VIPER ENVENOMING: EVALUATION OF TREATMENT BY RESTORATION OF HAEMOSTASIS AND VENOM CLEARANCE

Review article

VIPER ENVENOMING: EVALUATION OF TREATMENT BY RESTORATION OF HAEMOSTASIS AND VENOM CLEARANCE

R. D. G. THEAKSTON

, A. S. KAMIGUTI

1 Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK, 2 Department of Haematology, University of Liverpool, Royal Liverpool Hospital, Liverpool L69 3BX, UK.

ABSTRACT. Treatment of systemic envenoming in snakebite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralising potency of the antivenom used, the route by which it is administered and the dose. The use of techniques for evaluating the efficacy of antivenoms has proved to be very useful, as an adjunct to recognised clinical observations, for a more objective evaluation of antivenom efficacy and dosage. In patients bitten by many vipers, including the Brazilian pit vipers, the reversal of the venom-induced coagulopathy provides an ideal indicator as to the efficacy of an antivenom. Likewise, the development of enzyme immunoassay has permitted us to estimate levels of circulating specific venom and antivenom levels at any time after the bite in the patient's blood; the efficacy of an antivenom can thus be objectively assessed by measuring the neutralisation and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms, which results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported Indian antivenom is relatively ineffective in neutralising the procoagulant and other effects of the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved restoration of blood coagulability, the resolution of haemorrhagic disturbances and the increased rate of clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new smaller fragment Fab antivenoms have been developed and are now under clinical assessment. Such clinically based coagulation and immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enable us to assess the use of first aid measures such as tourniquets.

KEY WORDS: snakebite, therapy, haemostasis, enzyme immunoassay.

INTRODUCTION

For many years conventional antivenoms have been prepared by immunising large animals, usually horses, with an individual venom (monospecific antivenom) or a range of different venoms (polyspecific antivenom) obtained from large venom pools to eliminate intraspecific variation. They usually comprise a concentrated F(ab')2 fragment of the IgG molecule separated from the Fc component by pepsin digestion. However, recent developments in immunotherapy include the use of smaller, possibly less reactive Fab fragments prepared from the whole IgG molecule by papain digestion (41,47). Although such an antivenom has some theoretical advantages over an F(ab')2 antivenom, there are also some important disadvantages.

Treatment of envenomed victims has, in the past, been carried out without any real scientific criteria as to the optimal dose of antivenom. The dose given to a patient usually still depends on the individual clinician's experience in assessing the severity of envenoming based on clinical signs (e.g. local and systemic haemorrhage, incoagulable blood, neurotoxicity, myotoxicity, local swelling and necrosis and a range of other effects) (2). More recently, however, more objective methods of assessment involving the observation of restoration of venom-induced coagulability after antivenom therapy in victims of envenoming by many vipers and enzyme immunoassay (EIA) for studying the pharmacokinetics of envenoming and therapy have been evolved. Such studies are also useful in assessing the efficacy of first aid and traditional methods of treatment.

Countries where snakebite is a problem, and where the systems referred to above for assessing antivenom efficacy, include Brazil, Sri Lanka and Nigeria. In Brazil, patients bitten by the jararaca (Bothrops jararaca) are treated with a starting dose of 4 (40 ml), 8 (80 ml) or more ampoules of Bothrops polyspecific antivenom (13,46). The dose usually depends on the severity of envenoming as assessed by the individual clinician treating the patient (2).

In Sri Lanka, the incidence of snake bite is currently one of the highest in the world with a mortality of 6/100,000/year and an incidence exceeding 400/100,000/year (29). The most important species is Russell's viper, Daboia russelli russelli, and systemic envenoming involving haemorrhage, coagulopathy, neurotoxicity and myotoxicity, is normally treated with a starting dose of 5-15 ampoules (50-150 ml) of imported Indian (Haffkine or Serum Institute of India) polyspecific antivenom.(46) In Sri Lanka, problems encountered during antivenom therapy relate to the relative inefficacy of an imported antivenom, not highly active against the venom of the same subspecies in Sri Lanka.

