Snakebite Envenoming Diagnosis and Diagnostics - DTU Orbit

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Snakebite Envenoming Diagnosis and Diagnostics

Knudsen Cecilie Juumlrgensen Jonas A Foslashns Sofie M Haack Aleksander U W Friis Rasmus DamSoslashren H P Bush Sean White Julian Laustsen Andreas H

Published inFrontiers in Immunology

Link to article DOI103389fimmu2021661457

Publication date2021

Document VersionPublishers PDF also known as Version of record

Link back to DTU Orbit

Citation (APA)Knudsen C Juumlrgensen J A Foslashns S M Haack A U W Friis R Dam S H P Bush S White J ampLaustsen A H (2021) Snakebite Envenoming Diagnosis and Diagnostics Frontiers in Immunology 12[661457] httpsdoiorg103389fimmu2021661457

Frontiers in Immunology | wwwfrontiersin

Edited byManuela Berto Pucca

Federal University of RoraimaBrazil

Reviewed bySakthivel Vaiyapuri

University of ReadingUnited Kingdom

Clara Guerra-DuarteEzequiel Dias Foundation (FUNED)

Brazil

CorrespondenceCecilie KnudsenCecknudtudk

Andreas H LaustsenAholabiodtudk

Specialty sectionThis article was submitted to

Vaccines and Molecular Therapeuticsa section of the journal

Frontiers in Immunology

Received 30 January 2021Accepted 29 March 2021Published 28 April 2021

CitationKnudsen C Juumlrgensen JA Foslashns S

Haack AM Friis RUW Dam SHBush SP White J and Laustsen AH

(2021) Snakebite EnvenomingDiagnosis and DiagnosticsFront Immunol 12661457

doi 103389fimmu2021661457

REVIEWpublished 28 April 2021

doi 103389fimmu2021661457

Snakebite Envenoming Diagnosisand DiagnosticsCecilie Knudsen12 Jonas A Juumlrgensen1 Sofie Foslashns1 Aleksander M Haack1Rasmus U W Friis1 Soslashren H Dam1 Sean P Bush3 Julian White4 and Andreas H Laustsen1

1 Department of Biotechnology and Biomedicine Technical University of Denmark Kongens Lyngby Denmark 2 BioPortoDiagnostics AS Hellerup Denmark 3 Department of Emergency Medicine Yale School of Medicine New HavenCT United States 4 Toxinology Department Womenrsquos and Childrenrsquos Hospital North Adelaide SA Australia

Snakebite envenoming is predominantly an occupational disease of the rural tropicscausing death or permanent disability to hundreds of thousands of victims annually Thediagnosis of snakebite envenoming is commonly based on a combination of patienthistory and a syndromic approach However the availability of auxiliary diagnostic tests atthe disposal of the clinicians vary from country to country and the level of experiencewithin snakebite diagnosis and intervention may be quite different for clinicians fromdifferent hospitals As such achieving timely diagnosis and thus treatment is a challengefaced by treating personnel around the globe For years much effort has gone intodeveloping novel diagnostics to support diagnosis of snakebite victims especially in ruralareas of the tropics Gaining access to affordable and rapid diagnostics could potentiallyfacilitate more favorable patient outcomes due to early and appropriate treatment Thisreview aims to highlight regional differences in epidemiology and clinical snakebitemanagement on a global scale including an overview of the past and ongoing researchefforts within snakebite diagnostics Finally the review is rounded off with a discussionon design considerations and potential benefits of novel snakebite diagnostics

Keywords envenoming clinical toxinology diagnosis diagnostics ophidism snakebite managementsyndromic approach

INTRODUCTION

Every year people lose their livelihoods limbs and lives to a disease that is as neglected as it isancient Snakebite envenoming The exact burden of snakebite envenoming is notoriously difficultto assess because data on envenoming prevalence are scarce and the available data points are ofteninaccurate or not representative for broader geographical areas (1ndash4) Nonetheless studies suggestthat mortality due to snakebite envenoming may exceed 125000 deaths per year globally while thenumber of people suffering permanent sequelae may be around 400000 and the toll of associateddisability-adjusted life years might add up to a total of over 6 million (4ndash8) To make matters worsesnakebite envenoming is both a disease mainly affecting the poor and a disease that leads to furtherimpoverishment (7 9ndash12) In spite of the immense burden of snakebite envenoming on victimstheir families and local communities the disease remains largely neglected and has historically onlyreceived few resources and limited efforts have gone into the development of better treatments anddiagnostics (13)

org April 2021 | Volume 12 | Article 6614571

Knudsen et al Snakebite Envenoming Diagnosis and Diagnostics

Once diagnosed the mainstay treatment of severeenvenoming is antivenom in combination with auxiliarytreatment (7 14 15) Monovalent antivenoms can be usedwhen the species of the offending snake is known whilepolyvalent antivenoms are useful in cases where the snakespecies has not been identified However the ability ofpolyvalent antivenoms to neutralize a broad range of venomsmight come at the cost of decreased efficacy as the relativeproportion of antibodies in a polyvalent antivenom that targetstoxins of a specific snake venom is often not as great as theproportion of antibodies in a monovalent antivenom targetingthe same venom toxins Therefore it can become necessary toadminister a greater dose of a polyvalent antivenom than of amonovalent antivenom in order to treat a given envenoming (716) Increasing the dose can in turn affect the cost of treatmentand the risk of developing adverse reactions to the antivenom (7)Unfortunately polyvalent antivenoms are favored in manyplaces either due to the lack of monovalent antivenoms or dueto the difficulty in choosing which monovalent antivenom toadminister in the absence of reliable information on theperpetrating snake species (17)

In addition to enabling the administration of monovalentantivenoms where available identifying the offending snakespecies or the type of venom might enable clinicians to predictand prepare for the development of clinical manifestations Toaid clinicians in this task there is a common and deceptivelysimple categorization of elapid venoms as being primarilyneurotoxic and viperid venoms as being primarily cytotoxicandor hemotoxic (here understood as toxicity directed towardblood and the cardiovascular system including hemostasis) (7)While this simplification does represent a general trend it cancause clinical misinterpretation and there are several importantexceptions to the rule For example many major Australianelapid snakes commonly cause coagulopathy often withoutevident neurotoxicity while some important viperids causeminimal cytotoxicity yet important neurotoxicity (18)Similarly bites from some elapid species of the cobra genusparticularly spitting cobras can cause strong cytotoxic symptomswithout causing neurotoxicity (19) which may be confused withviper envenomings Matters are further complicated by the factthat snake venom composition can vary within genera and evenwithin species due to ontogeny and geographical distribution(20ndash23) As such the variability of clinical manifestations ofenvenoming and the time courses of their developmentcomplicate the treatment of snakebite (7) Thus whilesnakebite is generally well-handled in many areas room forinnovation and improvement still exists Encouragingly recentyears have seen a renewed interest in such innovations andimprovements with much research being published not only onnovel treatment modalities (eg recombinant antivenoms andsmall molecule inhibitors) but also on novel diagnostics (egenzyme-linked immunosorbent assays lateral flow assaysimpedimetric immunoassays infrared imaging and polymerasechain reaction-based methods) [see Table 1 and (104ndash108)] Intime some of these diagnostic tools may enter the clinic wherethey could be utilized to obtain valuable information such as the

Frontiers in Immunology | wwwfrontiersinorg 2

identity of the perpetrating snake species or genus allowing useof monovalent antivenoms or quantitative measures of thedegree of envenoming Additionally if implemented in ruralsettings diagnostic kits may guide treatment decision for lessexperienced clinicians enabling proper management ofsnakebite victims at rural facilities This information mightsupport clinical management of snakebite envenoming andepidemiological studies of relevance to antivenomdevelopment resource management and advocacy forincreased attention to snakebite

COMMONLY ADOPTED APPROACHESFOR DIAGNOSIS OF SNAKEBITEENVENOMING

A basic diagnosis of snakebite envenoming requires a thoroughpatient history targeted examination and appropriate laboratoryinvestigations (109) Taking a detailed history includes askingabout the circumstances of the bite (eg geography time of theincident activity and number of bites) details of the snake (ifseen brought or photographed) clinical manifestations ofenvenoming (including time of onset) first aid applied andpast medical history (eg allergies prior snakebites relevantmedications and pre-existing medical conditions) (109)Laboratory investigations almost always include an evaluationof the blood clotting profile to screen for venom-inducedcoagulopathies In its simplest form a blood clotting test canbe carried out in the form of a 20 minute whole blood clottingtest (20WBCT) If more sophisticated equipment is available it iscommon to run repeated tests of the International NormalizedRatio (INR) of blood clotting activated partial thromboplastinetime (aPTT) D-dimer andor fibrinogen degradation products(FDP) supplemented by hemograms and potentially also byelectrocardiograms Acute falls in hemoglobin and hematocritvalues may indicate internal bleeding and a drop in fibrinogenlevels might be indicative of coagulopathy (7 110 111) Bloodsamples are usually also screened for creatine kinase (CK) levelselectrolytes urea nitrogencreatinine which together withurinalysis (hematuria proteinuria urea levels and urineoutput) can be used to assess venom-induced rhabdomyolysisand associated complications such as myoglobinuric renalfailure or polyuria oliguria or anuria (110 111) See Table 2for a list of these laboratory investigations Based on the patienthistory and laboratory tests trained toxinologists may be able toinfer the offending snake species and this can in turn guide thechoice of treatment

Diagnostic algorithms summarize much of the knowledgerequired to diagnose snakebites They have been developed forsome settings and regions to provide support for doctors andother healthcare workers tasked with frontline management ofpatients with suspected envenoming Here it is acknowledgedthat frontline staffs will often have limited training in managingenvenoming and limited rapid access to clinical toxinologyexpertise to guide their important treatment decisions in the

April 2021 | Volume 12 | Article 661457

TABLE 1 | Overview of snakebite diagnostics capable of differentiating snake venoms

Notes Year Ref

1957 (24)

Only abstract available 1967 (25)

1968 (26)

Sensitivity 396 (40101) 1974 (27)

1974 (28)

1975 (29)

Cross-reactivity to the following speciestested A rhodostoma B gabonica Cadamanteus E schistosa N haje N najaN nigricollis O scutellatus V berus

1977 (30)

1977 (3132)

1978 (33)


1982 (35)



(Continued)

Knudsen

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Snakebite

Envenoming

Diagnosis

andDiagnostics

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Type Subtype Abs Area Snake(s) targeted LoD Assay duration Tested onpatient

samples

Sample matrix

Immuno-assay

Immuno-diffusion

Equine Cobra spp (possiblyO hannah)

1100000000dilution

Patients 1(case study)

Tissue homogenate

Immuno-diffusion

Australia A antarcticusN scutatusP porphyriacusP textilis

gt3 hours Serum exudate(guineapig)

Agglutinationtest

Caprine ampequine

California C v helleri gt2 hours Patients 16 Serum

Immuno-diffusion

Leporine Africa B arietansC maculatusE carinatusN hajeN melanoleucaN nigricollis

48 hours(immuno-difussion) amp gt1hour (immuno-electrophoresis)

Patients 101 Wound aspiratesblister fluids seraurine

RIA Australia N scutatusP textilis

lt10 ng gt24 hours Patients 2(also testedon rabbitserum)

Serum sample buffer


gt24 hours Patients 3 Tissue samplesfluids

ELISA Leporine ampequine IgGs

Multiple B arietansC maculatusE carinatus

1-5 ngmL ON incubation No ndash Testedin rodents

Serum (human andrat)

RIA Leporine Australia N scutatus ON incubation Patients 3 Urine serumclothing tissuesamples

RIA LeporineIgG

Australia A antarcticusA superbaN scutatusO scutellatusP australisP porphyriacusP textilis

01-04 ngmL gt20 hours Unpublished Tissue samples

ELISA LeporineIgG

05-2 ngmL 30-90 minutes

Enzymeimmunoassay

LeporineIgG

Australia Acantophis sppNotechis sppOxyuranus sppPseudoechis sppPseudonaja spp

5-15 ngmL 20-40 minutes Patients 43 Whole blood urinewound swab

ELISA 1 ngmL gt3 hours No - Testedin rabbits

Blood urine exudate

ELISA LeporineIgG

A rhodostomaN naja

78-156 ngmL 35-45 minutes Yes Serum

TABLE 1 | Continued

Notes Year Ref

ss-reactions with venoms of otherkes were extensive at highercentrations

1984 (38)

cross-reactivity found to B fasciatus N O hannah T gramineus

1984 (39)

1986 (40)1987 (41)

1987 (42)

1987 (43)

1987 (44)

wn to cross-react with cobra venom 1987 (45)

cificity mentioned as being a problem 1988 (46)

y abstract available No cross-reactivity t tigrinus venom

1988 (47)

ted for cross-reactivity to Bothrops spptalus spp Lachesis spp and Tityusulatus venom

1990 (48)

y abstract available 1990 (49)

sitivity of 525 Tested for hook effectinterference from sample matrices

1991 (50)

1991 (51)

cificity 88 (14 false positives118 negatives) Tested for

1991 (52)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

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Immunology

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April2021

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6614574

Crosnacon

Nonaja

Kno

Spe

Onlto RTesCroserrOnl

Senand

Spefrom


samples

Sample matrix

ELISA LeporineIgG

USA A contortrixC atroxC scutulatus

01-01 microgmL ON incubation No - Testedin animals

Sero-sanguineousfluid blood urineperitoneal fluidpleural fluid lungkidney

ELISA Equine Myanmar D russelii 10 ngmL ON incubation Yes Serum

ELISA Leporine Thailand N kaouthia 2 ngwell Yes SerumEnzyme-linkedcoagulationassay

MurinemonoclonalIgG

D russelii 2-10 pgmL No

ELISA Nigeria B arietansC maculatusE carinatus

ON incubation Patients 31 Blood serum urinesputum bite siteaspirates

ELISA Philippines N n philippinensis ON incubation Patients 1(postmortem)

Blood

ELISA LeporineIgG

Thailand C rhodostomaD russeliiN kaouthiaT albolabris

10-20 ngmL ON incubation Patients 251 Serum

RIA MurinemonoclonalAbs

Thailand D russelii 4-20 ngmLdepending onmatrix

ON incubation Patients 4 Serum urine

ELISA Equine Europe V ammodytes lt1 ngmL lt20 minutes No - Testedin rabbits

Blood

ELISA Asia A b blomhoffii 54 ngwell No - Testedin mice

Serum

ELISA LeporineIgG

Brazil B jararaca 146 ngmL ON incubation No ndash Testedin mice

Serum

ELISA A b blomhoffii No - Testedin rats amprabbits

Serum

Agglutinationtest

LeporineIgG

Thailand B fasciatusC rhodostomaD russeliiN kaouthiaN n siamensisO hannahT albolabris

016-12 microgmL 40 minutes Serumsamples 59Woundswabs 26

Serum woundswabs

ELISA Equine F(abrsquo)2

Brazil C d terrificus 1-3 pgmL ON incubation No ndash Testedin mice

Serum (mice)sample buffer

ELISA LeporineIgG

Myanmar D russelii 10 ngmL ON incubation Patients 311Controls 118

Serum

TABLE 1 | Continued

Notes Year Ref

reactivity to B fasciatusuthianahhrurusfor cross-reactivity toraca and C d terrificus

1992 (53ndash56)

1992 (57)

reactivity to 26 venoms tested 1992 (58)

ivity of 813 for serum samples andfor wound swabs Cross reactivity at

ntrations at least 62 times higher

1993 (59)

1993 (60)

sted in mice 1993 (61)

bstract available Shown to cross-ith several other venoms

1993 (62)

1994 (63)

1994 (64)

1994 (65)

1995 (66)

1996 (67)

bstract available 1996 (68)

(Continued)

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Snakebite

Envenoming

Diagnosis

andDiagnostics

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samples

Sample matrix

crossN kaoO haT ery


Europe Vipera spp 2-7 ngmL(depending onsample matrix)

gt45 hours Yes Blood serum urine TesteB jara

ELISA LeporineIgG

Australia A antarcticusN scutatusO scutellatusP australisP textilis

25 ngmL ON incubation Unpublished Sample buffer

LeporineIgG

SouthernThailand

C rhodostoma 5 ngmL 50 minutes No Sample buffer Cross

Agglutinationtest

LeporineIgG

Thailand B fasciatusC rhodostomaD russellliN kaouthiaO hannahT albolabris

2-635 ngmL 60-120 minutes Serumsamples 59Woundswabs 26

Serum wound swab Sensit615conce

ELISA LeporineIgG

Brazil B alternatusB atroxB jararacaBjararacussuB moojeniB neuwediC d terrificusC d collineatusL muta

lt001-01 microgmL ON incubation No Sample buffer serum(non-envenomedhumans)

ELISA LeporineIgG

Brazil B atroxL m muta

20 ngmL 2 hours Yes Serum Also t

FluorogenicELISA

D russelii 01 pgmL Only areact

ELISA PapuaNewGuinea

P papuanus ON incubation Patients 9 Serum urine woundaspirates

ELISA LeporineIgG

Tunisia E pyramidum lt10 ngmL ON Yes Serum

ELISA NorthAmerica

Agkistrodon spp 2 ngmL gt5 hours No ndash Testedin rabbits

Serum

ELISA Myanmar O hannah lt20 ngmL Patients 2(casestudies)

