Clinical Toxicology (2014), 52, 187–191 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2014.887725
CRITICAL CARE
Rapid diagnosis of Naja atra snakebites D. Z. HUNG,1,2 J. H. LIN,1 J. F. MO,1 C. F. HUANG,3 and M. Y. LIAU4 1Division
of Toxicology, China Medical University Hospital, Taichung, Taiwan Institute of Clinical Medical Science, College of Medicine, China Medical University, Taichung, Taiwan 3School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan 4Department of Biotechnology, Fooyin University, Kaohsiung, Taiwan Clinical Toxicology Downloaded from informahealthcare.com by University of Massachusetts on 04/02/15 For personal use only.
2Graduate
Background. The clinical diagnosis of snakebites is critical and necessary in many parts of the world, especially in Southeastern Asia, where venomous snakebites are a burden on public health. It is difficult to define or recognize the species of venomous snake because of the overlapping clinical manifestations of envenomations. A quick and reliable method for identifying the snake species is necessary. We designed and tested a strip of lateral flow system for the diagnosis of cobra snake bites in Taiwan. Methods. We developed a kit based on an immunochromatographic method for rapid detection of cobra (Naja atra) venom in human serum. The test and control lines composed of 1 mg/ml polyclonal duck antivenom and 0.5 mg/ml goat anti-rabbit immunoglobulin antibody solutions, respectively, were coated on nitrocellulose strips. Colloidal gold was conjugated with rabbit polyclonal anti-cobra venom antibodies. From July 2007 to December 2012, we used the kit to test serum from snakebite patients and to examine the agreement between our rapid test and the currently used sandwich enzyme-linked immunosorbent assay (ELISA). Results. Our kit was able to detect cobra venom in serum samples in 20 minutes with a detection limit of 5 ng/ml. An absence of cross-reactivity with other non-cobra venoms from Taiwan was noted in vitro. A total of 88 snakebite patients (34 cobra and 54 other non-cobra) were tested. The sensitivity of the strips based on the ELISA results was 83.3% and the specificity was 100%. There was a strong agreement between the results of the ELISA and immunochromatographic strips (κ ⫽ 0.868). Discussion and conclusions. This data indicates that an immunochromatographic strip might be suitable for cobra venom detection and could be used as a quick diagnostic tool in cases of N. atra snakebite. Keywords
Immunochromatography; Cobra; Snake bite
Introduction
In Taiwan, there are four types of antivenom produced to treat six species of venomous snake bites, one for the Elapidae family and three for Viperidae. Among the Viperidae snakes, envenomation by Deinagkistrodon acutus, Protobothrops mucrosquamatus, Trimeresurus stejnegeri, and Daboia siamensis can lead to local tissue swelling, erythematous changes, ecchymosed skin, systemic bleeding, and even local necrosis.4,8 Ovophis monticola and Oxyus gracilis are other two kinds of vipers, rarely found but with clinically similar to P. mucrosquamatus snakebite. In addition to its neurotoxic character, Naja atra envenomation, unlike that of another Elapidae member, Bungarus multicinctus, has frequently been reported to induce local tissue swelling, erythema, and tissue necrosis. Cobra snakebite is frequently misdiagnosed and can lead to worsened local destructive lesions if left without specific antivenom treatment.9 It should be diagnosed and antivenom therapy should be given as early as possible to reduce advanced tissue necrosis and possible repeated surgeries.2,3,9 Snake venoms largely consist of polypeptide toxins and other proteins with specificity for a wide range of tissue receptors, making them scientifically fascinating but clinically challenging. There are numerous serological procedures available for the detection of snake venom, including radioimmunoassay, agglutination assay, fluorescence immunoassay, and
Snake envenomation has been recently recognized as one of the neglected tropical diseases by the World Health Organization and urgently requires international support to assess the advanced epidemiological studies, engage health education, and produce effective antivenom.1 Snakebite envenomations occur frequently and pose serious hazards, especially in rural areas of tropical developing countries, such as those in Southern Asia, Africa, and Latin-America.2,3 Death or disability from snakebite envenomation is often preventable if an effective antivenom is administered appropriately. However, multiple species of venomous snakes are found in most areas of the world. For example, there are about six species of clinically important venomous snakes in Taiwan. It can be noted that the bites of different snakes may cause similar or overlapping clinical manifestations. Correct diagnosis and prompt antivenom treatment are therefore critical for improving patient outcome.2–7 Received 13 November 2013; accepted 22 January 2014. Address correspondence to Dong-Zong Hung, M.D., Ph.D., Division of Toxicology, China Medical University Hospital and Graduate Institute of Clinical Medical Science, College of Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: ⫹ 886-4-22333916. E-mail: [email protected]
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enzyme-linked immunosorbent assay (ELISA).10 ELISA has a greater practical use than other tests.10–12 ELISA has been used in the clinical diagnosis of snakebites, to monitor the antivenom dose, to study the clinical syndromes associated with envenomation, to detect venom in forensic cases, and to evaluate first aid techniques.10–14 However, the technique is not easy to apply in the field as it is time consuming and requires special equipment and reagents.2,9–12 The immunochromatographic test (ICT) has been popularly applied for the serological diagnosis of many protein-based diseases because of its userfriendly platform, rapid result generation, and high degrees of detection sensitivity and specificity.15–18 In this study, we developed a rapid diagnostic test using an immunochromatographic method to detect cobra venom in serum samples from snakebite patients. We tested the sensitivity and specificity of the rapid cobra test as comparing with the ELISA method.
