Q J Med 2001; 94: 551-559
© 2001 Association of Physicians
Coagulopathy following lethal and non-lethal envenoming of humans by the South American rattlesnake (Crotalus durissus) in Brazil
From the Laboratório de Fisiopatologia, Instituto Butantan, São Paulo, Brazil, 1 Hospital João XXIII, Minas Gerais, Belo Horizonte, Brazil, 2 Departamento de Medicina Interna, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 3 University Department of Haematology, Royal Liverpool Hospital, Liverpool, UK, 4 Centre for Tropical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, UK and 5 Liverpool School of Tropical Medicine, Liverpool, UK
Received 16 May 2001 and in revised form 3 August 2001
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Summary |
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The South American tropical rattlesnake (Crotalus durissus subspp) is responsible for
10% of bites from venomous snakes in Brazil. We studied 24 victims of bites by this species over 3 years, in south-eastern Brazil, particularly investigating haemostatic alterations. Thirteen patients were defined as moderately envenomed and 11 as severe. There were two deaths, which were not attributed to venom-induced haemostatic disturbances. However, envenoming by C. durissus is frequently associated with haemostatic disorders, which are probably attributable mainly to the action of the thrombin-like enzyme, with possible additional effects secondary to the powerful myotoxic activity of the venom. |
Introduction |
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The Crotalus durissus complex, comprising the South American tropical rattlesnakes, is responsible for approximately 10% of cases of envenoming by snakes in Brazil; the majority of these are caused by the cascavel, Crotalus durissus terrificus. The venom of this species possesses neurotoxic,1 myotoxic2 and thrombin-like activities.3
The principal toxins present in the venom include crotoxin,4 crotamine,5 convulxin,6 a thrombin-like enzyme7 and gyroxin.8,9 The high toxicity of the whole venom is attributable to crotoxin, a phospholipase A2 complex, which is the principal component of the venom.4 Experimentally, crotoxin in combination with crotamine exerts a neurotoxic effect on peripheral nerves10 and a myonecrotic effect on muscle.11 The crotamine content of C. durissus venoms varies between different populations of snakes in Brazil and Argentina. Crotamine was absent from populations in north and eastern Brazil, present in north-western São Paulo State and adjacent areas of Paraná and Minas Gerais, and in Ceará there were mixed populations, some secreting crotamine and some not.12 An intriguing possible clinical correlation with the presence of crotamine is the ‘broken neck’ sign resulting from paralysis of the cervical flexor muscles, possibly through direct action of crotamine. This feature has been reported from various parts of Latin America.13 Convulxin causes convulsions and respiratory and circulatory disturbances in mice, dogs, cats and guinea pigs; it also causes in vitro platelet aggregation in the platelet rich plasma of many mammalian species.6 Gyroxin, when injected intravenously, induces episodes of opisthotonos and rotation of the animal's body in the longitudinal axis.8,9 These signs are not observed in human victims. A thrombin-like enzyme, first isolated and characterized by Raw et al.,7 is responsible for the coagulant action of the venom. More recently, a comparative study of the biological activities of the venoms of three different Brazilian subspecies of C. durissus (C. d. terrificus, C. d. collilineatus and C. d. cascavella) proved that most of these activities were common to all these subspecies.14
In most respects, Rosenfeld's clinical descriptions of envenoming by Crotalus durissus ssp in Brazil have not been bettered.15 Local symptoms at the site of the bite include pain, paraesthesiae such as formication or anaesthesia, but little or no swelling and no local necrosis. Rosenfeld denied erythema, but we have observed this (Figure 1
), for example in patients 3 and 13 (below). There is a similar lack of effect when the venom is injected subcutaneously, intramuscularly or intradermally in experimental animals.16 This contrasts with bites by many Crotalus species in North and Central America, which commonly cause severe local necrosis.17,18