In Nigeria, bites by the carpet viper (Echis ocellatus) constitute a major problem in farming communities (32,52). Envenoming by this species results in local and systemic haemorrhage, incoagulable blood and local necrosis. On the basis of these signs, two ampoules (20 ml) of a monospecific Echis antivenom (South African Institute for Medical Research) or four ampoules (40 ml) of either a French (Institute Pasteur) or German (Behringwerke) polyspecific antivenom have been recommended as a starting dose in the past (53,54). The main problems in Nigeria relate to the frequent lack of availability of effective antivenom in areas where snakebite is a problem; for example, in the north of the country there is a real need for a new antivenom against the venom of E. ocellatus.

TECHNIQUES USED FOR ASSESSING ANTIVENOM EFFICACY

RESTORATION OF HAEMOSTASIS. Depending on the species of snake which has caused envenoming, the usual method by which the clinician assesses antivenom efficacy is by observing the reversal of systemic signs such as venom-induced coagulopathy, haemorrhage or neurotoxicity. For example, in South America most accidents are due to types of vipers (pit vipers) whose venoms contain enzymes capable of activating clotting factors. Therefore, both in South America and in other areas of the world where bites by vipers are common, envenomed patients often develop consumption coagulopathy accompanied by local or systemic haemorrhage (2,14,15,16,23). Coagulopathy results from the action of venom enzymes acting on fibrinogen (thrombin-like enzyme or TLE) or activating prothrombin and/or factor X (10,20,26). Therefore, low levels of circulating fibrinogen and other clotting factors with elevated levels of fibrinogen degradation products undoubtedly characterise pit viper bites in most parts of South America (2,13), and many viper bites in other areas of the world including Nigeria and Sri Lanka. Other common clinical features in these cases are the rapid development of intense local haemorrhage and systemic bleeding. These effects are caused by venom haemorrhagic factors (haemorrhagins), potent metalloproteinases which degrade proteins of the matrix (1) and which can also affect platelet function (17,27,43). Fortunately, all these haemostatic alterations are efficiently corrected by the administration of good-quality antivenom. Because of the observation that the normalisation of blood coagulation in victims of many viper bites correlates well with effective antivenom therapy, an in vitro method for the determination of the blood coagulability, the whole blood clotting test (WBCT), has been adopted routinely to evaluate the treatment. The WBCT was established many years ago and consists of drawing 2 ml of blood from the victim and placing it in a clean glass tube. Then, at minute intervals, the tube is gently tilted at about an angle of 45°. The blood runs freely in the first few minutes and when it stops, the whole blood clotting time is recorded. This is a simple method that can be easily performed at the patient's bedside. Because many factors can influence the WBCT measurement (e.g. temperature and inadequate handling of the test tube), the test was modified to record clot formation in the tube left undisturbed for 20 min, in an all-or-nothing fashion (WBCT20) (53,55). Therefore, if on admission, the patient presents with non-clotting blood, antivenom is administered and every 6 h following antivenom, the WBCT20 is determined. The 6-hour interval for checking blood coagulability was introduced by Rosenfeld and colleagues (35) who observed that 50% fibrinogen levels were restored following effective antivenom therapy in patients with incoagulable blood bitten by B. jararaca in Brazil. Antivenom infusion is continued if the blood is still non-clotting. Measurements of other haemostatic parameters in the laboratory are time consuming and frequently doctors need a quick answer to decide whether or not more antivenom should be administered. The WBCT20 has been successfully used recently in a large number of victims of B. jararaca envenoming in Brazil, in E. ocellatus bites in Nigeria and in envenoming by D. r. russelli in Sri Lanka. Good correlation has been demonstrated between the recorded WBCT20 and fibrinogen levels following treatment with polyvalent Bothrops antivenom in Brazil (37), and evidence suggests that this is also true of bites by vipers whose venom contains procoagulant and TLE activities in other parts of the world. However, it should be stressed that not all viper bites result in blood coagulation alterations. Therefore, ideally what is required is a specific and more rapid EIA than is currently available, as discussed below. Systemic bleeding, such as the frequently observed gingival haemorrhage, results either from low levels of clotting factors, thrombocytopenia and/or platelet defects. In B. jararaca envenoming, mild thrombocytopenia has been reported with defective platelet responses to agonists (e.g. ADP, ristocetin and collagen) (12,38). The defective platelet responses have been attributed either to high levels of fibrin(ogen) degradation products, due to their anticoagulant properties, or to RGD-containing venom disintegrins. Disintegrins are non-enzymatic polypeptides (<10kDa) which possess high affinity binding to platelet integrin, the fibrinogen receptor (39). Some of these defects can be attributed also to the venom haemorrhagins which are responsible for degradation of the matrix surrounding vessel walls. One such factor purified from B. jararaca venom, jararhagin (28), can also impair platelet function by interfering not only with plasma proteins but also by directly affecting platelet surface integrin responsible for collagen binding (17,18). Again, all these signs can be resolved by an efficient antivenom. However, in comparison with the WBCT, observation of cessation of bleeding may not be entirely reliable as an indicator for antivenom therapy.

