toxinologist
Active Member
Hi folks,
I've posted this elsewhere, but thought it might be interesting to some of the people here...
The current claims that brown snake antivenom is ineffective are an extrapolation from invitro studies that bear no resemblance to the synergistics of real-life invivo envenomation. I would even go so far as to say that the statements which have appeared in the media as gross exaggerations of the clinical reality.
There are two issues, and these should not be confused, although ultimately they may be found to be intrinsically linked:
1. Antivenom dosages
Clinicians in various parts of Australia have expressed concerns in the past that a single vial of antivenom was insufficient to reverse the clinical effects of envenoming, and in some cases, patients have needed to be given multiple vials. In a review of medical charts from 35 brown snake bites in Western Australia, patients received from 1-23 vials of antivenom with an average of 5 vials. Multiple vials were often given because patients had remained afibrinogenaemic (absent fibrinogen - a substance needed to form clots) after initial antivenom. Normal fibrinogen levels were typically present within 10 hours of initial treatment.
Afibrinogenaemia is a consequence of the activation of prothrombin by toxins in snake venom, and in the snake bitten patient it can lead to catastrophic spontaneous bleeding - from the gums, bite sites, mucosal linings of tissues (such as the stomach), eyes, nose etc. Intracranial bleeding (into the brain tissue) may occur and the consequences of this range from physical or intellectual retardation to death. The syndrome of bleeding seen after snakebite is often referred to as disseminated intravascular coagulopathy (DIC).
In DIC due to brown snake envenoming, the toxin relies on the presence in blood of a substrate - typically phospholipid membranes such as the walls of platelets etc - and Calcium ions - in order to activate prothrombin cleavage to form thrombin which precipitates the conversion of fibrinogen to fibrin. Fibrin is the fundamental building block for clot formation, and the sudden triggering of systemic fibrin production in turn activates the bodies clot dissolving system in which plasminogen converts to plasmin and clots are removed. The net result in snakebite is that the body overcompensates and normal clotting becomes impossible due to the elevated plasmin levels produced in response to the fibrin overproduction. The patient bleeds.
An important limiting factor in all of this is the exhaustion of available phospholipid substrate and/or circulating Ca2+ ions. What we see in patients with DIC due to Papuan taipan bite in PNG is that once these are exhausted, the toxin cannot continue to activate prothrombin and as the liver naturally replaces depleted clotting factors over a period of 6-9 hours, normal coagulation becomes spontaneously restored (even if no antivenom has been given at all!). In other words many patients who may have had a significant coagulopathy after the snakebite, who subsequently present at hospital 10-15 hours later, may have restored haemostasis even though the other effects of the venom such as neurotoxicity are still present. Of course giving antivenom early is undoubtedly highly desirable as a means of quickly arresting DIC and minimising the potential risk of severe effects like intracranial haemorrhage.
In Australia the practice in some places (Western Australia & parts of Queensland for example) that doctors continue giving antivenom until no evidence of coagulopathy is present. Based on evidence from PNG it could well be that in these Australian brown snake cases the original early doses of antivenom may have been sufficient to arrest further progression of the DIC as well, and that time is required for natural restoration of clotting factor levels (in the absence of the ability to transfuse these factors).
One of the commonly quoted explanations for a "need" to give more antivenom is that traditionally we have underestimated the amount of venom injected by brown snakes, and this may well be true. The standard antivenom dose is based on providing the volume of antibodies needed to neutralise the amount of venom injected in an average brown snake bite. The average has for many years been considered to be from 2-4 milligrams. In reality we now know that many brown snakes can inject significantly more venom than this - in some cases 20-25 times this amount. It would therefore seem logical in these cases to administer a larger dose of antivenom.
THE BOTTOM LINE HERE is that the physiological basis for the slow restoration of normal coagulation after brown snake bites needs to be investigated, and we need to develop an understanding of why it takes 10 hours or so before normal values are seen even when antivenom has been given. We also need to reconsider the currently recommendations for initial doses of antivenom in brown snake envenoming and perhaps increase the quantities of antibody present in each vial of antivenom accordingly. Rather than 1000 neutralising units per vial, it may be necessary to package 10000 neutralising units into each vial.
