Poor continuity of care for TB diagnosis and treatment in Zambian Prisons: a situation analysis.

OBJECTIVES: Prisons act as infectious disease reservoirs. We aimed to explore the challenges of TB control and continuity of care in prisons in Zambia. METHODS: We evaluated treatment outcomes for a cohort of inmates diagnosed with TB during a TB REACH funded screening programme initiated by the Zambia Prisons Service and the Centre for Infectious Disease Research in Zambia. RESULTS: Between October 2010 and September 2011, 6282 inmates from six prisons were screened for TB, of whom 374 (6.0%) were diagnosed. TB treatment was initiated in 345 of 374 (92%) inmates. Of those, 66% were cured or completed treatment, 5% died and 29% were lost to follow-up. Among those lost to follow-up, 11% were released into the community and 13% were transferred to other prisons. CONCLUSIONS: Weak health systems within the Zambian prison service currently undermines continuity of care, despite intensive TB screening and case-finding interventions. To prevent TB transmission and the development of drug resistance, we need sufficient numbers of competent staff for health care, reliable health information systems including electronic record keeping for prison facilities, and standard operating procedures to guide surveillance, case-finding and timely treatment initiation and completion.

Mating system and early viability resistance to habitat fragmentation in a bird-pollinated eucalypt.

Habitat fragmentation has been shown to disrupt ecosystem processes such as plant-pollinator mutualisms. Consequently, mating patterns in remnant tree populations are expected to shift towards increased inbreeding and reduced pollen diversity, with fitness consequences for future generations. However, mating patterns and phenotypic assessments of open-pollinated progeny have rarely been combined in a single study. Here, we collected seeds from 37 Eucalyptus incrassata trees from contrasting stand densities following recent clearance in a single South Australian population (intact woodland=12.6 trees ha(-1); isolated pasture=1.7 trees ha(-1); population area=10 km(2)). 649 progeny from these trees were genotyped at eight microsatellite loci. We estimated genetic diversity, spatial genetic structure, indirect contemporary pollen flow and mating patterns for adults older than the clearance events and open-pollinated progeny sired post-clearance. A proxy of early stage progeny viability was assessed in a common garden experiment. Density had no impact on mating patterns, adult and progeny genetic diversity or progeny growth, but was associated with increased mean pollen dispersal. Weak spatial genetic structure among adults suggests high historical gene flow. We observed preliminary evidence for inbreeding depression related to stress caused by fungal infection, but which was not associated with density. Higher observed heterozygosities in adults compared with progeny may relate to weak selection on progeny and lifetime-accumulated mortality of inbred adults. E. incrassata appears to be resistant to the negative mating pattern and fitness changes expected within fragmented landscapes. This pattern is likely explained by strong outcrossing and regular long-distance pollen flow.

A variant in long palate, lung and nasal epithelium clone 1 is associated with cholera in a Bangladeshi population.

Vibrio cholerae causes a dehydrating diarrheal illness that can be rapidly fatal in the absence of specific treatment. The organism is an historic scourge and, like similar infectious diseases, may have influenced the evolution of the human genome. We report here the results of the first candidate gene association study of cholera. In a family-based study of 76 pedigrees from Dhaka, Bangladesh, we evaluated the association between cholera and five candidate genes-the cystic fibrosis transmembrane receptor; lactoferrin; long palate, lung and nasal epithelium clone 1 (LPLUNC1); estrogen-related receptor alpha and calcium-activated chloride channel 1. We found a significant association with a marker in the promoter region of LPLUNC1 (rs11906665), a member of a family of evolutionarily conserved innate immunity proteins. An earlier microarray-based study of duodenal biopsies showed significantly increased expression of LPLUNC1 in cholera patients compared with healthy control subjects. Our results suggest that variation in host innate immune responses may influence the outcome of exposure to V. cholerae in an endemic setting.

Early lessons from the integration of tuberculosis and HIV services in primary care centers in Lusaka, Zambia.

