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Myotoxicity is an important toxidrome that can occur with envenoming from multiple Australian snake types. Early antivenom administration is an important strategy to reduce the incidence and severity of myotoxicity. The current gold standard biomarker, serum creatine kinase activity, does not rise early enough to facilitate early antivenom administration. Several other skeletal muscle biomarkers have shown promise in other animal models and scenarios. The aim of this study was to examine the predictive values of six skeletal muscle biomarkers in a rat model of Australian snake myotoxicity. Sprague-Dawley rats were anaesthetised and administered either Pseudechis porphyriacus (red-bellied black snake) or Notechis scutatus (tiger snake) venom, or normal saline via intramuscular injection. Blood samples were collected. Assays were performed for serum creatine kinase skeletal muscle troponin-I concentration, skeletal muscle troponin-C concentration, myoglobin activity, skeletal muscle myosin light chain-1 concentration, and creatine kinase-MM activity. Serum markers were plotted against time, with comparison of area under the concentration (or activity)-time curve. The predictive values of six skeletal muscle biomarkers were examined using receiver operating characteristic curves. There was no difference in area under the serum creatine kinase activity-time curve between venom and control groups. Serum creatine kinase-MM activity rose early in the venom treated rats, which had a significantly greater area under the serum activity-time curve. No difference in area under the serum concentration-time curve was demonstrated for the other biomarkers. Creatine kinase-MM activity had a superior predictive values than creatine kinase activity at 0–4 hours and 0–10 hours after venom administration, as indicated by area under the receiver operating characteristic curves (95 per cent confidence intervals) of 0.91 (0.78–1.00) and 0.88 (0.73–1.00) versus 0.79 (0.63–0.95) and 0.66 (0.51–0.80). The limitations of serum creatine kinase activity in early detection of myotoxicity were demonstrated in this rat model. Serum creatine kinase-MM activity was superior for early detection of Australian myotoxic snake envenoming. [ABSTRACT FROM AUTHOR]

Antivenom is widely accepted as an effective treatment for snake envenomation. This is despite very limited evidence supporting clinical effectiveness for major envenomation syndromes, and is mainly based on pre-clinical studies and observational studies without control groups. Although antivenom exhibits efficacy by binding to snake toxins and preventing toxic injury in animals if pre-mixed with venom, this efficacy does not always translate to clinical effectiveness. There are many irreversible venom mediated effects that antivenom cannot neutralise or reverse, such as pre-synaptic neurotoxicity and myotoxicity. Fortunately, early antivenom appears to prevent some of these. With good evidence that early antivenom prevents some envenomation syndromes, the time between bite and antivenom administration must be reduced. This requires improving the initial assessment of snakebite patients, and improving early decision making based on clinical effects. Until there are improved, simplified, easy to use, rapid and inexpensive tests, whether available in the laboratory or preferably at the bedside that identify systemic envenomation, the key to early antivenom administration is early assessment and decision making based on systemic symptoms, including nausea, vomiting, headache and abdominal pain. [ABSTRACT FROM AUTHOR]

The Australasian region is home to the most diverse elapid snake radiation on the planet (Hydrophiinae). Many of these snakes have evolved into unique ecomorphs compared to elapids on other continents; however, their venom compositions are poorly known. The Australian elapid Hoplocephalus stephensii (Stephen's banded snake) is an arboreal snake with a unique morphology. Human envenoming results in venom-induced consumption coagulopathy, without neurotoxicity. Using transcriptomics and a multi-step fractionation method involving reverse-phase high-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis and bottom-up proteomics, we characterized the venom proteome of H. stephensii. 92% of the total protein component of the venom by weight was characterized, and included all dominant protein families and 4 secondary protein families. Eighteen toxins made up 76% of the venom, four previously characterized and 14 new toxins. The four dominant protein families made up 77% of the venom, including snake venom metalloprotease (SVMP; 36.7%; three identified toxins), phospholipase A2 (PLA2; 24.0%; five identified toxins), three-finger toxin (3FTx; 10.2%; two toxins) and snake venom serine protease (SVSP; 5.9%; one toxin; Hopsarin). Secondary protein families included L-amino acid oxidase (LAAO; 10.8%; one toxin), natriuretic peptide (NP; 0.8%; two toxins), cysteine-rich secretory protein (CRiSP; 1.7%; two toxins), c-type lectin (CTL; 1.1%; one toxin), and one minor protein family, nerve growth factor (NGF; 0.8%; one toxin). The venom composition of H. stephensii differs to other elapids, with a large proportion of SVMP and LAAO, and a relatively small amount of 3FTx. H. stephensii venom appeared to have less toxin diversity than other elapids, with only 18 toxins making up three-quarters of the venom. [ABSTRACT FROM AUTHOR]

In agreement with this, recent studies have demonstrated that the outcome of rodent lethality assays are heavily influenced by the -neurotoxin activity of snake venoms, when -neurotoxins are present in the venoms ([13]; [14]). Keywords: lethality; snake venom; antivenom; LD50; ED50; rodent; mouse EN lethality snake venom antivenom LD50 ED50 rodent mouse 1 4 4 02/08/22 20220203 NES 220203 Introduction Snakebite is a major public health problem in the tropics, being closely associated with agricultural economy, poverty and underdevelopment. Procoagulant Snake Venoms Have Differential Effects in Animal Plasmas: Implications for Antivenom Testing in Animal Models. However, sustaining sufficient -neurotoxin concentrations at the neuromuscular junction to maintain inhibition of the nAChR depends on the relative abundance of the -neurotoxins in the particular snake venom as well as the amount of venom injected during the snakebite. [Extracted from the article]

Understanding snake venom proteomes is becoming increasingly important to understand snake venom biology, evolution and especially clinical effects of venoms and approaches to antivenom development. To explore the current state of snake venom proteomics and transcriptomics we investigated venom proteomic methods, associations between methodological and biological variability and the diversity and abundance of protein families. We reviewed available studies on snake venom proteomes from September 2017 to April 2021. This included 81 studies characterising venom proteomes of 79 snake species, providing data on relative toxin abundance for 70 species and toxin diversity (number of different toxins) for 37 species. Methodologies utilised in these studies were summarised and compared. Several comparative studies showed that preliminary decomplexation of crude venom by chromatography leads to increased protein identification, as does the use of transcriptomics. Combining different methodological strategies in venomic approaches appears to maximize proteome coverage. 48% of studies used the RP-HPLC →1D SDS-PAGE → in-gel trypsin digestion → ESI -LC-MS/MS pathway. Protein quantification by MS1-based spectral intensity was used twice as commonly as MS2-based spectral counting (33–15 studies). Total toxin diversity was 25–225 toxins/species, with a median of 48. The relative mean abundance of the four dominant protein families was for elapids; 3FTx–52%, PLA2–27%, SVMP–2.8%, and SVSP–0.1%, and for vipers: 3FTx–0.5%, PLA2–24%, SVMP–27%, and SVSP–12%. Viper venoms were compositionally more complex than elapid venoms in terms of number of protein families making up most of the venom, in contrast, elapid venoms were made up of fewer, but more toxin diverse, protein families. No relationship was observed between relative toxin diversity and abundance. For equivalent comparisons to be made between studies, there is a need to clarify the differences between methodological approaches and for acceptance of a standardised protein classification, nomenclature and reporting procedure. Correctly measuring and comparing toxin diversity and abundance is essential for understanding biological, clinical and evolutionary implications of snake venom composition. [ABSTRACT FROM AUTHOR]

