Transactions of the Royal Society of Tropical Medicine and Hygiene (2006) 100, 1173—1182

available at www.sciencedirect.com

journal homepage: www.elsevierhealth.com/journals/trst

Efficacy and safety of two whole IgG polyvalent

antivenoms, refined by caprylic acid fractionation
with or without ␤-propiolactone, in the treatment
of Bothrops asper bites in Colombia
Rafael Otero a,∗, Guillermo León b, José Marı́a Gutiérrez b, Gustavo Rojas b,
Marı́a Fabiola Toro a, Jacqueline Barona a, Verónica Rodrı́guez a, Abel Dı́az a,
Vitelbina Núñez a, Juan Carlos Quintana a, Shirley Ayala c, Diana Mosquera c,
Lesdy L. Conrado c, Diego Fernández d, Yobana Arroyo d, Carlos A. Paniagua e,
Mercedes López e, Carlos E. Ospina f, Claudia Alzate f, Jorge Fernández g,
Jazmı́n J. Meza g, Juan F. Silva g, Patricia Ramı́rez g, Patricia E. Fabra g,
Eugenio Ramı́rez h, Elkin Córdoba h, Ana B. Arrieta h, David A. Warrell i,
R. David G. Theakston j

a
Programa de Ofidismo/Escorpionismo, Facultad de Medicina, Universidad de Antioquia, A.A. 1226, Medellı́n, Colombia
b
Instituto Clodomiro Picado, Facultad de Microbiologı́a, Universidad de Costa Rica, San José, Costa Rica
c
Hospital San Francisco de Ası́s, Quibdó, Colombia
d
Hospital San Sebastián, Necoclı́, Colombia
e
Hospital Francisco Valderrama, Turbo, Colombia
f
Hospital Marı́a Auxiliadora, Chigorodó, Colombia
g
Hospital La Anunciación, Mutatá, Colombia
h
Hospital César Uribe Piedrahita, Caucasia, Colombia
i
Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 9DU, UK
j
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK

Received 24 October 2005; received in revised form 26 December 2005; accepted 4 January 2006
Available online 12 May 2006

KEYWORDS Summary The efficacy and safety of two whole IgG polyvalent antivenoms (A and B) were com-
Bothrops asper; pared in a randomised, blinded clinical trial in 67 patients systemically envenomed by Bothrops
Envenomation; asper in Colombia. Both antivenoms were fractionated by caprylic acid precipitation and had
Serotherapy similar neutralising potencies, protein concentrations and aggregate contents. Antivenom B was
additionally treated with ␤-propiolactone to lower its anticomplementary activity. Analysing all

∗ Corresponding author. Fax: +57 4 263 1914.

E-mail address: rafaotero@epm.net.co (R. Otero).

0035-9203/$ — see front matter © 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.trstmh.2006.01.006
1174 R. Otero et al.

treatment regimens together, there were no significant differences between the two antiven-
Caprylic acid;
␤-Propiolactone; oms (A = 34 patients; B = 33 patients) in the time taken to reverse venom-induced bleeding and
Anticomplementary coagulopathy, to restore physiological fibrinogen concentrations and to clear serum venom antige-
activity; naemia. Blood coagulability was restored within 6—24 h in 97% of patients, all of whom had normal
Colombia coagulation and plasma fibrinogen levels 48 h after the start of antivenom treatment. Two patients
(3.0%) had recurrent coagulopathy and eight patients suffered recurrence of antigenaemia within
72 h of treatment. None of the dosage regimens of either antivenom used guaranteed resolution
of venom-induced coagulopathy within 6 h, nor did they prevent recurrences. A further dose of
antivenom at 6 h also did not guarantee resolution of coagulopathy within 12—24 h in all patients.
The incidence of early adverse reactions (all mild) was similar for both antivenoms (15% and 24%;
P > 0.05).
© 2006 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights
reserved.

1. Introduction ity. Bites by snakes of the genera Porthidium and Both-

riechis cause less serious but similar clinical effects to those
described above. Porthidium spp. venom is devoid of a defib-
Every year in Colombia, 90—95% of the 3000 snakebites
rinating effect (Otero et al., 1992a, 1992b, 1996, 1999,
reported are caused by pit vipers of the genera Bothrops,
2002a, 2002b; Otero-Patiño et al., 1998; Warrell, 2004).
Porthidium and Bothriechis. Most bites occur in Antioquia
Three randomised clinical trials have been performed in
and Chocó (Figure 1), where there are 500—700 cases annu-
Antioquia and Chocó in patients bitten by Bothrops spp.
ally, mostly caused by Bothrops species. These are associ-
(mainly B. asper) using antivenoms from Brazil, Costa Rica
ated with a high case fatality (5—9%) and sequelae (6—10%)
(Otero et al., 1992a, 2001). Bothrops asper (Figure 2A), a
species widely distributed in areas of tropical rain forest
up to 1200 m above sea level, is responsible for 50—70%
of the bites. The venom of this species induces conspic-
uous local effects (oedema, haemorrhage, blistering, der-
monecrosis, myonecrosis) (Figure 2B) and life-threatening
systemic effects such as defibrination, haemorrhage distant
from the bite site, coagulopathy, shock and nephrotoxic-

