Current Treatment for Venom-Induced Consumption Coagulopathy Resulting from Snakebite

Venomous snakebite is considered the single most important cause of human injury from venomous animals worldwide. Coagulopathy is one of the commonest important systemic clinical syndromes and can be complicated by serious and life-threatening haemorrhage. Venom-induced consumption coagulopathy (VICC) is the commonest coagulopathy resulting from snakebite and occurs in envenoming by Viperid snakes, certain elapids, including Australian elapids, and a few Colubrid (rear fang) snakes. Procoagulant toxins activate the clotting pathway, causing a broad range of factor deficiencies depending on the particular procoagulant toxin in the snake venom. Diagnosis and monitoring of coagulopathy is problematic, particularly in resource-poor countries where further research is required to develop more reliable, cheap clotting tests. MEDLINE and EMBASE up to September 2013 were searched to identify clinical studies of snake envenoming with VICC. The UniPort database was searched for coagulant snake toxins. Despite preclinical studies demonstrating antivenom binding toxins (efficacy), there was less evidence to support clinical effectiveness of antivenom for VICC. There were no placebo-controlled trials of antivenom for VICC. There were 25 randomised comparative trials of antivenom for VICC, which compared two different antivenoms (ten studies), three different antivenoms (four), two or three different doses or repeat doses of antivenom (five), heparin treatment and antivenom (five), and intravenous immunoglobulin treatment and antivenom (one). There were 13 studies that compared two groups in which there was no randomisation, including studies with historical controls. There have been numerous observational studies of antivenom in VICC but with no comparison group. Most of the controlled trials were small, did not use the same method for assessing coagulopathy, varied the dose of antivenom, and did not provide complete details of the study design (primary outcomes, randomisation, and allocation concealment). Non-randomised trials including comparison groups without antivenom showed that antivenom was effective for some snakes (e.g., Echis), but not others (e.g., Australasian elapids). Antivenom is the major treatment for VICC, but there is currently little high-quality evidence to support effectiveness. Antivenom is not risk free, and adverse reactions can be quite common and potentially severe. Studies of heparin did not demonstrate it improved outcomes in VICC. Fresh frozen plasma appeared to speed the recovery of coagulopathy and should be considered in bleeding patients.     

Toxicological and immunological aspects of scorpion venom (Tytius pachyurus): neutralizing capacity of antivenoms produced in Latin America

The toxicity and immunochemical properties of Tityus pachyurus Pocock scorpion venom was characterized, as well as the neutralization capacity against it by three anti-scorpion antivenoms (Alacramyn, Instituto Bioclón, México; Suero antiescorpiónico, Instituto Butantán, Sao Paulo, Brasil; and Suero antiescorpiónico, Centro de Biotecnología, Universidad Central de Venezuela, Caracas, Venezuela). The venom yield, obtained by manual milking, 680+/-20 microg venom, a 50% lethal dose in mice was 4.8 microg/kg (90 microg for an 18-20 g mouse). The most common symptoms of venom poisoning in mice were sialorrhea, respiratory distress, profuse sweating, ataxia, behavior alterations (restlessness, somnolence) and hyperglycemia at 3 and 24 hours after subcutaneous venom injection (0.5 LD50). The neutralizing capacity of Bioclón (México City) and Butantán (Sao Paulo) antivenoms (for a 50% effective dose) was 330 and 292 microg venom/ml antivenom, respectively. The Biotecnología (Caracas) antivenom did not neutralize the lethal effect of venom. By electrophoresis (SDS-PAGE) was demonstrated that the venom contains proteins from less than 14 kd to 97 kd. The Western blots indicated immunological reactivity of the three antivenoms with most of venom components, including proteins of low molecular mass (<14 kd). The results allow to conclude that T. pachyurus venom is neutralized efficiently by anti-scorpion antivenoms produced in México and Brasil.     

The "echis carinatus venom" prothrombin assay in coumarin treated patients. A comparison with one-stage and immunological assays.

