Transactions of the Royal Society of Tropical Medicine and Hygiene (2004) 98, 28—42

Clinical trial of two antivenoms for the treatmentof Bothrops and Lachesis bites in the northeastern Amazon region of Brazil

Pedro Pereira de Oliveira Pardala, Suzana Medeiro Souzaa,Maria Rita de Cássia da Costa Monteiroa, Hui Wen Fanb,João Luiz Costa Cardosob, Francisco Oscar Siqueira Françab,Sandra Corrallo Tomyc, Ida S. Sano-Martinsc,Maria Cristina Cirillo de Sousa-e-Silvac, Monica Colombinid,Nancy F. Koderad, Ana Maria Moura-da-Silvad, Diva Ferreira Cardosod,1,David Toledo Velardee, Aura S. Kamiguti f, Robert David G. Theakstong,David A. Warrell g,h,*

a Hospital Universitário João de Barros Barreto, Belém-Pará, Brazilb Hospital Vital Brazil, Instituto Butantan, São Paulo, SP, Brazilc Laboratório de Fisiopatologia, Instituto Butantan, São Paulo, Brazild Laboratório de Imunopatologia, Instituto Butantan, São Paulo, SP, Brazile Fundação Ezequiel Dias, Divisão de Imunobiológicos, Belo Horizonte, MG, Brazilf Department of Haematology, University of Liverpool, Liverpool, UKg Liverpool School of Tropical Medicine, Liverpool, UKh Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington,University of Oxford, Oxford OX3 9DU, UK

Received 5 March 2003 ; received in revised form 31 March 2003; accepted 23 June 2003

KEYWORDSSnakebite;Antivenom;Clinical trial;Bothrops atrox;Lachesis muta;Amazonia;Brazil

Summary The efficacies of specific Bothrops atrox-Lachesis and standard Bothrops-Lachesis antivenoms were compared in the north eastern Amazon region of Brazil.The main aim was to investigate whether a specific antivenom raised against thevenom of B. atrox, the most important Amazon snake species from a medical point ofview, was necessary for the treatment of patients in this region. Seventy-four patientswith local and systemic effects of envenoming by Bothrops or Lachesis snakes wererandomly allocated to receive either specific (n = 38) or standard (n = 36) antiven-oms. In 46 cases (24 in the standard antivenom group, 22 in the other) the snake wasidentified either by enzyme immunoassay or by examination of the dead snake, asB. atrox in 45, L. muta in one. Patients were similar in all clinical and epidemiologi-cal respects before treatment. Results indicated that both antivenoms were equallyeffective in reversing all signs of envenoming detected both clinically and in the labo-ratory. Venom-induced haemostatic abnormalities were resolved within 24 h after the

*Corresponding author. Tel.: +44-1865-221-332/220-968; fax: +44-1865-220-984.E-mail address: david.warrell@ndm.ox.ac.uk (D.A. Warrell).1Deceased.

0035-9203/$ — see front matter © 2003 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.doi:10.1016/S0035-9203(03)00005-1

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Clinical trial of two antivenoms for the treatment of Bothrops and Lachesis bites 29

start of antivenom therapy in most patients. The extent of local complications, suchas local skin necrosis and secondary infection, was similar in both groups. There wereno deaths. The incidence of early anaphylactic reactions was 18% and 19%, respec-tively for specific and standard antivenoms; none was life-threatening. Measurementof serum venom concentrations by enzyme immunoassay (EIA) confirmed that bothantivenoms cleared venom antigenaemia effectively. EIA also revealed that one pa-tient had been bitten by Lachesis muta, although the clinical features in this casewere not distinctive.© 2003 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. Allrights reserved.

1. Introduction

Snake bites are an important cause of morbidityand mortality in tropical countries. In 1999, 17 704snake bites were registered in Brazil, of which 20%occurred in the Amazon region (Brasil Ministério daSaúde, unpublished), but it seems likely that thisfigure is an underestimate as many victims fail toreach hospital, and may die unrecorded in rural ar-eas (WHO, 1981). The estimated mortality amongrubber tappers and indigenous peoples reaches400/100 000 population in some areas of the rainforest (Pierini et al., 1996). In the Amazon region,the genus Bothrops (lance-headed pit vipers) is im-plicated in most human snakebites. Bothrops atrox(barba amarilla), a highly adaptable and widely dis-tributed species, causes most of these bites (Pardalet al., 1997b; Silva, 1989). The genus Lachesis(bushmasters) inhabits tropical rain forests fromNicaragua south through Central and South America(Campbell et al., 2003). Most species do not appearto be aggressive and rarely bite humans (Oteroet al., 1993; Pierini et al., 1996; Warrell, 2003).Lachesis and Bothrops envenoming may result insimilar clinical features, characterised by localsigns, coagulopathy and a bleeding diathesis. Localnecrosis may lead to functional sequelae and, oc-casionally, leads to amputation of the bitten limb.A syndrome of nausea, vomiting, abdominal colic,diarrhoea, sweating, hypotension, bradycardia andshock is sometimes observed in patients bitten byLachesis muta. The presence of these autopharma-cological or autonomic disturbances indicates se-vere envenoming (Borges et al., 1999; Jorge et al.,1997). Distinguishing between Bothrops and Lach-esis bites depends on identification of the snake orby enzyme immunoassay diagnosis (EIA), which isnot generally available in Brazil (Chavez-Olorteguiet al., 1993; Theakston et al., 1977).In Brazil, antivenom was first produced at

the beginning of the twentieth century (Brazil,1901). Production methods have scarcely changedsince then, although modernisation of techni-cal schedules has improved antivenom quality