Serum

ELISA amp RIA Ovine Fab Europe Vipera spp 08 ngmL (ELISA)amp 2 ngmL (RIA)

gt3 hours (ELISA) ampON incubation (RIA)

Yes Plasma urine

ELISA Leporine F(abrsquo)2

India B caerulusD russelii

1 ngmL 30 min Patients 27 Blood serum urinewound swab

Only a

-

ntd

-

e

w

TABLE 1 | Continued

Notes Year Ref

ivity 46 specificity 88 1997 (69)

1997 (70)

1999 (71)


reactivity to 11 venoms and toxins

2002 (73)

it uncertain 2004 (74)

2004 (75)

2006 (76)

2007 (77)

2008 (78)

sted in rat serum and for cross-ity with Australian snake venoms

2010 (79)

city 100 2012 (80)

s of 25-28 depending on the 2013 (81)


2016 (83)

2017 (84)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

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Immunology

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April2021

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6614576


samples

Sample matrix

E carinatusN naja


Martinique B lanceolatus 3 hours Patients 40Controls120

Serum Sensit

ELISA ampagglutinationassay

Equine CentralAmerica

Micrurus spp 03 mgmL(agglutinationassay) amp 4 ngmL(ELISA)

gt5 minutes(agglutinationtest) amp ONincubation(ELISA)

No ndash Testedin rabbitsand mice

Serum plasma

ELISA Caprine ampleporine IgG

India Bungarus sppDaboia sppEchis sppNaja spp

01 ngmL gt5 hours Yes(postmortemonly)

Tissue samples

ELISA Taiwan Cobra spp 05 ngmL 6 hours Calf serum andhuman urine

Only a

Opticalimmunoassay

LeporineIgG

Asia B multicinctus 25-10 ngmL 25 minutes No ndash Testedin mice

Blood tissuesamples

Crosstested

Agglutinationtest

Equine Venezuela Bothrops sppCrotalus spp

167 microgmL 10 minutes No Sample buffer LoD u

Opticalimmunoassay

LeporineIgG

Vietnam C rhodostomaN kaouthiaO hannahT albolabris

02-08 ngmLdepending on thevenom andsample matrix

40 minutes Patients 83Samples125

Serum urine woundexudate

ELISA Avian IgY ampleporine IgG

India N naja 01-300 ng ON incubation Patients 12(live)Patients 7(postmortem)

Skin bloodcerebrospinal fluid

ELISA LeporineIgG

India B caeruleusN naja

ON incubation Samples 22(postmortem)

Skin blood

Immuno-flourescence

N kaouthia 5ndash10 ngmL 3 hours No Sample buffer

ELISA LeporineIgG

Australasia Oxyuranus spp 015 ngmL ON incubation Patients 17 Serum Also treactiv

ELISA LeporineIgG

Colombia L acrochorda 39 ngmL No Sample buffer Speci

ELISA LeporineIgG

Egypt N hajeN nigricollisW aegyptia

lt10 ngwell ON incubation No Sample buffer Aviditivenom

LFA Avian Taiwan N atra 5 ngmL 20 minutes Patients 88(34 cobraand 54 non-cobra)

Serum Sensit

LFA Equine ampleporine IgG

India Daboia spp Naja spp 01 ngmL 10 minutes No - Testedin mice

Plasma

ELISA LeporineIgG

India Bungarus sppDaboia spp

1 ngmL 20-25 minutes No - Testedin mice

Sample buffer

-

n

e

fi

e

TABLE 1 | Continued

Notes Year Ref

nsitivity and specificity of 100 forurotoxic venoms Sensitivity of 364 formorrhagic venoms

2018 (85)

sted for cross-reactivity to C d terrificusd M leminiscatus

2018 (86)

antitative Cross-reactivity between thenoms was tested

2020 (87)

2020 (88)

sed on (82) 2020 (89)

o tested on venom from B fasciatus Cdostma D russelii O Hannah

2001 (90)

2015 (91)

ecificity 100 2016 (92)

2014 (93)

2017 (94)

2018 (95)

ssay with an incubation time of 3 hours For other unreportedbeen marked ldquordquo Studies describing the detection of venom-re not included in this table Abs antibodies ELISA enzyme-munoassay

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

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Immunology

|wwwfrontiersinorg

April2021

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6614577


samples

Sample matrix

Echis sppNaja spp

ELISA amp LFA Equine Taiwan Neurotoxic vshemorrhagic venom

LoD of 5-50 ngmL (LFA) amp LoQof 039-078 ngmL (ELISA)

10-15 minutes Patients 21 Serum Senehe

Impedimetricimmunoassay

Equine Brazil Bothrops spp 027 ugmL gt25 minutes No Sample buffer Tean

ELISA Leporine Sri Lanka B caeruleusD russeliiH hypnaleN naja

019-156 ngmL(depending onthe venom)

gt2 hours Patients 19Controls 20

Serum Quve

LFA Avian ampequine

South-East Asia

Daboia sppNaja spp

10 ngmL (invitro)

25-30 minutes Samples 5 Serum

LFA Leporine ampequine

Asia ampAfrica

Naja spp 5-10 ngmL forAsian cobras andlt500 ngmL forAfrican cobras

gt20 minutes No Serum (fetal bovine) Ba

Molecularbiology

PCR NA Thailand N kaouthia gt2 hours No - Testedon mice

Wound swabs (mice) Alrh

PCR NA Thailand B fasciatusC rhodostomaD siamensisHydrophiinae sppNaja sppO HannahTrimeresurus spp

0025 ngmL gt67 minutes No Saliva (snake)

PCR NA Nepal Bungarus sppNaja sppO hannahO moticolaTrimeresurus spp

ON incubation Patients 565 Wound swab Sp

Misc Enzymaticactivity assay

NA Sri Lankaamp Australia

B caeruleusD russeliiH hypnaleN najaP porphyriacus

Patients 108 Serum

Infraredthermography

NA Brazil B moojeniC d terrificusB jararaca

gt15 minutes Patients 8

Enzymaticactivity assayamp ELISA

NA Australia Elapid spp 01-02 ngmL(ELISA)

Patients 115Controls 80

Serum

Many studies did not report on the duration of the diagnostic procedure In such cases assay duration was reported in this table as ldquogt total incubation timerdquo eg ldquogt3 hoursrdquo for anvalues the corresponding fields were left empty In some cases only the abstracts of the studies were available to us and in these cases values not reported in the abstract havespecific antibodies in snakebite victims [eg (96ndash98)] or the detection of toxins or toxin activities for the purpose of venom characterization rather than diagnosis [eg (99ndash103)] welinked immunosorbent assay F(abrsquo)2 fragment antigen binding 2 IgG immunoglobulin G LFA lateral flow assay ON overnight PCR polymerase chain reaction RIA radioim

sa

a


critical early hours after a bite The purpose of such diagnosticalgorithms is to synthesize and distill the knowledge and experienceof experts in clinical toxinology into a readily and rapidly accessibleformat to guide less experienced health professionals towardoptimal care of bitten and envenomed patients Formallyassessing the effectiveness of diagnostic and treatment algorithmsfor envenoming is a challenge with no clear published researchavailable However experience in countries such as Australia (firstworld) and Myanmar (developing world) appears to indicate thatdiagnostic algorithms developed for snakebite individualized foreach country or region are both widely used and accepted InMyanmar snakebite diagnostic algorithms were developed by ateam of health professionals through a series of drafts tested byfrontline healthcare workers and a final version was adopted androlled out nationally by the Ministry of Health Feedback fromfrontline healthcare workers in Myanmar was strongly positiveDiagnostic algorithms do not replace expertise in clinicaltoxinology but can be an important part of an optimal carepathway However it must always be acknowledged in suchalgorithms that they are merely a guide and cannot cater for everypossible clinical scenario and presentation

As previously mentioned it is also a common procedure toask whether the patient saw the snake and if so what it lookedlike (112) However using the victimrsquos description of the snake isoften not in isolation a reliable diagnostic method foridentifying the snake although for some snakes in somecountries (eg Russellrsquos viper in Myanmar) it may bereasonably reliable In some cases the biting snake is not seenand even if it has been spotted the victimrsquos description canpotentially be misleading (113) While it may be easier to identifythe snake if it is brought to the hospital for identification even inthese cases misidentification can occur and in somecommunities there is an unwillingness among hospital staff toinspect or handle the snake (114) As an example hump-nosed


pit vipers (H hypnale) are often misidentified as saw-scaledvipers (E carinatus) in India resulting in administration ofineffective antivenom (115) Even if the snake has beenpositively identified by an expert herpetologist the clinicalpresentation of the patient is pivotal as different specimens ofvenomous snakes (eg from different regions) can cause differentclinical envenoming syndromes (109 116) Another caveat ofthis approach is the inherent risk of attempting to capture or killthe snake which can lead to further envenoming of the victim ora helper attempting to catch the snake however if a snake hasalready been killed this is a potentially valuable diagnostic aidHospital staff should be encouraged to examine and preserve allsuch presented snakes as this can allow retrospective studiesclearly defining medically relevant species for a particular regionIn most settings 70 ethanol is an appropriate preservative fordead snakes immersing the entire snake and injecting thepreserving fluid into the body cavity It is critically importantthat preserved snakes are adequately labeled and thatpreservation methods that will not lead to deterioration of thelabel (in the preserving fluid) are employed so that subsequentexamination can unequivocally link the dead snake to aparticular snakebite patient

SNAKEBITE DIAGNOSIS IN AUSTRALIA

In Australia snakebites are diagnosed based on patient historyand laboratory investigations as described above Incombination with neurological assessment it is possible toidentify most severe envenoming cases within 12 hours of thebite (117) after which patients with confirmed or suspectedenvenoming can be discharged if clinical findings andlaboratory test results indicate no envenoming has occurredIn rare cases envenoming ndash in particular by death adders ndashdoes not manifest itself before 24 hours post-bite though it isunclear whether such late-presenting envenomings canprogress to severe or life-threatening envenomings insubsequent hours

Precise epidemiological data on snakebites in Australia arenot available however one estimate suggests between 500 and3000 snakebites occur annually (118) while another studyreported 6123 hospital admissions due to contact withvenomous snakes in a period from August 2001 to May 2013or an average of about 500 cases per year (119) Snakebiteenvenoming in Australia is not common but can be severewith an average of 22 deaths annually in the past 15 years without-of-hospital cardiac arrest being the most common cause(120 121) Most medically significant snakebites can beattributed to five terrestrial snake groups Brown snakes(Pseudonaja spp) members of the tiger snake group (Notechisscutatus Tropidechis carinatus Austrelaps spp andHoplocephalus spp) black snakes and mulga snakes(Pseudechis spp) taipans (Oxyuranus spp) and death adders(Acanthophis spp) (18 109 121) Some of these snakes can easilybe confused by people without experience in identifying snakesFor example a snakebite victim may report to have been bittenby ldquoa brown snakerdquo which could belong to any number of

TABLE 2 | Examples of auxilliary tests that are frequently performed forsuspected snakebite victims

Auxilliary tests

Type Subtype

Hemograms Platelet countBlood count (hemoglobin white cell count absolutelymphocyte count)Examination of blood film for evidence of intravascularhemolysis (schistocytes spherocytes etc)

Clotting profile Fibrinogen levelProthrombin timeINR of blood clottingActivated partial thromboplastine time (aPTT)D-dimerfibrinogen degradation products (FDP)

Serumbiochemistry

ElectrolytesBilirubinLiver function testsCreatine kinase (CK CPK)

Urinalysis HematuriaMyoglobinuria

Renal function Serum creatinineUreaGlomerular filtration rateUrine output (polyuria oliguria anuria)

Electrocardiagram



species eg the eastern brown snake (Pseudonaja textilis) or theking brown snake (Pseudoechis australis) (see Figures 1A B) theclinical significance and treatment of which would differDiagnostic algorithms can aid clinicians in determining thesnake species most likely to have caused the bite asexemplified in Figure 2 (109 116)

Major local effects such as hemorrhagic blebs and necrosisafter snakebites are rare in Australia and minimal for the brownsnakes that cause most cases of snakebite Nonetheless somespecies may cause at least moderate local swelling and localbruising can uncommonly occur following bites by those speciescausing defibrination coagulopathy (116) Systemic effects varydepending on species and may include neurotoxic flacciddescending paralysis systemic myolysis defibrinationcoagulopathy anticoagulant coagulopathy acute kidney injury(AKI) sudden collapse cardiac collapsearrest anaphylaxis andmicroangiopathic hemolytic anemia (MAHA) (109 116) Deathadders taipans tiger snakes and the rough scaled snakecommonly cause neurotoxicity however tiger snakes therough scaled snake and taipans can also cause myotoxicityBlack snakes and mulga snakes cause myotoxicity andanticoagulant coagulopathy while defibrination coagulopathy(referred to by some authors as ldquovenom-induced consumptioncoagulopathyrdquo ldquoVICCrdquo) is frequent for brown snakes tigersnakes rough sca led snake broad headed snakes(Hoplocephalus spp) and taipans (116) Defibrinationcoagulopathy can be diagnosed based on an elevated INR ofblood clotting and aPTT and grossly elevated D-dimer the lattermay be the first evidence of developing coagulopathy before anychanges in INR of blood clotting and aPTT occur (109) In caseof anticoagulant coagulopathy fibrinogen and degradationproducts are at normal levels and aPTT and possibly INR ofblood clotting can be prolongedelevated whereas defibrinationcoagulopathy leads to decreased or undetectable levels offibrinogen and elevated levels of degradation products both D-dimer and FDP (109 116) Typically symptoms of coagulopathyare seen early sometimes upon arrival to the emergencydepartment while neurotoxicity and myotoxicity take hours todevelop with CK levels peaking between 24-48 hours after thebite (116)

If severe envenoming is diagnosed diagnostic algorithms inconjunction with the Seqirus (formerly Commonwealth SerumLabs CSL) Snake Venom Detection Kit (SVDK) can helpdetermine which snake venom immunotype is involved (109)The SVDK is a non-laboratory rapid freeze-driedimmunoassay kit developed for Australian and some PapuaNew Guinea snake venoms that uses bite site swabs oralternatively a urine sample to detect the venom immunotypeThe SVDK is widely distributed and available in Australia but itsusage has declined in part because of concerns over accuracy andreliability The reliability of the SVDK is debated due to a highrisk of false positives when the SVDK is inappropriately tested onnon-envenomed patients the occurrence of false negatives withenvenomed patients (121) and the presence of a hook effect (alsoknown as prozone effect ndash an effect which describes how themeasured analyte concentration can decrease even as the actual


concentration increases) (122) The propensity for false positiveshas proven especially problematic as the SVDK has frequentlybeen used inappropriately for all suspected snakebitessometimes as a screening tool ndash a function that it was notdesigned for and is not suitable for One study suggests thatfalse negatives are often a result of operator errors (123) andperhaps for this reason there is now an annual quality assuranceprocess for all laboratories using the SVDK to minimize thelikelihood of operator errors Antivenom is available at 750hospitals across Australia and if an immunotype can bedetermined via the SVDK or otherwise the appropriateldquomonovalentrdquo antivenom can be selected as treatment (109) Inaddition to the five terrestrial ldquomonovalentrdquo antivenoms apolyvalent antivenom against the five snake groups is alsoavailable (121) If the diagnostic algorithms and the SVDKresults are in conflict then either polyvalent antivenom or anappropriate mixture of two ldquomonovalentrdquo antivenoms should beused but the large volume of antivenom needed particularly ifusing polyvalent represents a potential increased risk of adversereactions (7 109) If a clinician is in doubt when handlingsuspected or confirmed snakebite cases then advice may besought via the antivenom producer (Seqirus Melbourne) orthrough the Clinical Toxinology service (Womenrsquos ampChildrenrsquos Hospital Adelaide) (109)

From 2005 to 2015 the median dose of antivenomadministered to Australian snakebite victims has decreasedfrom four vials to one vial with debated implications fortreatment (121 124) Meanwhile the median time to firstantivenom administration has remained unchanged at 43hours (121) despite increasing evidence of a more favorableoutcome when antivenom is administered early (125 126) Thislack of change in time to antivenom administration might bebecause Australia covers a large landmass with many areas beingremote from major health services making delays in treatingsnakebites more likely to occur particularly in remote siteswhere antivenom is not stocked and aeromedical retrieval isrequired Competing demands on aeromedical retrieval servicescan exacerbate delays Additionally many dangerouslyvenomous snakes in Australia only cause envenoming in aminority of cases As prophylactive antivenom administrationcan negatively affect the patient antivenom should not beadministered until it is certain that the patient has beenenvenomed this can necessitate further delays in antivenomadministration as symptom development is monitored For thesereasons it seems unlikely that the time to antivenomadministration will improve significantly in Australia

