1. Progress and challenges on the way to improving the diagnosis of snakebite Department of Tropical Medicine and Public Health Institute of Occupational Medicine, Social Medicine and Environmental Medicine Goethe University Frankfurt am Main, Germany Ulrich Kuch

2. Knowing the species of snake involved in bites is important for: Treatment decisions (antivenom: which one, when, how much?) Prognosis, preparing for/preventing complications, referral Epidemiology, resource allocation, design of better antivenoms Community education, prevention Clinical trials of (antivenom) treatment and diagnostic tools

4. Voucher specimens: hard evidence for snake bite research Must be properly labeled with (link to) patient data Best preserved in >70% ethanol

5. Photos by Aniruddha Ghose Syndromic approaches to snakebite diagnosis

6. Ariaratnam et al. Am J Trop Med Hyg 2009;81(4): 725-31 Syndromic approach: Sri Lanka

7. Immunodiagnosis: detection of snake venom antigens Retrospective screening of large samples Rapid diagnosis Development of bed-side tests

8. Immunodiagnosis: requirements and limitations Requires availability of local snake venoms to raise (diagnostic) antisera in animals –several snakes per species –from various geographic localities and regions Non-envenoming bites not identified Can only find known species Complicated where many species

9. Lateral Flow Assay to detect Russell‘s viper venom prototype, clinical validation study in preparation rapid – 20 min specific and sensitive – limit of detection 10 ng / ml Aye Aye Myint et al. (in prep.)

10. Forensic toxinology: molecular diagnosis of snakebite DNA extraction PCR / nested PCR Sequencing Sequence comparison: Identification

11. Sample collection from the bite sites using a cotton-bud swab-stick

12. Stabbing does leave trace DNA: utility of PCR for diagnosing bites by long-fanged viper species

13. PCR for diagnosing bites by long-fanged viper species: laboratory experiments 78 pitvipers from 5 species provoked, allowed to bite one dead mouse each Majority were Bothrops asper and Crotalus simus of different sizes also Bothriechis lateralis, Cerrophidion godmani, Porthidium ophryomegas

15. Annual incidence of: -Snakebite: up to 1’162/100’000 -Deaths due to snakebite: 162/100’000 (Sharma et al. 2004) Clinical study on snakebite diagnosis in southern Nepal

16.  To identify the snake species responsible for envenoming and non- envenoming bites in southeastern Nepal  Snake identification  To develop and/or validate tools to identify snake species  History of bite and clinical features  PCR + DNA sequencing on material collected at the bite site  Development of LFA on serum Sanjib K. Sharma, Ulrich Kuch, Patrick Höde, Laura Bruhse, Deb Pandey, François Chappuis, Emilie Alirol (submitted) Study objectives

17.  Prospective study in 3 treatment centres of southern Nepal  Inclusion criteria:  History of snakebite with or without (Damak, Charali) sign(s) of envenoming  ≥ 5 years  No antivenom prior to admission  Informed consent signed

18.  Dead snakes brought by victims  Preserved in ethanol and labelled  Identification by a blinded taxonomist  Questionnaire  Circumstances of bite  Demographic & clinical characterictics  Molecular analysis:  Rubbing one cotton swab at bite site  PCR & nested PCR, sequencing  Collection of serum

19.  749 patients with H/O snakebite (2010-2012)  52.5% males, median age: 29 years  264 (35.2%) with local/systemic signs of envenoming  Swabs for PCR done in 568 patients (76%)  Snake species identified for 194 patients (25.9%)  62 dead snakes identified (8.3%)  153 had a positive PCR (26.9%)  Positive snake ID and DNA sequence in 21 patients: 100% concordance

21. Comparison of baseline characteristics of 55 patients bitten by kraits and cobras with neurotoxic envenoming

22.  Serious mismatch between snake species targeted by antivenom and venomous species identified in southern Nepal  Russell’s viper, saw-scaled viper not seen  Pit vipers and krait species other than B. caeruleus identified  PCR from swabs at bite site  Limited sensitivity (26.9%)  not for individual diagnosis  High concordance with species ID (100%) but n=21  Snake bite history and clinical features strongly associated with cobra or krait bites  Clinical score (Pathmeswaran et al. Trans Roy Soc Trop Med Hyg 2006;100:874-8)  Syndromic approach (Ariaratnam et al. Am J Trop Med Hyg 2009;81(4): 725-31)

23.  New diagnostic study in Nepal and Myanmar (2015-2016)  Further validation of clinical features  Clinical score & algorithm  Further validation of PCR as diagnostic tool  Reference standard  Validation of «low-tech» DNA-based test (e.g., LAMP)  Development & validation of LFA for Russell’s viper and cobra/krait Next steps

24. (1) Adaptation to existing systems species-specific PCR LFA detection of PCR product no gel-electrophoresis, no sequencing (2) LAMP experiments

25. 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min neg. LAMP test: isothermal amplification of DNA LAMP test for Bungarus caeruleus

26. Sanjib K. Sharma, B.P. Koirala Inst. of Health Sci. Anup Ghimire, B.P. Koirala Inst. of Health Sci. Mamit Rai, B.P. Koirala Inst. of Health Sci. Emilie Alirol, Geneva University Hospitals & MSF Gabriel Alcoba, Geneva Univ. Hospitals & MSF Benoit Ehrensberger, Geneva Univ. Hospitals François Chappuis, Geneva Univ. Hospitals Mahmood Sasa, Instituto Clodomiro Picado Fabian Bonilla M., Instituto Clodomiro Picado Health assistants, doctors and nurses of Damak Snakebite Treatment Centre, Charali Snakebite Clinic, Bharatpur Hospital, B.P. Koirala Institute of Health Sciences; Nepal Health Research Council Acknowledgements Friederike Bock Laura Bruhse Patrick Höde Christian Melaun Deb Pandey Antje Werblow Tun Pe, DMR Myanmar Aye Aye Myint, DMR miprolab GmbH Frank Gessler Annette Leunig Sibylle Pagel-Wieder Patrick Schindler Grant support: UBS Optimus Foundation Swiss National Funds VFF Goethe University LOEWE Programme