Variability in antivenom neutralization of Mexican viperid snake venoms.

BACKGROUND: Each year, 3,800 cases of snakebite envenomation are reported in Mexico, resulting in 35 fatalities. The only scientifically validated treatment for snakebites in Mexico is the use of antivenoms. Currently, two antivenoms are available in the market, with one in the developmental phase. These antivenoms, produced in horses, consist of F(ab')2 fragments generated using venoms from various species as immunogens. While previous studies primarily focused on neutralizing the venom of the Crotalus species, our study aims to assess the neutralization capacity of different antivenom batches against pit vipers from various genera in Mexico. METHODOLOGY: We conducted various biological and biochemical tests to characterize the venoms. Additionally, we performed neutralization tests using all three antivenoms to evaluate their effectiveness against lethal activity and their ability to neutralize proteolytic and fibrinogenolytic activities. RESULTS: Our results reveal significant differences in protein content and neutralizing capacity among different antivenoms and even between different batches of the same product. Notably, the venom of Crotalus atrox is poorly neutralized by all evaluated batches despite being the primary cause of envenomation in the country's northern region. Furthermore, even at the highest tested concentrations, no antivenom could neutralize the lethality of Metlapilcoatlus nummifer and Porthidium yucatanicum venoms. These findings highlight crucial areas for improving existing antivenoms and developing new products. CONCLUSION: Our research reveals variations in protein content and neutralizing potency among antivenoms, emphasizing the need for consistency in venom characteristics as immunogens. While Birmex neutralizes more LD50 per vial, Antivipmyn excels in specific neutralization. The inability of antivenoms to neutralize certain venoms, especially M. nummifer and P. yucatanicum, highlights crucial improvement opportunities, given the medical significance of these species.

Probable Transmission of SARS-CoV-2 from African Lion to Zoo Employees, Indiana, USA, 2021.

We describe animal-to-human transmission of SARS-CoV-2 in a zoo setting in Indiana, USA. A vaccinated African lion with physical limitations requiring hand feeding tested positive for SARS-CoV-2 after onset of respiratory signs. Zoo employees were screened, monitored prospectively for onset of symptoms, then rescreened as indicated; results were confirmed by using reverse transcription PCR and whole-genome virus sequencing when possible. Traceback investigation narrowed the source of infection to 1 of 6 persons. Three exposed employees subsequently had onset of symptoms, 2 with viral genomes identical to the lion's. Forward contact tracing investigation confirmed probable lion-to-human transmission. Close contact with large cats is a risk factor for bidirectional zoonotic SARS-CoV-2 transmission that should be considered when occupational health and biosecurity practices at zoos are designed and implemented. SARS-CoV-2 rapid testing and detection methods for big cats and other susceptible animals should be developed and validated to enable timely implementation of One Health investigations.

Single-Arm, Multicenter Phase I/II Clinical Trial for the Treatment of Envenomings by Massive Africanized Honey Bee Stings Using the Unique Apilic Antivenom.

We evaluated the safety, optimal dose, and preliminary effectiveness of a new-approach Africanized honeybee (Apis mellifera) Antivenom (AAV) in a phase I/II, multicenter, non-randomized, single-arm clinical trial involving 20 participants with multiple stings. Participants received 2 to 10 vials of AAV depending on the number of stings they suffered, or a predefined adjuvant, symptomatic, and complementary treatment. The primary safety endpoint was the occurrence of early adverse reactions within the first 24 h of treatment. Preliminary efficacy based on clinical evolution, including laboratory findings, was assessed at baseline and at various time points over the four following weeks. ELISA assays and mass spectrometry were used to estimate venom pharmacokinetics before, during, and after treatment. Twenty adult participants, i.e., 13 (65%) men and 7 (35%) women, with a median age of 44 years and a mean body surface area of 1.92 m2 (median = 1.93 m2) were recruited. The number of stings ranged from 7 to > 2,000, with a median of 52.5. Symptoms of envenoming were classified as mild, moderate, or severe in 80% (16), 15% (3), and 5% (1) of patients, respectively; patients with mild, moderate, or severe envenoming received 2, 6, and 10 vials of AAV as per the protocol. None of the patients had late reactions (serum sickness) within 30 d of treatment. There was no discontinuation of the protocol due to adverse events, and there were no serious adverse events. One patient had a moderate adverse event, transient itchy skin, and erythroderma. All participants completed the intravenous antivenom infusion within 2 h, and there was no loss to follow-up after discharge. ELISA assays showed venom (melittin and PLA2) concentrations varying between 0.25 and 1.479 ng/mL prior to treatment. Venom levels decreased in all patients during the hospitalization period. Surprisingly, in nine cases (45%), despite clinical recovery and the absence of symptoms, venom levels increased again during outpatient care 10 d after discharge. Mass spectrometry showed melittin in eight participants, 30 d after treatment. Considering the promising safety results for this investigational product in the treatment of massive Africanized honeybee attack, and its efficacy, reflected in the clinical improvements and corresponding immediate decrease in blood venom levels, the AAV has shown to be safe for human use. Clinical Trial Registration: UTN: U1111-1160-7011, identifier [RBR-3fthf8].