Materials and methods Ethics statement The study protocol was approved by the Institutional Review Board (IRB) of Taichung Veteran General Hospital (registration number: 930219/298) in 2007 and China Medical University (registration number:DMR97-IRB-037), Taiwan in 2008. Informed consent documents to perform the quick test for the study were signed by patients or by their parents for patients under 18 years of age. The data were analyzed anonymously. Antibodies and antigens Both rabbit polyclonal and duck polyclonal antibodies raised against Taiwan cobra (N. atra) venom were donated by the Center of Disease Control (CDC), Taiwan.19 These antivenoms were affinity purified with Protein A Sepharose 4B (Zymed Laboratories, Inc., USA) and dialyzed overnight against PBS. Antivenom concentration was determined and then adjusted to 1 mg/ml before use. Purified goat anti-rabbit immunoglobulin antibody was purchased from Jackson ImmunoResearch Laboratories, Inc., USA. Pooled venoms of N. atra, B. multicinctus, D. acutus, P. mucrosquamatus, T stejnegeri, and D. siamensis were all donated by the CDC, Taiwan.
wavelength scan. The pH of the colloidal gold solution was adjusted to 7.0 with 0.1 M carbonate buffer (pH 9.6). Conjugation of antibodies to colloidal gold was performed by incubating 2 μg rabbit polyclonal anti-N. atra venom antibody and 1 ml of gold (A540 ⫽ 3) for 1 h with gentle stirring. After blocking with 20 mM borate buffer (pH 8) containing 1% BSA, the preparation was centrifuged at 39,000 ⫻ g for 30 min. The pellet was then re-suspended in PBS containing 2% sucrose and 1% BSA (A540 ⫽ 40) for storage at 4°C. Preparation of the immunochromatographic assay kit (ICT-Cobra) The test lines were prepared with 1 mg/ml duck polyclonal anti-N. atra venom solution, and the control lines with 0.5 mg/ml goat anti-rabbit immunoglobulin antibody solution. They were dispensed at a flow rate of 1 μl/cm on AE98 membranes supplied in laminated cards. Conjugate pads were soaked in buffer consisting of 0.1 M Borax buffer (pH 8.2), 0.25% Triton X-100 and 0.1% PVP-40, and then dried at room temperature overnight. The conjugated gold was dispensed at a flow rate of 1 μl/cm on pretreated conjugate pads and then dried at 37°C for 4 hours. After assembling all the components, the cards were cut into 0.4 mm-wide strips and inserted in a plastic housing (Fig. 1). The ready-for-use devices were stored in sealed aluminum bags with desiccant. Venom detection with the ICT-Cobra kit in vitro Venoms from six venomous Taiwanese snakes were dissolved in serum donated by laboratory staff at concentrations of 0, 1, 5, 10, 20, 50, and 100 ng/ml. ICT-Cobra kits were used to check the detection limit. The measurements for each concentration were performed in triplicate. Clinical study with the ICT-Cobra kit We performed the ICT-Cobra tests in an emergency room (ER). All patients with suspected snakebite admitted to the
Membranes and pads Nitrocellulose membranes (AE98) were used in the lateralflow assay (Whatman lnc., England). The membranes were laminated on GL-187 (G&L Precision Die Cutting Inc., USA) backing cards. The glass fiber for the conjugate pad was grade 6613 (Ahlstrom Corp., Finland), the sample pad grade 8964 (Ahlstrom Corp., Finland), and the adsorbent pad grade 205 (Whatman lnc., England). Colloidal gold-antibody conjugate preparation Colloidal gold was prepared by reduction of tetrachloroauric acid with trisodium citrate; particles were approximately 30 nm in diameter as determined by a spectrophotometric
Fig. 1. The cobra immunochromatographic strip. Samples with a venom concentration of 5 and 50 ng/ml (T) produced a positive line (light red) (colour version of this figure can be found in the online version at www.informahealthcare.com/ctx). Clinical Toxicology vol. 52 no. 3 2014
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Immunochromatographic detection of cobra venom 189 ER were enrolled in the study. After informed consent was obtained from patients or their parents (in the case of 2 girls ages 9 and 13), 5 ml of blood was collected in serum separation tube and centrifuged to obtain the serum for the quick test. The remaining samples were sent to our laboratory and stored at ⫺ 25°C for further ELISA tests as described previously.9,20 All patients were treated with specific antivenoms based on the patients’ history, clinical presentations, and the results of the ICT-Cobra rapid test, and their clinical results were followed up by a toxicologist. The final diagnosis or confirmation of the offending snakes was performed using the captured snakes and/or the results of the ELISA. The specificity and sensitivity of the rapid kit as compared to the final diagnosis of the snakebites were calculated and analyzed. The kappa statistic was used to assess the agreement between the results of the immunochromatographic strips and ELISA. Kappa statistic values more than 0.75, 0.40–0.75, and less than 0.40 represented excellent agreement, good to fair agreement, and poor agreement, respectively.21
