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Abstract Background Severe psychomotor agitation and aggression often require immediate pharmacological intervention, but clear evidence-based recommendations for choosing among the multiple options are lacking. To address this gap, we plan a systematic review and individual-participant-data network meta-analysis to investigate their comparative effectiveness in real-world emergency settings with increased precision. Methods We will include randomized controlled trials investigating intramuscular or intravenous pharmacological interventions, as monotherapy or in combination, in adults with severe psychomotor agitation irrespective of the underlying diagnosis and requiring rapid tranquilization in general or psychiatric emergency settings. We will exclude studies before 2002, those focusing on specific reasons for agitation and placebo-controlled trials to avoid concerns related to the transitivity assumption and potential selection biases. We will search for eligible studies in BIOSIS, CENTRAL, CINAHL Plus, Embase, LILACS, MEDLINE via Ovid, PubMed, ProQuest, PsycINFO, ClinicalTrials.gov, and WHO-ICTRP. Individual-participant data will be requested from the study authors and harmonized into a uniform format, and aggregated data will also be extracted from the studies. At least two independent reviewers will conduct the study selection, data extraction, risk-of-bias assessment using RoB 2, and applicability evaluation using the RITES tool. The primary outcome will be the number of patients achieving adequate sedation within 30 min after treatment, with secondary outcomes including the need for additional interventions and adverse events, using odds ratios as the effect size. If enough individual-participant data will be collected, we will synthesize them in a network meta-regression model within a Bayesian framework, incorporating study- and participant-level characteristics to explore potential sources of heterogeneity. In cases where individual-participant data are unavailable, potential data availability bias will be explored, and models allowing for the inclusion of studies reporting only aggregated data will be considered. We will assess the confidence in the evidence using the Confidence in Network Meta-Analysis (CINeMA) approach. Discussion This individual-participant-data network meta-analysis aims to provide a fine-tuned synthesis of the evidence on the comparative effectiveness of pharmacological interventions for severe psychomotor agitation in real-world emergency settings. The findings from this study can greatly be provided clearer evidence-based guidance on the most effective treatments. Systematic review registration PROSPERO CRD42023402365.

AbstractMethotrexate toxicity following intravenous (IV) methotrexate is well reported, but there is less information on accidental daily dosing of a prescribed weekly dose of oral methotrexate. A 70 year old female presented following accidentally taking 20 mg methotrexate for five days, with multiple oral ulcers, thrombocytopenia (platelets 80 x 109/L [reference range[RR]:150-400 x 109/L]) and an alanine aminotransferase 820 U/L [RR:10-35 U/L]. Methotrexate was undetectable [

Antivenom is the main treatment for snake envenoming and there are ongoing concerns about availability in resource poor regions of the world. However, effective antivenom treatment for snake envenoming requires more than improved availability of safe and efficacious antivenoms. Most importantly, antivenom must be administered as early as possible, and within 2–6 h of the bite in Australia. At the same time, it is also important that antivenom not be given to all patients indiscriminately with a suspected snakebite, because of the risk of anaphylaxis. Delays in the administration of antivenom are a significant impediment to effective antivenom treatment and can be divided into pre-hospital and in-hospital delays. These range from delays due to remoteness of snakebite, to delays in diagnosis and administration of antivenom once in hospital. In Australia, antivenom is readily available in most hospitals, and a large portion of patients present to hospital within 2 h of the bite. However, there is on average a further delay of 2.5 h before antivenom is administered. Early diagnosis with accurate bedside tests and rapid clinical assessment of patients with snakebite are key to improving the effective use of antivenom.

Abstract MicroRNAs in biofluids are potential biomarkers for detecting kidney and other organ injuries. We profiled microRNAs in urine samples from patients with Russell’s viper envenoming or acute self-poisoning following paraquat, glyphosate, or oxalic acid [with and without acute kidney injury (AKI)] and on healthy controls. Discovery analysis profiled for 754 microRNAs using TaqMan OpenArray qPCR with three patients per group (12 samples in each toxic agent). From these, 53 microRNAs were selected and validated in a larger cohort of patients (Russell’s viper envenoming = 53, paraquat = 51, glyphosate = 51, oxalic acid = 40) and 27 healthy controls. Urinary microRNAs had significantly higher expression in patients poisoned/envenomed by different nephrotoxic agents in both discovery and validation cohorts. Seven microRNAs discriminated severe AKI patients from no AKI for all four nephrotoxic agents. Four microRNAs (miR-30a-3p, miR-30a-5p, miR-92a, and miR-204) had > 17 fold change (p 0.72. Pathway analysis of target mRNAs of these differentially expressed microRNAs showed association with the regulation of different nephrotoxic signaling pathways. In conclusion, human urinary microRNAs could identify toxic AKI early after acute injury. These urinary microRNAs have potential clinical application as early non-invasive diagnostic AKI biomarkers.