Figure 2 (A) Bothrops asper from Antioquia (photo by D. War-

Figure 1 Study area. Antioquia: 1 = Necoclı́; 2 = Turbo; rell). (B) Severe envenomation caused by B. asper with intense
3 = Chigorodó; 4 = Mutatá; 5 = Caucasia; Chocó: 6 = Quibdó. swelling, blisters and necrosis (photo by R. Otero).
Whole IgG polyvalent antivenoms for Bothrops asper bites in Colombia 1175

Table 1 Grade of envenomation and specific treatment

Grade of envenomation Clinical featuresa Treatment (no. of vials)

Phase I (n = 30)b Phase II (n = 37)c

Group I Group II
(n = 15) (n = 15)

Mild Incoagulable blood, local swelling involving one or two 12 12 + 3 3, 4 or 5

segments of the bitten limb, no systemic bleeding and no
local necrosis
Moderate Incoagulable blood and local swelling involving three 12 12 + 3 6, 8 or 10
segments of the bitten limb, local or systemic bleeding, no
hypotension and no local necrosis
Severe Incoagulable blood and local necrosis or local swelling 12 12 + 3 10
extending beyond the bitten limb, local and systemic
bleeding and/or hypotension, renal failure, disseminated
intravascular coagulation or cerebral haemorrhage
n: number of patients.
a Skin or conjunctival sensitivity tests were not performed. Antihistamines or other pre-medications were not used and all patients were

regarded as potentially reactive to antivenom therapy.

b During phase I, all patients received an initial dose of 12 vials (antivenom A or B, randomly assigned), independent of the grade of

envenomation on admission. Group II patients also received one additional dose of three vials at 6 h.
c During phase II, patients were stratified into three groups (mild envenomation, n = 12; moderate envenomation, n = 19; severe enven-

omation, n = 6) and the antivenom dose was randomly assigned according to the grade of envenomation on admission.

