The development of Jack Jumper ant venom immunotherapy: our 25 years' experience.

Jack Jumper ant venom allergy is a uniquely Australian medical issue. The stinging ant is a leading cause of insect venom allergy in south-eastern Australia. An effective venom immunotherapy-based treatment was successfully developed by the Tasmanian Jack Jumper Allergy Research group. This paper provides a synopsis of our 25 years' research journey in developing this evidence-based treatment modality.

Snakebite-associated thrombotic microangiopathy: an Australian prospective cohort study [ASP30].

BACKGROUND: Snakebite-associated thrombotic microangiopathy (TMA) occurs in a subset of patients with venom-induced consumption coagulopathy (VICC) following snakebite. Acute kidney injury (AKI) is the commonest end-organ manifestation of TMA. The epidemiology, diagnostic features, outcomes, and effectiveness of interventions including therapeutic plasma-exchange (TPE), in snakebite-associated TMA are poorly understood. METHODS: We reviewed all patients with suspected or confirmed snakebite recruited to the Australian Snakebite Project (2004-2018 inclusive), a prospective cohort study, from 202 participating Australian hospitals across the country. TMA was defined as anemia with schistocytosis. RESULTS: 2069 patients with suspected snakebite were enrolled, with 1158 (56.0%) systemically envenomed, of which 842 (72.7%) developed VICC, from which 104 (12.4%) developed TMA. Of those systemically envenomed, TMA occurred in 26% (13/50) taipan, 17% (60/351) brown, and 8% (16/197) tiger snakebites. Thrombocytopenia was present in 90% (94/104) of TMA cases, and a further eight (8%) had a > 25% decrease in platelets from the presentation. Patients with TMA were significantly older than non-TMA patients with VICC (53 [35-61] versus 41 [24-55] years, median [IQR], p < 0.0001). AKI developed in 94% (98/104) of TMA patients, with 34% (33/98) requiring dialysis (D-AKI). There were four deaths. In D-AKI TMA cases, eventual dialysis-free survival (DFS) was 97% (32/33). TPE was used in five D-AKI cases, with no significant difference in DFS or time to independence from dialysis. >90-day follow-up for 25 D-AKI cases (130 person-years) showed no end-stage kidney disease but 52% (13/25) had ≥ stage 3 chronic kidney disease (CKD). CONCLUSION: Our findings support a definition of snakebite-associated TMA as anemia with schistocytosis and either thrombocytopenia or >25% drop in platelet count. AKI occurring with snakebite-associated TMA varied in severity, with most achieving DFS, but with a risk of long-term CKD in half. We found no evidence of benefit for TPE in D-AKI.

Global View on Ant Venom Allergy: from Allergenic Components to Clinical Management.

Hymenoptera venom allergy is characterised by systemic anaphylactic reactions that occur in response to stings from members of the Hymenoptera order. Stinging by social Hymenoptera such as ants, honeybees, and vespids is one of the 3 major causes of anaphylaxis; along with food and drug exposure, it accounts for up to 43% of anaphylaxis cases and 20% of anaphylaxis-related fatalities. Despite their recognition as being of considerable public health significance, stinging ant venoms are relatively unexplored in comparison to other animal venoms and may be overlooked as a cause of venom allergy. Indeed, the venoms of stinging ants may be the most common cause of anaphylaxis in ant endemic areas. A better understanding of the natural history of venom allergy caused by stinging ants, their venom components, and the management of ant venom allergy is therefore required. This article provides a global view on allergic reactions to the venoms of stinging ants and the contemporary approach to diagnose and manage ant venom allergy.

Phospholipase A2 (PLA2) as an Early Indicator of Envenomation in Australian Elapid Snakebites (ASP-27).

