Venomous Snakes Research

Snake bites by European vipers in Mainland France in 2017-2018: comparison of two antivenoms Viperfav® and Viperatab®.

Context: Today, immunotherapy with Fab or F(ab’)2 fragments is considered as a gold standard treatment for patients bitten by vipers. We compared the efficiency of two antivenoms, Viperfav® and Viperatab®, in mainland France in 2017-2018 with data provided by the French poison control centre (PCC).Methods: Patients with a moderate (2a and 2b) or severe (3) envenomation after a European viper bite and treated with immunotherapy were included and the markers chosen were the risk of post-antivenom treatment worsening, duration of hospital stay and persistent functional discomfort on day 15. Statistical studies were based on multivariate data analysis.Results: Two hundred and ninety-seven cases were recorded. One hundred and eighty-two (61.3%) patients received Viperfav® and 115 (38.7%) received Viperatab®.

Compared to Viperfav®, use of Viperatab® significantly increased the risk of post-antivenom treatment worsening (OR* 12.05; 95%CI [3.11; 46.70]; p < .001). No significant difference between these antivenoms was recorded with respect to the duration of hospital stay and persistent functional discomfort on day 15. Viperfav® and Viperatab® have a similar tolerance (p > .21). Otherwise, duration of hospitalisation was significantly increased by a delay of immunotherapy infusion of more than 12 h (OR 2.70; 95%CI [1.45-5.06]; p = .002) or a preventive administration of LMWH (OR 6.55; 95%CI [1.58-27.13]; p=.02).Discussion: While Viperfav® and Viperatab® have a similar tolerance, our data show that Viperatab® was associated with a higher risk of post-antivenom treatment worsening compared to Viperfav®. Furthermore, this study confirms that the antivenom should be used as soon as possible. Indeed, patients receiving the immunotherapy infusion from the grade 2b presented significantly more frequent exacerbated symptoms (OR 3.99; 95%CI [1.16-13.73]; p=.028) after the antivenom infusion compared to grade 2a group.Conclusions: Whereas no significant difference between these antivenoms was recorded with respect to the duration of hospital stay and persistent functional discomfort on day 15, use of Viperatab®, compared to Viperfav®, significantly increased the risk of post-antivenom treatment worsening (OR* 12.05; 95%CI [3.11; 46.70]; p < .001). Taken together, these data show that Viperfav® is the treatment of choice for the management of snake bites in France.


Resistance of South American opossums to vWF-binding venom C-type lectins.

Opossums in the clade Didelphini are well known to be resistant to snake venom due to endogenous circulating inhibitors which target metalloproteinases and phospholipases.

However, the mechanisms through which these opossums cope with a variety of other damaging venom proteins are unknown. A protein involved in blood clotting (von Willebrand Factor) has been found to have undergone rapid adaptive evolution in venom-resistant opossums. This protein is a known target for a subset of snake venom C-type lectins (CTLs), which bind it and then induce it to bind platelets, causing hemostatic disruption. Several amino acid changes in vWF unique to these opossums could explain their resistance; however, experimental evidence that these changes disrupt venom CTL binding was lacking. We used platelet aggregation assays to quantify resistance to a venom-induced platelet response in two species of venom-resistant opossums (Didelphis virginiana, Didelphis aurita), and one venom-sensitive opossum (Monodelphis domestica). We found that all three species have lost nearly all their aggregation response to the venom CTLs tested.

Using washed platelet assays we showed that this loss of aggregation response is not due to inhibitors in the plasma, but rather to the failure of either vWF or platelets (or both) to respond to venom CTLs. These results demonstrate the potential adaptive function of a trait previously shown to be evolving under positive selection. Surprisingly, these findings also expand the list of potentially venom tolerant species to include Monodelphis domestica and suggest that an ecological relationship between opossums and vipers may be a broader driver of adaptive evolution across South American marsupials than previously thought.


Identifying the snake: First scoping review on practices of communities and healthcare providers confronted with snakebite across the world.

Snakebite envenoming is a major global health problem that kills or disables half a million people in the world’s poorest countries. Biting snake identification is key to understanding snakebite eco-epidemiology and optimizing its clinical management. The role of snakebite victims and healthcare providers in biting snake identification has not been studied globally.This scoping review aims to identify and characterize the practices in biting snake identification across the globe.