and Colombia. It was observed that at doses of two to three
vials (20—30 ml) for mild, four to six vials (40—60 ml) for
moderate and six to nine vials (60—90 ml) for severe enven-
oming (severity evaluated on admission), bleeding stopped
in 80% of patients and blood coagulation status was restored
in 30—50% of them within 6 h. Bleeding ceased within 12 h
and blood coagulation was restored within 24 h in all patients
(Otero et al., 1996, 1999; Otero-Patiño et al., 1998). On
the other hand, in patients bitten by other pit viper species
from South America and Southeast Asia, blood coagulation
was restored within 6 h of treatment in 67—96% of patients
using antivenom doses of 2 to 12 vials according to the sever-
ity of envenoming determined on admission (Cardoso et al.,
1993; Ho et al., 1986, 1990; Jorge et al., 1995; Pardal et
al., 2004; Smalligan et al., 2004). Normal plasma fibrinogen
concentrations were restored within 6—24 h.
Early antivenom reactions (EAR) develop in a number of
patients treated with antivenom. The incidence of these
reactions varies greatly between different products (Lalloo
and Theakston, 2003). It has been suggested that the anti-
complementary activity of antivenoms, probably caused by
the presence of aggregates of IgG fragments, plays a key role
in the development of such adverse reactions (Lalloo and
Theakston, 2003; Malasit et al., 1986; Sutherland, 1977).
Therefore, the use of improved fractionation protocols may
result in a decrease in EARs. A recent experimental study
showed that treatment of equine antivenom antibodies with
␤-propiolactone resulted in a significant reduction in their
in vitro anticomplementary activity (León et al., 2005). It is
therefore important to assess whether such treatment also
reduces EARs in patients.
Here we describe a randomised, blinded, comparative
clinical trial of two whole IgG antivenoms in patients system-
ically envenomed by B. asper. The aims of this investigation
were: (1) to determine whether the administration of one
relatively large initial dose of equine whole IgG antivenom
would stop local and systemic bleeding and restore blood
coagulation completely in all patients within 6 h of treat-
ment and prevent later recurrences of local and systemic
envenomation. This initial dose would be based on the sever-
ity of envenomation (Table 1), the median effective dose
(ED50 ) in mice, the results of three previous clinical trials
performed in this region with antivenoms of similar poten-
cies from Costa Rica (see above) and the calculated maxi-
mum amount of venom injected by a bite; (2) if objective
(1) was unsuccessful, to assess whether one additional dose
of three vials of the antivenom, administered 6 h after the
onset of antivenom therapy, would achieve a cure; and (3)
to determine whether the addition of ␤-propiolactone after
fractionation of horse plasma reduced the frequency of EARs
in caprylic acid-fractionated whole IgG antivenoms.
2. Patients and methods
2.1. Animals, venoms and antivenoms
Swiss Webster mice (18—20 g body weight) were used for
in vivo experiments. The venom used was a pool obtained
from more than 40 adult specimens of B. asper collected
in Antioquia and Chocó and maintained in the serpentarium
of the Universidad de Antioquia. Venom was lyophilised and
stored at −20 ◦ C until used. The project was approved by
the animal ethics committee of the university.
Two batches (3450402 LQ-A and 3450402 LQ-B, both with
expiry dates of April 2005) of the commercial equine poly-
valent antivenom (antibothropic, anticrotalic, antilachetic)
from the Instituto Clodomiro Picado, San José, Costa Rica,
1176
were produced by immunising horses subcutaneously with
increasing amounts (0.75—45.0 mg) of equal parts of the
venoms of B. asper, Crotalus durissus durissus and Lachesis
stenophrys from Costa Rica at intervals of 10 days (Angulo
et al., 1997; Otero et al., 1997). Plasma was fractionated by
caprylic acid precipitation of non-Ig proteins as described by
Rojas et al. (1994), but one of the two batches was addition-
ally treated with ␤-propiolactone (León et al., 2005). The
median effective doses (ED50 ) of these antivenoms against
the lethal effect of B. asper venom from Costa Rica and
Colombia were assessed in mice by intraperitoneal injec-
tion following the Spearman—Karber method (WHO, 1981)
and results were analysed by the computer program Toxi-
calc (Robles and Gene, 1990). Additionally, antibody titres
against B. asper venom were determined by ELISA (León et
al., 2005).
Additional pre-clinical experimental studies on the
antivenoms included determination of: protein concentra-
tion using the Biuret method (Schosinsky et al., 1983); tur-
bidity by recording the absorbance at 590 nm (Rojas et al.,
1994); protein aggregate content by fast protein liquid chro-
matography (FPLC) using a Superdex 200 column (Pharma-
cia, Uppsala, Sweden) (Otero et al., 1999); and anticom-
plementary activity using the complement haemolytic assay
(León et al., 2005; Otero et al., 1999).
Antivenoms that fulfilled all the quality control require-
ments established at the Instituto Clodomiro Picado were
dispensed in 10 ml vials and packaged in cardboard boxes
containing a variable number of vials (depending on the