Prothrombin (factor II) was assayed in a group of coumarin treated patients using the Echis carinatus venom as thromboplastin. The levels obtained were comparable to those observed using the classical one-stage method. A good correlation was in fact observed between the two methods. The levels observed by the Echis carinatus method were definitely lower than those obtained using two immunological methods indicating that Echis carinatus venom activated, in our system, only normal prothrombin. However, even the levels obtained immunologically were slightly decreased, regardless of the method used, as compared to pooled normal plasma. In congenital prothrombin deficiency (homozygotes and heterozygotes) the level obtained by the Echis carinatus method was comparable to that observed by the one-stage method. On the contrary, in a congenital dysprothrombinemia (prothrombin Padua) a normal level was observed whereas the one-stage and two-stage methods yielded constantly levels of about 50% of normal.     

The effect of several viper venoms on prothrombin Padua.

The effect of four viper venoms (Oxyuranus scutellatus, Notechis scutatus scutatus, Echis carinatus, Naja nigricollis) on prothrombin Padua has been studied. Using Oxyuranus scutellatus venom and Notechis scutatus scutatus venom, prothrombin activity resulted to be moderately decreased similarly to what observed with other one-stage and two-stage methods. On the contrary, using Echis carinatus venom a normal level was obtained. No clotting was observed using the Naja nigricollis venom, regardless of the concentration used. The normal level of factor II obtained with Echis carinatus venom as compared with the low levels obtained with the other venoms, suggests that it acts on a different site of the prothrombin molecule.     

Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity.

In the presence of a procoagulant fraction (Echis carinatus procoagulant) isolated from the venom of the saw-scaled viper Echis carinatus sochureki, purified human prothrombin (P1) is completely converted to thrombin. The first step is the removal of an NH2-terminal peptide (F1) representing approximately one-third of the prothrombin molecule. The remaining peptide (P2) is then cleaved by the action of E.c. procoagulant to yield a two-chain, disulfide-bridged protein (P'2) which has the same molecular weight as P2. P'2 has enzymic (thrombin) activity, as evidence by incorporation of radiolabeled diisopropylphosphate into its heavy chain (TB), hydrolysis of p-toluenesulfonylarginine methyl ester, and clotting of fibrinogen. Relative to thrombin, its esterolytic activity greatly exceeds its clot-promoting activity. Examination of the polypeptide chains obtained by reducing P'2 has shown that its larger chain (TB) is indistinguishable from the heavy chain of thrombin. Its other chain (F2TA) consists of the light chain (TA) of thrombin bound by peptide linkage to the protion of the prothrombin molecule which had been adjacent to F1. Removal of this portion (F2) is catalyzed by thrombin (and, evidently, by P'2), but not by the E.c. procoagulant. When F2 is removed from P'2, the remaining two-chian protein is indistinguishable from thrombin by any of the criteria applied--molecular weight, subunit chain composition, or enzymic activity. Polyacrylamide gel electrophoresis was carried out in sodium dodecyl sulfate before and after disulfide reduction of samples generated in the presence and in the absence of diisopropylphosphorofluoridate, which inhibits thrombin but not the E.c. procoagulant. Such experiments showed that thrombin (and probably P'2), as well as E.c. procoagulant, catalyzes the release of F1. Furthermore, thrombin brings about the cleavage of F1 to yield a two-chain, disulfidebridged protein (F'1). These observations, particularly those made in the course of characterizine P'2, have led to the conclusion that cleavage of the peptide bond connecting the TA and TB portions of the prothrombin molecule (or its derivatives) produces a serine active center and, hence, a molecule possessing thrombin activity. This cleavage is catalyzed by the E.c. procoagulant but not by thrombon itself.     

Pre-clinical assays predict pan-African Echis viper efficacy for a species-specific antivenom

Background

Snakebite is a significant cause of death and disability in subsistent farming populations of sub-Saharan Africa. Antivenom is the most effective treatment of envenoming and is manufactured from IgG of venom-immunised horses/sheep but, because of complex fiscal reasons, there is a paucity of antivenom in sub-Saharan Africa. To address the plight of thousands of snakebite victims in savannah Nigeria, the EchiTAb Study Group organised the production, testing and delivery of antivenoms designed to treat envenoming by the most medically-important snakes in the region. The Echis saw-scaled vipers have a wide African distribution and medical importance. In an effort to maximise the clinical utility of scarce antivenom resources in Africa, we aimed to ascertain, at the pre-clinical level, to what extent the E. ocellatus-specific EchiTAbG antivenom, which was designed specifically for Nigeria, neutralised the lethal activity of venom from two other African species, E. pyramidum leakeyi and E. coloratus.