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Systemic envenoming usually starts with the development of symmetrical ptosis, external ophthalmoplegia and facial weakness, resulting in the characteristic myopathic/neurotoxic facies. Paresis of the pupils may impair visual accommodation (responsible for patients’ complaints of difficulties with vision), loss of pupillary reflexes and mydriasis. Rosenfeld regarded mydriasis as a fatal prognostic sign.15 Rarely, respiratory muscle involvement may lead to respiratory failure.19 The venom induces generalized rhabdomyolysis, causing myalgias, a massive rise in serum myoglobin and creatine kinase (CK) levels, accompanied by myoglobinuria.2,20–23 Pain throughout the whole body, possibly explained by rhabdomyolysis, was the main symptom remembered by one of the first recorded victims of cascavel bite. Father Luis Rodrigues was bitten near Bahia in north-eastern Brazil at Christmas-time in 1560. He suffered terrible symptoms for the next 20 days.24
In the fatal case described in Londrina, São Paulo State there were clinical, electrocardiographic, biochemical and histological features suggestive of venom-induced myocardial damage.25
Blood coagulation disturbances have been described in about 50% of patients bitten by C. durissus subspp.26 Amaral et al.27 reported patients with afibrinogenaemia but without thrombocytopenia. Spontaneous bleeding has only been rarely observed in human patients. The aim of the present study was therefore to examine in detail the haemostatic disorders in patients bitten by this species, and to assess the ability of antivenom to reverse them.
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Patients
Twenty-four patients were admitted to Hospital João XXIII, Belo Horizonte, Minas Gerais State, Brazil. There were three females and 21 males, ages ranging from 11 to 74 years. All had been bitten by C. durissus subspecies (of which C. d. terrificus is the most prevalent subspecies in Minas Gerais) between January 1993 and November 1995. Diagnosis of Crotalus durissus bite was based on identification of the snake brought with the patient (four patients, C. d. terrificus in each case); detection of specific venom antigen in serum (11 patients) or on clinical features considered diagnostic at admission (nine patients). All of these last nine patients showed characteristic signs of systemic envenoming by C. durissus (e.g. ptosis, myalgia, diplopia or paraesthesia). As none of these signs occur following bites by any other venomous snake in this region, it is reasonable to include these cases in the study. The patients were divided into two entirely arbitrary groups—‘severe’ and ‘moderate’ envenoming—based on their presenting symptoms and signs. ‘Moderate envenoming’ was defined as disturbance of visual accommodation, ptosis or neurotoxic facies,15 mild myalgia, slight or absent urinary pigment with normal urine output, and altered or normal coagulation time. ‘Severe envenoming’ was defined by the presence of obvious neurotoxic signs, intense myalgia accompanied by pigmented urine with the presence or absence of oliguria/anuria and normal or altered coagulation time.28 Patients who had been treated with antivenom before admission to the hospital were excluded from the study. Informed consent to participate in the clinical study was obtained from the patients or, if young children were involved, from their parents. The study was approved by the Ethics Committee of Hospital João XXIII.
Blood coagulation
Venous blood was sampled on admission, at 6, 12 and 24 h after the end of antivenom therapy, and then daily until discharge from hospital. For coagulation and fibrinolysis assays, 9.0 ml blood was mixed with 0.8 ml 16 mM trisodium citrate containing 0.2 ml Instituto Butantan Crotalus monospecific antivenom29 to neutralize any venom present in the sample at the time of collection. The citrated blood was centrifuged at 2000 g for 15 min at 4 °C and the platelet-poor plasma was frozen in 0.5 ml aliquots at -20 °C until tested. Control samples, collected from people with no history of snakebite living in the same region as the victims were treated as described above.
