vaccines did not impact the immunogenicity of the 3 vaccines, except for the antipertussis toxoid vaccine response, which was slightly decreased. Hepatitis A virus vaccine was well tolerated in children 11 to 25 months of age. CONCLUSION: The administration of 2 doses of hepatitis A virus vaccine on a 0- and 6-month schedule starting at 11 to 13 months of age or at 15 to 18 months of age was as immunogenic and well tolerated as the administration of 2 doses in children 2 years of age. Immune responses to diphtheria-tetanus-acellular pertussis and H influenzae type b either given alone or coadministered with hepatitis A virus vaccine were similar except for antipertussis toxoid response.

Antibody Formation↗

Identification of risk factors for death from tetanus in Pernambuco, Brazil: a case-control study.

A case-control study was conducted to identify risk factors for death from tetanus in the State of Pernambuco, Brazil. Information was obtained from medical records of 152 cases and 152 controls, admitted to the tetanus unit in the State University Hospital, in Recife, from 1990 to 1995. Variables were grouped in three different sets. Crude and adjusted odds ratios, p-values and 95% confidence intervals were estimated. Variables selected in the multivariate analysis in each set were controlled for the effect of those selected in the others. All factors related to the disease progression - incubation period, time elapsed between the occurrence of the first tetanus symptom and admission, and period of onset - showed a statistically significant association with death from tetanus. Similarly, signs and/or symptoms occurring on admission or in the following 24 hours (second set): reflex spasms, neck stiffness, respiratory signs/symptoms and respiratory failure requiring artificial ventilation (third set) were associated with death from tetanus even when adjusted for the effect of the others.

Adolescent↗

Autonomic nervous system dysfunction in children with severe tetanus: dissociation of cardiac and vascular sympathetic control.

The medical records of ten pediatric patients with a clinical diagnosis of tetanus were reviewed retrospectively. The heart rate and blood pressure of all tetanus patients were measured noninvasively every hour during the first two weeks of hospitalization. Six of ten tetanus patients presented clinical evidence of sympathetic hyperactivity (group A) and were compared with a control group consisting of four children who required mechanical ventilation for diseases other than tetanus (group B). Heart rate and blood pressure simultaneously and progressively increased to a maximum by day 7. The increase over baseline was 43.70 +/- 11.77 bpm (mean +/- SD) for heart rate (P<0.01) and 38.60 +/- 26.40 mmHg for blood pressure (P<0.01). These values were higher and significantly different from those of the control group (group B) at day 6, which had an average heart rate increase over baseline of 19.35 +/- 12.26 bpm (P<0.05) and blood pressure of 10.24 +/- 13.30 mmHg (P<0.05). By the end of the second week of hospitalization, in group A the increase of systolic blood pressure over baseline had diminished to 9.60 +/- 15.37 mmHg (P<0.05), but the heart rate continued to be elevated (27.80 +/- 33.92 bpm, P = NS), when compared to day 7 maximal values. The dissociation of these two cardiovascular variables at the end of the second week of hospitalization suggests the presence of asymmetric cardiac and vascular sympathetic control. One possible explanation for these observations is a selective and delayed action of tetanus toxin on the inhibitory neurons which control sympathetic outflow to the heart.

Adolescent↗

Factors affecting prognosis of neonatal tetanus.

Neonatal tetanus is associated with a high mortality rate, despite intensive care. Although it can be prevented by immunization of expectant mothers and by good hygiene and asepsis during delivery, neonatal tetanus is still a common cause of neonatal mortality in underdeveloped countries. In our study, hospital records of 133 newborns with neonatal tetanus were evaluated to identify the possible factors affecting prognosis of neonatal tetanus; 79 were males and 56 females, and 54 (41.6%) of the 133 died. Sex and birth weight did not affect the prognosis. As a result of our study, we found that age on admission < 10 days, symptoms < 5 days and presence of risus sardonicus and fever indicate a poor prognosis in neonatal tetanus.