Results ICT-Cobra kit for detection of cobra venom in vitro The detection limit of the ICT strip from N. atra venom was 5 ng/ml in donated serum (Fig. 1). There was no crossreactivity with venom samples of up to 100 ng/ml of venoms from B. multicinctus, D. acutus, P. mucrosquamatus, T. stejnegeri, and D. siamensis. Stability of the ICT-Cobra kit The ICT-Cobra kits were stored at 60°C for 1 week. Such high temperature is believed to accelerate the deterioration of the strip, as 1 week of accelerated aging is equal to 1 year under standard storage conditions. The detection limit of the test strips was 20 ng/ml after 1 week of storage at 60°C. Therefore, the test strips can possibly be stored at room temperate for up to 1 year without any significant loss of activity. We recommend that the strips should be refrigerated to help preserve their stability.21 Clinical efficacy and cross-reactivity of the ICT-Cobra kit Ninety-one cases of suspected snakebite were sent to the ER from July 2007 to December 2012. Three cases were excluded due to other injuries and lack of blood samples. In total, 88 patients (41 females and 47 males, ages 7–81, age median: 54) were recruited. There were 34 cases of cobra envenomation, 24 of V. stejnegeri, 23 of P. mucrosquamatus, one of D. acutus and six of unknown or nonvenomous snakes. In all 54 cases of non-cobra snakebites, the ICT-Cobra test showed negative results and had a specificity of 100% [54/ (54 ⫹ 0) ⫻ 100% ⫽ 100%]. In the 34 cases with a final diagnosis of cobra snakebite, nine blood samples showed false negative ICT-Cobra test results (ELISA results: 0–6.6 ng/ ml). Three of these nine patients were admitted to the ER for wound management several days after the snakebite and antivenom (antivenom for B. multicinctus and N. atra, Copyright © Informa Healthcare USA, Inc. 2014
CDC, Taiwan) therapy, blood samples from four patients were taken just a few hours after antivenom administration, and two cases were noted to be dry bites. There were four cases with negative ELISA results (⬍ 1 ng/ml), including two cases of dry bites and two cases where antivenom was administered. Overall, 25 cases of cobra envenomation had positive ICT-Cobra test results, indicating that the sensitivity of ICT-Cobra strip was 73.5% (25/34 ⫻ 100% ⫽ 73.5%, 95% CI 0.80–1.10). The positive predictive value of the strip in this study was found to be 100% [25/(25 ⫹ 0) ⫻ 100%], and the negative predictive value 85.7% [(54/54 ⫹ 9) ⫻ 100%]. There was a strong correlation between the results of the ELISA and ICT-Cobra strips (κ ⫽ 0.868, 95% fiducial limits for kappa is 0.7571–0.9794, Table 1).
Discussion In this study, we found that an ICT could detect amounts of N. atra venom as low as 5 ng/ml in vitro, and lacked cross-reactivity with the venoms of other major venomous snakes in Taiwan. Using serum from patients, the ICT-Cobra test also produced promising results for the rapid diagnosis of cobra snakebites. Rapid assessment might give patients an opportunity for better recovery and less destructive consequences.22 Identification of envenoming species is important in management of venomous snake bites.2 Syndromatic approach of envenoming by analysis of a series of reliably identified cases might be useful in some regions.23 Captured or killed snake and recognition from pictures have been esteemed to be a guideline by some emergency physicians in Taiwan due to the distinct appearance and some generally recognizable behavioral characteristics of venomous snakes in this island. However, N. atra, as envenoming by some other Naja spp. (e.g., the Asian or common cobra, Naja naja) can often cause significant local tissue damage characterized by swelling, blistering, and ecchymosis, and thus making it difficult to differentiate from viper snakebites in the early stages.2,9,24,25 Misidentification and subsequently late antivenom treatment continue to be frequently encountered, leading to significant local destructive lesions. According to Warrel,2 antivenom is the only specific antidote to snake venom, and it should be given as soon as it is indicated. Also, appropriate antivenom therapy has been noted to reverse systemic envenoming as long as systemic abnormality persists; however, this Table 1. Agreement between the ICT-Cobra and ELISA tests. ELISA ICT-Cobra Positive Negative Total
Positive
Negative
κ
Total
25 5 30
0 58 58
0.868
25 63 88
Relative to ELISA, specificity of ICT-Cobra: 58/58 ⫻ 100% ⫽ 100%; sensitivity of ICT-Cobra: 25/30 ⫻ 100% ⫽ 83.33%; Observed agreement(Po) ⫽ (58 ⫹ 25)/88 ⫽ 94.32%; Expected agreement(Pe) ⫽ [(30/88) ⫻ (25/88) ⫹ (58/88) ⫻ (63/88)] ⫽ 0.569; Kappa ⫽ (Po-Pe)/(1-Pe) ⫽ (0.943–0.569)/(1–0.569) ⫽ 0.868. 95% fiducial limits 0.7571–0.9794.
