Transcript Folie 1

Progress and challenges on the way to improving
the diagnosis of snakebite
Ulrich Kuch
Department of Tropical Medicine and Public Health
Institute of Occupational Medicine, Social Medicine and Environmental Medicine
Goethe University
Frankfurt am Main, Germany
Knowing the species of snake involved in bites
is important for:
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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
Voucher specimens:
hard evidence for snake bite research
• Must be properly labeled with (link to) patient data
• Best preserved in >70% ethanol
Syndromic approaches to snakebite diagnosis
Photos by Aniruddha Ghose
Syndromic approach: Sri Lanka
Ariaratnam et al. Am J Trop Med Hyg 2009;81(4): 725-31
Immunodiagnosis:
detection of snake venom antigens
• Retrospective screening of large samples
• Rapid diagnosis
• Development of bed-side tests
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
Lateral Flow Assay to detect Russell‘s viper venom
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Russell's viper toxin [ng/ml]
• 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.)
Forensic toxinology: molecular diagnosis of snakebite
DNA extraction
PCR / nested PCR
Sequencing
Sequence
comparison:
Identification
Sample collection from the bite sites
using a cotton-bud swab-stick
Stabbing does leave trace DNA: utility of PCR for
diagnosing bites by long-fanged viper species
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
Clinical study on snakebite diagnosis in southern Nepal
Annual incidence of:
- Snakebite: up to
1’162/100’000
- Deaths due to
snakebite: 162/100’000
(Sharma et al. 2004)
Study objectives
 To identify the snake species responsible for envenoming and nonenvenoming 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)
 Prospective study in 3 treatment centres of southern Nepal
 Inclusion criteria:
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History of snakebite with or without (Damak, Charali) sign(s) of envenoming
≥ 5 years
No antivenom prior to admission
Informed consent signed
 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
 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
Comparison of baseline characteristics of 55 patients bitten by kraits and cobras with neurotoxic envenoming
 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)
Next steps
 New diagnostic study in Nepal and Myanmar (2015-2016)
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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
(1) Adaptation to existing systems
• species-specific PCR
• LFA detection of PCR product
• no gel-electrophoresis,
no sequencing
(2) LAMP experiments
LAMP test: isothermal amplification of DNA
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LAMP test for Bungarus caeruleus
Acknowledgements
Friederike Bock
Laura Bruhse
Patrick Höde
Christian Melaun
Deb Pandey
Antje Werblow
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
Tun Pe, DMR Myanmar
Aye Aye Myint, DMR
miprolab GmbH
Frank Gessler
Annette Leunig
Sibylle Pagel-Wieder
Patrick Schindler
Mahmood Sasa, Instituto Clodomiro Picado
Fabian Bonilla M., Instituto Clodomiro Picado
Grant support:
UBS Optimus Foundation
Swiss National Funds
VFF Goethe University
LOEWE Programme
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