Age Factors↗

Inhibitory activity of fenoterol on Dermatophagoides-, Parietaria-, tetanus-toxoid-, and Candida albicans-stimulated blood mononuclear cells: differences in beta2-adrenoreceptor stimulation but not in cell apoptosis.

beta2-adrenoreceptor agonists have the ability to downregulate in vitro the proliferative response of peripheral blood mononuclear cells (BMCs). This activity could be related to a variety of beta2-adrenoreceptor-mediated functions, including induction of cell apoptosis in activated T-cells. To test this hypothesis, BMCs from atopic subjects, sensitized to house dust mites (Dermatophagoides [Der p]) and/or to Parietaria were incubated with fenoterol (10(-8)-10(-5) M) in the presence of (a) purified allergen extracts (Der p [5 microg/mL] or Parietaria [5 microg/mL]) or (b) antigens (tetanus toxoid [1 microg/mL] or Candida albicans [5 x 10(5) bodies/mL]). The BMC proliferation was assessed by [3H] thymidine incorporation and cell apoptosis was assessed by evaluating DNA fragmentation by a fluorescence technique, using propidium iodide. In cultures stimulated with Der p or with Parietaria, fenoterol induced a dose-dependent inhibition of BMC proliferation, significant also at the lowest concentration tested (10(-8) M) (p < 0.05, each comparison). In contrast, the inhibitory activity of the drug on tetanus-toxoid-stimulated BMCs was significant only at the highest dose tested (10(-5)M) (p < 0.05), whereas no effect was seen when BMCs were stimulated with C. albicans extract (p > 0.05). The different inhibitory efficacy of fenoterol appeared to be related to the degree of activation of beta2-adrenoreceptors on the different BMC populations that responded to the different stimuli. Indeed, in the presence of fenoterol (10(-6) and 10(-5)M), a significant increase in cyclic adenosine monophosphate (cAMP) levels was seen in Der p- or Parietaria-stimulated cells (p < 0.05; each comparison), but not in cell cultures stimulated with tetanus toxoid or with C. albicans extracts (p > 0.05; each comparison). Finally, the percentage of cells with fragmented DNA was lower in cultures stimulated with Der p or Parietaria than in those stimulated with tetanus toxoid or C. albicans, and the presence of fenoterol did not modify cell apoptosis (p > 0.05; each comparison). Thus, the different inhibitory activity of fenoterol on BMCs activated by allergens (Der p or Parietaria) or by antigens (tetanus toxoid or C. albicans) seems to be related to differences in beta2-adrenoreceptor expression and/or function in the different antigen-specific T-cell subsets, but it is not influenced by changes in cell apoptosis.

Adrenergic beta-Agonists↗

Prospective study of tetanus-induced acute renal dysfunction: role of adrenergic overactivity.

To assess the mechanisms related to tetanus-induced acute renal failure (ARF), 30 patients with tetanus had their renal function prospectively studied and factors possibly related to renal changes were evaluated during four weeks of hospitalization. Fifty percent of these patients had a glomerular filtration rate (GFR) < or = 50 ml/min in the first or second week of hospitalization (Group I) and 50% had a GFR > 50 ml/min throughout the entire hospitalization period (Group II). Age, gender, tetanus incubation time and tetanus onset time, hospitalization time, use of nephrotoxic drugs, need for mechanical ventilation with intermittent positive pressure, and presence of systemic infection were similar in both groups. None of the patients presented with oliguria. Autonomic nervous system (ANS) overactivity, characterized by intense variations in systolic and diastolic blood pressure, by increased heart rate and elevated urinary metanephrine excretion, was higher in Group I compared with Group II. Plasma renin activity, serum creatinephosphokinase levels, and myoglobinuria were not significantly different between the two groups. These results strongly suggest that tetanus-induced ARF has a high prevalence, is characterized by early onset, and is probably related to ANS overactivity.