Standardized reagents purchased from Diagnostics Stago (France) were used to measure prothrombin time (PT) and the activated partial thromboplastin time (APTT). Levels of coagulation factors (factors II, VII, VIII, IX, X, XI, XII) were estimated using deficient plasmas, protein C (PC) (Staclot protein C assay), cross-linked fibrin fragment (D-dimer, DD) using immunoassay by latex agglutination, 
2-antiplasmin (2-AP) (Stachrom Antiplasmin) and serum fibrin(ogen) degradation products (FnDP/FgDP) using immunoassay (latex agglutination). Thrombin-antithrombin antigen (TAT) was determined using an EIA kit system from Behring (Enzygnost-TAT). Factor V was estimated using the standardized technique of Denson30 and fibrinogen levels were measured using the method of Ratnoff and Menzie.31
Haematological tests
Blood (2.5 ml) was mixed with 25 µl of 10% potassium EDTA and 50 µl Crotalus antivenom. Erythrocytes, leukocytes and platelets were counted using an automated cell counter (Coulter T 890). Differential leukocyte counts were carried out on blood films stained with panchromatic stain.
Biochemical tests
Blood was sampled for creatinine, urea and creatine kinase (CK, normal values: 10–120 U/L) enzyme activity determinations using Labtest kits. Serum myoglobin concentration was measured using the Rapitex-myoglobin kit (Behring).
Venom antigen levels
Enzyme-linked immunosorbent assay (ELISA) was used for detection and measurement of C. durissus venom in admission serum samples according to the technique described by Chávez-Olórtegui et al.32 A 96-well Microtitre plate (Hemobag Produtos Cirúrgicos) was coated overnight with 100 µl of a 5 µg/ml solution of anti-C. durissus IgG in carbonate buffer. After blocking and washing, 50 µl serum diluted 1:2 in dilution buffer was added (1 h, room temperature). The plates were washed and incubated with peroxidase coupled with anti-C. durissus IgG (1 h, room temperature). The wells were washed, and the assay was completed and stopped by the addition of 20 µl of a 1:20 dilution of sulphuric acid. A reference curve was obtained using dilutions of known concentrations (4–500 ng/ml) of crude venom from C. durissus terrificus. The baseline for the assay was established according to the recommendations of Theakston33 and Ho et al.34 A cut-off was established by testing sera from 103 controls of the same socio-economic group as the patients. The mean±SD OD at 492 nm was 0.037±0.016 for the whole venom assay. Using the mean plus 2 SD as the negative cut-off value for the assays, which corresponded to a whole venom concentration of 20 ng/ml, two of the 103 normal sera were positive for whole venom, resulting in a specificity of 98.1%.
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Results |
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Clinical observations
The time between bite and admission to hospital in the 24 envenomed patients ranged from 2 to 60 h. In 13/24 (54%) a tourniquet had been applied, 7/22 (32%) had paraesthesiae, 18/22 (82%) had ptosis, and 10/19 (53%) suffered from myalgia. Only 1/24 (4%) had respiratory insufficiency on admission. The basic information and clinical symptoms for each patient are shown in Table 1
.
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Laboratory findings
On admission and before antivenom therapy, 10/22 (45%) patients had a PT<10% and 12/22 (55%) had a prolonged APTT, with three patients having totally incoagulable blood (Table 1
). Assays of clotting and fibrinolytic factors showed fibrinogen and factor V consumption, consumption of 2-AP and consequent increase of FnDP/FgDP and D-DD and the formation of thrombin-antithrombin complex in some patients (Table 2). Only 3/5 patients studied had decreased levels of protein C (0%, 26%, 49%).
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There were no gross alterations in the haematological picture, although a slight polymorphonuclear leukocytosis was recorded in the majority of cases (Table 2
). Interestingly, there was no alteration in the platelet count in the majority of patients; thrombocytopenia was observed only in the two fatal cases.
Twelve hours after antivenom treatment, fibrinogen and FnDP/FgDP levels were within the normal haemostatic range. Fibrinogen levels increased, and this was paralleled by a decrease in FnDP/FgDP levels and normalization of DD levels in the plasma. Simultaneous consumption of 2-antiplasmin indicated secondary activation of the fibrinolytic system. All these levels had returned to normal by the time the patient was discharged from hospital (Figure 2).