Epidemiological studies of snakebite in humans that provide information on biting snake identification were systematically searched in Web of Science and Pubmed from inception to 2nd February 2019. This search was further extended by snowball search, hand searching literature reviews, and using Google Scholar. Two independent reviewers screened publications and charted the data.

We analysed 150 publications reporting 33,827 snakebite cases across 35 countries. On average 70% of victims/bystanders spotted the snake responsible for the bite and 38% captured/killed it and brought it to the healthcare facility. This practice occurred in 30 countries with both fast-moving, active-foraging as well as more secretive snake species. Methods for identifying biting snakes included snake body examination, victim/bystander biting snake description, interpretation of clinical features, and laboratory tests.

In nine publications, a picture of the biting snake was taken and examined by snake experts. Snakes were identified at the species/genus level in only 18,065/33,827 (53%) snakebite cases. 106 misidentifications led to inadequate victim management.

The 8,885 biting snakes captured and identified were from 149 species including 71 (48%) non-venomous species.Snakebite victims and healthcare providers can play a central role in biting snake identification and novel approaches (e.g. photographing the snake, crowdsourcing) could help increase biting snake taxonomy collection to better understand snake ecology and snakebite epidemiology and ultimately improve snakebite management.


Plasmapheresis in a case of acute kidney injury with severe hemolysis and thrombocytopenia due to hematotoxic (Russell’s viper) snake bite.

We present a case of a male patient after being bitten by a vasculotoxic snake (Russell’s viper) with severe hemolysis, thrombocytopenia, and acute kidney injury requiring hemodialysis. As attempt to administer anti-snake venom (ASV) failed because of development of anaphylactic reaction, a single session of plasmapheresis was done to stop hemolysis and fall in platelets, which was refractory to all other measures and proved to be a lifesaving procedure in this patient.

The role of plasmapheresis in the management of snakebite victims is yet to be established, but can be beneficial in snake bite victims refractory to ASV or nonavailability of ASV or intolerant to ASV as in this case.


Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM).

We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.


Drysdalin, an antagonist of nicotinic acetylcholine receptors highlights the importance of functional rather than structural conservation of amino acid residues.

Snake venom neurotoxins are potent antagonists of nicotinic acetylcholine receptors (nAChRs). Here, we describe a novel member of class 3c long-chain neurotoxin drysdalin from the venom of Drysdalia coronoides. Drysdalin lacks three of the eight conserved classical functional residues critical for nAChRs interaction. Despite such a drastic alteration of the functional site, recombinant drysdalin showed irreversible postsynaptic neurotoxicity with nanomolar potency and selectively antagonizes the rodent muscle (α1)2β1δε, and human α7 and α9α10 nAChRs, but had no significant activity at the human α3β2, α3β4, α4β2, and α4β4 nAChRs. Substitution of Leu34 and Ala37 residues with the conserved Arg had minimal impact on the potency whereas conserved Phe replacement of residue Arg30 substantially reduced or abolished inhibitory activity. In contrast, truncation of the 24-residue long C-terminal tail leads to complete loss in (a) activity at α9α10 nAChR; and (b) irreversibility with reduced potency at the muscle and α7 nAChRs. Overall, the non-conserved Arg30 residue together with the uniquely long C-terminal tail contribute to the inhibitory activity of drysdalin at the nAChRs suggesting, at least for drysdalin, functional rather than sequence conservation plays a critical role in determining the activity of the toxin.


Experimental model for removal of snake venom via hemoperfusion in rats.

To examine the efficiency of hemoperfusion in removing South American rattlesnake (Crotalus durissus terrificus) venom from rats compared with neutralization by antivenom.An exploratory experimental investigation in rats involving the injection of snake venom with or without subsequent hemoperfusion or antivenom administration.

Basic animal research laboratory in a private university.