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is controversial to local necrosis.2 In some cases of N. atra envenoming with detected high blood venom level, we had found that the patients suffered from less tissue injuries if they called on hospital earlier for treatment, which were also reported in cases of African cobra envenoming that antivenom administration might limit further tissue damage.9,26 Thereafter, early recognition and confirmation by immunological detection of venom antigens in patient’s blood or tissue fluids might save patients from advanced tissue necrosis and repeated surgical interventions. ELISA is useful for detection of snake venom in body fluid of victims, in differential diagnosis, and in assessing the severity of cobra snakebite,9–12 but it requires 5–6 hours to attain the result and is not always readily available. Some modified ELISA tests (e.g., snake venom detection kit, SVDK, CSL, Melbourne, Australia) are commonly used to confirm the identity of a venomous snake suspected of inflicting an envenomation. These kits also can provide a relatively rapid result in 30–45 min.27 But, their popularity is limited due to their complexity, species cross-reactivity, and regional limitations.28 In contrast, ICT tests have been commonly applied for the serodiagnosis of many diseases, including microorganismor drugs-induced. The most well-known application is the pregnancy test,29 the results of which are usually interpretable within 10–20 minutes. The ICT test is an inexpensive, rapid, disposable, membrane-based assay for many diseases, which provides visual evidence for the presence of a substance in a liquid sample.30–32 The sensitivity and selectivity of ICT are achieved using analyte-specific antibodies to label the analyte or recognition element. In cases of unwitnessed cobra snakebites, a quick strip test could avoid unnecessary antivenom usage and might prevent patients from undergoing several rounds of surgical debridement. Most importantly, the test can be used in remote areas or areas where snakebites are frequent, but are lacking in diagnostic facilities.31 Our ICT-Cobra kit was proven to have good specificity for N. atra venom. The detection limit was 5 ng/ml of venom in human serum. There was no cross-reactivity with the viperid venoms observed in patient samples and in vitro. However, ICT-Cobra test negative results occurred in patients (seven cases in this study) who had been administered antivenom treatment and could be due to the decreased venom concentration in the serum. Although the ELISA method had a lower detection limit and higher sensitivity, we found that there was an almost perfect agreement between the ELISA method and ICT-Cobra test for detecting N. atra venom in patient serum as determined by the Kappa test.33,34 Our strip test was proven to be useful for guiding clinical diagnosis and antivenom treatment in cases of Taiwan cobra snakebites. Other types of specimens, such as patient urine or wound discharge, have been suggested as a substitute for serum in some ELISA tests used to identify snake species,9–11 and could also be compatible with the ICT-Cobra test. However, the stability of our strip when used for other types of samples needs further investigation and verification. In conclusion, it is feasible to use an immunochromatographic strip to rapidly detect N. atra venom in patient
serum. Our ICT-Cobra strip detects only N. atra venom without cross-reactivity with other principal venomous species in Taiwan. The strips probably can be stored up to 1 year under standard storage conditions. This assay provides a rapid, simple, and accurate method for clinical identification of individuals envenomated by Naja atra.
Acknowledgments The authors thank the Vaccine Center of the Center for Disease Control (CDC) for their generous supply of lyophilized snakes’ antivenin.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. Part of this work was supported by the grants from the National Science Council, Taiwan (NSC 96-2314-B-039028 and 100-2923-B-039001).
References 1. Gutiérrez JM, Theakston RDG, Warrell DA. Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Med 2006; 3:e150. 2. Warrell DA. Snake bite. Lancet 2010; 375:77–88. 3. Alirol E, Sharma SK, Bawaskar HS, Kuch U, Chappuis F. Snake bite in South Asia: a review. PLoS Negl Trop Dis 2010; 4:e603. 4. Hung DZ. Taiwan’s venomous snakebite: epidemiological, evolution and geographic differences. Trans R Soc Trop Med Hyg 2004; 98:96–101. 5. Saravu K, Somavarapu V, Shastry AB, Kumar R. Clinical profile, species-specific severity grading, and outcome determinants of snake envenomation: An Indian tertiary care hospital-based prospective study. Indian J Crit Care Med 2012; 16:187–192. 6. Gold BS, Dart RC, Barish RA. Bites of venomous snakes. N Engl J Med 2002; 347:347–356. 7. Williams D, Gutiérrez JM, Harrison R, Warrell DA, White J, Winkel KD, Gopalakrishnakone P; Global Snake Bite Initiative Working Group, International Society on Toxinology. The Global Snake Bite Initiative: an antidote for snake bite. Lancet 2010; 375:89–91. 8. Chen YW, Chen MH, Chen YC, Hung DZ, Chen CK, Yen DHT, et al. Differences in Clinical Profiles of Patients with Protobothrops mucrosquamatus and Viridovipera stejnegeri Envenoming in Taiwan. Am J Trop Med Hyg 2009; 80:28–32. 9. Hung DZ, Liau MY, Lin-Shiau SY. The clinical significance of venom detection in patients of cobra snakebite. Toxicon 2003; 41:409–415. 10. Selvanayagam ZE, Gopalakrishnakone P. Tests for detection of snake venoms, toxins and venom antibodies: review on recent trends (1987– 1997). Toxicon 1999; 37:565–586. 11. Kulawickrama S, O’Leary MA, Hodgson WC, Brown SG, Jacoby T, Davern K, Isbister GK. Development of a sensitive enzyme immunoassay for measuring taipan venom in serum. Toxicon 2010; 55:1510–1518. 12. Dong LV, Quyen LK, Eng KH, Gopalakrishnakone P. Immunogenicity of venoms from four common snakes in the South of Vietnam and development of ELISA kit for venom detection. J Immunol Methods 2003; 281:13–31. 13. Norris RL, Pfalzgraf RR, Laing G. Death following coral snake bite in the United States—first documented case (with ELISA confirmation of envenomation) in over 40 years. Toxicon 2009; 53: 693–697. 14. Morais V, Berasain P, Ifrán S, Carreira S, Tortorella MN, Negrín A, Massaldi H. Humoral immune responses to venom and antivenom of patients bitten by Bothrops snakes. Toxicon 2012; 59:315–319. Clinical Toxicology vol. 52 no. 3 2014
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Immunochromatographic detection of cobra venom 191 15. Leung W, Chan P, Bosgoed F, Lehmann K, Renneberg I, Lehmann M, Renneberg R. One-step quantitative cortisol dipstick with proportional reading. J Immunol Methods 2003; 281:109–118. 16. Jin S, Chang ZY, Ming X, Min CL, Wei H, Sheng LY, Hong GX. Fast dipstick dye immunoassay for detection of immunoglobulin G (IgG) and IgM antibodies of human toxoplasmosis. Clin Diag Lab Immunol 2005; 12:198–201. 17. Smits HL, Eapen CK, Sugathan S, Kuriakose M, Gasem MH, Yersin C, et al. Lateral-flow assay for rapid serodiagnosis of human leptospirosis. Clin Diag Lab Immunol 2001; 8:166–169. 18. Posthuma-Trumpie GA, Korf J, van Amerongen A. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. a literature survey. Anal Bioanal Chem 2009; 393:569–582. 19. Chiou VY. The development of IgY(delta Fc) antibody based neuro toxin antivenoms and the study on their neutralization efficacies. Clin Toxicol (Phila) 2008; 46:539–544. 20. Huang YP, Yu YJ, Hung DZ. Sandwich enzyme-linked immunosorbent assay for Taiwan cobra venom. Vet Hum Toxicol 2002; 44:200–204. 21. Chen K, Zhao K, Song D, He W, Gao W, Zhao C, et al. Development and evaluation of an immunochromatographic strip for rapid detection of porcine hemagglutinating encephalomyelitis virus. Virol J 2012; 9:172. 22. Sankar J, Nabeel R, Sankar MJ, Priyambada L, Mahadevan S. Factors affecting outcome in children with snake envenomation: a prospective observational study. Arch Dis Child 2013; 98:596–601. 23. Ariaratnam CA, Sheriff MHR, Arambepola C, Theakston RDG, Warrell DA. Syndromic approach to treatment of snake bite in Sri Lanka based on results of a prospective national hospital-based survey of patients envenomed by identifi ed snakes. Am J Trop Med Hyg 2009; 81:725–731. 24. Warrell DA. Clinical toxicology of snakebite in Asia. In: Meier J, White J, eds. Handbook of Clinical Toxicology of Animal Venoms and Poisons. Boca Raton (Florida): CRC Press. 1995:493–594.