Early diagnosis of snake envenomation is essential, especially neurotoxicity and myotoxicity. We investigated the diagnostic value of serum phospholipase (PLA2) in Australian snakebites. In total, 115 envenomated and 80 non-envenomated patients were recruited over 2 years, in which an early blood sample was available pre-antivenom. Serum samples were analyzed for secretory PLA2 activity using a Cayman sPLA2 assay kit (#765001 Cayman Chemical Company, Ann Arbor MI, USA). Venom concentrations were measured for snake identification using venom-specific enzyme immunoassay. The most common snakes were Pseudonaja spp. (33), Notechis scutatus (24), Pseudechis porphyriacus (19) and Tropidechis carinatus (17). There was a significant difference in median PLA2 activity between non-envenomated (9 nmol/min/mL; IQR: 7-11) and envenomated patients (19 nmol/min/mL; IQR: 10-66, p < 0.0001) but Pseudonaja spp. were not different to non-envenomated. There was a significant correlation between venom concentrations and PLA2 activity (r = 0.71; p < 0.0001). PLA2 activity was predictive for envenomation; area under the receiver-operating-characteristic curve (AUC-ROC), 0.79 (95% confidence intervals [95%CI]: 0.72-0.85), which improved with brown snakes excluded, AUC-ROC, 0.88 (95%CI: 0.82-0.94). A cut-point of 16 nmol/min/mL gives a sensitivity of 72% and specificity of 100% for Australian snakes, excluding Pseudonaja. PLA2 activity was a good early predictor of envenomation in most Australian elapid bites. A bedside PLA2 activity test has potential utility for early case identification but may not be useful for excluding envenomation.

Conservative versus Interventional Treatment for Spontaneous Pneumothorax.

BACKGROUND: Whether conservative management is an acceptable alternative to interventional management for uncomplicated, moderate-to-large primary spontaneous pneumothorax is unknown. METHODS: In this open-label, multicenter, noninferiority trial, we recruited patients 14 to 50 years of age with a first-known, unilateral, moderate-to-large primary spontaneous pneumothorax. Patients were randomly assigned to immediate interventional management of the pneumothorax (intervention group) or a conservative observational approach (conservative-management group) and were followed for 12 months. The primary outcome was lung reexpansion within 8 weeks. RESULTS: A total of 316 patients underwent randomization (154 patients to the intervention group and 162 to the conservative-management group). In the conservative-management group, 25 patients (15.4%) underwent interventions to manage the pneumothorax, for reasons prespecified in the protocol, and 137 (84.6%) did not undergo interventions. In a complete-case analysis in which data were not available for 23 patients in the intervention group and 37 in the conservative-management group, reexpansion within 8 weeks occurred in 129 of 131 patients (98.5%) with interventional management and in 118 of 125 (94.4%) with conservative management (risk difference, -4.1 percentage points; 95% confidence interval [CI], -8.6 to 0.5; P = 0.02 for noninferiority); the lower boundary of the 95% confidence interval was within the prespecified noninferiority margin of -9 percentage points. In a sensitivity analysis in which all missing data after 56 days were imputed as treatment failure (with reexpansion in 129 of 138 patients [93.5%] in the intervention group and in 118 of 143 [82.5%] in the conservative-management group), the risk difference of -11.0 percentage points (95% CI, -18.4 to -3.5) was outside the prespecified noninferiority margin. Conservative management resulted in a lower risk of serious adverse events or pneumothorax recurrence than interventional management. CONCLUSIONS: Although the primary outcome was not statistically robust to conservative assumptions about missing data, the trial provides modest evidence that conservative management of primary spontaneous pneumothorax was noninferior to interventional management, with a lower risk of serious adverse events. (Funded by the Emergency Medicine Foundation and others; PSP Australian New Zealand Clinical Trials Registry number, ACTRN12611000184976.).

Pharmaceutical and preclinical evaluation of Advax adjuvant as a dose-sparing strategy for ant venom immunotherapy.

