Factors influencing the quality of Myrmecia pilosula (Jack Jumper) ant venom for use in in vitro and in vivo diagnoses of allergen sensitization and in allergen immunotherapy.

BACKGROUND: Allergen immunotherapy uses pharmaceutical preparations derived from naturally occurring source materials, which contain water-soluble allergenic components responsible for allergic reactions. The success of in vivo and in vitro diagnoses in allergen sensitization and allergen immunotherapy largely depends on the quality, composition and uniformity of allergenic materials used to produce the active ingredients, and the formulation employed to prepare finished products. OBJECTIVES: We aimed to examine the factors influencing batch-to-batch consistency of Jack Jumper (Myrmecia pilosula) ant venom (JJAV) in the form of active pharmaceutical ingredient (AI) and informed whether factors such as temperature, artificial light and container materials influence the quality of JJAV AIs. We also aimed to establish handling and storage requirements of JJAV AIs to ensure preservation of allergenic activities during usage in the diagnosis of allergen sensitization and in allergen immunotherapy. METHODS: The quality and consistency of JJAV AIs were analysed using a combination of bicinchoninic acid assay for total protein quantification, HPLC-UV for JJAV allergen peptides quantification, ELISA inhibition for total allergenic potency, SDS-PAGE, AU-PAGE and immunoblot for qualitative assessment of JJAV components, and Limulus Amebocyte Lysate assay for the quantification of endotoxin concentration. API-ZYM and Zymogram assays were used to probe the presence of enzymatic activities in JJAV. RESULTS: Pharmaceutical-grade JJAV for allergen immunotherapy has good batch-to-batch consistency. Temporary storage at 4°C and light exposure do not affect the quality of JJAV. Exposure to temperature above 40°C degrades high MW allergens in JJAV. Vials containing JJAV must be stored frozen and in upright position during long-term storage. CONCLUSIONS AND CLINICAL RELEVANCE: We have identified factors, which can influence the quality and consistency of JJAV AIs, and provided a framework for appropriate handling, transporting and storage of JJAV to be used for the diagnosis of allergen sensitization and in AIT.

Neutrophil activation during acute human anaphylaxis: analysis of MPO and sCD62L.

BACKGROUND: The mechanisms involved in the amplification of the mast cell response during anaphylaxis are unclear. Mouse models of anaphylaxis demonstrate the critical involvement of neutrophils. These innate immune cells are highly abundant in peripheral blood and can be rapidly activated to trigger both local and systemic inflammation. OBJECTIVE: To investigate neutrophil activation in peripheral blood during acute human anaphylaxis. METHODS: Patients presenting to the emergency department with anaphylaxis underwent blood sampling upon enrolment and at up to three subsequent time-points. Traditional anaphylaxis biomarkers, histamine and mast cell tryptase, were measured by ELISA and ImmunoCAP, respectively. Plasma myeloperoxidase concentrations were measured by ELISA, serum soluble CD62L concentrations by cytometric bead array, and both compared to healthy controls. RESULTS: In 72 patients, 37 (51%) had severe anaphylaxis, 33 (60%) were histamine positive, and 47 (70%) were mast cell tryptase positive. At enrolment, myeloperoxidase concentrations were 2.9- (95% CI: 1.3, 6.5) and 5.0- (95% CI: 2.4, 10.5) fold higher in moderate and severe patients, respectively, compared with healthy controls, and remained stable over the first 5 h following symptom onset. At enrolment, soluble CD62L was 29% (95% CI: 19, 38) and 31% (95% CI: 22, 40) lower in moderate and severe patients, respectively, than healthy controls, and was stable over the first 5 h. There were no associations between myeloperoxidase or soluble CD62L concentrations and either histamine or mast cell tryptase concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: These results provide compelling evidence for the involvement of neutrophils during acute human anaphylaxis, suggesting they are activated early in the reaction, regardless of mast cell activation. This important finding increases our understanding of the basic mechanisms of anaphylaxis, a necessary precursor to improving treatment and prevention.