Understanding the time course of venom exposure in snakebite patients is important for the optimisation of treatment including antivenom dose and timing. We aimed to investigate the pharmacokinetics of red-bellied black snake (RBBS; Pseudechis porphyriacus) venom in envenomed patients. Timed venom concentration data were obtained from patients with RBBS envenomation recruited to the Australian Snakebite Project (ASP), including demographics and antivenom treatment. Venom concentrations were measured using an enzyme immunoassay. Data were modelled using NONMEM version 7.3. Uncertainty in venom "dose" was accounted for by arbitrarily fixing the average amount to 1 mg and incorporating between-subject variability on relative bioavailability. A scale parameter for venom clearance was implemented to account for the rapid venom clearance following antivenom dosing. A sensitivity analysis was performed to determine the magnitude of venom clearance amplification. There were 457 venom concentrations in 114 patients (median age 41, 2–90 y; 80 male). Antivenom was administered to 54 patients a median of 4.2 h post-bite (0.67 to 32 h). A one-compartment model with first-order absorption and elimination provided the best description of the data. The estimated clearance and volume of distribution were 5.21 L/h and 39.9 L, respectively. The calculated elimination half-life of P. porphyriacus venom from the final pharmacokinetic model was 5.35 ± 0.36 h. The variability in the relative dose of injected venom was 140%. Antivenom administration increased venom clearance by 40-fold. Ten patients showed evidence of a double peak in the absorption profile. The information on the exposure time of venom in the body following envenomation will help improve treatment and the timing of antivenom. [ABSTRACT FROM AUTHOR]

Introduction: The major systemic manifestation of hemotoxicity in human snakebite envenoming is venom‐induced consumption coagulopathy (VICC). A subset of patients with VICC develop thrombotic microangiopathy (TMA), in which acute kidney injury (AKI) occurs. We aimed to investigate the association between schistocytosis in snakebite patients with VICC and AKI, compared to non‐envenomed patients. Methods: Serial blood films collected from a prospective cohort of snakebite patients (Australian Snakebite Project) were examined. Cases were classified a priori as non‐envenomed snakebites (normal controls), envenomed without VICC, partial VICC without AKI, complete VICC without AKI, and VICC with AKI based on defined clinical and laboratory criteria. The percentage of schistocytes between groups was compared and correlated by Kendall's tau b test. Results: Seven hundred and eighty blood films from 234 snakebite cases were analyzed. There was a statistically significant correlation (τ =.69, SE.03, P <.001) for schistocytosis between the ordered groups of non‐envenomed snakebites, envenomed without VICC, partial VICC without AKI, complete VICC without AKI, and VICC with AKI groups. Patients with VICC and AKI had a platelet nadir median of 42 × 109/L (interquartile range [IQR] :25‐130 × 109/L), hemoglobin nadir of median 107 g/L (IQR 66‐122 g/L), and maximum LDH median of 1128 U/L (IQR 474‐3255 U/L). A 1.0% threshold for schistocytosis yielded 90% sensitivity (95% CI: 67%‐98%) and 71% specificity (95% CI: 62%‐79%) for predicting AKI in patients with VICC. Conclusion: Schistocyte quantitation has good diagnostic utility in snakebite patients with VICC. A definition of snakebite TMA as MAHA with ≥1.0% schistocytes and thrombocytopenia, would appear to be appropriate. [ABSTRACT FROM AUTHOR]

Background: Myotoxicity is one of the common clinical manifestations of red-bellied black snake (Pseudechis porphyriacus) envenomation characterised by elevated creatine kinase (CK) concentrations of greater than 1000 U/L. This study aimed to investigate the occurrence of myotoxicity in patients following envenomation. Methods/Principal findings: Patient characteristics and serial blood samples (timed venom concentrations and CK concentrations, pre- and post- antivenom) from 114 patients (median age 41, 2-90y; 80 male) were extracted from the Australian Snakebite Project database. Patients were categorised into three groups based on peak CK concentrations [no myotoxicity (<1000 U/L), mild (1000–10,000 U/L) and severe (>10,000 U/L)]. The odds of (mild or severe) myotoxicity was lower in patients that received early antivenom (within 6 hours post-bite) compared to those that received late or no antivenom (odd ratio was 0.186; 95% confidence interval, 0.052–0.664). A population pharmacokinetic-pharmacodynamic (PKPD) model was developed to describe the relationship between the time course of venom (a mixture of toxins) and effect (elevated CK). In addition, a kinetic-pharmacodynamic (KPD) model was developed to describe the relationship between time course of a theoretical toxin and effect. Model development and parameter estimation was performed using NONMEM v7.3. No single set of parameter values from either the PKPD or KPD models were found that could accurately describe the time course of different levels of severity of myotoxicity. The predicted theoretical toxin half-life from the KPD model was 11 ± 3.9 hours compared to the half-life of venom of 5.3 ± 0.36 hours. This indicates that the putative causative toxin's concentration-time profile does not parallel that of venom. Conclusion: Early antivenom administration reduces the incidence of myotoxicity. The venom concentration profile does not appear to be the driver for myotoxicity following envenomation. Additional factors that affect the sensitivity of the patient to snake venom/toxins must be explored to understand the relationship with myotoxicity. [ABSTRACT FROM AUTHOR]

Snakebite is a major public health issue in the rural tropics. Antivenom is the only specific treatment currently available. We review the history, mechanism of action and current developments in snake antivenoms. In the late nineteenth century, snake antivenoms were first developed by raising hyperimmune serum in animals, such as horses, against snake venoms. Hyperimmune serum was then purified to produce whole immunoglobulin G (IgG) antivenoms. IgG was then fractionated to produce F(ab) and F(ab')2 antivenoms to reduce adverse reactions and increase efficacy. Current commercial antivenoms are polyclonal mixtures of antibodies or their fractions raised against all toxin antigens in a venom(s), irrespective of clinical importance. Over the last few decades there have been small incremental improvements in antivenoms, to make them safer and more effective. A number of recent developments in biotechnology and toxinology have contributed to this. Proteomics and transcriptomics have been applied to venom toxin composition (venomics), improving our understanding of medically important toxins. In addition, it has become possible to identify toxins that contain epitopes recognized by antivenom molecules (antivenomics). Integration of the toxinological profile of a venom and its composition to identify medically relevant toxins improved this. Furthermore, camelid, humanized and fully human monoclonal antibodies and their fractions, as well as enzyme inhibitors have been experimentally developed against venom toxins. Translation of such technology into commercial antivenoms requires overcoming the high costs, limited knowledge of venom and antivenom pharmacology, and lack of reliable animal models. Addressing such should be the focus of antivenom research. [ABSTRACT FROM AUTHOR]

We investigated the cardiovascular effects of venoms from seven medically important species of snakes: Australian Eastern Brown snake (Pseudonaja textilis), Sri Lankan Russell's viper (Daboia russelii), Javanese Russell's viper (D. siamensis), Gaboon viper (Bitis gabonica), Uracoan rattlesnake (Crotalus vegrandis), Carpet viper (Echis ocellatus) and Puff adder (Bitis arietans), and identified two distinct patterns of effects: i.e. rapid cardiovascular collapse and prolonged hypotension. P. textilis (5 µg/kg, i.v.) and E. ocellatus (50 µg/kg, i.v.) venoms induced rapid (i.e. within 2 min) cardiovascular collapse in anaesthetised rats. P. textilis (20 mg/kg, i.m.) caused collapse within 10 min. D. russelii (100 µg/kg, i.v.) and D. siamensis (100 µg/kg, i.v.) venoms caused 'prolonged hypotension', characterised by a persistent decrease in blood pressure with recovery. D. russelii venom (50 mg/kg and 100 mg/kg, i.m.) also caused prolonged hypotension. A priming dose of P. textilis venom (2 µg/kg, i.v.) prevented collapse by E. ocellatus venom (50 µg/kg, i.v.), but had no significant effect on subsequent addition of D. russelii venom (1 mg/kg, i.v). Two priming doses (1 µg/kg, i.v.) of E. ocellatus venom prevented collapse by E. ocellatus venom (50 µg/kg, i.v.). B. gabonica, C. vegrandis and B. arietans (all at 200 µg/kg, i.v.) induced mild transient hypotension. Artificial respiration prevented D. russelii venom induced prolonged hypotension but not rapid cardiovascular collapse from E. ocellatus venom. D. russelii venom (0.001–1 μg/ml) caused concentration-dependent relaxation (EC50 = 82.2 ± 15.3 ng/ml, Rmax = 91 ± 1%) in pre-contracted mesenteric arteries. In contrast, E. ocellatus venom (1 µg/ml) only produced a maximum relaxant effect of 27 ± 14%, suggesting that rapid cardiovascular collapse is unlikely to be due to peripheral vasodilation. The prevention of rapid cardiovascular collapse, by 'priming' doses of venom, supports a role for depletable endogenous mediators in this phenomenon. [ABSTRACT FROM AUTHOR]