Single-arm, multicenter phase I/II clinical trial for the treatment of envenomings by massive africanized honey bee stings using the unique apilic antivenom

We evaluated the safety, optimal dose, and preliminary effectiveness of a new-approach Africanized honeybee (Apis mellifera) Antivenom (AAV) in a phase I/II, multicenter, non-randomized, single-arm clinical trial involving 20 participants with multiple stings. Participants received 2 to 10 vials of AAV depending on the number of stings they suffered, or a predefined adjuvant, symptomatic, and complementary treatment. The primary safety endpoint was the occurrence of early adverse reactions within the first 24 h of treatment. Preliminary efficacy based on clinical evolution, including laboratory findings, was assessed at baseline and at various time points over the four following weeks. ELISA assays and mass spectrometry were used to estimate venom pharmacokinetics before, during, and after treatment. Twenty adult participants, i.e., 13 (65%) men and 7 (35%) women, with a median age of 44 years and a mean body surface area of 1.92 m2 (median = 1.93 m2) were recruited. The number of stings ranged from 7 to > 2,000, with a median of 52.5. Symptoms of envenoming were classified as mild, moderate, or severe in 80% (16), 15% (3), and 5% (1) of patients, respectively; patients with mild, moderate, or severe envenoming received 2, 6, and 10 vials of AAV as per the protocol. None of the patients had late reactions (serum sickness) within 30 d of treatment. There was no discontinuation of the protocol due to adverse events, and there were no serious adverse events. One patient had a moderate adverse event, transient itchy skin, and erythroderma. All participants completed the intravenous antivenom infusion within 2 h, and there was no loss to follow-up after discharge. ELISA assays showed venom (melittin and PLA2) concentrations varying between 0.25 and 1.479 ng/mL prior to treatment. Venom levels decreased in all patients during the hospitalization period. Surprisingly, in nine cases (45%), despite clinical recovery and the absence of symptoms, venom levels increased again during outpatient care 10 d after discharge. Mass spectrometry showed melittin in eight participants, 30 d after treatment. Considering the promising safety results for this investigational product in the treatment of massive Africanized honeybee attack, and its efficacy, reflected in the clinical improvements and corresponding immediate decrease in blood venom levels, the AAV has shown to be safe for human use. Clinical Trial Registration: UTN: U1111-1160-7011, identifier [RBR-3fthf8].

Neotropical Rattlesnake (Crotalus simus) Venom Pharmacokinetics in Lymph and Blood Using an Ovine Model.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

The Bold and the Beautiful: a Neurotoxicity Comparison of New World Coral Snakes in the Micruroides and Micrurus Genera and Relative Neutralization by Antivenom.