There is however insufficient quantifiable evidence to show that a failing of the antivenom itself is the reason why DIC resolves slowly, and a need for larger doses to neutralise large volumes of antivenom is NOT a failure of the product itself.
2. The current claims of "ineffective antivenom"
Professor George Jelinek is a nice guy and a generally fastidious researcher, but there are some major flaws in the study they have done. I was at the Australian College of Emergency Medicine's 2005 Scientific Meeting in Melbourne a couple of weeks ago when this paper was presented and the consensus of the doctors there who have experience managing snakebite was that the evidence was tenuous at best, and incorrectly interpreted at worst.
Essentially the WA research group had performed a technique known as 2-dimensional polyacrylamide gel electrophoresis (2D PAGE) to separate the components of three brown snake venoms (P. affinis, P. nuchalis and P.textilis) according to both the toxin molecular size, and charge (isoelectric) properties. They then used what is called Western Blotting to determine which of the individually separated toxins could be bound by antivenom. What they reported was that toxins of between 6 and 32 Kilodaltons (kDa) in size did not seem to bind to antivenom at all - hence the claim that antivenom is ineffective.
The problem with reaching this conclusion is that first of all the 6-32 kDa components are not the only components. The toxins responsible for the bleeding in brown snake envenomation for example are a multi-subunit complex comprised of Factor X-like (51-52 kDa) and Factor V-like (166 kDa) subunits - significantly larger than 6-32 kDa. Similarly the major presynaptic neurotoxin (textilotoxin) is another multi-subunit toxin - 4 subunits for a total size of 55-57 kDa. Interestingly enough neurotoxicity is a rare manifestation of brown snake envenomation, and it is typically the clinical syndrome of bleeding that dominates.
There are several brown snake venom components such as Textilinins (9 kDa), postsynaptic neurotoxins like pseudonajatoxin b (7.7 kDa) and individual phospholipases A2 (from 13-17 kDa) that do fall in the 6-32 kDa size range that appeared not to be bound by antivenom, but the reality is we know little of their actual clinical significance, and the group who presented the research did a poor job of providing any evidence for the toxins on this size range having any substantial role in the clinical outcomes of envenomation in snakebite patients.
Data was presented on tests carried out using rat aorta preparations to test for inhibition of toxin activity by antivenom, and it was claimed that the results showed that the antivenom was ineffective. The problem here is that they used whole venom - not isolated toxins - this means they have no way of knowing which component is causing the effect they observed.
We know that the presynaptic neurotoxins and the postsynaptic neurotoxins target receptors on either the motor neurons or the corresponding skeletal muscle neurotransmitter receptors, so it struck me as rather curious that whole venom was used in a smooth muscle assay system instead!
Certainly the whole venom had an effect on smooth muscle that was not attenuated by antivenom, but the key question - and the one which these researchers made no attempt to answer - is what clinical relevance does this have in relation to survival of patients after brown snake envenoming???
THE BOTTOM LINE HERE is that the study only shows that SOME components of brown snake venom do not bind with antivenom under extremely artificial laboratory conditions. It does not show that CSL brown snake antivenom cannot neutralise the toxins responsible for the important clinical effects of envenomation. It does not show that patients with brown snake envenoming obtained no benefit from receiving the current CSL brown snake antivenom, and most importantly it does not show that the antivenom is CLINICALLY ineffective.
Consequently the claims the media should be seen as having been inaccurate exaggerations of basic research findings. The unfortunate thing is that these comments are inflammatory and derisive and promote a lack of confidence in CSL's brown snake antivenom that is thoroughly undeserved.
I would suggest that people disregard the media reports completely.
Cheers
David Williams
Australian Venom Research Unit
Department of Pharmacology
School of Medicine
University of Melbourne
Parkville Vic 3010 AUSTRALIA
I've posted this elsewhere, but thought it might be interesting to some of the people here...
