THIS ST UDY WAS COMPLETED
AT THE INST ITUTE FOR SCIENTIFIC RESEARCH
MINISTRY OF TRAINING & EDUCATION

MINIST RY OF DEFENCE

ON CLINICAL MEDICINE AND PHARMACY 108

INS TITU TE FO R SC IENTIFIC RES EARCH
ON C LINIC AL MEDICINE AND PHARMACY 108

Scientific instructors:
1. Associate prof. Pham Due, PhD
2. Dr. To Vu Khuong, PhD

NGUYEN TRUN G NGUYEN

Scientific critic 1: ……………………………………………
Scientific critic 2: ……………………………………………

CLINICAL FEATURES,
BLOOD VENOM LEVELS
AND THE ROLE OF COBRA RAPID TEST®
IN THE DIAGNOSIS AND TREATMENT OF
COBRA BITTEN PATIENTS
Expe rtise: Anaesthesia & Inte nsive care
Code: 62720122

Scientific critic 3: ……………………………………………

This thesis is planned to be defended before institutional thesis

There have been certain studies on snakebite in V ietnam.
However, no study focusing on cobra bite spcifying the exposure factors,
clinical and laboratory features of the evenomation, especially the
evaluation on the relationships between these parameters and blood
venom levels in all patients, has been carried out.
The currently known 61 species of venomous snakes have
overlapping evenomation clinical conditions challenging the diagnosis
approach by syndromes, leading to delayed, incorrect or missed
diagnosis and affecting emergency management. A rapid cobra venom
test is thefore very necessary to be available to assist the diagnosis and
treatment.
The specific treatment with snake antivenom f or the neutralization
of venom is the optimal solution. There has been only a monovalent
cobra antivenom (made from the venom of Naja kaouthia in the South of
Vietnam) so far in the country for the treatment of evenomation by cobra
species. There have been also no studies on the evaluation of the protocol
and effectiveness of this antivenom on the evenomation by cobra species
in the North of Vietnan.
This study is aimed at:
1. Specifying c linical fearures, blood venom levels in the cobra
bitten patients treated at the Poison control center of Bach Mai hospita l.
2. Eva luating the value of Cobra Rapid Test in the diagnosis
and treatment w ith antive nom in patients with cobrabite.

1.1. Cobra:
1.1.1. Epide miology:
According to an evaluation on the global snakebite burden,
Vietnam belongs to one of 21 areas with the highest number of snakebite
victims and one of 4 zones with the highest death rate.
Researches and reports from Bach Mai hosita l and Cho Ray

1.2.3.Diagnosis of s nake bite:
1.2.3.1. Diagnosis of s pecific snake species:
a. Syndrome approach: practical, symple, rapid and must be
applied from the beginning but difficult to identify specific species.
b. Identification of culprit snake: common and gold standard.
c. Immunologic tests:
 Rapid test kits: there have been rapid tests in the world but very
few have been commercially available.
Immunochromatographic rapid test for cobra venom:
The test kit Cobra Rapid Test (CRT ®) develope d by D.Z.Hung et
al (2005) for the dia gnosis of snakebite by N. atra in Ta iwan,
detection threshold for the blood cobra venom is 5ng/ml. Test is
performe d by dropping plasma, urine or fluid from bitten site on the
test kit. The result is observe d after 20 minutes with the sensitivity of
88% and spec ificity of 100%.
 Qualification and quantification of ve nom with Enzyme
linked immunosorbe nt assay (ELISA):
Patient sample is incubated with anti-snake venom specific
antibody which is to be analyzed, the incubated environment w ill change
the color when the antibody combines with the antigen. The intensity of
the color is equivalent to the venom concentration in the sample.
Detection threshold is from 1 to 5ng/ml and performing duration is
within 3 hours.
d. Ide ntification of snake species with gene analysis.
1.2.3.2. Diagnosis of severity of the snakebite:
Common classification of poisoning severity - Poisoning
Severity Score (PSS): recommended by the Wold Health Organization.
Classification of poisoning severity specicially for snake bite:
The classicication help the direction of management, especially for the
use of antivenom and monitoring the patients’ condition. There have

Rapid tests for snake venoms have been use to support the
indication of antivenom only and the nonitoring of the patients’
conditions rely totally on clinical situation which may make clinical
doctors meet difficult.