(Raw et al.,1991; Theakston et al., 2003). Clinicaltrials have confirmed the efficacy of antivenom inthe treatment of Bothrops envenoming (Cardosoet al., 1993; Jorge et al., 1995; Otero et al., 1996;Otero et al., 1998). Brazilian regulations for thepreparation of Bothrops-Lachesis antivenom stipu-late immunisation with a mixture of L. muta venomand different species of Bothrops venoms (BrasilMinistério da Saúde, 1996). Despite the recognisedimportance of B. atrox throughout the Amazonregion, difficulties in obtaining a standardised B.atrox venom and the high levels of experimen-tal cross-neutralization between Bothrops venoms(Dias da Silva et al., 1989) have led to its exclusionfrom the antigen pool used in antivenom produc-tion. No B. atrox antivenom manufactured in Brazilhas ever before been tested clinically. The aimof the present study was to compare the effica-cies of a specific B. atrox-Lachesis and standardBothrops-Lachesis antivenoms in reversing the clin-ical effects of envenoming in the Amazon region ofBrazil.

2. Patients and methods

2.1. Patients

Between 1998 and 2000, a randomised open clinicaltrial was carried out at the Hospital UniversitárioJoão de Barros Barreto, Belém, Pará, a tertiary re-ferral hospital for the metropolitan region of Belémin the north eastern Amazonian region of Brazil. Itspopulation comprises indigenous Amerindian com-munities, long-term migrants from other northernregions and subsistence farmers. B. atrox (Fig. 1)causes most snake bites (Borges et al., 1999; Pardalet al., 1993; Pardal et al., 1997b) but the very sim-ilar Bothrops marajoensis, whose status as a sepa-rate species is questionable, occurs in Marajó Islandand L. muta (Fig. 2) also occurs in the study area.Patients, aged between 5 and 68 years old, with

signs and symptoms compatible with Bothropsenvenoming (local pain and swelling, local and

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Fig. 1 Bothrops atrox from Mosqueiro Island nearBelem, Para State.

systemic bleeding) were considered eligible for thestudy whether or not the snake had been identi-fied. Informed consent was obtained from patientsor their relatives for inclusion in the protocol.Patients excluded from the study were those ad-mitted more than 48 h after bite, those who hadbeen treated with antivenom before admission tothe referral hospital, pregnant women and childrenunder 5-years-old.

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Fig. 3 Levels of B. atrox venom in sera of patients on admission (T0), and 6 h (T6), 12 h (T12), 24 h (T24) andat discharge (Tdis), after receiving B. atrox-Lachesis (left) or Bothrops-Lachesis (right) antivenoms. Horizontal linecorresponds to the background value obtained with serum of non-bitten individuals from the same region as thepatients.

Fig. 2 Lachesis muta from Quebrangulo, Alagoas State,Brazil.

2.2. Identity of snakes responsiblefor the bites

Dead snakes brought to the hospital with the vic-tims were preserved in formalin or 70% ethanoland identified at the Museu Emılio Goeldi, Belém,Pará. Where direct identification was impossible,EIA was used to detect specific circulating B. atrox

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or L. muta venoms in admission (preantivenom)serum samples, wound and blister aspirates (seeEIA section). This assay does not distinguish be-tween B. atrox and B. marajoensis and so, forthe purposes of this study, and accepting the un-certainties about the specific status of the lattertaxon, bites in the Western forested part of MarajóIsland were attributed to B. atrox while those inthe seasonally flooded relatively open Eastern sidewere attributed to B. marajoensis.

2.3. Clinical and epidemiological data

These were collected on standard forms on admis-sion and at least daily thereafter until dischargefrom hospital. Evidence of local swelling, bruising,blistering and signs of infection and local necrosiswere recorded, as were signs of spontaneous sys-temic bleeding from the gums or skin and haema-turia. Extension of local swelling was graded 1—5,according to the number of segments involved.Necrosis was defined as dead, discoloured, anaes-thetic tissue; ‘abscess’ as a fluctuant inflammatoryswelling from which pus could be aspirated.

2.4. Patient management

Patients were stratified into three arbitrary groupsaccording to clinical severity on admission (BrasilMinistério da Saúde, 1998). ‘Mild’ patients hadlocal swelling of no more than two segments oftheir bitten limb, with or without coagulopathyand systemic bleeding; ‘moderate’ patients hadlocal swelling involving three or four segments ormild systemic bleeding (from gums, skin, nose)and ‘severe’ patients presented with swelling ofthe whole bitten limb or profuse systemic bleed-ing or hypotension (supine systolic blood pressure<70mm Hg on at least two occasions 10min aparton admission with impaired peripheral circula-tion). Patients received 4 to 12 ampoules of eitherantivenom depending on this assessment of theirseverity.

