Margaret A. O'Leary

Development of a sensitive enzyme immunoassay for measuring taipan venom in serum.

The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis.

Macrocyclic tetraamines from reaction of the (1,10-diamino-4,7-diazadecane(copper(II) cation with formaldehyde and the carbon acids nitroethane and diethylmalonate: variability in reactivity

Abstract Reaction of (1,10-diamino-4,7-diazadecane)copper(II) in basic methanol with formaldehyde and the carbon acid nitroethane leads to the facile and high-yielding synthesis of the (10-methyl-10-nitro-1,4,8,12-tetraazacyclopentadecane)copper(II) cation. The similar reaction with diethylmalonate, a weaker carbon acid, yields the (10,10-dicarboxyethyl-1,4,8,12-tetraazacyclopentadecane)copper(II) cation in only low yield.

Clinical Effects and Antivenom Dosing in Brown Snake (Pseudonaja spp.) Envenoming — Australian Snakebite Project (ASP-14)

Background Snakebite is a global health issue and treatment with antivenom continues to be problematic. Brown snakes (genus Pseudonaja) are the most medically important group of Australian snakes and there is controversy over the dose of brown snake antivenom. We aimed to investigate the clinical and laboratory features of definite brown snake (Pseudonaja spp.) envenoming, and determine the dose of antivenom required. Methods and Finding This was a prospective observational study of definite brown snake envenoming from the Australian Snakebite Project (ASP) based on snake identification or specific enzyme immunoassay for Pseudonaja venom. From January 2004 to January 2012 there were 149 definite brown snake bites [median age 42y (2–81y); 100 males]. Systemic envenoming occurred in 136 (88%) cases. All envenomed patients developed venom induced consumption coagulopathy (VICC), with complete VICC in 109 (80%) and partial VICC in 27 (20%). Systemic symptoms occurred in 61 (45%) and mild neurotoxicity in 2 (1%). Myotoxicity did not occur. Severe envenoming occurred in 51 patients (38%) and was characterised by collapse or hypotension (37), thrombotic microangiopathy (15), major haemorrhage (5), cardiac arrest (7) and death (6). The median peak venom concentration in 118 envenomed patients was 1.6 ng/mL (Range: 0.15–210 ng/mL). The median initial antivenom dose was 2 vials (Range: 1–40) in 128 patients receiving antivenom. There was no difference in INR recovery or clinical outcome between patients receiving one or more than one vial of antivenom. Free venom was not detected in 112/115 patients post-antivenom with only low concentrations (0.4 to 0.9 ng/ml) in three patients. Conclusions Envenoming by brown snakes causes VICC and over a third of patients had serious complications including major haemorrhage, collapse and microangiopathy. The results of this study support accumulating evidence that giving more than one vial of antivenom is unnecessary in brown snake envenoming.

Factor deficiencies in venom‐induced consumption coagulopathy resulting from Australian elapid envenomation: Australian Snakebite Project (ASP‐10)

Summary.  Background: Limited information exists on the dynamics of hemostasis in patients with venom‐induced consumption coagulopathy (VICC) from snake envenomation. Objective: The aim of the present study was to investigate specific factor deficiencies and their time course in Australasian elapid envenomation. Methods: We measured coagulation parameters and factor concentrations in patients recruited to the Australian Snakebite Project, an observational cohort study. There were 112 patients with complete VICC, defined as an international normalized ratio (INR) > 3, and 18 with partial VICC. Serial citrated plasma samples were collected from 0.5 to 60 h post‐bite. INR, activated partial thromboplastin time (aPTT), coagulation factors (F)I, II, V, VII, VIII, IX, X, von Willebrand factor antigen (VWF:Ag) and D‐dimer concentrations were measured. Results: Complete VICC was characterized by near/total depletion of fibrinogen, FV and FVIII, with an INR and aPTT that exceeded the upper limits of detection, within 2 h of snakebite. Prothrombin levels never fell below 60% of normal, suggesting that the toxins were rapidly eliminated or inactivated and re‐synthesis of clotting factors occurred irrespective of antivenom. Partial VICC caused limited depletion of fibrinogen and FV, and almost complete consumption of FVIII. Onset of VICC was more rapid with brown snake (Pseudonaja spp.) venom, which contains a group C prothrombin activator toxin, compared with the tiger snake group, which contains a group D prothrombin activator toxin and requires human FVa formation. Resolution of VICC occurred within 24–36 h irrespective of snake type. Conclusions: These results suggest that Australasian elapid prothrombin activators have a potent but short duration of action. Antivenom is unlikely to be administered in time to prevent VICC.