Methodology/Principal Findings

Despite apparently quite distinctive venom protein profiles, we observed extensive cross-species similarity in the immuno-reactivity profiles of Echis species-specific antisera. Using WHO standard pre-clinical in vivo tests, we determined that the monospecific EchiTAbG antivenom was as effective at neutralising the venom-induced lethal effects of E. pyramidum leakeyi and E. coloratus as it was against E. ocellatus venom. Under the restricted conditions of this assay, the antivenom was ineffective against the lethal effects of venom from the non-African Echis species, E. carinatus sochureki.

Conclusions/Significance

Using WHO-recommended pre-clinical tests we have demonstrated that the new anti-E. ocellatus monospecific antivenom EchiTAbG, developed in response to the considerable snakebite-induced mortality and morbidity in Nigeria, neutralised the lethal effects of venoms from Echis species representing each taxonomic group of this genus in Africa. This suggests that this monospecific antivenom has potential to treat envenoming by most, perhaps all, African Echis species.     

Venomics and antivenomics profiles of North African Cerastes cerastes and C. vipera populations reveals a potentially important therapeutic weakness

We report the proteomic analysis of the venom of the medically relevant snake, Cerastes cerastes, from Morocco, and the immunoreactivity profile of an experimental monospecific (CcMo_AV against Moroccan C. cerastes venom) and a commercial (Gamma-VIP against Tunisian C. cerastes and M. lebetina venoms) F(ab')(2) antivenoms towards geographic variants of C. cerastes and C. vipera venoms. The venom of C. cerastes is a low-complexity proteome composed of 25-30 toxins belonging to 6 protein families, mainly targetting the hemostatic system. This toxin arsenal explains the clinical picture observed in C. cerastes envenomings. Despite geographic compositional variation, the monospecific CcMo_AV and the Gamma-VIP divalent antivenom produced at Institut Pasteur de Tunis, showed similar immunocapturing capability towards Moroccan, Tunisian, and Egyptian C. cerastes venom proteins. Proteins partially escaping immunorecognition were all identified as PLA(2) molecules. Antivenomic analysis showed low degree of cross-reactivity of Moroccan CcMo_AV and Tunisian Gamma-VIP antivenoms towards C. vipera venom toxins. This study indicates that a more complete therapeutic cover could be achieved by including C. vipera venom in the formulation of venom immunization mixtures, thereby generating a pan-Cerastes antivenom.     

Inhibition of Sendai virus by various snake venom.

Viperid, elapid and crotalid snake venoms were screened in vitro for antiviral activity against Sendai virus. The hemolysis of 10(8) human erythrocytes in 1 ml, caused by 70 HAU of Sendai virus, was abolished when the virions were pretreated with 10 ug of the viperid venom of Echis coloratus, and was considerably diminished when pretreated with 10 ug of the venom of Echis carinatus sochureki, the cobra venoms of Naja atra and Naja nigricollis nigricollis. These venoms did not affect the erythrocytes but inhibited the virions themselves irreversibly. All other examined snake venoms had low or no antiviral activity. There was no correlation between the proteolytic and the antiviral activity of the venoms.     

Pulmonary fibrin microembolism with Echis carinatus venom in dogs: effects of a synthetic thrombin inhibitor

We produced pulmonary fibrin microembolism using an infusion of a prothrombin activator (Echis carinatus venom, 30 min, 0.5 NIH thrombin equivalent units/kg) in open-chest mongrel dogs. To determine the nonclotting effects of this venom on edemagenesis we infused an irreversible thrombin inhibitor, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK, 57 nmol X kg-1 X min-1 for 120 min), alone (n = 5) or with venom (Echis + PPACK, n = 5). The control group (n = 5) was given 1 ml of 0.9% NaCl. A decline in left atrial pressure (means +/- SE, 5.3 +/- 0.4 to 4.0 +/- 0.5 mmHg, P less than 0.05) and cardiac index (149 +/- 10 to 82 +/- 13 ml X min-1 X kg-1, P less than 0.01) in association with a marked increase in pulmonary arterial pressure (14.5 +/- 0.6 to 26.6 +/- 2.5 mmHg, P less than 0.001) and pulmonary vascular resistance (64 +/- 5 to 304 +/- 42 mmHg X ml-1 X min-1 X kg-1, P less than 0.001) was observed after 20 min of venom infusion. During this interval, pulmonary artery wedge pressure increased (4 +/- 1 to 12 +/- 4 mmHg, P less than 0.01) in four of eight animals. Fibrinogen declined below measurable levels and fibrin microemboli were seen in many pulmonary arterioles. These changes were not observed in the Echis + PPACK, PPACK, or control groups. Leukopenia and thrombocytopenia were observed in the Echis and Echis + PPACK groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment outcomes among snakebite patients in north-west Ethiopia—A retrospective analysis

