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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:
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
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:
To link to this article:
View supplementary material Published online: 06 Aug 2019.
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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
ao Pedro Tavares Pereira
, Hiochelson Najibe dos
Santos Ibiapina
, Lybia K
assia Santos Sarraff
audio Monteiro-J
, Handerson da Silva Pereira
Bruno Santos
, Valeria Mour~
ao de Moura
amella Silva de Oliveira
, Marcus Lacerda
Vanderson Sampaio
, Igor Luis Kaefer
, Jos
ıa Guti
, Paulo S
ergio Bernarde
, Hui Wen Fan
Jacqueline Sachett
, Ana Maria Moura da Silva
and Wuelton Marcelo Monteiro
Escola Superior de Ci^
encias da Sa
ude, Universidade Do Estado Do Amazonas, Manaus, Brazil;
Instituto de Pesquisa Cl
ınica Carlos
Borborema, Fundac¸~
ao de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Brazil;
Programa de P
ao em Zoologia,
Universidade Federal Do Amazonas, Manaus, Brazil;
Instituto de Sa
ude Coletiva, Universidade Federal Do Oeste Do Par
a, Santar
em, Brazil;
Instituto Le^
onidas & Maria Deane, Manaus, Brazil;
Sala de Situac¸~
ao em Sa
ude, Fundac¸~
ao de Vigil^
ancia em Sa
ude Do Amazonas, Manaus,
Instituto Clodomiro Picado, Facultad de Microbiolog
ıa, Universidad de Costa Rica, San Jos
e, Costa Rica;
orio de
Herpetologia, Centro Multidisciplinar, Campus Floresta, Universidade Federal Do Acre, Cruzeiro Do Sul, AC, Brazil;
Instituto Federal Do Acre,
Campus de Cruzeiro Do Sul, Cruzeiro Do Sul, Acre, Brazil;
ucleo Estrat
egico de Venenos e Antivenenos, Instituto Butantan, S~
ao Paulo,
Diretoria de Ensino e Pesquisa, Fundac¸~
ao Alfredo da Matta, Manaus, Brazil
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.
Received 9 May 2019
Revised 17 July 2019
Accepted 20 July 2019
Published online 2 August
Bothrops atrox; snakebite
envenomation; snake
morphometrics; coagulop-
athy; antivenom
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
, 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 Escola Superior de Ci^
encias da Sa
ude, Universidade Do Estado Do Amazonas, Manaus,
Supplemental data for this article can be accessed here.
ß2019 Informa UK Limited, trading as Taylor & Francis Group
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.
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).
Clinico-epidemiological information was collected through a
standardized questionnaire (Epi Info
, 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
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).
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
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 %
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
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
(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
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
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
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].
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.
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.
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
Yes 9 (6.8%) 8 (9.2%) Ref.
No 123 (93.2%) 79 (90.8%) 0.72 (0.26-1.95) 0.529
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.
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
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.
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.
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].
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.
This study was financially supported by Coordenac¸~
ao de
Aperfeic¸oamento de Pessoal de N
ıvel Superior.
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... In South America, an important aspect of Bothrops envenomings is that bites by juvenile snakes can cause no local or very mild local manifestations, which can be misdiagnosed as dry bites. However, 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. ...
... 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. ...
Full-text available
Snake 'dry bites' are characterized by the absence of venom being injected into the victim during a snakebite incident. The dry bite mechanism and diagnosis are quite complex, and the lack of envenoming symptoms in these cases may be misinterpreted as a miraculous treatment or as proof that the bite from the perpetrating snake species is rather harmless. The circumstances of dry bites and their clinical diagnosis are not well-explored in the literature, which may lead to ambiguity amongst treating personnel about whether antivenom is indicated or not. Here, the epidemiology and recorded history of dry bites are reviewed, and the clinical knowledge on the dry bite phenomenon is presented and discussed. Finally, this review proposes a diagnostic and therapeutic protocol to assist medical care after snake dry bites, aiming to improve patient outcomes.