A major challenge in broader clinical application of Jack Jumper ant venom immunotherapy (JJA VIT) is the scarcity of ant venom which needs to be manually harvested from wild ants. Adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend JJA supplies if they were to similarly enhance JJA VIT immunogenicity. The purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT formulated with a novel polysaccharide adjuvant referred to as delta inulin or Advax™. Jack Jumper ant venom (JJAV) protein stability was assessed by UPLC-UV, SDS-PAGE, SDS-PAGE immunoblot, and ELISA inhibition. Diffraction light scattering was used to assess particle size distribution of Advax; pH and benzyl alcohol quantification by UPLC-UV were used to assess the physicochemical stability of JJAV diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted VIT formulation. To assess the effect of adjuvant on JJA venom immunogenicity, mice were immunised four times with JJAV alone or formulated with Advax adjuvant. JJA VIT formulated with Advax was found to be physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25 °C with a trend for an increase in allergenic potency observed beyond 2 days of storage. Low-dose JJAV formulated with Advax adjuvant induced significantly higher JJAV-specific IgG than a 5-fold higher dose of JJAV alone, consistent with a powerful allergen-sparing effect. The pharmaceutical data provides important guidance on the formulation, storage and use of JJA VIT formulated with Advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of Advax adjuvant to achieve 4-fold JJAV dose sparing in JJA-allergic human patients.

Markers Involved in Innate Immunity and Neutrophil Activation are Elevated during Acute Human Anaphylaxis: Validation of a Microarray Study.

BACKGROUND: We have previously identified the upregulation of the innate immune response, neutrophil activation, and apoptosis during anaphylaxis using a microarray approach. This study aimed to validate the differential gene expression and investigate protein concentrations of "hub genes" and upstream regulators during anaphylaxis. METHODS: Samples were collected from patients with anaphylaxis on their arrival at the emergency department, and after 1 and 3 h. mRNA levels of 11 genes (interleukin-6 [IL-6], IL-10, oncostatin M [OSM], S100A8, S100A9, matrix metalloproteinase 9 [MMP9], FASL, toll-like receptor 4 [TLR4], MYD88, triggering receptor expressed on myeloid cells 1 [TREM1], and cluster of differentiation 64 [CD64]) were measured in peripheral blood leucocytes using qPCR. Serum protein concentrations were measured by ELISA or cytometric bead array for 6 of these candidates. RESULTS: Of 69 anaphylaxis patients enrolled, 36 (52%) had severe reactions, and 38 (55%) were female. Increases in both mRNA and protein of IL-10, S100A9, MMP9, and TREM1 were observed. OSM, S100A8, TLR4, and CD64 were upregulated and IL-6 protein concentrations were increased during anaphylaxis. Both FASL and soluble Fas ligand decreased during anaphylaxis. CONCLUSION: These results provide evidence for the involvement of innate immune pathways and myeloid cells during human anaphylaxis, validating previous microarray findings. Elevated S100A8, S100A9, TLR4, and TREM1 expression, and increased S100A9 and soluble TREM1 protein concentrations strongly suggest that neutrophils are activated during acute anaphylaxis.

Towards complete identification of allergens in Jack Jumper (Myrmecia pilosula) ant venom and their clinical relevance: An immunoproteomic approach.

BACKGROUND: The venomous stings of Jack Jumper ant (JJA; species of the Myrmecia pilosula taxonomic group) are a significant public health issue in parts of south-eastern and south-western Australia, causing anaphylaxis in approximately 3% of the population. Three allergenic peptides, Myr p 1, Myr p 2 and Myr p 3, and one histamine-releasing peptide, pilosulin 5, have been fully described, but there are at least 5 additional high molecular weight IgE-binding components that have not been identified. OBJECTIVE: To identify IgE-binding components in JJA venom (JJAV) and to relate the IgE recognition of these components to relevant clinical parameters. METHODS: Identification of IgE-binding components and determination of their sensitizing prevalence was performed using SDS-PAGE immunoblot assay and sera from 90 patients with confirmed allergy to JJAV. Tandem mass spectrometry was used for identification of novel JJAV components fractionated by size exclusion chromatography (SEC) and SDS-PAGE. RESULTS: Using SDS-PAGE immunoblot, 10 IgE-binding bands were identified in JJAV, two of which were recognized by 81% and 47% of the population studied. Mass spectrometry identified 17 novel JJAV proteins, including 2 glycoproteins, and confirmed the presence of 4 known Myr p and pilosulin peptides in JJAV. Most of the newly identified IgE-binding proteins were enzymes, including phospholipase A2 , hyaluronidase, arginine kinase and dipeptidyl peptidase IV. Correlations were found between recognition of certain IgE-binding bands with JJAV-specific IgE titre by ImmunoCAP, intradermal test threshold and treatment-related issues. CONCLUSIONS AND CLINICAL RELEVANCE: This study has for the first time revealed the identity of various proteins with IgE-binding capacity in the venom of JJA and demonstrated their clinical relevance in the diagnosis and treatment of JJAV allergy.

