Accepted Manuscript Fatal stroke after Bothrops snakebite in the Amazonas state, Brazil: A case report Sâmella Silva de Oliveira, Luciana Aparecida Freitas-de-Sousa, Eliane Campos Alves, Luiz Carlos de Lima Ferreira, Iran Mendonça da Silva, Marcus Vinícius Guimarães de Lacerda, Hui Wen Fan, Ana Maria Mourada-Silva, Wuelton Marcelo Monteiro PII:
S0041-0101(17)30266-0
DOI:
10.1016/j.toxicon.2017.08.021
Reference:
TOXCON 5705
To appear in:
Toxicon
Received Date: 27 June 2017 Revised Date:
18 August 2017
Accepted Date: 21 August 2017
Please cite this article as: Silva de Oliveira, Sâ., Freitas-de-Sousa, L.A., Alves, E.C., de Lima Ferreira, L.C., da Silva, Iran.Mendonç., de Lacerda, Marcus.Viní.Guimarã., Fan, H.W., Mourada-Silva, A.M., Monteiro, W.M., Fatal stroke after Bothrops snakebite in the Amazonas state, Brazil: A case report, Toxicon (2017), doi: 10.1016/j.toxicon.2017.08.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fatal stroke after Bothrops snakebite in the Amazonas state, Brazil: a case report.
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Sâmella Silva de Oliveiraa,bϕ, Luciana Aparecida Freitas-de-Sousac,dϕ, Eliane Campos Alvesa,b,
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Luiz Carlos de Lima Ferreiraa,b, Iran Mendonça da Silvaa,b, Marcus Vinícius Guimarães de
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Lacerdaa, Hui Wen Fane, Ana Maria Mourada-Silvac,d and Wuelton Marcelo Monteiroa,b*.
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a
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Amazonas, Brazil.
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Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado,
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Brazil.
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Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Amazonas,
Programa de Pós-Graduação em Ciências - Toxinologia, Instituto Butantan, São Paulo, Brazil. Laboratório de Imunopatologia, Instituto Butantan, São Paulo, Brazil.
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Núcleo Estratégico de Venenos e Antivenenos, Instituto Butantan, São Paulo, Brazil.
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*Corresponding author: Dr. Wuelton Marcelo Monteiro. Fundação de Medicina Tropical Dr.
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Heitor Vieira Dourado. Avenida Pedro Teixeira 25, Dom Pedro, 69040-000, Manaus, AM,
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Brazil. Phone: 55 92 2127-3430 e-mail: [email protected]
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These authors contributed equally to this work.
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Abbreviations
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IV: Intravenous.
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SVMPs: Snake Venom Metalloproteinases.
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SVSPs: Snake Venom Serine proteinases.
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PLA2: Phospholipases A2.
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Abstract
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Bothrops atrox is the snake responsible for the majority of snakebites in the Brazilian Amazon.
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Patients generally evolve to local manifestations such as edema, pain and ecchymoses. Systemic
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effects of B. atrox venom are usually restricted to blood incoagulability and spontaneous
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bleeding. However, in a few cases, bleeding in the central nervous system may occur, which can
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lead to sequels and deaths. Here, we report a case of a 59 year-old woman who presented edema,
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pain and ecchymoses on the right foot, headache, nausea, diarrhea, hypertension and blood
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incoagulability after the bite by Bothrops snake in the Brazilian Amazon. This case evolved with
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stroke resulting in death despite the antivenom and conservative therapy employed. In addition,
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we were able to identify the presence of venom in the patient's brain tissue after death. Direct
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action of toxins present in the snake’s venom in the induction of systemic hemorrhage allied to
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blood incoagulability and hypertension presented by the patient could be involved in the
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mechanism of stroke in this case.
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Keywords: hemorrhage, coagulopathy, Bothrops envenoming, stroke.
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1.
Introduction
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Bothrops envenoming usually result in local clinical features (i.e. edema, pain and ecchymoses)
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as well as systemics effects (i.e. coagulation disorders and systemic spontaneous bleeding)
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(Ribeiro et al. 1998, Ribeiro and Jorge 1990), in consequence mainly of the action of
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metalloproteinases, phospholipases A2 and serine proteinases (Calvete et al. 2011, Sousa et al.
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2013). These accidents may evolve with complications such as necrosis, secondary bacterial
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infection, compartment syndrome and acute renal failure (Ribeiro and Jorge 1990), being the
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main causes of death renal and respiratory failure, shock, sepsis and rarely hemorrhage in the
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central nervous system (Ribeiro et al. 1998). In a study on the prevalence of cerebrovascular
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complications in Bothrops envenoming, it was found that 2.6 % of the victims developed a
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cerebrovascular event, of which about 60 % died and 40 % remained with sequelae (Mosquera et
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al. 2003). Here, we describe a case of fatal stroke after Bothrops snakebite in the Brazilian
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Amazon.
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2.
Case Report
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A 59 year-old woman was bitten in the fifth right toe by a snake inside her house in the rural area
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of Manaus, Amazonas State, in Brazil. One hour and a half after the bite she was admitted at the
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reference hospital with intense local pain, swelling and ecchymosis on the right foot, and severe
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headache, nausea and episodes of diarrhea. No systemic bleeding was observed. Patient
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identified the snake as “surucucurana”, a local name for B. atrox, but did not bring it with her to
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the Health Facility. She denied previous diabetes and hypertension. On admission blood pressure
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was 200/110 mmHg and respiratory frequency was 18 bpm. Her blood was incoagulable and
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coagulogram was altered (Table 1). No other clinical manifestations were observed.
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Pre-medication was given (IV hydrocortizone 500 mg, IV promethazine 50 mg, IV
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dexchlorpheniramine and IV ranitidine 50 mg). Symptomatic medication for pain was also
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administered (IV dipirona 1 g). After premedication, the patient experienced episodes of
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vomiting and was administered IV metocloropramide 10 mg. Subsequently, she received
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sublingual captopril 50 mg. She received 80 mL de Bothrops antivenom after two hours and a
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half from the bite. However, approximately one hour after administration of antivenom, the
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patient experienced sweating, cold and clammy skin, tachycardia, sialorrhea, aphasia and
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decreased level of consciousness, not responding to verbal stimuli, but to painful stimuli. Blood
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pressure was 190/130 mmHg. The patient received IV furosemide 80 mg, IV omeprazole 40 mg
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and venous hydration. Administration of antivenom was discontinued. She was transferred
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comatose to the intensive care unit with 5 point on the Glasgow Coma Scale, isocorics pupils
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with photoreaction and required mechanical ventilation. Blood pressure was 230/130 mmHg.
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The patient received IV hydralazine 20 mg, IV midazolam 50 mg, IV fentanyl 2 mg and IV
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omeprazole 40 mg. Sedation was interrupted, being administered IV mannitol 20 g and IV
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hydantal 250 mg.
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On the second day after the bite, the patient remained comatose and on mechanical ventilation,
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evolving with mydriatic pupils without photoreaction, with 3 point on the Glasgow Coma Scale,
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hemodynamically unstable, hypothermia, bradycardia and anuria. She received IV hydantal 250
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mg and IV omeprazole 40 mg. On the third day after the bite, the patient showed no clinical
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improvement, showing hypothermia, bradycardia and hypotension. On this day, the patient had a
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cardiorespiratory arrest and died. The patient evolved without local complications, such as
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necrosis and secondary bacterial infection. Laboratory tests during hospitalization are shown in
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Table 1. Computed tomography was requested during the hospitalization period, but was not
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performed.