Spontaneous pneumothorax; a multicentre retrospective analysis of emergency treatment, complications and outcomes.

BACKGROUND: Spontaneous pneumothorax can be managed initially by observation, aspiration or chest drain insertion. AIMS: To determine the clinical features of spontaneous pneumothorax in patients presenting to the emergency department (ED), interventions, outcomes and potential risk factors for poor outcomes after treatment. METHODS: Retrospective chart review from ED of three major referral and two general hospitals in Australia of presentations with primary spontaneous pneumothorax (PSP) or secondary spontaneous pneumothorax (SSP). Main outcomes were prolonged air leak (>5 days) and pneumothorax recurrence within 1 year. RESULTS: We identified 225 people with PSP and 98 with SSP. There were no clinical tension pneumothoraces with hypotension. Hypoxaemia (haemoglobin oxygen saturation measured by pulse oximetry ≤92%) occurred only in SSP and in older patients (age >50 years) with PSP. Drainage was performed in 150 (67%) PSP and 82 (84%) SSP. Prolonged air leak occurred in 16% (95% confidence interval 10-23%) of PSP and 31% (21-42%) of SSP. Independent risk factors for prolonged drainage were non-asthma SSP and pneumothorax size >50%. Complications were recorded in 11% (7.5-16%) of those having drains inserted. Recurrences occurred in 5/91 (5%, 1.8-12%) of those treated without drainage versus 40/232 (17%, 13-23%) of those treated by drainage, of which half occurred in the first month after drainage. CONCLUSION: Pneumothorax drainage is associated with substantial morbidity including prolonged air leak. As PSP appears to be well tolerated in younger people even with large pneumothoraces, conservative treatment in this subgroup may be a viable option to improve patient outcomes, but this needs to be confirmed in a clinical trial.

Severe hypoglycaemia and cognitive impairment in older patients with diabetes: the Fremantle Diabetes Study.

AIMS/HYPOTHESIS: The aim was to investigate the relationship between severe hypoglycaemia and cognitive impairment in older patients with diabetes. METHODS: A sample of 302 diabetic patients aged >/=70 years was assessed for dementia or cognitive impairment without dementia in 2001-2002 and a subsample of non-demented patients (n = 205) was followed to assess cognitive decline. A history of severe hypoglycaemia was determined from self-reports, physician assessments and records of health service use for hypoglycaemia (HSH). Prospective HSH was determined up to 2006. Data analysis, including multiple logistic and Cox regression models, was used to determine whether: (1) there were cross-sectional associations between hypoglycaemia and cognitive status, (2) historical hypoglycaemia predicted cognitive decline, and (3) baseline cognitive status predicted subsequent HSH. RESULTS: There were significant cross-sectional associations between both cognitive impairment and dementia and hypoglycaemia. Independent risk factors for future HSH included dementia (hazard ratio 3.00, 95% CI 1.06-8.48) and inability to self-manage medications (hazard ratio 4.17, 95% CI 1.43-12.13). However, there were no significant associations between historical hypoglycaemia, incident HSH and cognitive decline. CONCLUSIONS/INTERPRETATION: Dementia is an important risk factor for hypoglycaemia requiring health service utilisation. We found no evidence that hypoglycaemia contributes to cognitive impairment in older patients with diabetes.

Adrenaline for the treatment of anaphylaxis: cochrane systematic review.

BACKGROUND: Anaphylaxis is a serious allergic reaction that is rapid in onset and may cause death. Adrenaline is recommended as the initial treatment of choice for anaphylaxis. OBJECTIVES: To assess the benefits and harms of adrenaline in the treatment of anaphylaxis. METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2007, Issue 1), MEDLINE (1966 to March 2007), EMBASE (1966 to March 2007), CINAHL (1982 to March 2007), BIOSIS (to March 2007), ISI Web of Knowledge (to March 2007) and LILACS (to March 2007). We also searched websites listing ongoing trials: http://www.clinicaltrials.gov/, http://www.controlledtrials.com and http://www.actr.org.au/ and contacted pharmaceutical companies and international experts in anaphylaxis in an attempt to locate unpublished material. Randomized and quasi-randomized controlled trials comparing adrenaline with no intervention, placebo or other adrenergic agonists were eligible for inclusion. Two authors independently assessed articles for inclusion. RESULTS: We found no studies that satisfied the inclusion criteria. CONCLUSIONS: On the basis of this review, we are unable to make any new recommendations on the use of adrenaline for the treatment of anaphylaxis. In the absence of appropriate trials, we recommend, albeit on the basis of less than optimal evidence, that adrenaline administration by intramuscular injection should still be regarded as first-line treatment for the management of anaphylaxis.

