Recombinant snake antivenoms get closer to the clinic.

Snakebite envenomings kill ~100 000 victims each year and leave many more with permanent sequelae. Antivenoms have been available for more than 125 years but are in need of innovation. A new study by Khalek et al. highlights broadly neutralizing human monoclonal antibodies (mAbs) that might be used to develop recombinant antivenoms with superior therapeutic benefits.

A new 3D model of L929 fibroblasts microtissues uncovers the effects of Bothrops erythromelas venom and its antivenom.

In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities.

Systematic review and meta-analysis on the efficacy of Indian polyvalent antivenom against the Indian snakes of clinical significance.

Snakebite in India is a severe problem as it causes a mortality rate of 58,000 and a disability rate of 140,000 every year which is the highest among any other country. Antivenom is the primary therapy for snakebite, and its manufacturing techniques have essentially stayed unaltered for over a century. Indian polyvalent antivenom, a scientifically validated medicine for treating the toxic effects of snakebites, is available against the venom of the so-called Big Four snakes namely Spectacled cobra (Naja naja), Saw-scaled viper (Echis carinatus), Russell's viper (Daboia russelli) and the Common krait (Bungarus caeruleus), responsible for majority of the deaths in India. India hosts many other species of snakes, including cobras, kraits, saw-scaled vipers, sea snakes, and pit vipers, responsible for clinically severe envenomation. Neutralization strategy has been applied to access the efficacy of antivenoms, crucial for reducing snake bite deaths and disabilities. This review aims to conduct a systematic review and meta-analysis on the neutralization efficiency of the Polyvalent Antivenom (PAV) and focus on the factors that may contribute to the poor recognition of the antivenom towards the venom toxins. Reports focusing on the investigation of antivenom efficacy were searched and collected from several databases. Preclinical studies that reported the neutralization efficacy of the commercial antivenom against the medically important snakes of India were included. The articles were screened based on the inclusion criteria and 8 studies were shortlisted for meta-analysis. Pooled proportion was calculated for the antivenom efficacy reported by the studies and was found to be statistically significant with a 95% confidence interval. The heterogenicity in the venom toxicity and neutralization potency of the antivenom was evident in the overall estimate (proportion) and individual data. We provide comprehensive evidence on antivenom efficacy against medically important snakes from various parts of India which may aid in identifying the gaps in snake envenomation therapy and the need for novel potentially improved treatment of snakebites.

Successful use of anti-venom cross-neutralization effects in the clinical management of Shore Pit Viper envenomation.

As the landscape becomes more urbanized, snakebites have increasingly become uncommon presentations to the emergency departments in Singapore, while snakebites causing significant envenomation are even rarer. In this case report, we discuss a 55-year-old man who had significant envenomation from a Shore Pit Viper (Trimeresurus Purpureomaculatus) and who was successfully treated with haemato-toxic polyvalent antivenom (HPAV). He initially presented with pain, swelling and bleeding over his wound. Due to a deterioration in his coagulation profile, he was given two doses of HPAV which is typically reserved for viperid snakes instead. Following administration of the anti-venom, the patient's coagulation profile improved, and the local soft tissue effects of the venom resolved. He did not manifest any adverse effects and was discharged uneventfully about 72 h after the snakebite. The cross-neutralization potential of HPAV for Shore Pit Viper (Trimeresurus Purpureomaculatus) venom in this case study suggests that there may be a possible common underlying chemical structure and pathophysiology among the venom proteins of various snake species. Given that Trimeresurus-specific antivenom is unavailable in most countries, this cross-neutralization strategy deserves further consideration and evaluation in similar circumstances.

Vulnerability factors of snake bite patients in China.

OBJECTIVE: To analyze the vulnerability factors of snakebite patients in China. METHODS: Multi-stage random sampling was used as the main sampling method and snowball sampling as the auxiliary sampling method. The knowledge, attitude and behavior of snakebite among Chinese residents were investigated. Non-parametric test was used to compare the percentage differences in residents' knowledge, attitude and behavior of snakebite, and generalized linear regression analysis was used to analyze the influencing factors, and the vulnerability factors of snakebite patients were comprehensively analyzed. RESULTS: A total of 6338 subjects were included in this study, of which 68.4% were males, and 58.6% were farmers, workers and service personnel. The median total score of knowledge, attitude, and behavior was 26 (22,36). The patients who were improperly treated after injury were ligation proximal to the affected area (23.43%), squeezing (21.82%), and oral and suction wounds (8.74%). Did not go to hospital due to poverty (1351 cases) and did not receive antivenom (2068 cases). There were 21.32% and 32.63%, respectively. Among 4270 patients injected with antivenom 30.7% were vaccinated within 2 h. Among the patients who went to the hospital for treatment (4987), 75.0% arrived at the hospital within 6 h; Among the 4,761 patients who made emergency calls, 37.4% were treated within 0.5 h. CONCLUSIONS: Snakebite patients in China have weak knowledge about snakebite, low awareness of medical treatment, lack of correct prevention and emergency treatment measures, dependence on folk remedies, poor housing and so on. In addition, there are low availability of antivenoms and unreasonable distribution of medical resources in some areas of China. Multisectoral and multidisciplinary cooperation should be developed to prevent and control snakebites in order to reduce the burden caused by snakebites.