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25. Liao WB, Lee CW, Tsai YS, Liu BM, Chung KJ. Influential factors affecting prognosis of snakebite patients management: Kaohsiung Chang Gung Memorial Hospital experience. Chang Gung Med J 2000; 23:577–583. 26. Muller GJ, Wium CA, Modler H, Veale DJ. Snake bite in southern Africa: diagnosis and management. CME 2012; 30:362–381. 27. Sutherland SK. Rapid venom identification: availability of kits. Med J Aust 1979; 2:602–603. 28. Steuten J, Winkel K, Carroll T, Williamson NA, Ignjatovic V, Fung K, et al. The molecular basis of cross-reactivity in the Australian Snake Venom Detection Kit (SVDK). Toxicon 2007; 50:1041–1952. 29. Leuvering JH, Goverde BC, Thal PJ, Schuurs AH. A homogeneous sol particle immunoassay for human chorionic gonadotrophin using monoclonal antibodies. J Immunol Methods 1983; 60:9–23. 30. Ching KH, Lin A, McGarvey JA, Stanker LH, Hnasko R. Rapid and selective detection of botulinum neurotoxin serotype-A and -B with a single immunochromatographic test strip. J Immunol Methods 2012; 380:23–29. 31. Stauffer WM, Cartwright CP, Olson DA, Juni BA, Taylor CM, Bowers SH, et al. Diagnostic performance of rapid diagnostic tests versus blood smears for malaria in US clinical practice. Clin Infect Dis 2009; 49:908–913. 32. Ahmed K, Wimalaratne O, Dahal N, Khawplod P, Nanayakkara S, Rinzin K, et al. Evaluation of a monoclonal antibody-based rapid immunochromatographic test for direct detection of rabies virus in the brain of humans and animals. Am J Trop Med Hyg 2012; 86:736–740. 33. Viera AJ, Garrett JM. Understanding interobserver agreement: the kappa statistic. Fam Med 2005; 37:360–363. 34. McGinn T, Wyer P, Newman T, Keitz S, Leipzig R, Guyatt G, and The evidence-based medicine teaching tips working group. Tips for learners of evidence-based medicine: 3. Measures of observer variability (kappa statistic). CMAJ 2004; 171:1369–1373.
CRITICAL CARE
Rapid diagnosis of Naja atra snakebites D. Z. HUNG,1,2 J. H. LIN,1 J. F. MO,1 C. F. HUANG,3 and M. Y. LIAU4 1Division
of Toxicology, China Medical University Hospital, Taichung, Taiwan Institute of Clinical Medical Science, College of Medicine, China Medical University, Taichung, Taiwan 3School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan 4Department of Biotechnology, Fooyin University, Kaohsiung, Taiwan Clinical Toxicology Downloaded from informahealthcare.com by University of Massachusetts on 04/02/15 For personal use only.