phase of the study) labelled either A or B and were randomly
assigned to the patients. Medical, nursing and laboratory
personnel involved in the study or in the analysis of results
were unaware of the identity of the antivenoms labelled A
and B. The code was kept at the Production Division of Insti-
tuto Clodomiro Picado in a sealed envelope kept in a locked
box; the code was broken only after statistical analysis of the
final results of the research had been completed. Antivenom
A was fractionated by caprylic acid precipitation, whereas
antivenom B was fractionated by caprylic acid precipitation
and treated with ␤-propiolactone.
2.2. Epidemiological and clinical data
Patients were enrolled in the study if: (1) they had suffered
a confirmed (dead snake reliably identified taxonomically)
or suspected (snake seen and described but not captured) B.
asper bite; (2) they arrived at the local hospital of the towns
of Caucasia, Chigorodó, Mutatá, Turbo, Necoclı́ and Quibdó
(Figure 1) less than 48 h after the bite; (3) they presented
with incoagulable blood on admission; and (4) they had not
received antivenom before arrival at the hospital. Epidemi-
ological studies carried out in the region suggest that there
are no bites caused by Bothriechis schlegelii in the six towns
selected for this study (Otero et al., 1992a, 2001). The Insti-
tuto Nacional de Vigilancia de Medicamentos y Alimentos
(INVIMA) of the Colombian Ministry of Health approved the
protocol of the study and the antivenoms used in October
2002 (Record of Proceedings No. 28) before the start of the
study and after the approval of the human ethics committee
of the University of Antioquia and of the hospitals involved
in the research.
R. Otero et al.
Informed consent was obtained from all patients or their
relatives. Data recorded included age, site of the bite,
and time between bite, admission and start of antivenom
therapy; any treatment by traditional healers; length of
the snake; local and systemic signs of envenoming; results
of haematological, bacteriological and clinical chemistry
laboratory tests and diagnostic imaging; blood coagulation
(the 20 min whole blood clotting test) (Ho et al., 1986;
Sano-Martins et al., 1994; Warrell, 1995); plasma fibrino-
gen concentration (normal range 146—380 mg/dl) using a
colorimetric assay (Fibriquik reactive; bioMerieux, Durham,
NC, USA) on admission and at 6, 12, 24, 48, 72 and 96 h
after antivenom therapy; dose and type of antivenom (A or
B) administered and ancillary treatments (e.g. antibiotics,
surgery); and complications and outcome.
2.3. Clinical assessment of envenoming and
treatment
Patients were divided into three arbitrary groups (mild,
moderate and severe envenomation on admission) follow-
ing previously described criteria (Otero-Patiño et al., 1998;
Table 1) and were randomised into two groups according
to the antivenom administered (A or B). During the first 6
months of the study (phase I), patients were also randomly
allocated into two groups (I and II), both receiving one rela-
tively large initial dose (12 vials) of antivenom, hoped to be
sufficient to neutralise permanently all the injected venom,
but with patients in group II receiving an additional dose of
three vials of the same antivenom at 6 h. During phase II (9
months), patients received different antivenom doses (three
to ten vials) according to the envenoming grade on admis-
sion (Table 1). In addition, during phase II patients bitten by
B. asper whose total length exceeded 1 m were treated as
severe cases, independently of the grade of envenomation
on admission, if treatment was administered within the first
6 h of the bite. This was done because of the statistical asso-
ciation (P < 0.001) demonstrated in previous studies between
a bite by a B. asper adult specimen and the presence of
local necrosis (Otero et al., 1999, 2002a; Otero-Patiño et
al., 1998).
Skin or conjunctival sensitivity tests were not performed
because they have no predictive value for early anaphy-
lactoid reactions (Cupo et al., 1991; Malasit et al., 1986;
Warrell, 1995). Prophylaxis with antihistamines or other pre-
medications was not used and all patients were regarded
as potentially reactive to antivenom therapy and so were
closely observed for 24 h, with special attention being paid
during the first 2 h after the start of the antivenom infu-
sion, when EARs are most likely to occur (Otero et al.,
1996, 1999; Otero-Patiño et al., 1998). EARs were treated
conventionally using adrenaline, corticosteroids and anti-
histamines (Fan and França, 1992; Otero-Patiño et al.,
1998).
Cessation of local and systemic haemorrhage within the
first 6 h of treatment and permanent recovery of blood coag-
ulability no later than 6 h after the onset of serotherapy
were considered as the criteria of efficacy of the initial
antivenom dose (Warrell, 1995) during phase II of the study.
If these criteria were not fulfilled, an additional dose of
three vials of the same antivenom was administered.
Whole IgG polyvalent antivenoms for Bothrops asper bites in Colombia
2.4. Measurement of serum venom and antivenom
concentrations
Serum venom and antivenom concentrations were deter-
mined using the ELISA technique of Theakston et al. (1977,
