ANTISNAKE VENOM.pptx

Toxicology, Agadatantra, Ayurveda

1. ANTISNAKE VENOM Dr Anu Mariam Varghese 2nd yr MD Scholar Dept of Agadatantra GAVC, TVM

2. contents Prevalence of snake bite Venom- its composition Why ASV- Definition and Types History Preparation-collection , hyper immunization Why horses as host animal Lyophilisation Centers of manufacture Composition of ASV

3. Indications Administration and doses Contraindication Antivenom reactions Preventions and treatment of antivenin reactions Limitations contents

4. WORLD WIDE PREVALENCE OF SNAKEBITE

5. An estimated 5.4 million people are bitten each year with up to 2.7 million envenomation. Around 81 000 to 138 000 people die each year because of snake bites. India is the most heavily affected country in the world (at least 81,000 snake envenomations and 11,000 fatalities ). India has witnessed an estimated 1.2 million deaths due to snakebites between 2001-2020.(a new study, published in the journal eLife , claims 17-Jul-2020 dated)

6. SNAKE VENOM Complex cocktails of numerous bioactive proteins Secreted by special glands near the upper jaw of the snakes which are akin to human parotid glands.

7. COMPOSTION OF VENOM ENZYMES Phospholipase A2 Phosphodiestrase Phosphomomoestrases L-amino acid oxidase PROTEINS Disintegrin Ancrod ORGANIC COMPOUNDS Citrate Nucleosides Acetyl choline PEPTIDES Bradykinin- Potentiating peptides(BPP)

8. WHY ASV A ccording to the WHO Guidelines, Antivenom is the only specific and scientifically proved antidote to snake venom. A most important decision in the management of a snakebite victim is, whether or not to administer antivenom. D EFINITION Antivenom is a immunoglobulin (usually pepsin refined F(ab’) fragment of whole IgG) purified from the plasma of horse, mule or donkey( equine) or sheep( ovine) that has been immunised with the venoms of one or more species of snake.

9. TYPES Monovalent (monospecific) antivenom – neutralizes the venom of only one species of snake . Polyvalent (Polyspecific ) antivenom – neutralizes the venom of different species of snakes, usually the most important species . In India only Polyvalent ASV is available using venom of Big 4 as they are increasingly challenged . Monovalent variants are available in some countries like Australia, Thailand..

10. HISTORY First introduced independently by Phisalix, Bertrand and Calmette in 1894 They presents the antitoxic properties of the serum of rabbits and guinea –pigs immunized against cobra and viper venoms respectively. First horse derived antivenom sera that he prepared clinically used in 1895 by Haffkine in India and by Lepiney in VietNam and first successful ASV therapy latter reported in patients in 1896.

11. PREPARATION VENOM COLLECTION (MILKING) HYPERIMMUNISATION COLLECTION OF SERUM LYOPHILIZATION

12. Venom is reconstituted as liquid and a very small quantity is introduced into horse Repeated injections given at periodic intervals ,dosage is increased gradually. Horse’s immune system produces antibodies in its blood At certain stage, horse’s blood is extracted and the blood serum contains antibodies is separated and purified This is Antivenin and final product is marketed in crystal form

13. Collection and storage of venom Venom is milked from the snake by mechanical pressure on the venom gland or by electrical stimulation of straited muscles surrounding the gland . The interval between extractions varies from every 2 or 3 weeks to every 3 months. Period of quarantine from 6 to 12 weeks Venom have saliva and other impurities purified by centrifuging and it is preserved at - 10⁰C .

14. ADJUVANT Some snake venom cause local or systemic toxicity at the beginning of immunization course. To avoid toxicity , inoculation is made with small dose of venom well-emulsified in adjuvant such as Freund’s complete, bentonite etc.. Adjuvant may be added to venom to modify the immune response by boosting it . Liberate a higher amount of antibodies and a long lasting protection Minimizing the amount of injected foreign material(venom )

15. 2% solution of Bentonite ( an absorbent Aluminum  phyllosilicate - impure clay ) Used to hold venom in tissue and slowly liberate antigen. Decrease acute toxicity of venom. Enhance immunogenicity of venom activity .