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Autopsy showed subarachnoid and intraparenchymal hemorrhage, as shown in Figures 1 and 2,
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respectively, and determined intracranial hypertension as cause of death. No other alteration was
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described in the necroscopic examination. Venom detection in brain tissue was performed by
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immunohistochemistry (Supplementary material) and is shown in Figure 3. The venom was
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immunolocalized in some areas of the patient's brain. The positive region is represented by the
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brown spots indicated with the arrows (Figure 3E and F) appearing only in the group where an
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antibody produced against the B. atrox venom was used. The groups which were treated only
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with the blocking solution (Figure 3A and B) or normal serum rabbit IgG (Figure 3C and D) not
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show brown spots. The tissue from patient's heart not was positive for venom (data not show).
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4. Discussion
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We describe the occurrence of stroke after snakebite in the Brazilian Amazon, an uncommon
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finding, which resulted in death. This snakebite was diagnosed and treated as an accident with
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snake in the genus Bothrops. Despite the patient not coming along with the dead snake to the
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Health Facility, she identified the snake as “surucucurana”, a popular name attributed to B.
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atrox, which is considered the main cause of snakebite in the Amazon (Cardoso et al. 2009).
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Accidents caused by B. atrox have more local clinical features such as blisters, necrosis and
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abscesses and systemic effects mostly related to blood coagulation disorders and spontaneous
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bleeding (Pardal et al. 2004). In Brazil, the specific treatment of Bothrops snakebites is the
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administration of the Bothrops, Bothrops-Lachesis or Bothrops-Crotalus antivenoms. Bothrops
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antivenom has been efficient in neutralizing the coagulant and hemorrhagic activities of B. atrox
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venom in pre-clinical assessments and clinical trials (Pardal et al. 2004, Sousa et al. 2013). In
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this present case, Bothrops antivenom was administered in time ˂ 6 hours after the bite and the
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coagulability was restored after 24 hours of the administration of antivenom.
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Signs of neurological damage after snakebite are most often related to the activities toxins
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present in the venoms, due to the anticoagulant/procoagulant activities and/or neurotoxicity of its
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components (Del Brutto and Del Brutto 2012). Death cases caused by cerebral hemorrhage in
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patients bitten by viper snakes have been described (Pinho and Burdmann 2001, Mosquera et al.
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2003, Kitchens and Eskin 2008, Kouyoumdjian et al. 1991). Autopsy of this patient showed
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subarachnoid and intraparenchymal hemorrhage. In a study with patients who presented stroke
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following Bothrops spp. snakebite, seven patients presented intracranial bleeding and
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hemorrhages were located in the subcortical white matter of the cerebral hemispheres, in the
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cerebellum and in the subarachnoid space (Mosquera et al. 2003). Patients bitten by viper snakes
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with intracranial hemorrhage presented generally coagulation disorders, such as increased
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clotting times, prolongation of the prothrombin and thromboplastin times, and decrease in the
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fibrinogen levels and number of platelets (Mosquera et al. 2003, Kitchens and Eskin 2008,
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Kouyoumdjian et al. 1991). Snake venom metalloproteinases (SVMPs), snake venom serine
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proteinases (SVSPs) and phospholipases A2 (PLA2) are the components most abundant of the
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Bothrops venom and are involved with hemostatics disorders in victims by Bothrops
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envenoming (Sousa et al. 2013). The SVMPs are the major toxins of the B. atrox venom from
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Brazilian Amazon (Calvete et al. 2011, Neiva et al. 2009, Sousa et al. 2013) and local and
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systemic hemorrhage is caused by the direct action of SVMPs in components of basement
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membrane of capillaries (Gutiérrez et al. 2005, Moura-da-Silva, Butera and Tanjoni 2007). The
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hydrolysis of basement membrane components (Shannon et al. 1989) leads to loss of mechanical
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stability of the capillaries and hemodynamic forces contribute to the distension and rupture of the
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capillary and consequent bleeding (Gutiérrez et al. 2005). Three major classes of SVMPs are
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described, however the hemorrhage is mainly associated with the PIII-class (Escalante et al.
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2006, Escalante et al. 2011). Unlike PI-class, PII-class and PIII-class present adhesive domain
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that act in anchoring to components basement membrane from capillaries which facilitates the
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hydrolysis and consequent hemorrhage (Baldo et al. 2010, Herrera et al. 2015).
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Venom was located in this patient's brain tissue after death. These findings agree with the study
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of (Selvanayagam et al. 1999), which found venom in the brain, heart, lungs, liver, spleen,
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kidneys and the bite area from tissue of the patient bite by Echis carinatus. (Escalante et al.
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2003), also found small amounts of Jarharagin, PIII-class SVMP majority of B. jararaca venom,
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in brain tissue of mice. The major component of B. atrox venom is Batroxrhagin, a PIII-class
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SVMP, which has a strong identity with Jarharagin (Freitas-de-Sousa et al. 2015). However,
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intracranial hemorrhage cannot be related only to PIII-class, since these hemorrhagic toxins (PII
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and PIII-class) were located mainly at the site of the bite by bind of non-catalytic domains to
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basement membrane components (Baldo et al. 2010, Herrera et al. 2015). On the other hand, our
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group recently isolated and characterized a PI-class SVMP, named Atroxlysin-Ia, from the
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venom of B. atrox that induces a strong hemorrhage comparable to a PIII-class SVMP (Freitas-
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de-Sousa et al. 2017). Furthermore, Atroxlysin-Ia not exhibit the adhesive domains present in the
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PIII-class SVMPs and most likely presents rapid tissue diffusion. Thus, these SVMPs probably
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contributed to the brain hemorrhage in this patient.
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This patient also was with incoagulable blood and altered coagulogram after the snakebite. Blood
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incoagulability observed in Bothrops envenoming is due to the consumption of fibrinogen and
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some coagulation factors. SVSPs are usually thrombin-like enzymes, which directly hydrolyze
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fibrinogen in fibrin, that are involved in the blood coagulation disorders observed in B. atrox
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envenoming (Stocker and Barlow 1976). B. atrox venom also contains SVMPs that activate
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factors II and X of coagulation, resulting in the formation of endogenous thrombin (Hofmann
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and Bon 1987a, Hofmann and Bon 1987b). In addition, PI-class SVMPs with fibrino(geno)lytic
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activity were isolated from the venom of B. atrox (Cintra et al. 2012, Patiño et al. 2010, Freitas-
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de-Sousa et al 2017). Others components from the B. atrox venom act on factors XIII
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(Niewiarowski et al. 1979) and V (Rosing et al. 2001) and platelets, causing inhibition (Rucinski
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et al. 1990, Freitas-de-Sousa et al. 2015). Therefore, the proteolytic action due to SVMPs allied
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to blood incoagulability could be involved into the patient's cerebral hemorrhage.