Coral snake envenomations are well characterized to be lethally neurotoxic. Despite this, few multispecies, neurotoxicity and antivenom efficacy comparisons have been undertaken and only for the Micrurus genus; Micruroides has remained entirely uninvestigated. As the USA's supplier of antivenom has currently stopped production, alternative sources need to be explored. The Mexican manufacturer Bioclon uses species genetically related to USA species, thus we investigated the efficacy against Micrurus fulvius (eastern coral snake), the main species responsible for lethal envenomations in the USA as well as additional species from the Americas. The use of Coralmyn® coral snake antivenom was effective in neutralizing the neurotoxic effects exhibited by the venom of M. fulvius but was ineffective against the venoms of Micrurus tener, Micrurus spixii, Micrurus pyrrhocryptus, and Micruroides euryxanthus. Our results suggest that the Mexican antivenom may be clinically useful for the treatment of M. fulvius in the USA but may be of only limited efficacy against the other species studied.

A clinical trial protocol to treat massive Africanized honeybee (Apis mellifera) attack with a new apilic antivenom.

BACKGROUND: Envenomation caused by multiple stings from Africanized honeybees Apis mellifera constitutes a public health problem in the Americas. In 2015, the Brazilian Ministry of Health reported 13,597 accidents (incidence of seven cases per 100,000 inhabitants) with 39 deaths (lethality of 0.25%). The toxins present in the venom, which include melittin and phospholipase A2, cause lesions in diverse organs and systems that may be fatal. As there has been no specific treatment to date, management has been symptomatic and supportive only. METHODS: In order to evaluate the safety and neutralizing capacity of a new apilic antivenom, as well as to confirm its lowest effective dose, a clinical protocol was developed to be applied in a multicenter, non-randomized and open phase I/II clinical trial. Twenty participants with more than five stings, aged more than 18 years, of both sexes, who have not previously received the heterologous serum against bee stings, will be included for 24 months. The proposed dose was based on the antivenom neutralizing capacity and the number of stings. Treatment will be administered only in a hospital environment and the participants will be evaluated for a period up to 30 days after discharge for clinical and laboratory follow-up. RESULTS: This protocol, approved by the Brazilian regulatory agencies for ethics (National Commission for Ethics on Research - CONEP) and sanitation (National Health Surveillance Agency - ANVISA), is a guideline constituted by specific, adjuvant, symptomatic and complementary treatments, in addition to basic orientations for conducting a clinical trial involving heterologous sera. CONCLUSIONS: This is the first clinical trial protocol designed specifically to evaluate the preliminary efficacy and safety of a new antivenom against stings from the Africanized honeybee Apis mellifera. The results will support future studies to confirm a new treatment for massive bee attack that has a large impact on public health in the Americas.

Thrombelastographic characterization of the thrombin-like activity of Crotalus simus and Bothrops asper venoms.

: Annually, thousands suffer venomous snake-bite from Crotalus simus and Bothrops asper vipers in central and South America. The goals of the present study were to generally characterize the thrombin-like effects of venom from these snakes in human plasma with viscoelastic methods. Human plasma was exposed to the venom of three different C. simus subspecies and venoms obtained from B. asper vipers located in three different locations in Mexico. To characterize the factor X-activating and thrombin-like activity of these venoms, plasma (normal or factor XIII deficient) was pretreated with a variety of additives (e.g., heparin) in the absence or presence of calcium prior to exposure to 2.0 µg/ml of each viper's venom. These profiles were compared with plasma without venom that had contact activation of coagulation. Coagulation kinetics were determined with thrombelastography. All venoms had thrombin-like activity, with C. s. simus creating a slow growing, weak clot that was likely mediated by metalloproteinases. In contrast, B. asper venoms had rapid onset of coagulation and a high velocity of thrombus growth. Further, B. asper venom activity was calcium-independent, activated prothrombin, activated factor XIII, and independently polymerized fibrinogen. The viscoelastic methods used were able to differentiate subspecies of C. simus and specimens of B. asper, and provide insight into the mechanisms by which the venoms acted on plasma. These methods may be useful in the profiling of similar venoms and perhaps can assist in the assessment of interventions designed to treat envenomation (e.g., antivenom).

