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Introduction: Snake venom composition shows significant inter- and intra-species variation. In the case of the viperid species Bothrops atrox, responsible for the majority of snakebites in the Amazon region, geographical and ontogenetic variables affect venom composition, with ecological and medical implications. Previous studies had shown that venom from neonate and juvenile Bothrops specimens have a higher in vitro coagulant activity. The aim of this investigation was to assess the association of clinical outcomes, such as venom-induced coagulopathy and local complications, with B. atrox ontogenetic variables. Methods: This study explored the relationship between some clinical parameters in patients suffering envenomations by B. atrox in the Amazon and several morphometric parameters of the snake specimens causing the bites. Results: There were 248 specimens confirmed as agents of envenomation, mostly female snakes (70.5%) and classified as juveniles (62.7%). Patients bitten by neonates compared to adult snakes [OR = 2.70 (95%CI 1.15-6.37); p = .021] and by snakes with white tail tip [OR = 1.98 (95%CI 1.15–3.41); p = .013] were more likely to develop coagulopathy. Time from patient admission to the unclottable blood reversion was not affected by the snake gender (p = .214) or age (p = .254). Patients bitten by neonate (p = .024) or juvenile snakes (p < .0001) presented a lower frequency of moderate to severe edema, as compared to those bitten by adult snakes. In agreement with experimental observations, patients bitten by neonates and by snakes with a white tail tip were more likely to develop coagulopathy than those bitten by adult snakes. In contrast, envenomations by adult snakes were associated with a higher incidence of severe local edema. Conclusion: Despite these variations, no difference was observed in the time needed to recover blood clotting in these patients after Bothrops antivenom administration.
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Clinical Toxicology
ISSN: 1556-3650 (Print) 1556-9519 (Online) Journal homepage: https://www.tandfonline.com/loi/ictx20
Bad things come in small packages”: predicting
venom-induced coagulopathy in Bothrops atrox
bites using snake ontogenetic parameters
Jorge Carlos Contreras Bernal, Pedro Ferreira Bisneto, João Pedro Tavares
Pereira, Hiochelson Najibe dos Santos Ibiapina, Lybia Kássia Santos Sarraff,
Cláudio Monteiro-Júnior, Handerson da Silva Pereira, Bruno Santos, Valeria
Mourão de Moura, Sâmella Silva de Oliveira, Marcus Lacerda, Vanderson
Sampaio, Igor Luis Kaefer, José María Gutiérrez, Paulo Sérgio Bernarde, Hui
Wen Fan, Jacqueline Sachett, Ana Maria Moura da Silva & Wuelton Marcelo
Monteiro
To cite this article: Jorge Carlos Contreras Bernal, Pedro Ferreira Bisneto, João Pedro Tavares
Pereira, Hiochelson Najibe dos Santos Ibiapina, Lybia Kássia Santos Sarraff, Cláudio Monteiro-
Júnior, Handerson da Silva Pereira, Bruno Santos, Valeria Mourão de Moura, Sâmella Silva
de Oliveira, Marcus Lacerda, Vanderson Sampaio, Igor Luis Kaefer, José María Gutiérrez,
Paulo Sérgio Bernarde, Hui Wen Fan, Jacqueline Sachett, Ana Maria Moura da Silva & Wuelton
Marcelo Monteiro (2019): “Bad�things�come�in�small�packages”: predicting venom-induced
coagulopathy in Bothrops�atrox bites using snake ontogenetic parameters, Clinical Toxicology, DOI:
10.1080/15563650.2019.1648817
To link to this article: https://doi.org/10.1080/15563650.2019.1648817
View supplementary material Published online: 06 Aug 2019.
