Scorpion Stings and Antivenom Use in Arizona.

BACKGROUND: Arizona's rugged desert landscape harbors many venomous animals, including a small nocturnal scorpion, Centruroides sculpturatus, whose venom can cause severe neuromotor disturbance. An effective antivenom is available at selected health care facilities in the state. METHODS: We analyzed 4398 calls of scorpion stings to the Arizona Poison and Drug Information Center (APDIC) in Tucson over a period of 3 years, from January 2017 to December 2019. RESULTS: We followed 1952 (44.4%) of the victims to resolution. We excluded 2253 callers with minimal effects of the sting and 193 victims with possible toxic effects who were lost to follow-up. The most common complaints among callers were pain at the sting site in 88.9% and local numbness in 62.2%. Detailed clinical information was obtained from 593 calls from a health care facility. Neuromotor signs consistent with C. sculpuratus envenomation included nystagmus in 163 (27.5%), hypersalivation in 91 (15.3%), and fasciculations in 88 (14.8%). Antivenom (Anascorp; Rare Disease Therapeutics, Inc., Franklin, Tenn) was administered to 145 patients. Most were children <5 years old (n = 76, or 54.4%); 27 (18.6%) were 5-9 years old and 42 (30.0%) were ≥10 years of age. About half, 79 of 145 (54.5%) victims who received antivenom, met the APDIC recommended use criteria. CONCLUSIONS: Patients treated with antivenom exhibited a rapid resolution of symptoms without immediate or delayed hypersensitivity reactions. We recommend broadened availability of antivenom at sites where it is most needed.

Comparing clinical outcomes between two scorpion antivenom dosing strategies in children.

Background and Objectives: The clinical course from scorpion envenomation can range from mild to life threatening, particularly in younger children. The F(ab')2 antivenom currently available in the United States is extremely effective for countering the neurotoxic effects but extremely expensive. This dose comparison study assesses clinical outcomes between two antivenom dosing strategies. Methods: This was a retrospective review of medical records of pediatric patients treated in the pediatric emergency department (PED) with grade 3 or 4 envenomation requiring antivenom. Treatments rendered at two time-periods were assessed: 3-vial first dose (May 2007-August 2011) and single-vial-serial dose (September 2011-June 2016). Primary outcome was the proportion of patients who achieved complete symptom resolution within 4 h post antivenom dose. Results: One hundred and forty-one children met entry criteria, 76 in 3-vial first dose and 65 in single-vial-serial dose. Median age was 4 years (Q1:2-Q3:7), 56.2% males. There were no demographic and differences in clinical severity at presentation between the two dosing groups. All children, irrespective of group assignment, achieved the primary end-point of symptom resolution within 4 h. Median time to complete resolution of symptoms was longer for the single-vial-serial-dosing group vs. the 3-vial-first dose group [90 min (Q1:63-Q3:124) vs. 62 min (Q1:40-Q3:90), p = 0.002]. There were no statistically significant differences between the two groups regarding clinical outcomes including PED discharge, intubation, hospitalization, or death. Conclusion: In this retrospective analysis, children in both single-vial-serial dosing group, and 3-vial-full dosing group, achieved symptom resolution within 4 h of initiating therapy with no additional complications or adverse clinical outcomes.

Nanobodies as novel therapeutic agents in envenomation.

BACKGROUND: An effective therapy against envenoming should be a priority in view of the high number scorpion stings and snakebites. Serum therapy is still widely applied to treat the envenomation victims; however this approach suffers from several shortcomings. The employment of monoclonal antibodies might be an outcome as these molecules are at the core of a variety of applications from protein structure determination to cancer treatment. The progress of activities in the twilight zone between genetic and antibody engineering have led to the development of a unique class of antibody fragments. These molecules possess several benefits and lack many possible disadvantages over classical antibodies. Within recombinant antibody formats, nanobodies or single domain antigen binding fragments derived from heavy chain only antibodies in camelids occupy a privileged position. SCOPE OF REVIEW: In this paper we will briefly review the common methods of envenomation treatment and focus on details of various in vivo research activities that investigate the performance of recombinant, monoclonal nanobodies in venom neutralization. MAJOR CONCLUSIONS: Nanobodies bind to their cognate target with high specificity and affinity, they can be produced in large quantities from microbial expression systems and are very robust even when challenged with harsh environmental conditions. Upon administering, they rapidly distribute throughout the body and seem to be well tolerated in humans posing low immunogenicity. GENERAL SIGNIFICANCE: Scorpion and snake envenomation is a major issue in developing countries and nanobodies as a venom-neutralizing agent can be considered as a valuable and promising candidate in envenomation therapy.