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In addition, factors associated in victims bitten by snakes have also been observed in cases of
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stroke. Some case reports show that intracranial bleeding can be associated with arterial
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hypertension (Machado et al. 2010, Allen et al. 2012, Berling et al. 2015). In the Pará State, in
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Brazil, a case report described a hemorrhagic stroke in Bothrops envenoming associated with
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severe hypertension, who received discharge for healing, suggesting synergism hypertension-
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venom as a determining factor for cerebral hemorrhage (Machado et al. 2010). In the present
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case report, severity of cerebral injury was evidenced by the Cushing response, characterized by
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increase of systemic arterial pressure, bradycardia and irregular respirations. The Cushing
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response is assumed to have a protective function for the brain, raising cerebral perfusion
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pressure in critical conditions, thus allowing an increase of cerebral blood flow. However, the
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arterial hypertension may induce a proportional increase in intracranial pressure, and may raise
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pressure in cerebral arteries and capillaries, increasing the risk of cerebral hemorrhage or edema,
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and the mechanisms that triggers a vicious circle between intracranial and systemic arterial
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pressure. Other factor associated with stroke is older age. Stroke following Bothrops snakebite
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was observed in victims with a mean age 51.9 years (Mosquera et al. 2003). Case reports of
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stroke involving victims >60 years have also been reported in Bothrops snakebite in others
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regions of Brazil (Santos-Soares et al. 2007). These characteristics of fatal victims after Bothrops
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snakebites reveal more vulnerable groups requiring appropriate and early treatment to avoid
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serious complications.
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This case report shows the importance of identifying risk factors for the development of this
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complication in Bothrops snakebite and appropriate and early antivenom terapy and prompt
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supportive treatment to avoid progression of systemic complications. Moreover, presents the first
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evidence of the direct involvement of some venom components in the induction mechanisms of
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systemic hemorrhage.
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Ethical statement
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This patient was enrolled in a study approved by the Ethics Review Board of the Fundação de
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Medicina Tropical Dr. Heitor Vieira Dourado, with a waiver of informed consent (CAAE
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53192516.8.0000.0005).
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Acknowledgments
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We thank to Department of Archive and Statistic Services of Fundação de Medicina Tropical
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Dr. Heitor Vieira Dourado for the permits to access the medical records. This study was
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supported by PROCAD - CAPES AuxPE 2992/2014.
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Conflict of Interest
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The authors declare that there is no conflict of interest.
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Referências
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Allen, G. E., S. G. Brown, N. A. Buckley, M. A. O'Leary, C. B. Page, B. J. Currie, J. White, G. K. Isbister & A. Investigators (2012) Clinical effects and antivenom dosing in brown snake (Pseudonaja spp.) envenoming-Australian snakebite project (ASP-14). PLoS One, 7, e53188. Baldo, C., C. Jamora, N. Yamanouye, T. M. Zorn & A. M. Moura-da-Silva (2010) Mechanisms of vascular damage by hemorrhagic snake venom metalloproteinases: tissue distribution and in situ hydrolysis. PLoS Negl Trop Dis, 4, e727. Berling, I., S. G. Brown, F. Miteff, C. Levi & G. K. Isbister (2015) Intracranial haemorrhages associated with venom induced consumption coagulopathy in Australian snakebites (ASP-21). Toxicon, 102, 8-13. Calvete, J. J., L. Sanz, A. Pérez, A. Borges, A. M. Vargas, B. Lomonte, Y. Angulo, J. M. Gutiérrez, H. M. Chalkidis, R. H. Mourão, M. F. Furtado & A. M. Moura-Da-Silva (2011) Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management. J Proteomics, 74, 510-27. Cardoso, J. L. C., Francisco Oscar de Siqueira FranÇa, F. H. Wen, C. M. S. A. Málaque & V. H. Jr. 2009. Animais Peçonhentos no Brasil. In Serpentes peçonhentas do Brasil, ed. A. R. MELGAREJO, 550. Cintra, A. C., L. G. De Toni, M. A. Sartim, J. J. Franco, R. C. Caetano, M. T. Murakami & S. V. Sampaio (2012) Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity. Toxicon, 60, 70-82. Del Brutto, O. H. & V. J. Del Brutto (2012) Neurological complications of venomous snake bites: a review. Acta Neurol Scand, 125, 363-72. Escalante, T., J. Núñez, A. M. Moura da Silva, A. Rucavado, R. D. Theakston & J. M. Gutiérrez (2003) Pulmonary hemorrhage induced by jararhagin, a metalloproteinase from Bothrops jararaca snake venom. Toxicol Appl Pharmacol, 193, 17-28. Escalante, T., N. Ortiz, A. Rucavado, E. F. Sanchez, M. Richardson, J. W. Fox & J. M. Gutiérrez (2011) Role of collagens and perlecan in microvascular stability: exploring the mechanism of capillary vessel damage by
AC C
221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244
RI PT
211
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EP
TE D
M AN U
SC
RI PT