1992) with minor modifications (Ho et al., 1986; Otero et
al., 1996). Blood samples (3 ml) were collected in clean,
dry, glass tubes on admission and at 1, 6, 12, 24, 48, 72 and
96 h after the onset of serotherapy. Once obtained, serum
was stored at —20 ◦ C until used. Results were expressed as
microlitres of antivenom per millilitre of serum with ref-
erence to two standard curves prepared with known con-
centrations of horse IgG (venom-specific and non-specific)
obtained from both antivenoms (A and B), ranging from
0.8 ␮l/ml to 50 ␮l/ml, using as diluent a 1:1000 dilution of
normal human serum from 113 healthy donors of the same
socioeconomic group from the study region. Admission sam-
ples were taken as individual controls for the antivenom
assay.
The baseline for the venom assay was determined by
assaying the 113 serum samples of healthy donors with
reference to a standard curve prepared with known con-
centrations of B. asper venom. Absorbance at 492 nm was
0.081 ± 0.029 (mean ± SD). Using 0.14 as a negative cut-off
value (mean + 2 SD), 6 of the 113 normal serum were posi-
tive. The specificity was 94.7% and the lower limit for venom
detection was 5.8 ng/ml.
2.5. Statistical analysis
A data file was prepared for the information on the
patients and the analysis was performed using STATISTICA
98 program (StatSoft Inc., Tulsa, OK, USA). The associa-
tion between qualitative variables was established by the
Freeman—Halton test and the computer program StatXact
4.0. Quantitative variables were compared by Student’s
t-test. The mean levels of venom and antivenom were
analysed by one-way ANOVA on admission time and were
compared by the Newman—Keuls test. The mean levels at
later times were compared by repeated measure multi-
variate analysis. Differences were considered significant at
P < 0.05.
3. Results
3.1. Characterisation of antivenoms
The two antivenoms were similar in protein concentration,
aggregate content, neutralising potency (ED50 ) and ELISA
antibody titres against B. asper venom from Costa Rica
(Table 2) and Colombia (ED50 = 4.0 mg venom/ml antivenom
for both antivenoms). However, antivenom B treated with ␤-
propiolactone had significantly lower (P < 0.05) turbidity and
anticomplementary activity than antivenom A fractionated
with caprylic acid only (Table 2).
3.2. Epidemiological and clinical data
From April 2003 to July 2004, 96 patients with snake bites
sought medical attention at the six hospitals, however 29 of
1177
Table 2 Features of the antivenoms
Parameter Antivenoma
A B
Protein (g/dl) 4.18 ± 0.13 3.88 ± 0.12
Aggregates (%)b 2.8 ± 0.4 3.2 ± 0.5
Turbidity (A590 )c 0.045 ± 0.002* 0.021 ± 0.003*
Antibody titre 1.408 ± 0.043 1.343 ± 0.058
(A492 )d
Potency (ED50 )e 2.71 (2.21—3.32) 3.0 (2.38—3.78)
Anticomplementary 42 ± 7* 10 ± 1*
activityf
a A = fractionated by caprylic acid precipitation; B = fractionated
by caprylic acid precipitation and treated with ␤-propiolactone.
b Percentage of antivenom protein corresponding to high molec-
ular mass aggregates, as judged by fast protein liquid chromatog-
raphy gel filtration.
c Turbidity was assessed by determining the absorbance at
590 nm of undiluted antivenom samples.
d Tested against Bothrops asper venom by ELISA. Results cor-
respond to the absorbance at 492 nm in samples in which
antivenom was diluted 1:9000.
e Median effective dose (mg venom/ml antivenom) against B.
asper venom from Costa Rica. Results in parentheses are 95% CI.
f Anticomplementary activity is expressed as the reciprocal of
the antivenom dilution that reduced haemolytic activity of com-
plement by 50%. Results expressed as mean ± SD (n = 3).
* P < 0.05 by Student’s t-test.
them did not fulfil the criteria to be included in the research.
Thus, 67 patients with B. asper bites (44 confirmed taxonom-
ically and 20 of 23 suspected B. asper bites confirmed by
ELISA = 96% confirmation) were recruited in the clinical trial
(20 in Quibdó, 15 in Necoclı́, 15 in Mutatá, 10 in Caucasia, 6
in Turbo and 1 in Chigorodó). Thirteen (19.4%) patients were
children ranging from 2 to 14 years old; 47 (70.1%) patients
were male; 42 (62.7%) patients sought medical treatment
within 6 h of the bite, 11 (16.4%) within the first hour and 8
(11.9%) within the second hour. Thirty-four patients received
antivenom A and 33 received antivenom B. The two groups
were similar (P > 0.05) in all clinical and epidemiological
characteristics (Table 3). More than 80% of the bites were
inflicted on the lower extremities, with 46.3% being on the
feet (Figure 2).
3.3. Clinical assessment of envenomation and
treatment
All patients had non-clottable blood on admission, 24 (35.8%)
had local haemorrhage and 27 (40.3%) had systemic haem-
orrhage, mainly gingival bleeding (37.3%). The severity of
envenoming on admission to hospital was mild in 17 (25.4%)
of the patients, moderate in 35 (52.2%) and severe in 15
(22.4%) (Table 3).
Thirty patients were recruited in phase I of the study
(15 into group I and 15 into group II) and 37 patients were
recruited in phase II (Table 1). The patients in phase II
received variable doses (three vials (n = 7); four vials (n = 7);
five vials (n = 4); six vials (n = 4); eight vials (n = 7); and ten
vials (n = 8)) according to the grade of envenomation on
1178 R. Otero et al.