Preparation of F(ab')2 antivenom in Iraq against scorpion (Hottentotta saulcyi) venom.

The aim of this study was to prepare specific F(ab')2 antivenom against Iraqi scorpion (Hottentotta saulcyi). Venom was obtained by electrical stimulation method, the scorpions venom storage capacity was 1.7 ±â€¯0.4 mg and LD50 found to be 1.07 mg/kg by subcutaneous (s.c) route. Three local horses aging 3-4 years were selected for immunization. During the schedule each horse received 0.5-14 mg venom in 49 days by s.c route. The horses immune response was monitored by Ouchterlony double immunodiffusion method, however, older horse A showed the highest antibody titer (1:1056576). Hyperimmune plasma was purified by modified World Health Organization (WHO) protocol using pepsin and ammonium sulfate, whereas high purity and total removal of albumin was detected by serum protein electrophoresis (SPEP) method on cellulose acetate. ED50 of the antivenom was determined via s.c route and found to be 83 LD50/ml, neutralizing 1.78 mg (venom)/ml (antivenom). Protein concentration of the antivenom was 30 mg/ml, less than limited value (100 mg/ml) by WHO. Abnormal toxicity test showed no abnormal signs when 0.2 ml of the antivenom injected intraperitoneally in mice.

Management of scorpion envenomation at the Faya-Largeau medical post, February-June, 2014.

Scorpion envenomation is common in northern Chad and associated with a high lethality rate. We report the management of 16 cases of scorpion envenomation in 2014 at our Faya-Largeau medical post. Our clinical experience revealed dissociated muscarinic symptoms in patients treated early in contrast to those treated later, who presented cardiogenic shock. In the absence of antivenom, patients with an isolated muscarinic syndrome received small doses of atropine, and their signs and symptoms improved afterwards. Although the use of atropine is controversial, the question here is about using it to treat muscarinic symptoms of scorpion envenomation in the absence of severe hypertension and with no signs of heart failure.

Centruroides sculpturatus envenomation in three adult patients requiring treatment with antivenom.

CONTEXT: Envenomation by Centruroides sculpturatus can manifest with cranial nerve dysfunction and neuromuscular hyperactivity. While these symptoms are most commonly seen in young children, they may also be seen in adults. CASE DETAILS: Three cases of adult patients are presented with grades III & IV scorpion envenomation. They reported symptoms including disconjugate, roving eye movements, and motor involvement. Also reported were hyposmia, difficulty with fine motor movements, and dysgeusia. All were first treated with benzodiazepines with little to no effect. They then received a three vial antivenom bolus with resolution of severe symptoms within 30-60 min. DISCUSSION: Severe Centruroides envenomation can occur in adults as well as children. These three cases demonstrate the usefulness, safety, and effectiveness of antivenom therapy to quickly relieve symptoms in adult patients with grades III & IV envenomations.

Curcumin Protects Mitochondria and Cardiomyocytes from Oxidative Damage and Apoptosis Induced by Hemiscorpius Lepturus Venom.

The main aim of the current study was to determine cardio-toxicity mechanisms of H. lepturus and protective effect of curcumin against this toxin in rats, using isolated heart mitochondria and cardiomyocytes. Our findings indicated that H. lepturus venom caused significantly ((P<0.05) cytotoxicity and caspase 3 activation in cardiomyocytes and mitochondrial dysfunction including increased mitochondrial ROS level, swelling in the mitochondria, decline in the mitochondria membrane potential (MMP), decrease in the cytochrome-c oxidase activity (complex IV), decrease ATP level and finally mitochondrial outer membrane (MOM) rupture in isolated mitochondria. Our results showed that the administration of curcumin efficiently decreased (P<0.05) cytotoxicity and caspase 3 activation, ROS formation, MMP collapse, mitochondrial swelling and mitochondrial outer membrane (MOM) rupture. Our findings suggest H. lepturus venom cusses a disruptive effect on mitochondrial respiratory chain, especially on complex II, and IV that predispose cardiomyocytes to ATP depletion and death signaling that could be protected with administration of curcumin.

Barbaloin inhibits ventricular arrhythmias in rabbits by modulating voltage-gated ion channels.