BackgroundVenom-induced consumption coagulopathy (VICC) is an important clinical consequence of Russell’s viper (Daboia russelii) envenoming. There is limited evidence for antivenom effectiveness in resolving VICC. We aimed to compare the recovery of VICC in patients who received and did not receive antivenom following Russell’s viper envenoming.Patients and MethodsThis was a non-randomized observational study comparing patients with VICC from Russell’s viper envenoming given antivenom for systemic envenoming and those not given antivenom. Antivenom administration was decided by the treating physicians. We included 44 patients with confirmed Russell’s viper bites with one or more International Normalized Ratio (INR) value ≥ 1.5 (VICC). We compared five patients who did not receive antivenom with 39 patients who did receive antivenom. The primary outcome was the proportion of patients with an INR < 1.5 by 48 h post-bite.ResultsThe antivenom group had higher peak serum venom concentrations [median (IQR) = 272 (96–1,076) ng/mL versus 21 (8–58) ng/mL] and more severe VICC compared to the no antivenom group. Twenty seven of 39 patients (69%) in the antivenom group had an INR < 1.5 at 48 h post-bite compared to none of the five patients (0%) in the no antivenom group (absolute difference: 69%; 95%CI: 13 to 83%; p = 0.006; Fisher’s exact test). The fibrinogen recovered in 32 of 39 patients (82%) in the antivenom group compared to one of five patients (20%) in the no antivenom group (absolute difference 62%; 95% CI: 28 to 95%; p = 0.001; Fisher’s exact test). Both INR and fibrinogen were significantly improved between 24 and 48 h post-bite in the antivenom group compared to the no antivenom group.ConclusionAntivenom accelerated the recovery of VICC in patients with Russell’s viper envenoming, compared to no recovery in a smaller group of patients with milder VICC not receiving antivenom. This supports the efficacy of antivenom in patients with VICC.

The King Cobra (Ophiophagus hannah) is the world’s largest venomous snake and has a widespread geographical distribution throughout Southeast Asia. Despite proteomic studies indicating the presence of postsynaptic neurotoxins in O. hannah venom, there are few pharmacological investigations of these toxins. We isolated and characterized α-elapitoxin-Oh3a (α-EPTX-Oh3a; 7,938 Da), a long-chain postsynaptic neurotoxin, which constitutes 5% of O. hannah venom. α-EPTX-Oh3a (100–300 nM) caused concentration-dependent inhibition of indirect twitches and inhibited contractile responses of tissues to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior incubation of tissues with Thai Red Cross Society King Cobra antivenom (1 ml/0.8 mg) prevented the in vitro neurotoxic effects of α-EPTX-Oh3a (100 nM). The addition of Thai Red Cross Society King Cobra antivenom (1 ml/0.8 mg), at the t90 time point partially reversed the in vitro neurotoxicity of α-EPTX-Oh3a (100 nM). Repeatedly washing the tissue did not allow significant recovery from the in vitro neurotoxic effects of α-EPTX-Oh3a (100 nM). α-EPTX-Oh3a demonstrated pseudo-irreversible antagonism of concentration-response curves to carbachol, with a pA2 of 8.99. De novo sequencing of α-EPTX-Oh3a showed a long-chain postsynaptic neurotoxin with 72 amino acids, sharing 100% sequence identity with Long neurotoxin OH-55. In conclusion, the antivenom is useful for reversing the clinically important long-chain α-neurotoxin-mediated neuromuscular paralysis.