CLEARANCE OF VENOM FROM THE CIRCULATION. Development of EIA for detection of specific venom antigen (8,48) and for detection of therapeutic antivenom (49) has enabled a more objective assessment of antivenom dosage and efficacy to be made. It is now possible, using this method, to detect and quantify specific venom in the blood or body fluids at any time after the bite, as well as to calculate the amount of therapeutic antivenom circulating at any time after antivenom administration. Figures 1 and 2 show how the kinetics of envenoming and therapy can be assessed when both a relatively ineffective (Figure 1) and an effective (Figure 2) antivenom is used.

Although the existing EIA is excellent for retrospective studies (2,8,9,25,29,30,55), what is really required is a specific, cheap and rapid EIA for use in developing countries. The clinician treating the patient will then be able to obtain a specific diagnosis within a very short time enabling him to treat the patient rapidly with the correct monospecific antivenom, thus avoiding the large volumes of antivenom often required when a polyspecific antivenom is used. This results in a decreased incidence of anaphylactic reactions. It is likely that high doses of antivenom may induce complement activation and formation of immune complexes (aggregates) that have been observed during the more severe clinical reactions associated with homologous immunoglobulin treatment (24). Following the initial studies on EIA as a tool for immunodiagnosis developed by the Liverpool group (48), the Commonwealth Serum Laboratories in Melbourne, Australia did develop such a test (4,50). Regrettably, however, although this is of use in developed countries such as Australia, it is far too expensive ($54/assay in 1995) for routine use in developing countries where snakebite is a major problem.

TREATMENT OF ENVENOMING IN DIFFERENT PARTS OF THE WORLD

BRAZIL. In Brazil, the three major polyspecific Bothrops antivenoms have been compared clinically in a randomised comparative trial (2,49). All the antivenoms produced rapid restoration of whole blood coagulability, a high degree of protection and a rapid rate of venom clearance after a single dose of the lowest amount of antivenom (4 ampoules; 40 ml) recommended for the treatment of moderately envenomed patients (2). A more recent study has demonstrated that even two ampoules (20 ml) is adequate in similarly envenomed patients (13). Restoration of haemostasis and the demonstration of high levels of antivenom in the circulation, even after the venom antigenaemia had been abolished, were usually recorded. In these patients antivenom was only finally cleared from the circulation after 37 days (49); this permitted immediate neutralisation of any additional venom entering the circulation from the depot at the bite site. The results of this particular study indicated, by the combined use of coagulation assays and immunoassay, that the patients had received more antivenom than was actually necessary for neutralising the circulating venom especially in the higher dose ranges (4, 8 and more ampoules) in cases of moderate envenoming.