Snake venom toxins: Potential anticancer therapeutics.

Snake venom contains a cocktail of compounds dominated by proteins and peptides, which make up the toxin. The toxin components of snake venom attack several targets in the human body including the neuromuscular system, kidney and blood coagulation system and cause pathologies. As such, the venom toxins can be managed and used for the treatment of these diseases. In this regard, Captopril used in the treatment of cardiovascular diseases was the first animal venom toxin-based drug approved by the US Food and Drug Administration and the European Medicines Agency. Cancers cause morbidity and mortality worldwide. Due to side effects associated with the current cancer treatments including chemotherapy, radiotherapy, immunotherapy, hormonal therapy and surgery, there is a need to improve the efficacy of current treatments and/or develop novel drugs from natural sources including animal toxin-based drugs. There is a long history of earlier and ongoing studies implicating snake venom toxins as potential anticancer therapies. Here, we review the role of crude snake venoms and toxins including phospholipase A2, L-amino acid oxidase, C-type lectin and disintegrin as potential anticancer agents tested in cancer cell lines and animal tumour models in comparison to normal cell lines. Some of the anti-tumour activities of snake venom toxins include induction of cytotoxicity, apoptosis, cell cycle arrest and inhibition of metastasis, angiogenesis and tumour growth. We thus propose the advancement of multidisciplinary approaches to more pre-clinical and clinical studies for enhanced bioavailability and targeted delivery of snake venom toxin-based anticancer drugs.

Transient Hematotoxicity After Emerald Horned Pitviper (Ophryacus smaragdinus) Envenomation: A Case Report.

BACKGROUND: A minority of snake envenomations in the United States involve non-native snakes. In this report, we describe what we believe is the first documented human envenoming from an emerald horned pitviper, Ophryacus smaragdinus. CASE REPORT: A previously healthy 36-year-old woman was bitten on her left index finger by a captive emerald horned pitviper she was medicating at work. Swelling to the entire hand was present on emergency department arrival. She had no systemic symptoms and her initial laboratory studies were unremarkable. The affected limb was elevated. We administered five vials of Antivipmyn TRIⓇ (Bioclon), which specifically lists Ophryacus among the envenomations for which it is indicated. She developed pruritus and was treated with IV diphenhydramine and famotidine. Her swelling improved, but her repeat laboratory studies were notable for a platelet count of 102 K/µL and a fibrinogen level of 116 mg/dL. She declined additional antivenom because of the previous allergic reaction. She was admitted for further monitoring and pain control. Subsequent laboratory tests were better, but a small hemorrhagic bleb developed at the bite site. She was discharged the next day and followed up as an outpatient. Her swelling had resolved, her bleb had healed, and her laboratory studies continued to improve. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency physicians may be required to treat bites from non-native snakes. Many of these bites will warrant treatment with non-U.S. Food and Drug Administration-approved antivenoms. Consultation with a regional poison center or medical toxicologist may be necessary to procure the proper antivenom.

A Cytotoxicity Assay as an Alternative to the Murine Model for the Potency Testing of Bothrops jararaca Venom and Antivenom: An Intralaboratory Pre-validation Study.

Antivenom therapy is the only specific treatment for snakebite envenomation, and antivenom potency determination is key in the efficacy assurance quality control process. Nowadays, this process relies on the in vivo murine model - thus, the development of alternative in vitro methods is imperative. In the current study, the principle of the proposed method is the ability of Bothrops venom to induce cytotoxic effects in Vero cells, and the capacity to evaluate the inhibition of this cytotoxicity by the respective antivenom. After exposure to the venom/antivenom, the relative proportions of adherent (viable) cells were evaluated by direct staining with Coomassie Blue. The optical density (OD) of the lysed cell eluate was directly proportional to the number of adherent cells. This cytotoxicity-based alternative method could represent a potential candidate for validation as a replacement for the current in vivo test. The in vitro-determined cytotoxicity of the Brazilian Bothrops reference venom (expressed as the 50% effective concentration; EC50) was 3.61 µg/ml; the in vitro-determined 50% inhibitory concentration (IC50) of the Brazilian Bothrops reference antivenom was 0.133 µl/ml. From these two values, it was possible to calculate the potency of the reference antivenom. The results from the assays exhibited a good linear response, indicating that the method could be a potential candidate replacement method for use in antivenom quality control prior to lot release, subject to further validation.