H1-antihistamines for the treatment of anaphylaxis with and without shock.

BACKGROUND: Anaphylaxis is an acute systemic allergic reaction, which can be life-threatening. H1-antihistamines are commonly used as an adjuvant therapy in the treatment of anaphylaxis. OBJECTIVES: To assess the benefits and harm of H1-antihistamines in the treatment of anaphylaxis. SEARCH STRATEGY: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), (The Cochrane Library), MEDLINE (1966 to June 2006);EMBASE (1966 to June 2006); CINAHL (1982 to June 2006) and ISI Web of Science (1945 to July 2006). We also contacted pharmaceutical companies and international experts in anaphylaxis in an attempt to locate unpublished material. SELECTION CRITERIA: Randomized and quasi-randomized controlled trials comparing H1-antihistamines with placebo or no intervention. DATA COLLECTION AND ANALYSIS: Two authors independently assessed articles for inclusion. MAIN RESULTS: We found no studies that satisfied the inclusion criteria. AUTHORS' CONCLUSIONS: Based on this review, we are unable to make any recommendations for clinical practice. Randomized controlled trials are needed, although these are likely to prove challenging to design and execute.

Thrombotic microangiopathy from Australian brown snake (Pseudonaja) envenoming.

BACKGROUND: Australian brown snake (genus Pseudonaja) envenoming causes a venom-induced consumptive coagulopathy (VICC). A proportion of cases go on to develop thrombotic microangiopathy characterized by thrombocytopenia, microangiopathic haemolytic anaemia (MAHA) and acute renal failure (ARF). AIM: The aim of the study was to define better the natural history and empirical treatments for thrombotic microangiopathy in brown snake envenoming. METHODS: A review of brown snake bites recruited to the Australian Snakebite Project (ASP), a national multicentre study of snake envenoming was undertaken. Serial data are recorded on clinical effects and laboratory results, including measurement of venom concentrations. We describe cases of thrombotic microangiopathy and compare these to cases without thrombotic microangiopathy. RESULTS: From 32 cases of brown snake envenoming with severe VICC, four (13%) developed thrombotic microangiopathy, we also included two cases of thrombotic microangiopathy from prior to ASP. All six developed severe thrombocytopenia (<20 x 10(-9)/L), worst 3 days after the bite and resolving over a week, MAHA with fragmented red blood cells on the blood film and five developed anuric ARF requiring dialysis and lasting 2-8 weeks. All six received antivenom, which was delayed compared with other brown snake-envenoming cases. Four were treated with plasmapheresis. The severity and recovery of the thrombocytopenia, anaemia and renal function were similar with and without plasmapheresis. The median length of stay for MAHA cases was 14 days (interquartile range (IQR) 12-14) compared to 1.8 days (IQR 1.3-2) for all other cases. CONCLUSION: Thrombotic microangiopathy resulting from brown snake bite appears to have a good prognosis and management should focus on early antivenom therapy and supportive care including dialysis. The role of plasmapheresis is yet to be defined.

Insect sting anaphylaxis; prospective evaluation of treatment with intravenous adrenaline and volume resuscitation.