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BASIC RESEARCH
Bad things come in small packages: predicting venom-induced coagulopathy in
Bothrops atrox bites using snake ontogenetic parameters
Jorge Carlos Contreras Bernal
a,b
, Pedro Ferreira Bisneto
c
,Jo
~
ao Pedro Tavares Pereira
b
, Hiochelson Najibe dos
Santos Ibiapina
a,b
, Lybia K
assia Santos Sarraff
b
,Cl
audio Monteiro-J
unior
b
, Handerson da Silva Pereira
b
,
Bruno Santos
b
, Valeria Mour~
ao de Moura
b,d
,S
^
amella Silva de Oliveira
a,b
, Marcus Lacerda
b,e
,
Vanderson Sampaio
b,f
, Igor Luis Kaefer
c
, Jos
eMar
ıa Guti
errez
g
, Paulo S
ergio Bernarde
h,i
, Hui Wen Fan
j
,
Jacqueline Sachett
a,k
, Ana Maria Moura da Silva
j
and Wuelton Marcelo Monteiro
a,b
a
Escola Superior de Ci^
encias da Sa
ude, Universidade Do Estado Do Amazonas, Manaus, Brazil;
b
Instituto de Pesquisa Cl
ınica Carlos
Borborema, Fundac¸~
ao de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil;
c
Programa de P
os-Graduac¸~
ao em Zoologia,
Universidade Federal Do Amazonas, Manaus, Brazil;
d
Instituto de Sa
ude Coletiva, Universidade Federal Do Oeste Do Par
a, Santar
em, Brazil;
e
Instituto Le^
onidas & Maria Deane, Manaus, Brazil;
f
Sala de Situac¸~
ao em Sa
ude, Fundac¸~
ao de Vigil^
ancia em Sa
ude Do Amazonas, Manaus,
Brazil;
g
Instituto Clodomiro Picado, Facultad de Microbiolog
ıa, Universidad de Costa Rica, San Jos
e, Costa Rica;
h
Laborat
orio de
Herpetologia, Centro Multidisciplinar, Campus Floresta, Universidade Federal Do Acre, Cruzeiro Do Sul, AC, Brazil;
i
Instituto Federal Do Acre,
Campus de Cruzeiro Do Sul, Cruzeiro Do Sul, Acre, Brazil;
j
N
ucleo Estrat
egico de Venenos e Antivenenos, Instituto Butantan, S~
ao Paulo,
Brazil;
k
Diretoria de Ensino e Pesquisa, Fundac¸~
ao Alfredo da Matta, Manaus, Brazil
ABSTRACT
Introduction: Snake venom composition shows significant inter- and intra-species variation. In the
case of the viperid species Bothrops atrox, responsible for the majority of snakebites in the Amazon
region, geographical and ontogenetic variables affect venom composition, with ecological and medical
implications. Previous studies had shown that venom from neonate and juvenile Bothrops specimens
have a higher in vitro coagulant activity. The aim of this investigation was to assess the association of
clinical outcomes, such as venom-induced coagulopathy and local complications, with B. atrox onto-
genetic variables.
Methods: This study explored the relationship between some clinical parameters in patients suffering
envenomations by B. atrox in the Amazon and several morphometric parameters of the snake speci-
mens causing the bites.
Results: There were 248 specimens confirmed as agents of envenomation, mostly female snakes
(70.5%) and classified as juveniles (62.7%). Patients bitten by neonates compared to adult snakes
[OR ¼2.70 (95%CI 1.15-6.37); p¼.021] and by snakes with white tail tip [OR ¼1.98 (95%CI 1.153.41);
p¼.013] were more likely to develop coagulopathy. Time from patient admission to the unclottable
blood reversion was not affected by the snake gender (p¼.214) or age (p¼.254). Patients bitten by
neonate (p¼.024) or juvenile snakes (p<.0001) presented a lower frequency of moderate to severe
edema, as compared to those bitten by adult snakes. In agreement with experimental observations,
patients bitten by neonates and by snakes with a white tail tip were more likely to develop coagulop-
athy than those bitten by adult snakes. In contrast, envenomations by adult snakes were associated
with a higher incidence of severe local edema.
Conclusion: Despite these variations, no difference was observed in the time needed to recover blood
clotting in these patients after Bothrops antivenom administration.