2.5. Randomisation

Patients fulfilling the entry criteria were randomlyallocated to receive either standard Bothrops-Lachesis antivenom or specific B. atrox-Lachesisantivenom. Antivenom administration and premed-ication schedules (promethazine 0.5mg/kg i.m.and hydrocortisone 10mg/kg i.v.) followed the con-troversial Brazilian Ministry of Health’s guidelines(Brasil Ministério da Saúde, 1998). Antivenom wasdiluted in saline and administered by intravenousinjection at a rate of 1.5—2.0mL/min. Patients

were monitored for 24 h after the start of an-tivenom and, in the event of a reaction, antivenominjection was interrupted and 0.1% adrenalinewas given subcutaneously. After the symptoms ofreaction had subsided, antivenom therapy wasrestarted. A second dose of four ampoules of an-tivenom was given if the patient’s blood remainedtotally incoagulable 24 h after the initial dose,again following the Brazilian Ministry of Health’sguidelines (Brasil Ministério da Saúde, 1998). As faras possible, medical staff were blinded to whichantivenom a particular patients had received.Analgesic drugs were given for pain, the bitten

limb was nursed in the most comfortable position,bullaewere aspirated, necrotic tissuewas surgicallydebrided, abscesses were drained and, if appropri-ate, antibiotic treatment was given.

2.6. Antivenoms

1. Standard Bothrops-Lachesis antivenom was pro-duced by the Instituto Butantan (batch 9606073,expiry May 1999) from equine plasma collectedafter hyperimmunization of horses with a poolof antigens containing venoms of L. m. muta(44%), B. jararaca (28%), B. moojeni (7%), B.neuwiedi sensu lato (7%), B. alternatus (7%) andB. jararacussu (7%), obtained from snakesmainlycollected in the southeast region of the coun-try. Its median effective dose (ED50) in mice, ex-pressed as mg of venom neutralised by each mLof antivenom, was 7.58mg/mL for the mixtureof Bothrops venoms and 6.0mg/mL for L. mutavenom.

2. B.atrox-Lachesis antivenom was produced byFundação Ezequiel Dias, Minas Gerais State,Brazil (batch ABL 970113-03, expiry October1999) using equine plasma collected from horseshyperimmunised with B. atrox (50%) and L. m.muta (50%) venoms obtained from snakes col-lected in the proximity of Belém, Pará. TheED50 of this new antivenom was 6.59mg/mL forBothrops and 3.12mg/mL for Lachesis venoms.

Both antivenoms were liquid (Fab’)2 antiven-oms prepared by similar fractionation techniquesafter pepsin digestion. The total protein contentof the antivenoms was 60mg/mL for standardBothrops-Lachesis antivenom and 97mg/mL forspecific B. atrox-Lachesis antivenom. The specificantivenom antibodies present per milligram pro-tein were equal in both antivenoms as assayed byELISA. The final products were aliquoted in 10mlampoules.

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2.7. Laboratory investigations

Venous blood was sampled on admission (T0), andat 6 (T6), 12 (T12) and 24 (T24) hours after the endof antivenom therapy and on discharge from hos-pital. Normal ranges for the coagulation tests andvenom detection assays were obtained from bloodsamples taken from patients’ relatives; these wereconsidered to be representative of the normal pop-ulation within the study area. Laboratory staff per-forming the assays were blinded to the treatmentgroups to minimise analytical bias.Blood coagulability was determined by the 20min

whole blood clotting test (Sano-Martins et al.,1994; Warrell et al., 1986, 1977). Results wererecorded and classified as coagulable when a solidclot was formed within 20min and incoagulablewhen no solid clot was formed within 20min.To assess plasma coagulant and fibrinolytic activi-

ties, venous blood was taken into plastic tubes con-taining 16mM sodium citrate, to which was added2% antivenom to neutralise any venom present inthe sample; samples were taken on T0, T12, T24and on discharge. Plasma samples were stored at−20 ◦C and sent, in dry ice, from Belém to theLaboratório de Fisiopatologia, Instituto Butantan,São Paulo where they were stored at −70 ◦C untiltested.Plasma fibrinogen was estimated colorimetri-

cally (Ratnoff and Menzie, 1951). Other tests wereperformed using commercial reagents (DiagnosticaStago, Asnieres-sur-Seines, France): �2-antiplasminwas determined using a chromogenic substrate sys-tem, and cross-linked fibrin fragment D (D-dimer)and serum fibrin(ogen) degradation products(FnDP/FgDP) were estimated using a monoclonalantibody-coated latex test.Renal function was assessed by blood urea,

measured using a colorimetric enzymatic method,and serum creatinine levels were measured usingJaffé’s reaction (Lustgarten and Wenk, 1972). Re-nal impairment was considered likely when ureawas >40mg/dL or creatinine >1.2mg/dL. Serumcreatine kinase was estimated using commercialkits (normal: <30UI/mL; Celm, Brazil).

2.8. Immunoassays

Serum samples, from the simple whole blood clot-ting test, and wound aspirates were obtainedon admission, at T6, T12, T24 and on discharge.Samples were stored for detection of therapeuticantivenom immunoglobulins and B. atrox and L.m. muta venom antigens by enzyme immunoassay(Theakston et al., 1977). For antivenom assays,specific antibodies were captured by a mixture of B.

atrox, L. m. muta and B. jararaca venoms coated onELISA plates. The negative sample was the prean-tivenom admission sample. The reaction, followingincubation with anti-horse IgG-peroxidase conju-gate and by addition of the enzyme substrate, wasrecorded. Values were calculated using as standardcurve known concentrations of specific and stan-dard antivenoms diluted in normal human serum.For venom detection, antigens were captured

with a mixture of equal proportions of both antiven-oms. Test specificity for detection of bound antigenwas controlled using antibodies raised in rabbitsagainst L. m. muta or B. atrox venoms. Thesewere isolated using Protein-A Sepharose columnsand adsorbed with the heterologous venom by im-munoaffinity using Sepharose coupled with B. atroxor L. m. muta venoms. The reaction was revealedby incubation with anti-rabbit IgG-peroxidaseconjugate followed by addition of the enzymesubstrate. Values were calculated using knownconcentrations of either B. atrox or L. m. mutavenoms diluted in normal human serum as standardcurves. Control absorbance for venom assays wasprovided by samples from 72 donors never beforeexposed to snake bite from the same socioeco-nomic group and geographical area as the patients.The cut-off level corresponded to the mean plus 2S.D. of values obtained with these control individ-uals (17.3 ng/mL for B. atrox and 10.2 ng/mL forL. m. muta).