BACKGROUND: Zambia faces overlapping tuberculosis (TB) and human immunodeficiency virus (HIV) epidemics; however, care for co-infected patients often occurs through separate, vertical programs. OBJECTIVE: To establish a program to integrate TB and HIV services in Lusaka primary care centers. METHODS: In collaboration with the Zambian Ministry of Health, TB-HIV integration activities began in December 2005 and were expanded to seven health centers by March 2007. Principal activities included developing staff capacity to manage co-infected patients, implementing HIV testing within TB departments and establishing referral systems between departments. RESULTS: Using a provider-initiated approach, 2053 TB patients were offered HIV testing. Seventy-seven per cent agreed to be tested; 69% of those tested were HIV-infected. Of these, 59% were enrolled in HIV care. The proportion of antiretroviral treatment (ART) program enrollees who were TB-HIV co-infected increased by 38% after program implementation. The median CD4 count among co-infected patients was 161 cells/microl, with 88% eligible for ART. CONCLUSION: Integration of HIV testing and referral services into urban primary care centers identified many co-infected patients and significantly increased the proportion of TB patients among people accessing HIV care. Ongoing challenges include maximizing the number of patients accepting HIV testing and overcoming barriers to enrollment into HIV care.

Bites by the Monocled Cobra, Naja kaouthia, in Chittagong Division, Bangladesh: Epidemiology, Clinical Features of Envenoming and Management of 70 Identified Cases.

AbstractWe describe 70 cases of monocled cobra (Naja kaouthia) bite admitted to Chittagong Medical College Hospital, Bangladesh. The biting snakes were identified by examining the dead snake and/or detecting N. kaouthia venom antigens in patients' serum. Bites were most common in the early morning and evening during the monsoon (May-July). Ligatures were routinely applied to the bitten limb before admission. Thirty-seven patients consulted traditional healers, most of whom made incisions around the bite site. Fifty-eight patients experienced severe neurotoxicity and most suffered swelling and pain of the bitten limb. The use of an Indian polyvalent antivenom in patients exhibiting severe neurotoxicity resulted in clinical improvement but most patients experienced moderate-to-severe adverse reactions. Antivenom did not influence local blistering and necrosis appearing in 19 patients; 12 required debridement. Edrophonium significantly improved the ability of patients to open the eyes, endurance of upward gaze, and peak expiratory flow rate suggesting that a longer-acting anticholinesterase drug (neostigmine) could be recommended for first aid. The study suggested that regionally appropriate antivenom should be raised against the venoms of the major envenoming species of Bangladesh and highlighted the need to improve the training of staff of local medical centers and to invest in the basic health infrastructure in rural communities.

Envenoming bites by kraits: the biological basis of treatment-resistant neuromuscular paralysis.

Beta-bungarotoxin, a neurotoxic phospholipase A2 is a major fraction of the venom of kraits. The toxin was inoculated into one hind limb of young adult rats. The inoculated hind limb was paralysed within 3 h, and remained paralysed for 2 days. The paralysis was associated with the loss of synaptic vesicles from motor nerve terminal boutons, a decline in immunoreactivity of synaptophysin, SNAP-25 and syntaxin, a loss of muscle mass and the upregulation of NaV(1.5) mRNA and protein. Between 3 and 6 h after the inoculation of toxin, some nerve terminal boutons exhibited clear signs of degeneration. Others appeared to be in the process of withdrawing from the synaptic cleft and some boutons were fully enwrapped in terminal Schwann cell processes. By 12 h all muscle fibres were denervated. Re-innervation began at 3 days with the appearance of regenerating nerve terminals, a return of neuromuscular function in some muscles and a progressive increase in the immunoreactivity of synaptophysin, SNAP-25 and syntaxin. Full recovery occurred at 7 days. The data were compared with recently published clinical data on envenoming bites by kraits and by extrapolation we suggest that the acute, reversible denervation caused by beta-bungarotoxin is a credible explanation for the clinically important, profound treatment-resistant neuromuscular paralysis seen in human subjects bitten by these animals.

High prevalence of tuberculosis in newly enrolled HIV patients in Zambia: need for enhanced screening approach.