Barbaloin (10-ß-D-glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthracenone) is extracted from the aloe plant and has been reported to have anti-inflammatory, antitumor, antibacterial, and other biological activities. Here, we investigated the effects of barbaloin on cardiac electrophysiology, which has not been reported thus far. Cardiac action potentials (APs) and ionic currents were recorded in isolated rabbit ventricular myocytes using whole-cell patch-clamp technique. Additionally, the antiarrhythmic effect of barbaloin was examined in Langendorff-perfused rabbit hearts. In current-clamp recording, application of barbaloin (100 and 200 µmol/L) dose-dependently reduced the action potential duration (APD) and the maximum depolarization velocity (Vmax), and attenuated APD reverse-rate dependence (RRD) in ventricular myocytes. Furthermore, barbaloin (100 and 200 µmol/L) effectively eliminated ATX II-induced early afterdepolarizations (EADs) and Ca2+-induced delayed afterdepolarizations (DADs) in ventricular myocytes. In voltage-clamp recording, barbaloin (10-200 µmol/L) dose-dependently inhibited L-type calcium current (ICa.L) and peak sodium current (INa.P) with IC50 values of 137.06 and 559.80 µmol/L, respectively. Application of barbaloin (100, 200 µmol/L) decreased ATX II-enhanced late sodium current (INa.L) by 36.6%±3.3% and 71.8%±6.5%, respectively. However, barbaloin up to 800 µmol/L did not affect the inward rectifier potassium current (IK1) or the rapidly activated delayed rectifier potassium current (IKr) in ventricular myocytes. In Langendorff-perfused rabbit hearts, barbaloin (200 µmol/L) significantly inhibited aconitine-induced ventricular arrhythmias. These results demonstrate that barbaloin has potential as an antiarrhythmic drug.

Cost Minimization Analysis of Different Strategies of Management of Clinically Significant Scorpion Envenomation Among Pediatric Patients.

OBJECTIVE: Scorpion antivenom was recently approved for use in patients with clinically significant scorpion envenomation in the United States; no formal economic analysis on its impact on cost of management has been performed. METHODS: Three different strategies of management of scorpion envenomation with systemic neurotoxic symptoms in children were compared for cost minimization from a societal perspective. In strategy I, patients were managed with supportive care only without antivenom. In strategy II, an aggressive strategy of full-dose antivenom (initial dose of 3 vials with the use of additional vials administered 1 vial at a time) was considered. In strategy III, a single-vial serial antivenom dosing strategy titrated to clinical response was considered. Clinical probabilities for the different strategies were obtained from retrospective review of medical records of patients with scorpion envenomation over a 10-year period at our institution. Baseline cost values were obtained from patient reimbursement data from our institution. RESULTS: In baseline analysis, strategy I of supportive care only with no antivenom was least costly at US $3466.50/patient. Strategy III of single-vial serial dosing was intermediate but less expensive than strategy II of full-dose antivenom, with an incremental cost of US $3171.08 per patient. In a 1-way sensitivity analysis, at a threshold antivenom cost of US $1577.87, strategy III of single-vial serial dosing became the least costly strategy. CONCLUSIONS: For children with scorpion envenomation, use of a management strategy based on serial dosing of antivenom titrated to clinical response is less costly than a strategy of initial use of full-dose antivenom.

Mapping the Interaction Anatomy of BmP02 on Kv1.3 Channel.

The potassium channel Kv 1.3 plays a vital part in the activation of T lymphocytes and is an attractive pharmacological target for autoimmune diseases. BmP02, a 28-residue peptide isolated from Chinese scorpion (Buthus martensi Karsch) venom, is a potent and selective Kv1.3 channel blocker. However, the mechanism through which BmP02 recognizes and inhibits the Kv1.3 channel is still unclear. In the present study, a complex molecular model of Kv1.3-BmP02 was developed by docking analysis and molecular dynamics simulations. From these simulations, it appears the large ß-turn (residues 10-16) of BmP02 might be the binding interface with Kv 1.3. These results were confirmed by scanning alanine mutagenesis of BmP02, which identified His9, Lys11 and Lys13, which lie within BmP02's ß-turn, as key residues for interacting with Kv1.3. Based on these results and molecular modeling, two negatively charged residues of Kv1.3, D421 and D422, located in turret region, were predicted to act as the binding site for BmP02. Mutation of these residues reduced sensitivity of Kv 1.3 to BmP02 inhibition, suggesting that electrostatic interactions play a crucial role in Kv1.3-BmP02 interaction. This study revealed the molecular basis of Kv 1.3 recognition by BmP02 venom, and provides a novel interaction model for Kv channel-specific blocker complex, which may help guide future drug-design for Kv1.3-related channelopathies.

Biotechnological Trends in Spider and Scorpion Antivenom Development.

Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest in using bioactive toxins from spiders and scorpions for drug discovery purposes and for solving crystal structures of membrane-embedded receptors. Additionally, the identification and isolation of a myriad of spider and scorpion toxins has allowed research within next generation antivenoms to progress at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation immunization strategies. The increasing number of discovery and development efforts within this field may point towards an upcoming transition from serum-based antivenoms towards therapeutic solutions based on modern biotechnology.

Morphine blocks the Mesobuthus tamulus venom-induced augmentation of phenyldiguanide reflex and pulmonary edema in anesthetized rats.

OBJECTIVE: Pulmonary edema, a manifestation of scorpion envenomation syndrome, is attributed to cardiogenic or noncardiogenic factors. Morphine is a drug used for cardiogenic pulmonary edema and its effect on Mesobuthus tamulus (MBT) venom-induced changes is not known. Therefore, we hypothesized that morphine blocks the MBT venom-induced augmentation of phenyldiguanide (PDG) reflex and pulmonary edema. MATERIALS AND METHODS: Experiments were performed on anesthetized adult female rats. Trachea and jugular vein were cannulated, and the electrocardiographic potentials were recorded by connecting needle electrodes in limb lead II configuration. PDG (10 ΅g/kg, IV, bolus injection) responses were elicited by bolus injection initially, after saline/morphine (1 mg/kg) and after injecting MBT venom (100 µg/kg). The time-response area of the PDG-induced bradycardiac response after treatment was calculated as % of the initial PDG response area. At the end of experiments, lungs were excised for determination of pulmonary water content. RESULTS: PDG produced bradycardiac response that lasted for >60 s. MBT venom augmented the PDG reflex response by 2.5 times. In morphine pretreated group, augmentation of bradycardiac response induced by MBT venom was absent. MBT venom increased the pulmonary water content, and the increase was absent in morphine pretreated animals. CONCLUSION: The results reveal that morphine prevents the MBT venom-induced augmentation of PDG reflex response and pulmonary edema. Thus, morphine can be useful in scorpion envenomation syndrome associated with pulmonary edema.

Camelid antivenom development and potential in vivo neutralization of Hottentotta saulcyi scorpion venom.

Scorpion envenoming is a serious health problem which can cause a variety of clinical toxic effects. Of the many scorpion species native to Iran, Hottentotta saulcyi is important because its venom can produce toxic effects in man. Nowadays, antivenom derived from hyper immune horses is the only effective treatment for sever scorpion stings. Current limitations of immunotherapy urgently require an efficient alternative with high safety, target affinity and more promising venom neutralizing capability. Recently, heavy chain-only antibodies (HC-Abs) found naturally in camelid serum met the above mentioned advantages. In this study, immuno-reactivities of polyclonal antibodies were tested after successful immunization of camel using H. saulcyi scorpion crude venom. The lethal potency of scorpion venom in C57BL/6 mice injected intraperitoneally was determined to be 2.7 mg/kg. These results were followed by the efficient neutralization of lethal activity of H. saulcyi scorpion venom by injection of antivenom and purified IgG fractions into mice intraperitonelly or intravenously, respectively. HC-Ab camelid antivenom could be considered as a useful serotherapeutics instead of present treatment for scorpion envenomation.

Development of protective agent against Hottentotta saulcyi venom using camelid single-domain antibody.

Hottentotta saulcyi, medically important scorpion species, causes some of harmful toxic exposure in Iran. Administrated, conventional antivenom-based immunotherapy is still limited and hardly meet ideal characteristic of effective treatment for scorpion envenomation. In this study we aimed to develop a neutralizing agent directed against scorpion venom based on VHH, variable domain of the Camelidae heavy chain antibody or Nanobody. This promising biomolecule is well-established as an advantageous tool for therapeutic purposes due to its small size, stability, monomeric performance and less immunogenicity. In this study, a large Nb library was constructed and phage displayed after successful camel immunization using H. saulcyi scorpion crude venom. After a series of biopanning rounds on Sephadex G50 purified venom fraction and screening by monoclonal phage ELISA, the best reactive Nb was retrieved and designated Nb12. The selected Nb was then expressed as soluble protein in Escherichia coli, purified and confirmed by SDS-PAGE analysis and western blotting. The lead candidate Nb12 bound scorpion venom with Kaff value of 5×10(7)M(-1). Nb12 was shown to be capable of neutralizing 2 LD50 of whole venom of scorpion toxin when injected in the ratio of the Nb/toxin of 1.4:1 into C57BL/6 mice. In challenge experiment, Nb succeeded to rescue all i.p. lethal dose injected mice even when administrated i.v., 20min after envenoming. These results with ease of production and superior neutralizing activity make Nb a suitable anti-toxin candidate for treatment of scorpion envenoming.