CHAPTER 2
RESEARCH METHOD
2.1. Patie nts:
 Inclusion criteria:
All snakebite patients were admitted to the Poison control center
of Bach Mai hospital from 01/01/2013 to 31/12/2015 and fullfilled the
both following criteria:
1) Patients give the samples or photographs of the culprit snakes to
the doctors at the Poison center for the identification of snake species by
morphology analysis (the photographs need to bee clear, the posteria
views of the hoods must be available if it is the case of cobrabite. 2)
Antivenom has noot been used before arrival.
2.2. Research me thod:
- This is a prospective and descritiptive study.
2.2.1.Sample size :
 Evaluation of the CRT on cobrabite patie nts:
The main purpose of this study is to evaluate the sensitivity of the
rapid test CRT is calculated by the rate (%) of the number of patients
with positive results over total number of the patients diagnosed
definitively with cobrabite.
Estimate n with the formula: n =
TP: true possitive
FN: false negative
Calculate TP + FN:

 Proposed classification of severity in this study and research
protocol for the use of antivenom: compatibility between the research
classification of severity and PSS.
d. Exploration of re lationships among risk factors, severity
and tre atment:
 Relationships among evenomation severity, exposure factors and
treatment.
 First aids applied prehospita lly and local tissue injuries.
 Blood venom levels, results of blood CRT and exposure factors,
severity and treatment.
2.2.2.3.
The role of CRT in the diagnosis and monitoring of
treame nt:


a. In the diagnosis:
- Sensitivity and specific ity of CRT, negative predictive value,
positive predictive value, rates of false negative and false positive results.
b. In the monitoring and evaluation of treatment:
 CRT:
- Negative or positive results are compared with the severity,
loca l tissue injuries, methods of first aids already applied prehospitally.
- Evaluation of 2 protocols for the monitoring of antivenom
therapy: group w ith the combination of clinical condition and blood CRT
or group w ith only c linical condition are compared on the local tissue
injuries, total dose of antivenom, duration of antivenom therapy,
hospitalization time.
 ELISA (blood venom levels): used to evaluate the effectiveness
of the first aids, treatment measures and outcomes.
c.Treatment outcomes:

antivenom was decided based on the resolution of clinical condition.
 The second phase when the CRT was added: the completion of
antivenom was decided based on the combination of negative repeated blood
CRT and resolution of clinical condition.
 ELISA (enzyme linked immunosorbent assay) to detect the
venom of N. atra:
- Blood samples taken on admission, before and after antivenom
therapy. Venom of Vietnam N. Atra, anti-cobra venom antibody
produced from rabbits by the Faculty of Immunology of the Academy of
Military Medicine and other materials.
- ELISA microplate reader: produced by Diagnostic
Automation/Cortez D iagnostics.
2.2.3.3. Mate rials for the treatme nt:
 N. k aouthia anrivenom produced by the Nationa l Institute for
Vaccines and Biologicals (IVAC).
2.3. Data analysis:
 Data are processed and analyzed with SPSS software.
 Results are presented as percentage for qualitative variables,
mean and standard deviation for quantitative variables. Comparision of
means and percentages with T-test, Fisher, 2 , significant leve l with p

Nauea, vomittng
22 (18,0)
9 (45,0)
0,05
Ptosis
4 (3,3)
3 (15,0)
>0,05
Difficult speech
6 (4,9)
2 (10,0)
>0,05
Pharyngeal paralysis
4 (3,3)
2 (10,0)
>0,05
Intercostal, diaphramic paralysis
4 (3,3)

hyponatremia 22,9%, hypokalemia 55,7%, CPK >1000 U/L 11,5%.
Blood venom levels 23,84 (0 - 996,18) ng/ml. N.kaouthia bite: increased
hematocrit 10%, hyponatremia 35%, hypokalemia 55%, CPK >1000 U/L
30%. Blood venom levels 165,38 (0,11-457) ng/ml. N.sumatrana
patient: total white blood cells >10,68 G/L; platelet 130 G/L; INR 1,32;