BackgroundMillions of people are bitten by venomous snakes annually, causing high mortality and disability, but the true burden of this neglected health issue remains unknown. Since 2015, Médecins Sans Frontières has been treating snakebite patients in a field hospital in north-west Ethiopia. Due to the poor market situation for effective and safe antivenoms for Sub-Saharan Africa, preferred antivenom was not always available, forcing changes in choice of antivenom used. This study describes treatment outcomes and the effectiveness and safety of different antivenoms used.Methodology / Principal findingsThis retrospective observational study included 781 snakebite patients presenting at the field hospital between 2015 and 2019. Adjusted odds ratios, 95%-CI and p-values were used to compare the treatment outcome of patients treated with Fav-Afrique (n = 149), VacSera (n = 164), and EchiTAb-PLUS-ICP (n = 156) antivenom, and to identify the risk of adverse reactions for each antivenom. Whereas only incidental snakebite cases presented before 2015, after treatment was made available, cases rapidly increased to 1,431 in 2019. Envenomation was mainly attributed to North East African saw-scaled viper (Echis pyramidum) and puff adder (Bitis arietans). Patients treated with VacSera antivenom showed lower chance of uncomplicated treatment outcome (74.4%) compared to Fav-Afrique (93.2%) and EchiTAb-PLUS-ICP (90.4%). VacSera and EchiTAb-PLUS-ICP were associated with 16- and 6-fold adjusted odds of treatment reaction compared to Fav-Afrique, respectively, and VacSera was weakly associated with higher odds of death.Conclusions / SignificanceSnakebite frequency is grossly underreported unless treatment options are available. Although EchiTAb-PLUS-ICP showed favorable outcomes in this retrospective analysis, prospective randomized trials are needed to evaluate the effectiveness and safety of the most promising antivenoms for Sub-Saharan Africa. Structural investment in sustained production and supply of antivenom is urgently needed.     

Efficacy of IgG and F(ab')2 antivenoms to neutralize snake venom-induced local tissue damage as assessed by the proteomic analysis of wound exudate

Proteomic analysis of wound exudates represents a valuable tool to investigate tissue pathology and to assess the therapeutic success of various interventions. In this study, the ability of horse-derived IgG and F(ab')(2) antivenoms to neutralize local pathological effects induced by the venom of the snake Bothrops asper in mouse muscle was investigated by the proteomic analysis of exudates collected in the vicinity of affected tissue. In experiments involving the incubation of venom and antivenom prior to injection in mice, hemorrhagic activity was completely abolished and local muscle-damaging activity was significantly reduced by the antivenoms. In these conditions, the relative amounts of several intracellular and extracellular matrix proteins were reduced by the action of antivenoms, whereas the relative amounts of various plasma proteins were not modified. Because not all intracellular proteins were reduced, it is likely that there is a residual cytotoxicity not neutralized by antivenoms. In experiments designed to more closely reproduce the actual circumstances of envenoming, that is, when antivenom is administered after envenomation, the number of proteins whose amounts in exudates were reduced by antivenoms decreased, underscoring the difficulty in neutralizing local pathology due to the very rapid onset of venom-induced pathology. In these experiments, IgG antivenom was more efficient than F(ab')(2) antivenom when administered after envenomation, probably as a consequence of differences in their pharmacokinetic profiles.     