Snake venom α-neurotoxins potently inhibit rodent nicotinic acetylcholine receptors (nAChRs), but their activity on human receptors and their role in human paralysis from snakebite remain unclear. We demonstrate that two short-chain α-neurotoxins (SαNTx) functionally inhibit human muscle-type nAChR, but are markedly more reversible than against rat receptors. In contrast, two long-chain α-neurotoxins (LαNTx) show no species differences in potency or reversibility. Mutant studies identified two key residues accounting for this. Proteomic and clinical data suggest that paralysis in human snakebites is not associated with SαNTx, but with LαNTx, such as in cobras. Neuromuscular blockade produced by both subclasses of α-neurotoxins was reversed by antivenom in rat nerve-muscle preparations, supporting its effectiveness in human post-synaptic paralysis. [ABSTRACT FROM AUTHOR]

Advances in the last decade combining transcriptomics with established proteomics methods have made possible rapid identification and quantification of protein families in snake venoms. Although over 100 studies have been published, the value of this information is increased when it is collated, allowing rapid assimilation and evaluation of evolutionary trends, geographical variation, and possible medical implications. This review brings together all compositional studies of snake venom proteomes published in the last decade. Compositional studies were identified for 132 snake species: 42 from 360 (12%) Elapidae (elapids), 20 from 101 (20%) Viperinae (true vipers), 65 from 239 (27%) Crotalinae (pit vipers), and five species of non-front-fanged snakes. Approximately 90% of their total venom composition consisted of eight protein families for elapids, 11 protein families for viperines and ten protein families for crotalines. There were four dominant protein families: phospholipase A2s (the most common across all front-fanged snakes), metalloproteases, serine proteases and three-finger toxins. There were six secondary protein families: cysteine-rich secretory proteins, L-amino acid oxidases, kunitz peptides, C-type lectins/snaclecs, disintegrins and natriuretic peptides. Elapid venoms contained mostly three-finger toxins and phospholipase A2s and viper venoms metalloproteases, phospholipase A2s and serine proteases. Although 63 protein families were identified, more than half were present in <5% of snake species studied and always in low abundance. The importance of these minor component proteins remains unknown. [ABSTRACT FROM AUTHOR]

Objective: To describe the epidemiology, treatment and adverse events after snakebite in Australia.Design: Prospective, multicentre study of data on patients with snakebites recruited to the Australian Snakebite Project (2005-2015) and data from the National Coronial Information System. Setting, participants: Patients presenting to Australian hospitals with suspected or confirmed snakebites from July 2005 to June 2015 and consenting to participation.Main Outcome Measures: Demographic data, circumstances of bites, clinical effects of envenoming, results of laboratory investigations and snake venom detection kit (SVDK) testing, antivenom treatment and adverse reactions, time to discharge, deaths.Results: 1548 patients with suspected snakebites were enrolled, including 835 envenomed patients (median, 87 per year), for 718 of which the snake type was definitively established, most frequently brown snakes (41%), tiger snakes (17%) and red-bellied black snakes (16%). Clinical effects included venom-induced consumption coagulopathy (73%), myotoxicity (17%), and acute kidney injury (12%); severe complications included cardiac arrest (25 cases; 2.9%) and major haemorrhage (13 cases; 1.6%). There were 23 deaths (median, two per year), attributed to brown (17), tiger (four) and unknown (two) snakes; ten followed out-of-hospital cardiac arrests and six followed intracranial haemorrhages. Of 597 SVDK test results for envenomed patients with confirmed snake type, 29 (4.9%) were incorrect; 133 of 364 SVDK test results for non-envenomed patients (36%) were false positives. 755 patients received antivenom, including 49 non-envenomed patients; 178 (24%), including ten non-envenomed patients, had systemic hypersensitivity reactions, of which 45 (6%) were severe (hypotension, hypoxaemia). Median total antivenom dose declined from four vials to one, but median time to first antivenom was unchanged (4.3 hours; IQR, 2.7-6.3 hours).Conclusions: Snake envenoming is uncommon in Australia, but is often severe. SVDKs were unreliable for determining snake type. The median antivenom dose has declined without harming patients. Improved early diagnostic strategies are needed to reduce the frequently long delays before antivenom administration. [ABSTRACT FROM AUTHOR]

Antivenomtherapy is currently the standard practice for treating neuromuscular dysfunction in snake envenoming. We reviewed the clinical and experimental evidence-base for the efficacy and effectiveness of antivenom in snakebite neurotoxicity. The main site of snake neurotoxins is the neuromuscular junction, and themajority are either: (1) pre-synaptic neurotoxins irreversibly damaging the presynaptic terminal; or (2) post-synaptic neurotoxins that bind to the nicotinic acetylcholine receptor. Pre-clinical tests of antivenom efficacy for neurotoxicity include rodent lethality tests, which are problematic, and in vitro pharmacological tests such as nerve-muscle preparation studies, that appear to provide more clinically meaningful information. We searched MEDLINE (from 1946) and EMBASE (from 1947) until March 2017 for clinical studies. The search yielded no randomised placebo-controlled trials of antivenom for neuromuscular dysfunction. There were several randomised and non-randomised comparative trials that compared two or more doses of the same or different antivenom, and numerous cohort studies and case reports. The majority of studies available had deficiencies including poor case definition, poor study design, small sample size or no objective measures of paralysis. A number of studies demonstrated the efficacy of antivenom in human envenoming by clearing circulating venom. Studies of snakes with primarily pre-synaptic neurotoxins, such as kraits (Bungarus spp.) and taipans (Oxyuranus spp.) suggest that antivenom does not reverse established neurotoxicity, but early administrationmay be associatedwith decreased severity or prevent neurotoxicity. Small studies of snakes with mainly post-synaptic neurotoxins, including some cobra species (Naja spp.), provide preliminary evidence that neurotoxicity may be reversed with antivenom, but placebo controlled studies with objective outcome measures are required to confirm this. [ABSTRACT FROM AUTHOR]

Background: Sri Lankan Russell’s viper (Daboia russelii) envenoming is reported to cause myotoxicity and neurotoxicity, which are different to the effects of envenoming by most other populations of Russell’s vipers. This study aimed to investigate evidence of myotoxicity in Russell’s viper envenoming, response to antivenom and the toxins responsible for myotoxicity. Methodology and Findings: Clinical features of myotoxicity were assessed in authenticated Russell’s viper bite patients admitted to a Sri Lankan teaching hospital. Toxins were isolated using high-performance liquid chromatography. In-vitro myotoxicity of the venom and toxins was investigated in chick biventer nerve-muscle preparations. Of 245 enrolled patients, 177 (72.2%) had local myalgia and 173 (70.6%) had local muscle tenderness. Generalized myalgia and muscle tenderness were present in 35 (14.2%) and 29 (11.8%) patients, respectively. Thirty-seven patients had high (>300 U/l) serum creatine kinase (CK) concentrations in samples 24h post-bite (median: 666 U/l; maximum: 1066 U/l). Peak venom and 24h CK concentrations were not associated (Spearman’s correlation; p = 0.48). The 24h CK concentrations differed in patients without myotoxicity (median 58 U/l), compared to those with local (137 U/l) and generalised signs/symptoms of myotoxicity (107 U/l; p = 0.049). Venom caused concentration-dependent inhibition of direct twitches in the chick biventer cervicis nerve-muscle preparation, without completely abolishing direct twitches after 3 h even at 80 μg/ml. Indian polyvalent antivenom did not prevent in-vitro myotoxicity at recommended concentrations. Two phospholipase A2 toxins with molecular weights of 13kDa, U1-viperitoxin-Dr1a (19.2% of venom) and U1-viperitoxin-Dr1b (22.7% of venom), concentration dependently inhibited direct twitches in the chick biventer cervicis nerve-muscle preparation. At 3 μM, U1-viperitoxin-Dr1a abolished twitches, while U1-viperitoxin-Dr1b caused 70% inhibition of twitch force after 3h. Removal of both toxins from whole venom resulted in no in-vitro myotoxicity. Conclusion: The study shows that myotoxicity in Sri Lankan Russell’s viper envenoming is mild and non-life threatening, and due to two PLA2 toxins with weak myotoxic properties. [ABSTRACT FROM AUTHOR]