2.9. Statistical analysis

A database was constructed using Epi-Info 6.04csoftware (CDC, Atlanta, GA, USA). Discrete vari-ables were compared using Yates correctedchi-square test or Fisher exact test. Continuousvariables were compared by Student’s-t test andKruskal-Wallis for non-parametric data. Differenceswere considered significant when P < 0.05.

3. Results

Seventy four patients were recruited to the study.

3.1. Identity of snakes responsiblefor the bites

Only four patients brought the dead snake. All wereidentified as B. atrox. In 3 of these, EIA had not pro-vided the diagnosis. In the rest, diagnosis was basedon clinical features, confirmed retrospectively bydetection of specific venom in serum or wound as-pirate.

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3.2. Venom antigen detection

Of the 74 patients, 71 blood samples and 37 blisteraspirates were collected on admission and assayedfor L. m. muta and B. atrox venom. EIA confirmeda genus diagnosis in 43 cases (42 Bothrops, 1 Lach-esis). Of the four patients who brought the snakes,EIA confirmed Bothrops envenoming in one, two hadlevels below the background values and one samplewas not obtained (Table 1).Twenty-one patients were positive for B. atrox

venom in each group (Table 1). Venom antigenconcentrations increased according to severity ofenvenoming: 24 ± 28, 50 ± 43 and 113 ± 70 ng/mLin mild, moderate and severe groups on admis-sion. One patient was positive for L. muta venom(183 ng/mL). Seventeen out of the 37 blister aspi-rates tested were positive, 16 for B. atrox and onefor L. muta venom. The level of venom in the blis-ter aspirates varied considerably reaching levelsas high as 8.2�g/mL. However, as expected, theselevels showed no correlation with the severity (onadmission) of envenoming.Venom antigen concentrations had decreased 6 h

after antivenom treatment and remained low un-til discharge Fig. 3 except in 7 patients (3 whohad received standard antivenom and 4 specific an-tivenom). Six of these patients had B. atrox venomantigen concentrations slightly above background

Table 1 Comparison between groups of snake bite patients before treatment with standard Bothrops-Lachesisand specific B. atrox-Lachesis anti-venoms

Variable Standard Bothrops-LachesisAV (n = 36)

Specific B. atrox-LachesisAV (n = 38)

P

Number of patients 36 (48.6%) 38 (51.4%)Snake responsible: BothropsConfirmed: by EIA 21 21 0.79By dead snake only(EIA negative/not done)

2 1

Snake responsible: Lachesis 1 0Age (mean ± S.D.) years (range) 26 ± 4.1 (7—55) 32 ± 7.7 (5—68) 0.11Male/female 33:3 31:7 0.48Interval accident/AV (h) (range) 11.7 ± 10.3 (3.3—36.3) 10.8 ± 10.5 (3.0—48.0) 0.72

Site of biteFoot/leg 32 33Hand/arm 3 4 0.54Other 1 1

Severity on admissionMild 21 22Moderate 12 15 0.52Severe 3 1

Use of tourniquet 14 14 0.95Incoagulable blood 17 14 0.50

(control absorbance) levels on discharge. The L. m.muta patient also had detectable venom antigen ondischarge.Of the 5 patients bitten on Marajó Island, one

(Case 2) was confirmed by EIA to have been enven-omed by L. muta and 4 by B. atrox/B. marajoensis.The 3 cases bitten in the east (at Cachoeira do Arari,Ponta de Pedras and Soure) were assumed to havebeen bitten by B. marajoensis and the one bitten inthe west (Case 1) by B. atrox.

3.3. Randomisation and clinical features

Thirty-six patients were randomised to treat-ment with standard Bothrops-Lachesis antivenomand thirty eight to treatment with specific B.atrox-Lachesis antivenom. Patients in the twogroups were similar in all epidemiological andclinical aspects on admission before receiving an-tivenom (Tables 1 and 2).Pain and swelling were present in all 74 patients,

local warmth (on palpation) in 54, regional lym-phadenopathy in 33, bleeding from fang marks in29, redness in 19, ecchymosis in 13, blistering in 3and necrosis in 2. Non-specific symptoms, such asheadache and nausea occurred in 30 and 24 patientsin the standard Bothrops-Lachesis group and inthe specific B. atrox-Lachesis group, respectively.Spontaneous systemic bleeding was observed in 13

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Table 2 Clinical features in 74 snake bite patients on admission before antivenom treatment

Sign/symptom Standard Bothrops-LachesisAV (n = 36)

Specific B. atrox-LachesisAV (n = 38)

LocalPain 36 38Swelling 36 38Fever 23 31Lymphadenopathy (regional) 15 18Bleeding from fang punctures 12 17Redness 10 9Ecchymosis 6 7Blistering 2 1Necrosis 1 1

Spontaneous systemic bleedingGingival 5 6Haemoptysis 2 4Macrohaematuria 4 1Ecchymosis 2 2Haematemesis 0 1

Other systemic symptomatologySweating 6 14Headache 12 18Nausea 10 14Vomiting 11 4Diarrhoea 2 0Colicky abdominal pain 7 7

patients; these were gingival (11) (Fig. 4), haemop-tysis (6), macrohaematuria (5) (see Table 2), pur-pura (4), conjunctival (1) and haematemesis (1).No difference was observed between the groups inseverity of local or systemic signs (Table 2). Thenumber of patients with incoagulable blood wasalso similar (17 in Bothrops-Lachesis group and 14in B. atrox-Lachesis group (P = 0.50; Table 1).Clinically, on hospital admission, none of the

patients were suspected to have been bitten byLachesis. However in one, envenoming by L. m.muta was confirmed following detection of specificvenom antigen in the serum.