Pathology-specific experimental antivenoms for haemotoxic snakebite: The impact of immunogen diversity on the in vitro cross-reactivity and in vivo neutralisation of geographically diverse snake venoms

BackgroundSnakebite is a neglected tropical disease that causes high global rates of mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapeutic for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions.Methodology/Principal findingsIn this study we explored the feasibility of generating globally effective pathology-specific antivenoms to counteract the haemotoxic signs of snakebite envenoming. Two different immunogen mixtures, consisting of seven and twelve haemotoxic venoms sourced from geographically diverse and/or medically important snakes, were used to raise ovine polyclonal antibodies, prior to characterisation of their immunological binding characteristics and in vitro neutralisation profiles against each of the venoms. Despite variability of the immunogen mixtures, both experimental antivenoms exhibited broadly comparable in vitro venom binding and neutralisation profiles against the individual venom immunogens in immunological and functional assays. However, in vivo assessments using a murine preclinical model of antivenom efficacy revealed substantial differences in venom neutralisation. The experimental antivenom generated from the seven venom immunogen mixture outperformed the comparator, by providing protective effects against venom lethality caused by seven of the eight geographically diverse venoms tested, including three distinct venoms that were not used as immunogens to generate this antivenom. These findings suggest that a core set of venom immunogens may be sufficient to stimulate antibodies capable of broadly neutralising a geographically diverse array of haemotoxic snake venoms, and that adding additional venom immunogens may impact negatively on the dose efficacy of the resulting antivenom.Conclusions/SignificanceAlthough selection of appropriate immunogens that encapsulate venom toxin diversity without diluting antivenom potency remains challenging and further optimisation is required, the findings from this pilot study suggest that the generation of pathology-specific antivenoms with global utility is likely to feasible, thereby highlighting their promise as future modular treatments for the world’s tropical snakebite victims.     

Comparative biochemistry of disintegrins isolated from snake venom: consideration of the taxonomy and geographical distribution of snakes in the genus Echis

Species in the genus Echis have been classified mainly based on their morphological appearance and the analytical patterns of their serum. However, re-classification of the genus Echis has recently been suggested by taxonomists, toxicologists, and clinicians, since there have been problems with the current classification, such as the efficacy of antivenoms used for treating bites and the broad geographical distribution of Echis snakes. In this study, we purified five novel disintegrins, the platelet aggregation inhibitors pyramidin A and B from the venom of Echis pyramidum, ocellatin from the venom of Echis ocellatus, and leucogastin A and B from the venom of Echis leucogaster, to compare their sequences and allow us to re-evaluate the classification of various species in the genus Echis. Comparison of the amino acid sequences of five new and four known isolated disintegrins from snake venoms of six Echis species and their distribution strongly support the recent re-classification of the genus Echis.     

Proteins from Mucuna pruriens and enzymes from Echis carinatus venom: characterization and cross-reactions

Mucuna pruriens seeds have been widely used against snakebite in traditional medicine. The antivenin property of a water extract of seeds was assessed in vivo in mice. The serum of mice treated with extract was tested for its immunological properties. Two proteins of Echis carinatus venom with apparent molecular masses of 25 and 16 kDa were detected by Western blot analysis carried out using IgG of mice immunized with extract or its partially purified protein fractions. By enzymatic in-gel digestion and electrospray ionization-mass spectrometry/mass spectrometry analysis of immunoreactive venom proteins, phospholipase A(2,) the most toxic enzyme of snake venom, was identified. These results demonstrate that the observed antivenin activity has an immune mechanism. Antibodies of mice treated with non-lethal doses of venom reacted against some proteins of M. pruriens extract. Proteins of E. carinatus venom and M. pruriens extract have at least one epitope in common as confirmed by immunodiffusion assay.     

Bites by foreign venomous snakes in Britain.

In 1970-7 17 people in Britain were the victims of 32 bites by foreign venomous snakes. Crotalus atrox caused eight of these bites, Bitis arietans five, and the remaining 19 bites were caused by 12 different species. All the victims were bitten while handling the snake, and 24 bites were incurred by private individuals in their own homes. Poisoning was negligible in 17 of the 32 bites but life-threatening in at least two cases. Thus in the early stages snake bite may be unpredictable as a clinical problem. All victims of snake bite should be observed for at least 12 hours to assess the severity of poisoning and to ensure rational treatment. Local necrosis developed in six cases and resulted in prolonged illness in five of these cases; local incision was carried out and many have been a casual factor. Comprehensive stocks of antivenoms for treating bites by foreign venomous snakes are held by the National Health Services in Liverpool and London. Antivenom is indicated (a) for potentially serious systemic poisoning, as evidenced by hypotension, electrocardiographic changes, neurtrophilia, and acidosis (after viper or elapid bites), abnormal bleeding or non-clotting blood after viper bites; and ptosis or glossopharyngeal palsy after elapid bites; and (b) for bites from snakes whose venom causes local necrosis, to prevent or minimise this unpleasant complication. For effective antivenom treatment intravenous infusion is mandatory.     