SNAKEBITE DIAGNOSIS IN ASIA

The epidemiology of snakebite envenoming differs across Asia asa result of high inter- and intra-species diversity and varyingpopulation density of venomous snakes The impact of snakebiteis relatively high in many countries in South- and Southeast Asiawhere the overall estimated mortality rate is 105 to 542 deathsper 100000 people (4) This includes the Philippines Thailand



Vietnam Laos Cambodia Malaysia Myanmar Nepal PakistanSri Lanka and India where envenomings are predominantlyincurred from the following snakes Cobras (Naja spp) kraits(Bungarus spp) Russellrsquos vipers (Daboia spp) saw-scaled vipers


(Echis spp) Malayan pit viper (Calloselasma rhodostoma)hump-nosed pit vipers (Hypnale spp) and green pit vipers(Trimeresurus spp) (114 127ndash129) In Japan Korea HongKong Taiwan and Indonesia most envenomings are caused

FIGURE 1 | Comparison of venomous snakes with similar names andor appearance andor clinical syndromes Visual comparison of two Australian snakes withsimilar names and appearances (A) A king brown snake (Pseudonaja textilis) which belongs to the black snake genus and (B) an eastern brown snake(Pseudonaja textilis) which belongs to the brown snake genus Visual comparison of two venomous snake species from Brazil (C) Bothrops sp and (D) Lachesissp Species from these genera can appear similar to those not trained in snake identification can cause similar clinical manifestations and are both locally known aslsquosurucucursquo in certain parts of Brazil Visual comparison of (E) a puff adder (Bitis arietans) and (F) a horned viper (Cerastes cerastes) the venoms of which can causesimilar clinical manifestations Figures 1A C D copyright copy Prof Julian White Figure 1B copyright copy of Prof Sean Bush 1E 1F were found on WikiMediaCommons are are copyright copy of the user 4028mdk09 and the user Broobas respectively



FIGURE 2 | Diagnostic algorithm for snakebite envenoming in South Australia Algorithm copyright copy Prof Julian White

Frontiers in Immunology | wwwfrontiersinorg April 2021 | Volume 12 | Article 66145711


by pit vipers (subfamily Crotalinae) which might be associatedwith lower mortality rates (130ndash134) Data on the epidemiologyof snakebite in Central- West- and North Asia including Russiaand the Middle East are limited but estimates suggest that therates are low compared to the subtropical and tropical regions ofSouth- and Southeast Asia (4) Similarly data on theepidemiology in China are limited with one study suggestingthat mortality rates in East Asia including China range from0033 to 0347 per 100000 people (4)

The majority of physicians in South- and Southeast Asia relyon the circumstances of the bite and clinical manifestations todiagnose the victim (135) Syndromic diagnostic tools andalgorithms are available for Southeast Asia in general (SEAROguide (136)) and for some countries in particular (eg Myanmar(9 137 138)) Similar to Australia a thorough patient history canbe helpful in identifying the type of snake involved in theaccident Eg if a victim has been bitten in a house during thenight and has developed paralysis the culprit is more likely to bea krait (Bungarus spp) while a bite sustained from a venomoussnake in a tree might suggest a green pit viper (Trimeresurusspp) (135 139 140) Systemic signs of envenoming can also behelpful in clinical practice as the venom of most species inSouth- and Southeast Asia are mainly toxic to eitherneuromuscular or hemostatic systems Neurotoxicity is oftenrelated to bites by cobras king cobras and kraits whilehemotoxicity usually indicates envenoming by a true viper(subfamily Viperinae) or pit viper although in rare cases itmay indicate envenoming by a colubrid such as a red-neckedkeelback (Rhabdophis subminiatus) or a tiger keelback(R tigrinus) (139) It can sometimes be difficult to differentiatebetween neurotoxic envenoming by cobras and kraits based onclinical signs However krait bites are often associated withdelayed onset and prolonged total period of paralysis whilecobra bites are often associated with significant local evidence ofenvenoming (136 141) Behavioral differences of the snakesmight further elucidate the matter as krait bites primarilyoccur at night while cobra bites are much more likely to occurduring the day (135 140) When documenting the clinicalmanifestations of envenoming some clinicians use astandardized questionnaire based on national snakebitemanagement guidelines to support the diagnostic process(142) A systematic syndromic approach combined with ascoring system based on clinical manifestations has beenproposed to assist clinicians in identifying the offending snakespecies (143) but sufficient data on envenoming profiles to createsuch systems are lacking for many species throughout South-and Southeast Asia (137 143)

India has more snakebites and snakebite-related deaths peryear than any other country in the world (135 144) It is home to52 venomous snake species out of which the Russellrsquos viper(D russelii) the common krait (B Caeruleus) the Indian cobra(N naja) and the saw-scaled viper (E Carinatus) known as theldquoBig Fourrdquo are considered the most medically important Bothpolyvalent and monovalent antivenoms are available in India butthey do not cover all venomous species Furthermore there is aneed for a standardized quality control process for manufacturing


of snakebite antivenoms to ensure that they are safe and effective(145) National guidelines for management of snakebites in Indiado exist and some states have developed their own protocolsHowever in many cases these protocols are not followed strictlyleading to misdiagnosis and inappropriate management (146) Asan example hump-nosed pit vipers (H hypnale) are oftenmisidentified as saw-scaled vipers (E carinatus) resulting inadministration of ineffective antivenom (141) Furthermoresome doctors and hospitals are unwilling to manage snakebitevictims causing potentially critical treatment delays (147) Adiagnostic tool for identification of the offending snake speciescombined with a coordinated approach to ensure that healthcareworkers across India have adequate knowledge skills andconfidence to manage snakebite patients could potentiallyreduce this problem

Snakebite envenoming remains an important health issue inmany regions of Asia especially throughout South- andSoutheast Asia where incidence and mortality rates are amongthe highest in the world The high species diversity complicatesclinical management although this problem is alleviatedsomewhat by the widespread use of polyvalent antivenomsWhile these polyvalent antivenoms are convenient forphysicians they may arguably be disadvantageous for overallpatient outcomes as they can be a disincentive for qualityepidemiologic and clinical envenoming studies Uncertaintyabout the offending snake species may result in masking ofldquonewrdquo envenoming syndromes thereby hampering the inclusionof new species into antivenom immunization protocols Thecontinuing absence of Hypnale spp from the immunizing mixfor Indian polyvalent antivenoms (148ndash150) can be mentionedas an example of this

SNAKEBITE DIAGNOSIS IN THE UNITEDSTATES AND CANADA

In the United States (US) and Canada around 6500 people sufferfrom snakebites annually resulting in 5-6 deaths (5 151 152)The US has about 26 indigenous venomous snake species whererattlesnakes (Crotalus spp) moccasins (Agkistrodon spp) andpygmy rattlesnakes (Sistrurus spp) all of which belong to the pitviper (Crotalinae) subfamily are the main genera implicated insnakebites Coral snakes (Micrurus spp) are also present in alimited southern distribution but do not account for many bites(153) with an estimated 70-80 annual cases reported to theAmerican Association of Poison Control Centers In Canadarattlesnakes are the only medically relevant snake species andwith a very limited distribution the risk of snakebite is relativelysmall (154)

Pit vipers are the most prolific group of snakes involved insnakebite accidents in the US and Canada therefore whenmanaging a snakebite patient it is important to keep in mindthat less than 10 percent of pit viper bites are dry bites (155 156)Pit viper venom typically contains hemotoxins causing direct orindirect lysis of fibrinogen thrombocytopenia and vascularendothelial damage (157 158) thereby emphasizing the



importance of carefully monitoring the patientrsquos bloodcoagulation profile through laboratory tests Furthermore thepresence of Mojave toxin in Mojave rattlesnake (C scutulatus)and southern pacific rattlesnake (C helleri) venoms causespotentially severe systemic neurotoxicity including cranialneuropathy and flaccid paralysis Severe neurotoxic clinicalmanifestations when present are a relevant diagnosticindicator (159ndash161) It is recommended to perform laboratorytests of the patient every 6-8 hours and twice prior to dischargingthe patient in order to follow the progression of theenvenoming (110)

Coral snake envenoming generally causes only mild localeffects while the systemic manifestations can include euphorialethargy nausea vomiting excessive salivation ptosis dyspneaconvulsions abnormal reflexes and motor weakness or paralysisleading to respiratory paralysis which is lethal in absence ofclinical intervention (162ndash165) In case of a coral snakeenvenoming serum creatine kinase activity may rise andmyoglobin may be detected in the urine (164 166) butcoagulopathy is not a feature (165ndash168) The observation timein the hospital depends on the severity of the envenoming theage of patient and the location of the bite wound ranging fromat least 8 hours to 12-24 hours for mild envenomings whererepeated laboratory evaluations are advised (110) The markedvisual appearance and clinical presentation of coral snakeenvenomings in the US make coral snake envenomings easy todistinguish from pit viper envenomings The genus-specificantivenom Pfizer Antivenin has been available for thetreatment of coral snake envenoming but is currently in veryshort supply resulting in rationing (162 169) though the recentrecommencement of production should alleviate this shortage

Like several other countries the US also has a treatmentalgorithm The unified treatment algorithm published in 2011with the purpose of streamlining the management and diagnosisof snakebites in the US (110) However since the algorithm waspublished a new antivenom has become available and thealgorithm has not yet been updated accordingly As Canadadoes not have indigenous snake species that are different fromthose in the US it is likely that this algorithm is applicable toassess snakebite cases in Canada as well

Clinicians in the US will often factor in information providedby the victim or bystanders about the identity of the snake Astudy comparing the snake identifications of expertherpetologists with those of snakebite victims witnesses andhealthcare providers in southern parts of the US found that 40of the specimens identified as copperheads (A contortrix) wereactually cottonmouths (A piscivorous) with juvenile snakesbeing particularly difficult to identify leading to confusion(170) While other species were less frequently confused itmight be problematic that (possibly erroneous) snakeidentifications are used by poison control centers whenrecommending treatment (170) Although pit viper bites in theUS are treated with polyvalent antivenom (CroFab or AnaVip)when required misidentification of pit vipers might stillnegatively impact treatment For example AnaVip which isbased on equine F(abrsquo)2 antibodies has proven more efficient in


treatment of late onset and recurrent coagulopathy than CroFabwhich is based on ovine Fab antibodies (171) This difference inefficacy versus coagulopathies might be related to the differenthalf-lives of Fab and F(abrsquo)2 antivenoms (171) Both CroFab andAnaVip are recommended for treatment of rattlesnakeenvenoming in North America but AnaVip has not receivedFDA-approval for treatment of bites by cottonmouths andcopperheads Conversely CroFab works well for treatment ofcopperhead (A contortrix) bites by decreasing limb disabilitysubsequent to bites (172) and being associated with fewerpatients using opioids to treat pain related to the envenoming(173) It has additionally been demonstrated that earlyadministration of CroFab for copperhead bites results in fasterlimb recovery than does late administration (174) Thus in casesof copperhead envenomings it might be especially beneficial torapidly identify the culprit species so the optimal polyvalentantivenom can be administered early on

SNAKEBITE DIAGNOSISIN LATIN AMERICA

In Latin America and the Caribbean islands 80000-129000snakebite envenomings occur each year leading to an estimated540-2300 deaths (4) Throughout the Latin American countriesbites from lanceheads (Bothrops spp) are the most prevalentRattlesnakes (Crotalus spp) bushmasters (Lachesis spp) andCoral snakes (Micrurus spp) are also present but especially thelatter two are far less common causes of snakebites (175 176) InCentral America snakebites are also caused by moccasins(Agkistrodon spp) jumping pit vipers (Atropoides spp) palmpit vipers (Bothriechis spp) montane pit vipers (Cerrophidionspp) and hog-nosed pit vipers (Porthidium spp) The venomsof pit vipers indigenous to Central America can be treated withpolyvalent antivenom (175) The clinical utilization of polyvalentantivenom makes diagnosis at a species or even genus level lessimportant as noted earlier for Asia and the US and CanadaHowever it is important to determine which family (viperelapid or other) the perpetrating snake species belongs towhether an envenoming has taken place and the severity ofthe envenoming (175) For South American countries bothpolyvalent and genus-specific antivenoms are available (111176ndash179)

Several Latin American countries have protocols for diagnosisand treatment of snakebite envenoming describing the use of thesyndromic approach and the laboratory investigationsmentioned in Table 2 (111 175 176 178ndash180) Several ofthese protocols mention coagulation time as a commonlyinvestigated parameter for early detection of a pit viperenvenoming (111 175 176 178ndash180) Often in pit viperenvenomings the 20WBCT is positive (no clot at 20 minutes)while for elapids it remains negative (normal clot at 20 mins)(165ndash168) In Mexico where rattlesnakes are the predominantgenus the Lee-White clotting time (LWCT) is utilized todetermine the presence of coagulation disorders which can in



turn give an indication of the urgency of commencing treatment(180) LWCT is fundamentally similar to the 20WBCT describedearlier with the only difference being that the LWCT is observedonce per minute after an initial incubation time of five minutes(181) The effectiveness of LWCT was assessed in Brazil for itssensitivity toward detecting coagulopathy in lanceheadenvenomings and was considered a valuable tool in evaluatingthe need for antivenom therapy (181)

Pit viper envenomings may cause both local and systemiceffects but there are two distinct patterns Most Central andSouth American pit vipers cause moderate to severe local effectsand coagulopathy often with hemorrhagic features Theexception is rattlesnakes which are more likely to cause majorsystemic effects including neurotoxicity rhabdomyolysis andcoagulopathy while only causing mild local effects Local effectsfollowing pit viper bite with the exception of rattlesnakes mayinclude edema severe local pain swelling local hemorrhageinflammatory erythema lymphangitis bleeding from the bitewound blistering ecchymosis tissue necrosis and secondaryinfections (7 111 175 176 178ndash180 182ndash185) Systemic effectsmay include early syncope confusion transient loss of vision ordarkening of vision hypotension shock renal damage cardiactachyarrhythmia or bradyarrhythmia coagulopathy andsystemic hemorrhage (7 111 175 176 178ndash180 182 183185) With the knowledge of which snakes induce whichclinical manifestations the syndromic approach works welland is widely used However the approach requires thoroughknowledge of the different venomous snakes (7 186) and relieson the presence of polyvalent antivenoms targeting the venomsof one or multiple genera

The similarity between the local effects of lancehead speciesand bushmaster species makes differentiating the two achallenging task which can be further complicated by the factthat in some regions both genera are known locally asldquosurucucurdquo (see Figures 1C D) However the vagomimeticeffects sometimes induced by bushmaster venom on thegastrointestinal system may cause diarrhoea thus indicatingthe most likely genus of the culprit snake Although this can be astrong indicator the lack of such effects does not exclude thepresence of a lachetic envenoming (151 176) nor do theirexistence confirm it

Unlike bites from lanceheads and bushmasters manyrattlesnake bites are more easily recognized by the neurotoxiceffects that they can inflict South American rattlesnakes(C durissus) generally do not cause severe local manifestationsbut instead induce neurotoxicity resulting in neuromuscularparalysis (183 184 187) caused by neurotoxic crotamines andcrotoxins present in the venoms Envenomings by SouthAmerican rattlesnakes often lead to mild to severe neurotoxicmanifestations in the patient which are clinical hallmarks thatmay guide the physician toward a correct diagnostic assessment(111 159ndash161 176 178 180) However it has been reported thatenvenomings by juvenile South American rattlesnakes can resultin coagulopathy as the main systemic manifestation instead ofneurotoxicity which may lead to misdiagnosis andadministration of wrong antivenom (188)


Coral snake envenomings are associated with very differentclinical manifestations such as local paresthesias vomitingmuscle paralysis including paralysis of respiratory musclesptosis ophthalmoplegia diplopia and late manifestationsincluding secondary renal damage and respiratory failure (111168 175 176 178ndash180 183 184) Although clinicalmanifestations may overlap with those of some rattlesnakesthe recognizable color schemes of coral snakes make a strongcase for coral snake envenoming Coral snakes found in most ofthe Pan-American countries are visually very distinct from pitvipers However nonvenomous snake species mimicking thevenomous coral snakes exist (eg milk snakes Lampropeltistriangulum) These are difficult to distinguish by a non-professional but guidelines based on the color scheme of thesnakes can be found that aid in the differentiation (162 175 189)

Snake biodiversity varies significantly throughout LatinAmerica from Argentina inhabited by three medicallyimportant snake genera (Bothrops Crotalus and Micrurus) tothe plethora of medically important species found in Mexico andthe Central American countries (190) This shift in indigenoussnake species greatly impacts the diagnostic approach where thesevere local effects of lancehead envenomings become a specificindicator in Argentina (111) but is easily confused for a lacheticenvenoming in Brazil (176) Polyvalent antivenoms alleviatethe dependence on successful determination of the species ofthe culprit snake by simply requiring successful assessment of thesnake family involved However as discussed previously theremay be disadvantages to being restricted to polyvalentantivenoms and different polyvalent antivenoms may performdifferently in a given clinical case