2Graduate
Background. The clinical diagnosis of snakebites is critical and necessary in many parts of the world, especially in Southeastern Asia, where venomous snakebites are a burden on public health. It is difficult to define or recognize the species of venomous snake because of the overlapping clinical manifestations of envenomations. A quick and reliable method for identifying the snake species is necessary. We designed and tested a strip of lateral flow system for the diagnosis of cobra snake bites in Taiwan. Methods. We developed a kit based on an immunochromatographic method for rapid detection of cobra (Naja atra) venom in human serum. The test and control lines composed of 1 mg/ml polyclonal duck antivenom and 0.5 mg/ml goat anti-rabbit immunoglobulin antibody solutions, respectively, were coated on nitrocellulose strips. Colloidal gold was conjugated with rabbit polyclonal anti-cobra venom antibodies. From July 2007 to December 2012, we used the kit to test serum from snakebite patients and to examine the agreement between our rapid test and the currently used sandwich enzyme-linked immunosorbent assay (ELISA). Results. Our kit was able to detect cobra venom in serum samples in 20 minutes with a detection limit of 5 ng/ml. An absence of cross-reactivity with other non-cobra venoms from Taiwan was noted in vitro. A total of 88 snakebite patients (34 cobra and 54 other non-cobra) were tested. The sensitivity of the strips based on the ELISA results was 83.3% and the specificity was 100%. There was a strong agreement between the results of the ELISA and immunochromatographic strips (κ ⫽ 0.868). Discussion and conclusions. This data indicates that an immunochromatographic strip might be suitable for cobra venom detection and could be used as a quick diagnostic tool in cases of N. atra snakebite. Keywords
Immunochromatography; Cobra; Snake bite
Introduction
In Taiwan, there are four types of antivenom produced to treat six species of venomous snake bites, one for the Elapidae family and three for Viperidae. Among the Viperidae snakes, envenomation by Deinagkistrodon acutus, Protobothrops mucrosquamatus, Trimeresurus stejnegeri, and Daboia siamensis can lead to local tissue swelling, erythematous changes, ecchymosed skin, systemic bleeding, and even local necrosis.4,8 Ovophis monticola and Oxyus gracilis are other two kinds of vipers, rarely found but with clinically similar to P. mucrosquamatus snakebite. In addition to its neurotoxic character, Naja atra envenomation, unlike that of another Elapidae member, Bungarus multicinctus, has frequently been reported to induce local tissue swelling, erythema, and tissue necrosis. Cobra snakebite is frequently misdiagnosed and can lead to worsened local destructive lesions if left without specific antivenom treatment.9 It should be diagnosed and antivenom therapy should be given as early as possible to reduce advanced tissue necrosis and possible repeated surgeries.2,3,9 Snake venoms largely consist of polypeptide toxins and other proteins with specificity for a wide range of tissue receptors, making them scientifically fascinating but clinically challenging. There are numerous serological procedures available for the detection of snake venom, including radioimmunoassay, agglutination assay, fluorescence immunoassay, and
Snake envenomation has been recently recognized as one of the neglected tropical diseases by the World Health Organization and urgently requires international support to assess the advanced epidemiological studies, engage health education, and produce effective antivenom.1 Snakebite envenomations occur frequently and pose serious hazards, especially in rural areas of tropical developing countries, such as those in Southern Asia, Africa, and Latin-America.2,3 Death or disability from snakebite envenomation is often preventable if an effective antivenom is administered appropriately. However, multiple species of venomous snakes are found in most areas of the world. For example, there are about six species of clinically important venomous snakes in Taiwan. It can be noted that the bites of different snakes may cause similar or overlapping clinical manifestations. Correct diagnosis and prompt antivenom treatment are therefore critical for improving patient outcome.2–7 Received 13 November 2013; accepted 22 January 2014. Address correspondence to Dong-Zong Hung, M.D., Ph.D., Division of Toxicology, China Medical University Hospital and Graduate Institute of Clinical Medical Science, College of Medicine, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: ⫹ 886-4-22333916. E-mail: [email protected]
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enzyme-linked immunosorbent assay (ELISA).10 ELISA has a greater practical use than other tests.10–12 ELISA has been used in the clinical diagnosis of snakebites, to monitor the antivenom dose, to study the clinical syndromes associated with envenomation, to detect venom in forensic cases, and to evaluate first aid techniques.10–14 However, the technique is not easy to apply in the field as it is time consuming and requires special equipment and reagents.2,9–12 The immunochromatographic test (ICT) has been popularly applied for the serological diagnosis of many protein-based diseases because of its userfriendly platform, rapid result generation, and high degrees of detection sensitivity and specificity.15–18 In this study, we developed a rapid diagnostic test using an immunochromatographic method to detect cobra venom in serum samples from snakebite patients. We tested the sensitivity and specificity of the rapid cobra test as comparing with the ELISA method.
Materials and methods Ethics statement The study protocol was approved by the Institutional Review Board (IRB) of Taichung Veteran General Hospital (registration number: 930219/298) in 2007 and China Medical University (registration number:DMR97-IRB-037), Taiwan in 2008. Informed consent documents to perform the quick test for the study were signed by patients or by their parents for patients under 18 years of age. The data were analyzed anonymously. Antibodies and antigens Both rabbit polyclonal and duck polyclonal antibodies raised against Taiwan cobra (N. atra) venom were donated by the Center of Disease Control (CDC), Taiwan.19 These antivenoms were affinity purified with Protein A Sepharose 4B (Zymed Laboratories, Inc., USA) and dialyzed overnight against PBS. Antivenom concentration was determined and then adjusted to 1 mg/ml before use. Purified goat anti-rabbit immunoglobulin antibody was purchased from Jackson ImmunoResearch Laboratories, Inc., USA. Pooled venoms of N. atra, B. multicinctus, D. acutus, P. mucrosquamatus, T stejnegeri, and D. siamensis were all donated by the CDC, Taiwan.
