MINISTRY OF TRAINING & EDUCATION MINIST RY OF DEFENCE INS TITU TE FO R SC IENTIFIC RES EARCH ON C LINIC AL MEDICINE AND PHARMACY 108 NGUYEN TRUN G NGUYEN CLINICAL FEATURES, BLOOD VENO
Trang 1MINISTRY OF TRAINING & EDUCATION MINIST RY OF DEFENCE
INS TITU TE FO R SC IENTIFIC RES EARCH
ON C LINIC AL MEDICINE AND PHARMACY 108
NGUYEN TRUN G NGUYEN
CLINICAL FEATURES, BLOOD VENOM LEVELS
IN THE DIAGNOSIS AND TREATMENT OF
COBRA BITTEN PATIENTS
Expe rtise: Anaesthes ia & Inte ns ive care
Code: 62720122
ABS TRACT OF DOCTORAL THES IS
HANOI - 2019
THIS ST UDY WAS COMPLETE D
AT THE INST ITUTE FOR SCIENTIFIC RESEARCH
ON CLINICAL MEDICINE AND PHARMACY 108
Scientific instructors:
1 Associate prof Pham Due, PhD
2 Dr To Vu Khuong, PhD
Scientific critic 1: ……… Scientific critic 2: ……… Scientific critic 3: ………
This thesis is planned to be defended before institutional thesis evaluation board at: time day month year
The thesis can be assessed at:
1 National library
2 The library of the Institute for scientific research on clinical medicine and pharmacy 108
Trang 2INTRODUCTION
Snakebite is a common emergency condition It has been inc luded
in the list of neglected tropical diseases requiring adequate attention
Cobra bite is the most frequent in the snakebite cases and causes many
types of injuries, demands emergency hospita l admission and has high
risk of death, longterm sequalae or disability
There have been certain studies on snakebite in V ietnam
However, no study focus ing 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
diagnos is 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 neutra lization
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
CHAPTER 1 OVERVIEW 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 hos ita l and Cho Ray hospital show t hat snakebite is a common emergency in which cobrabite
is the highest
1.1.2 Naja genus:
a In the world:
There have been totally 26 species of cobra identified so far
inc luding 11 species in Asia and 15 others in Africa
b In Vie tnam: N atra (Cantor, 1842): “rắn hổ mang miền Bắc”,
in V ietnamese Distribution: widely distributed in t he North of V ietnam
N kaouthia (Lesson, 1831): in Vietnamese, “rắn hổ đất” Distribut ion:
widely distributed in the South of Vietnam N siamensis (Laurenti,
1768): in V ietnamese, “rắn hổ mèo”, only distributed in the South
1.2 Disgnos is and treatment of cobrabite:
1.2.1 Signs and s ymptoms :
a Snakebite by N atra, N.kaouthia:
- Local: there is ussually mechanical injury at the bitten s ite Redness, swelling, necrosis , vesicles may occur and progress gradually Local lymphatic nodes can enlarge
- Systematic: paralysis may occur, flaccid paralysis beginning at cranial nerves and decending to limbs Paralysis may lead to respiratory failure and death if untreated Other signs and symptoms can exist: abdominal pa in, nausea and vomitting, diarrhea, rhabomyolysis, renal failure
b Snakebite by N siamensis:
Signs and symptoms are similar to the conditions caused by the 2 above 2 mentioned sepcies but more frequent and severe Paralysis has
Trang 3not been seen
1.2.2 Complications of snakebite: respiratory failure , infection,
renal failure, compartment syndrome, scar, amputation, disability,
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 s nakebite 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 s ite 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
linke d immunosorbe nt assay (ELISA):
Patient sample is incubated with anti-snake venom specific
antibody which is t o be analyzed, the incubated environment w ill change
the color when the antibody combines with the antigen The intens ity 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 s pecies with gene analys is
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 s pecicially for s nake bite:
The classicication help the direction of management, especially for the
use of antivenom and monitoring the patients’ condition There have
been classification of snakebite severity commonly applied for cobrabite
in V ietnam and the world
1.2.4 Management of s nake bite:
1.2.4.1 Antivenom the rapy:
Evidence has shown that antivenom therapy can prevent toxic effects of the venom if it is used early and can limit these effects even if
used when clinical condition became clear
Indication of antivenom therapy by the WHO: it is appled for all snakebites This guideline, however, does not mention the necrosis, the cause of severe outcomes and sequalae for the patients Therefore this
issue needs to be studied
Specific situdies on the use of cobra antivenom are required in Vietnam so that results of treatment can be improved