ARTICLE HISTORY
Received 9 May 2019
Revised 17 July 2019
Accepted 20 July 2019
Published online 2 August
2019
KEYWORDS
Bothrops atrox; snakebite
envenomation; snake
morphometrics; coagulop-
athy; antivenom
Introduction
Bothrops atrox, the Amazonian lancehead, is the main species
responsible for snakebites in the Amazon, causing 8090% of
the envenomations in the region [1]. This species inhabits
mostly forests, although it may be occasionally found in agri-
cultural and urban environments [2,3]. B. atrox venom
includes metalloproteinase as the major toxin family followed
by phospholipases A
2
, serine proteinases, cysteine-rich secre-
tory proteins, L-amino acid oxidases and C-type lectin-like
toxins [46]. Hypofibrinogenemia is a major systemic
complication from B. atrox bites, affecting more than 80% of
the patients [7]. This condition results from the action of ser-
ine proteinases having thrombin-like activity, which converts
fibrinogen to fibrin, and also due to the procoagulant activity
of metalloproteinases, which activate factors II and X of the
coagulation cascade, resulting in the formation of endogen-
ous thrombin [8]. In addition, PI and P-III metalloproteinases,
such as batroxase [9], Atroxlysin-Ia [10] and Batroxrhagin
[11], cause microvascular damage by proteolytic degradation
of basement membrane [12]. These processes combined are
CONTACT Wuelton Marcelo Monteiro wueltonmm@gmail.com Escola Superior de Ci^
encias da Sa
ude, Universidade Do Estado Do Amazonas, Manaus,
Brazil
Supplemental data for this article can be accessed here.
ß2019 Informa UK Limited, trading as Taylor & Francis Group
CLINICAL TOXICOLOGY
https://doi.org/10.1080/15563650.2019.1648817
responsible for systemic bleeding observed in B. atrox snake-
bites [7,13].
The variability in Bothrops venom is related to the age [14],
sex [15], geographical variation [5,16] and diet [17] of the
snakes. B. atrox venom variability is mostly related to the
expression level of each group of toxins rather than to the
presence or absence of major families of venom proteins [5].
Remarkable differences in biological activities of juvenile and
adult pit vipers have been observed, with venoms from juve-
niles having higher coagulant and lower proteolytic activities
as compared to those of adults [18]. Some reports described
that coagulopathy is more frequent in envenomation caused
by juveniles, whereas local effects, including edema and
necrosis, are more common in envenomations by adult snakes
[1921]. In general, this type of study is of dubious interpret-
ation, since the number of envenomations with snake charac-
terization is small, and snake morphometrics to confirm the
ontogenetic stage are poorly assessed. The aim of this study
is to assess the association of clinical outcomes, such as
venom-induced coagulopathy and edema with ontogenetic
stages of the B. atrox specimens perpetrating the bites.
Methods
Study population
We analyzed museum-preserved snake specimens causing
human envenomations in the Fundac¸~
ao de Medicina Tropical
Doutor Heitor Vieira Dourado (FMT-HVD), from 2009 to 2017.
By consecutive sampling, every patient who brought the
snake to the hospital, with full corresponding medical infor-
mation obtained in previous prospective studies, was
included. This study was approved by the Ethical Committee
of the FMT-HVD (approval number 53192516.8.0000.0005).
Patientsinformation
Clinico-epidemiological information was collected through a
standardized questionnaire (Epi Info
TM
, CDC). Clinical severity
of envenomation was classified according to the Brazilian
Ministry of Health guidelines [22]: (i) mild envenomation,
with pain and edema in one segment of the bitten limb; (ii)
moderate envenomation, with evident edema involving two
segments of the limb and local or systemic bleeding without
hemodynamic repercussion; (iii) severe envenomation, with
severe pain, severe edema in the limb, severe hemorrhagic
conditions with hemodynamic repercussion, compartment
syndrome and renal failure. Clotting time was evaluated by
the modified Lee-White clotting time test (LWCT) [22]. With a
plastic syringe, 1 mL of venous blood was collected and
placed into a glass tube (1375 mm) without anticoagulants,
at 25 C. Using a stopwatch, timing started as soon as the
blood was drawn into the tube. The tube was left undis-
turbed for 5 min and then checked for clots every minute
by gently tilting the tube. Unclottable blood was defined
when the blood was not clotted until 10 min [7].
Thrombocytopenia was defined as platelet counts <150,000
platelets/mL. Mild thrombocytopenia was defined by a
platelet count of 100,000 to 150,000/lL, moderate thrombo-
cytopenia as a platelet count of 50,000 to 100,000/lL and
severe thrombocytopenia as a platelet count below 50,000/lL.
