Bite Me!An Overview of AustralianSnakebite Envenomation

Paul Harris

Introduction

Australia has a lot of dangerous creaturesA lot of these are venomousA lot of these venomous creatures are snakesA lot of these venomous snakes look a bit similarA lot of these similar looking, venomous snakeshave potentially lethal bites and different treatmentsA lot of this makes managing them all a bit tricky

Contents of PresentationEpidemiology of snake bite in WASnake ClassificationComposition of Snake VenomPhysiology of NMJ and Coagulation CascadeAction and clinical picture of envenomation fromdifferent snake speciesPractical management of snake biteVenom detection and anti-venomsSummary

Australian Poison CentreBites/Stings Information

Spiders: 54%5-10,000 bites/yearMostly Red Back spider bites (<20% requiring specific treatment)Funnel Web spider bites (<50/yr)Necrotic Arachnadism (<100/yr)Paralysis Ticks (very common but paralysis very few)

Insects: 38%Ants, bees, wasps

Marine: 4%Box Jellyfish (<50/yr)Blue-ringed Octopus (10/yr)Venomous fish stings (Stonefish <10/yr)

Snake Bite InformationAustralian Poisons Centre:

1,000 – 3,000 cases per year = 3%Up to 200 requiring anti-venomNot a notifiable diseaseSutherland SK & Leonard RL. MJA 1995;163:616-618

World Health Organisation Data:Global mortality 50-100,000 hospital deaths / yearSignificant chronic disability in survivors

Physical handicap (necrosis, amputations)Systemic disease

Size of the ProblemDespite advances in the past 20 years with first-aid, venomdetection, supportive care and the use of anti-venom, thenumber of snakebite fatalities appears to have risen

White J. Emergency Med 2000;12:204-6

Between 1982 – 1992, 1.8 deaths per yearSutherland SK. MJA 1992;157:734-739.

Between 1992-1994, 3.7 deaths per yearSutherland SK, Leonard RL. MJA 1995;163:616-8

Epidemiology ofSnake Bite in Perth

Admissions for suspected snake bite to the Perth adultteaching hospitals, 1979 to 1988

Jelinek A. et al. Med J Aust 1991 155;(2):761-499 definite bites, 193 cases total

53 envenomations (3 in snake handlers)30 equivocal envenomationsDugite commonest snake bite (often coagulopathy only)Tiger and Gwardar common

36% of snake genus identified by venom detection kitsAll brown or tiger snake genus

At least 5 fatalities in last 10 years

Epidemiology ofSnake Bite in Perth

Use of Anti-venom:RPH:46 patients received anti-venom34 patients definitely envenomedSCGH:15 patients received anti-venom13 patients definitely envenomedFH:11 patients received anti-venom11 patients definitely envenomed

Snake ClassificationKingdom: AnimaliaPhylum: ChordataSubphylum: VertebrataClass: ReptiliaOrder: SquamataSuborder: Serpentes

Serpent Superfamilies

Henophidia:Boas, pythons andrelatives

Typhlopoidea:Blind snakes

Xenophidia:Colubridae (back fanged)Elapidae (front fanged)Hydrophiidae

Australian Species86 species of elapids33 species of hydrophiidae seasnakes2 species of acrochordidae seasnakes11 species of colubridae15 species of boas31 insectivorous blind snakes178 in total

Australian Snakes

25% of world land snakes = venomous70% of Australian land snakes = venomous

200 species of elapids, worldwide86 of these are specific to Australia20 of these are deadly12+ are potentially dangerous

Elapid Land Snakes of Australia

Brown snakesBlack snakesTiger snakesTaipansDeath adders

All ‘orrible

Snake VenomVenom is a substance which is capable ofproducing toxic reactions when introduced intoanother animalPurpose:

Incapacitates preyAids digestion of preyDeterrent to predators

50% snake bites are dry bites (no venom injected)