... Ontogenetic venom variation has been shown to affect envenomation syndromes. For example, coagulopathy was reportedly more pronounced for neonate envenomations by some Bothrops species (Bernal et al., 2020;Guercio et al., 2006;Milani Junior et al., 1997). Amongst the most extreme ontogenetic venom variation is that seen in the Australian elapid genus Pseudonaja (brown snakes), whereby neonates lack coagulotoxins present in adult venoms, being instead potently neurotoxic (Cipriani et al., 2017;Jackson et al., 2016). ...
Major variations in venom composition can occur between juvenile and adult venomous snakes. However, due to logistical constraints, antivenoms are produced using adult venoms in immunising mixtures, possibly resulting in limited neutralisation of juvenile snake venoms. Daboia russelii is one of the leading causes of snakebite death across South Asia. Its venom is potently procoagulant, causing stroke in prey animals but causing in humans consumptive coagulopathy—a net anticoagulant state—and sometimes death resulting from hemorrhage. In this in vitro study, we compared the venom activity of—and antivenom efficacy against—six 2-week-old D. russelii relative to that of their parents. Using a coagulation analyser, we quantified the relative coagulotoxicity of these venoms in human, avian, and amphibian plasma. The overall potency on human plasma was similar across all adult and neonate venoms, and SII (Serum Institute of India) antivenom was equipotent in neutralising these coagulotoxic effects. In addition, all venoms were also similar in their action upon avian plasma. In contrast, the neonate venoms were more potent on amphibian plasma, suggesting amphibians make up a larger proportion of neonate diet than adults. A similar venom potency in human and avian plasmas but varying selectivity for amphibian plasma suggests ontogenetic differences in toxin isoforms within the factor X or factor V activating classes, thereby providing a testable hypothesis for future transcriptomics work. By providing insights into the functional venom differences between adult and neonate D. russelii venoms, we hope to inform clinical treatment of patients envenomated by this deadly species and to shed new light on the natural history of these extremely medically important snakes.
... These proteomic differences have clinical consequences. Envenomings inflicted by adult B. atrox snakes cause more severe local inflammatory effects, whereas venom-induced coagulopathy is more frequent in envenomings caused by juvenile specimens [114]. Similarly, for an equal mass of venom from a small snake than a large one, more antivenom seems to be needed to correct coagulopathy [110]. ...
Full-text available
Toxins from Bothrops venoms targeting hemostasis are responsible for a broad range of clinical and biological syndromes including local and systemic bleeding, incoagulability, thrombotic microangiopathy and macrothrombosis. Beyond hemostais disorders, toxins are also involved in the pathogenesis of edema and in most complications such as hypovolemia, cardiovascular collapse, acute kidney injury, myonecrosis, compartmental syndrome and superinfection. These toxins can be classified as enzymatic proteins (snake venom metalloproteinases, snake venom serine proteases, phospholipases A2 and L-amino acid oxidases) and non-enzymatic proteins (desintegrins and C-type lectin proteins). Bleeding is due to a multifocal toxicity targeting vessels, platelets and coagulation factors. Vessel damage due to the degradation of basement membrane and the subsequent disruption of endothelial cell integrity under hydrostatic pressure and tangential shear stress is primarily responsible for bleeding. Hemorrhage is promoted by thrombocytopenia, platelet hypoaggregation, consumption coagulopathy and fibrin(ogen)olysis. Onset of thrombotic microangiopathy is probably due to the switch of endothelium to a prothrombotic phenotype with overexpression of tissue factor and other pro-aggregating biomarkers in association with activation of platelets and coagulation. Thrombosis involving large-caliber vessels in B. lanceolatus envenomation remains a unique entity, which exact pathophysiology remains poorly understood.
... Several studies have shown experimentally that in different species of viperid snakes, the venoms of juvenile individuals are more pro-coagulant than venoms from adults [57,[65][66][67]. The higher pro-coagulant activity of venoms from small snakes was also reported in human cases of snakebites [68,69]. The rapid blood clotting is related to a consumption coagulopathy characterized by the conversion of fibrinogen to fibrin and activation of coagulation factors, as well as interference in platelet function and fibrinolysis [70,71]. ...
Full-text available
Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.
... It commonly arises in the case of microvascular damage by proteolytic degradation of the basement membrane [21]. The involved enzymes are PI and P-III snake venom metalloproteinases (examples in the venom of B. atrox are Batroxase [22], Atroxlysin-Ia [23] and Batroxrhagin [24]). Systemic bleeding observed in snakebites was reported in 3.6-15.3% of patients [15,25]. ...