Acute Kidney Injury↗

Tetanus caused by occupational accidents.

The role of occupational accidents as a source of tetanus is poorly known. In Finland, during the period 1969-1985, 28 (26%) of the 106 cases of tetanus were caused by occupational accidents, 16 of which occurred in agriculture and forestry. Twenty-one of the patients were men and seven were women. The mean annual incidence of tetanus was 1 per 100,000 occupational accidents during the study period. The cases were concentrated in summer and autumn. Most of the primary injuries were minor, 61% of the injuries occurring in the hands and fingers. Forty-three percent of the patients had not been immunized against tetanus, and 46% were unaware of their state of immunization. The systematic immunization of the population against tetanus is important because the disease is often caused by slight injuries not requiring treatment by health care personnel. This need is emphasized for work in agriculture, forestry, and other branches in which contact with soil or animals occurs.

Accidents, Occupational↗

[Prevalence of antibodies against tetanus, diphtheria and Bordetella pertussis in health care professionals].

AIM: The aim of the present study was to determine the prevalence of antibodies versus tetanus, diphtheria and Bordetella pertussis in a population of health care personnel. PATIENTS AND METHODS: A transversal study was carried out in a population of healthy health care professionals from two hospitalary centers in the Community of Madrid (Spain). The ELISA method was used for antibody determinations. The determination of antibodies versus tetanus and diphtheria was performed in a total of 344 individuals (mean age 32 years) and that of whooping cough in 104 subjects (mean age 23 years). RESULTS: The global prevalence of tetanus, diphtheria and whooping cough was of 76.5, 48 and 57%, respectively. The prevalence of antibodies versus tetanus was significantly greater in males than in females. CONCLUSIONS: Revaccination versus tetanus and diphtheria is recommended every 10 years to guarantee protector antibody concentrations in adults. With regard to whooping cough, revaccination could be a strategy to consider in the future, particularly in risk groups in contact with cases of this disease.

Adult↗

Influenza, pneumococcal, and tetanus toxioid vaccination of adults--United States, 1993-7.

PROBLEM/CONDITION: An increasing proportion of adults have received recommended vaccinations against influenza, pneumococcal infection, and tetanus. However, in 1995, fewer than 60% of adults were vaccinated as recommended. REPORTING PERIOD COVERED: 1993-1997. DESCRIPTION OF SYSTEM: Data were obtained from the state-based Behavioral Risk Factor Surveillance System (BRFSS) for 1993, 1995, and 1997 and from the National Health Interview Survey (NHIS) for 1995 to describe national, regional, and state-specific patterns of use of influenza and pneumococcal vaccines and tetanus toxoid among noninstitutionalized adults aged > or = 18 years. RESULTS: Among adults aged > or = 65 years in 1995, 58% reported receiving an influenza vaccination during the previous 12 months, and 34% reported ever receiving a pneumococcal vaccination. In this age group, non-Hispanic whites were more likely to report receipt of influenza (61%) and pneumococcal vaccines (36%) than non-Hispanic blacks (40% and 22%, respectively) and Hispanics (50% and 23%, respectively). Among the 50 states and the District of Columbia, the median vaccination level among older adults (i.e., persons aged > or = 65 years) increased from 51% in 1993 to 66% in 1997 for influenza vaccine, and from 28% in 1993 to 46% in 1997 for pneumococcal vaccine. Adults with chronic medical conditions had low vaccination levels. Those aged 50-64 years were more likely than those aged 18-49 years to report influenza (38% versus 20%) and pneumococcal vaccination (20% versus 12%). In 1995, the proportion of adults who reported receiving a tetanus vaccination during the previous 10 years decreased with age, from 65% among those aged 18-49 years to 54% among those aged 50-64 years and to 40% among those aged > or = 65 years. In each age group, women were less likely than men to report receiving tetanus toxoid; and among adults aged > or = 65 years, Hispanics and Asians/Pacific Islanders were least likely among all racial/ethnic groups to report receiving tetanus toxoid. INTERPRETATION: By 1995, the Healthy People 2000 objective to increase to at least 60% the proportion of persons aged > or = 65 years who had received annual influenza vaccination had been achieved among non-Hispanic whites (objective 20.11). However, substantial improvement is needed among non-Hispanic blacks, Hispanics, and adults aged < 65 years with high-risk medical conditions. PUBLIC HEALTH ACTIONS: Continued surveillance of vaccine coverage among adults will direct attention to undervaccinated populations that may be disproportionately affected by vaccine-preventable diseases. Vaccination coverage data can be used to guide efforts to increase awareness among health-care providers and the public about the benefits of vaccination, establish systems to ensure that every contact with the health-care system is used to update vaccinations, and further support financial mechanisms to increase vaccine delivery.

