SNAKE BITE STUDY - PROTOCOL DEVELOPMENT WORKSHOP11-12 March, 2013

Organized byChristian Medical College, Vellore

Presentation by

Dr. A. K. MukherjeeProfessor, 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:

1. No proper /methodical (computerized) record of snakebite data in most of the Govt.

hospitals: lack of region specific true snakebite data.

2. Geographical variation in snake venom composition and a detail study on region specific

clinical manifestations following envenomation is lacking

3. Lack of reliable venom detection kit : failure to understand which species of snake is bitten?

4. Snakebite treatment protocol-which guideline is to be followed?

5. Quality control (efficiency) and supply of antivenom: Who have been taking care?

6. Monovalent Vs Polyvalent antivenom in snakebite therapy: Superiority of treatment Vs

treatment cost needs to be worked out.

7. Less attention has been paid for very poorly immunogenic, low molecular mass venom

components which are difficult to neutralize by antivenom.

8. Venom quality control for efficient antivenom production: should be addressed

Clinicians

ToxinologistsAntivenom manufacturers

All these problems have aroused because the lack of a proper coordination

amongst……

To address all these issues, “Toxinological Society of India” was established in 2011

Snakebite Management

Laboratory based venom and

antivenom analysis

Hospital based clinical work

For Efficient Management of Snakebite, the Following Scheme is Proposed

Improvement of treatment & quality

of antivenom

Laboratory Based Snakebite Management

Venomics Antivenomics Venom detection kitA 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

1. 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.

2. 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.

3. 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

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.

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

Naja Naja venom

Naja Kaouthiavenom

Properties Naja naja Naja kaouthi

aLethality (mg/body weight of mouse)

0.40 ± 0.08 0.7 ± 0.09

Hemorrhagic activity (20μg crude venom)

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

Table: A Comparison of Pathophysiology between N. naja and N. kaouthia

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Lethality Edemainducing

Directhemolysis

indirecthemolysis

Myotoxicity

N. naja

N. kaouthia

Neutralization of lethality and pathophysiology of N. naja and N. Kaouthia Venom samples by a commercial N. naja antivenom

Neu

tral

izati

on p

oten

cy

(mg

veno

m /

100

mg

antiv

enom

)

Antivenomics: Application of Proteomic Tools for Production of Efficient

Antivenom for Proper Management of Snakebite Patients

1. 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.

2. 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)

Source: Ibrahim et al. Toxicon 63 (2013) 88-97

Identification and Discrimination of Snake Venoms from Egyptian Cobras

IMPROVEMENT OF SNAKEBITE TREATMENT: SUMMARY OF SUGGESTIONS

1. To identify PI’s from different fields but having a similar research interest.

2. To identify coordinator(s) from each group, may be one from each region of India

3. Snakebite data collection from different parts of country-permission from Govt.

4. Permission to collect snake venom from different geographical location

5. Approach to Govt. of India for allocation of more money in snakebite research

6. Organization of workshop for project writing

7. Organization of regular meetings amongst the interested PIs.

8. Strengthening of Toxinological Society of India (TSI) and its activities