Full-text available
The management of snakebite (SB) envenoming in French Guiana (FG) is based on symptomatic measures and antivenom (AV) administration (Antivipmyn Tri®; Instituto Bioclon—Mexico). Our study aimed to assess clinical manifestations, the efficacy, and safety of Antivipmyn Tri® in the management of SB. Our study is a prospective observational work. It was conducted in the Intensive Care Unit (ICU) of Cayenne General Hospital between 1 January 2016 and 31 December 2019. We included all patients hospitalized for SB envenoming. Our study contained three groups (without AV, three vials, and six vials Antivipmyn Tri®). During the study period, 133 patients were included. The main clinical symptoms were edema (98.5%), pain (97.7%), systemic hemorrhage (18%), blister (14.3%), and local hemorrhage (14.3%). AV was prescribed for 83 patients (62.3%), and 17 of them (20%) developed early adverse reactions. Biological parameters at admission showed defibrinogenation in 124 cases (93.2%), International Normalized Ratio (INR) > 2 in 104 cases (78.2%), and partial thromboplastin time (PTT) > 1.5 in 74 cases (55.6%). The time from SB to AV was 9:00 (5:22–20:40). The median time from SB to achieve a normal dosage of fibrinogen was 47:00 vs. 25:30, that of Factor II was 24:55 vs. 15:10, that of Factor V was 31:42 vs. 19:42, and that of Factor VIII was 21:30 vs. 10:20 in patients without and with AV, respectively, (p < 0.001 for all factors). Patients receiving Antivipmyn Tri® showed a reduction in the time to return to normal clotting tests, as compared to those who did not. We suggest assessing other antivenoms available in the region to compare their efficacy and safety with Antivipmyn Tri® in FG.
... The search did not identify a study that has evaluated, validated or used the original Lee-White method in the diagnosis of VICC. However, a slightly modified Lee-White clotting time has been used in diagnosing VICC in Bothrops atrox-envenomed patients from Brazil [22,23]. This modified test is performed by placing 1 mL of venous blood in a glass tube and leaving it undisturbed for 5 min. ...
Full-text available
Venom-induced consumption coagulopathy is the most important systemic effect of snake envenoming. Coagulation tests are helpful to accurately and promptly diagnose venom-induced consumption coagulopathy and administer antivenom, which is the only specific treatment available. However, bedside clotting tests play a major role in diagnosing coagulopathy in low-income settings, where the majority of snakebites occur. We conducted a literature search in MEDLINE® from 1946 to 30 November 2019, looking for research articles describing clinical studies on bedside coagulation tests in snakebite patients. Out of 442 articles identified, 147 articles describing bedside clotting assays were included in the review. Three main bedside clotting tests were identified, namely the Lee-White clotting test, 20-min whole blood clotting time and venous clotting time. Although the original Lee-White clotting test has never been validated for snake envenoming, a recently validated version has been used in some South American countries. The 20-min whole blood clotting time test is the most commonly used test in a wide range of settings and for taxonomically diverse snake species. Venous clotting time is almost exclusively used in Thailand. Many validation studies have methodological limitations, including small sample size, lack of case-authentication, the inclusion of a heterogeneous mix of snakebites and inappropriate uses of gold standard tests. The observation times for bedside clotting tests were arbitrary, without proper scientific justification. Future research needs to focus on improving the existing 20-min whole blood clotting test, and also on looking for alternative bedside coagulation tests which are cheap, reliable and quicker.
... Differences have been observed in the coagulant activity of venom from B. atrox snakes from distinct locations [21,22], habitats [23], and at different ontogenetic stages [24,25], as well as when compared to the venom of other Bothrops species [26]. There is little information regarding how clotting factors and other components are affected in Bothrops snakebite patients in the Amazon who evolve to systemic or local bleeding. ...