Background: Sea snakes are venomous snakes found in the warm parts of the Indo-Pacific, including around Australia. Most sea snake envenoming causes myotoxicity, but previous Australian case reports describe neurotoxicity. We aimed to describe the epidemiology and clinical presentation of Australian sea snake envenoming and the effectiveness of antivenom.Methods: Patients were recruited to the Australian Snakebite Project (ASP), an Australia-wide prospective observational study recruiting all patients with suspected or confirmed snakebite >2 years. Information about demographics, bite circumstances, species involved, clinical and laboratory features of envenoming, and treatment is collected and entered into a purpose-built database.Results: Between January 2002 and August 2020, 13 patients with suspected sea snake bite were recruited to ASP, 11 were male; median age was 30 years. Bites occurred in Queensland and Western Australia. All patients were in or around, coastal waters at the time of bite. The species involved was identified in two cases (both Hydrophis zweifeli). Local effects occurred in 9 patients: pain (5), swelling (5), bleeding (2), bruising (1). Envenoming occurred in eight patients and was characterised by non-specific systemic features (6) and myotoxicity (2). Myotoxicity was severe (peak CK 28200 and 48100 U/L) and rapid in onset (time to peak CK 13.5 and 15.1 h) in these two patients. Non-specific systemic features included nausea (6), headache (6), abdominal pain (3), and diaphoresis (2). Leukocytosis, neutrophilia, and lymphopenia occurred in both patients with myotoxicity and was evident on the first blood test. No patients developed neurotoxicity or coagulopathy. Early Seqirus antivenom therapy was associated with a lower peak creatine kinase.Conclusion: While relatively rare, sea snake envenoming is associated with significant morbidity and risk of mortality. Early antivenom appears to have a role in preventing severe myotoxicity and should be a goal of therapy.

The Indian Cobra (Naja naja) is among the “Big Four” responsible for most of the snakebite envenoming cases in India. Although recent proteomic studies suggest the presence of postsynaptic neurotoxins in N. naja venom, little is known about the pharmacology of these toxins. We isolated and characterized α-Elapitoxin-Nn2a (α-EPTX-Nn2a; 7020 Da) and α-Elapitoxin-Nn3a (α-EPTX-Nn3a; 7807 Da), a short-chain and long-chain postsynaptic neurotoxin, respectively, which constitute 1 and 3% of N. naja venom. α-EPTX-Nn2a (100–300 nM) and α-EPTX-Nn3a (100–300 nM) both induced concentration-dependent inhibition of indirect twitches and abolished contractile responses of tissues to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior incubation of tissues with Indian polyvalent antivenom (1 ml/0.6 mg) prevented the in vitro neurotoxic effects of α-EPTX-Nn2a (100 nM) and α-EPTX-Nn3a (100 nM). The addition of Indian polyvalent antivenom (1 ml/0.6 mg), at the t90 time point, could not reverse the in vitro neurotoxicity of α-EPTX-Nn2a (100 nM). The in vitro neurotoxicity of α-EPTX-Nn3a (100 nM) was partially reversed by the addition of Indian polyvalent antivenom (1 ml/0.6 mg), as well as repeated washing of the tissue. α-EPTX-Nn2a displayed non-competitive antagonism of concentration-response curves to carbachol, with a pA2 of 8.01. In contrast, α-EPTX-Nn3a showed reversible antagonism of concentration-response curves to carbachol, with a pA2 of 8.17. De novo sequencing of α-EPTX-Nn2a and α-EPTX-Nn3a showed a short-chain and long-chain postsynaptic neurotoxin, respectively, with 62 and 71 amino acids. The important observation made in this study is that antivenom can reverse the neurotoxicity of the clinically important long-chain neurotoxin, but not the short-chain neurotoxin, from N. naja venom.

Abstract Background The relative influence of diet and phylogeny on snake venom activity is a poorly understood aspect of snake venom evolution. We measured the activity of two enzyme toxin groups – phospholipase A2 (PLA2), and L-amino acid oxidase (LAAO) – in the venom of 39 species of Australian elapids (40% of terrestrial species diversity) and used linear parsimony and BayesTraits to investigate any correlation between enzyme activity and phylogeny or diet. Results PLA2 activity ranged from 0 to 481 nmol/min/mg of venom, and LAAO activity ranged from 0 to 351 nmol/min/mg. Phylogenetic comparative methods, implemented in BayesTraits showed that enzyme activity was strongly correlated with phylogeny, more so for LAAO activity. For example, LAAO activity was absent in both the Vermicella and Pseudonaja/Oxyuranus clade, supporting previously proposed relationships among these disparate taxa. There was no association between broad dietary categories and either enzyme activity. There was strong evidence for faster initial rates of change over evolutionary time for LAAO (delta parameter mean 0.2), but no such pattern in PLA2 (delta parameter mean 0.64). There were some exceptions to the phylogenetic patterns of enzyme activity: different PLA2 activity in the ecologically similar sister-species Denisonia devisi and D. maculata; large inter-specific differences in PLA2 activity in Hoplocephalus and Austrelaps. Conclusions We have shown that phylogeny is a stronger influence on venom enzyme activity than diet for two of the four major enzyme families present in snake venoms. PLA2 and LAAO activities had contrasting evolutionary dynamics with the higher delta value for PLA2 Some species/individuals lacked activity in one protein family suggesting that the loss of single protein family may not incur a significant fitness cost.

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.