A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma

INTRODUCTION Assessment of the procoagulant effect of snake venoms is important for understanding their effects. The aim of this study was to develop a simple automated method to measure clotting times to assess procoagulant venoms. METHODS A turbidimetric assay was developed which monitors changes in optical density when plasma and venom are mixed. Plasma was added simultaneously to venom solutions in a 96 well microtitre plate. After mixing, the optical density at 340 nm was monitored in a microplate reader every 30 s over 30 min. The clotting time was defined as the lag time until the absorbance sharply increased. The turbidimetric method was compared to manual measurement of the clotting time defined as the time when a strand of fibrin can be drawn out of the mixture. The two methods were done simultaneously, with the same venom and plasma, and compared by plotting the manual versus turbidimetric clotting times. Within-day and between-day runs were done and the coefficient of variation (CV) was calculated. RESULTS Plots comparing manual clotting times to the lag time in the turbidimetric assay showed good correlation between the two methods for brown snake (Pseudonaja textilis) venom, including 24 determinations in triplicate over six days for seven different venom concentrations. Good correlation was also found for four other venoms: tiger snake (Notechis scutatus), Carpet viper (Echis carinatus), Russells viper (Daboia russelii) and Malaysian pit piper (Calloselasma rhodostoma). Between-day CV was in the range 10-20% for both methods, while within-day CV <10%. DISCUSSION The turbidimetric assay appears to be a simple and convenient automated method for the measurement of clotting times to assess the effects of procoagulant venoms.

Commercial monovalent antivenoms in Australia are polyvalent.

Monovalent antivenoms have a lower volume of specific antibodies that may reduce reactions but require accurate snake identification to be used. Polyvalent antivenoms are larger volume and may have a higher reaction rate. However, they avoid the problem of snake identification and may be more cost-effective to manufacture. We have previously shown cross-neutralisation of two Australian elapid venoms, tiger snake (Notechis scutatus) and brown snake (Pseudonaja textilis) venoms, by their respective monovalent antivenoms. In this study enzyme immunoassays were used to quantify the amount of monovalent antivenom (quantity of monovalent antibodies to a specific snake venom) in vials of commercially produced antivenom in Australia. All antivenoms tested appeared to be polyvalent and contain varying amounts of all five terrestrial snake monovalent antibodies based on their binding to the five representative venoms. Redback spider antivenom did not have any measurable binding affinity for any of the five snake venoms, showing that the observed binding is not due to non-specific interactions with equine protein. The antivenoms had expire dates over a 15 year period, suggesting that the antivenoms have been mixtures for at least this time. This study cannot be used to rationalise hospital stocks of antivenom in Australia because there is no guarantee that the antivenoms will remain as mixtures. However, it would be possible for the manufacturer to reduce the number of types of snake antivenoms available in Australia to two polyvalent antivenoms which would simplify treatment of snakebite.

Synthesis of a thirteen-membered tetra-azamacrocycle employing formaldehyde and nitroalkanes directed by metal ions. Crystal structures of (12-methyl-12-nitro-1,4,7,10-tetra-azacyclotridecane)copper(II) perchlorate and µ-chloro-1,1,1-trichloro-2-(12-methyl-12-nitro-1,4,7,10-tetra-azacyclotridecane)dicopper(II)

Reaction of formaldehyde and nitroethane or nitropropane with the copper(II) or nickel(II) complexes of 1,8-diamino-3,6-diazaoctane in methanol yields the complexed macrocycle 12-methyl-12-nitro-1,4,7,10-tetra-azacyclotridecane (L1) or the 12-ethyl analogue (L2). Both copper(II) and nickel(II) complexes were isolated as perchlorate salts. The latter exists in the singlet ground state, but tetrahydroborato and dithiocyanate nickel(II) compounds with triplet ground states were also isolated. The perchlorate salt of [Cu(L1)]2+, defined by a structure determination, consists of a linear polymer with bridging perchlorate groups occupying axial sites about a square plane of the macrocycle primary amines and the metal ion; Cu–N distances are 1.943(7) and 1.96(1)A, with Cu–O at 2.62(1) and 2.65(2)A. Slow crystallization of [Cu(L1)]2+ from chloride ion solution yielded a dark green complex of L1 which contained an equimolar amount of the [CuCl4]2– anion. An X-ray structure analysis defined the molecule as the binuclear neutral compound [Cu(L1)Cl(CuCl3)], with a single bridging chloride ion linking copper(II) ions in either a tetrahedron of chloride ions or a square-based pyramid of four nitrogen donors [Cu–N 2.006(4) and 2.022(3)A] with an apical chloride ion [Cu–Cl 2.507(2)A] which bridges to the second copper(II) ion. The Cu ⋯ Cu distance is 3.928(2)A, and there are no antiferromagnetic exchange interactions.