Adult↗

[Value of intrathecal baclofen in the treatment of severe tetanus in the tropical milieu].

Tetanus continues to be a cause of high mortality in developing countries, where resources for muscle relaxation and respiratory support are not readily available. Baclofen, a GABAB receptor agonist, directly restores physiologic inhibition of alpha motoneuron, that is blocked by tetanus toxin. Its use has been suggested at high dosage by intrathecal (i.t.) route, as part of the treatment of tetanus-induced contractures, and to limit the need for general anesthesia and for tracheal intubation in afflicted patients. This review reports personal experience and focuses on published data about i.t. baclofen for severe tetanus. Although statistical analysis are difficult to perform, i.t. baclofen appears to be effective in resolving muscle rigidity and in avoiding the need for deep sedation and for tracheal intubation, thus achieving lower mortality. Nevertheless, i.t. baclofen has a narrow therapeutic range, and a large interindividual pharmacodynamic variability. Thus, its use should be reserved for patients who would have been intubated without it. I.t. baclofen is a fairly simple and cost-effective modality, with significant advantages for management of severe tetanus, especially in tropical environment.

Adult↗

Collaborative study for the establishment of tetanus vaccine (adsorbed) Third International Standard and European Pharmacopoeia Biological Reference Preparation batch no. 2.

The aim of the present collaborative study was to calibrate two candidate replacement standards for tetanus vaccine for human use in the terms of the Second International Standard (IS) for Tetanus Toxoid, Adsorbed (TEXA-2) using challenge potency assays in guinea pigs and mice and to establish the 3rd IS and the Ph. Eur. BRP batch 2. Two test preparations (coded B and C) were included as candidate replacement for TEXA-2 and Ph. Eur. BRP batch 1 (EBRP-1). This project was run as a joint WHO/Ph. Eur. study. Twenty-seven laboratories representing nineteen countries participated in the study. Twenty-three laboratories performed assays in mice (11 laboratories performed lethal, 11 paralysis, and one serology assays) and fourteen laboratories performed assays in guinea pigs (10 laboratories performed lethal and 4 paralysis assays). Estimates of potency, expressed in ampoule of TEXA-2 per ampoule of candidate replacement standards were two-fold higher in mice than in guinea pigs. However, using the assumed relationship between TEXA-1 and TEXA-2 in mice and guinea pigs (Lyng and Nyerges, 1984), potency estimates for candidate replacement standards (sample coded B and C), in terms of TEXA-1, gives estimates which are similar in both animal models. Stability was assessed within the collaborative study for candidate standard B. On the basis of this collaborative study, the ECBS of WHO has established the candidate material coded B as the Third IS for Tetanus Toxoid, Adsorbed, with an assigned unitage of 469 IU/ampoule, based on its calibration by guinea pig challenge assays (WHO report BS/00.1932). Further studies were organised to assess which of the two candidate standards is most suitable as replacement for EBRP-1. Manufacturers were asked to include candidate B and candidate C in their routine quality control potency assays of tetanus vaccines in order to assess the impact of the use of either preparation on their calibration results. The features and results of this additional phase are described at the end of this report in a separate section entitled "Choice of replacement batch for EBRP-1". Based on these results, at the session in March 2001, the Ph. Eur. Commission adopted candidate B as the Tetanus vaccine (adsorbed) Ph. Eur. BRP Batch 2 with assigned potencies of 469 IU/ampoule for the guinea pig assays and 496 IU/ampoule for the mouse assays.