SETTING: Tuberculosis (TB) remains a leading cause of morbidity and mortality in sub-Saharan Africa. In Zambia, smear microscopy and chest radiography (CXR) are the primary TB diagnostic tools, and most cases are not bacteriologically confirmed. OBJECTIVE: We implemented enhanced screening to determine the TB burden among new human immunodeficiency virus (HIV) clinic enrollees. DESIGN: Consecutive adult HIV clinic enrollees were screened, regardless of symptoms. All underwent microscopy (Ziehl-Neelsen/fluorescence microscopy) on three sputum specimens, physical examination, and digital CXR. Sputum, blood and urine specimens were cultured. Xpert(®) MTB/RIF testing was performed retrospectively. RESULTS: From July 2011 to April 2012, 399 patients were enrolled. The median age was 34.4 years; body mass index was 20.8 kg/m(2), CD4 count was 202 cells/µl and 86% were symptomatic. Culture-confirmed TB was diagnosed in 72/399 (18%) patients; an additional 31/399 (8%) were culture-negative but diagnosed clinically. Symptom screening for any cough, fever, weight loss or night sweats had high sensitivity (95%) but low specificity (14%) for detecting culture-confirmed cases. Among culture-confirmed cases, 35/72 (49%) were missed clinically and detected only by culture. Xpert was 64% sensitive and 98% specific. CONCLUSIONS: High TB prevalence was found in Zambians newly enrolled into HIV care. Screening with sensitive diagnostics should be considered with culture when feasible in this population.

Equine grass sickness, but not botulism, causes autonomic and enteric neurodegeneration and increases soluble N-ethylmaleimide-sensitive factor attachment receptor protein expression within neuronal perikarya.

REASONS FOR PERFORMING STUDY: Equine grass sickness (EGS) is of unknown aetiology. Despite some evidence suggesting that it represents a toxico-infection with Clostridium botulinum types C and/or D, the effect of EGS on the functional targets of botulinum neurotoxins, namely the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, is unknown. Further, while it is commonly stated that, unlike EGS, equine botulism is not associated with autonomic and enteric neurodegeneration, this has not been definitively assessed. OBJECTIVES: To determine: 1) whether botulism causes autonomic and enteric neurodegeneration; and 2) the effect of EGS on the expression of SNARE proteins within cranial cervical ganglion (CCG) and enteric neuronal perikarya. STUDY DESIGN: Descriptive study. METHODS: Light microscopy was used to compare the morphology of neurons in haematoxylin-eosin stained sections of CCG and ileum from 6 EGS horses, 5 botulism horses and 6 control horses. Immunohistochemistry was used to compare the expression of synaptosomal-associated protein-25, synaptobrevin (Syb) and syntaxin within CCG neurons, and of Syb in enteric neurons, from horses with EGS, horses with botulism and control horses. The concentrations of these SNARE proteins in extracts of CCG from EGS and control horses were compared using quantitative fluorescent western blotting. RESULTS: EGS, but not botulism, was associated with autonomic and enteric neurodegeneration and with increased immunoreactivity for SNARE proteins within neuronal perikarya. Quantitative fluorescent western blotting confirmed increased concentrations of synaptosomal-associated protein-25, Syb and syntaxin within CCG extracts from EGS vs. control horses, with the increases in the latter 2 proteins being statistically significant. CONCLUSIONS: The occurrence of autonomic and enteric neurodegeneration, and increased expression of SNARE proteins within neuronal perikarya, in EGS but not botulism, suggests that EGS may not be caused by botulinum neurotoxins. Further investigation of the aetiology of EGS is therefore warranted.

Myotoxic activity of the toxic phospholipase, notexin, from the venom of the Australian tiger snake.

Notexin is a neurotoxic and myotoxic phospholipase A2 derived from the venom of the Australian tiger snake, Notechis scutatus. Though the phospholipase activity has been implicated in the toxicity of notexin, little is understood of its site and mode of action. In this study we investigated the myotoxicity of notexin on the skeletal muscle of the rat. Using immunogold labeling both in vivo and in vitro, we demonstrated that notexin bound exclusively to the sarcolemma. At the early stages when notexin was injected into the muscle there was no evidence of internalization, though at more advanced degeneration when muscle fibers were necrotic, the toxin was able to penetrate the interior of the fibers to exhibit nonspecific labeling. We also showed the toxin was able to bind to glycolytic muscle fibers, which are known to be resistant to the myotoxic effects of notexin. Electron microscopy confirmed the localization of the binding site to the sarcolemma. Scanning electron microscopy showed that the primary pathological changes associated with exposure to notexin were the appearance of areas of hypercontraction along the muscle fibers associated with small lesions in the sarcolemma. At more advanced stages large tears appeared in the sarcolemma between adjacent areas of hypercontraction, revealing apparently intact myofibrils below. We conclude that the sarcolemma is the binding site for the toxin. We propose that the hydrolytic activity causes the appearance of small lesions in the membrane, the loss of ion gradients, and hypercontraction. The weakened area between sites of hypercontraction is then ruptured, leading ultimately to the degeneration of the muscle fibers.