OBJECTIVES: To assess a protocol for treatment of sting anaphylaxis. DESIGN: Prospective assessment of treatment with oxygen, intravenous infusion of adrenaline (epinephrine), and volume resuscitation with normal saline. SETTING: Sub-study of a venom immunotherapy trial. PARTICIPANTS: 21 otherwise healthy adults with systemic allergic reactions to diagnostic sting challenge. MAIN OUTCOME MEASURES: Response to treatment, total adrenaline dose and infusion duration, recurrence of symptoms after stopping the infusion, and additional volume resuscitation. RESULTS: 19 participants required intervention according to the protocol. All received adrenaline, and five received volume resuscitation. In nine cases, physical signs of anaphylaxis recurred after initial attempts at stopping adrenaline but resolved after recommencing the infusion. The median total dose and infusion duration were 590 micro g and 115 minutes respectively, but were significantly higher for eight patients who had hypotensive reactions (762 micro g and 169 minutes respectively). Hypotension was always accompanied by a relative bradycardia, which was severe and treated with atropine in two patients. Widespread T wave inversion occurred, before starting treatment with adrenaline, in one person with an otherwise mild reaction. All patients fully recovered and were fit for same day discharge, apart from the person with ECG changes who was observed overnight and discharged the following day. CONCLUSIONS: Carefully titrated intravenous adrenaline combined with volume resuscitation is an effective strategy for treating sting anaphylaxis, however severe bradycardia may benefit from additional treatment with atropine. Cardiac effects of anaphylaxis, perhaps including neurocardiogenic mechanisms, may be an important factor in some lethal reactions.

Is comorbid status the best predictor of one-year mortality in patients with severe sepsis and sepsis with shock?

Understanding longer term outcomes in critically ill patients will assist treatment decisions, allocation of scarce resources and clinical research in that population. The aim of this study was to compare a well-validated means of determining comorbidity, the Charlson Comorbidity Score, to other verified risk stratification models in predicting one-year mortality and other outcomes in emergency department patients with severe sepsis and sepsis with shock. We conducted a planned subgroup analysis of a prospective observational study, the Critical Illness and Shock Study, in adult patients with sepsis meeting study criteria for critical illness. From emergency department arrival, patients were prospectively enrolled with data collected for a minimum of one year post-enrolment. Scoring systems were derived from this data and compared using receiver-operating characteristic curves. One hundred and four patients were enrolled. The 28-day mortality was 18% and one-year mortality 40%. For predicting one-year mortality, the area under the receiver-operating characteristic curve for age-weighted Charlson Comorbidity Score (0.71, 95% confidence interval 0.61 to 0.81) was at least as good or superior to other scoring systems analysed. The intensive care unit admission rate was 45% and the median hospital length-of-stay was eight days. We conclude that in patients who present to the emergency department with severe sepsis or sepsis with shock, age-weighted Charlson Comorbidity Score is a predictor of one-year mortality that is simple to calculate and at least as accurate as other validated scoring systems.

A randomized controlled trial of fresh frozen plasma for treating venom-induced consumption coagulopathy in cases of Australian snakebite (ASP-18).

BACKGROUND: Venom-induced consumption coagulopathy (VICC) is a major effect of snake envenoming. OBJECTIVES: To investigate whether fresh frozen plasma (FFP) given after antivenom resulted in more rapid correction of coagulation. PATIENTS/METHODS: This was a multicenter open-label randomized controlled trial in patients with VICC of FFP vs. no FFP within 4 h of antivenom administration. Patients (> 2 years) recruited to the Australian snakebite project with VICC (International Normalized Ratio [INR] > 3) were eligible. Patients were randomized 2 : 1 to receive FFP or no FFP. The primary outcome was the proportion with an INR of < 2 at 6 h after antivenom administration. Secondary outcomes included time from antivenom administration to discharge, adverse effects, major hemorrhage, and death. RESULTS: Of 70 eligible patients, 65 consented to be randomized: 41 to FFP, and 24 to no FFP. Six hours after antivenom administration, more patients randomized to FFP had an INR of < 2 (30/41 [73%] vs. 6/24 [25%]; absolute difference, 48%; 95% confidence interval 23-73%; P = 0.0002). The median time from antivenom administration to discharge was similar (34 h, range 14-230 h vs. 39 h, range 14-321 h; P = 0.44). Seven patients developed systemic hypersensitivity reactions after antivenom administration - two mild and one severe (FFP arm), and three mild and one severe (no FFP). One serious adverse event (intracranial hemorrhage and death) occurred in an FFP patient with pre-existing hypertension, who was hypertensive on admission, and developed a headache 6 h after FFP administration. Post hoc analysis showed that the median time from bite to FFP administration was significantly shorter for non-responders to FFP than for responders (4.7 h, interquartile range [IQR] 4.2-6.7 h vs. 7.3 h, IQR 6.1-8 h; P = 0.002). CONCLUSIONS: FFP administration after antivenom administration results in more rapid restoration of clotting function in most patients, but no decrease in discharge time. Early FFP administration (< 6-8 h) post-bite is less likely to be effective.