snake venom metalloproteinases. PLoS One, 6, e28017. Escalante, T., J. Shannon, A. M. Moura-da-Silva, J. M. Gutiérrez & J. W. Fox (2006) Novel insights into capillary vessel basement membrane damage by snake venom hemorrhagic metalloproteinases: a biochemical and immunohistochemical study. Arch Biochem Biophys, 455, 144-53. Freitas-de-Sousa, L. A., D. R. Amazonas, L. F. Sousa, S. S. Sant'Anna, M. Y. Nishiyama, S. M. Serrano, I. L. Junqueirade-Azevedo, H. M. Chalkidis, A. M. Moura-da-Silva & R. H. Mourão (2015) Comparison of venoms from wild and long-term captive Bothrops atrox snakes and characterization of Batroxrhagin, the predominant class PIII metalloproteinase from the venom of this species. Biochimie, 118, 60-70. Freitas-de-Sousa, L. A., Colombini, M., Lopes-Ferreira, M., Serrano, S. M. T., Moura-da-Silva, A.M. (2017) Insights into the Mechanisms involved in strong hemorrhage and dermonecrosis induced by Atroxlysin-Ia, a PIClass snake venom metalloproteinase. Toxins, 9, 239. Gutiérrez, J. M., A. Rucavado, T. Escalante & C. Díaz (2005) Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon, 45, 997-1011. Herrera, C., T. Escalante, M. B. Voisin, A. Rucavado, D. Morazán, J. K. Macêdo, J. J. Calvete, L. Sanz, S. Nourshargh, J. M. Gutiérrez & J. W. Fox (2015) Tissue localization and extracellular matrix degradation by PI, PII and PIII snake venom metalloproteinases: clues on the mechanisms of venom-induced hemorrhage. PLoS Negl Trop Dis, 9, e0003731. Hofmann, H. & C. Bon (1987a) Blood coagulation induced by the venom of Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator. Biochemistry, 26, 772-80. --- (1987b) Blood coagulation induced by the venom of Bothrops atrox. 2. Identification, purification, and properties of two factor X activators. Biochemistry, 26, 780-7. Kitchens, C. & T. Eskin (2008) Fatality in a case of envenomation by Crotalus adamanteus initially successfully treated with polyvalent ovine antivenom followed by recurrence of defibrinogenation syndrome. J Med Toxicol, 4, 180-3. Kouyoumdjian, J. A., C. Polizelli, S. M. Lobo & S. M. Guimares (1991) Fatal extradural haematoma after snake bite (Bothrops moojeni). Trans R Soc Trop Med Hyg, 85, 552. Machado, A. S., F. B. Barbosa, G. a. S. Mello & P. P. Pardal (2010) [Hemorrhagic stroke related to snakebite by bothrops genus: a case report]. Rev Soc Bras Med Trop, 43, 602-4. Mosquera, A., L. A. Idrovo, A. Tafur & O. H. Del Brutto (2003) Stroke following Bothrops spp. snakebite. Neurology, 60, 1577-80. Moura-da-Silva, A. M., D. Butera & I. Tanjoni (2007) Importance of snake venom metalloproteinases in cell biology: effects on platelets, inflammatory and endothelial cells. Curr Pharm Des, 13, 2893-905. Neiva, M., F. B. Arraes, J. V. de Souza, G. Rádis-Baptista, A. R. Prieto da Silva, M. E. Walter, M. e. M. Brigido, T. Yamane, J. L. López-Lozano & S. Astolfi-Filho (2009) Transcriptome analysis of the Amazonian viper Bothrops atrox venom gland using expressed sequence tags (ESTs). Toxicon, 53, 427-36. Niewiarowski, S., E. P. Kirby, T. M. Brudzynski & K. Stocker (1979) Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors. Biochemistry, 18, 3570-7. Pardal, P. P., S. M. Souza, M. R. Monteiro, H. W. Fan, J. L. Cardoso, F. O. França, S. C. Tomy, I. S. Sano-Martins, M. C. de Sousa-e-Silva, M. Colombini, N. F. Kodera, A. M. Moura-da-Silva, D. F. Cardoso, D. T. Velarde, A. S. Kamiguti, R. D. Theakston & D. A. Warrell (2004) Clinical trial of two antivenoms for the treatment of Bothrops and Lachesis bites in the north eastern Amazon region of Brazil. Trans R Soc Trop Med Hyg, 98, 28-42. Patiño, A. C., J. A. Pereañez, V. Núñez, D. M. Benjumea, M. Fernandez, A. Rucavado, L. Sanz & J. J. Calvete (2010) Isolation and biological characterization of Batx-I, a weak hemorrhagic and fibrinogenolytic PI metalloproteinase from Colombian Bothrops atrox venom. Toxicon, 56, 936-43. Pinho, F. M. & E. A. Burdmann (2001) Fatal cerebral hemorrhage and acute renal failure after young Bothrops jararacussu snake bite. Ren Fail, 23, 269-77. Ribeiro, L. A., M. J. Albuquerque, V. A. de Campos, G. Katz, N. Y. Takaoka, M. L. Lebrão & M. T. Jorge (1998) [Deaths caused by venomous snakes in the State of São Paulo: evaluation of 43 cases from 1988 to 1993]. Rev Assoc Med Bras (1992), 44, 312-8. Ribeiro, L. A. & M. T. Jorge (1990) [Epidemiology and clinical picture of accidents by adult and young snakes Bothrops jararaca]. Rev Inst Med Trop Sao Paulo, 32, 436-42.
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Rosing, J., J. W. Govers-Riemslag, L. Yukelson & G. Tans (2001) Factor V activation and inactivation by venom proteases. Haemostasis, 31, 241-6. Rucinski, B., S. Niewiarowski, J. C. Holt, T. Soszka & K. A. Knudsen (1990) Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin. Biochim Biophys Acta, 1054, 257-62. Santos-Soares, P. C., A. Bacellar, H. P. Povoas, A. F. Brito & D. L. Santana (2007) Stroke and snakebite: case report. Arq Neuropsiquiatr, 65, 341-4. Selvanayagam, Z. E., S. G. Gnanavendhan, K. A. Ganesh, D. Rajagopal & P. V. Rao (1999) ELISA for the detection of venoms from four medically important snakes of India. Toxicon, 37, 757-70. Shannon, J. D., E. N. Baramova, J. B. Bjarnason & J. W. Fox (1989) Amino acid sequence of a Crotalus atrox venom metalloproteinase which cleaves type IV collagen and gelatin. J Biol Chem, 264, 11575-83. Sousa, L. F., C. A. Nicolau, P. S. Peixoto, J. L. Bernardoni, S. S. Oliveira, J. A. Portes-Junior, R. H. Mourão, I. Lima-dosSantos, I. S. Sano-Martins, H. M. Chalkidis, R. H. Valente & A. M. Moura-da-Silva (2013) Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of bothrops complex. PLoS Negl Trop Dis, 7, e2442. Stocker, K. & G. H. Barlow (1976) The coagulant enzyme from Bothrops atrox venom (batroxobin). Methods Enzymol, 45, 214-23.
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Table 1. Laboratory tests during hospitalization. D1
D2
D3
2.58x106
3.01 x106
7.8
8.9
22.9
26.4
3.65x106
Hemoglobin g/dL
11.1
Hematocrit %
31.7
Leukocytes /µL
8,800
2,700
900
89
67.7
59
4
23.2
40
242,000
148,000
143,000
Prothrombin activity (%)
27
85.5
61.9
INR
2.59
1.10
1.36
241
195
196
Creatinine mg/dL
-
-
2.2
Urea mg/dL
-
41
46
Calcium mg/dL
-
8.5
10.4
Na+ mEq/L
-
157
157
K+ mEq/L
-
1.8
2
Cl- mEq/L
-
125
125
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Erythrocyte /µL
Neutrophils % Lymphocytes %
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Glycemia mg/dL
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Platelets /µL
317
References ranges: erythrocyte (4.7-6.1x106 /µL); hemoglobin (13-16 g/dL); hematocrit (40-52
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%); leukocytes (4,000-10,800 /µL); neutrophil (42.1-5.2 %); lymphocytes (25-40 %) platelets
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(130,000-400,000 /µL); prothrombin activity (100%); INR (1); glycemia (70-110 mg/dL);
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creatinine (0.7-1.5 mg/dL); urea (15-40 mg/dL); calcium (8.5-10.1 mg/dL); Na+ (136-145
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mEq/L); K+ (3.5-5.1 mEq/L); Cl- (98-107 mEq/L). D1: in the first day after snakebite; D2 and
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D3: 2 and 3 days after snakebite.
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Legend of Figures:
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Figure 1: Subarachnoid hemorrhage after fatal snakebite by Bothrops atrox.
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Figure 2: Subarachnoid (A) and intraparenchymatous (B) hemorrhage after fatal snakebite by
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Bothrops atrox. Sections embedded in paraffin of human brain tissue were stained with
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hematoxylin and eosin, x100; x400, respectively. The arrows indicate the hemorrhagic areas. The
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sections were analyzed in at least 10 different fields in light microscopy.
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Figure 3: Localization of venom from Bothrops atrox in the brain tissue. Sections embedded in
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paraffin of human brain tissue were stained with anti-B. atrox polyclonal antibody by
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immunohistochemistry as described in the Supplementary material. The groups which were
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treated only with the blocking solution (A and B), normal serum rabbit IgG (C and D) and
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antibody produced against the B. atrox venom (E and F). The arrows indicate the brown spots
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that are positive for the venom. The sections were analyzed in at least 10 different fields in light
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microscopy.