Snake Venom Toxicity

Standard measure = LD50 in miceVarious routes of administrationNumber / weights of test animalsVenom binding to lab glasswareMilking may not produce in vivo venom compositionQuantity of venom important

Australia has dubious distinction of holding mostdangerous snakes in the world (18/25):

Inland Taipan LD50 = 0.025mg/Kg (250,000 mice, 100 men)Common Brown snake = 0.053mg/KgTaipan = 0.099mg/Kg

Composition ofSnake Venom

Enzymes:PhospholipasesPhosphodiesterasesAmino acid oxidasesAcetylcholinesteraseProteolytic enzymesArginine esterasesNucleosidasesHyaluronidase

PeptidesNeurotoxinsCytotoxinsMyotoxinsCardiotoxinsDisintegrins

Snake Venom ToxicityLocal effectsSystemic effects:

NeurotoxicityPre- and post-synaptic

HaemotoxicityMyotoxicityNephrotoxicity(Cardiotoxicity)

Neuromuscular Junction:Summary

Interchange between somatic nerve ending and muscularend-plateNerve synthesises Acetylcholine and stores it withinvesiclesNerve stimulation causes vesicle migration to nervesurface, rupture and ACh release into cleftACh receptors open post-junctional voltage gated Na+

channelsNa+ influx initiates skeletal muscle contractionACh detaches from receptor and is destroyed by acetylcholinesterase

NMJ MorphologyMotor neurones (group α fibres) run from ventral horn of spinalcord (monosynaptic)Branches repeatedly on muscle approach = motor unitMyelin sheath lost and replaced by Schwann cell coverNerve separated from muscle by 20nm junctional cleftNerve and muscle held in tight alignment by basal laminaMuscle layer corrugated into primary and secondary cleftsACh receptors on cleft shoulders (5 million per junction)Na+ channels exist deep within the clefts

NMJ: Nerve Action PotentialNerve action potential begins with sodium propagationdown axon = saltatory conductionThis depolarising voltage opens Ca2+ channelsCa2+ influx into cell causes ACh release into synaptic spacevia mobilisation of vesiclesECF [Ca2+ ] controls quantal release of AChCa2+ release is limited by Ca2+-activated K+ channelsCa2+ entry explains post-tetanic potentiationCalcium channels (P-type and L-type) responsible for AChrelease

NMJ: Calcium ChannelsVoltage dependent P channels are found only at nerveterminalsLocated immediately adjacent to active zonesTargeted in Eaton-Lambert SyndromeAntagonised by bivalent inorganic cations eg Mg2+

Explains rationale behind Mg2+ use in pre-eclampsiaNot affected by calcium channel blockers, which blockL-type channels? Prolonged n.m block with non-depolarising agents andcalcium channel blockers

NMJ: Synaptic Vesicles

2 pools of vesicles:VP1 = reserve storeVP2 = readily releasable store

Calcium docks to vesicle wall and causes ACh exocytosisMost vesicles fuse with release sites (VP2 stores)SNARE protein mediated (soluble N-ethylmaleimide-sensitive attachment protein receptors)

Synaptobrevin = vesicle proteinSyntaxins = plasma membrane proteinsSynaptotagmin = neuronal calcium receptor bridge

NMJ: ACh Release

NMJ: Acetylcholine Receptor

Mature / junctional form:2 α, β, δ and ε sub-unitsε = shorter open times, high-amplitude channel currents

Immature / extra-junctional form:2 α, β, γ and δ sub-unitsγ = long open times, low amplitude channel currents

Receptor = 250,000Da molecular weightα units = ACh binding sitesAnchored to end-plate membrane by protein = rapsyn

ACh Receptor

NMJ: Electrophysiology ofNeurotransmission

ACh receptor normally closed by approximation of sub-unitsACh binds both α sub-units to open channelCations flow down concentration gradients:

Influx: Na+, Ca2+

Outflux: K+

Anions excluded from channel

Ion current depolarises muscle membraneEach quantum of ACh opens 500,000 channels (picoamps)Non-depolarising blockers competitively block this action