For edema assessment, the bitten limb was divided into
three segments. The upper limb was divided into (i) hand
and wrist; (ii) forearm and elbow, and (iii) arm. Lower limb
was divided into (i) foot and ankle; (ii) leg and knee and (iii)
thigh. The presence of edema only in the segment of the
bite was considered as mild, in two segments as moderate
and in three segments as severe [23]. Secondary bacterial
infection was defined according the guidelines of the
Infectious Diseases Society of America [24]. Cellulitis was
defined by the presence of local inflammation signs (ery-
thema, edema, bruising and pain) associated with fever,
leukocytosis, lymphangitis and/or lymphadenitis. An abscess
was characterized by individual injuries, floating, presenting
purulent or serous-purulent secretion. For edema assessment
and secondary infection diagnosis, two independent exam-
iners blinded to snakesstage evaluated the patients and
came to a final agreement. Acute renal failure was defined
according to the Acute Kidney Injury Network (AKIN), as the
increase of at least >0.3 mg/dL or up to 199% of baseline
creatinine levels [25].
Morphometric characterization of snakes
Sex was verified by examination of the reproductive organs
through an incision in the first subcaudal scales [26]. The fol-
lowing morphometric variables were recorded: snout-vent
length (SVL), tail length (TaL), total length (ToL), head length
(HL), head width (HW), nasal-ocular distance (OND), ocular-
loreal distance (OLD), loreal-nasal distance (LND), inter-fang
distance (IFD), ventral-symphysal distance (VSD), rostral-labial
distance (RLD), loreal width (LW), nasal width (NW), ocular
width (OW) and cloacal width (CW) (Figure 1). SVL, TaL and
ToL were obtained using a measuring tape, whereas the
remaining variables with analogue digital caliper. The individ-
uals were grouped by age as follows: neonate males and
females (SVL <300 mm), juvenile males (SVL between 300-
460 mm) and juvenile females (SVL between 300-800 mm),
adult males (SVL >470 mm) and adult females (SVL >
850mm) [27]. Snake weight (g) (W) was obtained by a digital
precision scale. An incision was made on the ventral side of
the snake to check the presence of the gastric and intestinal
contents. Presence of white tail tip (WTT), an indicative of
juvenile stage, was recorded. The measurement of the snakes
was made by a single trained herpetologist. Three measure-
ments were made for each parameter and the mean
obtained was used in this study. Some snakes were brought
to the hospital with damaged structures, impairing the meas-
urement of all morphometric variables. However, the pos-
sible measures to obtain from these specimens were used in
the analysis.
Statistical analyses
The primary endpoint was a prolonged clotting time. Snake
variables were compared between patients presenting
2 J. C. C. BERNAL ET AL.
unclottable or clottable blood and edema severity using Chi-
square test. Odds ratios with the corresponding confidence
interval were presented. Snake morphometrics were com-
pared between patients presenting unclottable or clottable
blood using Studentsttest. A classification model for clot-
ting status was performed based on the creation of decision
trees using snake morphometrics and weight as independent
variables. Receiver operating characteristic (ROC) analysis was
conducted to verify potential morphometric markers. Time
until unclottable blood reversion was analyzed using Kaplan-
Meier estimates, using snake sex and age as independent
variables. All the analyses considered a 5% significance level
and were conducted using the STATA package version 13
(Stata Corp. 2013).
Results
Patientsparameters
Table 1 presents the characteristics of the 247 patients
included in the study.
The most frequent local manifestations were pain
(87.9%) and edema (83.9%). Systemic bleeding was
observed in 3.6% of the patients. Snakebites were mostly
classified as moderate (50.2%). Secondary infection (21%)
was the most frequent local complication observed. Acute
kidney injury was observed in 12.8% of the patients.
Unclottable blood was observed in 61.1% of the patients
(Figure 2). Mild thrombocytopenia was reported in 4.7% of
the patients. Case fatality rate was 0.4% during
hospitalization.
Snake descriptive analysis
Out of 247 B. atrox individuals confirmed as agents of
envenomation, sex was identified in 193, being 57 males
Figure 1. Description of Bothrops atrox morphometrics: Left part of picture; head length (HL), nasal-ocular distance (OND), ocular-loreal distance (OLD), loreal-nasal
distance (LND), rostral-labial distance (RLD), tail length (TaL), snout-vent length (SVL) and cloacal width (CW). Right upper corner of the picture, (A) head width
(HW), ocular width (OW), loreal width (LW) and nasal width (NW). (B) ventral view of head: ventral-symphysal distance (VSD). (A and B) ventral and dorsal view of
non-white tail, and white tail (TW), respectively. Total length (ToL), is the sum of TaL and SVL.