A pharmacological approach to first aid treatment for snakebite.

Snake venom toxins first transit the lymphatic system before entering the bloodstream. Ointment containing a nitric oxide donor, which impedes the intrinsic lymphatic pump, prolonged lymph transit time in rats and humans and also increased rat survival time after injection of venom. This pharmacological approach should give snakebite victims more time to obtain medical care and antivenom treatment.

Mulga snake (Pseudechis australis) envenoming: a spectrum of myotoxicity, anticoagulant coagulopathy, haemolysis and the role of early antivenom therapy - Australian Snakebite Project (ASP-19)

Context. Mulga snakes (Pseudechis australis) are venomous snakes with a wide distribution in Australia. Objective. The objective of this study was to describe mulga snake envenoming and the response of envenoming to antivenom therapy. Materials and methods. Definite mulga bites, based on expert identification or venom-specific enzyme immunoassay, were recruited from the Australian Snakebite Project. Demographics, information about the bite, clinical effects, laboratory investigations and antivenom treatment are recorded for all patients. Blood samples are collected to measure the serum venom concentrations pre- and post-antivenom therapy using enzyme immunoassay. Results. There were 17 patients with definite mulga snake bites. The median age was 37 years (6–70 years); 16 were male and six were snake handlers. Thirteen patients had systemic envenoming with non-specific systemic symptoms (11), anticoagulant coagulopathy (10), myotoxicity (7) and haemolysis (6). Antivenom was given to ten patients; the median dose was one vial (range, one–three vials). Three patients had systemic hypersensitivity reactions post-antivenom. Antivenom reversed the coagulopathy in all cases. Antivenom appeared to prevent myotoxicity in three patients with high venom concentrations, given antivenom within 2 h of the bite. Median peak venom concentration in 12 envenomed patients with samples was 29 ng/mL (range, 0.6–624 ng/mL). There was a good correlation between venom concentrations and the area under the curve of the creatine kinase for patients receiving antivenom after 2 h. Higher venom concentrations were also associated with coagulopathy and haemolysis. Venom was not detected after antivenom administration except in one patient who had a venom concentration of 8.3 ng/ml after one vial of antivenom, but immediate reversal of the coagulopathy. Discussion. Mulga snake envenoming is characterised by myotoxicity, anticoagulant coagulopathy and haemolysis, and has a spectrum of toxicity that is venom dose dependant. This study supports a dose of one vial of antivenom, given as soon as a systemic envenoming is identified, rather than waiting for the development of myotoxicity.

Endogenous Thrombin Potential as a novel method for the characterization of procoagulant snake venoms and the efficacy of antivenom

Venom-induced consumption coagulopathy occurs in snake envenoming worldwide but the interaction between procoagulant snake venoms and human coagulation remains poorly understood. We aimed to evaluate an assay using endogenous thrombin potential (ETP) to investigate the procoagulant properties of a range of Australian whole venoms in human plasma and compared this to traditional clotting and prothrombinase activity studies. We developed a novel modification of ETP using procoagulant snake venoms to trigger thrombin production. This was used to characterise the relative potency, calcium and clotting factor requirements of five important Australian snake venoms and efficacy of commercial antivenom, and compared this to prothrombinase activity and clotting assays. All five venoms initiated thrombin generation in the absence and presence of calcium. Pseudonaja textilis (Brown snake; p<0.0001), Hoplocephalus stephensii (Stephens-banded snake; p<0.0001) and Notechis scutatus (tiger snake; p=0.0073) all had statistically significant increases in ETP with calcium. Venom potency varied between assays, with ETP ranging from least potent with Oxyuranus scutellatus (Taipan) venom to intermediate with N. scutatus and H. stephensii venoms to most potent with P. textilis and Tropidechis carinatus (Rough-scale snake) venoms. ETPs for N. scutatus, T. carinatus and H. stephensii venoms were severely reduced with factor V deficient plasma. Antivenom neutralized the thrombin generating capacity but not prothrombin substrate cleaving ability of the venoms. Contrary to previous studies using clotting tests and factor Xa substrates, these venoms differ in calcium requirement. ETP is a useful assay to investigate mechanisms of other procoagulant venoms and is a robust method of assessing antivenom efficacy.