Table 3 Clinical and epidemiological features on admission of 67 patients bitten by Bothrops asper

Antivenoma Total
A B

No. of patients 34 33 67 (100.0%)

Time from bite to antivenom therapy (h) (mean ± SEM) 8.6 ± 1.8 7.0 ± 1.3 7.9 ± 1.1
Traditional medical attention (no. of patients) 15 17 32 (47.8%)
Age (years)
<15 6 7 13 (19.4%)
15—44 23 21 44 (65.7%)
≥45 5 5 10 (14.9%)
Sex
Male 23 24 47 (70.1%)
Female 11 9 20 (29.9%)
Length of the snake (cm)
≤50 1 3 4 (6.0%)
51—100 9 7 16 (23.9%)
>100 13 11 24 (35.8%)
Unknown 11 12 23 (34.3%)
Severity of envenomation
Mild 9 8 17 (25.4%)
Moderate 18 17 35 (52.2%)
Severe 7 8 15 (22.4%)
a A = fractionated by caprylic acid precipitation; B = fractionated by caprylic acid precipitation and treated with ␤-propiolactone.

admission. Twenty-nine patients received an additional dose between 12 h and 24 h after the start of antivenom therapy
(three vials) of either antivenom (A or B), 26 of them after (Figure 3). There were no significant differences (P > 0.05)
6 h of therapy (15 during phase I (group II); 11 due to per- in plasma fibrinogen concentrations at any time either
sistence of coagulopathy at 6 h during phase II) and 3 others between the two groups of patients treated with antiven-
due to persistence of coagulopathy at 12 h (1 patient) or 24 h oms A and B (results not shown) or between patients
(2 patients) in phase II.
Local and systemic haemorrhage stopped sometime
within the first 6 h in 96% and 89% of patients treated with Table 4 Restoration of blood coagulation after antivenom
antivenoms A and B, respectively. Twelve hours after the therapy
onset of serotherapy, bleeding stopped in all patients. There Antivenom A Antivenom B Total
was no significant difference (P > 0.05) in the time required (n = 34)a (n = 33)a (n = 67)
to stop bleeding by either antivenom.
Blood coagulation was restored within 6 h in 17 (25.4%) Patients (n = 30) receiving one relatively large initial dose of
of the patients, within 12 h in 44 (65.7%) and within 24 h in 12 vials on admission (phase I)
65 (97.0%) (Table 4). Two patients with unclottable blood 6h 2 0 2 (6.7%)
at 24 h also had fibrinogen consumption. They received 12 12 h 8 8 16 (53.3%)
and 8 vials of antivenom A on admission, respectively. There 24 h 16 13 29 (96.7%)
was no significant difference (P > 0.05) between the two 48 h 17b 13 30 (100.0%)
antivenoms in the time required to restore normal blood Patients (n = 37) receiving three to ten vials on admission
coagulation. Additionally, two other patients who received (phase II)c
antivenom A on admission (12 vials, phase I, group I) suf- 6h 8 7 15 (40.5%)
fered a recurrence of coagulopathy at 24 h (one patient) 12 h 14 14 28 (75.7%)
and at 48 h (one patient), along with low fibrinogen con- 24 h 16 20 36 (97.3%)
centration (116 mg/dl and 98 mg/dl, respectively). Never- 48 h 17b 20 37 (100.0%)
theless, none of the 26 patients who received an additional
a A = fractionated by caprylic acid precipitation; B = fractionated
dose of antivenom at 6 h had a recurrence of coagulopathy
by caprylic acid precipitation and treated with ␤-propiolactone.
within 48 h (P = 0.33) and only 1 presented with incoagulable b The two patients who could not achieve clottable blood at
blood at 24 h (P = 0.82); these differences were not signifi-
48 h received 12 or 8 vials on admission. They had moderate
cant when compared with patients who did not receive an envenomation.
additional dose of antivenom at 6 h. c Patients receiving on admission three vials (n = 7), four vials
There was a progressive increase in plasma fibrinogen (n = 7), five vials (n = 4), six vials (n = 4), eight vials (n = 7) and
concentrations (only measured in 40 patients) within the ten vials (n = 8). All numbers and percentages of the results were
first 24 h of treatment, and normal values were achieved accumulated vertically.
Whole IgG polyvalent antivenoms for Bothrops asper bites in Colombia
Figure 3 Plasma fibrinogen concentration (mg/dl) in patients
of both groups (antivenoms A and B) who received on admission
one relatively large initial dose (12 vials) of antivenom (phase
I) or a variable dose (three to ten vials) according to the grade
of envenomation on admission (phase II). Results expressed as
mean ± SEM. Antivenom was administered at time zero.
who received one relatively large initial dose (12 vials) of
antivenom on admission (phase I) and those who received
variable doses (three vials (n = 5); four vials (n = 3); five vials
(n = 3); six vials (n = 2); eight vials (n = 5); and ten vials (n = 6))
according to the grade of envenomation on admission (phase
II) (Figure 3). By the time blood clotting was corrected by
antivenom, these patients had plasma fibrinogen concentra-
tions >120 mg/dl.
3.4. Serum venom and antivenom concentrations
Serum venom and antivenom concentrations were deter-
mined in 52 patients, 26 in each group (A and B), 20
of whom were suspected cases of B. asper bite. Serum
venom concentrations (mean ± SEM) were significantly dif-
ferent (P < 0.05) for 14 patients with mild (8.1 ± 1.6 ng/ml),
27 with moderate (39.3 ± 3.7 ng/ml) and 11 patients with
severe (128.5 ± 44.5 ng/ml) envenomation on admission.
Serum venom levels decreased significantly (P < 0.05) for all
patients within the first hour of treatment, remaining below
the cut-off value at later times for most of them (Figure 4a).