Table 1. Epidemiological characteristics of the 247 patients.
Variables Cases %
Sex
Male 195 79
Female 52 21
Age (years)
010 20 8.1
1120 42 17.0
2130 57 23.1
3140 50 20.2
4150 31 12.6
5060 34 13.8
>60 13 5.3
Area of occurrence
Rural 188 78.7
Urban 33 13.8
Others 27 10.9
Anatomical region of the bite
Foot 145 61.2
Leg 41 17.3
Toe 26 11
Hand 14 5.9
Others 22 8.9
Time from bite to medical assistance (hrs)
03 154 72
463215
712 18 8.4
>12 10 4.7
Use of topical medications
Yes 30 12.4
Use of oral medications
Yes 46 18.6
Use of tourniquet
Yes 24 9.7
Incision in the bite site
Yes 10 4.1
CLINICAL TOXICOLOGY 3
(29.5%) and 136 females (70.5%). Detailed morphometric
information is presented in Supplementary file 1. Most of the
snakes were classified as juveniles (62.7%). Considering
males, 68.5% of the snakes were adults, while females were
mostly juveniles (57.9%). White tail tip was present in 56.3%
of the snakes. A proportion of 24.2% of the snakes had gas-
tric or intestinal contents (Figure 3).
Variables associated with coagulopathy
Patients bitten by neonates compared to adult snakes
[OR ¼2.70 (95%CI 1.15-6.37); p¼.021] and by snakes with
white tail tip [OR ¼1.98 (95%CI 1.15-3.41); p¼.013] pre-
sented a higher probability of developing coagulopathy
(Table 2). No snake variable was found to be associated with
thrombocytopenia.
Predicting coagulopathy from snake morphometrics
For female snakes, mean weight and most morphometric
variables distinguished between the two types of patients
except NOD, OLD, LDN and NW (Figure 4). For patients bit-
ten by male snake specimens, the only variable significantly
different between the two groups of patients was LND, pre-
senting good diagnostic ability to classify blood clotting sta-
tus (AUC ¼0.802 (95%CI 0.676-0.928)) (Figure 5).
Decision trees generated from total and female snakes
presented a greater number of branches in relation to those
from male snakes. Supplementary file 2 presents rules with
90% of discriminatory power and at least five patients in
the branch. Snake weight was the most discriminatory attri-
bute in patients bitten by female snakes. Snake LND was the
most discriminatory attribute in patients bitten by male
snakes.
Time until restoration of blood coagulability
Time from patient admission to the unclottable blood rever-
sion was not affected by the snake sex (p¼.214) nor age
(p¼.254) (Figure 6).
Snake age and local complications
Patients bitten by neonate (p¼.024) or juvenile snakes
(p<.0001) presented a lower frequency of moderate/severe
edema, as compared to those bitten by adult snakes (Figure
7). Although in low frequency for robust analysis, other local
complications such as blistering, compartment syndrome and
necrosis were not seen among patients bitten by neonate
snakes.
In this study, one patient died. A 91 year-old male was bit-
ten in the right hand and the right foot. Immediately after
the bite the patient reported an intense acute pain in the
bitten sites. Eleven hours after the snakebite, patient was
hospitalized presenting intense pain in the right foot and
edema extending to the whole limb. Eight vials (80 mL) of
Bothrops antivenom were administered. He presented with
acute renal injury. Patients health status deteriorated and
Figure 2. Clinical characteristics of patients. Blood was considered unclottable when sample lasted more than 9 minutes to clot in the modified LeeWhite clotting
time (LWCT) method [7]. Clinical classification was made according to the guidelines of the Brazilian Ministry of Health [6]. Acute kidney injury was defined accord-
ing to the Acute Kidney Injury Network (AKIN) Guideline [25].