SNAKEBITE DIAGNOSIS IN AFRICA

The extent of the snakebite problem in Africa is difficult to assessdue to the scarcity of epidemiological data (182) However of allthe African regions affected snakebite is most commonlyobserved in sub-Saharan Africa where an estimated 90000-420000 envenomings occur annually resulting in 3000-32000deaths (5) In comparison an estimated number of 3000-80000bites occur in North Africa and the Middle East combinedleading to 4000-8000 deaths annually (5) To the best of ourknowledge a combined mortality rate for all of Africa has notbeen recorded but it has been estimated that some of thepopulations most vulnerable to snakebite worldwide are foundin Africa (191) The snakes that are responsible for the majorityof bites and are associated with serious or life-threateningenvenomings are saw-scaled vipers (Echis spp) large Africanadders or vipers (Bitis spp) spitting or cytotoxic cobras (Najaspp) neurotoxic cobras (Naja spp) and mambas (Dendroaspisspp) (192) In addition to the potency of the snake venomsthemselves factors potentially contributing to the high mortalityrate may include scarcity of antivenoms (partially due to the highcost of antivenoms relative to personal income levels) lowquality inappropriate or counterfeit antivenoms suboptimalhealth services difficulties with quick access to health centers



and insufficient training in clinical snakebite managementincluding a lack of diagnostic training andor tests (2 193ndash196)

In many African cases appropriate clinical management ofsnakebite patients requires identification of the distinctiveclinical syndrome based on epidemiological clinical andlaboratory data (eg 20WBCT) and consequently thesyndromic approach is often recommended (15) Researchersand clinicians have sought to objectively quantify the severity ofsnakebite envenoming to minimize confusion due to theambiguity of the definitions offered by current guidelines(197) In Southern Africa five main clinical syndromes ofsnakebite envenoming are recognized and often these guidediagnosis Local pain and progressive swelling (cytotoxicity)progressive paralysis (neurotoxicity) incoagulable blood(hemotoxicity) moderate to marked local swelling (associatedwith otherwise neurotoxic bites) and mild to moderate swellingwith negligible or absent systemic effects (neurotoxicity andcytotoxicity) (15) However with the syndromic approach it ispossible to misidentify snake species due to the similaritybetween symptoms that develop following envenoming fromdifferent types of snakes For instance mixed hemorrhagic andcytotoxic symptoms develop following envenoming caused bysaw-scaled vipers puff adders (Bitis arietans) and horned desertvipers (Cerastes cerastes) (see Figures 1E F) (192)

Several polyvalent and a few monovalent antivenoms havebeen marketed for the treatment of envenomings caused byAfrican snake species but the antivenoms are not necessarilyequally appropriate for the treatment of bites from a given genusor species in spite of being marketed as such (198ndash201) Theantivenoms are also not evenly distributed throughout thecontinent and some areas have been plagued by antivenomshortages (2 193 196 202) It might therefore be expected thatthe disparity in the availability and types of antivenoms in Africais reflected by a variability in the demands for diagnosisHowever to be diagnosed or treated the patient must maketheir way to either a health center or a properly trained clinicianwhich may often be difficult or result in long delays A studypublished in 2015 estimated that about 29 of the population inAfrica are geographically marginalized from emergency medicalcare and live more than two hours from the nearest publichospital (203) The same study found that only 16 of 48 countrieshave more than 80 of their population living within two hoursrsquotravel time of emergency hospital care (203) Thus it is nosurprise that many snakebite victims in rural communities resortto seeking out traditional healers rather than trained physicians(195) This trend is also observed outside of Africa when lookingat other rural parts of the world that are heavily burdened bysnakebite (204ndash209) This delay in receiving proper medical carewill in most cases worsen the symptoms and thus increase thelikelihood of a poorer clinical outcome (206 210)

For diagnosis management and treatment of snakebitevictims in Africa to improve it will be essential to address theknowledge gap between the health institutions ruralcommunities and their local traditional healers (206) Onestrategy to approach this is via outreach and educationprograms promoting snakebite prevention and first aid (211)


Such programs could even include traditional healers in anattempt to utilize their status as authority figures (211) ratherthan attempting to fight strongly-held community culturalbeliefs Also increased availability of mobile phones withinbuilt cameras could facilitate the involvement of (distantlylocated) expert herpetologists in snake species identificationwithout the need to capture or kill the snake (206 212)

SNAKEBITE DIAGNOSIS IN EUROPE

Snakebite incidents are a relatively rare occurrence in Europewith an incidence rate of 106 bites per 100000 people and about4 deaths annually (213) Contrary to what some believesnakebites from species indigenous to Europe can cause severeenvenoming and require immediate medical attention Allsignificantly venomous snakes in Europe belong to theViperinae subfamily with the common European adder(Vipera berus) European asps (V aspis) and common sandadder (V ammodytes) being responsible for the largestproportion of severe envenomings (213 214)

Many areas of Europe are inhabited by only one species ofvenomous snake especially in Northern and Central Europe(213 214) If diagnosis is necessary in areas with more than onespecies it is usually based on witness statements a picture of theculprit snake or the snake itself brought by the victim (215ndash217)In severe cases the presence of neurotoxicity can be anindication that the envenoming was caused by either acommon sand adder or a European asp as these two speciesare the most common causes of neurotoxicity due toenvenoming by indigenous European snakes Additionallybecause these species have disjunct distributions neurotoxicitycan help pinpoint exactly which species caused the bite (213218) However the absence of neurotoxicity does not excludeEuropean asp bites as most subpopulations do not possessneurotoxic venom (219) Neurotoxic clinical features have alsooccasionally been reported after envenoming by the commonEuropean adder but this has been limited to a few geographicalareas in Eastern Europe and has mostly been caused by thesubspecies known as the Bosnian viper (V berus bosniensis) (216217 220) For this reason in most of Europe elaboratelaboratory tests for diagnosis of the culprit snake species is alow priority However laboratory tests are used to assess theseverity of envenoming and thereby the need for antivenom(214 221) Clinical manifestations monitored includehypotension neurologic or gastrointestinal symptoms edemaand leukocytosis A full overview of clinical manifestations isgiven elsewhere (222)

Snakes inject a variable amount of venom and dry bites canoccur (223ndash226) Victims are normally admitted for observationfor 24 hours to monitor possible symptom progression (225227) Despite the impracticality of using clinical signs fordiagnosing the species involved in most European snakebitesthe severity of the symptoms and signs can be used to determinethe need of antivenom administration in moderate to severeenvenomings A grading system for assessing the severity of an



envenoming has been proposed based on data on the appearanceof clinical manifestations from common European adder andEuropean asp cases and has been used as a guideline in researchand in certain clinical settings (53 221 225 228)

Despite the close phylogenetic relationship between Viperaspp inter- and intraspecific venom variability might occur bothwith regard to the toxins present and their individualabundances which in turn may affect antivenom efficacy(213 229ndash231) However available monospecific antivenomsmay still show cross-reactivity between venoms and studieshave shown that antivenom raised against venom from onespecies can in some cases have clinical efficacy againstvenoms from other vipers indigenous to Europe (214 215 232)

Bites by exotic snakes are not as prevalent as those byindigenous species However they are still the cause of a fewsevere bites around Europe mostly affecting amateur snakekeepers (233ndash235) In these cases rapid identification of theresponsible species is important as it can help predict clinicalmanifestations and aid symptomatic treatment As the snake isnot endemic to the country clinicians will usually rely onstatements from witnesses for identification and requiredantivenom should be sourced as soon as possible as it mightnot be stocked in the given country (236) (exotic antivenombanks exist in a few countries eg the Netherlandsand Germany)

POTENTIAL BENEFITS OF NOVELSNAKEBITE DIAGNOSTICS

Studies find that early treatment of Australian and NorthAmerican snakebite victims is linked to faster recovery andshorter time to hospital discharge (126 174) In a similar veinof inquiry it was established that delays in treatment increase therisk of acute kidney injury in snakebite victims in Myanmar andthe risk of acute renal failure and the overall severity ofenvenoming in snakebite victims in Brazil (237ndash239) One ofthe studies also found that patients who developed acute renalfailure required more antivenom and were hospitalized for alonger period of time than those who did not (238) These studiespoint to the unsurprising conclusion that delays in treatmentoften negatively impact patient outcome which in turn can resultin prolonged hospitalization time and increased resourceconsumption at the treatment facility It thus seems plausiblethat improved diagnostics might enable rapid diagnosis andthereby facilitate early and correct treatment as well asimproved patient outcomes This is backed by a recent studyof 742 snakebite patients in Sri Lanka which argues that delays inantivenom administration reflect an absence of diagnostics forearly detection of envenoming and that such diagnostics arerequired for improved early treatment with antivenom (240)Novel diagnostics will likely have the greatest impact in areaswhere transportation to the treatment facility and antivenomavailability are not limiting factors areas with many differentindigenous snake species that are visually difficult to discernareas where monovalent antivenoms are available and areas with


medical or paramedical personnel with limited training inclinical management of snakebite envenoming

In addition to their utility in supporting clinicians indiagnosing snakebite patients and choosing the correctantivenom on a case-by-case basis novel snakebite diagnosticscould also be of interest on a grander scale They could enableepidemiologists to map patterns of snakebite incidence In turnknowing which snake species are responsible for the majority ofbites in an area can help authorities manage their resourceswhen deciding which antivenoms to procure in which quantitiesand where to deploy them within a healthcare system (241)Improved diagnostics might also inform the design of novelantivenoms and they could become indispensable tools forclinical trials of future generations of antivenoms and later (ifadopted as companion diagnostics) in clinical snakebitemanagement Based on the potential use cases and benefitslisted above it is perhaps hardly surprising that researchersand physicians have indicated the need for improved diagnosisof snakebite victims for decades (7 83 240 242ndash248)

SNAKEBITE DIAGNOSTICS REPORTED INTHE LITERATURE

Several diagnostic assays have been developed to meet thedemand for improved diagnosis of snakebite victims Thediagnostics rely on techniques varying from immunoassays(typically ELISAs) over enzymatic activity assays to forensicgenetic methods (see Table 1) These studies demonstrate thatsnakebite envenoming can be diagnosed using varioustechnologies and they showcase the development of snakebitediagnostics throughout the past six decades As evident fromTable 1 there has been a gradual shift in the preferredmethodologies from radioimmunoassays and agglutinationtests over the ever-popular ELISA format toward an increasedfocus on LFAs and more diverse non-immunological methodsAs a reflection of this technological progression theexperimental diagnostics reported in literature have becomefaster over time although interestingly their limits of detectiondo not seem to have improved significantly One hypothesisexplaining this could be that while faster immunoassays havebeen developed the antibodies at the core of these assays areessentially unchanged with most still being derived from horsesrodents and lagomorphs (see Table 1)

Many of the earliest reported diagnostic tests for snakebiteswere developed for first-world countries with Australia beingprominently featured (see Table 1) However this trend haschanged and snakebite diagnostics have now been developed forcountries all over the world As an example in Brazil an ELISA-based diagnostic tool has been utilized experimentally to aiddifferential diagnosis on a genus level (176) Similar assays havebeen developed that make it possible to evaluate the effectivenessof the antivenom administered to neutralize the venom (176)More recent examples of innovation within snakebite diagnosticsin Brazil include an impedimetric immunosensor based onelectrochemical impedance spectroscopy (86) and the use of



infrared thermography (94) Meanwhile in Asia Hung et aldeveloped a sandwich-type enzyme-linked immunosorbent assay(ELISA) capable of detecting Taiwan cobra (N atra) venom inbiological samples with a detection limit of 1 ngmL (72) Thesame group later developed an immunochromatographic strip todetect Taiwan cobra venom in patient serum in only 20 minutes(82) while a different group similarly developed an ELISA andan immunochromatographic strip for diagnosis of snake speciesin Taiwan (85) A number of other molecular diagnostic PCR-based tests for stratifying venom from Asian snake species havealso been reported However these tests typically take at least 3-4hours to complete and have lower specificity compared toimmunoassays (90ndash92 249) Generally issues with cross-reactivity of the tests toward several species remains a problemfor rapid diagnosis of snakebite envenoming and many reportedrapid diagnostic methods are not reliable enough for clinical useand can only be used for research purposes (30 40 71 7376 77)

Although the studies referenced above clearly demonstratethat snakebite diagnostics can be developed for the stratificationof many snake species and using many methods to the best ofour knowledge the SVDK is the only snakebite diagnostic tohave been adopted in the clinical setting The success of theSVDK in Australia may reflect the preference there for usingmonovalent antivenoms unlike many other countries whichrely on polyvalent antivenoms This reliance could create abarrier for adoption of venom detection tests Generallyspeaking the reason for the low adoption rate for noveldiagnostic assays is not entirely clear but a variety ofexplanations of both technical financial and implementationalnature are likely to be part of the underlying cause (250) Theantivenom market is notoriously financially unstable in manyregions (196) and if this is any indication it leaves little financialincentive for marketing snakebite diagnostics To exacerbate theproblem snakebite diagnostics are perhaps above all else neededby clinicians in remote healthcare facilities with no training inclinical snakebite management A lack of education in snakebitemanagement among the users of future diagnostics mightcomplicate the implementation of the diagnostics Even if theseand other financial and implementational challenges can besurmounted a number of technical pitfalls still exist that oneneeds to be aware of Below follows a discussion of some of thesepitfalls and design considerations that developers of snakebitediagnostics should take into account to avoid them

DESIGN CONSIDERATIONS FORSNAKEBITE DIAGNOSTICS

When developing a diagnostic for a Neglected Tropical Diseaseone of the most important factors to consider is affordability Theassociation between snakebite envenoming and poverty greatlyaffects the availability of treatment (2 7 11 12 251 252) andthis link between affordability and availability is likely to alsoexist for diagnostics Affordability may place restrictions on thetypes of equipment required to use the diagnostic especially at


small remote treatment facilities where access to electricity canbe unreliable and for point-of-care testing Point-of-care testingadditionally requires greater user-friendliness as the personcarrying out the test may have received only limited or notraining in its use For these reasons a PCR with a low limit ofdetection and a requirement for specialized laboratoryequipment and knowhow such as that developed bySupikamolseni et al (91) and a user-friendly lateral flow assaywith a higher limit of detection such as that developed by Liuet al (85) may be differentially suited for use at centralizedtreatment facilities and point-of-care settings respectivelyHowever with the implementation of different types of PCR[see eg (253ndash255)] it will likely be possible to make fast user-friendly PCR-based diagnostics for point-of-care testing inthe future

The sample matrix and sampling method should also beconsidered and as far as possible be adapted to the intendeduse case In a healthcare facility it may be convenient to useblood samples for diagnostics as it is a standard procedure totake blood samples from snakebite patients for use in the existinglaboratory diagnosis (192 256) However in point-of-care usecases wound swabs and exudates may be more readily availableWhile the sampling method affects user-friendliness the samplematrix may affect the technical specifications of the diagnostic asdifferent sample types are likely to contain differentconcentrations of the analyte at different time points as well asdifferent concentrations of interfering substances (ie substancesthat alter the detected concentration of the analyte) For exampleblood samples have notoriously complex compositionscompared to eg urine samples and this increases the risk ofblood samples containing interferants Conversely the collectionof blood samples at healthcare facilities is a highly standardizedprocedure unlike the collection of wound swab samples whichmay additionally be affected by subjection of the bite wound toinappropriate first aid methods or other forms of tamperingBeing collected from the surface of the body wound swabsamples may not be representative of the amount of venomactually delivered into the body of a victim although they maystill provide valuable information about the type of snakeinvolved As demonstrated in Table 1 diagnostics have beendeveloped and tested on various different sample matricesincluding blood (and as derivatives hereof Plasma and serum)urine tissue samples wound exudate and wound swabsPreferences for the sample matrix vary with some researchersplacing more emphasis on standardization and how well thevenom content in the sample type reflects the venom content atthe active sites in the body while others emphasize user-friendliness and a low risk of interference from other samplecomponents Perhaps to account for the advantages anddisadvantages of the various sample types some assays eg theSVDK from Australia function with multiple different samplematrices (257)

An additional factor to consider is the time required to use thediagnostic Because snakebite envenoming is acute in naturewith some toxins exerting their effects within minutes it wouldlikely be beneficial for a diagnostic device intended for clinical



use to function on a timescale of minutes rather than hoursConversely for forensic and purely epidemiological studiesrapid assay time may not be a requirement Therefore time-consuming diagnostics such as ELISAs with overnightincubations may be as well-suited for retrospective diagnosisas more rapid assays Furthermore it is advantageous for thediagnostic tool to be stable over a wide range of temperatures andenvironmental conditions as the provision of cold-storage maybe problematic in some areas of the world (16 241)