Venom detection is crucial for confirmation of envenomation and snake type in snake-bite patients. Enzyme immunoassay (EIA) is used to detect venom, but antivenom in samples prevents venom detection. We aimed to detect snake venom in post-antivenom samples after dissociating venom-antivenom complexes with glycine-HCl (pH 2.2) and heating for 30 min at 950 °C. Serum samples underwent dissociation treatment and then Russell's viper venom or Australian elapid venom measured by EIA. In confirmed Russell's viper bites with venom detected pre-antivenom (positive controls), no venom was detected in untreated post-antivenom samples, but was after dissociation treatment. In 104 non-envenomed patients (negative controls), no venom was detected after dissociation treatment. In suspected Russell's viper bites, ten patients with no pre-antivenom samples had venom detected in post-antivenom samples after dissociation treatment. In 20 patients with no venom detected pre-antivenom, 13 had venom detected post-antivenom after dissociation treatment. In another 85 suspected Russell's viper bites with no venom detected pre-antivenom, 50 had venom detected after dissociation treatment. Dissociation treatment was also successful for Australian snake envenomation including taipan, mulga, tiger snake and brown snake. Snake venom can be detected by EIA in post-antivenom samples after dissociation treatment allowing confirmation of diagnosis of envenomation post-antivenom. [ABSTRACT FROM AUTHOR]

Brown snake ( Pseudonaja spp.)-induced early cardiovascular collapse is a life-threatening medical emergency in Australia. We have previously shown that this effect can be mimicked in animals and is mediated via the release of endogenous mediators. In the present study, we aimed to purify and characterize the component in Pseudonaja textilis venom which induces cardiovascular collapse following envenoming. The component (fraction 3) was isolated using a combination of techniques including hydroxyapatite and reverse phase chromatography. Fraction 3 (10 or 20 μg/kg, i.v.) produced a rapid decrease in mean arterial pressure (MAP) followed by cardiovascular collapse. Fraction 3-induced early collapse was abolished by prior administration of smaller priming doses of fraction 3 (i.e. 2 and 5 μg/kg, i.v.) or heparin (300 units/kg, i.v.). P. textilis whole venom (1 and 3 μg/ml), but not fraction 3 (1 or 3 μg/ml), induced endothelium-dependent relaxation in isolated rat mesenteric arteries. SDS-PAGE gel indicated the presence of 9–10 protein bands of fraction 3. Using proteomic based analysis some protein bands of fraction 3 were identified as subunits of venom prothrombin activator, pseutarin C of P. textilis venom. Our results conclude that prothrombin activator-like toxin is likely to be a contributor to the rapid collapse induced by P. textilis venom. [ABSTRACT FROM AUTHOR]

Venomous snakebite is considered the single most important cause of human injury from venomous animals worldwide. Coagulopathy is one of the commonest important systemic clinical syndromes and can be complicated by serious and life-threatening haemorrhage. Venom-induced consumption coagulopathy (VICC) is the commonest coagulopathy resulting from snakebite and occurs in envenoming by Viperid snakes, certain elapids, including Australian elapids, and a few Colubrid (rear fang) snakes. Procoagulant toxins activate the clotting pathway, causing a broad range of factor deficiencies depending on the particular procoagulant toxin in the snake venom. Diagnosis and monitoring of coagulopathy is problematic, particularly in resource-poor countries where further research is required to develop more reliable, cheap clotting tests. MEDLINE and EMBASE up to September 2013 were searched to identify clinical studies of snake envenoming with VICC. The UniPort database was searched for coagulant snake toxins. Despite preclinical studies demonstrating antivenom binding toxins (efficacy), there was less evidence to support clinical effectiveness of antivenom for VICC. There were no placebo-controlled trials of antivenom for VICC. There were 25 randomised comparative trials of antivenom for VICC, which compared two different antivenoms (ten studies), three different antivenoms (four), two or three different doses or repeat doses of antivenom (five), heparin treatment and antivenom (five), and intravenous immunoglobulin treatment and antivenom (one). There were 13 studies that compared two groups in which there was no randomisation, including studies with historical controls. There have been numerous observational studies of antivenom in VICC but with no comparison group. Most of the controlled trials were small, did not use the same method for assessing coagulopathy, varied the dose of antivenom, and did not provide complete details of the study design (primary outcomes, randomisation, and allocation concealment). Non-randomised trials including comparison groups without antivenom showed that antivenom was effective for some snakes (e.g., Echis), but not others (e.g., Australasian elapids). Antivenom is the major treatment for VICC, but there is currently little high-quality evidence to support effectiveness. Antivenom is not risk free, and adverse reactions can be quite common and potentially severe. Studies of heparin did not demonstrate it improved outcomes in VICC. Fresh frozen plasma appeared to speed the recovery of coagulopathy and should be considered in bleeding patients. [ABSTRACT FROM AUTHOR]

Background: This study examines the use of topical pharmacological agents as a snakebite first aid where slowing venom reaching the circulation prevents systemic toxicity. It is based on the fact that toxin molecules in most snake venoms are large molecules and generally first enter and traverse the lymphatic system before accessing the circulation. It follows on from a previous study where it was shown that topical application of a nitric oxide donor slowed lymph flow to a similar extent in humans and rats as well as increased the time to respiratory arrest for subcutaneous injection of an elapid venom (Pseudonaja textilis, Ptx; Eastern brown snake) into the hind feet of anaesthetized rats. Methodology/Principal Findings: The effects of topical application of the L-type Ca2+ channel antagonist nifedipine and the local anesthetic lignocaine in inhibiting lymph flow and protecting against envenomation was examined in an anaesthetized rat model. The agents significantly increased dye-measured lymph transit times by 500% and 390% compared to controls and increased the time to respiratory arrest to foot injection of a lethal dose of Ptx venom by 60% and 40% respectively. The study also examined the effect of Ptx venom dose over the lethal range of 0.4 to 1.5 mg/kg finding a negative linear relationship between increase in venom dose and time to respiratory arrest. Conclusions/Significance: The findings suggest that a range of agents that inhibit lymphatic flow could potentially be used as an adjunct treatment to pressure bandaging with immobilization (PBI) in snakebite first aid. This is important given that PBI (a snakebite first aid recommended by the Australian National Health and Medical research Council) is often incorrectly applied. The use of a local anesthetic would have the added advantage of reducing pain. Author Summary: Snakebite remains a major problem worldwide causing death or serious illness in many tens of thousands of victims annually. An approach to reduce the burden of envenoming is to provide optimum first aid procedures. We have previously shown that topical application of a nitric oxide (NO) donor slowed lymph flow to similar extent in humans and rats as well as increased the time to respiratory arrest by ∼50% for subcutaneous injection of eastern brown snake venom into the hind feet of anaesthetized rats. The present study examines the use of several other topical pharmacological agents that aim to slow venom toxins reaching the circulation through the lymphatic system. The study found that the agents examined were similarly effective to that previously found for the NO donor. The fact that one of these is a commonly used topical local anesthetic may be an ideal adjunct first aid, as it provides first aid while reducing pain. [ABSTRACT FROM AUTHOR]