APTT 0,94; fibrinogen 4,43g/L; CPK 8910 U/L; blood venom level
351,14 ng/ml.
N.kaouthia bite have higher hematocrit, CPK compared to those in
N.atra bite (P
50

Severe

0

0

4

32

36

Total

23

37

51

32

143

Kappa = 0,933 (P
15
165,38 (0,11-457)
97
23,48 (0- 996,18)
16 183,74 (0,58- 463,38)
53
21,92 (0- 633,7)
55
63,32 (0- 996,18)
19
86,87 (0,14- 633,7)
9
123,47 (0- 351,14)
103
55,29 (0- 996,18)
11

p
>0,05
0,05
>0,05

(0-8,21)

Mild2
(n=30)

Mode rate 3
(n=41)

Severe 4
(n=27)

De ath
(n=1)

9,31
84,96
180,88
257,22
(0,02(0,32(0299,92)
634,54)
996,18)
p (1-2)

Figure 3.1: Blood ve nom levels in groups of admiss ion time afte r the
accident
Comment: Blood venom increased early within the first 6 hours,
increased further rapidly and peaked at 6-12 hours. It then began to
decrease quickly and to very low level after 24 hours.

Bungarus multicinctus (Chinese krait)

12

11

1

Protobothrops mucrosquamatus (Habu)

2

2

0

Rhabdophis subminiatus (Red necked keel back)

2

2

0


0

41

37

4

Total
3.3. Application of CRT in the diagnosis and treatment:
3.3.1. CRT in the diagnosis:
3.3.1.1. Se nsitivity:
 Blood samples:
119/143 patients were found to have venom in the blood w ith
ELISA.
Overall sensitivity of CRT in all cobrabite patients is 108/119
(90,8%), the sensitivity in only N.atra 92/102 (90,2%).
Sensitivity of CRT in N.atra bite patients both have symptoms and

Comment: 37/41 (90,24%) patients had negative blood CRT,
specific ity of 90,24%.
3.3.1.2. Results of CRT in the evaluation of injuries:
20/21(95,2%) patients w ith negative blood CRT only had necrosis
square 10cm2 came days after accident and blood venom level of 0 ng/ml.
3.3.2. Blood CRT and quantitative ELISA in the evaluation
and monitoring of treatment:
3.3.2.1. Evaluation of first aids:



Bitten site washing
32.203
loca l application or ingestion of traditional medicine 35.474

>0,05
>0,05

Dependent variable: blood venom levels (ng/ml)
Comment: The blood venom level of patients being applied
constricted band decreased by 88,119 ng/ml compared to that in patients
not being applied the measure (difference with p

Note: Data are presented as n(%)
Comment:
- Total dose of antivenom, decrease in the necrosis and
hospitalization time were lower significantly than those in groups not
used CRT to guide antivenom therapy.
Table 3.37: Blood venom leve ls on admiss ion and tre atme nt
outcomes
Admitte d ≤ 12h
Admitte d > 12h
Tre atment outcomes
r
p
r
p
Total dose of antivenom
0,161
0,463
0,365
0,002
(via ls)
Total volume of IV fluid on
0,158
0,472
0,605
0,000
the first day (litres)
Hospita lization time (days) 0,424
0,000
0,491