The venom of the Russell’s viper (Daboia siamensis) contains neurotoxic and myotoxic phospholipase A2 toxins which can cause irreversible damage to motor nerve terminals. Due to the time delay between envenoming and antivenom administration, antivenoms may have limited efficacy against some of these venom components. Hence, there is a need for adjunct treatments to circumvent these limitations. In this study, we examined the efficacy of Chinese D. siamensis antivenom alone, and in combination with a PLA2 inhibitor, Varespladib, in reversing the in vitro neuromuscular blockade in the chick biventer cervicis nerve-muscle preparation. Pre-synaptic neurotoxicity and myotoxicity were not reversed by the addition of Chinese D. siamensis antivenom 30 or 60 min after venom (10 µg/mL). The prior addition of Varespladib prevented the neurotoxic and myotoxic activity of venom (10 µg/mL) and was also able to prevent further reductions in neuromuscular block and muscle twitches when added 60 min after venom. The addition of the combination of Varespladib and antivenom 60 min after venom failed to produce further improvements than Varespladib alone. This demonstrates that the window of time in which antivenom remains effective is relatively short compared to Varespladib and small-molecule inhibitors may be effective in abrogating some activities of Chinese D. siamensis venom.

Abstract 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 Background Thrombotic microangiopathy is an uncommon complication of snake envenoming associated with a subset of snakebite cases presenting with venom-induced consumption coagulopathy. It presents with microangiopathic haemolytic anaemia and thrombocytopenia. Available studies are predominantly small retrospective observational studies only. Renal end organ injury appears common. It has variably been likened to either thrombotic thrombocytopenic purpura or atypical haemolytic uraemic syndrome. Many studies report acute kidney injury, with dialysis required in a subset of patients. Some studies suggest a role for treatment with plasmapheresis. Interpretation of the literature is complicated by variable nomenclature and historically poor case definitions of both venom-induced consumption coagulopathy and thrombotic microangiopathy associated with snake envenoming. Methods The main objective of this systematic review is to synthesise the worldwide experience of snakebite-associated thrombotic microangiopathy with respect to its features and outcomes, and collate any evidence for the role of plasmapheresis as treatment. A predetermined case definition of confirmed or suspected snakebite with either blood film evidence of microangiopathic haemolytic anaemia or histological evidence of thrombotic microangiopathy will be used. This case definition will determine the search terms and study inclusion criteria. Relevant studies will be identified by electronic database, published conference abstracts, and grey literature search. This systematic review will be performed and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses checklist. The quality of included studies will be assessed by a proposed tool for evaluating the methodological quality of case reports and case series. Results will be reported by a descriptive synthesis with a focus on the presenting features; outcomes of acute kidney injury, dialysis-free survival, other end organ damage, and overall survival; and any evidence of a role for treatment with plasmapheresis. The quality of accumulated evidence will be assessed via the Grading of Recommendations, Assessment, Development, and Evaluations framework. Discussion It is anticipated that eligible studies will consist of small observational studies. Evidence gathered from this review will provide the first broader understanding of the clinical features, outcomes, prognosis, and management of snakebite-associated thrombotic microangiopathy. Systematic review registration PROSPERO CRD42019121436

Daboia siamensis (Russell’s viper) is a highly venomous and medically important snake in China, as well as much of Asia. There is minimal information on the pharmacological activity of the venom of the Chinese species, and currently no commercially available specific antivenom in China. This has led to the use of non-specific antivenoms to treat D. siamensis envenomation. In this study, the in vitro neurotoxicity and myotoxicity of D. siamensis venom was examined and the efficacy of four antivenoms was investigated, including the recently developed Chinese D. siamensis monovalent antivenom (C-DsMAV) and three commercially available antivenoms (Thai D. siamensis (Thai-DsMAV) monovalent antivenom, Deinagkistrodon acutus monovalent antivenom (DaAV), and Gloydius brevicaudus monovalent antivenom (GbAV). D. siamensis venom (10–30 µg/mL) caused the concentration-dependent inhibition of indirect twitches in the chick biventer cervicis nerve muscle preparation, without abolishing contractile responses to exogenous agonists ACh or CCh, indicating pre-synaptic neurotoxicity. Myotoxicity was also evident at these concentrations with inhibition of direct twitches, an increase in baseline tension, and the partial inhibition of ACh, CCh, and KCl responses. The prior addition of C-DsMAV or Thai-DsMAV prevented the neurotoxic and myotoxic activity of D. siamensis venom (10 µg/mL). The addition of non-specific antivenoms (GbAV and DaAV) partially prevented the neurotoxic activity of venom (10 µg/mL) but failed to neutralize the myotoxic effects. We have shown that D. siamensis venom exhibits in vitro weak presynaptic neurotoxicity and myotoxicity, which can be prevented by the pre-addition of the Chinese and Thai Russell’s viper antivenoms. Non-specific antivenoms were poorly efficacious. There should be further development of a monospecific antivenom against D. siamensis envenomation in China.