3.4. Efficacy of antivenom treatment

There were no deaths in the study and no differ-ences between the two groups in the evolutionof clinical features after antivenom treatment(Table 3). A minority of patients developed signs orsymptoms after antivenom treatment. The degreeof local swelling, assessed by the number of seg-ments affected (extent) and increase in circumfer-ence of the bitten limb (intensity), reached a peakwithin 24 h of admission. Seven patients developedlocal abscesses and two had necrosis requiring sur-gical debridement (Figs. 5 and 6); no amputationwas performed. On discharge, 23 patients had per-

sistent pain and 20 persistent local swelling. Localand systemic bleeding usually ceased within 6 h ofstarting treatment; only one patient in the specificB. atrox-Lachesis group had persistent local bleed-ing and macrohaematuria that stopped 24 h aftertreatment without additional antivenom. Blood co-agulability returned to normal within 6 h in 64 (86%)patients. Two patients in the B. atrox-Lachesisgroup required a second dose of antivenom after

Fig. 4 Spontaneous bleeding from gingival margins 6 hafter a bite by B. atrox.

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Table 3 Clinical features in 74 patients after antivenom treatment

Standard Bothrops-LachesisAV (n = 36)

Specific B. atrox-Lachesis AV(n = 38)

P

Number of ampoules 5.8 ± 2.4 5. 8 ± 2.3 0.97Premedication 24 25Early antivenom reactions(with premedication)

7 (3) 7 (5) 0.93

Colicky abdominal pain 3 5Urticaria 3 1Nausea/vomiting 2 1Pruritus 1 1Cough 1 1Dispnoea 1 0

the initial dose, one because of persistence andthe other because of recurrence of incoagulableblood. In both groups coagulability was restoredwithin 6 h after the second dose of antivenom.

3.5. Early antivenom reactions

Premedication with i.v. hydrocortisone and promet-hazine was given to 49 patients, 24 in the Bothrops-

Fig. 5 Necrosis of finger tip 10 days after a bite by B.atrox near Belem.

Fig. 6 Surgical debridement 27 days after a bite by B.marajoensis in Eastern Marajo Island. The anterior tibialcompartment contains only necrotic material and pus.

Lachesis group and 25 in the B. atrox-Lachesisgroup. Early anaphylactic reactions were observedin 14 of them (7 in each group); most were mild(restricted urticaria, facial flushing, dry cough andhoarseness) or moderate (extensive urticaria, nau-sea, vomiting, abdominal cramps, diarrhoea andbronchospasm). There was no association betweenuse of premedication and the frequency of earlyanaphylactic reactions (Tables 4 and 5).

3.6. Blood biochemistry

Mean serum creatine kinase (CK) levels remainedwithin the normal range on admission, 24 h after an-tivenom treatment and at discharge in both groups(P = 0.55), and were not associated with severityof envenoming (P = 0.39).Twenty-nine patients presented on admission

or during hospitalisation with serum creatininelevels above 1.2mg/dL (1.3 to 7.1mg/dL); 11of these were in the standard Bothrops-Lachesisgroup and 18 in the specific B. atrox-Lachesisgroup. Mean creatinine concentrations were similarin the two groups (P = 0.95). No patient requireddialysis.

3.7. Blood coagulation and haematologicalchanges

Fibrinogen concentrations, FnDP/FgDP, D-dimer and�2-antiplasmin levels are shown in Fig. 7. On hospi-tal admission, there were no significant differencesin coagulation variables between the two groups.Plasma fibrinogen levels were low (below or closeto minimum haemostatic levels) (Fig. 7A) as werelevels of �2-antiplasmin (Fig. 7D), while FnDP/FgDPand D-dimer levels were elevated (Fig. 7B and C).Blood coagulation abnormalities observed on admis-sion reversedwithin 24 h after antivenom treatmentin both groups.

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Table 4 Antivenom treatment and early anaphylactic reactions in 74 patients

Feature Standard Bothrops-LachesisAV (n = 36)

Specific B. atrox-LachesisAV (n = 38)

P

Time in hospital (days)(range)

3.6 ± 2.8 (1—15) 4.2 ± 3.8 (1—16) 0.43

Local complicationAbscess 3 4 1.00Necrosis 1 1 1.00

Incoagulable blood atT6 4 6 0.74T12 1 3 0.61T24 0 2 0.49

Table 5 Antivenom treatment and early anaphylactic reactions in 74 snake bite patients

Standard Bothrops-LachesisAV (n = 36)

Specific B. atrox-LachesisAV (n = 38)

P

Number of ampoules 5.8 ± 2.4 5. 8 ± 2.3 0.97Premedication 24 25Early antivenom reactions(with premedication)

7 (3) 7 (5) 0.93

Colicky abdominal pain 3 5Urticaria 3 1Nausea/vomiting 2 1Pruritus 1 1Cough 1 1Dispnoea 1 0

Fig. 7 Mean values (± SEM) of plasma fibrinogen (A), fibrin/fibrinogen degradation products (FnDP/FgDP) (B), crosslinked fibrin fragment D-dimer (C) and �-2-antiplasmin (D) in patients treated with polyvalent Bothrops-Lachesisantivenom (�) or specific B. atrox-Lachesis antivenom ( ). Numbers over bars = number of patients.

