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: Associate prof Pham Due, PhD 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 evaluation board at: time day month year The thesis can be assessed at: National library The library of the Institute for scientific research on clinical medicine and pharmacy 108 ABS TRACT OF DOCTORAL THES IS HANOI - 2019 INTRODUCTION CHAPTER OVERVIEW 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 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: Specifying c linical fearures, blood venom levels in the cobra bitten patients treated at the Poison control center of Bach Mai hospita l 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 zones with the highest death rate Researches and reports from Bach Mai hosita l and Cho Ray hospital show that 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 Vietnam: N atra (Cantor, 1842): “rắn hổ mang miền Bắc”, in V ietnamese Distribution: widely distributed in the North of V ietnam N kaouthia (Lesson, 1831): in Vietnamese, “rắn hổ đất” Distribution: widely distributed in the South of Vietnam N siamensis (Laurenti, 1768): in V ietnamese, “rắn hổ mèo”, only distributed in the South 1.2 Disgnosis and treatment of cobrabite: 1.2.1 Signs and symptoms: a Snakebite by N atra, N.kaouthia: - Local: there is ussually mechanical injury at the bitten site 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 above mentioned sepcies but more frequent and severe Paralysis has not 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 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 to 5ng/ml and performing duration is within 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 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 symptoms 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 situation which may make clinical doctors meet difficult CHAPTER 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: TP+FN = Z2 : constant of normal distribution, with α = 0,05, Z = 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 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 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:  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 the Taiwan colleagues according to the co-operative program between the 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 decision of the completion of antivenom therapy ­ The study was devided in to 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 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 < 0,05 Re lationships between factors or variables are explored Odd ratio (OR) to evaluate the risks of getting more severe condition CHAPTER RESULTS 3.1 Characte ristics of patie nts: 1297 snakebite patients were admitted at the poison center from 1/2013 to 12/2015 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 Exposure, 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%, vesicles 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 systematic signs & symptoms of cobra species Cobra species (n, %) P Syste matic signs, symptoms N atra N kaouthia (n=122) (n=20) Nauea, vomittng 22 (18,0) (45,0) 0,05 Ptosis (3,3) (15,0) >0,05 Difficult speech (4,9) (10,0) >0,05 Pharyngeal paralysis (3,3) (10,0) >0,05 Intercostal, diaphramic paralysis (3,3) (10,0) >0,05 Limb paralysis (3,3) (5,0) >0,05 Respiratory failure (due to (3,3) (10,0) >0,05 paralysis) Dilated pupils (0) (0) >0,05 Sinus tachycardia 24 (19,7) (25,0) >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 symptoms are see n in all bite s by c obra spec ies: nausea, vomitting, abdomina l pa in, dia rrhea Less freque nt signs, symptoms: paralysis of pharynx, respira 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 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 (P0,05 0,05 >0,05