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ACCEPTED MANUSCRIPT Highlights • We report a case of stroke after Bothrops snakebite in the Brazilian Amazon; • The case evolved with death despite the antivenom and conservative therapy employed; was
found
in
the
patient's
brain
tissue
after
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immunohistochemistry.
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S0041-0101(17)30266-0
DOI:
10.1016/j.toxicon.2017.08.021
Reference:
TOXCON 5705
To appear in:
Toxicon
Received Date: 27 June 2017 Revised Date:
18 August 2017
Accepted Date: 21 August 2017
Please cite this article as: Silva de Oliveira, Sâ., Freitas-de-Sousa, L.A., Alves, E.C., de Lima Ferreira, L.C., da Silva, Iran.Mendonç., de Lacerda, Marcus.Viní.Guimarã., Fan, H.W., Mourada-Silva, A.M., Monteiro, W.M., Fatal stroke after Bothrops snakebite in the Amazonas state, Brazil: A case report, Toxicon (2017), doi: 10.1016/j.toxicon.2017.08.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fatal stroke after Bothrops snakebite in the Amazonas state, Brazil: a case report.
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Sâmella Silva de Oliveiraa,bϕ, Luciana Aparecida Freitas-de-Sousac,dϕ, Eliane Campos Alvesa,b,
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Luiz Carlos de Lima Ferreiraa,b, Iran Mendonça da Silvaa,b, Marcus Vinícius Guimarães de
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Lacerdaa, Hui Wen Fane, Ana Maria Mourada-Silvac,d and Wuelton Marcelo Monteiroa,b*.
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a
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Amazonas, Brazil.
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b
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Diretoria de Ensino e Pesquisa, Fundação de Medicina Tropical Dr. Heitor Vieira Dourado,
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Brazil.
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c
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d
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e
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Escola Superior de Ciências da Saúde, Universidade do Estado do Amazonas, Amazonas,
Programa de Pós-Graduação em Ciências - Toxinologia, Instituto Butantan, São Paulo, Brazil. Laboratório de Imunopatologia, Instituto Butantan, São Paulo, Brazil.
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Núcleo Estratégico de Venenos e Antivenenos, Instituto Butantan, São Paulo, Brazil.
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*Corresponding author: Dr. Wuelton Marcelo Monteiro. Fundação de Medicina Tropical Dr.
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Heitor Vieira Dourado. Avenida Pedro Teixeira 25, Dom Pedro, 69040-000, Manaus, AM,
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Brazil. Phone: 55 92 2127-3430 e-mail: [email protected]
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These authors contributed equally to this work.
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Abbreviations
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IV: Intravenous.
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SVMPs: Snake Venom Metalloproteinases.
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SVSPs: Snake Venom Serine proteinases.
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PLA2: Phospholipases A2.
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Abstract
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Bothrops atrox is the snake responsible for the majority of snakebites in the Brazilian Amazon.
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Patients generally evolve to local manifestations such as edema, pain and ecchymoses. Systemic
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effects of B. atrox venom are usually restricted to blood incoagulability and spontaneous
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bleeding. However, in a few cases, bleeding in the central nervous system may occur, which can
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lead to sequels and deaths. Here, we report a case of a 59 year-old woman who presented edema,
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pain and ecchymoses on the right foot, headache, nausea, diarrhea, hypertension and blood
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incoagulability after the bite by Bothrops snake in the Brazilian Amazon. This case evolved with
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stroke resulting in death despite the antivenom and conservative therapy employed. In addition,
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we were able to identify the presence of venom in the patient's brain tissue after death. Direct
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action of toxins present in the snake’s venom in the induction of systemic hemorrhage allied to
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blood incoagulability and hypertension presented by the patient could be involved in the
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mechanism of stroke in this case.
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Keywords: hemorrhage, coagulopathy, Bothrops envenoming, stroke.
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1.
Introduction
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Bothrops envenoming usually result in local clinical features (i.e. edema, pain and ecchymoses)
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as well as systemics effects (i.e. coagulation disorders and systemic spontaneous bleeding)
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(Ribeiro et al. 1998, Ribeiro and Jorge 1990), in consequence mainly of the action of
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metalloproteinases, phospholipases A2 and serine proteinases (Calvete et al. 2011, Sousa et al.
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2013). These accidents may evolve with complications such as necrosis, secondary bacterial
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infection, compartment syndrome and acute renal failure (Ribeiro and Jorge 1990), being the
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main causes of death renal and respiratory failure, shock, sepsis and rarely hemorrhage in the
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central nervous system (Ribeiro et al. 1998). In a study on the prevalence of cerebrovascular
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complications in Bothrops envenoming, it was found that 2.6 % of the victims developed a
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cerebrovascular event, of which about 60 % died and 40 % remained with sequelae (Mosquera et
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al. 2003). Here, we describe a case of fatal stroke after Bothrops snakebite in the Brazilian
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Amazon.
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2.
Case Report
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A 59 year-old woman was bitten in the fifth right toe by a snake inside her house in the rural area
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of Manaus, Amazonas State, in Brazil. One hour and a half after the bite she was admitted at the
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reference hospital with intense local pain, swelling and ecchymosis on the right foot, and severe
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headache, nausea and episodes of diarrhea. No systemic bleeding was observed. Patient
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identified the snake as “surucucurana”, a local name for B. atrox, but did not bring it with her to
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the Health Facility. She denied previous diabetes and hypertension. On admission blood pressure
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was 200/110 mmHg and respiratory frequency was 18 bpm. Her blood was incoagulable and
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coagulogram was altered (Table 1). No other clinical manifestations were observed.
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Pre-medication was given (IV hydrocortizone 500 mg, IV promethazine 50 mg, IV
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dexchlorpheniramine and IV ranitidine 50 mg). Symptomatic medication for pain was also
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administered (IV dipirona 1 g). After premedication, the patient experienced episodes of
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vomiting and was administered IV metocloropramide 10 mg. Subsequently, she received
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sublingual captopril 50 mg. She received 80 mL de Bothrops antivenom after two hours and a
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half from the bite. However, approximately one hour after administration of antivenom, the
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patient experienced sweating, cold and clammy skin, tachycardia, sialorrhea, aphasia and
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decreased level of consciousness, not responding to verbal stimuli, but to painful stimuli. Blood
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pressure was 190/130 mmHg. The patient received IV furosemide 80 mg, IV omeprazole 40 mg
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and venous hydration. Administration of antivenom was discontinued. She was transferred
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comatose to the intensive care unit with 5 point on the Glasgow Coma Scale, isocorics pupils
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with photoreaction and required mechanical ventilation. Blood pressure was 230/130 mmHg.
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The patient received IV hydralazine 20 mg, IV midazolam 50 mg, IV fentanyl 2 mg and IV
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omeprazole 40 mg. Sedation was interrupted, being administered IV mannitol 20 g and IV
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hydantal 250 mg.
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On the second day after the bite, the patient remained comatose and on mechanical ventilation,
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evolving with mydriatic pupils without photoreaction, with 3 point on the Glasgow Coma Scale,
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hemodynamically unstable, hypothermia, bradycardia and anuria. She received IV hydantal 250
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mg and IV omeprazole 40 mg. On the third day after the bite, the patient showed no clinical
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improvement, showing hypothermia, bradycardia and hypotension. On this day, the patient had a
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cardiorespiratory arrest and died. The patient evolved without local complications, such as
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necrosis and secondary bacterial infection. Laboratory tests during hospitalization are shown in
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Table 1. Computed tomography was requested during the hospitalization period, but was not
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performed.