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Table 6 Haematological findings (mean±SEM) in 74 snake bite patients on admission, 24 h after antivenomtreatment and on discharge

Standard Bothrops-Lachesis AV (n = 36) Specific B. atrox-Lachesis AV (n = 38)

T0 T24 Discharge T0 T24 Discharge

Haemoglobin (g/L) 129 ± 3 117 ± 3 115 ± 4 131 ± 3 121 ± 3 126 ± 4PCV (%) 40 ± 1 37 ± 1 36 ± 1 40 ± 1 38 ± 1 39 ± 1RBC (x 1012/L) 4.5 ± 0,1 4.2 ± 0,1 4.1 ± 0.1 4.5 ± 0.1 4.2 ± 0.1 4.4 ± 0.1WBC (x 109/L) 13.7 ± 0.7 10.4 ± 0.4 9.4 ± 1.1 12.8 ± 0.6 10.2 ± 0.4 8.7 ± 0.5Platelets (x 109/L) 314 ± 29 319 ± 18 311 ± 28 299 ± 22 322 ± 15 373 ± 21

P>0.05 for ANOVA two-way comparison between groups and time.

Haematological findings did not change signif-icantly (P>0.05) after treatment (Table 6). Theslight peripheral leucocytosis on admission de-creased towards normal 24 h after treatment andwas normal on discharge (Table 6). Haematocritlevels and haemoglobin concentrations fell slightlyand remained low until discharge in both groups;there was no correlation with bleeding. The meanplatelet count was normal in both groups, although5 out 36 patients of standard Bothrops-Lachesisgroup and 4 out 38 of specific B. atrox-Lachesisgroup had values below 150 × 109/L (Table 6).

3.8. Serum concentrations oftherapeutic antivenom

Patients receiving 4 ampoules of either specific B.atrox-Lachesis or standard Bothrops-Lachesis an-tivenoms showed similar serum concentrations ofserum therapeutic antibodies at the different timestested. However, patients receiving 8 ampoules ofspecific antivenom had concentrations of serum an-tibodies significantly higher at 6 and 24 h after thestart of antivenom (P < 0.05) than those receiving8 ampoules of standard antivenom (specific: T6,2.71 ± 1.31mg IgG/mL; T24, 1.51 ± 0.90mg/mL;discharge, 0.66 ± 0.49mg/mL; standard: T6,1.35 ± 1.17mg IgG/mL; T24, 0.91 ± 0.50mg/mL;discharge, 0.47 ± 0.29mg/mL). These differencesmay be attributed to the higher protein con-centration of the specific B. atrox-Lachesis an-tivenom (97mg/mL) compared with standardBothrops-Lachesis antivenom (60mg/mL), althoughthe concentration of specific venom antibodies/mgprotein was the same for both antivenoms. On dis-charge, there was no significant difference betweenserum levels of the two antivenoms (P = 0.25). Inspite of the difference in the total protein levelsof the two antivenoms, there was no correlationwith clinical evolution or number of antivenomreactions.

3.9. Case history 1. Envenomingby B. atrox

A 48 year-old farmer was bitten at 07:00 on May 26,1996 on his left knee while farming, near his housein Anajás, a town on the western side of MarajóIsland, approximately 85 km from Belém. He identi-fied the snake as ‘surucucú’ (a local name for largelance-headed vipers and bushmasters in this re-gion), but did not bring it with him to the hospital.No tourniquet or traditional therapies were appliedto the area. It took him 34 h to reach the hospitaldue to local transportation problems. Before ar-riving at the hospital, relatives reported that hefainted but recovered consciousness a few minuteslater. He had been bitten by snakes five times be-fore, but had never received antivenom. On admis-sion, he was sweating and complaining of abdominalpain. His leg was tender and swollen up to the anklewith bleeding from fang marks. There were ecchy-moses and blisters on the bitten limb and tenderenlarged inguinal lymph nodes on the affected side.He was given 80mL of standard Bothrops-Lachesisantivenom by slow intravenous infusion withoutpremedication. No adverse reaction was recorded.Blood remained coagulable throughout the ad-mission but he had hypofibrinogenaemia (fibrino-gen 0.95 g/L) and thrombocytopenia (plateletcount 75 × 109/L). No systemic bleeding was ob-served. Laboratory tests on admission showed ahaemoglobin of 8.0 g/L, haematocrit 25%. Urineoutput was decreased and the serum creatinineconcentration was 7.0mg/dL. He had the highestB. atrox venom antigen concentration recorded inthis study (188.4 ng/mL) which cleared during thefirst 6 h after antivenom therapy. Lachesis venomantigen concentration remained under the controlabsorbance during his admission. Local swellingincreased up to the third day after the bite and su-perficial necrosis developed at the sites of the blis-ters. The patient was rehydrated intravenously andrenal function returned to normal in 2 days without

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dialysis. He was discharged after 8 days with de-creasing swelling, no other complications and withthe results of laboratory tests returning towardsnormal.