The expression of dystrophin-associated glycoproteins during skeletal muscle degeneration and regeneration. An immunofluorescence study.

The distribution and expression of dystrophin and three of the dystrophin-associated glycoproteins (DAG), alpha-dystroglycan (156 kDa DAG), beta-dystroglycan (43 kDa DAG) and adhalin (50 kDa DAG) in rat skeletal muscle were studied during a controlled cycle of degeneration and regeneration induced by the injection of a snake venom. Cryosections of muscle at various stages of degeneration and regeneration were labeled using monoclonal antibodies to the three glycoproteins and examined at fixed time points after venom injection. Adhalin and alpha-dystroglycan remained present at the sarcolemma throughout the entire cycle of degeneration and regeneration. beta-Dystroglycan, on the other hand, was lost from the sarcolemma by 12 hours and reappeared 2 days after venom injection when new muscle fibers were being formed. Dystrophin was not lost from the sarcolemma until 24 hours after venom injection and did not reappear at the membrane until 4 days. It is suggested that dystrophin and the glycoprotein complex are synthesized separately, both temporally and spatially, and only become associated at the plasma membrane during the later stages of regeneration. The expression of beta-dystroglycan in the regenerating muscle fibers was first seen at sites of newly forming plasma membrane that were closely associated with the old basal lamina tube. The basal lamina may therefore have a regulatory or modulatory role in the expression of the DAG.

Improved diagnosis of Becker muscular dystrophy by dystrophin testing.

We assessed the quantity (relative cellular abundance) and quality (approximate molecular weight) of dystrophin in muscle biopsies from 97 patients with a diagnosis of possible Becker muscular dystrophy. Fifty-four (all male) had dystrophin abnormalities and were deemed to have true Becker muscular dystrophy. The other 43 patients (14 female, 29 male) had no detectable dystrophin abnormalities. Of the dystrophin-verified Becker dystrophy patients, 35% (19/54) had a family history consistent with X-linked recessive inheritance. On the other hand, none of the 43 patients with apparently normal dystrophin had a clear X-linked family history, suggesting that few of these 43 actually had a form of Becker dystrophy. The data suggest that of all patients with a clinical picture consistent with Becker dystrophy but no family history, about 60% will be true Becker patients. The correlation of both the biochemical and clinical data suggests that Duchenne/Becker dystrophy can be divided into 4 clinically useful categories: Duchenne dystrophy (wheelchair at about age 11 years; dystrophin quantity less than 3% of normal); severe Becker dystrophy (wheelchair age 13 to 20 years; dystrophin 3% to 10%); and moderate/mild Becker dystrophy (wheelchair greater than 20 years; dystrophin quantity greater than or equal to 20%). Given the observed clinical variability of Becker dystrophy, it appears that dystrophin analysis is required for accurately distinguishing between Becker dystrophy and clinically similar autosomal recessive myopathies.

Effect of growth promoters on pig muscle structural protein and proteolytic enzyme levels in vivo and in vitro.

To elucidate the biochemical mechanism by which the growth hormone porcine somatotrophin (PST) promotes skeletal muscle growth, we have determined the activity of a comprehensive range of protein catabolizing proteolytic enzymes and structural proteins (determined by analytical electrophoresis, SDS-PAGE) in longissimus dorsi muscle from control and PST treated pigs. There was no significant difference in the levels of muscle structural proteins, or in the activity of muscle proteolytic enzymes at point of slaughter in control or PST treated animals. Similarly, in post-mortem muscle proteolysis time course experiments at pH 5.5 or pH 7.5, there was no significant difference in the rate of structural protein degradation by endogenous muscle proteases (determined via SDS-PAGE) using muscle from control or PST treated animals. In addition, investigation of a range of beta-agonist related drugs (clenbuterol, salbutamol, pirbuterol, fenoterol) showed no effect (10(-4)-10(-8) M) in vitro on the activity of individual protease types in control muscle, or on the degradation rate of muscle structural proteins by endogenous proteases in time course experiments at pH 5.5 or pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin isoform transitions and physiological properties of regenerated and re-innervated soleus muscles of the rat.