4 J. C. C. BERNAL ET AL.
48 hours from admission he was diagnosed with compart-
ment syndrome and was submitted to an extensive fasciot-
omy in the right leg. Secondary bacterial infection was
diagnosed and clindamycin was started. After two days, the
patient died of septic shock. Abnormalities in clotting time
or platelet counts and bleeding were not observed during
hospitalization. The snake brought by the patient was an
adult female.
Discussion
Several studies have analyzed the epidemiological and clin-
ical aspects of snakebite patients, but did not include the
characteristics of snakes responsible for the bites. With the
current knowledge about the variability in the composition
of snake venoms, the characteristics of the offending snake
are relevant for clinical studies.
One hypothesis was that male snakes cause more bites
than females, since males move around more often than
females, especially during the reproductive season [28,29].
Otherwise, in this work it was shown that most of the bites
were caused by female snakes (2.4 females for each male).
However, since only snakes captured and brought by the
patients to the hospital were included in this study, a selec-
tion bias may have occurred since males, smaller and faster
than females, might escape from the patient more often
than females, particularly the pregnant ones [29]. Another
possible explanation for this finding is that there are more
females than males in nature, which is suggested in some
studies [30,31]. Moreover, females of B. atrox tend to be in
general larger than males [27], thus having a higher capacity
to reach a person during a bite. On the other hand,
Figure 3. Morphometrics, age group, presence of white tail tip and intestinal contents of Bothrops atrox snakes which caused the envenomations. Neonate males
and females (SVL <300 mm); juvenile males (SVL 300-460 mm) and juvenile females (SVL 300-800mm); adult males (SVL >470 mm) and adult females
(SVL >850mm) [27].
Table 2. Variables associated with coagulopathy.
Variable
Unclottable
blood
Clottable
blood OR (CI95%) p
Patients age (years)
010 16 (11.9%) 4 (4.2%) Ref.
1120 20 (14.8%) 15 (15.8%) 3.00 (0.83-10.83) 0.096
2140 55 (40.7%) 45 (47.4%) 3.27 (1.02-10.48) 0.038
4160 38 (28.1%) 24 (25.3%) 2.52 (0.75-8.46) 0.132
>60 6 (4.4%) 7 (7.4%) 4.66 (0.99-21.89) 0.060
Patients gender
Male 105 (77.8%) 76 (80.0%) Ref.
Female 30 (22.2%) 19 (20.0%) 0.87 (0.46-1.66) 0.693
Area of occurence
Rural 108 (86.4%) 75 (85.2%) Ref.
Urban 17 (12.6%) 13 (14.8%) 1.10 (0.50-2.40) 0.806
Time to medical assistance (hours)
03 94 (73.3%) 68 (72.3%) Ref.
46 22 (16.9%) 15 (16.0%) 0.94 (0.45-1.94) 0.880
712 12 (9.2%) 6 (6.4%) 0.69 (0.24-1.93) 0.498
>12 2 (1.5%) 5 (5.3%) 3.45 (0.65-18.34) 0.151
Thrombocytopenia
Yes 9 (6.8%) 8 (9.2%) Ref.
No 123 (93.2%) 79 (90.8%) 0.72 (0.26-1.95) 0.529
Hemoglobin
Above normal values 19 (14.5%) 11 (12.8%) Ref.
Normal values 89 (67.9%) 66 (76.7%) 1.28 (0.57-2.87) 0.548
Below normal values 23 (17.6%) 9 (10.5%) 0.68 (0.23-1.97) 0.472
Snake gender
Male 33 (30.6%) 23 (29.1%) Ref.
Female 75 (69.4%) 56 (70.9%) 1.07 (1.57-2.02) 0.832
Snake age
Neonates 25 (19.4%) 10 (11.2%) Ref.
Juveniles 67 (51.9%) 40 (44.4%) 1.49 (0.65-3.43) 0.343
Adults 37 (28.7%) 40 (44.4%) 2.70 (1.15-6.37) 0.021
White tail tip
Yes 76 (57.6%) 37 (40.7%) Ref.
No 56 (42.4%) 54 (59.3%) 1.98 (1.15-3.41) 0.013
Feeding status
Fed 29 (21.7%) 29 (31.2%) Ref.
Unfed 103 (78.0%) 64 (68.8%) 0.62 (0.34-1.13) 0.125
Bold values represent statistical differences.
