Progress and challenges on the way to improving the diagnosis of snakebite

Department of Tropical Medicine and Public HealthInstitute of Occupational Medicine, Social Medicine and Environmental Medicine

Goethe UniversityFrankfurt am Main, Germany

Ulrich Kuch

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

Voucher specimens:hard evidence for snake bite research

• Must be properly labeled with (link to) patient data• Best preserved in >70% ethanol

Photos by Aniruddha Ghose

Syndromic approaches to snakebite diagnosis

Ariaratnam et al. Am J Trop Med Hyg 2009;81(4): 725-31

Syndromic approach: Sri Lanka

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

• 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.)

Russell's viper toxin [ng/ml]

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Forensic toxinology: molecular diagnosis of snakebite

DNA extractionPCR / nested PCRSequencing

Sequencecomparison: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

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

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

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

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)

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

(1) Adaptation to existing systems• species-specific PCR

• LFA detection of PCR product• no gel-electrophoresis, no sequencing

(2) LAMP experiments

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LAMP test: isothermal amplification of DNA

LAMP test for Bungarus caeruleus

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 & MSFGabriel Alcoba, Geneva Univ. Hospitals & MSFBenoit Ehrensberger, Geneva Univ. HospitalsFrançois Chappuis, Geneva Univ. Hospitals

Mahmood Sasa, Instituto Clodomiro PicadoFabian 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

AcknowledgementsFriederike BockLaura BruhsePatrick HödeChristian MelaunDeb PandeyAntje Werblow

Tun Pe, DMR MyanmarAye Aye Myint, DMR

miprolab GmbHFrank GesslerAnnette LeunigSibylle Pagel-WiederPatrick Schindler

Grant support:

UBS Optimus FoundationSwiss National FundsVFF Goethe UniversityLOEWE Programme