16. WHY HORSES AS HOST ANIMAL Horses are docile, thrive in most climates and yield a large volume of plasma. Easy to breed and handle. Amount of serum in one bleeding is larger than any of other animal. Antivenoms made from horse plasma have proven over time to have a satisfactory safety and efficacy prolife.

17. hyperimmunization Antivenom is prepared by hyper immunizing horses against venom of four common poisonous snakes- Big four. Cobra Common k rait Russel’s viper Saw scaled viper Creates an immunological response that produces large numbers of neutralizing antibodies against various components (toxins) of the venom.

18. BIG FOUR Cobra Naja naja Russel’s Viper Vipera russelli Common krait Bangarus caeruleus Saw Scaled Viper Echis carinatus

19. IN HORSES- HYPERIMMUNIZATION Areas to be immunized should thoroughly scrubbed with disinfectant, shaved and rubbed with 70 % ethanol. Site of immunization – close to major lymph nodes, preferably animal’s neck and back Route of injection – Subcutaneous Initial dose of each venom -1-4 mg/ horse, with a total combined volume of injection of about 2 ml Immunogen filled in a 1 –ml glass syringe with 18G needle. Subcutaneous injections of 100-200 Ül at each site, up to as may 8-12 sites.

20. After 2 weeks booster injection with same venom Subsequent booster immunizations at 2 week intervals can made with higher dose 5-10mg Blood is drawn before each immunization .

21. COLLECTION OF PLASMA Animals are bled by venipuncture from external jugular vein. Area should shaved and disinfected In one bleeding session 13-15ml of blood per kilogram body weight are collected. Blood is collected , ideally , in disposable plastic bags containing sterile citrate anticoagulant. Put in refrigerated room ( 2-8⁰ C) for plasma and blood cells separation procedure i.e validated centrifugation or sedimentation procedure.

22. LYOPHILISATION (FREEZE DRYING) Plasma obtained is concentrated and purified . The serum is lyophilised by drying it from frozen state under high vacuum . A dehydration process typically used to preserve a perishable material or make the material more convenient for transport In the presence of calcium and phosphoric acid. Four stages are there Freezing: Freeze-drying works by freezing the material

23. Vacuum processing : reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase . Heating : to aid vaporization Condensation: vaporized serum passed through low temperature condenser plates to change vapor to powder. Antivenom is available in the form of lyophilised powder Unstable at room temperature, requires 0-4 degree It has the shelf life of 5 yrs.

24. CENTRES OF ASV MANUFACTURING Haffkine Biopharmaceuticals Ltd institute, Mumbai King institute of Preventive medicine , Chennai Serum institute of India, Pune Central research institute Kasauli Bharat Serums and Vaccines Ltd, Mumbai ‘The Irula Snake Catcher’ Industrial Cooperative Society, Vadnemeli , Tamil Nadu Vins Bioproduct Ltd , Hyderabad Biological ‘E’ Ltd, Hyderabad Bengal chemicals & pharmaceuticals , Calcutta

25. Haffkine institute, Mumbai King institute, Chennai CENTRES OF ASV MANUFACTURING

26. SERUM INSTITUTE, PUNE VINS BIOPRODUCTS LIMITED, HYDERABAD

27. composition Each ml of ASV neutralizes the following quantities of standard venom tested in mice by IV route. Cobra…………………………………………………...0.6 mg Common Krait……………………………………….…0.45mg Russell’s Viper………………………..………..………0.6mg Saw-scaled Viper.....................................................0.45mg Preservative: Cresol I.P NMT………………………....0.25%v/v Stabilizer : Gycine I.P, Excipients : Mannitol I.P and Sodium Chloride I.P