Scorpion toxin BmK I directly activates Nav1.8 in primary sensory neurons to induce neuronal hyperexcitability in rats.

Voltage-gated sodium channels (VGSCs) in primary sensory neurons play a key role in transmitting pain signals to the central nervous system. BmK I, a site-3 sodium channel-specific toxin from scorpion Buthus martensi Karsch, induces pain behaviors in rats. However, the subtypes of VGSCs targeted by BmK I were not entirely clear. We therefore investigated the effects of BmK I on the current amplitude, gating and kinetic properties of Nav1.8, which is associated with neuronal hyperexcitability in DRG neurons. It was found that BmK I dose-dependently increased Nav1.8 current in small-sized (<25 µm) acutely dissociated DRG neurons, which correlated with its inhibition on both fast and slow inactivation. Moreover, voltage-dependent activation and steady-state inactivation curves of Nav1.8 were shifted in a hyperpolarized direction. Thus, BmK I reduced the threshold of neuronal excitability and increased action potential firing in DRG neurons. In conclusion, our data clearly demonstrated that BmK I modulated Nav1.8 remarkably, suggesting BmK I as a valuable probe for studying Nav1.8. And Nav1.8 is an important target related to BmK I-evoked pain.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified.

Evolution of alternative methodologies of scorpion antivenoms production.

Scorpionism represents a serious public health problem resulting in the death of children and debilitated individuals. Scorpion sting treatment employs various strategies including the use of specific medicines such as antiserum, especially for patients with severe symptoms. In 1909 Charles Todd described the production of an antiserum against the venom of the scorpion Buthus quinquestriatus. Based on Todd's work, researchers worldwide began producing antiserum using the same approach i.e., immunization of horses with crude venom as antigen. Despite achieving satisfactory results using this approach, researchers in this field have developed alternative approaches for the production of scorpion antivenom serum. In this review, we describe the work published by experts in toxinology to the development of scorpion venom antiserum. Methods and results describing the use of specific antigens, detoxified venom or toxins, purified toxins and or venom fractions, native toxoids, recombinant toxins, synthetic peptides, monoclonal and recombinant antibodies, and alternative animal models are presented.

Neolignans from Aristolochia elegans as antagonists of the neurotropic effect of scorpion venom.

ETHNOPHARMACOLOGICAL RELEVANCE: The high frequency of poisoning by sting or bite from venomous animals has begun to be a serious public health problem in Mexico where scorpion sting is the most common. Because of this, there is the need to seek active substances in plant species with an antagonistic effect against neurotropic activity of scorpion venom. The aim of this work was to demonstrate which of the compounds contained in the n-hexane extract from Aristolochia elegans roots display activity against scorpion venom. MATERIAL AND METHODS: Antagonist activity displayed by extract, fractions and isolated compounds obtained from Aristolochia elegans was guided by the inhibition of smooth muscle contraction induced by scorpion venom (Centruroides limpidus limpidus) in a model of isolated guinea pig ileum. The neolignans obtained from this extract were isolated and analyzed by chromatographic methods including HPLC. The chemical characterization of these compounds was performed by the analysis of (1)H and (13)C NMR spectra. RESULTS: The bio-guided chromatographic fractionation allowed us to isolate 4 known neolignans: Eupomatenoid-7 (1), licarin A (2), licarin B (3), eupomatenoid-1 (4) and other new neolignan which was characterized as 2-(3'-hydroxy-4'-methoxyphenyl)-3-methyl-5-[(E)-α-propen-γ-al]-7-methoxy-benzo [b] furan (5). This compound was named as eleganal. Compounds 1 and 2 were purified from the most active fraction AeF3 (EC50 of 149.9µg/mL, Emax of 65.66%). A doses-response analysis of eupomatenoid-7(1) and licarin A(2) allowed us to establish EC50 values (65.96µg/mL and 51.96µg/mL) respectively. CONCLUSIONS: The antagonistic effect against Centuroides limpidus limpidus scorpion venom displayed by the n-hexane extract from Aristolochia elegans roots is due to the presence of neolignans 1-2 contained in the fraction AeF3. Chemical analysis of fraction AeF2 allowed the isolation of a new compound which was identified as 2-(3'-hydroxy-4'-methoxyphenyl)-3-methyl-5-[(E)-α-propen-γ-al]-7-methoxy-benzo[b]furan (5), denominated as eleganal.