1.2.4.2 Supportive care , tre atme nt of s ymptoms and complications
1.3 Studies in Vie tnam and the world:
The definitive diagnosis of snakebite by a specific species: this is a
gold criteria but researches have not been carried out adequately
There have been at least 61 identified species of venomous snakes with overlapping evenoming conditions challenging clinical approach There needs to be other diagnostic methods , e.g immunologic techniques
There have been very few studies on the necrosis Moreover,
documents states that this injury does not respond to antivenom
Clinical researches usually lack blood venom leve l as a eveluation tool Whereas blood venom levels help the identification of culprit snake species, progress of evenomation, risk factors, degrees of evenomation and effectiveness of first aids and treatment in the hospital including antivenom therapy
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 s ituation which may make clinical doctors meet difficult
Trang 4CHAPTER 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 s ize :
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:
TP+FN =
Z2: constant of normal distribution, with α = 0,05, Z 2 = 1,96
SN: minimal sensitivity, 75% (0,75), W : odd of the probability
of true positive results and probability of true negative results,
select w = 0,1
Therefore: TP + FN = (1,96 x 0,75 x 0,25)/ (0,1 x 0,1) = 36,75
Based on the numbers of snakebite patients admitted to the
Poison center, the minimal of cobrabite is 33%
So n = 36,75/0,33 = 111,36 We select that n =112
2.2.2 Research paramete rs : 2.2.2.1 Demographic data:
Age, gender, occupation, geographic information
2.2.2.2 Clinical, laboratory fe atures and blood venom levels:
a Clinical condition:
Situation of accident, characteristics of culprit snake, reason for accident, applied firstaids, treatment at previous hospitals
Signs, symptoms Local: pain score, swelling, incomleted and completed necrosis, erytherma, vesicles, local enlarged lymphatic nodes, compartment syndrome Systemic : abdominal pain, nausea and vomitting, diarrhea, pulse rate, cardiac rythms, blood pressure, body temperature, paralysis, respiratory fa ilure
b Laboratory tests :
Blood count, coagulation tests, urea, creatinine, glucose, electrolytes, GOT, GPT, CPK, procalcitonin
Results of CRT: blood, fluid of fang marks, urine
Results of ELISA: blood venom levels on admission and after antivenom
c.Evaluation of evenomation severity:
Classification according to PSS
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 lations hips 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:
Trang 5a In the diagnos is :
- 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 t ime
ELISA (blood venom levels): used to evaluate the effectiveness
of the first aids, treatment measures and outcomes
c.Treatment outcomes:
Change in the local tissue injuries, total dose and duration of
antivenom therapy, complications, bedtime, final treatment results
2.2.3 Evaluation tools :
2.2.3.1 Clinical evaluation tools :
Patient records: history, physical exams, laboratory tests,
treatment measures and results Tools for the assessment of cobrabite
injuries: pain scales, tape measure: circumference measured across bitten
site, extension of swelling, transparent square ruler on which one unit
equals to 1cm2, permanent marker
2.2.3.2 Laboratory tests and imaging:
a Routine laboratory tests : blood cell ana lyzers, biochemical
analyzer Electrocardiographer, dopper ultrasound for the blood flows to
bitten areas
b CRT and quantitative ELISA of blood cobra venom:
CRT:
Sample : blood (for a ll pa tients on a dmission, before and
ntive nom thera py, after a ntive nom for the group used CRT to
combined with c linical c ondition): 3ml of blood is drawe d to the
unc oagula te d tube , centrifuged to collect plasma Fluid from bitten site a nd urine are a lso c olle cte d
Research condition:
+ CRT test strip was supplied by t he Taiwan colleagues according
to the co-operative program between the 2 sides The number of CRT was limited initially because it was planned to be evaluated the diagnostic role The CRT thereafter shown advantages when studied so the study group required additional number of CRT to support the monitoring and decis ion of the completion of antivenom therapy
The study was devided in to 2 phases:
The first phase when the test strip was not added: completion of 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 admiss ion, 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 analys is:
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 < 0,05 Re lationships between factors or variables are explored Odd ratio (OR) to evaluate the risks of getting more severe condition
Trang 6CHAPTER 3 RESULTS
3.1 Characte ristics of patie nts:
1297 snakebite patients were admitted at the poison center from
1/2013 to 12/2015 đã có 1297 BN, 184 patients were enrolled, including
143 cobrabites, 41 other snakebites
Male: 117/143 (81,8%), female: 26/143 (18,2%) Age: 43,60
16,58 (4 – 89) years
3.2 Expos ure, clinical and laboratory fe atures and blood
ve nom levels in cobrabite patie nts :
Species of cobra: N atra accounted for 85,3% (122/143),
N.kaouthia accounted for 13,9% (20/143), either encountered in both
natural or human made environment One case bitten by N Sumatrana
was seen
Types of snake s amples : 114/143 (79,7%) of patients had intact
snake bodies, 2/143 (1,4%) had head and necks of snakes, 27/143 (18,9
%) had photographs of snakes
Snake weights : were measured in 132 patients: N atra: 496,45
380,12 g (10-2000g) N kaouthia: 851,05 495,89g (20-2000g), N
sumatrana: 1000g
Local tissue injuries: bite by N atra: pain at bittent area 85,2%,
swelling 85,2%, necrosis 60,7%, ves icles 20,5%, regiona l enlarged
lymphatic nodes 3,3% N.kaouthia bite: local pain 90%, swelling 90%,
necrosis 65%, vesic le 20%, enlarged lymphatic nodes 15% N.sumatrana
bite : pain, swelling, necrosis, vesicles
Table 3.6: Rate of s ys tematic s igns & symptoms of cobra
species
Syste matic s igns , symptoms
Cobra s pecies (n, %)
P
N atra (n=122)
N kaouthia (n=20)
Pharyngeal paralysis 4 (3,3) 2 (10,0) >0,05 Intercostal, diaphramic paralysis 4 (3,3) 2 (10,0) >0,05
Respiratory failure (due to paralysis)
4 (3,3) 2 (10,0) >0,05
Note: data are presented as n(%)
Patient bitten by N.sumatrana: nausea, vomitting, abdominal
pain, generalized pain but no diarrhea, no paralysis , normal pupils and heart rate
Comment::
Signs a nd s ymptoms are see n in all bite s by c obra s pec ies: nausea, vomit ting, abdomina l pa in, dia rrhea Less freque nt s igns , symptoms : paralys is of phar ynx, res pira tion or limbs Nausea,
vomitting and abdomina l pain were more frequent in N.kaouthia bite compared to rắn N.atra bite
Laboratory tests : N.atra bite : increased hematocrit 0%,
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
patie nt: total white blood cells >10,68 G/L; platelet 130 G/L; INR 1,32;
Trang 7APTT 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<0,05)
Table 3.9: Compatibilty of propose d class ification of seve rity with
Poisoning Severity Score PSS
Proposed class
Non
Severe or
Kappa = 0,933 (P<0,001)
Comment: the proposed classification has high compatility w ith
PSS
We ights of s nake and severity: patients with snake weighted more than
500 grams have probability of severe evenomation or death 4,51 times
higher than whom with smaller snake (CI =1,77-11,46), P = 0,001)
Source of snake and seve rity: patients bitten by raised snakes have the
probability of severe evenomation or death 4,99 times higher than snakes
in natura l environment (CI=1,74-14,32, P=0,001)
Mechanis m of bite and severity: 14/15 (93,3%) of the patients bitten
through a layer of bag or clothes had only mild or no evenomation
Patients bitten through such layer had higher rate of no evenomation
compared to that in the patients bitten on bare skin body parts, 6/15
(40%) versus 17/128 (13,3%), respectively (P<0,05)
Admiss ion time and severity: patients admitted at ≥12 after the accident
have the probability of severe evenomation or death 8,91 times higher
than those admitted within the first 12 hours (CI=3,44-23,06; P<0,001)
Table 3.19: B lood venom levels on admiss ion and snakes’s factors
Median (min-max)
p
Cobra species
Sources of snakes
Natural 97 23,48 (0- 996,18) <0,05
Snake weights
Digesting condition
Hungry 19 86,87 (0,14- 633,7) >0,05 After eating prey 9 123,47 (0- 351,14)
Mechanism
of bite
Direct on bare skin
103 55,29 (0- 996,18) <0,05 Through a layer 11 4,51 (0-451,16)
Comment: Patients bitten by raised snakes had blood venom levels
higher than those bitten by natura l snakes Striking through a layer of clothes or bag led to decreased blood venom levels
Table 3.23: B lood ve nom levels on admiss ion and PSS:
PSS No signs
or
s ymptoms
1
(n=15)
Mild 2
(n=30)
Mode rate 3
(n=41)
Severe 4
(n=27)
De ath (n=1)
Venom levels ng/ml
0,21 (0-8,21)
9,31
(0,02-299,92)
84,96 (0,32-634,54)
180,88 (0-996,18)
257,22
p<0,001 p (1-2) <0,001; p (1-3) <0,001; p (1-4) <0,001; p (2-3) <0,001;
p (2-4) <0,001; p (3-4) >0,05
Data presented as median (min-max) Comment: blood venom leve ls are different among groups except