The current claims that brown snake antivenom is ineffective are an extrapolation from invitro studies that bear no resemblance to the synergistics of real-life invivo envenomation. I would even go so far as to say that the statements which have appeared in the media as gross exaggerations of the clinical reality.
There are two issues, and these should not be confused, although ultimately they may be found to be intrinsically linked:
1. Antivenom dosages
Clinicians in various parts of Australia have expressed concerns in the past that a single vial of antivenom was insufficient to reverse the clinical effects of envenoming, and in some cases, patients have needed to be given multiple vials. In a review of medical charts from 35 brown snake bites in Western Australia, patients received from 1-23 vials of antivenom with an average of 5 vials. Multiple vials were often given because patients had remained afibrinogenaemic (absent fibrinogen - a substance needed to form clots) after initial antivenom. Normal fibrinogen levels were typically present within 10 hours of initial treatment.
Afibrinogenaemia is a consequence of the activation of prothrombin by toxins in snake venom, and in the snake bitten patient it can lead to catastrophic spontaneous bleeding - from the gums, bite sites, mucosal linings of tissues (such as the stomach), eyes, nose etc. Intracranial bleeding (into the brain tissue) may occur and the consequences of this range from physical or intellectual retardation to death. The syndrome of bleeding seen after snakebite is often referred to as disseminated intravascular coagulopathy (DIC).
In DIC due to brown snake envenoming, the toxin relies on the presence in blood of a substrate - typically phospholipid membranes such as the walls of platelets etc - and Calcium ions - in order to activate prothrombin cleavage to form thrombin which precipitates the conversion of fibrinogen to fibrin. Fibrin is the fundamental building block for clot formation, and the sudden triggering of systemic fibrin production in turn activates the bodies clot dissolving system in which plasminogen converts to plasmin and clots are removed. The net result in snakebite is that the body overcompensates and normal clotting becomes impossible due to the elevated plasmin levels produced in response to the fibrin overproduction. The patient bleeds.
An important limiting factor in all of this is the exhaustion of available phospholipid substrate and/or circulating Ca2+ ions. What we see in patients with DIC due to Papuan taipan bite in PNG is that once these are exhausted, the toxin cannot continue to activate prothrombin and as the liver naturally replaces depleted clotting factors over a period of 6-9 hours, normal coagulation becomes spontaneously restored (even if no antivenom has been given at all!). In other words many patients who may have had a significant coagulopathy after the snakebite, who subsequently present at hospital 10-15 hours later, may have restored haemostasis even though the other effects of the venom such as neurotoxicity are still present. Of course giving antivenom early is undoubtedly highly desirable as a means of quickly arresting DIC and minimising the potential risk of severe effects like intracranial haemorrhage.
In Australia the practice in some places (Western Australia & parts of Queensland for example) that doctors continue giving antivenom until no evidence of coagulopathy is present. Based on evidence from PNG it could well be that in these Australian brown snake cases the original early doses of antivenom may have been sufficient to arrest further progression of the DIC as well, and that time is required for natural restoration of clotting factor levels (in the absence of the ability to transfuse these factors).
One of the commonly quoted explanations for a "need" to give more antivenom is that traditionally we have underestimated the amount of venom injected by brown snakes, and this may well be true. The standard antivenom dose is based on providing the volume of antibodies needed to neutralise the amount of venom injected in an average brown snake bite. The average has for many years been considered to be from 2-4 milligrams. In reality we now know that many brown snakes can inject significantly more venom than this - in some cases 20-25 times this amount. It would therefore seem logical in these cases to administer a larger dose of antivenom.
THE BOTTOM LINE HERE is that the physiological basis for the slow restoration of normal coagulation after brown snake bites needs to be investigated, and we need to develop an understanding of why it takes 10 hours or so before normal values are seen even when antivenom has been given. We also need to reconsider the currently recommendations for initial doses of antivenom in brown snake envenoming and perhaps increase the quantities of antibody present in each vial of antivenom accordingly. Rather than 1000 neutralising units per vial, it may be necessary to package 10000 neutralising units into each vial.