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As expected, the increased levels of myoglobin in serum, highest on admission, decreased rapidly following antivenom therapy (Figure 3a
). Serum CK levels were significantly elevated compared with the normal range (10–120 U/l) from admission to 24 h after the start of antivenom (Figure 3b). However, the differences in CK levels on admission and 12 and 24 h after antivenom were not significant (p>0.05).
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Admission venom levels were measured in 19/24 patients. The mean venom antigen level in the 11 moderately envenomed patients was 38.4 ng/ml±20.0 ng/ml (SEM) and in the eight severely envenomed patients it was 251.2 ng/ml±86.9&!thinsp;ng/ml (SEM) (Table 1
). The difference between these levels was statistically significant (p=0.022). The majority of the severely envenomed patients (6/9, 67%) had an intense coagulopathy (PT<10%) on admission, whereas only 4/13 (31%) of the moderately envenomed patients had evidence of severe coagulopathy (PT<10%) on admission (Table 1).
Severe coagulopathy in two fatal cases
Among the 24 patients, there were two deaths (8.3%). A 15-year-old boy (patient 3) was bitten on the back of the hand and, 8 h later, was admitted to a local hospital, prostrated, sweating, hypotensive and in urinary retention. Urethral catheterization yielded 1000 ml of cloudy urine. He was transferred to Hospital João XXIII, arriving 25 h after the bite. He was comatose and dehydrated, with gingival bleeding, and oedema and redness at the site of the bite. He was tachycardic (120 bpm) and tachypnoeic (28 breaths/min) with nasal flaring and audible rhonchi. Blood pressure was 100/60 mmHg. His blood was incoagulable, with a grossly prolonged prothrombin time, fibrinogen 0.023 g/l, platelets 166x109/l, leukocytes 12.3x109/l (83% polymorphs), CK 96 000 U/l (3.9% CKMB), haemoglobin 115 g/l, haematocrit 35%. The venom antigen level was 325 ng/ml. Urine was strongly positive for blood, haemoglobin or myoglobin. Twenty ampoules of Crotalus antivenom (Anticrotálico, Instituto Butantan) was given 27 h after the bite. Two hours after admission, the patient's respiration had become shallow and he was intubated, transferred to the intensive care unit and mechanically ventilated, but he may have aspirated at this time. There was radiographic evidence of right basal consolidation. There was evidence of renal impairment (urea 760 mg/l, creatinine 20 mg/l) and mild metabolic acidosis. Despite treatment with intravenous fluids, furosamide and dopamine, urine output was only 500 ml in 24 h, and the patient became more comatose with neck rigidity and extensor plantar responses. A brain scan showed no evidence of intracranial haemorrhage. At 42 h after the bite, 12 h after antivenom treatment, blood urea had risen to 1450 mg/l, creatinine to 36 mg/l, potassium to 7 mequiv/l and the leukocyte count to 16.3x109/l (77% polymorphs). The prothrombin ratio had improved to 42%, activated partial prothrombin time (APPT) 50/32 seconds. Following initiation of peritoneal dialysis, the patient's level of consciousness improved. The pupils were equal in size and reacted to light. The platelet count had fallen to 85x109/l. Eighty hours after the bite, the patient remained oliguric on peritoneal dialysis. Diffuse consolidation developed in both lungs, with progressive deterioration in respiratory and haemodynamic function. He died 110 h after the bite. The key features of this fatal progression were early shock, coagulopathy with spontaneous bleeding, renal failure, respiratory failure, rhabdomyolysis and finally, progressive respiratory failure compounded by aspiration pneumonia, adult respiratory distress syndrome and possibly pulmonary haemorrhage.