3.10. Case history 2. Envenoming by L. muta

A 25 year-old farmer was bitten on his right anklewhile cutting palm trees in the forest. Immedi-ately after the bite he applied a tourniquet whichwas maintained for about 5 h until he reachedthe nearest town (Anajás, Marajó Island). Hewas transferred to Belém, arriving at the hos-pital 26 h after the bite. On admission, he hadprominent local swelling and pain, mild bleedingfrom the fang punctures, ecchymosis and blistersat the site of bite, mild gingival haemorrhageand haemoptysis, salivation and abdominal pain,but no sweating, diarrhoea, bradycardia or hy-potension. His blood was incoagulable. The snakeresponsible was not identified but clinically thediagnosis was moderate envenoming by Bothrops.No premedication was given before he received80mL of standard Bothrops-Lachesis antivenom;no adverse reactions were observed. Systemicbleeding stopped minutes after the end of an-tivenom infusion and blood was coagulable sixhours later. Haematological tests remained abnor-mal for 24 h after hospitalization (on admission,haemoglobin was 11.3 g/L and haematocrit 40%;at T24, haemoglobin was 8.1 g/L and haematocrit25%). Serum creatinine concentrations increasedfrom 4.8 to 5.7mg/dL during the first day return-ing to normal by discharge; urine output remainednormal throughout. The patient was discharged6 days later without any local or systemic com-plications. Immunoassay results, available monthsafter the accident, revealed a serum Lachesisvenom antigen concentration of 183.0 ng/mL, in-dicating envenoming by this species. After an-tivenom therapy, venom antigen concentrationdecreased to 0.01 ng/mL (T6) and remained undercontrol absorbance until discharge 6 days after en-venoming (T12 = 1.35 ng/mL, T24 = 2.21 ng/mL,discharge = 1.08 ng/mL). B. atrox venom antigenconcentration remained below control absorbanceon admission (1.25 ng/mL), T12 (1.49 ng/mL) andT24 (1.98 ng/mL).

4. Discussion

This randomised comparative clinical trial com-pared the efficacies of specific B. atrox-Lachesisand standard Bothrops-Lachesis antivenoms in thetreatment of snake bites in the Amazon region of

Pará State, Brazil. In both groups neutralisationof venom-induced haemorrhage and coagulopathywas evident, since systemic bleeding invariablyceased and blood coagulability was usually re-stored after antivenom therapy. These effects ofenvenoming are potentially life-threatening be-cause of the risk of massive/fatal haemorrhage(Kouyoumdjian et al., 1991; Pardal et al., 1997a)and renal failure (Amaral et al., 1986). Abnormalserum creatinine concentrations were fairly com-mon, reflecting the risk of renal damage whenantivenom treatment is delayed. The restorationof blood coagulability has been proposed as a goodindicator of venom neutralisation in snake bites(Ho et al., 1986; Sano-Martins et al., 1994). Ourresults indicated that an improvement in coagu-lation status may be expected within 6—12 h inmost patients and within 24 h in nearly all patientsreceiving a single dose of antivenom (Cardosoet al., 1993; Otero et al., 1998). Blood coagulabil-ity should be monitored every 6 h after the initialdose of antivenom (Phillips et al., 1988; Warrellet al., 1986), and further doses of antivenom shouldbe administered, if necessary, until coagulabilityis restored (Cardoso et al., 1993; Jorge et al.,1995; Otero et al., 1998). In this way, the du-ration of life-threatening coagulopathy can beminimised.The efficacy of antivenom in neutralizing lo-

cal effects was difficult to assess because manypatients were admitted to hospital late whenvenom-induced tissue destruction had alreadybecome established; progression of oedema andcutaneous necrosis may also involve indirect in-flammatory mechanisms that are independent ofantivenom-mediated neutralization (Chaves et al.,1995; Farsky et al., 1997; Trebien and Calixto,1989). Local necrotic effects and secondary in-fection may lead to chronic physical handicap,particularly when the bite involves digits or thecontents of fascial compartments. In our study,there was no significant difference in the frequencyof these complications between the two treatmentgroups. Progression of local effects could continuedespite antivenom therapy and the presence ofsuch signs should be interpreted with caution as acriterion for recommending an additional dose ofantivenom.Haemostatic disturbances in victims of B.

atrox/B. marajoensis envenoming were similar tothose observed in patients bitten by B. jararaca(Kamiguti et al., 1986; Maruyama et al., 1990).Both antivenoms administered to our patients wereefficient in reversing systemic disturbances, such asthe blood incoagulability caused by venom-inducedfibrinogen consumption and secondary activation

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of the fibrinolytic system. Most patients had nor-mal coagulation on discharge and no differenceswere recorded in measures of coagulation betweenthe two groups, except for marginally significantdifferences in FnDP/FgDP levels.The standard Bothrops-Lachesis antivenom used