between moderate and severe groups
Trang 8Figure 3.1: B lood 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
3.3 Application of CRT in the diagnos is and treatment:
3.3.1 CRT in the diagnosis:
3.3.1.1 Se ns itivity:
Blood samples:
119/143 patients were found to have venom in the blood w ith
ELISA
Overall sens itivity 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
and venom in the blood (ELISA) is 89/91 BN (97,8%) Rate of false negative: 11/119 (9,2%) patients with venom in the blood but had negative CRT
N.kaouthia bite : 93,8% of patients had positive blood CRT
One N.sumatrana bite patient: pos itive blood CRT
Fluids from bitte n site : overall sensitivity was 98,4%
Urine : Overall sensitivity was 89,5%
3.3.2.1 Specificity:
Table 3.28: Specificity of CRT on blood
(Evaluated on samples of patients bitten by non-cobra snakes)
(-) (+)
Comment: 37/41 (90,24%) patients had negative blood CRT,
specific ity of 90,24%
3.3.1.2 Res ults of CRT in the evaluation of injuries :
20/21(95,2%) patients w ith negative blood CRT only had necrosis square <1 cm2 1 patient w ith negative blood CRT and necrosis aquare
>10cm2 came days after accident and blood venom level of 0 ng/ml
3.3.2 B lood CRT and quantitative ELISA in the evaluation and monitoring of treatment:
3.3.2.1 Evaluation of first aids:
<6h 6-12h >12h-24h >24h
Time since a ccide nt (hours)
Trang 9Inte rfe re nce of the first aids on the res ults of CRT tested on
fluid samples from bitte n s ite: The rate of positive or negative result
CRT were not different among f irst aid pat ients or non-first aid patients,
in term of squeezing or sucking of blood from bitten site , incis ion,
constricted band, bitten site sterlization, washing, local application or
ingestion of traditiona l medicine
Table 3.33: Influe nce of applied first aids on the blood venom
leve ls on admission (n=114) (Multiple linear regression)
loca l application or ingestion of traditional medicine 35.474 >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<0,05)
3.3.2.2 CRT and blood venom levels in the evaluation and
ins truction on tre atment me as ures in hos pital:
Change in the incomplete d and completed ne crosis : median
(min-max)
Square of necrosis after antivenom therapy: 1(0 - 769) cm2,
decreased significantly compared to that measured before the treatment
(0 - 839) cm2 (P=0,005)
Table 3.36: Outcomes of patie nts be ing used CRT and not being used CRT for the s upport of monitoring of antivenom therapy
Outcomes
Patient groups
P
Clinical + CRT (n=85)
Clinical only (n=26)
Median (Min-Max) Duration of antivenom
therapy (hours)
10 (0,5-120) 7 (1-23) >0,05 Total dose of antivenom
(via ls)
21 (0,5-70) 38 (4-72) <0,01 Decrease in the necrosis
after antivenom (cm2)
0,5 (0,5 -116)
0 (-42 -89) <0,05 Hospita lization time (days) 2 (1-10) 3 (1-13) <0,05
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: B lood venom leve ls on admiss ion and tre atme nt
outcomes
Tre atment outcomes Admitte d ≤ 12h Admitte d > 12h
Total dose of antivenom
Total volume of IV fluid on the first day (litres) 0,605 0,000 0,158 0,472 Hospita lization time (days) 0,424 0,000 0,491 0,017
Comment: in patients admitted within the first 12 hours after
accident, the higher the blood venom leve l, the higher dose of antivenom
or IV fluid volume and the longer hospitalization time are
- In patients admitted after accident more than 12 hours, t he higher blood venom, the longer hospita lization time is
Trang 10Table 3.38: Comparision of total dose and duration of antivenom
be tween seve rity groups (PSS)
Non-symptom 1
(n=23)
Mil d 2 (n=28)*
Moderate 3 (n=51)
Severe 4 (n=31)
Death (n=1 )
Total dose
of
antrivenom
(via ls)
p<0,001
(0,5-30)
30 (1-60)
40 (10-72)
60
p (2-3) <0,001; p (2-4) <0,001; p (3-4) <0,001 Duratrion
of
antivenom
(hours)
P<0,001
(0,5-77)
10 (0,5-120)
12 (3-50)
6
p (2-3) <0,001; p (2-4) <0,001; p (3-4) >0,05
Data are presented as median (Min-Max)
Comment: Total dose and duration of antivenom therapy were
different among severity group
B lood venom levels and tre atme nt outcomes: completed
recovery group 7,79(0-634,54) ng/ml, lower than those in group with
loca l tissue sequalae 88,04 (0-996,18) ng/ml (P<0,001)
Figure 3.13 Change in the blood ve nom levels before and afte r
antive nom the rapy
Blood venom level before antivenom: 85,92 (0,11-996,18) ng/ml,
blood venom level after antivenom: 0,21 (0-1,1) ng/ml (P<0,001)
Comment: Blood venom levels were very scattered before
antivenom, however they all decreased to below 1ng/ml (a lmost approaching the level of 0 ng/ml)