Harnessing the Cross-Neutralisation Potential of Existing Antivenoms for Mitigating the Outcomes of Snakebite in Sub-Saharan Africa.

Over 32,000 individuals succumb to snake envenoming in sub-Saharan Africa (sSA) annually. This results from several factors, including a lack of antivenom products capable of neutralising the venoms of diverse snake species in this region. Most manufacturers produce polyvalent antivenoms targeting 3 to 16 clinically important snake species in sSA. However, specific products are unavailable for many others, especially those with a restricted geographic distribution. While next-generation antivenoms, comprising a cocktail of broadly neutralising antibodies, may offer an effective solution to this problem, given the need for their clinical validation, recombinant antivenoms are far from being available to snakebite victims. One of the strategies that could immediately address this issue involves harnessing the cross-neutralisation potential of existing products. Therefore, we assessed the neutralisation potency of PANAF-Premium antivenom towards the venoms of 14 medically important snakes from 13 countries across sSA for which specific antivenom products are unavailable. Preclinical assays in a murine model of snake envenoming revealed that the venoms of most snake species under investigation were effectively neutralised by this antivenom. Thus, this finding highlights the potential use of PANAF-Premium antivenom in treating bites from diverse snakes across sSA and the utility of harnessing the cross-neutralisation potential of antivenoms.

Phosphate-Buffered Saline and Dimethyl Sulfoxide Enhance the Antivenom Action of Ruthenium Chloride against Crotalus atrox Venom in Human Plasma-A Preliminary Report.

Ruthenium chloride (RuCl3) is widely utilized for synthesis and catalysis of numerous compounds in academia and industry and is utilized as a key molecule in a variety of compounds with medical applications. Interestingly, RuCl3 has been demonstrated to modulate human plasmatic coagulation and serves as a constituent of a compounded inorganic antivenom that neutralizes the coagulopathic effects of snake venom in vitro and in vivo. Using thrombelastography, this investigation sought to determine if RuCl3 inhibition of the fibrinogenolytic effects of Crotalus atrox venom could be modulated by vehicle composition in human plasma. Venom was exposed to RuCl3 in 0.9% NaCl, phosphate-buffered saline (PBS), or 0.9% NaCl containing 1% dimethyl sulfoxide (DMSO). RuCl3 inhibited venom-mediated delay in the onset of thrombus formation, decreased clot growth velocity, and decreased clot strength. PBS and DMSO enhanced the effects of RuCl3. It is concluded that while a Ru-based cation is responsible for significant inhibition of venom activity, a combination of Ru-based ions containing phosphate and DMSO enhances RuCl3-mediated venom inhibition. Additional investigation is indicated to determine what specific Ru-containing molecules cause venom inhibition and what other combinations of inorganic/organic compounds may enhance the antivenom effects of RuCl3.

Successful Management of Severe Unresponsive Snake Bite Envenomation Using Plasmapheresis and Corticosteroid at Egyptian National Environmental and Clinical Toxicology Research Center: A Case Report.

Snakebite is a significant public health issue in which venom-induced consumption coagulopathy is a common and serious complication that results from the activation of the coagulation pathway by snake toxins. We report a male patient, 56 y old, who was thought to have been bitten by a snake on his left foot. He was transported to a nearby hospital where he received analgesics and 3 snake polyvalent antivenom vials, and then he was transported to our hospital after 12 h. He presented with 2 small puncture wounds, pain, blistering, and edema of the left foot. On the 2nd day, the patient developed gingival bleeding and hematuria. Laboratory investigations upon admission revealed prothrombin time (PT) of more than 3 min, prothrombin concentration (PC) of less than 2.5%, and an international normalized ratio (INR) of 23.43. Further investigation of urine showed more than 100 RBCs. Despite receiving 16 packs of plasma and 40 snake polyvalent antivenom vials manufactured by VACSERA over 3 days, hemoglobin concentration and platelet count decreased with the appearance of jaundice, lactate dehydrogenase was 520, and reticulocytes were 3.5%. PT was more than 300 s, and INR was still over range. Plasmapheresis and corticosteroids were provided, which improved the patient's general condition, PT, PC, and INR, and the patient was discharged after 6 days of hospital stay. This case report indicated that plasmapheresis and corticosteroids were clinically efficient approaches in the management of snake envenomation unresponsive to antivenom.