28. INDICATIONS SYSTEMIC ENVENOMING Hemostatic abnormalities -Spontaneous systemic bleeding, coagulopathy. Bleeding time > 20 minutes Thrombocyte < 10,000/cu mm Neurotoxic signs-Ptosis, paralysis , external opthalmoplegia CVS abnormalities- Hypotension, shock, cardiac arrhythmia, abnormal ECG. Evidences of intravascular hemolysis. Acute kidney injury-Oliguria/Anuria Hyperkalemia Dark brown urine, Muscle aches etc LOCAL ENVENOMING Local swelling involving more than half of bitten limb. Rapid extension of swelling. Enlarged tender lymph node draining the bitten limb

29. aDMINISTRATION The lyophilised powder is diluted in 500ml of distilled water or normal saline and infused over a period of one hour . Skin test is conducted for detection of sensitivity ,A subcutaneous injection of a minute dose of antivenin 0.1 ml dilute 1:10 ) ASV is administered intravenously either diluted at the rate of not more than 1 ml per minute or is diluted in 500ml of IV fluid( either sodium chloride injection or 5% Dextrose Injection) and administered rapidly as tolerated over 1-2 hours . Antiserum, mix by gentle swirling rather than shaking to avoid foaming.

30. SENSITIVITY TEST 0.02 - 0.5 ml of 1:10 dilution of serum is injected intradermaly . If the reaction is positive an urticarial wheel of 1cm diameter surrounded an erythema of about the same width develops within 5-20 minutes . In non-allergic individuals usual dose is given.

31. DESENSITISATION 0.1 ml of Adrenaline in 1:1000 dilution is given subcutaneously . Then ASV is induced in increased doses at every 15 minutes Then repeat the same regimen with 1:10 dilution At last with undiluted ASV Another method intravenous administration of anti histamine followed by infusion of adrenaline ( 1mg dissolved in 100ml saline ).

32. PROCEDURE Dissolve the antivenin in distilled water or normal saline Administer the appropriate dose as an infusion in 500ml of saline in 15-20 drops / minute The rate can be progressively increased so that infusion is completed in 1-2 hours Children requires the same dose ASV injection repeated after 1 hr , if the symptom persists Further doses repeated every 6 hrs till symptom disappears completely

33. DOSAGE . I nitial dose recommended in severe poisoning is 100 ml as in a moderately severe bite 60 mg of venom injected (10 vials in the case of unidentified snake bites) When local reaction only present 3-6 vials after test dose( one vial of ASV diluted in 200ml of normal saline slowly), rest of dose is given as infusion in 20 minutes and observe for other signs of envenomation. When systemic envenomation is present 10 vials of ASV is given as infusion in 20-30 minutes and simultaneously start six vials of ASV in 5% glucose as a drip to be run in 4-6 hours.

34. Maximum dose of ASV Mild envenomation ……….3 to 5 vials M oderate envenomation……..5 to 10 vials S evere envenomation ………10 to 20 vials (20 vials in case of neurotoxic bites and 30 vials for hemotoxic bites) C lotting time is to be repeated every four to six hour and ASV repeated if necessary B etter late than never is the policy in ASV therapy if signs of systemic envenomation are present even in cases presenting late after a bite. D ose in children exactly same as that of adults.

35. GENERAL MEASURES AFTER ASV Inject tetanus antitoxin or booster dose of tetanus toxoid. Broad spectrum of anti biotics used. Antihistamine & cortisone help in relieving symptoms. In severe poisoning infusion of normal saline or transfusion of blood or plasma useful. Renal dialysis and peritoneal dialysis if needed.

36. ADMINISRATION CRITERIA ASV is a scarce, costly ,commodity and should only be administered when there are definite signs of envenomation. Unbound, free flowing venom can only be neutralized when it is in the blood stream or tissue fluid. If a patient has evidence to suggest systemic envenoming or severe local envenoming then only ASV will be administered.