We studied the neutralisation of Sri Lankan Russell’s viper (Daboia russelii) and Australian mulga snake (Pseudechis australis) venom-induced myotoxicity by Indian (Vins and Bharat) and Australian (Seqirus) polyvalent antivenoms, using the in vitro chick biventer skeletal muscle preparation. Prior addition of Bharat or Vins antivenoms abolished D. russelii venom (30 µg/mL)-mediated inhibition of direct twitches, while Australian polyvalent antivenom was not protective. Bharat antivenom prevented, while Vins and Australian polyvalent antivenoms partially prevented, the inhibition of responses to exogenous KCl. Myotoxicity of Mulga venom (10 µg/mL) was fully neutralised by the prior addition of Australian polyvalent antivenom, partially neutralised by Vins antivenom but not by Bharat antivenom. Although the myotoxicity of both venoms was partially prevented by homologous antivenoms when added 5 min after the venom, with an increasing time delay between venom and antivenom, the reversal of myotoxicity gradually decreased. However, antivenoms partially prevented myotoxicity even 60 min after venom. The effect of antivenoms on already initiated myotoxicity was comparable to physical removal of the toxins by washing the bath at similar time points, indicating that the action of the antivenoms on myotoxicity is likely to be due to trapping the toxins or steric hindrance within the circulation, not allowing the toxins to reach target sites in muscles.

Despite antivenoms being the only established specific treatment for neuromuscular paralysis arising from snake envenoming, their ability to reverse the post-synaptic neurotoxicity in snake envenoming is poorly understood. We investigated the ability of five commercial antivenoms i.e., King cobra monovalent, Thai cobra monovalent, Thai neuro polyvalent, Indian polyvalent and Australian polyvalent antivenoms to reverse neurotoxicity induced by the venoms of King cobra (Ophiophagus hannah, 3 µg/mL), Indian cobra (Naja naja, 5 µg/mL) and Thai cobra (Naja kaouthia, 3 µg/mL) using the in vitro chick-biventer cervicis nerve–muscle preparation. All three venoms displayed post-synaptic neurotoxicity, which was prevented by all tested antivenoms (40 µL/mL) added to the bath prior to venom. All antivenoms partially reversed the established post-synaptic neuromuscular block after the addition of the three venoms during a 180 min observation period, but to varying degrees and at different rates. The neurotoxic effects of O. hannah venom recovered to a greater magnitude (based on twitch height restoration) and faster than the neurotoxicity of N. kaouthia venom, which recovered to a lower magnitude more slowly. The recovery of post-synaptic neurotoxicity by N. naja venom was hindered due to the likely presence of cytotoxins in the venom, which cause direct muscle damage. The observations made in this study provide further evidence that the commercial antivenoms are likely to actively reverse established α-neurotoxin-mediated neuromuscular paralysis in snake envenoming, and there is cross-neutralisation with different antivenoms.

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.

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 (9/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.

Bites by many Asiatic and African cobras (Genus: Naja) cause severe local dermonecrosis and myonecrosis, resulting in permanent disabilities. We studied the time scale in which two Indian polyvalent antivenoms, VINS and Bharat, remain capable of preventing or reversing in vitro myotoxicity induced by common cobra (Naja naja) venom from Sri Lanka using the chick biventer cervicis nerve-muscle preparation. VINS fully prevented while Bharat partially prevented (both in manufacturer recommended concentrations) the myotoxicity induced by Naja naja venom (10 µg/mL) when added to the organ baths before the venom. However, both antivenoms were unable to reverse the myotoxicity when added to organ baths 5 and 20 min post-venom. In contrast, physical removal of the venom from the organ baths by washing the preparation 5 and 20 min after the venom resulted in full and partial prevention of the myotoxicity, respectively, indicating the lag period for irreversible cellular injury. This suggests that, although the antivenoms contain antibodies against cytotoxins of the Sri Lankan Naja naja venom, they are either unable to reach the target sites as efficiently as the cytotoxins, unable to bind efficiently with the toxins at the target sites, or the binding with the toxins simply fails to prevent the toxin-target interactions.

