- Email: [email protected]
232. Death Adder Envenoming Causes Neurotoxicity not Reversed by Antivenom - Australian Snakebite Project (ASP-16)
214
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
neglected tropical pathology of snakebite envenoming. A deep knowledge of the toxin composition and the immunological profile of venoms are central for developing polyspecific antivenoms exhibiting broad paraspecificity and cross-reactivity against the most medically-relevant snakes of a given geographical area. Preclinical neutralization tests and antivenomic assessments are necessary to demonstrate antivenom safety and efficacy prior to clinical trials. Antivenomics is a proteomic tool for the qualitative and quantitative analysis of the immunoreactivity of antivenoms. The original (first generation, 1G) antivenomics protocol is based on the immunodepletion of toxins upon incubation of whole venom with purified antivenom IgGs, followed by the addition of a secondary antibody or immobilized IgGbinding moiety. Antivenom immunoreactivity is inferred indirectly through the proteomic characterization of the toxin fraction that remains in solution after immunoprecipitation. This antivenomic approach is not appropriate for F(ab’)2 antivenoms. Objective: (i) To design a new method based on affinity chromatography which allows assessing F(ab’)2 antivenoms, employing two F(ab’)2 monospecific (antiCerastes cerastes and anti-Macrovipera mauritanica) antivenoms; (ii) to compare the 1G, immunoprecipitation protocol, and the new, second generation (2G, affinity capture) antivenomic approaches. These would assess the immunological profile of the pan-African EchiTAb-PlusICPÒ whole IgG antivenom, whose immunoreactivity characteristics have been previously tested towards the venoms of a panel of African viperid snakes and spitting cobras. Methods: F(ab’)2 fragments or whole purified IgG molecules were coupled to a NHS-activated SepharoseÒ. After incubating the matrix with the venom, the nonretained fraction and the immunospecific venom components were analyzed and quantified by reverse-phase HPLC followed by venomic analysis. Results: The affinity capture protocol allowed analyzing both types of antivenoms. Furthermore the pan-African EchiTAb-Plus-ICPÒ antivenom showed qualitatively similar immunoreactivity patterns using either antivenomic approach. Although quantitative departures were noticed between both methods, these may be ascribed to differences in calculating the relative amounts of the nonrecognized venom proteins. Discussion: Our results indicate that both 1G and 2G antivenomic methods can be used interchangeably to investigate the in vitro immunoreactivity of antivenoms. An advantage of the affinity approach is the reusability of the affinity columns. Furthermore, the smoother baseline in RP-HPLC chromatograms of affinity column fractions allowed better resolution and a more accurate quantification of the antivenomic outcome than the original 1G protocol. These features contribute to the generalization, economy and reproducibility of the method. Keywords: snake antivenom, antivenomics, immunodepletion, immunoaffinity protocol. 10.1016/j.toxicon.2012.04.232
232. Death Adder Envenoming Causes Neurotoxicity not Reversed by Antivenom - Australian Snakebite Project (ASP-16) Christopher I. Johnston 1, Margaret A. O'Leary 2, Simon G.A. Brown 3, Bart J. Currie 4, Geoffrey K. Isbister 2 for the ASP investigators 1 School of Medicine Sydney, the University of Notre Dame Australia, Darlinghurst, NSW, Australia 2 Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle and the University of Newcastle, Newcastle, NSW, Australia 3 Centre for Clinical Research in Emergency Medicine, Western Australian Institute for Medical Research, Royal Perth Hospital and University of Western Australia, Australia 4 Tropical Toxinology Unit, Menzies School of Health Research, Charles Darwin University, Darwin, Australia
E-mail address: [email protected] (C.I. Johnston).