Normal, healthy male Wistar rats (0.29-0.40 kg, 3-6 months old) from a commercial breeder.Four experimental groups of randomly allocated rats (n = 3/group) were studied: Group 1: rats were injected with a single dose of venom (5 mg/kg, IM, in the right thigh) with no other treatment; blood samples were collected minutes before death to determine leukocyte, platelet, and erythrocyte counts; Group 2 (Control): rats underwent hemoperfusion alone for 60 min using a hemoperfusion cartridge designed for protein adsorption (by granulated charcoal) and protein precipitation (by tannic acid); Group 3 (Venom + antivenom): rats were injected with venom (5 mg/kg, IM) and, 10 min later, were treated with antivenom at the venom:antivenom ratio recommended by the manufacturer; Group 4 (Venom + hemoperfusion): Rats were injected with venom (5 mg/kg, IM) and, 10 min later, were hemoperfused for 60 min. In groups 2-4, blood samples were collected for leukocyte, platelet, and erythrocyte counts 24 h after venom.Rats injected with venom alone (Group 1) developed signs of neurotoxicity and ataxia and died in 9.0 ± 0.43 h but showed no changes in leukocyte or erythrocyte counts. In contrast, there were no deaths in groups 2-4. The lack of deaths in Groups 3 and 4 indicated that antivenom and hemoperfusion, respectively, protected against the lethal effects of the venom.Hemoperfusion with a double-action hemoperfusion cartridge capable of protein adsorption and precipitation protected rats against C. d. terrificus venom.


Development of a generic high-throughput screening assay for profiling snake venom protease activity after high-resolution chromatographic fractionation.

Snakebite causes upwards of 1.8 million envenomings, 138,000 deaths and 500,000 cases of long term morbidity each year.

Viper snake venoms (family Viperidae) generally contain a high proportion of proteases, which can cause devastating effects, such as hemorrhage, coagulopathy, edema, necrosis, and severe pain, in envenomed victims. In this study, analytical techniques were combined with enzymatic assays to develop a novel method for the detection of snake venom protease activity by using rhodamine-110-peptide substrate. In the so called at-line nanofractionation set up, crude venoms were first separated with reversed phase liquid chromatography, after which fractions were collected onto 384-well plates. Protease activity assays were then performed in the 384-well plates and bioassay chromatograms were constructed revealing protease activity. Parallel obtained UV absorbance, MS and proteomics data from a previous study facilitated toxin identification. The application of the rhodamine-110-peptide substrate assay showed significantly greater sensitivity compared to prior assays using casein-FITC as the substrate.

Moreover, cross referencing UV and MS data and resulted in the detection of a number of tentative proteases suspected to exhibit protease activity, including snake venom serine proteases from Calloselasma rhodostoma and Daboia russelli venom and a snake venom metalloproteinase from the venom of Echis ocellatus. Our data demonstrate that his methodology can be a useful tool for selectively identifying snake venom proteases, and can be applied to provide a better understanding of protease-induced pathologies and the development of novel therapeutics for treating snakebite.


BoaγPLI: Structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor.

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor’s presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory.

Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction.

The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.


An assessment of medical students’ proficiency in the diagnosis and management of snakebites: a cross-sectional study from Palestine.

Snakebites are emergent and life-threatening injuries that may require intensive care. Physicians face difficulties in dealing with snakebite injuries due to the knowledge gaps in the diagnosis and management of snakebites. The study aimed to assess medical students’ knowledge about the diagnosis and management of snakebite injuries, as well as their proficiency in first aid methods in case of snakebite and perception regarding snakes and snakebite injuries.


Methods


A cross-sectional study conducted among 200 medical students in their clinical years at An-Najah National University. A questionnaire was developed and distributed among those students. The questionnaire assessed the students’ knowledge regarding the diagnosis and management of snakebites and their attitude regarding snakes and snakebites. The total scores of knowledge were obtained and tested based on the participants’ demographic characteristics using the Kruskal-Wallis test and the Mann-Whitney U test. P-values of < 0.05 were considered to be statistically significant.


Results


The mean age of participating medical students was 22.2 ± 2.4 (year). Half of these medical students were in there final year of study (sixth year). After the analysis was done, we found a knowledge deficit in snakebite diagnosis and management among medical students. The mean knowledge scores regarding Vipera palaestinae, signs and symptoms, laboratory investigations, anti-venom, and first aid were 3.8/13, 8.2/16, 6.1/10, 3.6/11 and 8.3/15 respectively for medical students. It was found that medical students in higher years of study had a higher knowledge of laboratory investigation, and males were more knowledgeable in the correct way for first aid methods than females (p < 0.036).


The level of knowledge regarding the diagnosis and management of snakebites was not good enough among most of the students. In order to improve their knowledge, snakebite diagnosis and management should be introduced and focused on in medical curriculum.

What should be the training to cure snake bites?

Also, formal first aid training classes for medical students should be introduced in order to teach them the correct and updated methods of first aid as they will be the future health care providers and proper first aid will effectively decrease morbidity and mortality of snakebites.

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