Bungarus multicinctus, the Chinese krait, is a highly venomous elapid snake which causes considerable morbidity and mortality in southern China. B. multicinctus venom contains pre-synaptic PLA2 neurotoxins (i.e., β-bungarotoxins) and post-synaptic neurotoxins (i.e., α-bungarotoxins). We examined the in vitro neurotoxicity of B. multicinctus venom, and the efficacy of specific monovalent Chinese B. multicinctus antivenom, and Australian polyvalent elapid snake antivenom, against venom-induced neurotoxicity. B. multicinctus venom (1–10 μg/mL) abolished indirect twitches in the chick biventer cervicis nerve-muscle preparation as well as attenuating contractile responses to exogenous ACh and CCh, but not KCl. This indicates a post-synaptic neurotoxic action but myotoxicity was not evident. Given that post-synaptic α-neurotoxins have a more rapid onset than pre-synaptic neurotoxins, the activity of the latter in the whole venom will be masked. The prior addition of Chinese B. multicinctus antivenom (12 U/mL) or Australian polyvalent snake antivenom (15 U/mL), markedly attenuated the neurotoxic actions of B. multicinctus venom (3 μg/mL) and prevented the inhibition of contractile responses to ACh and CCh. The addition of B. multicinctus antivenom (60 U/mL), or Australian polyvalent snake antivenom (50 U/mL), at the t90 time point after the addition of B. multicinctus venom (3 μg/mL), did not restore the twitch height over 180 min. The earlier addition of B. multicinctus antivenom (60 U/mL), at the t20 or t50 time points, also failed to prevent the neurotoxic effects of the venom but did delay the time to abolish twitches based on a comparison of t90 values. Repeated washing of the preparation with physiological salt solution, commencing at the t20 time point, failed to reverse the neurotoxic effects of venom or delay the time to abolish twitches. This study showed that B. multicinctus venom displays marked in vitro neurotoxicity in a skeletal muscle preparation which is not reversed by antivenom. This does not appear to be related to antivenom efficacy, but due to the irreversible/pseudo-irreversible nature of the neurotoxins.

We read with interest the article by Manuwar et al [...]

Early diagnosis of snake envenomation is essential, especially neurotoxicity and myotoxicity. We investigated the diagnostic value of serum phospholipase (PLA2) in Australian snakebites. In total, 115 envenomated and 80 non-envenomated patients were recruited over 2 years, in which an early blood sample was available pre-antivenom. Serum samples were analyzed for secretory PLA2 activity using a Cayman sPLA2 assay kit (#765001 Cayman Chemical Company, Ann Arbor MI, USA). Venom concentrations were measured for snake identification using venom-specific enzyme immunoassay. The most common snakes were Pseudonaja spp. (33), Notechis scutatus (24), Pseudechis porphyriacus (19) and Tropidechis carinatus (17). There was a significant difference in median PLA2 activity between non-envenomated (9 nmol/min/mL; IQR: 7–11) and envenomated patients (19 nmol/min/mL; IQR: 10–66, p < 0.0001) but Pseudonaja spp. were not different to non-envenomated. There was a significant correlation between venom concentrations and PLA2 activity (r = 0.71; p < 0.0001). PLA2 activity was predictive for envenomation; area under the receiver-operating-characteristic curve (AUC-ROC), 0.79 (95% confidence intervals [95%CI]: 0.72–0.85), which improved with brown snakes excluded, AUC-ROC, 0.88 (95%CI: 0.82–0.94). A cut-point of 16 nmol/min/mL gives a sensitivity of 72% and specificity of 100% for Australian snakes, excluding Pseudonaja. PLA2 activity was a good early predictor of envenomation in most Australian elapid bites. A bedside PLA2 activity test has potential utility for early case identification but may not be useful for excluding envenomation.

Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10–15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.