SRI LANKA. In Sri Lanka, imported Indian (Haffkine or Serum Institute of India) polyspecific antivenom is used for treating envenoming by Russell's viper (D. r. russelli), saw-scaled viper (Echis carinatus), the Indian and Ceylon krait (Bungarus caeruleus, B. ceylonicus) and the Sri Lankan cobra (Naja naja naja). These antivenoms are not very effective against the venom of Sri Lankan D. r. russelli (Figure 1) because they are prepared against the venom of Indian D. r. russelli (29,46). Sri Lankan Russell's viper contains some different components from the Indian venom such as a presynaptically-acting phospholipase A2 (PLA2), which is also myotoxic and not neutralised by the imported antivenom (29). The reversion to the incoagulable state following therapy in many patients treated with the Indian antivenom also supported this (29).

NIGERIA. In northern Nigeria, the best antivenom used in the past has proved to be the monospecific South African Institute for Medical Research (SAIMR, now SA Vaccine Producers (Pty) Ltd.) antivenom raised against the venom of Kenyan Echis, probably E.pyramidum leakeyi (46). This, in most cases, cleared E. ocellatus venom from the circulation within 4-8 hours of intravenous administration of antivenom (Figure 2) with simultaneous permanent resolution of the clinical signs of envenoming such as haemorrhage and blood incoagulability (30,36). The polyspecific German (North and West Africa) and French (Pasteur 'Bitis-Echis-Naja' and Ipser Africa) antivenoms tested were less effective and, although their use resulted in an initial decrease in venom antigenemia and temporary resolution of the clinical signs after admission, this was often followed by an increase in levels of circulating venom and recurrence of clinical symptoms such as incoagulable blood, due presumably to further influx of venom from a depot area at the bite site (5,25,53,54). Owing to an unsustained and inadequate level of antivenom in the circulation, this additional venom was not neutralised and a further dose (or doses) of antivenom was then required. In a case of envenoming by a Tunisian snake of the Echis pyramidum complex, a total dose of over 300 ml of three different antivenoms (one monospecific and two polyspecific) with activity against Echis species failed to neutralise the toxins present in the circulating venom in the patient (Figure 3), demonstrating the lack of specificity of these antivenoms against this particular venom (6).

RECENT ADVANCES IN PATIENT TREATMENT

All these antivenoms are F(ab')2 fragment preparations (21,46,49). A novel ovine Fab antivenom, purified by papain digestion, raised against Nigerian E. ocellatus venom (22,41,47) has recently been assessed. Preliminary studies have yielded promising results (25). Such an antivenom, being a smaller fragment of the IgG molecule than F(ab')2, has a larger volume of distribution and should theoretically also be less immunoreactive. A major disadvantage of this type of small fragment antivenom may be that it is cleared more rapidly via the kidney. In a recent study, the elimination half-time of a Pasteur F(ab')2 antivenom was 18.0 hours compared with 4.1 hours in the case of a Fab antivenom (25). However, the latter antivenom may have the advantage of being more potent than other available products because it is a monospecific preparation raised against the venom of local E. ocellatus; it may also be more effective against local venom effects because of the relatively small size of the Fab fragment. Further studies, designed to fully assess this type of antivenom both in Nigeria and Sri Lanka, are currently in progress.

ANTIVENOM THERAPY FOR TREATMENT OF LOCAL VENOM EFFECTS

The rationale for giving exceptionally high doses of antivenom in Brazil is that some clinicians consider that there may be some beneficial effect in reducing the extent of the local necrosis caused by the cytolytic enzymes present in Bothrops (and other) venoms. Both experimental (11) and clinical (Warrell and Theakston, unpublished observations) studies, however, have shown that although a small amount of F(ab')2 antivenom does gain eventual access to the area of local necrosis, this is well after the initial rapid and irreversible changes have occurred. In human victims, who usually arrive at hospital hours or even days after the bite, there appears to be no real effect on local lesions (33).