A clinical trial protocol to treat massive Africanized honeybee (Apis mellifera) attack with a new apilic antivenom

Background Envenomation caused by multiple stings from Africanized honeybees Apis mellifera constitutes a public health problem in the Americas. In 2015, the Brazilian Ministry of Health reported 13,597 accidents (incidence of seven cases per 100,000 inhabitants) with 39 deaths (lethality of 0.25%). The toxins present in the venom, which include melittin and phospholipase A2, cause lesions in diverse organs and systems that may be fatal. As there has been no specific treatment to date, management has been symptomatic and supportive only. Methods In order to evaluate the safety and neutralizing capacity of a new apilic antivenom, as well as to confirm its lowest effective dose, a clinical protocol was developed to be applied in a multicenter, non-randomized and open phase I/II clinical trial. Twenty participants with more than five stings, aged more than 18 years, of both sexes, who have not previously received the heterologous serum against bee stings, will be included for 24 months. The proposed dose was based on the antivenom neutralizing capacity and the number of stings. Treatment will be administered only in a hospital environment and the participants will be evaluated for a period up to 30 days after discharge for clinical and laboratory follow-up. Results This protocol, approved by the Brazilian regulatory agencies for ethics (National Commission for Ethics on Research - CONEP) and sanitation (National Health Surveillance Agency - ANVISA), is a guideline constituted by specific, adjuvant, symptomatic and complementary treatments, in addition to basic orientations for conducting a clinical trial involving heterologous sera. Conclusions This is the first clinical trial protocol designed specifically to evaluate the preliminary efficacy and safety of a new antivenom against stings from the Africanized honeybee Apis mellifera. The results will support future studies to confirm a new treatment for massive bee attack that has a large impact on public health in the Americas.(AU)

A clinical trial protocol to treat massive Africanized honeybee (Apis mellifera) attack with a new apilic antivenom

Abstract Background Envenomation caused by multiple stings from Africanized honeybees Apis mellifera constitutes a public health problem in the Americas. In 2015, the Brazilian Ministry of Health reported 13,597 accidents (incidence of seven cases per 100,000 inhabitants) with 39 deaths (lethality of 0.25%). The toxins present in the venom, which include melittin and phospholipase A2, cause lesions in diverse organs and systems that may be fatal. As there has been no specific treatment to date, management has been symptomatic and supportive only. Methods In order to evaluate the safety and neutralizing capacity of a new apilic antivenom, as well as to confirm its lowest effective dose, a clinical protocol was developed to be applied in a multicenter, non-randomized and open phase I/II clinical trial. Twenty participants with more than five stings, aged more than 18 years, of both sexes, who have not previously received the heterologous serum against bee stings, will be included for 24 months. The proposed dose was based on the antivenom neutralizing capacity and the number of stings. Treatment will be administered only in a hospital environment and the participants will be evaluated for a period up to 30 days after discharge for clinical and laboratory follow-up. Results This protocol, approved by the Brazilian regulatory agencies for ethics (National Commission for Ethics on Research CONEP) and sanitation (National Health Surveillance Agency ANVISA), is a guideline constituted by specific, adjuvant, symptomatic and complementary treatments, in addition to basic orientations for conducting a clinical trial involving heterologous sera. Conclusions This is the first clinical trial protocol designed specifically to evaluate the preliminary efficacy and safety of a new antivenom against stings from the Africanized honeybee Apis mellifera. The results will support future studies to confirm a new treatment for massive bee attack that has a large impact on public health in the Americas.

Venom yield and its relationship with body size and fang separation of pit vipers from Argentina.

The amount of venom that a snake can inject is related to its body size. The body size is related to head size and to the distance between fangs. To correlate snake body size, distance between fangs and distance between puncture wounds with the venom yield (and consequently with the venom dose potentially injected in a single snakebite), we studied these variables in two species of public health importance in South America, Bothrops (Rhinocerophis) alternatus, and Crotalus durissus terrificus. In all cases a positive correlation was observed between body length, fang separation distance, distance between puncture wounds and venom yield, with a regression coefficient over 0.5 for Bothrops alternatus and over 0.6 for Crotalus durissus terrificus in all cases, being the relation distance between punctures wounds and venom yield of 0.54 and 0.69 respectively. The difference between fang separation and puncture separation was never greater than 30%, with a mean difference around 13%. The strong relationships between body size, fang separation and venom yield may be useful for planning potential venom production in serpentariums. In addition, because puncture mark separation gives an approximate idea of the size of the snake, this provides a rough idea of the size of the snake that produced a bite and the potential amount of venom that could have been injected.