Snakebite is a significant and under-resourced global public health issue. Snake venoms cause a variety of potentially fatal clinical toxin syndromes, including venom-induced consumption coagulopathy (VICC) which is associated with major haemorrhage. A subset of patients with VICC develop a thrombotic microangiopathy (TMA). This article reviews recent evidence regarding snakebite-associated TMA and its epidemiology, diagnosis, outcomes, and effectiveness of interventions including antivenom and therapeutic plasma-exchange. Snakebite-associated TMA presents with microangiopathic haemolytic anaemia (evidenced by schistocytes on the blood film), thrombocytopenia in almost all cases, and a spectrum of acute kidney injury (AKI). A proportion of patients require dialysis, most survive and achieve dialysis free survival. There is no evidence that antivenom prevents TMA specifically, but early antivenom remains the mainstay of treatment for snake envenoming. There is no evidence for therapeutic plasma-exchange being effective. We propose diagnostic criteria for snakebite-associated TMA as anaemia with >1.0% schistocytes on blood film examination, together with absolute thrombocytopenia (<150 × 109/L) or a relative decrease in platelet count of >25% from baseline. Patients are at risk of long-term chronic kidney disease and long term follow up is recommended. [ABSTRACT FROM AUTHOR]

Background: Snake bite is one of the most neglected public health issues in poor rural communities worldwide. In addition to the clinical effects of envenoming, treatment with antivenom frequently causes serious adverse reactions, including hypersensitivity reactions (including anaphylaxis) and pyrogenic reactions. We aimed to investigate the immune responses to Sri Lankan snake envenoming (predominantly by Russell's viper) and antivenom treatment. Methodology/Principal Findings: Plasma concentrations of Interleukin (IL)-6, IL-10, tumor necrosis factor α (TNFα), soluble TNF receptor I (sTNFRI), anaphylatoxins (C3a, C4a, C5a; markers of complement activation), mast cell tryptase (MCT), and histamine were measured in 120 Sri Lankan snakebite victims, both before and after treatment with antivenom. Immune mediator concentrations were correlated with envenoming features and the severity of antivenom-induced reactions including anaphylaxis. Envenoming was associated with complement activation and increased cytokine concentrations prior to antivenom administration, which correlated with non-specific systemic symptoms of envenoming but not with coagulopathy or neurotoxicity. Typical hypersensitivity reactions to antivenom occurred in 77/120 patients (64%), satisfying criteria for a diagnosis of anaphylaxis in 57/120 (48%). Pyrogenic reactions were observed in 32/120 patients (27%). All patients had further elevations in cytokine concentrations, but not complement activation, after the administration of antivenom, whether a reaction was noted to occur or not. Patients with anaphylaxis had significantly elevated concentrations of MCT and histamine. Conclusions/Significance: We have demonstrated that Sri Lankan snake envenoming is characterized by significant complement activation and release of inflammatory mediators. Antivenom treatment further enhances the release of inflammatory mediators in all patients, with anaphylactic reactions characterised by high levels of mast cell degranulation but not further complement activation. Anaphylaxis is probably triggered by non allergen-specific activation of mast cells and may be related to the quality of available antivenom preparations, as well as a priming effect from the immune response to the venom itself. [ABSTRACT FROM AUTHOR]

Abstract: α-Neurotoxins have been isolated from hydrophid, elapid and, more recently, colubrid snake venoms. Also referred to as postsynaptic neurotoxins or ‘curare mimetic’ neurotoxins, they play an important role in the capture and/or killing of prey by binding to the nicotinic acetylcholine receptor on the skeletal muscle disrupting neurotransmission. They are also thought to cause respiratory paralysis in envenomed humans. This review will discuss the historical background into the discovery, isolation, structure and mechanism of action of the α-neurotoxins, including targets and cellular outcomes, and then will examine the potential uses of α-neurotoxins as pharmacological tools and/or as drug leads. [Copyright &y& Elsevier]

Abstract: Two common in vitro skeletal muscle preparations used for the study of venom neurotoxicity are the indirectly stimulated chick isolated biventer cervicis nerve-muscle preparation and the rat isolated phrenic nerve-diaphragm preparation. The aim of the current study was to compare the in vitro neurotoxicity of six Pseudechis spp. (Black snakes) venoms in both avian (chicken) and mammalian (rat) skeletal muscle preparations to determine differences in sensitivity. All Pseudechis spp. venoms significantly inhibited indirect twitches, in both preparations, indicating the presence of post synaptic neurotoxins. The inhibitory effects of all venoms were more rapid in the avian preparation, except for Pseudechis colletti venom where no significant difference was seen between the murine and avian muscles. Time taken to produce 50% reduction in stimulated twitches (i.e. t 50) was markedly shorter in the avian preparation. We have shown that the avian in vitro preparation is more sensitive to the neurotoxic activity of Pseudechis spp. than the murine preparation. This difference is likely to be due to species differences in the interaction between the neurotoxins and the nicotinic receptor binding sites as well as differences in the ‘safety factor’ between the preparations. [Copyright &y& Elsevier]

Cross-neutralisation has been demonstrated for haemorrhagic venoms including Echis spp. and Cerastes spp. and for Australia elapid procoagulant toxins. A previous study showed that commercial tiger snake antivenom ( TSAV) was able to neutralise the systemic effects of the Egyptian cobra, Naja haje, in vivo but it is unclear if this was true cross-neutralisation. The aim of the current study was to determine whether TSAV can neutralise the in vitro neurotoxic effects of N. haje venom. Both Notechis scutatus (10 μg/ml) and N. haje (10 μg/ml) venoms caused inhibition of indirect (supramaximal V, 0.1 Hz, 0.2 msec.) twitches of the chick biventer cervicis nerve-muscle preparation with t90 values (i.e. the time to produce 90% inhibition of the original twitch height) of 26 ± 1 min. (n = 4 ) and 36 ± 4 min.; (n = 4). This effect at 10 μg/ml was significantly attenuated by the prior addition of TSAV (5 U/ml). A comparison of the reverse-phase HPLC profiles of both venoms showed some similarities with peak elution times, and SDS- PAGE analysis elucidated comparable bands across both venoms. Further analysis using Western immunoblotting indicated TSAV was able to detect N. haje venom, and enzyme immunoassay showed that in-house biotinylated polyclonal monovalent N. scutatus antibodies were able to detect N. haje venom. These findings demonstrate cross-neutralisation between different and geographically separated snakes supporting potential immunological similarities in snake toxin groups for a large range of snakes. This provides more evidence that antivenoms could be developed against specific toxin groups to cover a large range of snakes. [ABSTRACT FROM AUTHOR]

Abstract: Snake venoms contain toxins that activate the coagulation network and cause venom-induced consumption coagulopathy. A previously developed mathematical model of the coagulation network was refined and used to describe and predict the time course of changes in the coagulation factors following envenomation by Brown snake (Pseudonaja spp.), Tiger snake (Notechis scutatus), Rough-scaled snake (Tropidechis carinatus) and Hoplocephalus spp. (Stephens banded, Pale headed and Broad headed). Simulations of the time course of the change in coagulation factors were compared to data obtained from a large prospective study of Australian snake bites – the Australian Snakebite Project. The model predictions were also compared against data for partial and complete VICC obtained from the same study. The model simulations were used to understand the differences in consumption and recovery of clotting factors in partial versus complete VICC as well as among bites from different snake types. The model suggested that the venoms were absorbed almost instantaneously and provided a reasonable prediction of the observed concentration of clotting factors over time in patients bitten by Australian elapid snakes. The model predictions suggested a higher consumption of factors (fibrinogen, II and IX in particular) in patients with complete VICC compared to those with partial VICC. The model also predicted that snakes with “Xa-like” venoms may produce a less severe VICC than snakes with “Xa:Va-like” venoms. [Copyright &y& Elsevier]