Important technical parameters which are not specific tosnakebite also need to be considered including specificitysensitivity and positive predictive value Low specificity (iethe number of true negatives divided by the total number ofindividuals not suffering from a condition) leads to falsepositives as demonstrated in a study by Ho et al (40) wherethe researchers set up an ELISA to study snakebites in ruralThailand Here non-specific reactions of ELISA reagents led to afalse positive rate of up to 75 (40) A study by Isbister et aldemonstrated how low sensitivity (ie the number of truepositives divided by the total number of individuals with thecondition) of the 20WBCT for Russellrsquos viper envenoming led toa high rate of false negatives which in some cases resulted indelayed antivenom administration (258) If the sensitivityspecificity and disease prevalence are known they can be usedto calculate the positive predictive value using the formula

Positive predictive value = sensitivity middot prevalence

sensitivity middot prevalence + (1 minus specificity) middot (1 minus prevalence)

The positive predictive value is an indication of how likelypatients with positive test results are to truly suffer from acondition (eg snakebite envenoming sustained by a cobra)Unfortunately although Ho et al argued the importance ofreporting these measurements of assay performance already in1986 (40) very few studies involving snakebite diagnostics containthese values and some diagnostics are not even tested on patientsamples (seeTable 1) The absence of positive predictive values forsnakebite diagnostics in the literature may be a reflection of thelack of available data on disease prevalence As moreepidemiological data hopefully becomes available it maybecome easier to evaluate the potential of novel diagnostics byusing the positive predictive value as a performance measurement

Additional technical parameters of importance include limitof detection (LoD) quantitativity and limit of quantitation(LoQ) In the literature snakebite diagnostics have beenreported with limits of detection (ie the lowest concentrationof a substance that can be distinguished from the absence of thesubstance) ranging from 01 pgmL to 03 mgmL (see Table 1)The limit of detection required depends on the pharmaco-toxicokinetics of the analyte For example if the analyte has ashort half-life and a high volume of distribution the limit ofdetection in a blood sample will need to be much lower than foran analyte with a long half-life and low volume of distributionThe influence of analyte kinetics is also relevant when discussingquantitative diagnostics Quantitative diagnostics are interestingbecause they can provide information not only about thepresence of an analyte but also about its abundance For


quantitative assays in addition to establishing the limit ofquantitation (the lowest amount of analyte that can bequantitatively measured with a certain precision and accuracy)it may also be important to establish threshold values Forexample if the analyte measured is a biomarker of kidneyinjury which is also found in low amounts in healthyindividuals it is important to determine a threshold value todistinguish patients with normal amounts of biomarker frompatients with abnormal amounts If the analyte is a snake venomtoxin it might be relevant to determine several thresholdscorresponding to commonly used categorizations of ldquomildrdquoldquomoderaterdquo and ldquosevererdquo envenomings which correspond todifferent treatment strategies Quantitative diagnostics couldpotentially also be used to evaluate the effectiveness with whichantivenoms sequester toxins by monitoring unbound toxins inblood samples from patients This could make quantitativediagnostics useful tools for antivenom performance evaluationand monitoring of patientsrsquo disease progressionenvenominggrade alike A low level of free toxins might mislead non-toxinologist clinicians into believing the patient can bedischarged but due to a depot effect and a mismatch betweenthe toxicokinetics of the venom and the pharmacokinetics of theantivenom symptoms can recur (259) Potentially if patients arekept under observation quantitative diagnostics will enableclinicians to detect an increase in free toxin levels beforesymptoms recur and prepare accordingly However theAchillesrsquo heel of diagnostics relying on toxin detection may bethe underlying assumption that the toxin concentration ismeasurable in a readily available sample and that thisconcentration is always reflective of the toxin concentration atthe site where the toxin exerts its effects While some studies havefound correlations between venom antigen concentration inpatient samples and certain clinical manifestations ofenvenoming others have found the opposite (52 53 69 260ndash262) This dichotomy underlines the complexity of therelationship between toxin concentration and distribution overtime (263) The matter is complicated further for both toxin andnon-toxin analytes if the influence of preexisting morbidities onthe analytersquos kinetics is factored in For instance diseases such aschronic kidney disease may alter the clearance or even thebaseline concentration of an analyte (if the analyte is anaturally occurring biomarker see (264) for examples) Thedisparity between measured analyte concentration and signs ofenvenoming might be alleviated by using samples of the affectedtissues instead of more distant tissues eg by using a musclebiopsy instead of using a wound swab if one is trying to assessmyotoxicity However for clinical (as opposed to forensic)samples this strategy could be highly problematic as the risksinvolved for the envenomed patient may outweigh the potentialbenefits If in spite of these challenges any meaningfulthresholds can be established for quantitative diagnostics itwill be important that assay precision and accuracy are high inthe surrounding range It should also be determined in whichrange there is a linear correlation between actual and measuredanalyte concentration and whether the assay is affected by thehook effect



As evidenced by Table 1 many snakebite diagnostics havebeen developed using immunological methods often usingequine l epor ine or murine IgGs For diagnost icimmunoassays it may be worth considering the format andorigin of the antibodies used While format and origin are keydecisions that greatly affect antivenom utility (177) theseantibody properties may be somewhat less influential indiagnostics because the diagnostic antibodies are not injectedthus rendering their pharmacokinetics and pharmacodynamicsirrelevant However it is still possible that endogenous factors ina patient sample (eg anti-idiotypic antibodies otherheterophilic antibodies or factors in a blood sample) can reactwith heterologous antibodies on a diagnostic test therebycausing a high background signal which needs to beaccounted for (40) In addition to establishing standard curvesand subtracting such background signals less laborious optionsare available For example it might be possible to use a differentsample type that does not contain the problematic factors or tofilter the problematic factors out of the originally selected sampletype especially if their sizes are very different from that of theanalyte [see eg (265 266)] Alternatively an excess of unspecificantibody could be added to outcompete the diagnostic antibodiesfor unspecific binding to the interfering factors The antibodyformat should also be considered as different antibody formatshave different avidities and different options for chemicalmodifications such as linkage to dyes and tags or attachmentto surfaces or larger particles (267 268)

It is also important to consider which information is of mostuse for treatment Several parameters exist that if measuredcould provide useful information to the treating physician Forexample measurement of biomarkers for the development ofclinical manifestations is already used to help physiciansidentify and predict pathologies such as coagulopathiesrhabdomyolysis and acute kidney injury (7 109 256)Detection of snake venom toxins andor identification ofsnake species is also of interest as knowing the snake speciesor type of venom injected into the victim can aid in decidingwhich antivenom (if any) is appropriate as well as it might helppredict later clinical manifestations It may be relevant todistinguish which snakes are of greatest interest to discernfrom an epidemiological and a clinical viewpoint respectivelyFor example snake stratification at a species level may be veryvaluable in epidemiological studies as it can uncover neglected


species which should be included in future antivenomsConversely stratification at the species level may be entirelyirrelevant from a clinical perspective if no species-specificantivenoms are available As such the taxonomic level atwhich snakes should be differentiated depends on theintended usage of the diagnostic and for diagnostics intendedto support clinicians in deciding on treatment it will depend onthe treatments available in that area Whichever analyte ischosen it will inform subsequent interference studies (iestudies that determine which if any substances from patientsamples interfere with the measured analyte concentration)Eg if the diagnostic measures the concentration of a cobracytotoxin in order to diagnose a patient with cytotoxic cobraenvenoming it should be investigated whether thediagnostic also reacts with cytotoxins from the venoms ofother snakes found in the same area Additionally in thisexample it should be tested whether antibiotics (which aresometimes administered to fight infections at the bitesite)prophylactically administered antivenom or other factorsfound in the sample matrix (eg anti-idiotypic antibodies asmentioned above or medications used to treat preexistingmorbidities) can interfere with analyte measurement eg bypotentiating the enzymatic activities of snake venom toxins(269) Several of the studies listed in Table 1 describeinvestigations of the potential for cross-reactivity with othersnake venoms with some studies having screened multiplevenoms and others only a few but none of the studies report abroader systematic screening for interferents

Ultimately the design of a novel diagnostic will be fraughtwith compromises as developers will have to weigh the pros andcons of diagnostic technologies for different applicationsComparatively slow and sensitive ELISAs may be ideal forcoroners and researchers attempting to retrospectively identifythe type of venom that caused a patientrsquos death while rapid anduser-friendly albeit potentially less sensitive LFAs may bepreferable to first-responders trying to decide on appropriatefirst aid (or maybe in the future to decide on whether to usefirst-line-of-defense drugs such as varespladib or batimastat)The most desirable properties of a diagnostic will thus always bedetermined by its intended usage and it is unlikely that there willbe a one-size-fits-all solution to developing snake venomdiagnostics Rather multiple technologies are likely to find usein various applications

TABLE 3 | Overview of clinically commonly used diagnostic methods for snakebite envenoming in different parts of the world

Syndromic approach Visual identification of the snake if available Patient history 20WBCT Lab tests Immuno-assays

Australia () Africa ()

Asia ()

Europe North America () Latin America ()

April 20

21 | Volume 12

Diagnostic methods can vary between and within countries in these regions and tick marks in parentheses indicate that the method is infrequently used or only used in relatively few areas

| Article 661457


Novel diagnostic tools for snakebite envenoming and snakeidentification do not have to come in the form of bioassaysRecently an alternative was proposed with the suggestion thatapps capable of recognizing photos of biting snakes andormatching syndromes to snakes could empower healthcareproviders and facilitate better treatment (212) Additionallyimproved diagnosis of snakebite victims will likely depend onother initiatives in addition to novel diagnostics For instancestudies from multiple different countries indicate thatmisidentification of snakes occurs and in some cases promptsinadequate treatment (112 115 170 270ndash273) Severalauthorities have therefore indicated the need for improvededucation and training of healthcare workers (7 9 244 252274ndash277) a sentiment that is echoed in the World HealthOrganizationrsquos 2019 strategy for snakebite envenoming (211)

CONCLUSION

Snakebite envenoming has long been neglected and the lack ofcare is ndash in the words of Williams et al ndash a cruel anachronism(278) This neglect affects education treatment and diagnosisnone of which have received the attention or resources theydeserve However with the reinstatement of snakebiteenvenoming on the World Health Organizationrsquos list of top-prioritized Neglected Tropical Diseases in 2017 snakebite isgarnering more attention funding and resources Thisconstitutes an excellent opportunity for scientists physiciansand other stakeholders (many of whom have been workingtirelessly for decades to alleviate the burden of snakebiteenvenoming) to critically revisit both current practices andnew efforts within clinical snakebite management In this


relation it is important to remember the symbioticrelationship between treatment and diagnostics Treatmentsshould only be administered when so indicated by differentialdiagnosis and the pertinence of diagnostics depends on theavailable treatments Currently the most widely used method ofdiagnosis of snakebite envenoming is the syndromic approach(see Table 3) This approach can be highly effective when thetreating physician possesses sufficient knowledge on snakes andhas been properly trained in providing correct differentialdiagnosis Unfortunately not all physicians possess thisknowledge and expertise and in some regions utilization ofthe syndromic approach is challenging If novel diagnostics couldbe implemented in such clinical settings they could support thestandardization of snakebite diagnosis In combination withimproved training of healthcare workers this could in turnfurther improve standardization of treatment Additionalbenefits could be reaped by using diagnostics to improve ourknowledge of prevalence and inform the design of antivenomsand resource management

AUTHOR CONTRIBUTIONS

AL CK and JJ conceptualized the manuscript AH CK JJ RFSF and SD prepared the original draft All authors contributed tothe article and approved the submitted version

FUNDING

We thank Innovation Fund Denmark (grant 9065-00007B) forfinancial support

REFERENCES

1 Mohapatra B Warrell DA Suraweera W Bhatia P Dhingra N Jotkar RMet al Snakebite mortality in India A nationally representative mortalitysurvey PloS Negl Trop Dis (2011) 51ndash8 doi 101371journalpntd0001018

2 Brown NI Consequences of neglect Analysis of the sub-saharan africansnake antivenommarket and the global context PloS Negl Trop Dis (2012) 6e1670 doi 101371journalpntd0001670

3 Fox S Rathuwithana AC Kasturiratne A Lalloo DG de Silva HJUnderestimation of snakebite mortality by hospital statistics in theMonaragala District of Sri Lanka Trans R Soc Trop Med Hyg (2006)100693ndash5 doi 101016jtrstmh200509003

4 Kasturiratne A Wickremasinghe AR De Silva N Gunawardena NKPathmeswaran A Premaratna R et al The global burden of snakebite Aliterature analysis and modelling based on regional estimates of envenoming anddeaths PloS Med (2008) 51591ndash604 doi 101371journalpmed0050218

5 Chippaux J-P Snake-bites appraisal of the global situationWHO Bull OMS(1998) 76515ndash24

6 Swaroop S Grab B Snakebite mortality in the world Bull World HealthOrgan (1954) 1035ndash76

7 Gutierrez JM Calvete JJ Habib AG Harrison RA Williams DJ Warrell DASnakebite envenoming Nat Rev Dis Prim (2017) 317063 doi 101038nrdp201763

8 Habib AG Kuznik A Hamza M Abdullahi MI Chedi BA Chippaux J-Pet al Snakebite is Under Appreciated Appraisal of Burden from WestAfrica PloS Negl Trop Dis (2015) 94ndash11 doi 101371journalpntd0004088

9 White J Mahmood MA Alfred S Thwin KT Kyaw KM Zaw A et al Acomprehensive approach to managing a neglected neglected tropicaldisease The Myanmar Snakebite Project (MSP) Toxicon X (2019)1100001 doi 101016jtoxcx2018100001

10 Mahmood MA Halliday D Cumming R Thwin KT Kyaw MMZ White Jet al Snakebite incidence in two townships in Mandalay Division MyanmarPloS Negl Trop Dis (2018) 121ndash12 doi 101371journalpntd0006643

11 Harrison RA Hargreaves A Wagstaff SC Faragher B Lalloo DG SnakeEnvenoming A disease of poverty PloS Negl Trop Dis (2009) 3e569doi 101371journalpntd0000569

12 Chaves LF Chuang T-W Sasa M Gutierrez JM Snakebites are associatedwith poverty weather fluctuations and El Nintildeo Sci Adv (2015) 1e1500249ndashe1500249 doi 101126sciadv1500249

13 Pucca MB Cerni FA Janke R Bermudez-mendez E Ledsgaard L BarbosaJE et al History of Envenoming Therapy and Current Perspectives FrontImmunol (2019) 10159 doi 103389fimmu20190159

14 World Health Organization Global snakebite burden World HealthOrganization (2018) Available at httpsappswhointgbebwhapdf_filesWHA71A71_17-enpdf

15 Muumlller GJ Modler H Wium CA Veale DJH Snake bite in southern Africadiagnosis and management Contin Med Educ J (2012) 30362ndash81doi 101111j1365-29231988tb00427x

16 World Health Organization Guidelines for the production control andregulation of snake antivenom immunoglobulins (2018) Available athttpswwwwhointbloodproductsAntivenomGLrevWHO_TRS_1004_web_Annex_5pdfua=1



17 Rojnuckarin P Clinical Uses of Snake Antivenoms Clin Toxinol Asia PacificAfrica2 (2015) 2437ndash52 doi 101007978-94-007-6386-9_24

18 White J Venomous animals clinical toxinology In A Luch editorMolecular Clinical and Environmental Toxicology Birkhaumluser Verlag AG(2010) p 233ndash91

19 Wilkins D Burns DS Wilson D Warrell DA Lamb LEM Snakebites inAfrica and Europe a military perspective and update for contemporaryoperations J R Army Med Corps (2018) 164(5)370ndash9 doi 101136jramc-2017-000883 jramc-2017-000883

20 Calvete JJ Sanz L Cid P de la Torre P Flores-Dıaz M Dos Santos MC et alSnake Venomics of the Central American Rattlesnake Crotalus simus andthe South American Crotalus durissus Complex Points to Neurotoxicity asan Adaptive Paedomorphic Trend along Crotalus Dispersal in SouthAmerica J Proteome Res (2010) 9528ndash44 doi 101021pr9008749

21 Calvete JJ Sanz L Perez A Borges A Vargas AM Lomonte B et al Snakepopulation venomics and antivenomics of Bothrops atrox Paedomorphismalong its transamazonian dispersal and implications of geographic venomvariability on snakebite management J Proteomics (2011) 74510ndash27doi 101016jjprot201101003

22 Madrigal M Sanz L Flores-Dıaz M Sasa M Nuntildeez V Alape-Giron A et alSnake venomics across genus Lachesis Ontogenetic changes in the venomcomposition of Lachesis stenophrys and comparative proteomics of thevenoms of adult Lachesis melanocephala and Lachesis acrochordaJ Proteomics (2012) 77280ndash97 doi 101016jjprot201209003

23 Chippaux J-P Williams V White J Snake venom variability methods ofstudy results and interpretation Toxicon (1991) 291279ndash303 doi 1010160041-0101(91)90116-9

24 Muelling RJ Samson RF Beven T The precipitin test in elucidating thecause of death Am J Clin Pathol (1957) 28489ndash94 doi 101093ajcp285489

25 Trethewie ER Rawlinson PA Immunological diagnosis of type of snake insnake-bite Med J Aust (1967) 2111ndash3 doi 105694j1326-53771967tb73915x