Full-text available
Bleeding is a common hemostatic disorder that occurs in Bothrops envenomations. We evaluated the changes in coagulation, fibrinolysis components, and platelets in Bothrops atrox envenomations with bleeding. This is an observational study with B. atrox snakebite patients (n = 100) treated in Manaus, Brazilian Amazon. Bleeding was recorded on admission and during hospitalization. We found that the platelet count in our patients presented a weak correlation to tissue factor, factor II, and plasminogen. Tissue factor presented weak correlation to factor V, II, D-dimer, plasminogen, alpha 2-antiplasmin, and moderate correlation to fibrinogen and fibrin/fibrinogen degradation product (FDP). Patients with systemic bleeding (n = 20) presented low levels of factor V, II, fibrinogen, plasminogen, and alpha 2-antiplasmin, and high levels of tissue factor and FDP compared to those without bleeding. Patients with only local bleeding (n = 41) and without bleeding showed similar levels of hemostatic factors. Thrombocytopenia was observed mainly in patients with systemic bleeding and increased levels of serum venom. No association was found between venom levels and systemic bleeding, or between venom levels and clinical severity of envenomation. This is the first report that shows the participation of the extrinsic coagulation pathway in the consumption coagulopathy of B. atrox envenomations with systemic bleeding due to tissue factor release.
Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B. jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin by as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes’ venoms acted quicker than large snakes’ venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscores the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.
Full-text available
Access to antivenoms is not guarranteed for vulnerable populations that inhabit remote areas in the Amazon. The study of therapeutic itineraries (TI) for treatment of snakebites would support strategies to provide timely access to users. A TI is the set of processes by which individuals adhere to certain forms of treatment, and includes the path traveled in the search for healthcare, and practices to solve their health problems. This study aims to describe TIs of snakebite patients in the Brazilian Amazon. This study was carried out at the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado , in Manaus, state of Amazonas, Brazil. The itinerary from the moment of the bite to the patient’s admission to the reference unit was analyzed. Sample size was defined by saturation. After an exploratory survey to collect epidemiological variables, in-depth interviews were conducted following a semi-structured guide. Patients originated from rural areas of 11 different municipalities, including ones located >500 kilometers from Manaus. A great fragmentation was observed in the itineraries, marked by several changes of means of transport along the route. Four themes emerged from the analysis: exposure to snakebite during day-to-day activities, use of traditional therapeutic practices, and personal perception of the severity, as well as the route taken and its contingencies. Access to healthcare requires considerable effort on the part of snakebite patients. Major barriers were identified, such as the low number of hospitals that offer antivenom treatment, poor access to healthcare due to long distances and geographic barriers, low acceptability of healthcare offered in countryside, lack of use of personal protective equipment, common use of ineffective or deleterious self-care practices, late recognition of serious clinical signs and resistance to seeking medical assistance. Health education, promotion of immediate transport to health centers and decentralization of antivenom from reference hospitals to community healthcare centers in the Brazilian Amazon are more effective strategies that would to maximize access to antivenom treatment.
In the Amazon, around 90% of snakebites are caused by the Bothrops genus. Complications arising from Bothrops envenomations result from the inflammatory and coagulotoxic activities of the venom. The aim of this study was to investigate the potential of cell-free DNA (cfDNA) as a biomarker of severity in Bothrops snakebites. Patients were treated at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), Manaus, in the Brazilian Amazon. cfDNA plasma levels were measured by amplifying the human telomerase reverse transcriptase (hTERT) sequence using quantitative RT-PCR. Median levels of cfDNA were compared between envenomed and healthy volunteers and among patients presenting different complications, such as renal failure, bleeding and infection. Of the 76 patients included, 82.9% were male, with a mean age of 32.8 years, and envenomations were mainly classified as severe (39.5%). ROC curve analysis showed a good accuracy of cfDNA levels (AUROC of 0.745) in envenomation diagnosis. A correlation analysis using laboratory variables showed positive correlation with lactate dehydrogenase (p=0.033) and platelet count (p=0.003). When cfDNA levels were compared with clinical complications, significant statistical differences were only found among individuals with mild and severe pain (p<0.05). In summary, our results demonstrated that cfDNA levels are sufficiently accurate for discriminating between envenomed and non-envenomed patients, but are not able to distinguish different complications and the level of severity among envenomed patients. Thus, the role of cfDNA in the pathogenesis of the snakebite envenomations needs to be further investigated.