Figure 4 (a) Venom and (b) antivenom serum concentrations in patients with moderate bothropic envenomation treated on
admission with one relatively large initial dose (12 vials) of either antivenom (A or B) (phase I) or with variable doses (four vials
(n = 1); six vials (n = 5); eight vials (n = 2); ten vials (n = 5)) (phase II). Results expressed as mean ± SEM. Antivenom was administered
at time zero. * P < 0.05.
1179
Nevertheless, two moderate cases in group A and six in group
B (three moderate and three severe cases) had a late rise in
venom antigen concentrations (10.0—26.6 ng/ml) between
6 h and 72 h of treatment. This rise was not associated with
recurrence of coagulopathy, and five of the eight patients
had received one relatively large initial dose of antivenom
on admission (phase I). On the other hand, the two patients
(phase I) with recurrence of coagulopathy at 24 h or 48 h did
not have recurrence of antigenaemia. There were no signif-
icant differences (P > 0.05) in serum venom antigen concen-
trations at any time either for groups A and B (results not
shown) or between patients receiving on admission a rel-
atively large dose (phase I) or a lower dose of antivenom
(phase II) (Figure 4a).
One hour after the start of serotherapy, antivenom con-
centrations were significantly slightly higher (P < 0.05) for
patients of phase I than for those of phase II (Figure 4b).
Concentrations decreased gradually during the period of
observation, remaining detectable at 96 h. There were no
differences in serum antivenom concentrations between the
two groups of patients (A and B) (results not shown).
3.5. Early antivenom reactions (EARs)
Thirteen patients (19.4%) had EARs within the first 2 h of
treatment, all of which were mild (cutaneous, gastrointesti-
nal, chills). Eight patients had received a relatively large ini-
tial dose of antivenom (phase I, 26.7%) and five had received
usual doses (phase II, 13.5%); these differences were not
significant (P > 0.05). There was no significant difference
(P > 0.05) in the frequency of EARs in patients who received
antivenom B (24.2%) and those treated with antivenom A
(14.7%). No life-threatening anaphylactic reactions were
recorded.
3.6. Complications and outcome
Twenty-seven (40.3%) patients had one or more complica-
tions; 21 (31.3%) had soft tissue infection (cellulitis), 3 of
them (4.5%) with local abscesses that required drainage.
Cultures to isolate bacteria were not performed. Eleven
patients (16.4%), two of whom were bitten by very young
(<50 cm body length) B. asper specimens, had acute renal
1180
failure, although none of them required dialysis. One preg-
nant (32 weeks) 20-year-old patient had moderate enven-
oming on admission but had no complications affecting the
foetus during envenoming or later during the birth of the
child. There were no deaths or amputations. One patient
(1.5%) had loss of muscle mass as a result of local necrosis.
4. Discussion
Haemorrhage caused by venom metalloproteinases, com-
bined with coagulopathy caused by the action of thrombin-
like enzymes, other activators of the coagulation cascade,
the associated thrombocytopenia and platelet dysfunction
are responsible for the main pathological effects of Bothrops
envenoming (Kamiguti and Cardoso, 1989). For this reason,
successful antivenom therapy must be aimed at rapid rever-
sal both of venom-induced haemorrhage and the associated
coagulopathy, ideally within 6 h of the start of treatment
(Warrell, 1995).
Table 3 shows that when all the different dosage regi-
mens are analysed together, the groups treated with each
of the two antivenoms (A and B) were not significantly dif-
ferent from each other. None of the dosage regimens used
restored blood coagulability within 6 h in all patients bit-
ten by B. asper in Colombia. As in previous studies using
initial doses of less than ten vials of various antivenoms
composed of whole IgG or F(ab )2 fragments, blood coagu-
lability was restored within 6—24 h of antivenom therapy in
97% of patients. Additionally, bleeding manifestations other
than haematuria ceased sometime within 6 h in more than
90% of patients, and within 12 h in all of them (Otero et
al., 1996, 1999; Otero-Patiño et al., 1998; this study). More-
over, administration of an additional dose of three vials of
antivenom at 6 h did not result in the restoration of blood
coagulation within 12 h in all patients. Partial (42—68%)
restoration of blood coagulability by 6 h and an almost com-
plete restoration by 24 h were described in a clinical trial
of three antivenoms in snake bite envenomings in Ecuador
(Smalligan et al., 2004). However, it must be noted that, in
the case of a Colombian polyvalent antivenom from Instituto
Nacional de Salud (INS, Bogotá), blood coagulability was per-
manently restored at 6 h in the majority of patients treated.
Recurrence phenomena involving local signs (oedema,
haemorrhage), coagulopathy (hypofibrinogenaemia), throm-
bocytopenia and temporally increasing serum venom con-
centrations have been reported in the treatment of severe
crotaline snake bites using whole IgG, F(ab )2 and Fab frag-
ment antivenoms (Gutiérrez et al., 2003). Recurrence of
coagulopathy appears to be more frequent with the use of
antivenoms composed of Fab (53%) or F(ab )2 (5—17%) frag-
ments than with IgG antivenoms (Boyer et al., 2001; Dart and