The Chinese Cobra (Naja atra) is an elapid snake of major medical importance in southern China. We describe the in vitro neurotoxic, myotoxic, and cytotoxic effects of N. atra venom, as well as examining the efficacy of three Chinese monovalent antivenoms (N. atra antivenom, Gloydius brevicaudus antivenom and Deinagkistrodon acutus antivenom) and an Australian polyvalent snake antivenom. In the chick biventer cervicis nerve-muscle preparation, N. atra venom (1–10 µg/mL) abolished indirect twitches in a concentration-dependent manner, as well as abolishing contractile responses to exogenous acetylcholine chloride (ACh) and carbamylcholine chloride (CCh), indicative of post-synaptic neurotoxicity. Contractile responses to potassium chloride (KCl) were also significantly inhibited by venom indicating myotoxicity. The prior addition of Chinese N. atra antivenom (0.75 U/mL) or Australian polyvalent snake antivenom (3 U/mL), markedly attenuated the neurotoxic actions of venom (3 µg/mL) and prevented the inhibition of contractile responses to ACh, CCh, and KCl. The addition of Chinese antivenom (0.75 U/mL) or Australian polyvalent antivenom (3 U/mL) at the t90 time point after the addition of venom (3 µg/mL), partially reversed the inhibition of twitches and significantly reversed the venom-induced inhibition of responses to ACh and CCh, but had no significant effect on the response to KCl. Venom (30 µg/mL) also abolished direct twitches in the chick biventer cervicis nerve-muscle preparation and caused a significant increase in baseline tension, further indicative of myotoxicity. N. atra antivenom (4 U/mL) prevented the myotoxic effects of venom (30 µg/mL). However, G. brevicaudus antivenom (24 U/mL), D. acutus antivenom (8 U/mL) and Australian polyvalent snake antivenom (33 U/mL) were unable to prevent venom (30 µg/mL) induced myotoxicity. In the L6 rat skeletal muscle myoblast cell line, N. atra venom caused concentration-dependent inhibition of cell viability, with a half maximal inhibitory concentration (IC50) of 2.8 ± 0.48 μg/mL. N. atra antivenom significantly attenuated the cytotoxic effect of the venom, whereas Australian polyvalent snake antivenom was less effective but still attenuated the cytotoxic effects at lower venom concentrations. Neither G. brevicaudus antivenom or D. acutus antivenom were able to prevent the cytotoxicity. This study indicates that Chinese N. atra monovalent antivenom is efficacious against the neurotoxic, myotoxic and cytotoxic effects of N. atra venom but the clinical effectiveness of the antivenom is likely to be diminished, even if given early after envenoming. The use of Chinese viper antivenoms (i.e., G. brevicaudus and D. acutus antivenoms) in cases of envenoming by the Chinese cobra is not supported by the results of the current study.

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.

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.

Russell’s viper (Daboia russelii) venom causes a range of clinical effects in humans. Hypotension is an uncommon but severe complication of Russell’s viper envenoming. The mechanism(s) responsible for this effect are unclear. In this study, we examined the cardiovascular effects of Sri Lankan D. russelii venom in anaesthetised rats and in isolated mesenteric arteries. D. russelii venom (100 μg/kg, i.v.) caused a 45 ± 8% decrease in blood pressure within 10 min of administration in anaesthetised (100 μg/kg ketamine/xylazine 10:1 ratio, i.p.) rats. Venom (1 ng/mL–1 μg/mL) caused concentration-dependent relaxation (EC50 = 145.4 ± 63.6 ng/mL, Rmax = 92 ± 2%) in U46619 pre-contracted rat small mesenteric arteries mounted in a myograph. Vasorelaxant potency of venom was unchanged in the presence of the nitric oxide synthase inhibitor, L-NAME (100 µM), or removal of the endothelium. In the presence of high K+ (30 mM), the vasorelaxant response to venom was abolished. Similarly, blocking voltage-dependent (Kv: 4-aminopryidine; 1000 µM) and Ca2+-activated (KCa: tetraethylammonium (TEA; 1000 µM); SKCa: apamin (0.1 µM); IKCa: TRAM-34 (1 µM); BKCa; iberiotoxin (0.1 µM)) K+ channels markedly attenuated venom-induced relaxation. Responses were unchanged in the presence of the ATP-sensitive K+ channel blocker glibenclamide (10 µM), or H1 receptor antagonist, mepyramine (0.1 µM). Venom-induced vasorelaxtion was also markedly decreased in the presence of the transient receptor potential cation channel subfamily V member 4 (TRPV4) antagonist, RN-1734 (10 µM). In conclusion, D. russelii-venom-induced hypotension in rodents may be due to activation of Kv and KCa channels, leading to vasorelaxation predominantly via an endothelium-independent mechanism. Further investigation is required to identify the toxin(s) responsible for this effect.