Background: Acanthophis spp (Death adders) occur in Australia, Papua New Guinea, and parts of eastern Indonesia and envenoming mainly cause neurotoxicity. The objectives of this study were to report the clinical syndrome of death adder envenoming and response to antivenom treatment. Methods: Definite bites were recruited from the Australian Snakebite Project (ASP) as defined by expert snake identification or detection of death adder venom in blood by enzyme immunoassay. Clinical effects and laboratory results were extracted from the ASP database, including the time course of neurotoxicity and response to treatment. Enzyme immunoassay was used to measure venom concentrations before and after administration of antivenom. Results: Twenty nine patients had definite death adder bites with a median age of 45 years (Range: 7 to 74y); 25 were male. The species of Death adder was determined in nine cases: four A. praelongus (Northern death adder), two A. antarcticus (Common death adder), two A. hawkei (Barkly Tableland death adder) and one A. rugosus (Rough-scaled death adder). Envenoming occurred in 14 patients. Two further patients had allergic reactions without envenoming; both were snake handlers with previous death adder bites. Of 14 envenomed patients, 12 developed neurotoxicity characterised by ptosis (12), diplopia (9), bulbar weakness (7), intercostal muscle weakness (3), limb weakness (6). Two required intubation. Only 2 of the 12 had non-specific systemic symptoms. One patient bitten by a Northern death adder developed myotoxicity and one patient only developed systemic symptoms (abdominal pain and vomiting) without neurotoxicity. No patients developed coagulopathy. The median peak venom concentration in 17 patients with pre-antivenom bloods available was 9.3ng/mL (interquartile range 4.4-24ng/mL, range 0.7245ng/mL). Antivenom was administered to 14 patients who all received an initial dose of one vial of death adder antivenom. Subsequent doses were administered in eight patients. In eight patients where post-antivenom blood samples were available, no venom was detectable after one vial of antivenom. In all 12 patients treated for neurotoxicity, persistent neurotoxicity occurred for 5 to 168 hours after antivenom.
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
Conclusion: Death adder envenoming is characterised by neurotoxicity. One vial of death adder antivenom was sufficient to bind all circulating venom. The persistence of neurotoxic effects after antivenom suggests that neurotoxicity may not be reversed by antivenom.
215
characterization of the epidemiology of snakebite epidemiology in the Western Development Region of Nepal. Antivenom use was associated with decreased mortality. Keywords: Envenomation, case fatality, anti-snake venom 10.1016/j.toxicon.2012.04.234
Keywords: Death adder, envenoming, antivenom, neurotoxicity 10.1016/j.toxicon.2012.04.233
234. Severity of Snakebites in Children in the United States: 2000-2009 233. Hospital Based Retrospective Study of Snakebite Epidemiology in Western Development Region of Nepal 1
2
Chhabi L Thapa , Kamal Devkota , Dev P. Pandey
3
1 District Health Office, Sindhulimadi, Sindhuli, Ministry of Health, Nepal Government, Nepal 2 Central Department of Zoology, Tribhuvan University, Kirtipur, Nepal 3 Biodiversity and Climate Research Center, Frankfurt, Germany E-mail address: [email protected] (D.P. Pandey).
Background: Snakebite is a common and life threatening public health problem in Nepal. Epidemiological data are fragmentary and sparse in Nepal and accurate, comprehensive epidemiological data on snakebites is still lacking. This study sought to characterize the snakebite epidemiology in Western Development Region of Nepal. Methods: A retrospective study of three years’ (20082010) snakebite data from medical records of 10 health institutes (1 Zonal Hospital, 3 District Hospital, 1 Private Hospital, 1 Mission Hospital, 2 Primary Healthcare Centers and 2 Army Camps) in the Western Development Region of Nepal was carried out during June 2011 to February 2012 by the use of pretested data sheets. Snakebite data were manually searched from those health institutions where snakebite victims used to be reported and government supplied anti-snake venom freely. Snakebite reported in medical records based on an eye witness and/or claim of snakebite victim, brought snakes, clear signs of snakebite wounds and symptoms of snakebite envenomation were included in the study. Those with a claim of snakebite with no or poor proof of snakebite were considered as suspected snakebite and not included in analysis. Statistical analysis was done by the application of MS Excel and R. The study was approved by the Ethical Clarence Review Board in Nepal Health Research Council. Results: The overall confirmed snakebite reported in three years was 6,993 of which 640 (9%) were envenomed and treated with antivenom (an average of 16 ASVS vials were administered to each victim) and the suspected snakebite cases were 2,562. The overall case fatality rate was 13% (10% in 2008, 16% in 2009, and 13% in 2010). July and August was the highly snakebite risk months (21% of overall snakebites in each month) in this region. The majority of snakebites were reported between 15:00 and 21:00 hours. Discussion: Snakebite and envenomation were greater in 2010 than in the two previous years. But the case fatality rate was greatest in 2009. This study detected decreased mortality associated with anti-snake antivenom use in this region. Conclusion: Snakebite records in the existing snakebite treatment center were poor and prevented complete
Scott A. Letbetter 1, Sharla A. Letbetter 1, 2, David L. Morgan 2 1 Scott and White Memorial Hospital, Dept of Emergency Medicine, Temple, TX, USA 2 Texas A&M Health Science Center, Temple, TX, USA E-mail address: [email protected] (S.A. Letbetter).