Neurotoxicityß-Neurotoxins

Highly specific pre-synaptic calcium channel blockers (P-type)Inhibit vesicle availability irreversiblyMultiple agents from different species:

Single chain: Notexin (tiger snake) Pseudexins (black snake) Acanthoxin (death adder)

Multi-chain: Taipoxin, Taicatoxin (taipan)Textilotoxin (brown snake)

All phospholipase A2 inhibitorsa-Neurotoxins

Non-lethal post-synaptic acetylcholine receptor antagonists“curare-mimetic” agents

Coagulation Cascade

Role of Clotting FactorsFactor I: Fibrinogen

Activates fibrin clotFactor II: Pro-thrombin

IIa activates factors I,V,VII,XIII, protein C, plateletsFactor III: Tissue factor

Co-factor for VIIaFactor IV: Calcium

Required for clotting factors to bind phospholipidFactor V: Pro-accelerin, labile factor

Co-factor of X, forms pro-thrombinase complexFactor VI: Defunct

Role of Clotting FactorsFactor VII: Stable factor (cothromboplastin)

Activates IX, XFactor VIII: Anti-haemophilic factor

Co-factor of IX, with which it forms the tenase complexFactor IX: Christmas factorFactor X: Stuart-Prower factor

Activates II, forming pro-thrombinase complex with VFactor XI: Plasma thromboplastin antecedent

Activates XII, IX and pre-kallikreinFactor XII: Hageman factor

Activates pre-kallikrein and fibrinolysisFactor XIII: Fibrin-stabilising factor

Cross-links fibrin

Role of Clotting Co-factorsVon Willebrand Factor:

Binds VIII, mediates platelet adhesion

Pre-Kallikrein: Activates XII and self; Cleaves HMWK

High Molecular Weight Kininogen (HMWK):Supports reciprocal activation of XII, XI and pre-kallikrein

Fibronectin:Mediates cell adhesion

Anti-thrombin III:Inhibits IIa, Xa and other proteases

Fibrinolysis

Role of Clotting Co-factorsProtein C:

Inactivates Va, VIIIaProtein S:

Co-factor for activated protein C (inactive bound to C4b)Protein Z:

Mediates thrombin adhesion to phospholipids, degrades XPlasminogen:

Converts to plasmin, lyses fibrin and other proteinsTissue plasminogen activator, urokinase

Activate plasmina2-antiplasmin

Inhibits plasmin

HaemotoxinsFibrinogen clotting enzymesFibrinogen degrading enzymesPlasminogen activatorsProtein C activatorsActivators of factors V, IX, X and prothrombinAnticoagulantsGIIb/IIIa receptor antagonismComplete defibrination can be seen in <20minutesProfound secondary haemorrhagic effects

Australian ElapidsGroup 1:

Eastern Brown snakeWestern Brown snake(Gwardar)DugitePeninsula Brown snakeSpeckled Brown snakeIngram’s Brown snake

Eastern Brown SnakePseudonaja textilis

Gwardar (Western Brown)Pseudonaja nuchalis

DugitePseudonaja affinis

Speckled Brown SnakePseudonaja guttata

Brown Snake VenomLeading cause of snakebite deaths throughout Australia

White J. Toxicon 1998;36:1483-1492.

Coagulopathy, renal failure and haemolytic anaemiaEarly deaths from shock and ?coronary thrombus

Tibballs J et al. Anaesthetics and Intensive Care 1989;17:466-469.

Tibballs J & Sutherland SK. Anaesthetics and Intensive Care1992;20:33-37.

Johnston M et al. MJA 2002; 177:646-649.

Paralysis and neurotoxicity incredibly rare

Brown Snake VenomVenom-induced activation of coagulation cascadeBrown snake venom contains a factor X-like protein thatactivates prothrombin in the presence of factor V, calciumand phospholipid.Leads to production of thrombin and consumption offibrinogen (i.e. consumption coagulopathy).