Long-term effects of envenoming compromise the quality of life of the survivors of snakebite. We searched MEDLINE (from 1946) and EMBASE (from 1947) until October 2018 for clinical literature on the long-term effects of snake envenoming using different combinations of search terms. We classified conditions that last or appear more than six weeks following envenoming as long term or delayed effects of envenoming. Of 257 records identified, 51 articles describe the long-term effects of snake envenoming and were reviewed. Disability due to amputations, deformities, contracture formation, and chronic ulceration, rarely with malignant change, have resulted from local necrosis due to bites mainly from African and Asian cobras, and Central and South American Pit-vipers. Progression of acute kidney injury into chronic renal failure in Russell’s viper bites has been reported in several studies from India and Sri Lanka. Neuromuscular toxicity does not appear to result in long-term effects. Endocrine anomalies such as delayed manifestation of hypopituitarism following Russell’s viper bites have been reported. Delayed psychological effects such as depressive symptoms, post-traumatic stress disorder and somatisation have been reported. Blindness due to primary and secondary effects of venom is a serious, debilitating effect. In general, the available studies have linked a clinical effect to a snakebite in retrospect, hence lacked accurate snake authentication, details of acute management and baseline data and are unable to provide a detailed picture of clinical epidemiology of the long-term effects of envenoming. In the future, it will be important to follow cohorts of snakebite patients for a longer period of time to understand the true prevalence, severity, clinical progression and risk factors of long-term effects of snake envenoming.

Understanding snake venom pharmacokinetics is essential for developing risk assessment strategies and determining the optimal dose and timing of antivenom required to bind all venom in snakebite patients. This review aims to explore the current knowledge of snake venom pharmacokinetics in animals and humans. Literature searches were conducted using EMBASE (1974–present) and Medline (1946–present). For animals, 12 out of 520 initially identified studies met the inclusion criteria. In general, the disposition of snake venom was described by a two-compartment model consisting of a rapid distribution phase and a slow elimination phase, with half-lives of 5 to 48 min and 0.8 to 28 h, respectively, following rapid intravenous injection of the venoms or toxins. When the venoms or toxins were administered intramuscularly or subcutaneously, an initial absorption phase and slow elimination phase were observed. The bioavailability of venoms or toxins ranged from 4 to 81.5% following intramuscular administration and 60% following subcutaneous administration. The volume of distribution and the clearance varied between snake species. For humans, 24 out of 666 initially identified publications contained sufficient information and timed venom concentrations in the absence of antivenom therapy for data extraction. The data were extracted and modelled in NONMEM. A one-compartment model provided the best fit, with an elimination half-life of 9.71 ± 1.29 h. It is intended that the quantitative information provided in this review will provide a useful basis for future studies that address the pharmacokinetics of snakebite in humans.

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

There is limited information on the cross-neutralisation of neurotoxic venoms with antivenoms. Cross-neutralisation of the in vitro neurotoxicity of four Asian and four Australian snake venoms, four post-synaptic neurotoxins (α-bungarotoxin, α-elapitoxin-Nk2a, α-elapitoxin-Ppr1 and α-scutoxin; 100 nM) and one pre-synaptic neurotoxin (taipoxin; 100 nM) was studied with five antivenoms: Thai cobra antivenom (TCAV), death adder antivenom (DAAV), Thai neuro polyvalent antivenom (TNPAV), Indian Polyvalent antivenom (IPAV) and Australian polyvalent antivenom (APAV). The chick biventer cervicis nerve-muscle preparation was used for this study. Antivenom was added to the organ bath 20 min prior to venom. Pre- and post-synaptic neurotoxicity of Bungarus caeruleus and Bungarus fasciatus venoms was neutralised by all antivenoms except TCAV, which did not neutralise pre-synaptic activity. Post-synaptic neurotoxicity of Ophiophagus hannah was neutralised by all antivenoms, and Naja kaouthia by all antivenoms except IPAV. Pre- and post-synaptic neurotoxicity of Notechis scutatus was neutralised by all antivenoms, except TCAV, which only partially neutralised pre-synaptic activity. Pre- and post-synaptic neurotoxicity of Oxyuranus scutellatus was neutralised by TNPAV and APAV, but TCAV and IPAV only neutralised post-synaptic neurotoxicity. Post-synaptic neurotoxicity of Acanthophis antarcticus was neutralised by all antivenoms except IPAV. Pseudonaja textillis post-synaptic neurotoxicity was only neutralised by APAV. The α-neurotoxins were neutralised by TNPAV and APAV, and taipoxin by all antivenoms except IPAV. Antivenoms raised against venoms with post-synaptic neurotoxic activity (TCAV) cross-neutralised the post-synaptic activity of multiple snake venoms. Antivenoms raised against pre- and post-synaptic neurotoxic venoms (TNPAV, IPAV, APAV) cross-neutralised both activities of Asian and Australian venoms. While acknowledging the limitations of adding antivenom prior to venom in an in vitro preparation, cross-neutralization of neurotoxicity means that antivenoms from one region may be effective in other regions which do not have effective antivenoms. TCAV only neutralized post-synaptic neurotoxicity and is potentially useful in distinguishing pre-synaptic and post-synaptic effects in the chick biventer cervicis preparation.

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.