wavelength scan. The pH of the colloidal gold solution was adjusted to 7.0 with 0.1 M carbonate buffer (pH 9.6). Conjugation of antibodies to colloidal gold was performed by incubating 2 μg rabbit polyclonal anti-N. atra venom antibody and 1 ml of gold (A540 ⫽ 3) for 1 h with gentle stirring. After blocking with 20 mM borate buffer (pH 8) containing 1% BSA, the preparation was centrifuged at 39,000 ⫻ g for 30 min. The pellet was then re-suspended in PBS containing 2% sucrose and 1% BSA (A540 ⫽ 40) for storage at 4°C. Preparation of the immunochromatographic assay kit (ICT-Cobra) The test lines were prepared with 1 mg/ml duck polyclonal anti-N. atra venom solution, and the control lines with 0.5 mg/ml goat anti-rabbit immunoglobulin antibody solution. They were dispensed at a flow rate of 1 μl/cm on AE98 membranes supplied in laminated cards. Conjugate pads were soaked in buffer consisting of 0.1 M Borax buffer (pH 8.2), 0.25% Triton X-100 and 0.1% PVP-40, and then dried at room temperature overnight. The conjugated gold was dispensed at a flow rate of 1 μl/cm on pretreated conjugate pads and then dried at 37°C for 4 hours. After assembling all the components, the cards were cut into 0.4 mm-wide strips and inserted in a plastic housing (Fig. 1). The ready-for-use devices were stored in sealed aluminum bags with desiccant. Venom detection with the ICT-Cobra kit in vitro Venoms from six venomous Taiwanese snakes were dissolved in serum donated by laboratory staff at concentrations of 0, 1, 5, 10, 20, 50, and 100 ng/ml. ICT-Cobra kits were used to check the detection limit. The measurements for each concentration were performed in triplicate. Clinical study with the ICT-Cobra kit We performed the ICT-Cobra tests in an emergency room (ER). All patients with suspected snakebite admitted to the
Membranes and pads Nitrocellulose membranes (AE98) were used in the lateralflow assay (Whatman lnc., England). The membranes were laminated on GL-187 (G&L Precision Die Cutting Inc., USA) backing cards. The glass fiber for the conjugate pad was grade 6613 (Ahlstrom Corp., Finland), the sample pad grade 8964 (Ahlstrom Corp., Finland), and the adsorbent pad grade 205 (Whatman lnc., England). Colloidal gold-antibody conjugate preparation Colloidal gold was prepared by reduction of tetrachloroauric acid with trisodium citrate; particles were approximately 30 nm in diameter as determined by a spectrophotometric
Fig. 1. The cobra immunochromatographic strip. Samples with a venom concentration of 5 and 50 ng/ml (T) produced a positive line (light red) (colour version of this figure can be found in the online version at www.informahealthcare.com/ctx). Clinical Toxicology vol. 52 no. 3 2014
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Immunochromatographic detection of cobra venom 189 ER were enrolled in the study. After informed consent was obtained from patients or their parents (in the case of 2 girls ages 9 and 13), 5 ml of blood was collected in serum separation tube and centrifuged to obtain the serum for the quick test. The remaining samples were sent to our laboratory and stored at ⫺ 25°C for further ELISA tests as described previously.9,20 All patients were treated with specific antivenoms based on the patients’ history, clinical presentations, and the results of the ICT-Cobra rapid test, and their clinical results were followed up by a toxicologist. The final diagnosis or confirmation of the offending snakes was performed using the captured snakes and/or the results of the ELISA. The specificity and sensitivity of the rapid kit as compared to the final diagnosis of the snakebites were calculated and analyzed. The kappa statistic was used to assess the agreement between the results of the immunochromatographic strips and ELISA. Kappa statistic values more than 0.75, 0.40–0.75, and less than 0.40 represented excellent agreement, good to fair agreement, and poor agreement, respectively.21
Results ICT-Cobra kit for detection of cobra venom in vitro The detection limit of the ICT strip from N. atra venom was 5 ng/ml in donated serum (Fig. 1). There was no crossreactivity with venom samples of up to 100 ng/ml of venoms from B. multicinctus, D. acutus, P. mucrosquamatus, T. stejnegeri, and D. siamensis. Stability of the ICT-Cobra kit The ICT-Cobra kits were stored at 60°C for 1 week. Such high temperature is believed to accelerate the deterioration of the strip, as 1 week of accelerated aging is equal to 1 year under standard storage conditions. The detection limit of the test strips was 20 ng/ml after 1 week of storage at 60°C. Therefore, the test strips can possibly be stored at room temperate for up to 1 year without any significant loss of activity. We recommend that the strips should be refrigerated to help preserve their stability.21 Clinical efficacy and cross-reactivity of the ICT-Cobra kit Ninety-one cases of suspected snakebite were sent to the ER from July 2007 to December 2012. Three cases were excluded due to other injuries and lack of blood samples. In total, 88 patients (41 females and 47 males, ages 7–81, age median: 54) were recruited. There were 34 cases of cobra envenomation, 24 of V. stejnegeri, 23 of P. mucrosquamatus, one of D. acutus and six of unknown or nonvenomous snakes. In all 54 cases of non-cobra snakebites, the ICT-Cobra test showed negative results and had a specificity of 100% [54/ (54 ⫹ 0) ⫻ 100% ⫽ 100%]. In the 34 cases with a final diagnosis of cobra snakebite, nine blood samples showed false negative ICT-Cobra test results (ELISA results: 0–6.6 ng/ ml). Three of these nine patients were admitted to the ER for wound management several days after the snakebite and antivenom (antivenom for B. multicinctus and N. atra, Copyright © Informa Healthcare USA, Inc. 2014
CDC, Taiwan) therapy, blood samples from four patients were taken just a few hours after antivenom administration, and two cases were noted to be dry bites. There were four cases with negative ELISA results (⬍ 1 ng/ml), including two cases of dry bites and two cases where antivenom was administered. Overall, 25 cases of cobra envenomation had positive ICT-Cobra test results, indicating that the sensitivity of ICT-Cobra strip was 73.5% (25/34 ⫻ 100% ⫽ 73.5%, 95% CI 0.80–1.10). The positive predictive value of the strip in this study was found to be 100% [25/(25 ⫹ 0) ⫻ 100%], and the negative predictive value 85.7% [(54/54 ⫹ 9) ⫻ 100%]. There was a strong correlation between the results of the ELISA and ICT-Cobra strips (κ ⫽ 0.868, 95% fiducial limits for kappa is 0.7571–0.9794, Table 1).