Background: Many of the 10,000 victims of snakebites each year in the US are under 18 years of age. The snakebite management of this population has not been studied as extensively as adults; however, previous studies have demonstrated that less than 2% of the pediatric victims result in major clinical effects. There may be particular characteristics specific to this population that could predict their overall outcomes. Treating physicians could use this information to direct emergency management of the pediatric victims of snakebites. Objective: Our goal was to determine the distinct characteristic differences of pediatric victims of snakebites between those who had major and those who had minor outcomes. Methods: Observational, case-control study of telephone calls to all US poison centers (National Poison Data System) for human victims of snakebites under 18 years from 2000 to 2009. Major outcome was defined as “significant disability” or death. Minor outcome was defined as “minimally bothersome” or no clinical effect. Those with moderate outcome were excluded. Results: There were 20,285 pediatric snakebites during the 10 year study period. Only 378 (1.9%) had a major outcome, 8,563 (42.2%) had a minor or no effect, and 11,344 (55.9%) had a moderate or unknown effect and were excluded. There were 3 deaths from snakebite. Most victims of major (65.9%) and minor bites (72.3%) were males (p ¼.008). Also, most victims of major (66.9%) and minor bites (78.9%) were over 5 years of age (p<.001). Seventeen States did not have a single snakebite that resulted in a major outcome. Four states (Texas, Florida, Georgia and California) represent 53.2 % of major outcomes. Most victims of major (78.8%) and minor bites (52.2%) were bitten by venomous snakes (P< .001). Nonvenomous snakes caused 1.5% of major and 9.6% of minor outcomes (p<.001). “Unknown snakes” accounted for 20.1% of major and 38.1% minor outcomes (p<.001). Rattlesnake bites produced 42.3% of major and 6.1% of minor outcomes (p<.001). Surprisingly, many major (44.1%) and minor bites (38.1%) occurred from September to April (p ¼.022). Conclusions: This is the largest outcome analysis of snakebites in pediatric victims. Almost half of these victims had only minor or no clinical effects. Significant disability or death of these victims was rare. Severity of outcome is associated with victim gender, age, geography, season, and type of
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
neglected tropical pathology of snakebite envenoming. A deep knowledge of the toxin composition and the immunological profile of venoms are central for developing polyspecific antivenoms exhibiting broad paraspecificity and cross-reactivity against the most medically-relevant snakes of a given geographical area. Preclinical neutralization tests and antivenomic assessments are necessary to demonstrate antivenom safety and efficacy prior to clinical trials. Antivenomics is a proteomic tool for the qualitative and quantitative analysis of the immunoreactivity of antivenoms. The original (first generation, 1G) antivenomics protocol is based on the immunodepletion of toxins upon incubation of whole venom with purified antivenom IgGs, followed by the addition of a secondary antibody or immobilized IgGbinding moiety. Antivenom immunoreactivity is inferred indirectly through the proteomic characterization of the toxin fraction that remains in solution after immunoprecipitation. This antivenomic approach is not appropriate for F(ab’)2 antivenoms. Objective: (i) To design a new method based on affinity chromatography which allows assessing F(ab’)2 antivenoms, employing two F(ab’)2 monospecific (antiCerastes cerastes and anti-Macrovipera mauritanica) antivenoms; (ii) to compare the 1G, immunoprecipitation protocol, and the new, second generation (2G, affinity capture) antivenomic approaches. These would assess the immunological profile of the pan-African EchiTAb-PlusICPÒ whole IgG antivenom, whose immunoreactivity characteristics have been previously tested towards the venoms of a panel of African viperid snakes and spitting cobras. Methods: F(ab’)2 fragments or whole purified IgG molecules were coupled to a NHS-activated SepharoseÒ. After incubating the matrix with the venom, the nonretained fraction and the immunospecific venom components were analyzed and quantified by reverse-phase HPLC followed by venomic analysis. Results: The affinity capture protocol allowed analyzing both types of antivenoms. Furthermore the pan-African EchiTAb-Plus-ICPÒ antivenom showed qualitatively similar immunoreactivity patterns using either antivenomic approach. Although quantitative departures were noticed between both methods, these may be ascribed to differences in calculating the relative amounts of the nonrecognized venom proteins. Discussion: Our results indicate that both 1G and 2G antivenomic methods can be used interchangeably to investigate the in vitro immunoreactivity of antivenoms. An advantage of the affinity approach is the reusability of the affinity columns. Furthermore, the smoother baseline in RP-HPLC chromatograms of affinity column fractions allowed better resolution and a more accurate quantification of the antivenomic outcome than the original 1G protocol. These features contribute to the generalization, economy and reproducibility of the method. Keywords: snake antivenom, antivenomics, immunodepletion, immunoaffinity protocol. 10.1016/j.toxicon.2012.04.232
232. Death Adder Envenoming Causes Neurotoxicity not Reversed by Antivenom - Australian Snakebite Project (ASP-16) Christopher I. Johnston 1, Margaret A. O'Leary 2, Simon G.A. Brown 3, Bart J. Currie 4, Geoffrey K. Isbister 2 for the ASP investigators 1 School of Medicine Sydney, the University of Notre Dame Australia, Darlinghurst, NSW, Australia 2 Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle and the University of Newcastle, Newcastle, NSW, Australia 3 Centre for Clinical Research in Emergency Medicine, Western Australian Institute for Medical Research, Royal Perth Hospital and University of Western Australia, Australia 4 Tropical Toxinology Unit, Menzies School of Health Research, Charles Darwin University, Darwin, Australia
E-mail address: [email protected] (C.I. Johnston).