in Brazil is produced by hyperimmunizing horsesagainst venoms from representative species ofBrazilian Bothrops snakes and L. m. muta venom.The venom composition is known to vary signifi-cantly among snakes of different species (Furtadoet al., 1991; Rodrigues et al., 1998). Marked dif-ferences in the ability of 6 different antivenoms toneutralize pharmacological and enzymatic effectsinduced by venom of B. atrox from Colombia havebeen demonstrated (Otero et al., 1995). However,the hypothesis that antigenic diversity among ven-oms of Bothrops species (Assakura et al., 1992),might render the standard Bothrops-Lachesis an-tivenom ineffective in neutralizing the effects ofenvenoming by B. atrox (Muniz et al., 2000) was notsupported by our data. The efficacy of antivenomcontaining 56% of standard Bothrops antigens (B.jarararaca 28%, B. jararacussu 7%, B. moojeni 7%,B. alternatus 7% and B. neuwiedi 7%) proved similarto that of specific B. atrox antivenom. Both pro-duced a satisfactory response judged by resolutionof the clinical signs of envenoming and laboratoryabnormalities. Our results are in agreement witha previous in vitro study which demonstrated thatBrazilian antivenom produced from 5 Bothrops ven-oms other than B. atrox was able to neutralize thelethal activity of the venom of this snake (Dias daSilva et al., 1989).The incidence of early anaphylactic reactions in

our patients was low (14/74 patients; 19%) com-pared with the observations of other investigatorswho reported incidences ranging from 25 to 87%(Cardoso et al., 1993; Fan et al., 1999; Malasitet al., 1986; Otero et al., 1996). Most adverse reac-tions were not life-threatening and were promptlyreversed by treatment with adrenaline and an-tihistamines. The Brazilian Ministry of Health’sguidelines are controversial and, although thereare no clinical trials to support the use of antihis-tamines and corticosterioids, premedication is stillpopular(Cupo et al., 1991). In our study, it seemsthat early reactions were not influenced by theuse of premedication but, since formal randomi-sation was not performed, firm conclusions areimpossible.The use of EIA to detect venom in biological

fluids (Ho et al., 1986; Theakston et al., 1977) isimportant in epidemiological studies, diagnosis ofenvenoming and pharmacological assessment ofantivenoms. In our study, EIA was unable to de-

tect serum B. atrox and L. muta venom antigens in29 out of 71 clinically envenomed patients whosesamples were collected, probably because of thehigh background absorbance values in non-bittencontrols from the same geographical areas andsocioeconomic groups who had never been bittenby snakes. However, a more sensitive and specificimmunoassay using monoclonal antibodies is cur-rently being developed (Colombini et al., 2001).Our EIA could not distinguish between venomsof the closely related species, B. atrox and B.marajoensis, but on geographical grounds alone,three of the bites could be attributed to the lattersince they occurred on the eastern part of MarajóIsland.EIA proved useful for distinguishing patients with

different degrees of clinical severity on admission.Clinical indicators of severity on admission were, inour cases, often associated with high serum venomconcentrations, supporting the proposal that theinitial dose of antivenom should be adjusted ac-cording to the clinical grade of severity. However,it must be stressed that envenoming is a highlydynamic process. The condition of patients admit-ted suffering from envenoming defined as mild ormoderate may rapidly deteriorate. Dogmatism indefining the severity of envenoming solely on thebasis of the clinical grade of severity on admis-sion to the hospital may therefore be dangerous.Our main interest in assaying the patients’ venomantigen concentrations was to compare venompharmacokinectics in the two groups after theyhad received an appropriate therapeutic dose ofantivenom. Like Theakston et al. (1992), we ob-served that 6 h after antivenom administration,serum levels of venom antigen were practicallyundetectable. The high serum therapeutic an-tivenom concentrations, which persisted well afterthe final permanent clearance of venom antige-naemia and after the discharge of the patients,demonstrated that the dosage regime was probablyadequate.EIA was positive for L. muta venom in only one

case confirming that this formidable but reclu-sive species rarely bites people in this part ofthe Amazon (Cunha and Nascimento, 1993). Theclinical features shown by our patient were in-distinguishable from those caused by the bitesof Bothrops and other genera of Latin Americanlance-headed pit vipers. Distinctive symptoms,such as profuse sweating, nausea, vomiting, ab-dominal cramps and watery diarrhoea (Jorge et al.,1997; Warrell, 2003) may not be manifested byall victims of Lachesis bites. The absence of au-tonomic nervous system excitation following en-venoming by L. acrochorda was also observed in

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western Colombia (Otero et al., 1993). Apart fromthe quantity of venom injected, geographical vari-ation in the venom composition of the differentLachesis species may explain differences in thefrequency and type of clinical features observed.Misdiagnosis between Lachesis and Bothrops en-venoming or lack of autonomic manifestationsin mild Lachesis envenoming are other possibil-ities. Elsewhere, other non-specific antivenomswith activity only against Bothrops species pro-duced unimpressive results in victims of L. mutaenvenoming (Bard et al., 1994; Torres et al.,1995).In conclusion, we have shown that while a spe-

cific B. atrox antivenom may not be essential,antivenoms with proven clinical efficacy againstLachesis and Bothrops venoms, such as the‘standard’ antivenom tested in this study, areneeded for the treatment of snakebites in this partof the Amazon region.

Acknowledgements

We are grateful to the nursing staff and laboratorystaff of the Hospital Universitário João de Bar-ros Barreto, Belém, Pará, for their help with thepatients and technical assistance. We also thankthe medical students of the Centro de Cienciasda Saúde da Universidade Federal do Pará fortheir help in collecting data; Ana Lúcia Costa Pru-dente, Museu Emılio Goeldi, for the identificationof the snakes; Anıbal R. Melgarejo, Instituto VitalBrazil, for the photo of Lachesis muta. Antiven-oms were donated by the Brazilian Ministry ofHealth and by the Fundação Ezequiel Dias, BeloHorizonte, MG. This study was supported by theEuropean Community (contract no. EC IC 18 CT960032). No author has an undisclosed conflict ofinterest.

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