ASSESSMENT OF FIRST AID MEASURES

Investigations in Burmese and Thai patients have attempted to assess the effectiveness of tourniquets as a first aid measure by estimating venom levels before and after tourniquet release (9), venom levels distal and proximal to the tourniquet (51) and admission venom levels in patients admitted with and without tourniquets (19). In these cases, tourniquets did not show any enhanced inhibition of spread of venom into the general circulation, but it should be borne in mind that, in the field, tourniquets are often not applied or managed correctly. In the Philippines, tourniquets properly applied have been shown to delay the spread of the major neurotoxin present in the venom of the Philippine cobra, Naja philippinensis, into the circulation until release in the hospital (56). Likewise, in Nigeria one patient admitted to the hospital with E. ocellatus bite, had no clinical signs and no detectable venom on admission, but developed significant clinical signs (incoagulable blood) with associated venom antigenaemia immediately after release of the tourniquet (30) (Figure 4). Another died, not because of envenoming, but due to pulmonary thromboembolus, preceded by thrombophlebitis, local necrosis and gas gangrene caused by the late removal of a tight tourniquet which had been in place for 48 hours after the bite (31).

The possibility that a smaller Fab IgG fragment, given by the intramuscular route, may be of use in pre-hospital treatment is currently under investigation. If this system works, it could represent a major advance in early therapy. Although preliminary results have indicated that absorption even of an Fab fragment into the circulation by this route is still too slow to be useful in early neutralisation of venom, further detailed studies are essential, as there is evidence that further modifications in the antivenom need to be undertaken before such a system could be successful.

Proposed future studies will involve the objective assessment of the so-called traditional and other untested remedies (such as the "black snake stone", high voltage/low current electric shock (7) and other somewhat suspect and unproven treatments) using EIA.

CONCLUSIONS

The first important stage in the evaluation of either a new or existing antivenom is its testing in an animal (usually rodent) model using assessment of protection against the lethal and other venom effects such as haemorrhagic, local necrotising, defibrinogenating and coagulant activities. Simple WHO recommended tests are available for this purpose (21,45,49,55,57), and tests involving the use of insensate tests such as the fertile hen's egg (40) are under development. However, it should be stressed that it is dangerous to extrapolate from animal results to the situation in man for a wide range of reasons (e.g. unrealistic routes of administration in animals of a pre-incubated mixture of venom and antivenom) (21,22,44). The second stage, which is the only truly meaningful method, is therefore testing the experimentally approved antivenom in envenomed humans using both clinical observations and objective measurements of both venom and antivenom levels.

An effective antivenom is one in which a high level of active circulating antibody is maintained in the circulation after initial venom clearance (e.g. Brazilian Bothrops antivenoms, SAIMR Echis antivenom in Nigeria) in human victims. This will be capable of neutralising any venom subsequently entering the circulation from a depot, or from other extravascular tissue compartments, thus preventing the recurrence of clinical signs of envenoming such as reversal to blood incoagulability. Experimental studies in rabbits have indicated that one effect of antivenom therapy is to cause redistribution of venom from the extravascular to the vascular compartment where, with a good antivenom, it will be immediately sequestered by venom antibodies (3,34). Optimum conditions for antivenom therapy are usually obtained when the venom or venoms used for producing the antivenom is obtained from snakes present in the country in which it is proposed to use the antivenom. Venoms, even within the same species, vary dramatically in composition from region to region (42,58), as is also shown in the case of the same subspecies of D. r. russelli from India and Sri Lanka (29).

The study carried out in Brazil strongly suggests that patients were receiving more antivenom than was necessary, thus resulting in a high incidence (37-87%) of early anaphylactic reactions, requiring urgent treatment with adrenaline and antihistamines (2). A lower but effective dose should decrease the extent of this problem (13), and also result in a reduction in cost: the latter is of major importance in developing countries. There is no real evidence that a high antivenom dose is effective in decreasing or eliminating local venom effects such as necrosis (11,13). Both clinical observations and EIA results support these observations.

There is also no convincing general evidence that tourniquets are effective as a first aid measure in delaying the absorption of venom into the circulation. As there are so many obvious variables, the results of studies using EIA are, not surprisingly, inconclusive (9,19,30,31,51,56). Likewise, further detailed studies need to be performed on the possibilities of using lower molecular weight Fab fragments or even smaller antibody components for early treatment following administration via the intramuscular route.