(via ls)
p
diagnosis of species specific snakebite. Patients were also tested CRT and
ELISA on blood samples.
Soources of cobra species: N. atra accounted for the highest rate
(85,3%), followed by N. kaouthia (13,9%), a patient was bitten by N.
Sumatrana cắn. This study confirms the inhabitance of N.kaouthia in the
North of Vietnam. This has been the first time for the species
N.sumatrana recorded to exist in Vietnam.
4.1.2. Clinical, laboratory and blood venom levels:
4.1.2.1. N.atra bite :
In 122 patients, signs and symptoms are similar to other studies
but the rate of necrosis in our patients (60,7%) is higher than that in
many other studies. Paralysis leading to respiratory fa ilure (3,3%) is new
compared to studies in other countries and the long common perception
clinically in our country. Hyponatremia seen in 22,9% of the patients,
maybe due to sodium diuretic peptide in the venom.
4.1.2.2. N. Kaouthia bite:
Bite by N.k aouthia in the North causes more severe necrosis
compared to that in the bite by the same species in the South.
Gastrointestinal symptoms are more frequent than those caused by N.atra
bite. Paralysis rate is 10%, lower than that in the bite by the same species
in the South (58,8% to 70,6%, Lê Khac Quyen). Increased hematocrit
and rhabdomyolysis are more frequent than those in N.atra bite.
4.1.2.3. N.sumatrana bite :
This only patient has all signs and symptoms similar to those in
N.atra, N. Kaouthia bites except paralysis.
4.1.2.4. Assessme nt of e venomation severity:

Classic ification of severity proposed in this study is compatible
to the PSS, Kappa = 0,993 (P

sensitivity of CRT in these samples. Sensitivity of CRT in fluids from
bitten sites in a ll N. atra bite is 98,4%. The sensitivity in symptomatic
N.atra bite is 100%.
 Urine : The re have not bee n studies on the use of CRT in
urine . The overall sensitivity of CRT in a ll cobrabite and symptomatic
cobrabite are 89,5% and 96,9%, respectively.
4.2.2. Specificity of CRT:
 Blood samples:
This evaluation is performed in non-cobra snakebite. Specific ity of
CRT in blood is 90,24%. CRT can cross-react with patients bitten by
krait, king cobra and bamboo viper.

4.2.3. CRT and local tissue injuries and severity: in early
admitted patients, negative blood CRT results suggest mild or nonevenomation.
4.2.4. CRT and quantitative ELISA in the diagnosis and
monitoring of treatment:
4.2.4.1. Assenssme nt of first aids:
a. CRT:
Results of CRT from local area fluid are not influenced by the
first aids applie d on the bite. This observation is very new to other
current understanding in rapid tests on loca bitten area fluid. Results
of blood CRT are not influenced by first aids.
b.Blood venom levels (ELISA):
Among the first a ids, only constricted band decreases the blood
venom levels signficantly. This results can help suggest venous
pressurized bandage to replace pressure immobilization technique which
is quite difficult to be deployed currently.
4.2.4.2. CRT and quantitative ELISA for the diagnosis and
monitoring of treatme nt:

- Species of cobra were seen in the North of Vietnam: N.atra
85,3%, N.kaouthia 13,9% and one patient bitten by N.sumatrana
(0,69%), both in naturally inhabited and raised conditions.
- N. atra bite : awelling 85,2%, incompleted and completed
necrosis 60,7%, vomitting 18 %, paralysis 3,3%, rhabdomyolysis 11,5%,
hyponatremia 22,9%, hypokalemia 55,7%, increased fibrinogen 11,5%.
- N.kaouthia bite: swelling 90%, incompleted necrosis and necrosis
65%, vomitting 45%, paralysis 10%, rhabdomyolysis 30%, hyponatremia
35%, hypokalemia 55%, increased fibrinogen 15%. Local tissue injuries
appear to be more severe than those in the same snakebite in other regions.
- Risk factors for severe evenomation: weight of snake more than
500 grams, bitten raised snakes, delayed admiss ion (more than ≥12 hours
since the accident).
- Classification of severity of cobrabite proposed in this study is
compatible with the PSS commonly recommended, but it is more simple
and clinically feasible.


Blood ve nom leve ls in the evaluation of progress ion, risk
factors, pre-hospital first aids:

- Blood venom level increases early after the bite and peaks at
approximately 6-12 giờ, gradua lly decreases thereafter and reach very
low level after 24 hours.
- Risk factors: Patients with blood venom leve l higher than
100ng/ml have more severe swelling, necrosis than those with lower
venom levels. Being bitten though a layer of bag or clothes may be a

protective factor and help patients have mild or nonevenomation.
- Among the first a ids, constricted band appears to be the only

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