Bridges between two medical realities: Perspectives of Indigenous medical and nursing students on snakebite care in the Brazilian Amazon.

In the Brazilian Amazon, snakebite envenomations (SBEs) disproportionately affect Indigenous populations, and have a significantly higher incidence and lethality than in non-Indigenous populations. This qualitative study describes the Indigenous and biomedical healthcare domains for SBE care from the perspective of the Indigenous medical and nursing students in Manaus, Western Brazilian Amazon. In-depth interviews were conducted with five Indigenous students from the Amazonas State University, between January and December 2021. The interviews were analyzed using inductive content analysis. We organized an explanatory model with five themes: (1) participants' identities; (2) causality levels in Indigenous and biomedical systems; (3) therapeutic itineraries in Indigenous and biomedical systems; (4) ideological implications of adding biomedical devices to Indigenous healing systems; and (5) therapeutic failure in and efficacy of Indigenous and biomedical systems. From a noncolonial perspective and seeking to increase the quality and acceptability of health care for the Indigenous populations of the Brazilian Amazon, the training of Indigenous health professionals presents itself as a promising strategy. For this goal, universities should serve as empowering settings for Indigenous health students that support them in their growth and development, raise their awareness of injustice, and catalyze change toward a culturally adapted and effective service for the users.

Snake Envenomation.

Snakebite envenomation (SBE) in horses can have devastating outcomes. Tissue damage, cardiotoxicity, coagulopathy, and neurotoxicity can be concerns with SBE. Understanding the actions of venom components is important in developing a successful treatment plan. Antivenom is the mainstay of treatment. Long-term deleterious effects can occur including cardiac dysfunction and lameness.

Experience of using expired lyophilized snake antivenom during a medical emergency situation in Lao People's Democratic Republic--A possible untapped resource to tackle antivenom shortage in Southeast Asia.

OBJECTIVES: To evaluate the safety and efficacy of expired lyophilized snake antivenom of Thai origin during a medical emergency in 2020/2021 in Lao People's Democratic Republic. METHODS: Observational case series of patients with potentially life-threatening envenoming who consented to the administration of expired antivenom between August 2020 and May 2022. RESULTS: A total of 31 patients received the expired antivenom. Malayan pit vipers (Calloselasma rhodostoma) were responsible for 26 (84%) cases and green pit vipers (Trimeresurus species) for two cases (6%). In three patients (10%) the responsible snake could not be identified. Of these, two presented with signs of neurotoxicity and one with coagulopathy. A total of 124 vials of expired antivenom were administered. Fifty-nine vials had expired 2-18 months earlier, 56 vials 19-36 months and nine vials 37-60 months before. Adverse effects of variable severity were observed in seven (23%) patients. All 31 patients fully recovered from systemic envenoming. CONCLUSIONS: Under closely controlled conditions and monitoring the use of expired snake antivenom proved to be effective and safe. Discarding this precious medication is an unnecessary waste, and it could be a valuable resource in ameliorating the current shortage of antivenom. Emergency use authorization granted by health authorities and preclinical testing of expired antivenoms could provide the support and legal basis for such an approach.

Production of recombinant scorpion antivenoms in E. coli: current state and perspectives.

Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms. KEY POINTS: • Commercial antivenoms consist of predominantly purified equine F(ab)'2fragments. • Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity. • Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.

Safe administration of Crotalidae equine immune F(ab')2 antivenom in a patient who suffered anaphylaxis from Crotalidae polyvalent immune Fab antivenom.

Allergic reactions to Crotalidae polyvalent immune Fab and Crotalidae immune F(ab')2 are uncommon but potentially life-threatening. It is unknown whether cross-reactivity reactions exist between these two antivenoms. We report a case of a patient who suffered anaphylaxis from Crotalidae polyvalent immune Fab but subsequently was safely administered Crotalidae immune F(ab')2 after a presumed Agkistrodon contortix (copperhead) envenomation. This single case supports the safety of Crotalidae immune F(ab')2 administration in patients with a history of anaphylaxis to Crotalidae polyvalent immune Fab treatment.

Comparing cost of F(.ab')2AV vs FabAV in the treatment of copperhead envenomation-One center's experience.