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Autopsy showed subarachnoid and intraparenchymal hemorrhage, as shown in Figures 1 and 2,
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respectively, and determined intracranial hypertension as cause of death. No other alteration was
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described in the necroscopic examination. Venom detection in brain tissue was performed by
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immunohistochemistry (Supplementary material) and is shown in Figure 3. The venom was
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immunolocalized in some areas of the patient's brain. The positive region is represented by the
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brown spots indicated with the arrows (Figure 3E and F) appearing only in the group where an
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antibody produced against the B. atrox venom was used. The groups which were treated only
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with the blocking solution (Figure 3A and B) or normal serum rabbit IgG (Figure 3C and D) not
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show brown spots. The tissue from patient's heart not was positive for venom (data not show).
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4. Discussion
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We describe the occurrence of stroke after snakebite in the Brazilian Amazon, an uncommon
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finding, which resulted in death. This snakebite was diagnosed and treated as an accident with
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snake in the genus Bothrops. Despite the patient not coming along with the dead snake to the
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Health Facility, she identified the snake as “surucucurana”, a popular name attributed to B.
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atrox, which is considered the main cause of snakebite in the Amazon (Cardoso et al. 2009).
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Accidents caused by B. atrox have more local clinical features such as blisters, necrosis and
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abscesses and systemic effects mostly related to blood coagulation disorders and spontaneous
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bleeding (Pardal et al. 2004). In Brazil, the specific treatment of Bothrops snakebites is the
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administration of the Bothrops, Bothrops-Lachesis or Bothrops-Crotalus antivenoms. Bothrops
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antivenom has been efficient in neutralizing the coagulant and hemorrhagic activities of B. atrox
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venom in pre-clinical assessments and clinical trials (Pardal et al. 2004, Sousa et al. 2013). In
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this present case, Bothrops antivenom was administered in time ˂ 6 hours after the bite and the
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coagulability was restored after 24 hours of the administration of antivenom.
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Signs of neurological damage after snakebite are most often related to the activities toxins
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present in the venoms, due to the anticoagulant/procoagulant activities and/or neurotoxicity of its
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components (Del Brutto and Del Brutto 2012). Death cases caused by cerebral hemorrhage in
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patients bitten by viper snakes have been described (Pinho and Burdmann 2001, Mosquera et al.
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2003, Kitchens and Eskin 2008, Kouyoumdjian et al. 1991). Autopsy of this patient showed
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subarachnoid and intraparenchymal hemorrhage. In a study with patients who presented stroke
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following Bothrops spp. snakebite, seven patients presented intracranial bleeding and
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hemorrhages were located in the subcortical white matter of the cerebral hemispheres, in the
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cerebellum and in the subarachnoid space (Mosquera et al. 2003). Patients bitten by viper snakes
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with intracranial hemorrhage presented generally coagulation disorders, such as increased
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clotting times, prolongation of the prothrombin and thromboplastin times, and decrease in the
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fibrinogen levels and number of platelets (Mosquera et al. 2003, Kitchens and Eskin 2008,
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Kouyoumdjian et al. 1991). Snake venom metalloproteinases (SVMPs), snake venom serine
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proteinases (SVSPs) and phospholipases A2 (PLA2) are the components most abundant of the
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Bothrops venom and are involved with hemostatics disorders in victims by Bothrops
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envenoming (Sousa et al. 2013). The SVMPs are the major toxins of the B. atrox venom from
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Brazilian Amazon (Calvete et al. 2011, Neiva et al. 2009, Sousa et al. 2013) and local and
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systemic hemorrhage is caused by the direct action of SVMPs in components of basement
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membrane of capillaries (Gutiérrez et al. 2005, Moura-da-Silva, Butera and Tanjoni 2007). The
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hydrolysis of basement membrane components (Shannon et al. 1989) leads to loss of mechanical
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stability of the capillaries and hemodynamic forces contribute to the distension and rupture of the
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capillary and consequent bleeding (Gutiérrez et al. 2005). Three major classes of SVMPs are
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described, however the hemorrhage is mainly associated with the PIII-class (Escalante et al.
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2006, Escalante et al. 2011). Unlike PI-class, PII-class and PIII-class present adhesive domain
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that act in anchoring to components basement membrane from capillaries which facilitates the
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hydrolysis and consequent hemorrhage (Baldo et al. 2010, Herrera et al. 2015).
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Venom was located in this patient's brain tissue after death. These findings agree with the study
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of (Selvanayagam et al. 1999), which found venom in the brain, heart, lungs, liver, spleen,
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kidneys and the bite area from tissue of the patient bite by Echis carinatus. (Escalante et al.
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2003), also found small amounts of Jarharagin, PIII-class SVMP majority of B. jararaca venom,
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in brain tissue of mice. The major component of B. atrox venom is Batroxrhagin, a PIII-class
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SVMP, which has a strong identity with Jarharagin (Freitas-de-Sousa et al. 2015). However,
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intracranial hemorrhage cannot be related only to PIII-class, since these hemorrhagic toxins (PII
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and PIII-class) were located mainly at the site of the bite by bind of non-catalytic domains to
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basement membrane components (Baldo et al. 2010, Herrera et al. 2015). On the other hand, our
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group recently isolated and characterized a PI-class SVMP, named Atroxlysin-Ia, from the
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venom of B. atrox that induces a strong hemorrhage comparable to a PIII-class SVMP (Freitas-
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de-Sousa et al. 2017). Furthermore, Atroxlysin-Ia not exhibit the adhesive domains present in the
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PIII-class SVMPs and most likely presents rapid tissue diffusion. Thus, these SVMPs probably
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contributed to the brain hemorrhage in this patient.
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This patient also was with incoagulable blood and altered coagulogram after the snakebite. Blood
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incoagulability observed in Bothrops envenoming is due to the consumption of fibrinogen and
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some coagulation factors. SVSPs are usually thrombin-like enzymes, which directly hydrolyze
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fibrinogen in fibrin, that are involved in the blood coagulation disorders observed in B. atrox
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envenoming (Stocker and Barlow 1976). B. atrox venom also contains SVMPs that activate
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factors II and X of coagulation, resulting in the formation of endogenous thrombin (Hofmann
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and Bon 1987a, Hofmann and Bon 1987b). In addition, PI-class SVMPs with fibrino(geno)lytic
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activity were isolated from the venom of B. atrox (Cintra et al. 2012, Patiño et al. 2010, Freitas-
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de-Sousa et al 2017). Others components from the B. atrox venom act on factors XIII
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(Niewiarowski et al. 1979) and V (Rosing et al. 2001) and platelets, causing inhibition (Rucinski
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et al. 1990, Freitas-de-Sousa et al. 2015). Therefore, the proteolytic action due to SVMPs allied
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to blood incoagulability could be involved into the patient's cerebral hemorrhage.