Discussion In this study, we found that an ICT could detect amounts of N. atra venom as low as 5 ng/ml in vitro, and lacked cross-reactivity with the venoms of other major venomous snakes in Taiwan. Using serum from patients, the ICT-Cobra test also produced promising results for the rapid diagnosis of cobra snakebites. Rapid assessment might give patients an opportunity for better recovery and less destructive consequences.22 Identification of envenoming species is important in management of venomous snake bites.2 Syndromatic approach of envenoming by analysis of a series of reliably identified cases might be useful in some regions.23 Captured or killed snake and recognition from pictures have been esteemed to be a guideline by some emergency physicians in Taiwan due to the distinct appearance and some generally recognizable behavioral characteristics of venomous snakes in this island. However, N. atra, as envenoming by some other Naja spp. (e.g., the Asian or common cobra, Naja naja) can often cause significant local tissue damage characterized by swelling, blistering, and ecchymosis, and thus making it difficult to differentiate from viper snakebites in the early stages.2,9,24,25 Misidentification and subsequently late antivenom treatment continue to be frequently encountered, leading to significant local destructive lesions. According to Warrel,2 antivenom is the only specific antidote to snake venom, and it should be given as soon as it is indicated. Also, appropriate antivenom therapy has been noted to reverse systemic envenoming as long as systemic abnormality persists; however, this Table 1. Agreement between the ICT-Cobra and ELISA tests. ELISA ICT-Cobra Positive Negative Total
Positive
Negative
κ
Total
25 5 30
0 58 58
0.868
25 63 88
Relative to ELISA, specificity of ICT-Cobra: 58/58 ⫻ 100% ⫽ 100%; sensitivity of ICT-Cobra: 25/30 ⫻ 100% ⫽ 83.33%; Observed agreement(Po) ⫽ (58 ⫹ 25)/88 ⫽ 94.32%; Expected agreement(Pe) ⫽ [(30/88) ⫻ (25/88) ⫹ (58/88) ⫻ (63/88)] ⫽ 0.569; Kappa ⫽ (Po-Pe)/(1-Pe) ⫽ (0.943–0.569)/(1–0.569) ⫽ 0.868. 95% fiducial limits 0.7571–0.9794.
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is controversial to local necrosis.2 In some cases of N. atra envenoming with detected high blood venom level, we had found that the patients suffered from less tissue injuries if they called on hospital earlier for treatment, which were also reported in cases of African cobra envenoming that antivenom administration might limit further tissue damage.9,26 Thereafter, early recognition and confirmation by immunological detection of venom antigens in patient’s blood or tissue fluids might save patients from advanced tissue necrosis and repeated surgical interventions. ELISA is useful for detection of snake venom in body fluid of victims, in differential diagnosis, and in assessing the severity of cobra snakebite,9–12 but it requires 5–6 hours to attain the result and is not always readily available. Some modified ELISA tests (e.g., snake venom detection kit, SVDK, CSL, Melbourne, Australia) are commonly used to confirm the identity of a venomous snake suspected of inflicting an envenomation. These kits also can provide a relatively rapid result in 30–45 min.27 But, their popularity is limited due to their complexity, species cross-reactivity, and regional limitations.28 In contrast, ICT tests have been commonly applied for the serodiagnosis of many diseases, including microorganismor drugs-induced. The most well-known application is the pregnancy test,29 the results of which are usually interpretable within 10–20 minutes. The ICT test is an inexpensive, rapid, disposable, membrane-based assay for many diseases, which provides visual evidence for the presence of a substance in a liquid sample.30–32 The sensitivity and selectivity of ICT are achieved using analyte-specific antibodies to label the analyte or recognition element. In cases of unwitnessed cobra snakebites, a quick strip test could avoid unnecessary antivenom usage and might prevent patients from undergoing several rounds of surgical debridement. Most importantly, the test can be used in remote areas or areas where snakebites are frequent, but are lacking in diagnostic facilities.31 Our ICT-Cobra kit was proven to have good specificity for N. atra venom. The detection limit was 5 ng/ml of venom in human serum. There was no cross-reactivity with the viperid venoms observed in patient samples and in vitro. However, ICT-Cobra test negative results occurred in patients (seven cases in this study) who had been administered antivenom treatment and could be due to the decreased venom concentration in the serum. Although the ELISA method had a lower detection limit and higher sensitivity, we found that there was an almost perfect agreement between the ELISA method and ICT-Cobra test for detecting N. atra venom in patient serum as determined by the Kappa test.33,34 Our strip test was proven to be useful for guiding clinical diagnosis and antivenom treatment in cases of Taiwan cobra snakebites. Other types of specimens, such as patient urine or wound discharge, have been suggested as a substitute for serum in some ELISA tests used to identify snake species,9–11 and could also be compatible with the ICT-Cobra test. However, the stability of our strip when used for other types of samples needs further investigation and verification. In conclusion, it is feasible to use an immunochromatographic strip to rapidly detect N. atra venom in patient
serum. Our ICT-Cobra strip detects only N. atra venom without cross-reactivity with other principal venomous species in Taiwan. The strips probably can be stored up to 1 year under standard storage conditions. This assay provides a rapid, simple, and accurate method for clinical identification of individuals envenomated by Naja atra.
Acknowledgments The authors thank the Vaccine Center of the Center for Disease Control (CDC) for their generous supply of lyophilized snakes’ antivenin.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. Part of this work was supported by the grants from the National Science Council, Taiwan (NSC 96-2314-B-039028 and 100-2923-B-039001).
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