MacNally, 2001; Ho et al., 1986; Otero et al., 1999; Otero-
Patiño et al., 1998; Seifert and Boyer, 2001; Warrell et al.,
1986) and is likely to depend on the different pharmacoki-
netic profiles (shorter elimination half-life) of these lower
molecular mass antibody fragments (Gutiérrez et al., 2003).
Administration of one relatively large initial dose of
antivenom composed of whole IgG in this study did not
completely prevent the development of recurrences (coagu-
lopathy 3%; venom antigenaemia 15.4%) in moderate/severe
cases of bothropic envenomation within the first 6—72 h of
R. Otero et al.
antivenom therapy. The clinical significance of these recur-
rences is uncertain, but none of our patients suffered obvi-
ous recurrent external or internal bleeding. There was no
statistical association (P > 0.05) of recurrences with either
antivenom A or B. However, regardless of the mechanisms
involved, any manifestation of recurrence of clinical enven-
oming demands additional administration of antivenom.
EARs are one of the main problems associated with
antivenom therapy. These may be due to (1) anaphylaxis, (2)
activation of the complement system (anaphylactoid reac-
tions), (3) the presence of antibody fragment aggregates
and/or Fc fragments or (4) the presence of pyrogens (endo-
toxins) owing to substandard manufacturing procedures.
Antivenoms composed of whole IgG and F(ab )2 fragments
have anticomplementary activity, inducing EARs in 10—82%
of patients, the incidence being higher for some IgG prepara-
tions purified by ammonium sulphate fractionation (Otero et
al., 1996, 1999; Otero-Patiño et al., 1998; Smalligan et al.,
2004). However, IgG antivenoms obtained by caprylic acid
precipitation of non-Ig proteins from plasma have no albu-
min, are of lower anticomplementary activity, have fewer
protein aggregates and have a lower total protein content
than those obtained by ammonium sulphate precipitation
of IgG (Otero et al., 1999). In consequence, IgG antiven-
oms obtained by caprylic acid fractionation are known to
induce a lower incidence of EARs (11—25%) than IgG antiven-
oms produced by ammonium sulphate precipitation and also
compared with some F(ab )2 fragment antivenoms (Cardoso
et al., 1993; Chippaux and Goyffon, 1998; Cupo et al., 1991;
Jurkovich et al., 1988; León et al., 2001; Malasit et al., 1986;
Morais et al., 1994; Otero et al., 1996, 1999; Otero-Patiño
et al., 1998; Rojas et al., 1994; Teixeira-Caiaffa et al., 1994;
WHO, 1981). The results presented here further support
these concepts, since the two caprylic acid-fractionated IgG
antivenoms induced a 19.4% EAR rate and most of these reac-
tions were mild.
Modification of the Fc portion of the IgG by ␤-
propiolactone in the production of human Ig prevents
non-specific complement activation without affecting the
integrity and biological properties of the IgG molecule
(Stephan, 1975). Treatment of IgG with ␤-propiolactone is
a simple chemical procedure that has been previously sug-
gested to be of possible use in the manufacture of safer
antivenoms (Guidolin et al., 1997). It was introduced in the
manufacture of antivenom B after caprylic acid fractiona-
tion of horse IgG. Despite having a lower anticomplementary
activity in vitro, antivenom B did not induce a significantly
lower incidence of EARs than a similar antivenom prepared
without treatment with ␤-propiolactone. In agreement with
these results, the haemolytic activity of serum complement
was not affected in rabbits after the administration of an
i.v. bolus of caprylic acid-fractionated antivenoms either
treated or not treated with ␤-propiolactone (León et al.,
2005). Our clinical and experimental findings therefore sug-
gest that in vitro anticomplementary activity of antivenoms
may not be a good predictor of their propensity to induce
EARs clinically.
In conclusion, the results of this study indicate that IgG
antivenoms fractionated by caprylic acid precipitation or by
caprylic acid precipitation and ␤-propiolactone treatment
are similarly effective and safe in the treatment of B. asper
envenomations in Colombia. The initial antivenom doses
Whole IgG polyvalent antivenoms for Bothrops asper bites in Colombia
used were insufficient to neutralise fully the antihaemo-
static components of B. asper venom within 6 h. A study to
assess this hypothesis is currently being performed in which
higher doses of antivenom are used. The introduction of ␤-
propiolactone in antivenom production did not reduce the
frequency of EARs in whole IgG equine preparations.
Conflicts of interest statement
The authors have no conflicts of interest concerning the work
reported in this paper.
Acknowledgements
We thank the personnel of the hospitals involved in the
study as well as Janeth Garcı́a and the staff of the Produc-
tion Division of Instituto Clodomiro Picado for their collab-
oration in different stages of this project. The support of
Instituto Colombiano para el Desarrollo de la Ciencia y la
Tecnologı́a Francisco José de Caldas (COLCIENCIAS), Univer-
sidad de Antioquia, CYTED (research project 206PI0281) and
Vicerrectorı́a de Investigación, Universidad de Costa Rica is
gratefully acknowledged. This work was performed in partial
fulfilment of the doctoral degree of G. León at the University
of Costa Rica.
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