A randomized-controlled trial of a patient-centred intervention in high-risk discharged older patients.

BACKGROUND: The risk of early reattendance after discharge has been proposed as a performance indicator for emergency departments (EDs), but is not uniform in all patients. Those individuals at the highest risk of reattendance may benefit from an intense intervention to reduce this risk, and our objective was to test this hypothesis in a clinical trial. METHODS: A randomized-controlled trial was conducted in the EDs of two hospitals. Very high-risk adults aged 65 years and older, identified using a validated risk-prediction nomogram and being discharged from ED, were randomized to receive a postdischarge patient-centred intervention or standard care. The intervention focused on identifying and supporting patients to address risk factors for future hospital presentation. The primary outcome measure was any unplanned ED reattendance within 28 days. Secondary outcomes included 28-day and 1-year hospital usage, institutionalization and death. RESULTS: We enrolled 164 patients, 82 in each study arm. There was an 8% absolute (95% confidence interval: -7%-20%) and a 20% relative risk reduction for an intervention patient making an unplanned ED reattendance within 28 days. This difference was not statistically significant (P=0.26). CONCLUSION: This postdischarge intervention was associated with only small and nonsignificant reductions in ED reattendance.

REstricted Fluid REsuscitation in Sepsis-associated Hypotension (REFRESH): study protocol for a pilot randomised controlled trial.

BACKGROUND: Guidelines recommend an initial intravenous (IV) fluid bolus of 30 ml/kg isotonic crystalloid for patients with sepsis and hypotension. However, there is a lack of evidence from clinical trials to support this. Accumulating observational data suggest harm associated with the injudicious use of fluids in sepsis. There is currently equipoise regarding liberal or restricted fluid-volume resuscitation as first-line treatment for sepsis-related hypotension. A randomised trial comparing these two approaches is, therefore, justified. METHODS/DESIGN: The REstricted Fluid REsuscitation in Sepsis-associated Hypotension trial (REFRESH) is a multicentre, open-label, randomised, phase II clinical feasibility trial. Participants will be patients presenting to the emergency departments of Australian metropolitan hospitals with suspected sepsis and a systolic blood pressure of < 100 mmHg, persisting after a 1000-ml fluid bolus with isotonic crystalloid. Participants will be randomised to either a second 1000-ml fluid bolus (standard care) or maintenance rate fluid only, with the early commencement of a vasopressor infusion to maintain a mean arterial pressure of > 65 mmHg, if required (restricted fluid). All will receive further protocolised fluid boluses (500 ml or 250 ml, respectively), if required during the 6-h study period. The primary outcome measure is total volume administered in the first 6 h. Secondary outcomes include fluid volume at 24 h, organ support 'free days' to day 28, 90-day mortality, and a range of feasibility and process-of-care measures. Participants will also undergo serial measurement, over the first 24 h, of biomarkers of inflammation, endothelial cell activation and glycocalyx degradation for comparison between the groups. DISCUSSION: This is the first randomised trial examining fluid volume for initial resuscitation in septic shock in an industrialised country. A pragmatic, open-label design will establish the feasibility of undertaking a large, international, multicentre trial with sufficient power to assess clinical outcomes. The embedded biomarker study aims to provide mechanistic plausibility for a larger trial by defining the effects of fluid volume on markers of systemic inflammation and the vascular endothelium. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Registry, ID: ACTRN12616000006448. Registered on 12 January 2016.