Research into Australian elapid venoms has mainly focused on the seven genera of greatest clinical significance: Acanthophis, Hoplocephalus, Notechis, Oxyuranus, Pseudechis, Pseudonaja and Tropidechis. However, even small species represent a potential for causing severe clinical envenoming. Further, owing to taxonomic distinctiveness, these species are a potential source of novel toxins for use in drug design and development. This is the first study to characterize the venoms of Cryptophis boschmai, Denisonia devisi, Echiopsis curta, Hemiaspis signata and Vermicella annulata. MALDI analysis of each venom, over the range of 4-40 kDa, indicated components in the weight range for three finger toxins (6-8 kDa) and phospholipase A2 ( PLA2; 12-14 kDA). Interestingly, C. boschmai venom was the only venom, which contained components > 25 kDa. All venoms (10 μg/ml) demonstrated in vitro neurotoxicity in the chick biventer cervicis nerve-muscle preparation, with a relative rank order of: H. signata ≥ D. devisi ≥ V. annulata = E. curta > C. boschmai. CSL polyvalent antivenom neutralized the inhibitory effects of C. boschmai venom but only delayed the inhibitory effect of the other venoms. All venoms displayed PLA2 activity but over a wide range (i.e. 1-621 μmol/min./mg). The venoms of C. boschmai (60 μg/kg, i.v.), D. devisi (60 μg/kg, i.v.) and H. signata (60 μg/kg, i.v.) produced hypotensive effects in vivo in an anaesthetized rat preparation. H. signata displayed moderate pro-coagulant activity while the other venoms were weakly pro-coagulant. This study demonstrated that these understudied Australian elapids have varying pharmacological activity, with notable in vitro neurotoxicity for four of the venoms, and may produce mild to moderate effects following systemic envenoming. [ABSTRACT FROM AUTHOR]

Abstract: Pseudonaja textilis (Eastern Brown snake) and Oxyuranus scutellatus scutellatus (Coastal taipan) are clinically important Australian elapid snakes, whose potent venoms contain the presynaptic (β) neurotoxins, textilotoxin and taipoxin, respectively, and a number of postsynaptic neurotoxins. However, while taipan envenoming frequently results in neurotoxicity, Brown snake envenoming causes an isolated coagulopathy and neurotoxicity is rare. This phenomenon is called the ‘Brown snake paradox’. This study compared the pharmacology of both venoms and their respective presynaptic neurotoxins to investigate this phenomenon. From size-exclusion high performance liquid chromatography (HPLC) analysis textilotoxin represents a significantly smaller proportion (5.7%) of P. textilis venom compared to taipoxin in O. s. scutellatus venom (20.4%). In the chick biventer cervicis nerve-muscle (CBCNM) preparation both venoms caused concentration-dependent neurotoxicity, with P. textilis venom being significantly more potent than O. s. scutellatus venom. Conversely, taipoxin was significantly more potent than textilotoxin when compared at the same concentration. Textilotoxin only partially contributed to the overall neurotoxicity of P. textilis venom, while taipoxin accounted for the majority of the neurotoxicity of O. s. scutellatus venom in the CBCNM preparation. Compared with taipoxin, textilotoxin is less potent and constitutes a smaller proportion of the venom. This is likely to be the reason for the absence of neurotoxicity in envenomed humans thus explaining the ‘Brown snake paradox’. [Copyright &y& Elsevier]

Abstract: Introduction: Acanthophis genus (i.e. death adders) and the Naja genus (i.e. cobras) belong to the family elapidae. The current study compared the in vitro cytotoxicity of venoms from four Acanthophis spp. and three Naja spp. on rat aortic smooth muscle cells, A7r5, and rat skeletal muscle cells, L6. The ability of CSL death adder antivenom and SAIMR antivenom, for Acanthophis spp. and Naja spp. venom respectively, to negate the cytotoxicity was also examined. Methods: A cell proliferation assay was used to determine cell viability following treatment with venom in the presence or absence of antivenom. Sigmoidal growth curves were obtained, and IC50 values were determined. Results: Acanthophis spp. and Naja spp. venoms produced concentration-dependent inhibition of cell proliferation in both cell lines. Naja spp. venoms were significantly more cytotoxic than the most potent Acanthophis venom (i.e. A. antarcticus) in both cell lines. Naja spp. venoms also displayed higher sensitivity in L6 cells. SAIMR antivenom significantly inhibited the cytotoxic actions of all Naja spp. venoms in both A7r5 and L6 cells. However, death adder antivenom (CSL Ltd) was unable to negate the cytotoxic effects of Acanthophis spp. venoms. Discussion: Concentrations of the predominantly cytotoxic Naja spp. venoms used were approximately three times less than the predominantly neurotoxic Acanthophis spp. venoms. SAIMR antivenom was partially effective in neutralising the effects of Naja spp. venoms. Death adder antivenom (CSL Ltd) was not effective in negating the cytotoxic effects of venom from Acanthophis spp. These results indicate that the cell-based assay is suited to the examination of cytotoxic snake venoms and may be used in conjunction with organ bath experiments to pharmacologically characterise snake venoms. Furthermore, the results suggest that the use of a skeletal muscle cell line is likely to be more clinically relevant for the examination of cytotoxic snake venoms. [Copyright &y& Elsevier]

Abstract: Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this to traditional clotting and prothrombinase activity studies. We developed a novel modification of ETP using procoagulant snake venoms to trigger thrombin production. This was used to characterise the relative potency, calcium and clotting factor requirements of five important Australian snake venoms and efficacy of commercial antivenom, and compared this to prothrombinase activity and clotting assays. All five venoms initiated thrombin generation in the absence and presence of calcium. Pseudonaja textilis (Brown snake; p <0.0001), Hoplocephalus stephensii (Stephen’s-banded snake; p <0.0001) and Notechis scutatus (tiger snake; p =0.0073) all had statistically significant increases in ETP with calcium. Venom potency varied between assays, with ETP ranging from least potent with Oxyuranus scutellatus (Taipan) venom to intermediate with N. scutatus and H. stephensii venoms to most potent with P. textilis and Tropidechis carinatus (Rough-scale snake) venoms. ETPs for N. scutatus, T. carinatus and H. stephensii venoms were severely reduced with factor V deficient plasma. Antivenom neutralized the thrombin generating capacity but not prothrombin substrate cleaving ability of the venoms. Contrary to previous studies using clotting tests and factor Xa substrates, these venoms differ in calcium requirement. ETP is a useful assay to investigate mechanisms of other procoagulant venoms and is a robust method of assessing antivenom efficacy. [Copyright &y& Elsevier]

Abstract: We report a 60 year old male bitten by snakes from the Acanthophis genus (Death adder) on two occasions who developed high titres of human IgG antibodies to Acanthophis venom detected at the time of the second bite. The patient was bitten by Acanthophis antarcticus (common death adder) on the first occasion, developed non-specific systemic effects and did not receive antivenom. Three months later he was bitten by Acanthophis praelongus (northern death adder) and he developed significant local myotoxicity associated with a moderate rise in the creatine kinase (maximum 4770U/L). He was given antivenom 55h after the bite and recovered over several days. Death adder venom was detected in serum at the time of the first bite, but not the second bite. Human IgG antibodies to death adder were detected on the second admission but not the first. However, despite the presence of antibodies to death adder venom and free venom not being detected, the patient still developed significant local myotoxicity. [Copyright &y& Elsevier]

Abstract: An understanding of the cross-neutralisation of snake venoms by antibodies is important for snake antivenom development. We investigated the cross-neutralisation of brown snake (Pseudonaja textilis) venom, taipan (Oxyuranus scutellatus) venom and death adder (Acanthophis antarcticus) with commercial antivenoms and monovalent anti-snake IgG, using enzyme immunoassays, in vitro clotting and neurotoxicity assays. Each commercial antivenom bound all three venoms, and neutralised clotting activity of brown snake and taipan venoms and neurotoxicity of death adder venom. The ‘in-house’ monovalent anti-snake venom IgG raised against procoagulant brown snake and taipan venoms, did not neutralise the neurotoxic effects of death adder venom. However, they did cross-neutralise the procoagulant effects of both procoagulant venoms. This supports the idea of developing antivenoms against groups of snake toxins rather than individual snake venoms. [Copyright &y& Elsevier]