Patient 13 was a 50-year-old man bitten on the dorsum of the foot. On admission to Hospital João XXIII 15 h after the bite, there was redness and swelling at the site of the bite, blurring of vision, bilateral ptosis, diplopia and passage of red urine. The blood was incoagulable, with prolonged prothrombin time and APPT and thrombocytopenia (78x109/l). Clotting factors V, VIII, VII and X were grossly depleted but with normal levels of factors II, IX, XI and XII, indicating disseminated intravascular coagulation. CK was 14400 U/l (5% CKMB), haemoglobin 147 g/l, haematocrit 46%, leukocytes 14.8x109/l (85% polymorphs). The urine was strongly positive for blood, haemoglobin or myoglobin, but there was no evidence of renal impairment. He was treated with 20 ampoules of Crotalus antivenom (Anticrotálico, Instituto Butantan) 18 h after the bite and remained haemodynamically stable, with normal urine output, no biochemical evidence of renal impairment, falling levels of CK and an increase in platelet count. His vision and ptosis improved. On the fourth day of admission (107 h after the bite), the patient developed visual and auditory hallucinations with paranoia. There was no biochemical or haemostatic abnormality. Ten days after the bite, the patient suffered a fatal cardiac arrest while taking a bath. Permission for autopsy was not granted by his family.
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Discussion |
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In this study, the severity of envenoming correlated with venom antigenaemia and haemostatic abnormalities on admission to hospital. The four highest venom antigen levels were found in the severely envenomed patients. However, in patient 21, in whom a negative admission venom level was recorded, it is likely that the blood sample was taken before release of the tourniquet. It is possible that only after release of the tourniquet were the clinical signs observed, probably due to the release of the venom, previously retained by the tourniquet, into the circulation.
Both the fatal cases recorded here had profound haemostatic abnormalities including thrombocytopenia. Among 12 cases of fatal envenoming by C. durissus in the State of São Paulo, 10 (83%) presented with coagulopathy.36 However, the likely cause of death in patient 3 in the present study was respiratory complications of probable respiratory muscle weakness that required intubation and mechanical ventilation. Other life-threatening effects of envenoming identified in this study were hypotension and shock, rhabdomyolysis, possible myocardial damage (patient 13), renal failure and spontaneous haemorrhage.
Alterations of the whole-blood clotting time have been observed in 48% (104/216) of patients bitten by C. durissus, and incoagulability in 38% (86/216).26 Some patients present with no detectable fibrinogen.27 In this study, consumption coagulopathy, characterized principally by the consumption of fibrinogen, factor V and protein C, was observed with slight depletion of factors II and VIII; however, levels of other coagulation factors were generally within the normal range. The coagulopathy was accompanied by an increase in levels of FnDP/FgDP and DD, and a significant consumption of 2-antiplasmin, indicating a secondary activation of the endogenous fibrinolytic system; this is consistent with evidence that the venoms of C. d. terrificus, C. d. collilineatus and C. d. cascavella do not have fibrinolytic activity in vitro.14 An increase in levels of the thrombin-antithrombin complex was also observed, indicating the formation of intravascular thrombin.
The decrease in fibrinogen levels soon after the bite can be explained by the thrombin-like enzyme in the venom. However, the alterations in the levels of some coagulation factors (II, V, VIII and protein C) and the formation of DD and thrombin-antithrombin complex, characteristic of physiological thrombin action, are difficult to explain as a direct effect of C. durissus venom, since no activator of factors II or X is present.14 It is possible that another, not yet identified, activator may be involved. Alternatively, the thrombin-like enzyme in C. durissus venom may be different from that present in Bothrops venoms, and act more like physiological thrombin. The thrombin-like enzyme of Bothrops jararaca venom is not capable of clotting rabbit fibrinogen,37 whereas C. d. terrificus venom clots both human (Minimum Coagulant Dose, MCD, 21.5 µg/ml) and rabbit (MCD 33.3 µg/ml) fibrinogen.14 This suggests that the thrombin-like component of the major Brazilian subspecies of C. durissus venom has an action similar to physiological thrombin. In C. durissus envenoming, all the changes in the coagulation system, with the exception of fibrinogen levels, are milder than in Bothrops envenoming, as we reported previously.38
The venom of C. durissus includes a powerful myotoxin which causes rhabdomyolysis in patients2 and damages the microvasculature of smooth muscle, especially that of endothelial cells lining the capillaries and arterioles.22 The changes in vascular smooth muscle may be due directly to the toxic effect of the venom components or indirectly to the ischaemia39 described in patients envenomed by South American rattlesnakes.26 It is possible that endothelial cells thus stimulated may release various constituents that can activate the coagulation system. The generation of intravascular thrombin in C. durissus envenoming could therefore be a consequence of the secondary activation of the coagulation system as suggested in this study.