Resistin and NGAL are associated with inflammatory response, endothelial activation and clinical outcomes in sepsis.

OBJECTIVE AND DESIGN: Resistin and neutrophil gelatinase-associated lipocalin (NGAL) are upregulated in circulating leucocytes in sepsis, but the significance of this is uncertain. We evaluated associations between Resistin and NGAL with endothelial cell activation and clinical outcomes in a prospective observational study in the Emergency Department (ED). METHODS: Serum levels of Resistin, NGAL, inflammatory cytokines (IL-6, IL-10) and soluble endothelial adhesion molecules (VCAM-1, ICAM-1) were measured at defined time points up to 24 h. Patterns and relationships between markers were investigated using linear mixed regression models. Predictive values for clinical outcomes for markers at enrollment were assessed by logistic regression and receiver operator characteristic (ROC) curves. RESULTS: 186 participants (89 septic-shock, 69 sepsis, 28 uncomplicated infection) were compared with 29 healthy controls. Median Resistin and NGAL were higher in uncomplicated infection compared to controls, and in septic shock compared to non-shock sepsis. Resistin and NGAL correlated with IL-6 and IL-10, with VCAM-1 and ICAM-1, and with organ failure. Resistin and NGAL were associated with septic shock but had limited predictive utility for mortality. CONCLUSION: Resistin and NGAL correlate with expression of endothelial cell adhesion molecules in sepsis. Further evaluation of the role of Resistin and NGAL in sepsis pathogenesis is warranted.

Australian taipan (Oxyuranus spp.) envenoming: clinical effects and potential benefits of early antivenom therapy - Australian Snakebite Project (ASP-25).

CONTEXT: Taipans (Oxyuranus spp.) are medically important venomous snakes from Australia and Papua New Guinea. The objective of this study was to describe taipan envenoming in Australian and its response to antivenom. METHODS: Confirmed taipan bites were recruited from the Australian Snakebite Project. Data were collected prospectively on all snakebites, including patient demographics, bite circumstances, clinical effects, laboratory results, complications and treatment. Blood samples were taken and analysed by venom specific immunoassay to confirm snake species and measure venom concentration pre- and post-antivenom. RESULTS: There were 40 confirmed taipan bites: median age 41 years (2-85 years), 34 were males and 21 were snake handlers. Systemic envenoming occurred in 33 patients with neurotoxicity (26), complete venom induced consumption coagulopathy (VICC) (16), partial VICC (15), acute kidney injury (13), myotoxicity (11) and thrombocytopenia (7). Venom allergy occurred in seven patients, three of which had no evidence of envenoming and one died. Antivenom was given to 34 patients with a median initial dose of one vial (range 1-4), and a median total dose of two vials (range 1-9). A greater total antivenom dose was associated with VICC, neurotoxicity and acute kidney injury. Early antivenom administration was associated with a decreased frequency of neurotoxicity, acute kidney injury, myotoxicity and intubation. There was a shorter median time to discharge of 51 h (19-432 h) in patients given antivenom <4 h post-bite, compared to 175 h (27-1104 h) in those given antivenom >4 h. Median peak venom concentration in 25 patients with systemic envenoming and a sample available was 8.4 ng/L (1-3212 ng/L). No venom was detected in post-antivenom samples, including 20 patients given one vial initially and five patients bitten by inland taipans. DISCUSSION: Australian taipan envenoming is characterised by neurotoxicity, myotoxicity, coagulopathy, acute kidney injury and thrombocytopenia. One vial of antivenom binds all measurable venom and early antivenom was associated with a favourable outcome.

Distinct inflammatory responses differentiate cerebral infarct from transient ischaemic attack.