The definitive treatment of North American crotalid snakebites is antivenin. In 2000, an FabAV antivenom (CroFab®) was introduced and in 2022, F(ab')2AV (Anavip®) was approved for treatment of copperhead bites. Our center that sees primarily copperhead snake bites added the recently approved treatment as a second option for the 2022 snake bite season. This brief report we describe our initial experience with the two antivenins via retrospective chart review: the cost, charge, laboratory differences, response to therapy, complications and duration of hospitalization of admitted patients with copperhead envenomation. Using three independent reviewers in this IRB exempt report we found 31 patients with copperhead bites (7 exclusions) leaving 19 adults and 7 children for analysis. We found there was no difference in age, sex, presence of lab abnormalities, total vials administered, or length of stay. There was significant differences in hospital costs and charges to the patient. Future research should include multi-center experiences comparing the two antivenins.

Advanced Situation with Recombinant Toxins: Diversity, Production and Application Purposes.

Today, the production and use of various samples of recombinant protein/polypeptide toxins is known and is actively developing. This review presents state-of-the-art in research and development of such toxins and their mechanisms of action and useful properties that have allowed them to be implemented into practice to treat various medical conditions (including oncology and chronic inflammation applications) and diseases, as well as to identify novel compounds and to detoxify them by diverse approaches (including enzyme antidotes). Special attention is given to the problems and possibilities of the toxicity control of the obtained recombinant proteins. The recombinant prions are discussed in the frame of their possible detoxification by enzymes. The review discusses the feasibility of obtaining recombinant variants of toxins in the form of protein molecules modified with fluorescent proteins, affine sequences and genetic mutations, allowing us to investigate the mechanisms of toxins' bindings to their natural receptors.

Delayed Compression Paralysis Following an Iliopsoas Hematoma 30 Days After Saw-Scaled Viper (Echis carinatus sochureki) Envenoming: A Case Report.

Snakebite envenoming is a neglected tropical disease disproportionately affecting the rural and marginalized population in low-middle-income countries. The saw-scaled viper (Echis carinatus) is a clinically important snake that causes serious morbidity and mortality in the Indian subcontinent. Even though it is within the so-called big-four snakes against which polyvalent antivenom is available throughout India, reports of antivenom ineffectiveness are emerging in saw-scaled viper envenoming, especially around Jodhpur, Rajasthan, India. This case report highlights a patient with saw-scaled viper envenoming with an ineffective antivenom response complicated by acute kidney injury as well as local and systemic bleeding complications, which subsequently resulted in a pelvic hematoma that compressed the lumbosacral nerves, causing lower-limb weakness and sensory deficits. He was successfully managed with hematoma aspiration and supportive care. This case brings into focus the challenges of managing saw-scaled viper envenoming in this region with antivenom ineffectiveness, resulting in delayed and significant coagulopathy and its complications leading to prolonged hospital stay and morbidity. Our report spotlights less emphasized aspects of long-term morbidity in snakebite survivors, such as loss of working days and productivity. We also highlight the need for an organized system of long-term follow-up of snakebite survivors to screen for possible complications and manage them early.

Snakebite Envenoming in Avian Species: A Systematic Scoping Review and Practitioner Experience Survey.

Snakebite envenoming in avian species is infrequently reported in the veterinary literature, although perhaps not as rarely as recent publications suggest. A systematic scoping review was performed on the topic using PubMed and Google Scholar, 21 veterinary textbooks, and 139 conference proceedings. A practitioner experience survey was also performed, with recruitment from Facebook groups for exotic animal practitioners and professional organization email listservs. Only 31 texts met our inclusion/exclusion criteria, which meant they described clinicopathologic signs of snakebite envenomation in avian species, the treatment of snakebite envenomation in avian species, or expanded the geographic range or the number of captive avian and snake species involved. Reports included approximately 15-20 different species of both snakes and birds worldwide; however, no reports described clinicopathologic signs of naturally occurring snakebites from Asia, Australasia, or Europe. The few responses from our practitioner experience survey suggest that snakebite envenomation may be more common than previously reported. Clinical signs of snake envenomation in birds appear to depend on the snake species involved but often include local swelling and subcutaneous edema or hemorrhage with paired fang marks; weakness, bleeding, neurologic deficits, and death may follow. A wide variety of treatment protocols have been used to counter snakebite envenomation in birds, including the successful use of antivenom. Based on this body of evidence, much remains to be learned about snakebite envenomation of birds, particularly about the efficacy of different treatment protocols.