Blood coagulation induced by the venom of Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator

In this paper, we show that the procoagulant action of Bothrops atrox venom is due in part to a protein component that activates prothrombin. The venom prothrombin activator was purified by ion-exchange chromatography and gel filtration. It was separated from a protease by affinity chromatography in a p-aminobenzamidine-CH-Sepharose column. It is a protein of about Mr 70,000, consisting of a single polypeptide chain. We have studied the kinetics of activation of prothrombin under different experimental conditions. The prothrombin activator from B. atrox venom is insensitive to reagents of serine and thiol proteases but is inactivated by ion chelators and by various divalent ions. These results suggest that it is a metalloenzyme. The prothrombin activator from B. atrox venom is inactive on the chromogenic substrates S-2337 and S-2238, and it is selective for prothrombin since it does not act on other blood coagulation factors such as fibrinogen and factor X. We have also studied the pattern of peptide cleavages produced in the human prothrombin molecule during the activation by the activator from B. atrox venom and compared it to that obtained with ecarin, a prothrombin activator from Echis carinatus venom. In the presence of thrombin inhibitors, e.g., hirudin, we found that the activators from B. atrox venom and ecarin act in a similar, or identical, manner by producing a thrombin intermediate, meizothrombin. In the absence of thrombin inhibitors, several peptides are generated, and alpha-thrombin is produced as a consequence of meizothrombin action.     

Snake venomics of Macrovipera mauritanica from Morocco, and assessment of the para-specific immunoreactivity of an experimental monospecific and a commercial antivenoms

Proteomic analysis of the venom of the medically relevant snake Macrovipera mauritanica from Morocco revealed a complex proteome composed of at least 45 toxins from 9 protein families targeting the hemostatic system of the prey or victim. The toxin profile of Moroccan M. mauritanica displays great similarity, but also worth noting departures, with the previously reported venom proteome of M. lebetina from Tunisia. Despite fine compositional differences between these Macrovipera taxa, their overall venom phenotypes explain the clinical picture observed in M. mauritanica and M. lebetina envenomings. However, M. mauritanica venom also contains significant amounts of orphan molecules whose presence in the venom seems to be difficult to rationalize in the context of a predator-prey arms race. The paraspecific immunoreactivity of an experimental monospecific (M. mauritanica) antivenom and a commercial bivalent antivenom, anti-C. cerastes and anti-M. lebetina, against the venoms of Moroccan M. mauritanica and Tunisian M. lebetina, was also investigated through an affinity chromatography-based antivenomics approach. Both antivenoms very efficiently immunodepleted homologous venom toxins and displayed a high degree of paraspecificity, suggesting the clinical utility of the two antivenoms for treating bites of both M. mauritanica or M. lebetina.     

Randomised Controlled Double-Blind Non-Inferiority Trial of Two Antivenoms for Saw-Scaled or Carpet Viper (Echis ocellatus) Envenoming in Nigeria

Background

In West Africa, envenoming by saw-scaled or carpet vipers (Echis ocellatus) causes great morbidity and mortality, but there is a crisis in supply of effective and affordable antivenom (ISRCTN01257358).

Methods

In a randomised, double-blind, controlled, non-inferiority trial, “EchiTAb Plus-ICP” (ET-Plus) equine antivenom made by Instituto Clodomiro Picado was compared to “EchiTAb G” (ET-G) ovine antivenom made by MicroPharm, which is the standard of care in Nigeria and was developed from the original EchiTAb-Fab introduced in 1998. Both are caprylic acid purified whole IgG antivenoms. ET-G is monospecific for Echis ocellatus antivenom (initial dose 1 vial) and ET-Plus is polyspecific for E. ocellatus, Naja nigricollis and Bitis arietans (initial dose 3 vials). Both had been screened by pre-clinical and preliminary clinical dose-finding and safety studies. Patients who presented with incoagulable blood, indicative of systemic envenoming by E. ocellatus, were recruited in Kaltungo, north-eastern Nigeria. Those eligible and consenting were randomly allocated with equal probability to receive ET-Plus or ET-G. The primary outcome was permanent restoration of blood coagulability 6 hours after the start of treatment, assessed by a simple whole blood clotting test repeated 6, 12, 18, 24 and 48 hr after treatment. Secondary (safety) outcomes were the incidences of anaphylactic, pyrogenic and late serum sickness-type antivenom reactions.