Ontogenic variations in snake venoms may be important.40 In the genus Bothrops, the venoms of the young snakes of the majority of species contain larger amounts of both factor II and factor X activators than those of adult snakes.41 This results in relatively more severe haemostatic disorders following envenoming by young snakes.42 In young Crotalus atrox, a direct thrombin-like clotting action on fibrinogen was observed in 2–8-month-old specimens; from 11 months onwards, the venoms of the same individual snakes no longer clotted fibrinogen directly.43 However, these age-related differences have not been described in envenoming by the South American rattlesnake,44 and there is no reference in the literature demonstrating ontogenic differences in coagulant activity of the venoms of either C. d. terrificus or C. d. collilineatus.
Venom-induced systemic haemorrhage is rarely reported following envenoming by C. durissus subspecies; slight bleeding at the site of the bite is usual.45 However, according to Jorge and Ribeiro,26 about 4.8% (12/249) patients had systemic bleeding; gingival haemorrhage, epistaxis, and vaginal bleeding. The figures in our study agree, with only one (4.2%) of our 24 patients having signs of spontaneous systemic haemorrhage, despite the prevalence of severe coagulopathy. Various snake venoms possess activators and inhibitors of platelets.46 In patients envenomed by Bothrops species, thrombocytopenia is very common, and is frequently associated with a bleeding tendency47 and platelet dysfunction.48 However, in C. durissus envenoming, thrombocytopenia is rarely recorded;27 our results support this observation. Although it is well known that convulxin is a potent platelet-aggregating agent6,49 and that crotoxin can also cause platelet aggregation,50 in our study only two patients developed thrombocytopenia. The polymorphic leukocytosis observed in C. durissus envenoming is similar to that seen after bites by many species of snakes, including Bothrops.38
Following treatment with specific C. d. terrificus antivenom,29 levels of fibrinogen, FnDP/FgDP, DD, 2-antiplasmin and myoglobin rapidly returned to normal. In fact, by 12 h after the start of antivenom, almost all laboratory haemostatic variables had returned to normal; however as would probably be expected, CK values remained elevated for over 24 h. We can thus confirm the efficacy of antivenom treatment, as with envenoming by B. jararaca.38
Clinically moderate and severe systemic envenoming by C. durissus is often associated with haemostatic disorders attributable to the action of the thrombin-like enzyme, possibly in association with the myotoxic component of the venom. However, the two fatalities in this series could not be attributed to bleeding or coagulation disturbances.
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Acknowledgments |
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Financial support was obtained from the Science and Technology for Development programme of the European Community (contract no. TS3-CT91-0024) and FAPEMIG (contract no. CBS 542/92). CFSA, NAR and ISS-M are recipients of CNPq fellowships. We wish to thank Neusa Tadeu Penas Picon for technical assistance.
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Address correspondence to Dr Ida S. Sano-Martins, Instituto Butantan, Laboratório de Fisiopatologia, Av. Vital Brazil, 1500, 05503 900, São Paulo (SP), Brazil. e-mail: lusiada{at}uol.com.br
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