We previously reported on a 26-year-old patient who presented early during a large and eventually fatal cerebral infarct. Microarray analysis of blood samples from this patient demonstrated initially up-regulated and subsequently down-regulated Granzyme B (GzmB) expression, along with progressive up-regulation of genes for S100 calcium binding protein A12 (S100A12) and matrix metalloproteinase 9 (MMP-9). To confirm these findings, we investigated these parameters in patients with suspected stroke presenting within 6h of symptom onset to a single centre. Blood samples were taken at enrolment, then 1h, 3h and 24h post-enrolment for the examination of cellular, protein and genetic changes. Patients with subsequently confirmed ischaemic (n=18) or haemorrhagic stroke (n=11) showed increased intracellular concentrations of GzmB in all cell populations investigated (CD8+, CD8- and Natural Killer [NK] cells). Infarct patients, however, demonstrated significantly reduced GzmB gene expression and increased circulating MMP-9 and S100A12 levels in contrast to transient ischaemic attack (TIA) patients or healthy controls. Furthermore, a pronounced neutrophilia was noted in the infarct and haemorrhage groups, while TIA patients (n=9) reflected healthy controls (n=10). These findings suggest a spectrum of immune response during stroke. TIA showed few immunological changes in comparison to infarct and haemorrhage, which demonstrated inhibition of GzmB production and a rise in neutrophil numbers and neutrophil-associated mediators. This implies a greater role of the innate immune system. These markers may provide novel targets for inhibition and reduction of secondary injury.

Study protocol for a randomised controlled trial of invasive versus conservative management of primary spontaneous pneumothorax.

INTRODUCTION: Current management of primary spontaneous pneumothorax (PSP) is variable, with little evidence from randomised controlled trials to guide treatment. Guidelines emphasise intervention in many patients, which involves chest drain insertion, hospital admission and occasionally surgery. However, there is evidence that conservative management may be effective and safe, and it may also reduce the risk of recurrence. Significant questions remain regarding the optimal initial approach to the management of PSP. METHODS AND ANALYSIS: This multicentre, prospective, randomised, open label, parallel group, non-inferiority study will randomise 342 participants with a first large PSP to conservative or interventional management. To maintain allocation concealment, randomisation will be performed in real time by computer and stratified by study site. Conservative management will involve a period of observation prior to discharge, with intervention for worsening symptoms or physiological instability. Interventional treatment will involve insertion of a small bore drain. If drainage continues after 1 hour, the patient will be admitted. If drainage stops, the drain will be clamped for 4 hours. The patient will be discharged if the lung remains inflated. Otherwise, the patient will be admitted. The primary end point is the proportion of participants with complete lung re-expansion by 8 weeks. Secondary end points are as follows: days in hospital, persistent air leak, predefined complications and adverse events, time to resolution of symptoms, and pneumothorax recurrence during a follow-up period of at least 1 year. The study has 95% power to detect an absolute non-inferiority margin of 9%, assuming 99% successful expansion at 8 weeks in the invasive treatment arm. The primary analysis will be by intention to treat. ETHICS AND DISSEMINATION: Local ethics approval has been obtained for all sites. Study findings will be disseminated by publication in a high-impact international journal and presentation at major international Emergency Medicine and Respiratory meetings. TRIAL REGISTRATION NUMBER: ACTRN12611000184976; Pre-results.

Changes in differential gene expression during a fatal stroke.

We present a young woman (with an identical twin sister) who arrived at the Emergency Department (ED) within 1 hour of her initial stroke symptoms. Previous microarray studies have demonstrated differential expression of multiple genes between stroke patients and healthy controls. However, for many of these studies there is a significant delay between the initial symptoms and collection of blood samples, potentially leaving the important early activators/regulators of the inflammatory response unrecognised. Blood samples were collected from the patient for an analysis of differential gene expression over time during the evolution of a fatal stroke. The time points for blood collection were ED arrival (T0) and 1, 3 and 24 hours post ED arrival (T1, T3 and T24). This was compared to her identical twin and an additional two age and sex-matched healthy controls. When compared to the controls, the patient had 12 mRNA that were significantly upregulated at T0, and no downregulated mRNA (with a cut off fold change value ±1.5). Of the 12 upregulated mRNA at T0, granzyme B demonstrated the most marked upregulation on arrival, with expression steadily declining over time, whereas S100 calcium-binding protein A12 (S100A12) gene expression increased from T0 to T24, remaining >two-fold above that in the healthy controls at T24. Other genes, such as matrix metalloproteinase 9, high mobility group box 2 and interleukin-18 receptor I were not upregulated at T0, but they demonstrated clear upregulation from T1­T3, with gene expression declining by T24. A greater understanding of the underlying immunopathological mechanisms that are involved during the evolution of ischaemic stroke may help to distinguish between patients with stroke and stroke mimics.