Biodistribution and Lymphatic Tracking of the Main Neurotoxin of Micrurus fulvius Venom by Molecular Imaging.

The venom of the Eastern coral snake Micrurus fulvius can cause respiratory paralysis in the bitten patient, which is attributable to ß-neurotoxins (ß-NTx). The aim of this work was to study the biodistribution and lymphatic tracking by molecular imaging of the main ß-NTx of M. fulvius venom. ß-NTx was bioconjugated with the chelator diethylenetriaminepenta-acetic acid (DTPA) and radiolabeled with the radionuclide Gallium-67. Radiolabeling efficiency was 60%-78%; radiochemical purity ≥92%; and stability at 48 h ≥ 85%. The median lethal dose (LD50) and PLA2 activity of bioconjugated ß-NTx decreased 3 and 2.5 times, respectively, in comparison with native ß-NTx. The immune recognition by polyclonal antibodies decreased 10 times. Biodistribution of ß-NTx-DTPA-(67)Ga in rats showed increased uptake in popliteal, lumbar nodes and kidneys that was not observed with (67)Ga-free. Accumulation in organs at 24 h was less than 1%, except for kidneys, where the average was 3.7%. The inoculation site works as a depot, since 10% of the initial dose of ß-NTx-DTPA-(67)Ga remains there for up to 48 h. This work clearly demonstrates the lymphatic system participation in the biodistribution of ß-NTx-DTPA-(67)Ga. Our approach could be applied to analyze the role of the lymphatic system in snakebite for a better understanding of envenoming.

Iron and carbon monoxide attenuate Crotalus atrox venom-enhanced tissue-type plasminogen activator-initiated fibrinolysis.

In addition to degrading fibrinogen as a source of consumptive coagulopathy, rattlesnake venom has also been demonstrated to enhance fibrinolysis and degrade alpha-2-antiplasmin. The goals of this investigation was to characterize the kinetic fibrinolytic profile of Crotalus atrox venom in the absence and presence of tissue-type plasminogen activator (tPA), and to also ascertain if iron and carbon monoxide (CO, a positive modulator of alpha-2-antiplasmin) could attenuate venom-enhanced fibrinolysis. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to C. atrox venom (0-2 µg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, either separately or in combination, plasma was exposed to iron (ferric chloride, 10 µmol/l) or CO (carbon monoxide-releasing molecule-2, 100 µmol/l) prior to incubation with venom; the plasma sample was subsequently subjected to thrombelastographic analysis with addition of tPA. Venom exposure in the absence of tPA did not result in detectable fibrinolysis. In the presence of tPA, venom markedly enhanced fibrinolysis. Iron and CO, markedly attenuated venom enhancement of fibrinolysis. C. atrox venom enhances tPA-mediated fibrinolysis, and interventions that enhance/protect alpha-2-antiplasmin activity significantly attenuate venom-enhanced fibrinolysis. Future preclinical investigation is required to determine if iron and CO can attenuate venom-mediated degradation of alpha-2-antiplasmin-dependent fibrinolytic resistance.

Iron and carbon monoxide attenuate degradation of plasmatic coagulation by Crotalus atrox venom.

Hypofibrinogenemia is an important clinical consequence following envenomation by Crotalus species, usually attenuated or prevented by administration of antivenom. It has been determined that iron and carbon monoxide (CO) enhance fibrinogen as a thrombin substrate, likely secondary to conformational changes in molecular structure. We tested the hypothesis that pretreatment of plasma with iron and CO could attenuate the effects of exposure to Crotalus atrox venom. Human plasma was exposed to 0 to 10 µmol/l ferric chloride (iron source) and 0 to 100 µmol/l CO-releasing molecule-2 (CO source) followed by exposure to 0 to 0.5 µg/ml venom for 5 to 20 min. Changes in coagulation kinetics were determined with thrombelastography. Iron and CO significantly attenuated venom-mediated degradation of plasmatic coagulation in terms of onset time, velocity of clot growth and final clot strength. Further preclinical investigation of iron and CO administration as a 'bridge-to-antivenom' to preserve plasmatic coagulation is justified.