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.

Bites in Australian snake handlers--Australian snakebite project (ASP-15).

BACKGROUND: Snakebites in snake handlers are an important clinical problem that may differ to bites in the general population. AIM: To investigate the epidemiology and clinical presentation of bites in snake handlers. DESIGN: Prospective observational study. METHODS: Bites in snake handlers recruited as part of the Australian Snakebite Project (ASP) from 2004 to 2011 were included in the study. Data were extracted from the ASP database, which included demographic and clinical information, laboratory tests and antivenom treatment. RESULTS: From 1089 snake bites recruited to ASP, there were 106 (9.7%) bites in snake handlers. The median age was 40 years (range: 16-81 years) and 104 (98%) were males. The commonest circumstances of the bites were handling snakes (47), catching snakes (22), feeding snakes (18) and cleaning cages (11). Bites were to the upper limb in 103 cases. Bites were most commonly by Red-bellied black snakes (20), Brown snakes (17), Taipan (15), Tiger snakes (14) and Death adders (14). Envenoming occurred in 77 patients: venom-induced consumption coagulopathy in 45 patients (58%), neurotoxicity in 10 (13%) and myotoxicity in 13 (17%). Systemic hypersensitivity reactions (SHSRs) to venom occurred in eight, satisfying clinical criteria for anaphylaxis in five, of which three were hypotensive. Antivenom was administered in 60 envenomed patients. SHSRs to antivenom occurred in 15 (25%; 95% CI:15-38%), including 2 (3%:1-13%) with severe (hypotensive) reactions. CONCLUSION: Bites in snake handlers remain a common, important problem involving a broad range of snakes. Neurotoxicity and myotoxicity are relatively common, consistent with the snakes involved. Venom anaphylaxis occured, despite previously being a poorly recognized problem in snake handlers. The incidence of SHSRs to antivenoms, including anaphylaxis, was not higher than that observed in non-snake handlers.

Histopathological analysis and in situ localisation of Australian tiger snake venom in two clinically envenomed domestic animals.

OBJECTIVE: To assess histopathological changes in clinically envenomed tiger snake patients and identify tissue specific localisation of venom toxins using immunohistochemistry. SAMPLES: One feline and one canine patient admitted to the Murdoch Pet Emergency Centre (MPEC), Murdoch University with tiger snake (Notechis sp.) envenoming. Both patients died as a result of envenomation. Non-envenomed tissue was also collected and used for comparison. METHODOLOGY: Biopsy samples (heart, lung, kidney andskeletal muscle tissue) were retrieved 1-2 h post death and processed for histopathological examination using Haemotoxylin and Eosin, Martius Scarlet Blue and Periodic Acid Schiff staining. Tissues were examined by light microscopy and tissue sections subjected to immunohistochemical staining using in-house generated monoclonal and polyclonal antibodies against Notechis venoms. RESULTS: Venom-induced pathological changes were observed in the lungs, kidneys and muscle tissue of both patients. Evidence, not previously noted, of procoagulant venom effects were apparent, with formed thrombi in the heart, lungs (small fibrillar aggregates and larger, discrete thrombi) and kidneys. Immunohistochemical assays revealed venom present in the pulmonary tissue, in and around the glomerular capsule and surrounding tubules in renal tissue and scattered throughout the Gastrocnemius muscle tissue. CONCLUSION: This work has shown pathological evidence of procoagulant venom activity supporting previous suggestions that an initial thrombotic state occurs in envenomed patients. We have shown that venom toxins are able to be localised to specific tissues, in this case, venom was detected in the lung, kidney and muscle tissues of clinically envenomed animals. Future work will examine specific toxin localisation using monoclonal antibodies and identify if antivenom molecules are able to reach their target tissues.