Incidence of serum sickness after the administration of Australian snake antivenom (ASP-22).

CONTEXT: Serum sickness is a delayed immune reaction resulting from the injection of foreign protein or serum. Antivenom is known to cause serum sickness but the incidence and characteristics are poorly defined. OBJECTIVE: To investigate the incidence and clinical features of serum sickness following the administration of Australian snake antivenoms. MATERIALS AND METHODS: This was a prospective cohort study of patients recruited to the Australian Snakebite Project who received snake antivenom from November 2012 to March 2014. Demographics, clinical information, laboratory tests and antivenom treatment were recorded prospectively. Patients administered antivenom were followed up at 7-10 days and 6 weeks' post-antivenom. The primary outcome was the proportion with serum sickness, pre-defined as three or more of: fever, erythematous rash/urticaria, myalgia/arthralgia, headache, malaise, nausea/vomiting 5-20 days post-antivenom. RESULTS: During the 16-month period, 138 patients received antivenom. 23 were not followed up (unable to contact, tourist, child, bee sting) and 6 died in hospital. Of 109 patients followed up, the commonest reason for antivenom was venom induced consumption coagulopathy in 77 patients. An acute systemic hypersensitivity reaction occurred post-antivenom in 25 (23%) and 8 (7%) were severe with hypotension. Serum sickness occurred in 32/109 (29%) patients, including 15/37 (41%) given tiger snake, 6/15 (40%) given polyvalent and 4/23 (17%) given brown snake antivenom. There was no association between the volume of antivenom and serum sickness, p = 0.18. The commonest effects were lethargy, headache, muscle/joint aches and fever. DISCUSSION: The incidence of serum sickness after snake antivenom in Australia was higher than earlier investigations which failed to define symptoms or follow-up patients, but similar to more recent studies of antivenoms in the United States. CONCLUSION: Serum sickness is common with Australian snake antivenom but does not appear to be predictable based on the volume of antivenom administered.

Changes in differential gene expression during a fatal stroke.

We present a young woman (with an identical twin sister) who arrived at the Emergency Department (ED) within 1hour of her initial stroke symptoms. Previous microarray studies have demonstrated differential expression of multiple genes between stroke patients and healthy controls. However, for many of these studies there is a significant delay between the initial symptoms and collection of blood samples, potentially leaving the important early activators/regulators of the inflammatory response unrecognised. Blood samples were collected from the patient for an analysis of differential gene expression over time during the evolution of a fatal stroke. The time points for blood collection were ED arrival (T0) and 1, 3 and 24hours post ED arrival (T1, T3 and T24). This was compared to her identical twin and an additional two age and sex-matched healthy controls. When compared to the controls, the patient had 12 mRNA that were significantly upregulated at T0, and no downregulated mRNA (with a cut off fold change value ±1.5). Of the 12 upregulated mRNA at T0, granzyme B demonstrated the most marked upregulation on arrival, with expression steadily declining over time, whereas S100 calcium-binding protein A12 (S100A12) gene expression increased from T0 to T24, remaining >two-fold above that in the healthy controls at T24. Other genes, such as matrix metalloproteinase 9, high mobility group box 2 and interleukin-18 receptor I were not upregulated at T0, but they demonstrated clear upregulation from T1-T3, with gene expression declining by T24. A greater understanding of the underlying immunopathological mechanisms that are involved during the evolution of ischaemic stroke may help to distinguish between patients with stroke and stroke mimics.