26 Boche RD Russell FE Passive hemagglutination studies with snake venomand antivenin Toxicon (1968) 6125ndash7 doi 1010160041-0101(68)90031-7

27 Greenwood BM Warrell DA Davidson NM Ormerod LD Reid HAImmunodiagnosis of Snake Bite Br Med J (1974) 4743ndash5 doi 101136bmj45947743

28 Coulter AR Sutherland SK Broad AJ Assay of snake venoms in tissue fluidsJ Immunol Methods (1974) 4297ndash300 doi 1010160022-1759(74)90072-6

29 Sutherland SK Coulter AR Broad AJ Human snake bite victims thesuccessful detection of circulating snake venom by radioimmunoassayMed J Aust (1975) 127ndash9 doi 105694j1326-53771975tb111208x

30 Theakston RDG Lloyd-Jones MJ Reid HA Micro-ELISA for detecting andassaying snake venom and venom-antibody Lancet (1977) 2639ndash41doi 101016S0140-6736(77)92502-8

31 Sutherland SK Coulter AR Snake bite detection of venom byradioimmunoassay Med J Aust (1977) 12683ndash4 doi 105694j1326-53771977tb99207x

32 Sutherland SK Coulter AR Three instructive cases of tiger snake (Notechisscutatus) envenomationndashand how a radioimmunoassay proved thediagnosis Med J Aust (1977) 6177ndash80 doi 105694j1326-53771977tb114543x

33 Coulter AR Fox JC Sutherland SK Waddell CJ A new solid-phasesandwich radioimmunoassay and its application to the detection of snakevenom J Immunol Methods (1978) 23241ndash52 doi 1010160022-1759(78)90198-9

34 Coulter AR Harris RD Sutherland SK Clinical Laboratory Enzymeimmunoassay for the rapid clinical detection of snake venom Med J Aust(1980) 1433ndash5 doi 105694j1326-53771980tb135003x

35 Chandler HM Hurrell JGR A new enzyme immunoassay system suitable forfield use and its application in a snake venom detection kit Clin Chim Acta(1982) 121225ndash30 doi 1010160009-8981(82)90062-6

36 Lwin KO Myint AA The use of enzyme-linked immunosorbent assay indetection of Russellrsquos viper venom in body fluid Snake (1983) 1477ndash82

37 Dhaliwal JS Lim TW Sukumaran KD A double antibody sandwich micro-ELISA kit for the rapid diagnosis of snake bite Southeast Asian J Trop MedPublic Heal (1983) 14367ndash73


38 Minton SA Weinstein SA Wilde CE3 An enzyme-linked immunoassay fordetection of North American pit viper venoms J Toxicol - Clin Toxicol(1984) 22303ndash16 doi 10310915563658408992562

39 Lwin KO Myint AA Pe T Nwe T Naing M Russellrsquos viper venom levels inserum of snake bite victims in Burma Trans R Soc Trop Med Hyg (1984)78165ndash8 doi 1010160035-9203(84)90267-0

40 Ho M Warrell MJ Warrell DA Bidwell D Voller A A critical reappraisal ofthe use of enzyme-linked immunosorbent assays in the study of snake biteToxicon (1986) 24211ndash21 doi 1010160041-0101(86)90147-9

41 Doellgast GJ Enzyme-linked coagulation assay Anal Biochem (1987)16797ndash105 doi 1010160003-2697(87)90137-0

42 Pugh RNH Theakston RDG A clinical study of viper bite poisoning AnnTrop Med Parasitol (1987) 81135ndash49 doi 10108000034983198711812106

43 Watt G Wurzel WD Theakston RDG Postmortem immunodiagnosis ofcobra bite in a Marine Mil Med (1987) 152209ndash10 doi 101093milmed1524209

44 Silamut K Ho M Looareesuwan S Viravan C Wuthiekanun V Warrell DADetection of venom by enzyme linked immunosorbent assay (ELISA) inpatients bitten by snakes in Thailand Br Med J (1987) 294402ndash4doi 101136bmj2946569402

45 Pukrittayakamee S Ratcliffe PJ McMichael AJ Warrell DA Bunnag D Acompetitive radioimmunoassay using a monoclonal antibody to detect thefactor X activator of Russellrsquos viper venom Toxicon (1987) 25721ndash9doi 1010160041-0101(87)90122-X

46 Labrousse H Nishikawa AK Boni C Avrameas S Development of a rapidand sensitive enzyme-linked immunosorbent assay (ELISA) for measuringvenom antigens after an experimental snake bite Toxicon (1988) 261157ndash67 doi 1010160041-0101(88)90300-5

47 Watanabe M Sakai A Kouda T Sawai Y Detection of Agkistrodonblomhoffii blomhoffii venom in serum of mice ny enzyme-linkedimmunosorbent assay (ELISA) Snake (1988) 2025ndash9

48 Barral-Netto M Schriefer A Vinhas V Almeida AR Enzyme-linkedimmunosorbent assay for the detection of Bothrops jararaca venomToxicon (1990) 281053ndash61 doi 1010160041-0101(90)90143-U

49 Hatsuse M Sakai A Sawai Y Detection and quantitative assay ofAgkistrodon blomhoffii blomhoffii venom and antivenom by enzyme-linkedimmunosorbent assay (ELISA) with special reference to in-vivo effect of theantivenom Snake (1990) 2220ndash7

50 Chinonavanig L Karnchanachetanee C Pongsettakul P RatanabanangkoonK Diagnosis of snake venoms by a reverse latex agglutination test J ClinToxicol (1991) 29493ndash503 doi 10310915563659109025746

51 Barral-Netto M von Sohsten RL Serum kinetics of crotoxin from Crotalusdurissus terrificus venom in mice evidence for a rapid clearance Toxicon(1991) 29527ndash31 doi 1010160041-0101(91)90028-P

52 Tun-Pe Ba-Aye Aye-Aye-Myint Tin-Nu-Swe Warrell DA Bites byRussellrsquos vipers (Daboia russelii siamensis) in Myanmar effect of thesnakersquos length and recent feeding on venom antigenaemia and severity ofenvenoming Trans R Soc Trop Med Hyg (1991) 85804ndash8 doi 1010160035-9203(91)90464-A

53 Audebert F Sorkine M Bon C Envenoming by viper bites in France Clinicalgradation and biological quantification by ELISA Toxicon (1992) 30599ndash609 doi 1010160041-0101(92)90854-X

54 Audebert F Grosselet O Sabouraud A Bon C Quantitation of VenomAntigens from European Vipers in Human Serum or Urine by ELISA J AnalToxicol (1993) 17236ndash40 doi 101093jat174236

55 Audebert F Sorkine M Robbe-Vincent A Bon C Viper bites in FranceClinical and biological evaluation Kinetics of envenomations Hum ExpToxicol (1994) 13683ndash8 doi 101177096032719401301006

56 Audebert F Urtizberea M Sabouraud A Scherrmann JM Bon CPharmacokinetics of Vipera aspis venom after experimental envenomationin rabbits J Pharmacol Exp Ther (1994) 2681512ndash7

57 Cox JC Moisidis AV Shepherd JM Drane DP Jones SL A novel format fora rapid sandwich EIA and its application to the identification of snakevenoms J Immunol Methods (1992) 146213ndash8 doi 1010160022-1759(92)90230-Q

58 Tan NH Yeo KH Nik Jaafar MI The use of enzyme-linked immunosorbentassay for the quantitation of Calloselasma rhodostoma (Malayan pit viper)



venom and venom antibodies Toxicon (1992) 301609ndash20 doi 1010160041-0101(92)90033-2

59 Kittigul L Ratanabanangkoon K Reverse passive hemagglutination tests forrapid diagnosis of snake envenomation J Immunoassay (1993) 14105ndash27doi 10108015321819308019844

60 Heneine LGD Catty D Species-specific detection of venom antigens fromsnakes of the Bothrops and Lachesis genera Toxicon (1993) 31591ndash603doi 1010160041-0101(93)90114-X

61 Chavez-Olortegui C Lopes CS Cordeiro FD Granier C Diniz CR Anenzyme linked immunosorbent assay (ELISA) that discriminates betweenBothrops atrox and Lachesis muta muta venoms Toxicon (1993) 31417ndash26doi 1010160041-0101(93)90177-K

62 Bhatti AR Wong JP Siddiqui YM Siddiqui S A sensitivte fluorogenicenzyme linked immunosorbent assay for the detection of Vipera russellivenom Nat Toxins (1993) 1277ndash82 doi 101002nt2620010504

63 Lalloo D Trevett A Black J Mapao J Naraqi S Owens D et alNeurotoxicity and haemostatic disturbances in patients envenomed by thePapuan black snake (Pseudechis papuanus) Toxicon (1994) 32927ndash36doi 1010160041-0101(94)90371-9

64 Gillissen A Theakston RDG Barth J May B Krieg M Warrell DANeurotoxicity haemostatic disturbances and haemolytic anaemia after abite by a Tunisian saw-scaled or carpet viper (Echis ldquopyramidumrdquo-complex)Failure of antivenom treatment Toxicon (1994) 32937ndash44 doi 1010160041-0101(94)90372-7

65 Li Q Ownby CL Development of an enzyme-linked immunosorbent assay(ELISA) for identification of venoms from snakes in the Agkistrodon genusToxicon (1994) 321315ndash25 doi 1010160041-0101(94)90404-9

66 Pe T Myint AA Warrell DA Myint T King cobra (Ophiophagus hannah)bites in Myanmar Venom antigen levels and development of venomantibodies Toxicon (1995) 33379ndash82 doi 1010160041-0101(94)00157-4

67 Sjostrom L Karlson-Stiber C Persson H Al-Abdulla IH Smith DCDevelopment and clinical application of immunoassays for Europeanadder (Vipera berus berus) venom and antivenom Toxicon (1996) 3491ndash8 doi 1010160041-0101(95)00110-7

68 De AK Development of an immunodiagnostic kit for species identificationof snakebite and studies on the cross-reacting venom antigens [PhD thesis]Bangalore Indian Institute of Science (1996) Available at httpsetdiiscacinstaticetdabstracts3956G14544-Abspdf

69 Bucher B Canonge D Thomas L Tyburn B Robbe-Vincent A Choumet Vet al Clinical indicators of envenoming and serum levels of venom antigensin patients bitten by Bothrops lanceolatus in Martinique Trans R Soc TropMed Hyg (1997) 91186ndash90 doi 101016S0035-9203(97)90219-4

70 Amuy E Alape-Giron A Lomonte B Thelestam M Gutierrez JMDevelopment of immunoassays for determination of circulating venomantigens during envenomations by coral snakes (Micrurus species)Toxicon (1997) 351605ndash16 doi 101016S0041-0101(97)00045-7

71 Selvanayagam ZE Gnanavendhan SG Ganesh KA Rajagopal D Rao PVELISA for the detection of venoms from four medically important snakes ofIndia Toxicon (1999) 37757ndash70 doi 101016S0041-0101(98)00215-3

72 Huang YP Yu YJ Hung D-Z Sandwich enzyme-linked immunosorbentassay for Taiwan cobra venom Vet Hum Toxicol (2002) 44200ndash2009

73 Dong LV Selvanayagam ZE Gopalakrishnakone P Eng KH A new avidin-biotin optical immunoassay for the detection of beta-bungarotoxin andapplication in diagnosis of experimental snake envenomation J ImmunolMethods (2002) 260125ndash36 doi 101016S0022-1759(01)00527-0

74 Lion V Rojas A Rodriguez-Acosta A Design of an agglutination latex test todetect venoms from viperidae snakes in patientsrsquo serum Rev Cuba Med Trop(2004) 5613ndash20

75 Van Dong L Khoo HE Quyen LK Gopalakrishnakone P Opticalimmunoassay for snake venom detection Biosens Bioelectron (2004)191285ndash94 doi 101016jbios200311020

76 Brunda G Sashidhar RB Sarin RK Use of egg yolk antibody (IgY) as animmunoanalytical tool in the detection of Indian cobra (Naja naja naja)venom in biological samples of forensic origin Toxicon (2006) 48183ndash94doi 101016jtoxicon200604011

77 Brunda G Sashidhar RB Epidemiological profile of snake-bite cases fromAndhra Pradesh using immunoanalytical approach Indian J Med Res (2007)125661ndash5


78 Gao R Zhang Y Gopalakrishnakone P Immuno-fluorescence detection ofsnake venom by using single bead as the assay platform J Exp Nanosci(2008) 3111ndash9 doi 10108017458080701713953

79 Kulawickrama S OrsquoLeary MA HodgsonWC Brown SGA Jacoby T DavernK et al Development of a sensitive enzyme immunoassay for measuringtaipan venom in serum Toxicon (2010) 551510ndash8 doi 101016jtoxicon201003003

80 Nuntildeez Rangel V Fernandez Culma M Rey-suarez P Pereantildeez JADevelopment of a sensitive enzyme immunoassay (ELISA) for specificidentification of Lachesis acrochorda venom J Venom Anim Toxins InclTrop Dis (2012) 18173ndash9 doi 101590S1678-91992012000200007

81 Ibrahim NM El-Kady EM Katamesh RA El-Borei IH Wahby AFIdentification and discrimination of snake venoms from Egyptian elapidsToxicon (2013) 6388ndash97 doi 101016jtoxicon201211020

82 Hung D-Z Lin J-H Mo JF Huang CF Liau MY Rapid diagnosis of Najaatra snakebites Clin Toxicol (2014) 52187ndash91 doi 103109155636502014887725

83 Pawade BS Salvi NC Shaikh IK Waghmare AB Jadhav NDWagh VB et alRapid and selective detection of experimental snake envenomation - Use ofgold nanoparticle based lateral flow assay Toxicon (2016) 119299ndash306doi 101016jtoxicon201610009

84 Shaikh IK Dixit PP Pawade BS Waykar IG Development of dot-ELISA forthe detection of venoms of major Indian venomous snakes Toxicon (2017)13966ndash73 doi 101016jtoxicon201710007

85 Liu CC Yu JS Wang PJ Hsiao YC Liu CH Chen YC et al Development ofsandwich ELISA and lateral flow strip assays for diagnosing clinicallysignificant snakebite in Taiwan PloS Negl Trop Dis (2018) 121ndash23doi 101371journalpntd0007014

86 Faria RAD Lins V de F Nappi GU Matencio T Heneine LGDDevelopment of an Impedimetric Immunosensor for Specific Detection ofSnake Venom Bionanoscience (2018) 8988ndash96 doi 101007s12668-018-0559-7 and 101007126682191-1649

87 Maduwage KP Gawarammana IB Gutierrez JM Kottege C Dayaratne RPremawardena NP et al Enzyme immunoassays for detection andquantification of venoms of Sri Lankan snakes Application in the clinicalsetting PloS Negl Trop Dis (2020) 141ndash17 doi 101371journalpntd0008668

88 Lin J-H Lo C-M Chuang S-H Chiang C-H Wang S-D Lin TY et alCollocation of avian and mammal antibodies to develop a rapid and sensitivediagnostic tool for Russellrsquos vipers snakebite PloS Negl Trop Dis (2020) 141ndash19 doi 101371journalpntd0008701

89 Lin J-H Sung W Liao J Hung D-Z A Rapid and International ApplicableDiagnostic Device for Cobra (Genus Naja) Snakebites Toxins (Basel) (2020)121ndash14 doi 103390toxins12090572

90 Suntrarachun S Pakmanee N Tirawatnapong T Chanhome L Sitprija VDevelopment of a polymerase chain reaction to distinguish monocellatecobra (Naja khouthia) bites from other common Thai snake species usingboth venom extracts and bite-site swabs Toxicon (2001) 391087ndash90doi 101016S0041-0101(00)00246-4

91 Supikamolseni A Ngaoburanawit N Sumnotha M Chanhome LSuntrarachun S Peyachoknagul S et al Molecular barcoding ofvenomous snakes and species-specific multiplex PCR assay to identifysnake groups for which antivenom is available in Thailand Genet Mol Res(2015) 1413981ndash97 doi 1042382015October2918

92 Sharma SK Kuch U Hoede P Brunhse L Pandey DP Ghimire A et al Useof Molecular Diagnostic Tools for the Identification of Species Responsiblefor Snakebite in Nepal A Pilot Study PloS Negl Trop Dis (2016) 10e0004620 doi 101371journalpntd0004620

93 Maduwage K OrsquoLeary MA Isbister GK Diagnosis of snake envenomationusing a simple phospholipase A2 assay Sci Rep (2014) 41ndash4 doi 101038srep04827

94 de Medeiros CR Brioschi ML de Souza SN Teixeira MJ Infraredthermography to diagnose and manage venomous animal bites andstings Rev Soc Bras Med Trop (2017) 50260ndash4 doi 1015900037-8682-0390-2016

95 Isbister GK Mirajkar N Fakes K Brown SGA Veerati PC PhospholipaseA2 (PLA2) as an early indicator of envenomation in elapid snakebites (ASP-27) Clin Toxicol (2018) 56919ndash20 doi 1010801556365020181506610