Clinical effects and treatment of envenoming by Hoplocephalus spp. snakes in Australia: Australian Snakebite Project (ASP-12).

There is limited information on envenoming by snakes of the genus Hoplocephalus from Eastern Australia. We investigated the clinical and laboratory features of patients with definite Hoplocephalus spp. bites including antivenom treatment, recruited to the Australian Snakebite Project. There were 15 definite Hoplocephalus spp. bites based on expert identification including eight by Hoplocephalus stephensi (Stephen's banded snakes), four by Hoplocephalus bungaroides (broad-headed snake) and three by H. bitorquatus (pale-headed snake). Envenoming occurred in 13 patients and was similar for the three species with venom induced consumption coagulopathy (VICC) in all envenomings. Seven patients had an INR >12 and partial VICC, with only incomplete fibrinogen consumption, occurred in three patients. Systemic symptoms occurred in eight patients. Myotoxicity and neurotoxicity did not occur. H. stephensi venom was detected in all three H. stephensi envenomings (1.1, 44 and 81 ng/mL) for whom pre-antivenom blood samples were available, and not detected in one without envenoming. In two cases with post-antivenom blood samples, venom was not detected after tiger snake antivenom (TSAV) was given. In vitro binding studies demonstrated that TSAV concentrations of 50mU/mL are sufficient to bind the majority of free H. stephensi venom components at concentrations above those detected in envenomed patients (100 ng/mL). Eleven patients received antivenom, median dose 2 vials (Range: 1 to 5 vials), which was TSAV in all but one case, where polyvalent antivenom was used. Immediate hypersensitivity reactions occurred in six cases including one case of anaphylaxis. Envenoming by Hoplocephalus spp. causes VICC and systemic symptoms, making it clinically similar to brown snake (Pseudonaja spp.) envenoming. Based on in vitro studies reported here, patients may be treated with one vial of TSAV, although one vial of brown snake antivenom may also be sufficient.

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

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.

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.

Failure of antivenom to improve recovery in Australian snakebite coagulopathy.

BACKGROUND: Venom-induced consumption coagulopathy (VICC) is an important feature of snake envenoming. AIM: To investigate the effect of antivenom and fresh frozen plasma (FFP) on recovery of VICC in Australian elapid snake envenoming. DESIGN: Prospective cohort study. METHODS: Patients with VICC were included from the Australian Snakebite Project (ASP). Time to recovery of VICC (defined as time until INR <2) was investigated using a time to event analysis in WinBUGS. The model considered the effects of age, sex, snake type, time of antivenom after bite, antivenom dose and use of FFP within 4 h. RESULTS: The study included 167 cases of VICC, median age being 41 [interquartile range (IQR): 28-53) years, and 130 (78%) were males. Antivenom was administered at a median of 3.6 (IQR: 2.2-5.6) h after the bite at a median dose of four vials (IQR: 2-6 vials). Thirteen patients received FFP within 4 h. Recovery of VICC occurred after a median of 14.4 (IQR: 11.5-17.5) h, and only the use of FFP within 4 h influenced the time to recovery. Neither antivenom dose nor time of antivenom administration had an effect on recovery of VICC. In patients administered with FFP, 12% [credible interval (CrI): 6-21%] and 81% (CrI: 61-94%) had recovered at 6 and 12 h, respectively, vs 2.5% (CrI: 1.5-4%) and 28% (CrI: 22-34%) not receiving FFP. DISCUSSION: Antivenom did not appear to be effective for the coagulopathy in snake envenoming in Australia. FFP appeared to shorten the time of VICC recovery.