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In addition, factors associated in victims bitten by snakes have also been observed in cases of
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stroke. Some case reports show that intracranial bleeding can be associated with arterial
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hypertension (Machado et al. 2010, Allen et al. 2012, Berling et al. 2015). In the Pará State, in
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Brazil, a case report described a hemorrhagic stroke in Bothrops envenoming associated with
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severe hypertension, who received discharge for healing, suggesting synergism hypertension-
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venom as a determining factor for cerebral hemorrhage (Machado et al. 2010). In the present
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case report, severity of cerebral injury was evidenced by the Cushing response, characterized by
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increase of systemic arterial pressure, bradycardia and irregular respirations. The Cushing
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response is assumed to have a protective function for the brain, raising cerebral perfusion
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pressure in critical conditions, thus allowing an increase of cerebral blood flow. However, the
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arterial hypertension may induce a proportional increase in intracranial pressure, and may raise
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pressure in cerebral arteries and capillaries, increasing the risk of cerebral hemorrhage or edema,
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and the mechanisms that triggers a vicious circle between intracranial and systemic arterial
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pressure. Other factor associated with stroke is older age. Stroke following Bothrops snakebite
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was observed in victims with a mean age 51.9 years (Mosquera et al. 2003). Case reports of
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stroke involving victims >60 years have also been reported in Bothrops snakebite in others
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regions of Brazil (Santos-Soares et al. 2007). These characteristics of fatal victims after Bothrops
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snakebites reveal more vulnerable groups requiring appropriate and early treatment to avoid
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serious complications.
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This case report shows the importance of identifying risk factors for the development of this
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complication in Bothrops snakebite and appropriate and early antivenom terapy and prompt
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supportive treatment to avoid progression of systemic complications. Moreover, presents the first
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evidence of the direct involvement of some venom components in the induction mechanisms of
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systemic hemorrhage.
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Ethical statement
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This patient was enrolled in a study approved by the Ethics Review Board of the Fundação de
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Medicina Tropical Dr. Heitor Vieira Dourado, with a waiver of informed consent (CAAE
207
53192516.8.0000.0005).
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Acknowledgments
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We thank to Department of Archive and Statistic Services of Fundação de Medicina Tropical
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Dr. Heitor Vieira Dourado for the permits to access the medical records. This study was
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supported by PROCAD - CAPES AuxPE 2992/2014.
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Conflict of Interest
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The authors declare that there is no conflict of interest.
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Referências
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Allen, G. E., S. G. Brown, N. A. Buckley, M. A. O'Leary, C. B. Page, B. J. Currie, J. White, G. K. Isbister & A. Investigators (2012) Clinical effects and antivenom dosing in brown snake (Pseudonaja spp.) envenoming-Australian snakebite project (ASP-14). PLoS One, 7, e53188. Baldo, C., C. Jamora, N. Yamanouye, T. M. Zorn & A. M. Moura-da-Silva (2010) Mechanisms of vascular damage by hemorrhagic snake venom metalloproteinases: tissue distribution and in situ hydrolysis. PLoS Negl Trop Dis, 4, e727. Berling, I., S. G. Brown, F. Miteff, C. Levi & G. K. Isbister (2015) Intracranial haemorrhages associated with venom induced consumption coagulopathy in Australian snakebites (ASP-21). Toxicon, 102, 8-13. Calvete, J. J., L. Sanz, A. Pérez, A. Borges, A. M. Vargas, B. Lomonte, Y. Angulo, J. M. Gutiérrez, H. M. Chalkidis, R. H. Mourão, M. F. Furtado & A. M. Moura-Da-Silva (2011) Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management. J Proteomics, 74, 510-27. Cardoso, J. L. C., Francisco Oscar de Siqueira FranÇa, F. H. Wen, C. M. S. A. Málaque & V. H. Jr. 2009. Animais Peçonhentos no Brasil. In Serpentes peçonhentas do Brasil, ed. A. R. MELGAREJO, 550. Cintra, A. C., L. G. De Toni, M. A. Sartim, J. J. Franco, R. C. Caetano, M. T. Murakami & S. V. Sampaio (2012) Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity. Toxicon, 60, 70-82. Del Brutto, O. H. & V. J. Del Brutto (2012) Neurological complications of venomous snake bites: a review. Acta Neurol Scand, 125, 363-72. Escalante, T., J. Núñez, A. M. Moura da Silva, A. Rucavado, R. D. Theakston & J. M. Gutiérrez (2003) Pulmonary hemorrhage induced by jararhagin, a metalloproteinase from Bothrops jararaca snake venom. Toxicol Appl Pharmacol, 193, 17-28. Escalante, T., N. Ortiz, A. Rucavado, E. F. Sanchez, M. Richardson, J. W. Fox & J. M. Gutiérrez (2011) Role of collagens and perlecan in microvascular stability: exploring the mechanism of capillary vessel damage by
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snake venom metalloproteinases. PLoS One, 6, e28017. Escalante, T., J. Shannon, A. M. Moura-da-Silva, J. M. Gutiérrez & J. W. Fox (2006) Novel insights into capillary vessel basement membrane damage by snake venom hemorrhagic metalloproteinases: a biochemical and immunohistochemical study. Arch Biochem Biophys, 455, 144-53. Freitas-de-Sousa, L. A., D. R. Amazonas, L. F. Sousa, S. S. Sant'Anna, M. Y. Nishiyama, S. M. Serrano, I. L. Junqueirade-Azevedo, H. M. Chalkidis, A. M. Moura-da-Silva & R. H. Mourão (2015) Comparison of venoms from wild and long-term captive Bothrops atrox snakes and characterization of Batroxrhagin, the predominant class PIII metalloproteinase from the venom of this species. Biochimie, 118, 60-70. Freitas-de-Sousa, L. A., Colombini, M., Lopes-Ferreira, M., Serrano, S. M. T., Moura-da-Silva, A.M. (2017) Insights into the Mechanisms involved in strong hemorrhage and dermonecrosis induced by Atroxlysin-Ia, a PIClass snake venom metalloproteinase. Toxins, 9, 239. Gutiérrez, J. M., A. Rucavado, T. Escalante & C. Díaz (2005) Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon, 45, 997-1011. Herrera, C., T. Escalante, M. B. Voisin, A. Rucavado, D. Morazán, J. K. Macêdo, J. J. Calvete, L. Sanz, S. Nourshargh, J. M. Gutiérrez & J. W. Fox (2015) Tissue localization and extracellular matrix degradation by PI, PII and PIII snake venom metalloproteinases: clues on the mechanisms of venom-induced hemorrhage. PLoS Negl Trop Dis, 9, e0003731. Hofmann, H. & C. Bon (1987a) Blood coagulation induced by the venom of Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator. Biochemistry, 26, 772-80. --- (1987b) Blood coagulation induced by the venom of Bothrops atrox. 