Decreased fibrinogen and platelets (sometimes)

Raised INR, APTT, FDPsOuyang C et al. Toxicon 1992;30:945-966.

Australian ElapidsGroup 2:

Common Tiger snakeBlack Tiger snakeWestern Tiger snakeLowlands CopperheadHighlands CopperheadPygmy CopperheadRough scaled snakeBroad-headed snakePale-headed snakeStephen’s Banded snakeEastern Small-eyed snake

Black Tiger SnakeNotechis ater

Mainland Tiger SnakeNotechis scutatus

Perth’s Tiger Snake

CopperheadAustrelaps superbus

Rough Scaled SnakeTropidechis carinatus

Broad Headed SnakeHoplocephalus bungaroides

Pale Headed SnakeHoplocephalus bitoquatus

Stephen’s Banded SnakeHoplocephalus stephensi

Eastern Small Eyed SnakeRhinocephalus nigrescens

Tiger Snake VenomVenom-induced activation of coagulation cascade

Rapid onset, factor V-like prothrombin activator

Isbister et al. Pathology 2002;34(6):588-590.

Neurotoxicity and paralysis

Slow in onset (up to 24 hours)

Associated with rise in creatine kinase

Descending flaccid paralysis

Anti-venom arrests progression but not reversible

Gavaghan CF & Sparkes G. Emerg Med 2003;15:497-499.

Australian ElapidsGroup 3:

Mulga (King Brown snake)Butler’s MulgaCollett’s snakeRed-bellied Black snakeSpotted / blue-bellied Black snake

Mulga (King Brown)Pseudechis australis

Red-Bellied Black SnakePseudechis porphyriacus

Collett’s snakePseudechis colletti

Blue-bellied Black SnakePseudechis guttatus

Black Snake VenomMild anti-coagulant effect but NO pro-coagulation

Normal fibrinogen and FDPsMildly raised INR, APTT

MyolysisCauses renal failureLocal swelling and necrosis at bite site

ParalysisVariable extent

Possible direct cardiotoxin with Mulga envenomation

Australian ElapidsGroup 4:

Common TaipanInland Taipan (fierce snake)

Group 5:Common Death AdderDesert Death AdderNorthern death Adder

Common TaipanOxyuranus scutellatus

Fierce Snake (Inland Taipan)Oxyuranus microlepidotus

Death AddersAcanthophis antarcticus, A. praelongus,A. pyrrhus

Other Elapid Snake Venoms

TaipansAggressive snakes with highrate of envenomationSimilar spectrum of toxicity totiger snakes

Activation of coagulationcascadeFactor V-like prothrombinactivationNeurotoxicity and progressiveflaccid paralysisMyotoxicity with raised CK

Death AddersNeurotoxicity

Potent post-synaptic blockadeNo CK rise

Paralysis:Reversed with anti-venomTemporarily reversed withneostigmine

No effect on coagulation cascadeNo myolytic toxinLittle M et al. MJA 1998;169: 229-30

Limitation of DataExtensively studied:

Common death adderMainland tiger snakeCoastal taipanMulgaEastern brown snake

Lethal and not studied:Black headed death adderNorthern death adderDesert death adderHighland copperheadIngram’s brown snakeBroad headed snakeStephen’s banded snakeButler’s snake

Clinical Manifestations of Snake Bite

Local effects:Local necrosisOedema

Systemic effects:Anti-haemostatic and thromboticCoagulopathyProgressive paralysisShock, hypovolaemiaDirect cardiac toxicityRhabdomyolysis, acute renal failure

Management of Snake BiteFirst aid:

Pressure-immobilisation principle (lymphatic spread)Pressure bandage over entire limbImmobilisation of limb and whole patientSutherland SK et al. Lancet 1979;1:183.