Animals↗

[Tetanus: epidemiological aspects at the Infectious Disease Clinics at the Fann University Hospital Center in Dakar].

This retrospective study was carried out to assess the recent epidemiological aspects of tetanus in Senegal. Based on the records of tetanus cases admitted at the Infectious Diseases Clinic in Dakar over a 7-year periode (1990-1996), this study showed that tetanus is still a major public health concern in Senegal. A global prevalence rate of 18.8% was found. Portals of entry were noticed to be: dirty wounds (48%), the umbilical stumps (19%), non sterile intra-muscular injections (4.5%), surgery (3.3%) and traditional practices (4.2%). Nevertheless, in a high proportion of cases (13.3%), the portal of entry was not found. A 30.3% case fatality rate was observed, that varied significantly according to portals of entry, reaching 60.4% in umbilical tetanus. It is recommended here that the immunization programme against tetanus be reinforced and supported by I E C Strategies together with retraining of medical personnel.

Academic Medical Centers↗

[Effect of regional (wound) revaccination with tetanus anatoxin on the functional activity of immunocompetent cells in the region].

The study made with the use of complex methods established that the local (wound) application of tetanus toxoid rapidly made the manifestation of the lysosomal apparatus more pronounced, increased the oxidizing activity (determined in the nitro blue tetrazolium test) and phagocytic activity of the mononuclear phagocytizing system in the wound and in the regional lymph nodes. The wound application of tetanus toxoid significantly increased blast transformation of T lymphocytes in guinea pigs simultaneously with tetanus wound infection. The study confirmed the pathogenetic expediency of the proposed method for the stimulation of anti-tetanus immunity by the application of tetanus toxoid on the wound which specifically inhibited the primary stage of the infectious process.

Administration, Topical↗

Case report: Diazepam in severe tetanus treatment.

The causes of death in tetanus are muscle spasms and spasm of the larynx, which are caused by blocking the release of inhibitory neurotransmitters in the spinal synapses, causing the uncontrolled spread of impulses. Diazepam controls the spasms by blocking the polysynaptic reflexes, working peripherally, without depressing the cortical center and has no cardiovascular or endocrine effects. High dose diazepam had been used and proved to be a good muscle relaxant. Diazepam seems to work better with tetanus than pancuronium bromide, but both drugs need mechanical ventilation. In cases where the dose exceeds 240 mg per day in a child, a ventilator should be on hand, and if the dose required is more than 480 mg per day, other drugs should be considered. In three cases of severe tetanus presented here, the first two were managed by diazepam and pancuronium bromide and the last case by high dose diazepam only. In the first case, the dose of diazepam was up to 480 mg/day. By using high dose diazepam in severe tetanus, management of the clinical manifestations of autonomic nerve involvement and the weaning process become easier. Most complications of severe tetanus became more manageable.

Anticonvulsants↗

Persistence of antibodies against tetanus upon revaccination.

To assess the interval since the last revaccination against tetanus the persistence of antibodies was determined using ELISA test in 128 subject. Levels of antibodies against tetanus higher than the protective level of 0.1 IU/ml were found not only in those revaccinated less than ten years ago, but also in those revaccinated more than ten years before. With increasing length of the interval since last revaccination the antibody levels against tetanus decreased gradually although they remained above the protective level. No basic dependence of that trend on gender or age has been found. In the whole series, antibodies against tetanus exceeded the protective level for up to 20-25 years post last revaccination. This finding together with a favorable epidemiological situation as regards the occurrence of tetanus could eventually enable to prolong the interval for revaccinating adults in the Czech Republic.

Adolescent↗