There is however insufficient quantifiable evidence to show that a failing of the antivenom itself is the reason why DIC resolves slowly, and a need for larger doses to neutralise large volumes of antivenom is NOT a failure of the product itself.
2. The current claims of "ineffective antivenom"
Professor George Jelinek is a nice guy and a generally fastidious researcher, but there are some major flaws in the study they have done. I was at the Australian College of Emergency Medicine's 2005 Scientific Meeting in Melbourne a couple of weeks ago when this paper was presented and the consensus of the doctors there who have experience managing snakebite was that the evidence was tenuous at best, and incorrectly interpreted at worst.
Essentially the WA research group had performed a technique known as 2-dimensional polyacrylamide gel electrophoresis (2D PAGE) to separate the components of three brown snake venoms (P. affinis, P. nuchalis and P.textilis) according to both the toxin molecular size, and charge (isoelectric) properties. They then used what is called Western Blotting to determine which of the individually separated toxins could be bound by antivenom. What they reported was that toxins of between 6 and 32 Kilodaltons (kDa) in size did not seem to bind to antivenom at all - hence the claim that antivenom is ineffective.
The problem with reaching this conclusion is that first of all the 6-32 kDa components are not the only components. The toxins responsible for the bleeding in brown snake envenomation for example are a multi-subunit complex comprised of Factor X-like (51-52 kDa) and Factor V-like (166 kDa) subunits - significantly larger than 6-32 kDa. Similarly the major presynaptic neurotoxin (textilotoxin) is another multi-subunit toxin - 4 subunits for a total size of 55-57 kDa. Interestingly enough neurotoxicity is a rare manifestation of brown snake envenomation, and it is typically the clinical syndrome of bleeding that dominates.
There are several brown snake venom components such as Textilinins (9 kDa), postsynaptic neurotoxins like pseudonajatoxin b (7.7 kDa) and individual phospholipases A2 (from 13-17 kDa) that do fall in the 6-32 kDa size range that appeared not to be bound by antivenom, but the reality is we know little of their actual clinical significance, and the group who presented the research did a poor job of providing any evidence for the toxins on this size range having any substantial role in the clinical outcomes of envenomation in snakebite patients.
Data was presented on tests carried out using rat aorta preparations to test for inhibition of toxin activity by antivenom, and it was claimed that the results showed that the antivenom was ineffective. The problem here is that they used whole venom - not isolated toxins - this means they have no way of knowing which component is causing the effect they observed.
We know that the presynaptic neurotoxins and the postsynaptic neurotoxins target receptors on either the motor neurons or the corresponding skeletal muscle neurotransmitter receptors, so it struck me as rather curious that whole venom was used in a smooth muscle assay system instead!
Certainly the whole venom had an effect on smooth muscle that was not attenuated by antivenom, but the key question - and the one which these researchers made no attempt to answer - is what clinical relevance does this have in relation to survival of patients after brown snake envenoming???
THE BOTTOM LINE HERE is that the study only shows that SOME components of brown snake venom do not bind with antivenom under extremely artificial laboratory conditions. It does not show that CSL brown snake antivenom cannot neutralise the toxins responsible for the important clinical effects of envenomation. It does not show that patients with brown snake envenoming obtained no benefit from receiving the current CSL brown snake antivenom, and most importantly it does not show that the antivenom is CLINICALLY ineffective.
Consequently the claims the media should be seen as having been inaccurate exaggerations of basic research findings. The unfortunate thing is that these comments are inflammatory and derisive and promote a lack of confidence in CSL's brown snake antivenom that is thoroughly undeserved.
I would suggest that people disregard the media reports completely.
Cheers
David Williams
Australian Venom Research Unit
Department of Pharmacology
School of Medicine
University of Melbourne
Parkville Vic 3010 AUSTRALIA