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ABSTRACT The pitviper Bothrops atrox is a widely distributed and medically important snake in Amazonia. Given that the reproductive and feeding biology of snakes varies geographically, we aimed to characterize the natural history of individuals from the central and southwestern portions of the biome. We analyzed museum-preserved specimens collected in the region between the metropolitan areas of the Brazilian cities of Manaus and Porto Velho. We took morphometric measurements and performed the analysis of gonads and digestive tracts of 109 individuals. Females attain sexual maturity with larger body sizes. Adults of the species show pronounced sexual dimorphism in body size, with females being larger. The reproductive cycle, especially in males, appears to be synchronized with regional rainfall, which markedly differed across the biome. The pitvipers had a generalist diet, consuming a wide range of prey such as centipedes, frogs, lizards, snakes and mammals. We provide the first record of predation of the snake Imantodes cenchoa (Dipsadidae) by B. atrox. There was an ontogenetic shift in the diet: smaller individuals fed mainly on ectothermic prey (centipedes, frogs, lizards and snakes), while adults tended to incorporate endothermic prey (mammals) to their food repertoire.
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Bothrops snakebites usually present systemic bleeding, and the clinical-epidemiological and laboratorial factors associated with the development of this manifestation are not well established. In this study, we assessed the prevalence of Bothrops snakebites with systemic bleeding reported at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, in Manaus, Amazonas State, Brazil, and the clinical-epidemiological and laboratorial factors associated with systemic bleeding. This is an observational, cross-sectional study carried out between August, 2013 and July, 2016. Patients who developed systemic bleeding on admission or during hospitalization were considered cases, and those with non-systemic bleeding were included in the control group. Systemic bleeding was observed in 63 (15.3%) of the 442 Bothrops snakebites evaluated. Bothrops snakebites mostly occurred in males (78.2%), in rural areas (89.0%) and in the age group of 11 to 30 years old (40.4%). It took most of the patients (59.8%) less than 3 h to receive medical assistance. Unclottable blood (AOR = 3.11 (95% CI = 1.53 to 6.31; p = 0.002)) and thrombocytopenia (AOR = 4.52 (95% CI = 2.03 to 10.09; p < 0.001)) on admission were independently associated with systemic bleeding during hospitalization. These hemostatic disorders on admission increase the chances of systemic bleeding during hospitalization. Prospective studies are needed to clarify the pathophysiology of systemic bleeding in Bothrops snakebites in the Amazon region. Key Contribution: Systemic bleeding observed after Bothrops snakebites in the Brazilian Amazon is an important systemic effect of the envenomation. Thrombocytopenia and unclottable blood on admission are factors associated with systemic bleeding during hospitalization.
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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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Snake envenomation is a major public health problem in Brazil. Systemic complications that may arise from snakebites are mainly related to coagulopathy. The Lee-White clotting time (LWCT) is a simple and inexpensive test and available even in remote health facilities. However, the diagnostic value of such test needs to be evaluated to accurately diagnose coagulopathy in the clinical practice. This study aimed to assess the reliability of the LWCT performed in hospital routine to diagnose venom-induced coagulopathy. We studied 186 patients admitted at the Tropical Medicine Foundation Dr. Heitor Vieira Dourado in Manaus, Amazonas, Brazil, withBothropsenvenomation diagnosis. At admission, blood samples were collected for performing LWCT and the concentration of fibrinogen. Sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratios, diagnostic odds ratio, and accuracy were calculated with 95% confidence intervals. From the total, 85.5% had hypofibrinogenemia. The sensitivity of the LWCT to the diagnosis of hypofibrinogenemia was 78.0% and the specificity 40.7%. The accuracy of the test was 72.6%, and patients with a prolonged LWCT had 2.4 higher odds of developing hypofibrinogenemia. In addition, the LWCT was also compared with venom antigen levels and systemic hemorrhage. The LWCT showed moderate sensitivity to detect consumption coagulopathy and constitutes a valuable tool for the diagnosis ofBothropssnake envenomation and indication of antivenom therapy.