Abstract: Monovalent antivenoms have a lower volume of specific antibodies that may reduce reactions but require accurate snake identification to be used. Polyvalent antivenoms are larger volume and may have a higher reaction rate. However, they avoid the problem of snake identification and may be more cost-effective to manufacture. We have previously shown cross-neutralisation of two Australian elapid venoms, tiger snake (Notechis scutatus) and brown snake (Pseudonaja textilis) venoms, by their respective monovalent antivenoms. In this study enzyme immunoassays were used to quantify the amount of monovalent antivenom (quantity of monovalent antibodies to a specific snake venom) in vials of commercially produced antivenom in Australia. All antivenoms tested appeared to be polyvalent and contain varying amounts of all five terrestrial snake monovalent antibodies based on their binding to the five representative venoms. Redback spider antivenom did not have any measurable binding affinity for any of the five snake venoms, showing that the observed binding is not due to non-specific interactions with equine protein. The antivenoms had expiry dates over a 15year period, suggesting that the antivenoms have been mixtures for at least this time. This study cannot be used to rationalise hospital stocks of antivenom in Australia because there is no guarantee that the antivenoms will remain as mixtures. However, it would be possible for the manufacturer to reduce the number of types of snake antivenoms available in Australia to two polyvalent antivenoms which would simplify treatment of snakebite. [Copyright &y& Elsevier]

Traditionally animals and cell cultures have been used to assess the toxic potential of xenobiotics on cell membranes. In search for more reproducible, quantitative, cost- and time-effective assays, toxicologists have recently become interested in biomimetic lipid vesicle-based test systems. Lipid vesicles (liposomes) have long been appreciated as simple cell membrane models in biochemical and biophysical studies providing a good understanding of the physicochemical properties of liposome systems. More recently a number of reports have been published on the interactions of toxic substances with vesicles. Literature reports on liposome assays have appeared for widely different classes of xenobiotics, such as dental materials, antibiotics, detergents, and peptides. In this review we focus on those reports that contain a quantitative and significant correlation with more established toxicological tests like cell culture assays. We provide an introduction to the structure and main characteristics of vesicles and related lipid aggregates. The two main assays presented are leakage of fluorescence dyes and differential scanning calorimetry (DSC) measurements of the solid-ordered/liquid-disordered main phase transition temperature (Tm). [ABSTRACT FROM AUTHOR]

Comments on Proteomic Investigations of Two Pakistani Naja Snake Venoms Species Unravel the Venom Complexity, Posttranslational Modifications, and Presence of Extracellular Vesicles. However, the venom proteome of I N. naja i from Pakistan has already been published by Wong et al. [[2]], who showed that the venom consists of 75% 3FTx and not the 21% claimed by Manuwar or the 15% that we recalculated when using their methods. Previously published venom proteomes of 11 species of I Naja i found that the abundance of 3FTxs in I Naja i venom proteomes was 56% to 84% of the total venom (depending on the species) [[7]]. [Extracted from the article]

Snake venom toxins first transit the lymphatic system before entering the bloodstream. Ointment containing a nitric oxide donor, which impedes the intrinsic lymphatic pump, prolonged lymph transit time in rats and humans and also increased rat survival time after injection of venom. This pharmacological approach should give snakebite victims more time to obtain medical care and antivenom treatment. [ABSTRACT FROM AUTHOR]

Intra-specific venom variation has the potential to provide important insights into the evolution of snake venom, but remains a relatively neglected aspect of snake venom studies. We investigated the venom from 13 individual coastal taipans Oxyuranus scutellatus from four localities on the north-east coast of Australia, spanning a distance of 2000 km. The intra-specific variation in taipan venom was considerably less than the inter-specific variation between it and the other Australian elapids to which it was compared. The electrophoretic venom profile of O. scutellatus was visually different to six other genera of Australian elapids, but not to its congener inland taipan O. microlepidotus. There was minimal geographical variation in taipan venom, as the intra-population variation exceeded the inter-population variation for enzymatic activity, procoagulant activity, and the abundance of neurotoxins. The pre-synaptic neurotoxin (taipoxin) was more abundant than the post-synaptic neurotoxins (3FTx), with a median of 11.0% (interquartile range (IQR): 9.7% to 18.3%; range: 6.7% to 23.6%) vs. a median of 3.4% (IQR: 0.4% to 6.7%; range: 0% to 8.1%). Three taipan individuals almost completely lacked post-synaptic neurotoxins, which was not associated with geography and occurred within two populations. We found no evidence of sexual dimorphism in taipan venom. Our study provides a basis for evaluating the significance of intra-specific venom variation within a phylogenetic context by comparing it to the inter-specific and inter-generic variation. The considerable intra-population variation we observed supports the use of several unpooled individuals from each population when making inter-specific comparisons. [ABSTRACT FROM AUTHOR]

Keywords: Snake bites; Antivenoms; Evidence-based medicine EN Snake bites Antivenoms Evidence-based medicine 45 45 1 07/07/20 20200701 NES 200701 To the Editor: We read the perspective by Weinstein and colleagues[1] with interest and agree that treatment of snake envenoming in Australia is complex, and that clinicians should seek expert advice in cases of severe or unusual envenoming. However, the cited study measured venom-antivenom complexes in vitro to determine the antivenom concentration at which every venom molecule is bound to at least one antivenom molecule, as a measure of efficacy.[4] It showed that this was similar to the manufacturer's original recommendation of a dose of a single vial of antivenom. [Extracted from the article]

Snake venom is comprised of a combination of different proteins and peptides with a wide range of molecular weights and different disposition processes inherent to each compound. This causes venom to have a complex exposure profile. Our study investigates 1) how each molecular weight fraction (toxin) of venom contributes to the overall time course of the snake venom, and 2) the ability to determine toxin profiles based on the profile of the overall venom only. We undertook an in silico simulation and modelling study. Sixteen variations of venom, comprising of two to nine toxins with different molecular weights were investigated. The pharmacokinetic parameters (i.e., clearance,  C L , and volume of distribution,  V ) of each toxin were generated based on a log-linear relationship with molecular weight. The concentration–time data of each toxin were simulated for 100 virtual patients using MATLAB and the total concentration–time data of each toxin were modelled using NONMEM. We found that the data of sixteen mixtures were best described by either two- or three-compartment models, despite the venom being made up of more than three different toxins. This suggests that it is generally not possible to determine individual toxin profiles based on measurements of total venom concentrations only. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which is concerned with the case study of a 43 year old Australian woman who presented to physicians for treatment of a snake bite.

A letter to the editor is presented in response to a report from J. Ou et al about the case of presumed brown snake envenoming.

The venoms of Australasian elapid snakes are known to possess coagulant activity, including some with strong procoagulant activity and others with anticoagulant activity, although the latter are less well known. This study investigates the anticoagulant activity of Australasian elapid snake venoms, and whether this activity is neutralised by commercial snake antivenom and varespladib (PLA 2 inhibiting agent). Clotting assays were completed for 34 species of Australasian elapids. Antivenom neutralisation assays with tiger snake antivenom (TSAV) were performed on five species to determine if there was cross-neutralisation. Varespladib neutralisation assays were also completed for the same five species. All Pseudechis species venoms had anticoagulant activity, except P. porphyriacus , which was procoagulant. Pseudechis species venoms had similar anticoagulant potency ranging from the most potent P. colletti venom to the least potent P. butleri venom. The three Austrelaps (copperhead) species venoms were the next most potent anticoagulants. Six further snakes, Elapognathus coronatus , Acanthophis pyrrhus, A. antarcticus, Suta suta , Denisonia devisi and D. maculata , had weaker anticoagulant activity, except for D. maculata which had similar anticoagulant activity to Pseudechis species. Tiger Snake Antivenom (1200mU/mL) neutralised the anticoagulant effect of P. australis for concentrations up to 1 mg/mL. TSAV (1200mU/mL) also neutralised P. colletti, D. maculata, A. superbus and A. pyrrhus venoms at their EC 50 , demonstrating cross neutralisation. Varespladib neutralised the anticoagulant effect of P. australis venom at 5 μM and for venoms of P. colletti, D. maculata, A. superbus and A. pyrrhus. We found anticoagulant activity to be present in six genera of Australasian snakes at low concentrations, which can be completely neutralised by both antivenom and varespladib. Anticoagulant activity in Australian elapid venoms was associated with species possessing high PLA 2 activity without procoagulant snake venom serine proteases. [Display omitted] • Australasian elapid snake venoms are known to have coagulant activity, procoagulant and anticoagulant activity. • We investigated the anticoagulant activity in a wide range of Austalasian elapids. • Pseudechis species (black snakes) had the most potent anticoagulant activity. • Anticoagulant activity was found in five other genera. • Anticoagulant activity was neutralised by commercial antivenom and a phospholipase A2 inhibitor for all genera. [ABSTRACT FROM AUTHOR]