37. RESPONSES GENERAL : Patient feels better partly placebo effect SPONTANEOUS SYSTEMIC BLEEDING : usually stops within 15-30 min BLOOD COAGULABILITY : usually restored in 3-9 hrs IN SHOCKED PATIENTS : BP may increase first 30-60 min , arrhythmias may resolve NEUROTOXIC ENVENOMING : of post synaptic type improve within 30 min after antivenom , with presynaptic toxins (kraits and sea snakes) will not respond. ACTIVE HAEMOLYSIS AND RHABDOMYOLYSIS : May cease within a few hrs and urine returns to its normal colour

38. CONTRAINDICATIONS There is no absolute contraindication to ant venom treatment Have reacted to horse(equine) or sheep(ovine) serum in past With strong history of atopic diseases( esp. severe asthma). High risk of severe reactions . High risk patients may be pretested empirically with subcutaneous epinephrine(adrenaline), IV antihistamines and corticosteroid. PREGNANCY IS NOT CONTRAINDICATED

39. ANTIVENOM REACTIONS More than 10 % of patients will develop reactions as Early anaphylactic reactions Usually within 10-180 mts of starting ASV Itch (often over scalp) Urticaria Fever, dry cough Nausea, Vomiting Abdominal colic, Diarrhea Tachycardia Shaking chills

40. Minority of these patients develop severe life threatening anaphylaxis, hypotension , bronchospasm and angioedema . These are not IgE mediated type 1 hypersensitivity reactions, mechanism more likely are Complement activation by IgE aggregate or residual fc fragments. Direct stimulation of mast cells or basophils by antivenom protein. 2. Pyrogenic(endotoxin reactions) usually develops 1-2 hrs after Rx due to pyrogenic contamination during manufacturing process. Shaking chills(rigors) Fever Vasodilation Fall in BP Febrile convulsions precipitated in children

41. 3.Late(Serum sickness type) reactions Develop 1-12 days after treatment Fever Nausea, vomiting Diarrhea Itching, recurrent urticaria. Arthralgia, myalgia Lymphadenopathy Periarticular swelling Mononeuritis multiplex Proteinuria with immune complex nephritis Rarely encephalopathy

42. PREVENTIONS AND TREATMENT OF ANTIVENOM REACIONS Prophylatic drugs - Adrenaline, Antihistamine anti H1 blockers, Corticosteroids . Speed and dilution of Intravenous antivenom administration (at the earliest signs of reaction) Administration must be temporarily suspended . Rx for early anaphylactic and pyrogenic reactions- Epinephrine (adrenaline) IM, 0.5mg for adults and 0.1mg/kg body wt. for children.

43. limitations Even among the same species of snakes, there are variations in the composition of venom produced by individuals in different geographical areas .This places limitations on the efficacy of antivenin . The polyvalent antivenin is not as effective as monovalent , thus necessitating larger dosages results in pushing up the cost of treatment. And polyvalent type cause more adverse side effects compared to a monovalent antivenin . Not effective against the bites of the King cobra , Hump-nosed pit vipers, sea snakes, Sochurek’s saw scaled viper (Rajasthan ). The Haffkine Anti Snake Venom (ASV) is thought to be not as effective in envenomed patients in Burma and Sri Lanka as in India.

44. REFERENCES The essentials of forensic medicine & Toxicology – Dr K S Narayan Reddy & Dr O P Murty 34 th edition Comprehensive Medical Toxicology – V V Pillay 3 rd edition , page no 1129-1134 WHO Guidelines for the management of snake bites - Annex 5 A Textbook of Agadatantra – Dr Sobha Bhat . K , page no 200-204 Textbook of forensic medicine and toxicology- V V Pillai -17 th edition Principles of forensic medicine including toxicology – Apurba Nandy Medical jurisprudence and Toxicology – Jaising P Modi

45. ANYTHING YOU CAN IMAGINE, YOU CAN CREATE