Background: Acanthophis spp (Death adders) occur in Australia, Papua New Guinea, and parts of eastern Indonesia and envenoming mainly cause neurotoxicity. The objectives of this study were to report the clinical syndrome of death adder envenoming and response to antivenom treatment. Methods: Definite bites were recruited from the Australian Snakebite Project (ASP) as defined by expert snake identification or detection of death adder venom in blood by enzyme immunoassay. Clinical effects and laboratory results were extracted from the ASP database, including the time course of neurotoxicity and response to treatment. Enzyme immunoassay was used to measure venom concentrations before and after administration of antivenom. Results: Twenty nine patients had definite death adder bites with a median age of 45 years (Range: 7 to 74y); 25 were male. The species of Death adder was determined in nine cases: four A. praelongus (Northern death adder), two A. antarcticus (Common death adder), two A. hawkei (Barkly Tableland death adder) and one A. rugosus (Rough-scaled death adder). Envenoming occurred in 14 patients. Two further patients had allergic reactions without envenoming; both were snake handlers with previous death adder bites. Of 14 envenomed patients, 12 developed neurotoxicity characterised by ptosis (12), diplopia (9), bulbar weakness (7), intercostal muscle weakness (3), limb weakness (6). Two required intubation. Only 2 of the 12 had non-specific systemic symptoms. One patient bitten by a Northern death adder developed myotoxicity and one patient only developed systemic symptoms (abdominal pain and vomiting) without neurotoxicity. No patients developed coagulopathy. The median peak venom concentration in 17 patients with pre-antivenom bloods available was 9.3ng/mL (interquartile range 4.4-24ng/mL, range 0.7245ng/mL). Antivenom was administered to 14 patients who all received an initial dose of one vial of death adder antivenom. Subsequent doses were administered in eight patients. In eight patients where post-antivenom blood samples were available, no venom was detectable after one vial of antivenom. In all 12 patients treated for neurotoxicity, persistent neurotoxicity occurred for 5 to 168 hours after antivenom.
Abstracts Toxins 2012 / Toxicon 60 (2012) 95–248
Conclusion: Death adder envenoming is characterised by neurotoxicity. One vial of death adder antivenom was sufficient to bind all circulating venom. The persistence of neurotoxic effects after antivenom suggests that neurotoxicity may not be reversed by antivenom.
215
characterization of the epidemiology of snakebite epidemiology in the Western Development Region of Nepal. Antivenom use was associated with decreased mortality. Keywords: Envenomation, case fatality, anti-snake venom 10.1016/j.toxicon.2012.04.234
Keywords: Death adder, envenoming, antivenom, neurotoxicity 10.1016/j.toxicon.2012.04.233
234. Severity of Snakebites in Children in the United States: 2000-2009 233. Hospital Based Retrospective Study of Snakebite Epidemiology in Western Development Region of Nepal 1
2
Chhabi L Thapa , Kamal Devkota , Dev P. Pandey
3
1 District Health Office, Sindhulimadi, Sindhuli, Ministry of Health, Nepal Government, Nepal 2 Central Department of Zoology, Tribhuvan University, Kirtipur, Nepal 3 Biodiversity and Climate Research Center, Frankfurt, Germany E-mail address: [email protected] (D.P. Pandey).