Serum sickness is a delayed immune reaction in which the immune system responds to a protein in antiserum as a potentially harmful substance and mounts an IgG-mediated antibody response. A 32 year-old female patient had systemic envenoming following a bite by a red-bellied black snake ( Pseudechis porphyriacus ). She was treated with Tiger snake antivenom and recovered over 24 h and did not develop myotoxicity. She then presented with local pain, itching and swelling, which was partially treated with antihistamines. Eleven days after the bite she presented again with symptoms of worsening serum sickness including rash on the upper legs, joint and muscle pain in arms, ankles and knees, and nausea. The patient was prescribed five days of prednisone 50 mg/day, antihistamine 10 mg/day and analgesia 1000 mg/day and improved over 2 days. She had no further problems on follow up at 4 months. This case highlights that serum sickness can cause significant effects after the treatment of snake envenoming. It develops 5–14 days after antivenom administration and has characteristic clinical and laboratory features. Severe cases of serum sickness can result in morbidity but it appears to respond well to corticosteroid treatment. [ABSTRACT FROM AUTHOR]

The Australian elapid snake radiation (Hydrophiinae) has evolved in the absence of competition from other advanced snakes. This has resulted in ecological specialisation in Australian elapids and the potential for venom proteomes divergent to other elapids. We characterised the venom of the Australian elapid Vermicella annulata (eastern bandy bandy). The venom was analysed using a two-dimensional fractionation process consisting of reverse-phase high-performance liquid chromatography then sodium dodecyl sulphate polyacrylamide gel electrophoresis, followed by bottom-up proteomics. Resulting peptides were matched to a species-specific transcriptome and 87% of the venom was characterised. We identified 11 toxins in the venom from six families: snake venom metalloproteinases (SVMP; 24.2%; two toxins) that are class P-III SVMPs containing a disintegrin-like domain, three-finger toxins (3FTx; 21.6%; five toxins), kunitz peptides (KUN; 19.5%; one toxin), cysteine-rich secretory proteins (CRiSP; 18%; one toxin), and phospholipase A2 (PLA2; 4%; two toxins). The venom had low toxin diversity with five protein families having one or two toxins, except for 3FTx with five different toxins. V. annulata expresses an unusual venom proteome, with high abundances of CRiSP, KUN and SVMP, which are not normally highly expressed in elapid venoms. This unusual venom composition could be an adaptation to its specialised diet. Although the Australian elapid radiation represents the most extensive speciation event of elapids on any continent, with 100 terrestrial species, the venom composition of these snakes has rarely been investigated, with only five species currently characterised. Here we provide the venom proteome of a sixth species, Vermicella annulata. The venom of this species could be particularly informative from an evolutionary perspective, as it is an extreme dietary specialist, only preying on blind snakes (Typhlopidae). We show that V. annulata expresses a highly unusual venom for an elapid, due to the high abundance of the protein families SVMP, CRiSP, and KUN, which together make up 61% of the venom. When averaged across all species, a typical elapid venom is 82% PLA 2 and 3FTx. This is the second recorded instance of an Australian elapid having evolved highly divergent venom expression. [Display omitted] • 87% of the venom of the snake Vermicella annulata was characterised by RP-HPLC and SDS-PAGE, and then bottom-up proteomics. • The venom contains at least 11 different toxins from six toxin families. • There was little toxin diversity except for having five different three-finger toxins • The venom differs to elapids worldwide being dominated by serine venom metalloproteinase, Kunitz and CRiSP protein families. [ABSTRACT FROM AUTHOR]

Background: Animal models are used to test toxic effects of snake venoms/toxins and the antivenom required to neutralise them. However, venoms that cause clinically relevant coagulopathy in humans may have differential effects in animals. We aimed to investigate the effect of different procoagulant snake venoms on various animal plasmas. Methods: Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer levels were measured in seven animal plasmas (human, rabbit, cat, guinea pig, pig, cow and rat). In vitro clotting times were then used to calculate the effective concentration (EC50) in each plasma for four snake venoms with different procoagulant toxins: Pseudonaja textilis, Daboia russelli, Echis carinatus and Calloselasma rhodostoma. Results: Compared to human, PT and aPTT were similar for rat, rabbit and pig, but double for cat and cow, while guinea pig had similar aPTT but double PT. Fibrinogen and D-dimer levelswere similar for all species. Human and rabbit plasmas had the lowest EC50 for P. textilis (0.1 and 0.4 µg/ml), D. russelli (0.4 and 0.1 µg/ml), E. carinatus (0.6 and 0.1 µg/ml) venoms respectively, while cat plasma had the lowest EC50 for C. rhodostoma (11 µg/ml) venom. Cow, rat, pig and guinea pig plasmas were highly resistant to all four venoms with EC50 10-fold that of human. Conclusions: Different animal plasmas have varying susceptibility to procoagulant venoms, and excepting rabbits, animal models are not appropriate to test procoagulant activity. In vitro assays on human plasma should instead be adopted for this purpose. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Point-of-care international normalised ratio (INR) has been suggested as a way to screen for venom-induced consumption coagulopathy following snakebite, but has not been validated for this. This study aimed to assess the diagnostic reliability of point-of-care INR for venom-induced consumption coagulopathy. Methods: This was a prospective study of snakebite patients recruited between January 2011 and May 2012 where a point-of-care INR was done and compared to an INR done on a laboratory coagulation analyser, as part of a quality assurance exercise. Data was obtained for each patient, including demographics, information on the snake bite, the point-of-care INR results and any laboratory derived coagulation studies. Snake identification was confirmed by expert identification or venom specific enzyme immunoassay. Results: There were 15 patients with a median age of 29years (2 to 68y) and 13 were male. Four of the 7 patients with venom-induced consumption coagulopathy had an abnormal point-of-care INR (3 false negatives) and 1 of the 7 non-envenomed patients had an abnormal point-of-care INR (1 false positive). The patient with a falsely elevated point-of-care INR was given antivenom prior to formal coagulation studies. The point-of-care INR was also negative in the patient with an anticoagulant coagulopathy. Conclusions: The study shows that point-of-care INR testing devices should not be used in suspected snakebite cases in Australia to diagnose venom-induced consumption coagulopathy. [Copyright &y& Elsevier]

Hump-nosed pit vipers (Genus Hypnale) are venomous snakes from South India and Sri Lanka. Envenoming by Hypnale species may cause significant morbidity and is characterized by local envenoming and less commonly coagulopathy and acute renal failure. Currently there are three nominal species of this genus: H. hypnale, H. zara and H. nepa. This study investigates the biochemical and pharmacological properties of the venoms from the three Hypnale species in Sri Lanka. The three Hypnale venoms had similar chromatographic profiles using reverse phase high performance liquid chromatography and fractions with procoagulant activity were identified. Hypnale venoms had potent cytotoxicity in cultured rat aorta smooth muscle cells with similar IC50 values. The venoms had weak neurotoxic and myotoxic activity in the isolated chick biventer muscle preparation. They had mild procoagulant activity with close MCC5 values and also phospholipase activity. Locally available polyvalent antivenom did not neutralise any venom effects. The study demonstrates that the three Hypnale venoms are similar and cytotoxicity appears to be the most potent effect, although they have mild procoagulant activity. These findings are consistent with clinical reports. [ABSTRACT FROM AUTHOR]

The article explains the need for effective and affordable antivenom for the treatment of snakebites in Nepal. A study identified Nepal as the country to have one of the highest snakebite fatality rates in south Asia. The concern on shortages of antivenom in the country is discussed, as well as the government's need to increase awareness and to improve political will in the effort to treat snakebites.

A correction to the article "The In Vitro Toxicity of Venoms From South Asian Hump-Nosed Pit Vipers (Viperidae: Hypnale)" that was published in the previous issue is presented.