96 Chippaux JP Theakston RD Epidemiological studies of snake bite in FrenchGuiana Ann Trop Med Parasitol (1987) 81301ndash4 doi 10108000034983198711812123

97 Pugh RNH Theakston RDG Incidence and mortality of snakebite inSavanna Nigeria Lancet (1980) 21181ndash3 doi 101016S0140-6736(80)92608-2

98 Theakston RDG Pugh RNH Reid HA Enzyme-linked immunosorbent-assay of venom-antibodies in human victims of snake bite J Trop Med Hyg(1981) 84109ndash12

99 Bober MA Glenn JL Straight RC Ownby CL Detection of myotoxin a-likeproteins in various snake venoms Toxicon (1988) 26665ndash73 doi 1010160041-0101(88)90248-6

100 Bober MA Ownby CL Use of affinity-purified antibodies to measure the invivo disappearance of antibodies to myotoxin a Toxicon (1988) 26301ndash8doi 1010160041-0101(88)90221-8

101 Alape-Giron A Stiles BG Gutierrez JM Antibody-mediated neutralizationand binding-reversal studies on a-neurotoxins from Micrurus nigrocinctusnigrocinctus (coral snake) venom Toxicon (1996) 34369ndash80 doi 1010160041-0101(95)00126-3

102 Stiles BG A non-radioactive receptor assay for snake venom postsynapticneurotoxins Toxicon (1991) 29503ndash10 doi 1010160041-0101(91)90024-L

103 Lomonte B Gutierrez J Romero M Nuntildeez J Tarkowski A Hanson LAring AnMTT-based method for the in vivo quantification of myotoxic activity ofsnake venoms and its neutralization by antibodies J Immunol Methods(1993) 161231ndash7 doi 1010160022-1759(93)90299-M

104 Laustsen AH Engmark M Milbo C Johannesen J Lomonte B Gutierrez JMet al From Fangs to Pharmacology The Future of Antivenoms Curr PharmDes (2016) 95270ndash93 doi 1021741381612822666160623073438

105 Laustsen AH Solagrave M Jappe EC Oscoz S Lauridsen LP Engmark MBiotechnological trends in spider and scorpion antivenom developmentToxins (Basel) (2016) 8226 doi 103390toxins8080226

106 Knudsen C Laustsen AH Recent Advances in Next Generation SnakebiteAntivenoms Trop Med Infect Dis (2018) 342 doi 103390tropicalmed3020042

107 Laustsen AH Karatt-Vellatt A Masters EW Arias AS Pus U Knudsen Cet al In vivo neutralization of dendrotoxin-mediated neurotoxicity of blackmamba (Dendroaspis polylepis) venom by mixtures of human IgGmonoclonal antibodies Nat Commun (2018) 93928 doi 101038s41467-018-06086-4

108 Albulescu L-O Xie C Ainsworth S Alsolaiss J Crittenden E Dawson CAet al A therapeutic combination of two small molecule toxin inhibitorsprovides broad preclinical efficacy against viper snakebite Nat Commun(2020) 111ndash14 doi 101038s41467-020-19981-6

109 White J A Clinicianrsquos Guide to Australian Venomous Bites and Stingsincorporating the updated CSL antivenom handbook (2013) Available athttpsbiomedicalsciencesunimelbeduaudataassetspdf_file00043216739A-Clinicians-Guide-to-Venomous-Bites-and-Stings-2013pdf

110 Lavonas EJ Ruha AM Banner W Bebarta V Bernstein JN Bush SP et alUnified treatment algorithm for the management of crotaline snakebite inthe United States Results of an evidence-informed consensus workshopBMC Emerg Med (2011) 112 doi 1011861471-227X-11-2

111 Orduna TA Lloveras SC de Roodt AR Garcia SI Haas AI Moreno I et alGuıa de Prevencion Diagnostico Tratamiento y Vigilancia Epidemiologica delos Envenenamientos Ofidicos Buenos Aires (2007) Available at httpwwwafamorgararticulos20sitio20web202016ofidismopdf

112 Bolon I Durso AM Mesa SB Ray N Alcoba G Chappuis F et al Identifyingthe snake First scoping review on practices of communities and healthcareproviders confronted with snakebite across the world PloS One (2020) 151ndash24 doi 101371journalpone0229989

113 Harris JB Faiz MA Rahman MR Jalil MM Ahsan MF Theakston RDGet al Snake bite in Chittagong Division Bangladesh A study of bittenpatients who developed no signs of systemic envenoming Trans R Soc TropMed Hyg (2010) 104320ndash7 doi 101016jtrstmh200912006

114 White J Alfred S Bates D Mahmood MA Warrell DA Cumming R et alTwelve month prospective study of snakebite in a major teaching hospital inMandalay Myanmar Myanmar Snakebite Project (MSP) Toxicon X (2018)1100002 doi 101016jtoxcx2018100002


115 Joseph JK Simpson ID Menon NCS Jose MP Kulkarni KJ RaghavendraGB et al First authenticated cases of life-threatening envenoming by thehump-nosed pit viper (Hypnale hypnale) in India Trans R Soc Trop Med Hyg(2007) 10185ndash90 doi 101016jtrstmh200603008

116 Isbister GK Brown SGA Page CB Snakebite in Australia a practicalapproach to diagnosis and treatment Med J Aust (2013) 199763ndash8doi 105694mja1211172

117 Ireland G Brown SGA Buckley NA Stormer J Currie BJ White J et alChanges in serial laboratory test results in snakebite patients when can wesafely exclude envenoming Med J Aust (2010) 193285ndash90 doi 105694j1326-53772010tb03909x

118 Isbister GK Snake bite A current approach to management Aust Prescr(2006) 29156ndash8 doi 1018773austprescr2006095

119 Welton RE Williams DJ Liew D Injury trends from envenoming inAustralia 2000-2013 Intern Med J (2016) 47170ndash6 doi 101111imj13297

120 Welton RE Liew D Braitberg G Incidence of fatal snakebite in Australia Acoronial based retrospective study (2000-2016) Toxicon (2017) 13111ndash5doi 101016jtoxicon201703008

121 Johnston CI Ryan NM Page CB Buckley NA Brown SGA OrsquoLeary MAet al The Australian snakebite project 2005ndash2015 (ASP-20) Med J Aust(2017) 207119ndash25 doi 105694mja1700094

122 Steuten J Winkel K Carroll T Williamson NA Ignjatovic V Fung K et alThe molecular basis of cross-reactivity in the Australian Snake VenomDetection Kit (SVDK) Toxicon (2007) 501041ndash52 doi 101016jtoxicon200707023

123 Nimorakiotakis V Winkel KD Prospective assessment of the false positiverate of the Australian snake venom detection kit in healthy human samplesToxicon (2016) 111143ndash6 doi 101016jtoxicon201512002

124 Weinstein SA Mirtschin PJ White J Risks and realities of single vialantivenom recommendations for envenoming by Australian elapid snakesMed J Aust (2019) 211492ndash3 doi 105694mja250314

125 Johnston CI Brown SGA OrsquoLeary MA Currie BJ Greenberg R Taylor Met al Mulga snake (Pseudechis australis) envenoming A spectrum ofmyotoxicity anticoagulant coagulopathy haemolysis and the role of earlyantivenom therapy-Australian Snakebite Project (ASP-19) Clin Toxicol(2013) 51417ndash24 doi 103109155636502013787535

126 Johnston CI Ryan NM OrsquoLeary MA Brown SGA Isbister GK Australiantaipan (Oxyuranus spp) envenoming clinical effects and potential benefits ofearly antivenom therapyndashAustralian Snakebite Project (ASP-25) ClinToxicol (2017) 55115ndash22 doi 1010801556365020161250903

127 Joseph JK Viperidae Envenomation in India In Gopalakrishnakone P Faiz AFernando R Gnanathasan C Habib A and Yang CC editors ToxinologyClinical Toxinology in Asia Pacific and Africa vol 2 Springer Dordecht (2015)pp 191ndash208 doi 101007978-94-007-6386-9_40

128 de Silva HJ Gunatilake SB Kularatne SA Sellahewa KH Anti-venom forsnakebite in Sri Lanka Ceylon Med J (2011) 4743ndash5 doi 104038cmjv47i23449

129 Chanhome L Cox MJ Wilde H Jintakoon P Chaiyabutr N Sitprija VVenomous snakebite in Thailand I Medically important snakes Mil Med(1998) 163310ndash7 doi 101093milmed1635310

130 Okamoto O Suzuki R Kusatsu M Nakashima R Inagaki N Kai Y et alMamushi (Gloydius blomhoffii) Snake Bites in Japan - Current Problems andClues to a Solution J Pharmacol Clin Toxicol (2018) 61103

131 Rha JJ Kwon SM Oh JR Han BK Lee KH Kim JH Snakebite in Korea AGuideline to Primary Surgical Management Yonsei Med J (2015) 561443ndash8doi 103349ymj20155651443

132 Fung HTJ Lam SKT Kam CW Simpson ID A survey of SnakebiteManagement Knowledge Amongst Select Physicians in Hong Kong andthe Implications for Snakebite Training Wilderness Environ Med (2009)20364ndash70 doi 1015801080-6032-0200040364

133 Mao Y-C Hung D-Z Epidemiology of snake envenomation in Taiwan ClinToxinol Asia Pacific Afr (2015) 3ndash22 doi 101007978-94-007-6386-9_45

134 Adiwinata R Nelwan EJ Snakebite in Indonesia Acta Med Indones (2015)47358ndash65

135 Alirol E Sharma SK Bawaskar HS Kuch U Chappuis F Snake bite in southasia A review PloS Negl Trop Dis (2010) 4e603 doi 101371journalpntd0000603



136 World Health Organization WHOSEARO Guidelines for the clinicalmanagement of snake bites in the South-East Asia Region Southeast AsianJ Trop Med Public Health (1999) 30 Suppl 11ndash85

137 World Health Organization Guidelines for the Clinical Management ofSnake bites in the South-East Asia Region Southeast Asian J Trop Med PublicHealth (2005) 301ndash85

138 Ghose A White J Asian Snakes In Brent J Burkhart K Dargan P Hatten BMegarbane B Palmer R et al editors Critical Care Toxicology Diagnosis andManagement of the Critically Poisoned Patient New YorkSpringerInternational Publishing (2017) p 2343ndash403 doi 101007978-3-319-17900-1

139 Rojnuckarin P Suteparak S Sibunruang S Diagnosis and management ofvenomous snakebites in Southeast Asia Asian BioMed (2012) 6795ndash805doi 1053721905-74150606125

140 Prasarnpun S Walsh J Awad SS Harris JB Envenoming bites by kraits thebiological basis of treatment-resistant neuromuscular paralysis Brain (2005)1282987ndash96 doi 101093brainawh642

141 Theakston RDG Phillips RE Warrell DA Galagedera Y Abeysekera DTDJDissanayaka P et al Envenoming by the common krait (Bungarus caeruleus)and Sri lankan cobra (Naja naja naja) Efficacy and complications of therapywith haffkine antivenom Trans R Soc Trop Med Hyg (1990) 84301ndash8doi 1010160035-9203(90)90297-R

142 Mondal R Chowdhury F Rani M Mohammad N Monjurul Islam MAshraful Haque M et al Pre-hospital and Hospital Management Practicesand Circumstances behind Venomous Snakebite in Northwestern Part ofBangladesh Asia Pacific J Med Toxicol (2012) 118ndash21

143 Pathmeswaran A Kasturiratne A Fonseka M Nandasena S Lalloo DG deSilva HJ Identifying the biting species in snakebite by clinical features anepidemiological tool for community surveys Trans R Soc Trop Med Hyg(2006) 100874ndash8 doi 101016jtrstmh200510003

144 Suraweera W Warrell D Whitaker R Menon GR Rodrigues R Fu SH et alTrends in snakebite deaths in India from 2000 to 2019 in a nationallyrepresentative mortality study Elife (2020) 9e54076 doi 107554eLife54076

145 Whitaker R Whitaker S Venom antivenom production and the medicallyimportant snakes of India Curr Sci (2012) 103635ndash43

146 Gupt A Bhatnagar T Murthy BN Epidemiological profile and managementof snakebite cases - A cross sectional study from Himachal Pradesh IndiaClin Epidemiol Glob Heal (2015) 3S96ndashS100 doi 101016jcegh201511007

147 Menon JC Joseph JK Jose MP Punde D Mazumdar DB Bawaskar HS et alManagement protocol of venomous snakebite in India a consensusstatement Toxin Rev (2016) 35147ndash51 doi 1010801556954320161185735

148 Ariaratnam CA Thuraisingam V Kularatne SAM Sheriff MHR TheakstonRDG de Silva A et al Frequent and potentially fatal envenoming by hump-nosed pit vipers (Hypnale hypnale and H nepa) in Sri Lanka lack of effectiveantivenom Trans R Soc Trop Med Hyg (2008) 1021120ndash6 doi 101016jtrstmh200803023

149 Sellahewa KH Gunawardena G Kumararatne MP Efficacy of antivenom inthe treatment of severe local envenomation by the hump-nosed viper(Hypnale hypnale) Am J Trop Med Hyg (1995) 53260ndash2 doi 104269ajtmh199553260

150 Villalta M Sanchez A Herrera M Vargas M Segura A Cerdas M et alDevelopment of a new polyspecific antivenom for snakebite envenoming inSri Lanka Analysis of its preclinical efficacy as compared to a currentlyavailable antivenom Toxicon (2016) 122152ndash9 doi 101016jtoxicon201610007

151 Rosenfeld G Symptomatology Pathology and Treatment of Snake Bitesin South America In Wolfgang B Buckley EE editors VenomousAnimals and Their Venoms Vol Ii Venomous Vertebrates Xxiv +687p Illus Maps New York NY USA London England AcademicPress (1971) pp 345ndash84

152 Langley RL Animal-related fatalities in the United States - an updateWilderness Environ Med (2005) 1667ndash74 doi 1015801080-6032(2005)16[67AFITUS]20CO2

153 OrsquoNeil ME Mack KA Wozniak EJ Snakebite injuries treated in UnitedStates emergency departments 2001-2004 Wilderness Environ Med (2007)18281ndash7 doi 10158006-WEME-OR-080R11


154 Graham DC Dickinson GT Godden JO Kelly AD Randall RL Wells TLSnake Bites in Canada Can Med Assoc J (1964) 901472ndash3

155 Russell FE Snake Venom Poisoning Annu Rev Med (1980) 31247ndash59doi 101016B978-0-323-04107-250231-3

156 Curry SC Horning D Brady P Requa R Kunkel DB Vance MV Thelegitimacy of rattlesnake bites in Central Arizona Ann Emerg Med (1989)18658ndash63 doi 101016S0196-0644(89)80523-2

157 Kitchens CS Hemostatic aspects of envenomation by North-Americansnakes Hematol Clin North Am (1992) 61189ndash95 doi 101016S0889-8588(18)30304-6

158 Hutton RA Warrell DA Action of snake venom components on thehaemostatic system Blood Rev (1993) 7176ndash89 doi 1010160268-960X(93)90004-N

159 Bush SP Siedenburg E Neurotoxicity associated with suspected sourthernPacific rattlesnake (Crotalus viridis helleri) envenomation WildernessEnviron Med (1999) 10247ndash9 doi 1015801080-6032(1999)010[0247NAWSSP]23CO2

160 Richardson WH Goto CS Gutglass DJ Williams SR Clark RF Rattlesnakeenvenomation with neurotoxicity refractory to treatment with crotaline Fabantivenom Clin Toxicol (2007) 45472ndash5 doi 10108015563650701338187

161 Clark RF Williams SR Nordt SP Boyer-Hassen LV Successful treatment ofcrotalid-induced neurotoxicity with a new polyspecific crotalid Fabantivenom Ann Emerg Med (1997) 3054ndash7 doi 101016S0196-0644(97)70111-2

162 Wyeth Laboratories Inc Antivenin (Micrurus fulvius) North American CoralSnake Antivenin (2001) pp 1ndash9 Available at httpswwwfdagovmedia82301download

163 Davidson TM Eisner J United States coral snakes Wilderness Environ Med(1996) 738ndash45 doi 1015801080-6032(1996)007[0038USCS]23CO2

164 Kitchens CS Van Mierop LH Envenomation by the Eastern coral snake(Micrurus fulvius fulvius) A study of 39 victims JAMA J Am Med Assoc(1987) 2581615ndash8 doi 101001jama258121615

165 Tan NH Ponnudurau G The biological properties of venoms of someAmerican coral snakes (Genus Micrurus) Comp Biochem Physiol - Part BComp Biochem (1992) 101471ndash4 doi 1010160305-0491(92)90029-Q

166 Campbell CH Symptomatology Pathology and Treatment of the Bites ofElapid Snakes In CY editor Snake Venoms Handbook of ExperimentalPharmacology (Continuation of Handbuch der experimentellenPharmakologie) vol 52 Berlin Heidelberg Springer (1979) pp 888ndash9 doi101007978-3-642-66913-2_24