2. Identification, purification, and properties of two factor X activators. Biochemistry, 26, 780-7. Kitchens, C. & T. Eskin (2008) Fatality in a case of envenomation by Crotalus adamanteus initially successfully treated with polyvalent ovine antivenom followed by recurrence of defibrinogenation syndrome. J Med Toxicol, 4, 180-3. Kouyoumdjian, J. A., C. Polizelli, S. M. Lobo & S. M. Guimares (1991) Fatal extradural haematoma after snake bite (Bothrops moojeni). Trans R Soc Trop Med Hyg, 85, 552. Machado, A. S., F. B. Barbosa, G. a. S. Mello & P. P. Pardal (2010) [Hemorrhagic stroke related to snakebite by bothrops genus: a case report]. Rev Soc Bras Med Trop, 43, 602-4. Mosquera, A., L. A. Idrovo, A. Tafur & O. H. Del Brutto (2003) Stroke following Bothrops spp. snakebite. Neurology, 60, 1577-80. Moura-da-Silva, A. M., D. Butera & I. Tanjoni (2007) Importance of snake venom metalloproteinases in cell biology: effects on platelets, inflammatory and endothelial cells. Curr Pharm Des, 13, 2893-905. Neiva, M., F. B. Arraes, J. V. de Souza, G. Rádis-Baptista, A. R. Prieto da Silva, M. E. Walter, M. e. M. Brigido, T. Yamane, J. L. López-Lozano & S. Astolfi-Filho (2009) Transcriptome analysis of the Amazonian viper Bothrops atrox venom gland using expressed sequence tags (ESTs). Toxicon, 53, 427-36. Niewiarowski, S., E. P. Kirby, T. M. Brudzynski & K. Stocker (1979) Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors. Biochemistry, 18, 3570-7. Pardal, P. P., S. M. Souza, M. R. Monteiro, H. W. Fan, J. L. Cardoso, F. O. França, S. C. Tomy, I. S. Sano-Martins, M. C. de Sousa-e-Silva, M. Colombini, N. F. Kodera, A. M. Moura-da-Silva, D. F. Cardoso, D. T. Velarde, A. S. Kamiguti, R. D. Theakston & D. A. Warrell (2004) Clinical trial of two antivenoms for the treatment of Bothrops and Lachesis bites in the north eastern Amazon region of Brazil. Trans R Soc Trop Med Hyg, 98, 28-42. Patiño, A. C., J. A. Pereañez, V. Núñez, D. M. Benjumea, M. Fernandez, A. Rucavado, L. Sanz & J. J. Calvete (2010) Isolation and biological characterization of Batx-I, a weak hemorrhagic and fibrinogenolytic PI metalloproteinase from Colombian Bothrops atrox venom. Toxicon, 56, 936-43. Pinho, F. M. & E. A. Burdmann (2001) Fatal cerebral hemorrhage and acute renal failure after young Bothrops jararacussu snake bite. Ren Fail, 23, 269-77. Ribeiro, L. A., M. J. Albuquerque, V. A. de Campos, G. Katz, N. Y. Takaoka, M. L. Lebrão & M. T. Jorge (1998) [Deaths caused by venomous snakes in the State of São Paulo: evaluation of 43 cases from 1988 to 1993]. Rev Assoc Med Bras (1992), 44, 312-8. Ribeiro, L. A. & M. T. Jorge (1990) [Epidemiology and clinical picture of accidents by adult and young snakes Bothrops jararaca]. Rev Inst Med Trop Sao Paulo, 32, 436-42.
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Rosing, J., J. W. Govers-Riemslag, L. Yukelson & G. Tans (2001) Factor V activation and inactivation by venom proteases. Haemostasis, 31, 241-6. Rucinski, B., S. Niewiarowski, J. C. Holt, T. Soszka & K. A. Knudsen (1990) Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin. Biochim Biophys Acta, 1054, 257-62. Santos-Soares, P. C., A. Bacellar, H. P. Povoas, A. F. Brito & D. L. Santana (2007) Stroke and snakebite: case report. Arq Neuropsiquiatr, 65, 341-4. Selvanayagam, Z. E., S. G. Gnanavendhan, K. A. Ganesh, D. Rajagopal & P. V. Rao (1999) ELISA for the detection of venoms from four medically important snakes of India. Toxicon, 37, 757-70. Shannon, J. D., E. N. Baramova, J. B. Bjarnason & J. W. Fox (1989) Amino acid sequence of a Crotalus atrox venom metalloproteinase which cleaves type IV collagen and gelatin. J Biol Chem, 264, 11575-83. Sousa, L. F., C. A. Nicolau, P. S. Peixoto, J. L. Bernardoni, S. S. Oliveira, J. A. Portes-Junior, R. H. Mourão, I. Lima-dosSantos, I. S. Sano-Martins, H. M. Chalkidis, R. H. Valente & A. M. Moura-da-Silva (2013) Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of bothrops complex. PLoS Negl Trop Dis, 7, e2442. Stocker, K. & G. H. Barlow (1976) The coagulant enzyme from Bothrops atrox venom (batroxobin). Methods Enzymol, 45, 214-23.
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Table 1. Laboratory tests during hospitalization. D1
D2
D3
2.58x106
3.01 x106
7.8
8.9
22.9
26.4
3.65x106
Hemoglobin g/dL
11.1
Hematocrit %
31.7
Leukocytes /µL
8,800
2,700
900
89
67.7
59
4
23.2
40
242,000
148,000
143,000
Prothrombin activity (%)
27
85.5
61.9
INR
2.59
1.10
1.36
241
195
196
Creatinine mg/dL
-
-
2.2
Urea mg/dL
-
41
46
Calcium mg/dL
-
8.5
10.4
Na+ mEq/L
-
157
157
K+ mEq/L
-
1.8
2
Cl- mEq/L
-
125
125
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Erythrocyte /µL
Neutrophils % Lymphocytes %
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Platelets /µL
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References ranges: erythrocyte (4.7-6.1x106 /µL); hemoglobin (13-16 g/dL); hematocrit (40-52
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%); leukocytes (4,000-10,800 /µL); neutrophil (42.1-5.2 %); lymphocytes (25-40 %) platelets
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(130,000-400,000 /µL); prothrombin activity (100%); INR (1); glycemia (70-110 mg/dL);
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creatinine (0.7-1.5 mg/dL); urea (15-40 mg/dL); calcium (8.5-10.1 mg/dL); Na+ (136-145
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mEq/L); K+ (3.5-5.1 mEq/L); Cl- (98-107 mEq/L). D1: in the first day after snakebite; D2 and
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D3: 2 and 3 days after snakebite.
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Figure 1: Subarachnoid hemorrhage after fatal snakebite by Bothrops atrox.
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Figure 2: Subarachnoid (A) and intraparenchymatous (B) hemorrhage after fatal snakebite by
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Bothrops atrox. Sections embedded in paraffin of human brain tissue were stained with
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hematoxylin and eosin, x100; x400, respectively. The arrows indicate the hemorrhagic areas. The
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sections were analyzed in at least 10 different fields in light microscopy.
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Figure 3: Localization of venom from Bothrops atrox in the brain tissue. Sections embedded in
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paraffin of human brain tissue were stained with anti-B. atrox polyclonal antibody by
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immunohistochemistry as described in the Supplementary material. The groups which were
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treated only with the blocking solution (A and B), normal serum rabbit IgG (C and D) and
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antibody produced against the B. atrox venom (E and F). The arrows indicate the brown spots
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that are positive for the venom. The sections were analyzed in at least 10 different fields in light
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microscopy.
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ACCEPTED MANUSCRIPT Highlights • We report a case of stroke after Bothrops snakebite in the Brazilian Amazon; • The case evolved with death despite the antivenom and conservative therapy employed; was
found
in
the
patient's
brain
tissue
after
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immunohistochemistry.
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• Venom
by