Soleus muscles in young female rats were destroyed by the local injection of the crude venom of the Australian tiger snake, Notechis scutatus and allowed to regenerate. The regenerated muscles consisted almost exclusively of type I muscle fibres, and histograms of fibre cross-sectional area were unimodal. In contrast the normal contralateral muscles consisted of a mixture of type I and IIa fibres, and histograms of fibre cross-sectional area were bimodal. There was no change in the ability of the regenerated muscles to generate tension in response to indirect stimulation, and muscle fibre number and motor unit index were similar to controls. The regenerated muscles often contained split muscle fibres, but if re-innervation of the regenerated muscles was delayed, splitting did not occur. The principal features of the regenerated muscles (fibre type homogeneity, unimodal histograms of muscle fibre area, normal numbers of muscle fibres and normal motor unit index) were reproduced in soleus muscles that were simply denervated by nerve crush and allowed to re-innervate. We conclude that the phenotypic homogeneity of the regenerated muscles is a consequence of the temporary disconnection of muscle from nerve, rather than of the cycle of degeneration/regeneration, and suggest that this disconnection results in the reprogramming of the soleus motor neurones.

Immunogold labelling of dystrophin in human muscle, using an antibody to the last 17 amino acids of the C-terminus.

Immunolabelling with a 10 nm gold probe was used to localize dystrophin at the ultrastructural level in human skeletal muscle. The primary antibody was raised against a synthetic peptide containing the last 17 amino acids at the C-terminus of dystrophin. Using this antibody, labelling was almost entirely confined to a narrow band enclosing 40 nm either side of the plasma membrane and including the membrane itself. Histograms of the position of the gold probe relative to the plasma membrane showed modes lying over the membrane itself or the extracellular face of the membrane. One interpretation of these results is that the C-terminus of dystrophin is inserted in the plasma membrane alongside the glycoproteins with which it is tightly associated. Histograms of the distances between gold probes displayed modes at approximately 120 nm in both transverse and longitudinal sections suggesting that dystrophin forms a lattice-like network adjacent to the plasma membrane.

Beta-bungarotoxin-induced depletion of synaptic vesicles at the mammalian neuromuscular junction.

The neurotoxic phospholipase A(2), beta-bungarotoxin, caused the failure of the mechanical response of the indirectly stimulated rat diaphragm. Exposure to beta-bungarotoxin had no effect on the response of the muscle to direct stimulation. Resting membrane potentials of muscle fibres exposed to the toxin were similar to control values, and the binding of FITC-labelled alpha-bungarotoxin to nAChR at the neuromuscular junction was unchanged. Motor nerve terminal boutons at a third of cell junctions were destroyed by exposure to beta-bungarotoxin leaving only a synaptic gutter filled with Schwann cell processes and debris. At other junctions, some or all boutons survived exposure to the toxin. Synaptic vesicle density in surviving terminal boutons was reduced by 80% and synaptophysin immunoreactivity by >60% in preparations exposed to beta-bungarotoxin, but syntaxin and SNAP-25 immunoreactivity was largely unchanged. Terminal bouton area was also unchanged. The depletion of synaptic vesicles was completely prevented by prior exposure to botulinum toxin C and significantly reduced by prior exposure to conotoxin omega-MVIIC. The data suggest that synaptic vesicle depletion is caused primarily by a toxin-induced entry of Ca(2+) into motor nerve terminals via voltage gated Ca(2+) channels and an enhanced exocytosis via the formation of t- and v-SNARE complexes.

The effect of some foreign anions on suxamethonium blockade of the isolated rat diaphragm preparation.

1. The pharmacology of suxamethonium blockade of the rat phrenic nerve-diaphragm preparation has been studied in the presence of some foreign anions.2. Blockade proceeded in two distinct phases in both normal and modified Krebs solution.3. In normal Krebs solution the characteristics bore no close resemblance to either depolarizing or competitive type blockade.4. In the presence of foreign anions the characteristics of the blockade more closely resembled those of depolarization.5. There was an increase in the sensitivity of the motor end-plate region of the muscle to the depolarizing action of acetylcholine, carbachol and suxamethonium in the presence of the anions.6. Although the anions enhanced the depolarizing activity of suxamethonium, the blocking potency of the drug was unaltered.7. It is suggested that end-plate depolarization plays little part in suxamethonium blockade of the isolated rat diaphragm and that desensitization is the primary cause of the blockade.