Background: Snakebite is a common and life threatening public health problem in Nepal. Epidemiological data are fragmentary and sparse in Nepal and accurate, comprehensive epidemiological data on snakebites is still lacking. This study sought to characterize the snakebite epidemiology in Western Development Region of Nepal. Methods: A retrospective study of three years’ (20082010) snakebite data from medical records of 10 health institutes (1 Zonal Hospital, 3 District Hospital, 1 Private Hospital, 1 Mission Hospital, 2 Primary Healthcare Centers and 2 Army Camps) in the Western Development Region of Nepal was carried out during June 2011 to February 2012 by the use of pretested data sheets. Snakebite data were manually searched from those health institutions where snakebite victims used to be reported and government supplied anti-snake venom freely. Snakebite reported in medical records based on an eye witness and/or claim of snakebite victim, brought snakes, clear signs of snakebite wounds and symptoms of snakebite envenomation were included in the study. Those with a claim of snakebite with no or poor proof of snakebite were considered as suspected snakebite and not included in analysis. Statistical analysis was done by the application of MS Excel and R. The study was approved by the Ethical Clarence Review Board in Nepal Health Research Council. Results: The overall confirmed snakebite reported in three years was 6,993 of which 640 (9%) were envenomed and treated with antivenom (an average of 16 ASVS vials were administered to each victim) and the suspected snakebite cases were 2,562. The overall case fatality rate was 13% (10% in 2008, 16% in 2009, and 13% in 2010). July and August was the highly snakebite risk months (21% of overall snakebites in each month) in this region. The majority of snakebites were reported between 15:00 and 21:00 hours. Discussion: Snakebite and envenomation were greater in 2010 than in the two previous years. But the case fatality rate was greatest in 2009. This study detected decreased mortality associated with anti-snake antivenom use in this region. Conclusion: Snakebite records in the existing snakebite treatment center were poor and prevented complete
Scott A. Letbetter 1, Sharla A. Letbetter 1, 2, David L. Morgan 2 1 Scott and White Memorial Hospital, Dept of Emergency Medicine, Temple, TX, USA 2 Texas A&M Health Science Center, Temple, TX, USA E-mail address: [email protected] (S.A. Letbetter).
Background: Many of the 10,000 victims of snakebites each year in the US are under 18 years of age. The snakebite management of this population has not been studied as extensively as adults; however, previous studies have demonstrated that less than 2% of the pediatric victims result in major clinical effects. There may be particular characteristics specific to this population that could predict their overall outcomes. Treating physicians could use this information to direct emergency management of the pediatric victims of snakebites. Objective: Our goal was to determine the distinct characteristic differences of pediatric victims of snakebites between those who had major and those who had minor outcomes. Methods: Observational, case-control study of telephone calls to all US poison centers (National Poison Data System) for human victims of snakebites under 18 years from 2000 to 2009. Major outcome was defined as “significant disability” or death. Minor outcome was defined as “minimally bothersome” or no clinical effect. Those with moderate outcome were excluded. Results: There were 20,285 pediatric snakebites during the 10 year study period. Only 378 (1.9%) had a major outcome, 8,563 (42.2%) had a minor or no effect, and 11,344 (55.9%) had a moderate or unknown effect and were excluded. There were 3 deaths from snakebite. Most victims of major (65.9%) and minor bites (72.3%) were males (p ¼.008). Also, most victims of major (66.9%) and minor bites (78.9%) were over 5 years of age (p<.001). Seventeen States did not have a single snakebite that resulted in a major outcome. Four states (Texas, Florida, Georgia and California) represent 53.2 % of major outcomes. Most victims of major (78.8%) and minor bites (52.2%) were bitten by venomous snakes (P< .001). Nonvenomous snakes caused 1.5% of major and 9.6% of minor outcomes (p<.001). “Unknown snakes” accounted for 20.1% of major and 38.1% minor outcomes (p<.001). Rattlesnake bites produced 42.3% of major and 6.1% of minor outcomes (p<.001). Surprisingly, many major (44.1%) and minor bites (38.1%) occurred from September to April (p ¼.022). Conclusions: This is the largest outcome analysis of snakebites in pediatric victims. Almost half of these victims had only minor or no clinical effects. Significant disability or death of these victims was rare. Severity of outcome is associated with victim gender, age, geography, season, and type of