Findings

Initial doses permanently restored blood coagulability at 6 hours in 161/194 (83.0%) of ET-Plus and 156/206 (75.7%) of ET-G treated patients (Relative Risk [RR] 1.10 one-sided 95% CI lower limit 1.01; P = 0.05). ET-Plus caused early reactions on more occasions than did ET-G [50/194 (25.8%) and 39/206 (18.9%) respectively RR (1.36 one-sided 95% CI 1.86 upper limit; P = 0.06). These reactions were classified as severe in 21 (10.8%) and 11 (5.3%) of patients, respectively.

Conclusion

At these doses, ET-Plus was slightly more effective but ET-G was slightly safer. Both are recommended for treating E. ocellatus envenoming in Nigeria.

Trial Registration

Current Controlled Trials ISRCTN01257358     

Development of passive haemagglutination (PHA) and haemagglutination inhibition (HAI) technique for potency estimation of Cobra Antisnake Venom Serum (ASVS).

Antibodies against snake venom or antivenom potency are assayed quantitatively by in-vivo neutralization test in mice, which requires large number of laboratory animals. In potency assays of biological substances such as antivenoms, it is highly desirable to avoid suffering and death of animals by substituting in-vivo method with in-vitro methods, provided such methods measure life-saving capability with precision similar to that of in-vivo method. The in-vitro tests determine the neutralizing power of antivenom by permitting the evaluation of a particular biological activity of the venom and its neutralization after mixing the venom with the antivenom [Theakston RDG, Reid HA. Development of simple standard assay procedures for the characterization of snake venom. Bull WHO 1983;61:949-956; Gutierrez JM, Rojas G, Lomonte B, Gene JA, Chaves F, Alvarado J, et al. Standardizing of assays for testing the neutralizing ability of antivenoms. Toxicon 1990;28:1127-1129; Theakston R.D.G. Comments on letter of Gutierrez et al. on standardization of assays for testing the neutralizing ability of antivenoms. Toxicon 1990;28:1131-1132; Harvey AL, Barfaraz A, Thomson E, Faiz A, Preston S, Harris JB. Screening of snake venom for neurotoxic and myotoxic effects using simple in-vitro preparation from rodents and chicks. Toxicon 1994;32:257-265; World Health Organization Progress in characterization of venom and standardization of anti-venoms. Geneva: WHO offset publication; 1981. p. 58.]. Hence, the ideal requirements for an assay in detecting venom and venom antibody include high level of sensitivity, specificity (ability to differentiate between venom and venom antibody produced by closely related species of snakes), reproducibility and simplicity. A new in-vitro procedure for quantitative analysis of potency of ASVS by passive haemagglutination (PHA) and haemagglutination inhibition (HAI) has been explored. The methods described are simple, rapid, economical, reproducible and useful in replacing the more expensive in-vivo neutralization assays. Moreover, it also eliminates the use of laboratory animals.     

Crystal structure of echicetin from Echis carinatus (Indian saw-scaled viper) at 2.4A resolution

Echicetin is a heterodimeric protein from the venom of the Indian saw-scaled viper, Echis carinatus. It binds to platelet glycoprotein Ib (GPIb) and thus inhibits platelet aggregation. It has two subunits, alpha and beta, consisting of 131 and 123 amino acid residues, respectively. The two chains are linked with a disulphide bond. The level of amino acid sequence homology between two subunits is 50%. The protein was purified from the venom of E.carinatus and crystallized using ammonium sulphate as a precipitant. The crystal structure has been determined at 2.4A resolution and refined to an R-factor of 0.187. Overall dimensions of the heterodimer are approximately 80Ax35Ax35A. The backbone folds of the two subunits are similar. The central portions of the polypeptide chains of alpha and beta-subunits move into each other to form a tight dimeric association. The remaining portions of the chains of both subunits fold in a manner similar to those observed in the carbohydrate-binding domains of C-type lectins. In echicetin, the Ca(2+)-binding sites are not present, despite being topologically equivalent to other similar Ca(2+)-binding proteins of the superfamily. The residues Ser41, Glu43 and Glu47 in the calcium-binding proteins of the related family are conserved but the residues Glu126/120 are replaced by lysine at the corresponding sites in the alpha and beta-subunits.