Reversal of paralysis in nerve-muscle preparations isolated from animals with hereditary motor endplate disease.

Motor endplate disease (med) in the mouse is an hereditary disorder of the skeletal neuromuscular system. Affected animals suffer a 'functional denervation' of skeletal muscle (Duchen & Stefani, 1971). Muscle fibres do not respond to indirect excitation, but motor nerve terminals release transmitter spontaneously. Spontaneous transmitter release can be enhanced by raising [K+]o or by exposing muscles to red-back spider venom and functional transmission following indirect stimulation may be restored by 4-aminopyridine.

Pharmacological aspects of neuromuscular transmission in the isolated diaphragm of the dystrophic (Rej 129) mouse.

1. Some aspects of the pharmacology of neuromuscular transmission have been studied in the isolated diaphragm of the normal and dystrophic mouse. 2. The effects of (+)-tubocurarine and atropine on the indirectly elicited twitch responses of the dystrophic diaphragm were indistinguishable from normal. 3. Intracellular recording techniques revealed no significant differences between the rise time, time to half decay, frequency and amplitude of miniature endplate potentials (m.e.p.ps) recorded in dystrophic muscle fibres, compared to those recorded in normal muscle fibres. 4. Transmitter null potential, the size of the available store of transmitter, the probability of release of the transmitter, and the characteristics of endplate potentials (e.p.ps) of dystrophic muscle fibres did not differ from normal. 5. The quantum contents of e.p.ps generated in response to nerve stimulation of 0.1 to 100 HZ were consistently larger in dystrophic muscle fibres than in normal muscle fibres, but the differences were not statistically significant under the conditions of the experiment.

The effects of a toxin isolated from Australian tiger snake (Notechis scutatus scutatus) venom on autonomic neuromuscular transmission.

1 The effects on mammalian autonomic neuromuscular transmission of a purified toxin from the crude venom of the Australian tiger snake, Notechis scutatus, have been investigated. 2 The toxin had no measurable effect on transmission in either the rat anococcygeus, the rat vas deferens, or the longitudinal muscle of the guinea-pig ileum. 3 The toxin induced a contraction of longitudinal smooth muscle of the ileum. The tissue relaxed in spite of the continued presence of the toxin, and remained insensitive to further doses. The contractile response was not mediated by either cholinergic or histaminergic mechanisms. 4 The toxin caused an inhibition of the response to field stimulation of the guinea-pig vas deferens and the guinea-pig seminal vesicle. The inhibition was spontaneously reversible, and the preparations remained insensitive to further doses fo the toxin. The effects of the toxin were not mediated by either prostaglandins or by noradrenaline. 5 The inhibitory effects of a variety of compounds known to act at presynaptic sites were also blocked by exposure to the toxin; inhibition caused by postsynaptic activity was unaffected by the toxin. 6 It is suggested that the toxin inhibits transmission in the guinea-pig vas deferens and seminal vesicle by a presynaptic mechanism. 7 The possibility that the mechanism involves phospholipase A2 activity is discussed.

The effects of the subcutaneous injection of the crude venom of the Australian common brown snake, Pseudonaja textilis on the skeletal neuromuscular system.

1 The effects of the crude venom of the Australian common brown snake on the mammalian neuromuscular system have been investigated. 2 The venom was injected subcutaneously into the dorso-lateral aspect of one hind limb of the rat. The limb was paralyzed within 90 min and remained paralysed for 2 to 3 days. 3 The exposed muscles failed to respond to indirect excitation, and individual fibres were not depolarized at the nerve-muscle junction by exposure to carbachol. 4 The wet weight, histological appearance, resting potential and input resistance of the muscle fibres and their ability to generate directly elicited action potentials were unaffected by exposure to the venom. 5 Administration of venom to isolated preparations caused a reduction in the amplitude of miniature endplate potentials, with no change in frequency. The quantal content of evoked endplate potentials was unchanged. 6 It was concluded that the crude venom was largely devoid of presynaptic activity and myotoxicity, and that its primary site of neurotoxicity was directed to the postsynaptic membrane.