CLINICAL TOXICOLOGY 5
considering female snakes in reproductive age, as previously
observed for B. jararaca [29], they present more thermo-
regulatory activity, needed for the development of the
embryos, in open sunny places more often frequented by
humans.
Systemic bleeding is a hallmark of the Bothrops envenom-
ation, with unclottable blood on admission as its major risk
factor [32]. Interestingly, coagulotoxic components are the
most variable in the venom of B. atrox. A drastic ontogenetic
change was observed in the proteome of B. atrox in the
region of Orinoquia, Venezuela where juveniles present PIII-
class metalloproteinases as major toxins in their venoms
while PI-class metalloproteinases and phospholipase A2 pre-
dominate in the venom of adult specimens [5]. Functional
variability on the coagulant activity has been demonstrated
in different ontogenetic stage of the snake [33]. For instance,
adult snakes from Central Amazonia present a venom with
lower lethality but higher dermonecrotic activity while ven-
oms from snakes from Maranh~
ao are more coagulotoxic,
with higher lethal activity [34]. It has been accepted that pro-
coagulant activity is a fundamental feature of pit-viper ven-
oms for the purpose of prey capture by small snakes [35].
In prey animals, the rapid formation of endogenous
thrombin by Bothrops venom could result in prey incapacita-
tion through stroke induction [35]. Dissimilar patterns were
observed in the main clot parameters in animal plasmas as
models of amphibian, mammalian and avian potential preys
[35]. Specific venom effects are consistent with the
Figure 4. Comparison of morphometrical variables from Bothrops atrox snakes according to blood clottability. Calculated by T-test comparing two independent
samples, considering p<.05 for significance. C: clottable blood, U: unclottable blood.
6 J. C. C. BERNAL ET AL.
evolutionary history of B. atrox populations regarding selec-
tion pressure of prey availability in various ecological niches
and ontogenetic stages [6]. Young Bothrops snakes preferen-
tially eat amphibians, lizards and birds, shifting to mammals
when they become adults [17]. Therefore, change in B. atrox
venom proteome is most likely related to the survival of the
snake by adaptation to particular preys. Experimentally, ven-
oms from juveniles displayed higher human plasma clotting
activity compared to venoms from adult specimens [33].
Venoms of juvenile specimens of B. atrox from the
Colombian Amazon possess higher hemorrhagic and coagu-
lant activities than venoms from adults [36].
In contrast to coagulopathy, local tissue damage was
more prevalent in patients bitten by adult snakes. Local signs
of Bothrops envenomations range from a painless injury to
intense pain and swelling at the bite site, evolving in some
cases to blistering, tissue necrosis and compartment syn-
drome [13,37]. Tissue damage is caused by metalloprotei-
nases, which cause the hydrolysis of extracellular matrix
components and disruption of capillary vessels. Moreover,
metalloproteinases and phospholipase A2 induce up-regula-
tion of pro-inflammatory mediators expression enhancing tis-
sue damage [38,39]. Phospholipases A2, namely BaPLA2I and
BaPLA2III, that cause edema and myonecrosis, and a dermo-
necrotic metalloproteinase, namely Atroxlysin-Ia, have been
isolated from B. atrox venom [40].
In this study, patients bitten by adult B. atrox presented a
higher frequency of moderate to severe edema, confirming
Figure 5. ROC curves of the discriminatory power of the morphometrics and weight for clotting blood status classification. The only morphometric variable pre-
senting good diagnostic ability to classify clotting blood status was the LND for snakebites caused by male specimens (AUC ¼0.802 (95%CI 0.6760.928)). The
other variables presented poor diagnostic performance.
Figure 6. Time until reversion of blood clottability after hospital admission and antivenom administration, according to snake gender and age. Survival analysis
demonstrated that the time from patient admission to the unclottable blood reversion was not affected by the morphological features of the snake.
CLINICAL TOXICOLOGY 7
previous observations from B. jararaca envenomations
[1921]. Moreover, the only death observed in this study was
from a patient that presented severe local tissue damage
and no manifestation of coagulopathy, which was bitten by
an adult snake. This finding is interpreted as a result of the
higher expression of metalloproteinases and phospholipases
A2 in venoms from adult pit vipers [36,41].