It should be stressed that in viper envenoming with coagulopathy, the most rapid method for assessment of antivenom efficacy and dosage is the WBCT20. This test is so simple to perform that it can be carried out in any rural tropical hospital lacking a proper laboratory. All it requires is a clean, dry, preferably new glass test tube or bottle. This test is more sensitive in detecting the presence of venom in patients with incoagulable blood than the immunoassay (Theakston, unpublished observations). Unfortunately, many doctors consider the test overly simplistic and therefore wrongly assume that it is inaccurate.

Using a combination of experimental, clinical and immunological assay systems, it is therefore possible to obtain a highly accurate assessment of the efficacy of antivenom and of current first aid procedures. The pharmacokinetic differences between F(ab')2 and Fab fragments are obviously connected with the pharmacokinetics of venom. For example, it has been shown in experimentally envenomed rabbits that the plasma redistribution and neutralisation of venom were lower with an Fab preparation that with one containing F(ab')2 (34). It is therefore possible that the "ideal" antivenom may be one which combines the less reactive properties, the increased volume of distribution and the more rapid tissue distribution of Fab fragments with the better plasma distribution and longer elimination time of F(ab')2 fragments.

FIGURE 1. Venom (unbroken line) and antivenom (broken line) levels in a patient following envenoming by Daboia russelli russelli in Sri Lanka. Doses of 5 ampoules (50 ml) of Haffkine polyspecific antivenom were given where indicated. Blood coagulability is recorded as 'NC' (no clot) and 'C' (clot). Note the transient rises in venom antigenaemia causing reoccurrence of clinical signs (e.g. incoagulable blood) requiring further doses of antivenom.

FIGURE 2. Venom (unbroken line) and antivenom (broken line) levels in a patient bitten by a Nigerian Echis ocellatus and treated with a monospecific African Institute for Medical Research (SAIMR) antivenom. Note the rapid venom clearance, restoration of blood coagulability and maintenance of high antivenom levels at the time of complete venom clearance. C = clotting blood and NC = non-clotting blood.

FIGURE 3.
Complete failure of antivenom therapy in a patient envenomed by a specimen of Echis pyramidum from Tunisia. Although there is a fall in venom levels (unbroken line), the patient failed to respond clinically to a total of 310 ml of three different antivenoms (broken line). The figures preceding the letters B (Behringwerke, North and West Africa polyspecific antivenom), S (SAIMR monospecific Echis antivenom) or P (Pasteur, Bitis-Echis-Naja antivenom) indicate the volume of antivenom (in millilitres) given on each occasion. Note also lack of restoration of coagulability in this patient. C = clotting and NC = non clotting blood.

FIGURE 4.
Apparent success of a properly applied tourniquet in delaying the absorption of Echis ocellatus venom into the circulation. Venom antigen levels are indicated by the unbroken line and antivenom levels by the broken line. The patient had no systemic signs on admission, but following release of the tourniquet signs of envenoming, including incoagulable blood, immediately developed necessitating the use of SAIMR Echis antivenom (AV SAIMR). ADM = Time of admission to hospital after the bite. C = clotting and NC = non-clotting blood.

ACKNOWLEDGEMENTS

We wish to thank all our colleagues in Liverpool, Oxford, Brazil, Nigeria, Sri Lanka and elsewhere, without whom it would not have been possible to carry out these and other projects. We are especially grateful to Dr G. D. Laing, Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine for critical reading of the manuscript. Funding agencies who have supported this work include the Medical Research Council, UK, the Wellcome Trust, the Leverhulme Trust, the European Community and Therapeutic Antibodies Ltd., UK.

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Received 11 December 1997

Accepted 16 December 1997

CORRESPONDENCE TO:

R. D. G. THEAKSTON - Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.

Publication Dates

• Publication in this collection
  26 Nov 1998
• Date of issue
  1998

History

• Received
  11 Dec 1997
• Accepted
  16 Dec 1997