SNAKE BITE STUDY - PROTOCOL DEVELOPMENT WORKSHOP 11-12 March, 2013 Organized by Christian Medical College, Vellore Presentation by Dr. A. K. Mukherjee Professor, Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam Head and Coordinator, DBT Nodal Center for Medical Colleges and Biomedical Research Institutes of NE India, T.U. Affiliate Professor, School of Biological Sciences, University of Northern Colorado, Greeley, USA

Strategies to Improve Snakebite Treatment: Challenges and Progress

Efficient Management of Snakebite in India: Problems Encountered The following constrains as listed below are based on my 18 years of research experience on snake venom and snakebite management in India: No proper /methodical (computerized) record of snakebite data in most of the Govt. hospitals: lack of region specific true snakebite data. Geographical variation in snake venom composition and a detail study on region specific clinical manifestations following envenomation is lacking Lack of reliable venom detection kit : failure to understand which species of snake is bitten? Snakebite treatment protocol-which guideline is to be followed? Quality control (efficiency) and supply of antivenom: Who have been taking care? Monovalent Vs Polyvalent antivenom in snakebite therapy: Superiority of treatment Vs treatment cost needs to be worked out. Less attention has been paid for very poorly immunogenic, low molecular mass venom components which are difficult to neutralize by antivenom. Venom quality control for efficient antivenom production: should be addressed

Antivenom manufacturers All these problems have aroused because the lack of a proper coordination amongst…… Clinicians Toxinologists Antivenom manufacturers To address all these issues, “Toxinological Society of India” was established in 2011

Hospital based clinical work For Efficient Management of Snakebite, the Following Scheme is Proposed Snakebite Management Laboratory based venom and antivenom analysis Hospital based clinical work Improvement of treatment & quality of antivenom

Laboratory Based Snakebite Management Venomics Antivenomics Venom detection kit A proteomics approach to study geographical variation in venom composition & pathophysiology of venom components Proteomics based immunochemical analysis of antivenom ELISA based affordable venom detection kits Molecular basis of taxanomic identification of Indian snakes

Venomics: A proteomics Approach for Analysis of Snake Venom Components Analysis of complex mixture of snake venom demands an approach that effectively separates a complex mixture of proteins and peptides with varying size and charge. Traditional methods of venom analysis does not provide understanding of the complexity of venom toxicity as well as elucidating the major biochemical differences among snake families. Efficiency of the current proteomic techniques in identifying pharmaceutically important venom proteins and a better correlation of geographical variation in venom composition with clinical manifestation in snakebite patients has been proved Advantages characterized snake venom proteomes from different parts of India would provide information useful with regard to the potential clinical manifestation of these bites in a particular location This will also guiding antivenom choices for proper treatment of snakebite victims.

Differences in the venom composition between two species of Indian Cobra (Naja naja and Naja kaouthia) Impact on the severity of pathogenesis and antivenom treatment

Pictures of two specis of Indian Cobra Naja naja Naja kaouthia

Although the biochemical composition of Naja naja from different parts of India is well Characterized, but no information was available on the biochemicalcomposition and biological activity of N. kaouthia venom from India. Therefore, effort was given to characterize the biochemical properties of N. kaouthia venom from eastern India and to compare these properties with the N. naja venom of the same origin. Further, potency of a commercial polyvalent antivenom raised against N. naja in neutralizing the lethality and various pathophysiological effects of N. kaouthiaVenom was also explored.

A comparison of enzyme activity present in N. naja and N. kaouthia venom samples from eastern India

Source: Mukherjee, A. K. and Maity, C. R Source: Mukherjee, A.K. and Maity, C.R. (2002) Comparative Biochemistry and Physiology 131 (B), 125-132

Comparison of gel-filtration pattern of N. naja and N. kaouthia venom samples from eastern India

Naja Naja venom Naja Kaouthia venom

Properties Naja naja Naja kaouthia Table: A Comparison of Pathophysiology between N. naja and N. kaouthia Properties Naja naja Naja kaouthia Lethality (mg/body weight of mouse) 0.40 ± 0.08 0.7 ± 0.09 Hemorrhagic activity (20μg crude venom) Nil Myotoxicity (CPK release) (1U/l) 28.5 ± 3 19.2 ± 2.1 Edema ratio (15μg crude venom) 148.0 ± 3.1 140.0 ± 3.1 Minimum edema inducing dose (μg) 5.0 4.8 Indirect hemolytic activity (% of Hb release by 100 μg of protein) 86 ± 4.1 80 ± 3.8 Direct hemolytic activity (% of Hb release by 100 μg of protein) 50 ± 2.1 39 ± 2.5 Neurotoxicity Present Source: Mukherjee, A.K. and Maity, C.R. (2002) Comparative Biochemistry and Physiology 131 (B), 125-132

Neutralization of lethality and pathophysiology of N. naja and N. Kaouthia Venom samples by a commercial N. naja antivenom (mg venom / 100 mg antivenom) Neutralization potency

Antivenomics: Application of Proteomic Tools for Production of Efficient Antivenom for Proper Management of Snakebite Patients Proteomics-based immunochemical analysis (antivenomics) provides relevant information for outlining which venom mixtures cross-react with the most important components in medically-relevant venoms from a particular region. This type of approach may set the basis for the development of effective antivenoms on an immunologically sound basis which in turn will be beneficial for better treatment of snakebite patients

Venom Detection Kits Background Information: The diagnosis of snake envenomation is based on sensitive immunoassays Geographical and species specific variation in snake venom composition as well as high-immunological cross-reactivity impose a great challenge for development of ELISA-based venom detection kits. Recently, Wahby and Ibrahim (2008) and ibrahim et al. (2013) have demonstrated avidity based VDK (Avidity: an inexact measure of the binding strength of antibodies to multiple antigenic determinants on natural antigens)

Identification and Discrimination of Snake Venoms from Egyptian Cobras Source: Ibrahim et al. Toxicon 63 (2013) 88-97

Permission to collect snake venom from different geographical location IMPROVEMENT OF SNAKEBITE TREATMENT: SUMMARY OF SUGGESTIONS To identify PI’s from different fields but having a similar research interest. To identify coordinator(s) from each group, may be one from each region of India Snakebite data collection from different parts of country-permission from Govt. Permission to collect snake venom from different geographical location Approach to Govt. of India for allocation of more money in snakebite research Organization of workshop for project writing Organization of regular meetings amongst the interested PIs. Strengthening of Toxinological Society of India (TSI) and its activities