Time from antivenom administration to the clotting activ-
ity restoration was not affected by the snake stage and the
antivenom was efficient for the treatment of B. atrox enveno-
mations although venom from juveniles is not used in the
immunization pool for antivenom production. In fact, previ-
ous in vitro studies showed a high efficacy of commercial
Bothrops antivenom to neutralize B. atrox venoms of different
populations, including from Manaus region [35], and the
antivenom efficacy was also observed in patients bitten by B.
atrox in Par
a State [13]. Once the antivenom is administered,
it effectively neutralizes the toxins of adult or juvenile snakes,
even considering the quantitative differences in the pro-
coagulant toxins from the venom, leading to a similar restor-
ation of coagulation parameters [6,34].
Conclusions
Venom-induced coagulopathy is more frequent in enveno-
mations caused by juvenile B. atrox, whereas envenomations
inflicted by adult snakes cause more severe local tissue dam-
age. These findings are attributed to venom ontogenetic
variability in B. atrox, since venoms of younger specimens
have higher procoagulant activity, whereas those of adults
exert stronger local tissue damage. Despite the observed
variation in the incidence of coagulopathy, clotting activity
restoration after antivenom administration was similar in the
two groups of patients.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was financially supported by Coordenac¸~
ao de
Aperfeic¸oamento de Pessoal de N
ıvel Superior.
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CLINICAL TOXICOLOGY 9
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... Once the physician is confident that envenoming can be ruled out and the patient can be discharged, the final diagnosis of a dry bite can be made. these cases require a careful examination of the patient's coagulation parameters, considering the predominance of coagulotoxins in the venom of these immature specimens, which may cause systemic bleeding [102,103]. Figure 2 shows the characteristics of a patient presenting mild, local manifestations and two true dry bites by snakes from the Bothrops genus. Once the physician is confident that envenoming can be ruled out and the patient can be discharged, the final diagnosis of a dry bite can be made. ...
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Lancehead pit-vipers (Bothrops genus) are an extremely diverse and medically important group responsible for the greatest number of snakebite envenomations and deaths in South America. Bothrops atrox (common lancehead), responsible for majority of snakebites and related deaths within the Brazilian Amazon, is a highly adaptable and widely distributed species, whose venom variability has been related to several factors, including geographical distribution and habitat type. This study examined venoms from four B. atrox populations (Belterra and Santarém, PA; Pres. Figueiredo, AM and São Bento, MA), and two additional Bothrops species (B. jararaca and B. neuwiedi) from Southeastern region for their coagulotoxic effects upon different plasmas (human, amphibian, and avian). The results revealed inter-and intraspecific variations in coagulotoxicity, including distinct activities between the three plasmas, with variations in the latter two linked to ecological niche occupied by the snakes. Also examined were the correlated biochemical mechanisms of venom action. Significant variation in the relative reliance upon the cofactors calcium and phospholipid were revealed, and the relative dependency did not significantly correlate with potency. Relative levels of Factor X or prothrombin activating toxins correlated with prey type and prey escape potential. The antivenom was shown to perform better in neutralising prothrombin activation activity than neutralising Factor X activation activity. Thus, the data reveal new information regarding the evolutionary selection pressures shaping snake venom evolution, while also having significant implications for the treatment of the envenomed patient. These results are, therefore, an intersection between evolutionary biology and clinical medicine.
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Significance: Analysis of individual transcripts and venom protein isoforms produced by specimens of a venomous snake (Bothrops atrox), from the Brazilian Amazon rainforest, revealed that transcriptional and translational mechanisms contribute to venom phenotypic variation. Our finding of evidence for high expression of toxin proteins with conserved function supports the hypothesis that the venom phenotype consists of two kinds of proteins: conserved "core function" proteins that provide essential functional activities with broader relevance and less conserved "adaptive" proteins that vary in expression and may permit customization of protein function. These observations allowed us to suggest that genetic mechanisms controlling venom variability are not restricted to selection of gene copies or mutations in structural genes but also to selection of the mechanisms controlling gene expression, contributing to the plasticity of this important phenotype for venomous snakes.
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Biological significance: In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (várzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not efficiently neutralized by Bothrops antivenom. Thus, using a functional proteomic approach, we highlighted intraspecific differences in B. atrox venom that could impact both in the ecology of snakes but also in the treatment of snake bite patients in the region.