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Hemorrhage is the most prominent effect of snake venom metalloproteinases (SVMPs) in human envenomation. The capillary injury is a multifactorial effect caused by hydrolysis of the components of the basement membrane (BM). The PI and PIII classes of SVMPs are abundant in viperid venoms and hydrolyze BM components. However, hemorrhage is associated mostly with PIII-class SVMPs that contain non-catalytic domains responsible for the binding of SVMPs to BM proteins, facilitating enzyme accumulation in the tissue and enhancing its catalytic efficiency. Here we report on Atroxlysin-Ia, a PI-class SVMP that induces hemorrhagic lesions in levels comparable to those induced by Batroxrhagin (PIII-class), and a unique SVMP effect characterized by the rapid onset of dermonecrotic lesions. Atroxlysin-Ia was purified from B. atrox venom, and sequence analyses indicated that it is devoid of non-catalytic domains and unable to bind to BM proteins as collagen IV and laminin in vitro or in vivo. The presence of Atroxlysin-Ia was diffuse in mice skin, and localized mainly in the epidermis with no co-localization with BM components. Nevertheless, the skin lesions induced by Atroxlysin-Ia were comparable to those induced by Batroxrhagin, with induction of leukocyte infiltrates and hemorrhagic areas soon after toxin injection. Detachment of the epidermis was more intense in skin injected with Atroxlysin-Ia. Comparing the catalytic activity of both toxins, Batroxrhagin was more active in the hydrolysis of a peptide substrate while Atroxlysin-Ia hydrolyzed more efficiently fibrin, laminin, collagen IV and nidogen. Thus, the results suggest that Atroxlysin-Ia bypasses the binding step to BM proteins, essential for hemorrhagic lesions induced by PII- and P-III class SVMPs, causing a significantly fast onset of hemorrhage and dermonecrosis, due to its higher proteolytic capacity on BM components.
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Background: Secondary bacterial infections from snakebites contribute to the high complication rates that can lead to permanent function loss and disabilities. Although common in endemic areas, routine empirical prophylactic use of antibiotics aiming to prevent secondary infection lacks a clearly defined policy. The aim of this work was to estimate the efficacy of amoxicillin clavulanate for reducing the secondary infection incidence in patients bitten by Bothrops snakes, and, secondarily, identify risk factors for secondary infections from snakebites in the Western Brazilian Amazon. Methods and findings: This was an open-label, two-arm individually randomized superiority trial to prevent secondary infection from Bothrops snakebites. The antibiotic chosen for this clinical trial was oral amoxicillin clavulanate per seven days compared to no intervention. A total of 345 patients were assessed for eligibility in the study period. From this total, 187 accomplished the inclusion criteria and were randomized, 93 in the interventional group and 94 in the untreated control group. All randomized participants completed the 7 days follow-up period. Enzyme immunoassay confirmed Bothrops envenoming diagnosis in all participants. Primary outcome was defined as secondary infection (abscess and/or cellulitis) until day 7 after admission. Secondary infection incidence until 7 days after admission was 35.5% in the intervention group and 44.1% in the control group [RR = 0.80 (95%CI = 0.56 to 1.15; p = 0.235)]. Survival analysis demonstrated that the time from patient admission to the onset of secondary infection was not different between amoxicillin clavulanate treated and control group (Log-rank = 2.23; p = 0.789).Secondary infections incidence in 7 days of follow-up was independently associated to fibrinogen >400 mg/dL [AOR = 4.78 (95%CI = 2.17 to 10.55; p<0.001)], alanine transaminase >44 IU/L [AOR = 2.52 (95%CI = 1.06 to 5.98; p = 0.037)], C-reactive protein >6.5 mg/L [AOR = 2.98 (95%CI = 1.40 to 6.35; p = 0.005)], moderate pain [AOR = 24.30 (95%CI = 4.69 to 125.84; p<0.001)] and moderate snakebites [AOR = 2.43 (95%CI = 1.07 to 5.50; p = 0.034)]. Conclusions/significance: Preemptive amoxicillin clavulanate was not effective for preventing secondary infections from Bothrops snakebites. Laboratorial markers, such as high fibrinogen, alanine transaminase and C-reactive protein levels, and severity clinical grading of snakebites, may help to accurately diagnose secondary infections. Trial registration: Brazilian Clinical Trials Registry (ReBec): RBR-3h33wy; UTN Number: U1111-1169-1005.
Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists.
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.
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.