Do not wash snake bite areaAim of first aid is to delay absorption of venom frombite site until the patient is in a facility that canadminister adequate doses of anti-venom if required

Sutherland SK & Leonard RL. MJA 1995;163:616-620.

Hospital ManagementFirst aid and non-removal of PIBTransfer to appropriate facility

Clinical expertise, laboratory facility, sufficient anti-venomAssessment of likely envenomation

Coagulation, neurological examination, respiratory functionEnvenomation is a clinical decision, not from venom detection kits

HistoryGeographical area, snake description, bite characteristics

Lab tests:Coagulation, CK, U&Es, myoglobinuria

Anti-venomAncillary therapy

Snake Species DetectionSnake captured or correctly identifiedGeographical locationLocal and systemic effectsPresence or absence of paralysisCoagulopathy (defibrination or anti-coagulation)Snake venom detection kits

ELISA test (affinity-purified venom specific antibodies)Urine / bite site / bloodDetects 2.5ng/ml venom in 25min

Australia has only commercially available test in worldSutherland SK & Leonard RL. MJA 1995;163:616-620.

VDK

Differences in Snake VenomBrown:

Life threatening coagulopathyParalysisThrombocytopenia

Black (including Mulga):No pro-coagulants in Mulga (anticoagulant toxin only)ParalysisMyolytic toxinsPossible cardiotoxin (Mulga)Less toxic but high volume

Differences in Snake VenomTiger snake:

Pro-coagulantParalysisMyolytic toxin

Taipan:Profound paralysisPotent pro-coagulantMyolytic toxin

Death Adder:Paralysis – commonCoagulopathy - rare

Anti-venomFab fraction of immunised horse serumFirst utilised in 189721 labs produce anti-venom worldwidePowerfully immunogenic to humans but more costeffective because of high yieldLiquid or lyophilised preparationMono-specific: Safer, smaller dose, species dependentPoly-specific: Higher risk of reaction, species independent

Anti-venom

Types available:Death adder (6,000u/vial: $1,582.25)Brown snake (1,000u/vial: $381.96)Black snake (18,000u/vial: $1,963.14)Taipan (12,000u/vial: $2,395.49)Tiger snake (3,000u/vial: $480.78)Sea snake (1,000u/vial: $1314.61)Polyvalent (40,000u/vial: $2,621.96)

Amount standardised to neutralise in vitro averageyield of venom for each snake

Indications for Anti-venomShockSpontaneous systemic bleedingIncoaguable bloodParalysisAcute renal failure or myoglobinuriaExtensive, progressive local swelling

Especially in digits or fascial compartments and necrotic venom

Anti-venoms all highly immunogenicControversy over use of adrenaline pre-medication

Anti-Venom Adverse Effects

Amount required highly variable depending on degree ofenvenomationEquine serum:

Risk of anaphylaxis highViral transmission possibleAnaphylactoid reactions with other equine serum

Complement fixationDelayed serum sickness

Anti-Venom Hypersensitivity1978 – 2003: 17 anti-venom adverse reactions

Polyvalent: 6Brown: 4Tiger: 3Black: 3Death Adder: 2Taipan: 0Sea snake: 2 minor

No deaths

Ancillary Therapy

Thorough supportive care required to maintaincardiovascular, respiratory and renal function whiledefinitive treatment (anti-venom) is given.ICU managementIntubation and ventilationCVVHF / haemodialysisControversies:

Use of blood productsAdrenaline and anaphylaxis precautions before anti-venomRole of fasciotomy in local necrosis

SummarySnakes are rare cause of venomous creature bites but carry highmortality if incorrectly treatedUp to 50% bites may be dry (no venom)6 broad categories of dangerous snakesIdentification of snake difficult and may be avoidableMixed neuro- and haemo-toxicity most serious effectsEnvenomation and need for anti-venom <50% casesManagement of envenomed patients is an enormous challengeand requires transfer to adequate facility and ICUAppropriate first aid therapy can be life savingLimited data available on venom composition and pharmacology

Any Questions?