167 Eagle H The coagulation of blood by snake venoms and its physiologicsignificance J Exp Med (1937) 65613ndash39 doi 101084jem655613

168 Sanchez EF Freitas TV Ferreira-Alves DL Velarde DT Diniz MR CordeiroMN et al Biological activities of venoms from South American snakesToxicon (1992) 3097ndash103 doi 1010160041-0101(92)90505-Y

169 US Food and Drug Administration Expiration Date Extension for NorthAmerican Coral Snake Antivenin (Micrurus fulvius) (Equine Origin) LotL67530 through January 31 2019 FDAgov (2018) Available at httpswwwfdagovvaccines-blood-biologicssafety-availability-biologicsexpiration-date-extension-north-american-coral-snake-antivenin-micrurus-fulvius-equine-origin-lot-0 (Accessed February 25 2020)

170 Cox RD Parker CS Cox ECE Marlin MB Galli RL Misidentification ofcopperhead and cottonmouth snakes following snakebites Clin Toxicol(2018) 561195ndash9 doi 1010801556365020181473583

171 Bush SP Ruha AM Seifert SA Morgan DL Lewis BJ Arnold TC et alComparison of F(abrsquo)2 versus Fab antivenom for pit viper envenomation Aprospective blinded multicenter randomized clinical trial Clin Toxicol(2015) 5337ndash45 doi 103109155636502014974263

172 Gerardo CJ Quackenbush E Lewis B Rutherfoord RS Greene S ToschlogEA et al The Efficacy of Crotalidae Polyvalent Immune Fab (Ovine)Antivenom Versus Placebo Plus Optional Rescue Therapy on RecoveryFrom Copperhead Snake Envenomation A Randomized Double-BlindPlacebo-Controlled Clinical Trial Ann Emerg Med (2017) 70233ndash44doi 101016jannemergmed201704034

173 Freiermuth CE Lavonas EJ Anderson VE Kleinschmidt KC Sharma KRapp-Olsson M et al Antivenom Treatment Is Associated with FewerPatients using Opioids after Copperhead Envenomation West J EmergMed (2019) 20497ndash505 doi 105811westjem2019342693



174 Anderson VE Gerardo CJ Rapp-Olsson M Bush SP Mullins ME Greene Set al Early administration of Fab antivenom resulted in faster limb recoveryin copperhead snake envenomation patients Clin Toxicol (2018) 01ndash6doi 1010801556365020181491982

175 Instituto Clodomiro Picado Envenenamiento Por Mordedura De SerpienteEn Centro America San Jose Universidad de Costa Rica (2013) doi 101021jp980047v

176 Funasa Manual de Diagnostico e Tratamento de Acidentes por AnimaisPeconhentos Brasılia Funasa Brazil (the Brazilian Ministry of Health)(2001)

177 Laustsen AH Gutierrez JM Knudsen C Johansen KH Mendez EB CerniFA et al Pros and cons of different therapeutic antibody formats forrecombinant antivenom development Toxicon (2018) 146151ndash75doi 101016jtoxicon201803004

178 Personas DGDSDL-DEDAIDS Norma Tecnica sobre Prevencion yTratamiento de Accidentes por Animales Ponzontildeosos Lima Minesterio deSalud Peru (the Peruvian Ministry of Health) (2005) Available at httpwwwessaludgobpedownloadsguias_emergencia_epidemiologicanormas_animales_ponzonosospdf

179 Direccion de normatizacion del sistema nacional de salud Manual de normasy procedimientos sobre prevencion y tratamiento de accidentes ocasionadospor morderura de serpientes Quito Ministerio de Salud Publica del Ecuador(Ministry of Health Ecuador) (2008) Available at httpsaplicacionesmspgobecsaludarchivosdigitalesdocumentosDireccionesdnnarchivosMANUAL20DE20NORMAS20PROCEDIM20PREVENCMORDEDURA20DE2020SERPIENTES(1)pdf

180 Centro de Informacion Toxicologica de Veracruz Guıa de diagnostico ytratamiento de intoxicacion por accidente ofıdico crotalico VeracruzAvailable at httpsdocplayeres7320740-Guia-de-diagnostico-y-tratamiento-de-intoxicacion-por-accidente-ofidico-crotalicohtml

181 Marques HO de Brito Sousa JD Fan HW de Oliveira SS Guimaratildees deLacerda MV Sachett JAG et al Accuracy of the Lee-White Clotting TimePerformed in the Hospital Routine to Detect Coagulapathy in Bothrops atroxEnvenomation Am J Trop Med Hyg (2018) 981547ndash51 doi 104269ajtmh17-0992

182 Mckessy SP Handbook of Venoms and Toxins of Reptiles 1st ed SP Mckessyeditor Boca Raton Florida CRC Press (2009) Available at httpswwwtaylorfranciscombooksmono1012019781420008661handbook-venoms-toxins-reptiles-stephen-mackessy-stephen-mackessy

183 Campbell JA Lamar WW The Venomous Reptiles of the WesternHemisphere 1st ed Ithaca United States of America Cornell UniversityPress (2004) Available at httpwwwcornellpresscornelledubookGCOI=80140100664830

184 de Oliveira ATAL de Sousa AFPB Alcantara I de CL de Miranda ITNMarques RB Acidentes com animais peconhentos no Brasil revisatildeo deliteratura Rev Intertox Toxicol RIsco Ambient E Soc (2018) 11119ndash36doi 1022280revintervol11ed3389

185 Otero-Patintildeo R Epidemiological clinical and therapeutic aspects of Bothropsasper bites Toxicon (2009) 54998ndash1011 doi 101016jtoxicon200907001

186 Warrell DA Thorpe RS Wuumlster W Malhotra A Venomous Snakes EcologyEvolution and Snakebite Clarendon Press (1997) 1997189ndash203 doi1023071447318

187 Gutierrez JM Snakebite envenoming in Latin America and the CaribbeanToxinology (2013)51ndash72 doi 101007978-94-007-6288-6_14-2

188 Bucaretchi F De Capitani EM Branco MM Fernandes LCR Hyslop SCoagulopathy as the main systemic manifestation after envenoming by ajuvenile South American rattlesnake (Crotalus durissus terrificus) Casereport Clin Toxicol (2013) 51505ndash8 doi 103109155636502013802796

189 Cribari C Management of Poisonous Snakebites (2004) pp 1ndash4 Availableat httpswwwfacsorg~mediafilesquality programstraumapublicationssnakebiteashx

190 Yantildeez-Arenas C Townsend Peterson A Rodrıguez-Medina K Barve NMapping current and future potential snakebite risk in the new world ClimChange (2016) 134697ndash711 doi 101007s10584-015-1544-6

191 Longbottom J Shearer FM Devine M Alcoba G Chappuis F Weiss DJ et alVulnerability to snakebite envenoming a global mapping of hotspots Lancet(2018) 392673ndash84 doi 101016S0140-6736(18)31224-8


192 World Health Organization Regional Office for Africa Guidelines for theprevention and clinical management of snakebite in Africa World HealthOrganization Regional Office for Africa (2010) Available at httpsappswhointirishandle10665204458

193 Theakston RDG Warrell DA Crisis in snake antivenom supply for AfricaLancet (2000) 3562104 doi 101016S0140-6736(05)74319-1

194 World Health Organization Rabies and envenomings A neglected publichealth issue World Health Organization (2007) Available at httpsappswhointirisbitstreamhandle10665438589789241563482_engpdfsequence=1ampisAllowed=y

195 Appiah B Snakebite neglect rampant in Africa Can Med Assoc J (2012)18427ndash8 doi 101503cmaj109-4046

196 Habib AG Brown NI The snakebite problem and antivenom crisis from ahealth-economic perspective Toxicon (2018) 150115ndash23 doi 101016jtoxicon201805009

197 Wood D Sartorius B Hift R Classifying snakebite in South AfricaValidating a scoring system South Afr Med J (2017) 10746ndash51doi 107196SAMJ2017v107i111361

198 Alirol E Lechevalier P Zamatto F Chappuis F Alcoba G Potet JAntivenoms for Snakebite Envenoming What Is in the Research PipelinePloS Negl Trop Dis (2015) 91ndash11 doi 101371journalpntd0003896

199 Warrell DA Unscrupulous marketing of snake bite antivenoms in Africa andPapua New Guinea choosing the right product-rsquoWhatrsquos in a namersquo Trans RSoc Trop Med Hyg (2008) 102397ndash9 doi 101016jtrstmh200712005

200 Rogalski A Soslashrensen CV op den Brouw B Lister C Dashvesky D ArbuckleK et al Differential procoagulant effects of saw-scaled viper (SerpentesViperidae Echis) snake venoms on human plasma and the narrowtaxonomic ranges of antivenom efficacies Toxicol Lett (2017) 280159ndash70doi 101016jtoxlet201708020

201 Abubakar IS Abubakar SB Habib AG Nasidi A Durfa N Yusuf PO et alRandomised controlled double-blind non-inferiority trial of two antivenomsfor Saw-scaled or carpet viper (Echis ocellatus) envenoming in Nigeria PloSNegl Trop Dis (2010) 48ndash17 doi 101371journalpntd0000767

202 Simpson ID Blaylock RSM The anti snake venom crisis in Africa Asuggested manufacturers product guide Wilderness Environ Med (2009)20275ndash82 doi 10158008-WEME-CON-296R11

203 Ouma PO Maina J Thuranira PN Macharia PM Alegana VA English Met al Access to emergency hospital care provided by the public sector in sub-Saharan Africa in 2015 a geocoded inventory and spatial analysis LancetGlob Heal (2018) 6e342ndash50 doi 101016S2214-109X(17)30488-6

204 Fry BG Snakebite When the Human Touch Becomes a Bad Touch Toxins(Basel) (2018) 101ndash23 doi 103390toxins10040170

205 Vaiyapuri S Vaiyapuri R Ashokan R Ramasamy K Nattamaisundar KJeyaraj A et al Snakebite and its socio-economic impact on the ruralpopulation of Tamil Nadu India PloS One (2013) 810ndash3 doi 101371journalpone0080090

206 Schioldann E Mahmood MA Kyaw MM Halliday D Thwin KT Chit NNet al Why snakebite patients in Myanmar seek traditional healers despiteavailability of biomedical care at hospitals Community perspectives onreasons PloS Negl Trop Dis (2018) 121ndash14 doi 101371journalpntd0006299

207 White P The concept of diseases and health care in African traditionalreligion in Ghana HTS Teol StudTheol Stud (2015) 711ndash7 doi 104102htsv71i32762

208 Hati AK Mandal M De MK Mukerjee H Hati RN Epidemiology of snakebite in the district of Burdwan West Bengal J Indian Med Asoc (1992)90145ndash7

209 Ediriweera DS Kasturiratne A Pathmeswaran A Gunawardena NKJayamanne SF Lalloo DG et al Health seeking behavior followingsnakebites in Sri Lanka Results of an island wide community basedsurvey PloS Negl Trop Dis (2017) 111ndash11 doi 101371journalpntd0006073

210 Chippaux J-P Snakebite in Africa current situation and urgent needs In SPMackessy editor Handbook of Venoms and Toxins of Reptiles London CRCPress (2010) p 453ndash73

211 World Health Organization Snakebite envenoming A strategy for preventionand control Geneva World Health Organization (2019)



212 de Castantildeeda RR Durso AM Ray N Fernandez JL Williams DJ Alcoba Get al Snakebite and snake identification empowering neglected communitiesand health-care providers with AI Lancet Digit Heal (2019) 1e202ndash3doi 101016S2589-7500(19)30086-X

213 Chippaux J-P Epidemiology of snakebites in Europe A systematic review ofthe literature Toxicon (2012) 59557ndash68 doi 101016jtoxicon201110008

214 Lamb T de Haro L Lonati D Brvar M Eddleston M Antivenom forEuropean Vipera species envenoming Clin Toxicol (2017) 55557ndash68doi 1010801556365020171300261

215 Brvar M Kurtovic T Grenc D Lang Balija M Krizaj I Halassy B Viperaammodytes bites treated with antivenom ViperaTAb a case series withpharmacokinetic evaluation Clin Toxicol (2017) 55241ndash8 doi 1010801556365020161277235

216 Varga C Malina T Alfoumlldi V Bilics G Nagy F Olah T Extending knowledgeof the clinical picture of Balkan adder (Vipera berus bosniensis) envenomingThe first photographically-documented neurotoxic case from South-WesternHungary Toxicon (2018) 129ndash35 doi 101016jtoxicon201712053

217 Malina T Krecsak L Warrell DA Neurotoxicity and hypertension followingEuropean adder (Vipera berus berus) bites in Hungary case report andreview QJM (2008) 101801ndash6 doi 101093qjmedhcn079

218 Latinovic Z Leonardi A Sribar J Sajevic T Zuzek MC Frangez R et alVenomics of Vipera berus berus to explain differences in pathology elicitedby Vipera ammodytes ammodytes envenomation Therapeutic implicationsJ Proteomics (2016) 14634ndash47 doi 101016jjprot201606020

219 Ferquel E de Haro L Jan V Guillemin I Jourdain S Teynie A et alReappraisal of Vipera aspis Venom Neurotoxicity PloS One (2007) 2e1194doi 101371journalpone0001194

220 Westerstroumlm A Petrov B Tzankov N Envenoming following bites by theBalkan adder Vipera berus bosniensis - first documented case series fromBulgaria Toxicon (2010) 561510ndash5 doi 101016jtoxicon201008012

221 Magdalan J Trocha M Merwid-Ld A Sozaski T Zawadzki M Viperaberus bites in the region of southwest Poland - A clinical analysis of 26cases Wilderness Environ Med (2010) 21114ndash9 doi 101016jwem201001005

222 Persson H Pathophysiology and Treatment of Envenomation by EuropeanVipers Springer Netherlands (2018) 357ndash75 doi 101007978-94-017-7438-3_9

223 Petite J Viper bites Treat or ignore Swiss Med Wkly (2005) 135618ndash25224 Karlson-Stiber C Salmonson H Persson H A nationwide study of Vipera

berus bites during one year - Epidemiology and morbidity of 231 cases ClinToxicol (2006) 4425ndash30 doi 10108015563650500394597

225 Hoslashnge BL Hedegaard SK Cederstroslashm S Nielsen H Hospital contacts afterbite by the European adder (Vipera berus) Dan Med J (2015) 621ndash6

226 Pucca MB Knudsen C Oliveira IS Rimbault C Cerni FA Wen FH et alCurrent Knowledge on Snake Dry Bites Toxins (Basel) (2020) 124ndash7doi 103390toxins12110668

227 Montag A Giftschlangen in Deutschland und Europa Zentralblatt fuumlrArbeitsmedizin Arbeitsschutz und Ergon (2018) 68287ndash308 doi 101007s40664-018-0301-x

228 Kollander Jakobsen L Lyhne JA Hugormebid symptomer undersoslashgelse ogbehandling (2018) Akutmodtagelse Thisted Aalborg Univ Reg NordAvailable at httpsprirndkSider16734aspx (Accessed May 31 2018)

229 Guiavarch M Medus M Tichadou L Glaizal M de Haro L Efficacite variablede lrsquoantivenin Viperfav pour traiter les envenimations viperines avecneurotoxicite Press Medicale (2011) 40654ndash6 doi 101016jlpm201101023

230 Zanetti G Duregotti E Locatelli CA Giampreti A Lonati D Rossetto Oet al Variability in venom composition of European viper subspecies limitsthe cross-effectiveness of antivenoms Sci Rep (2018) 81ndash9 doi 101038s41598-018-28135-0

231 Garcıa-Arredondo A Martınez M Calderon A Saldıvar A Soria RPreclinical assessment of a new polyvalent antivenom (Inoserp europe)against several species of the subfamily viperinae Toxins (Basel) (2019)11149 doi 103390toxins11030149

232 Casewell NR Al-Abdulla I Smith D Coxon R Landon J ImmunologicalCross-Reactivity and Neutralisation of European Viper Venoms with theMonospecific Vipera berus Antivenom ViperaTAb Toxins (Basel) (2014)62471ndash82 doi 103390toxins6082471


233 Valenta J Stach Z Michalek P Severe Snakebite Envenoming in IntensiveCare Prague Med Rep (2016) 117153ndash63 doi 101471223362936201616

234 Schaper A Desel H Ebbecke M De Haro L Deters M Hentschel H et alBites and stings by exotic pets in Europe An 11 year analysis of 404 casesfrom Northeastern Germany and Southeastern France Clin Toxicol (2009)4739ndash43 doi 10108015563650801954875

235 Schaper A De Haro L Desel H Ebbecke M Langer C Rattlesnake bites inEurope - Experiences from Southeastern France and Northern GermanyJ Toxicol - Clin Toxicol (2004) 42635ndash41 doi 101081CLT-200026962

236 Lonati D Butera R Cima M Cozzio S Locatelli C Manzo L Exotic snakes inEurope - A case of Mexican moccasin (Agkistrodon bilineatus) snakebitePress Medicale (2004) 331582ndash4 doi 101016S0755-4982(04)98997-X

237 Alfred S Bates D White J Mahmood MA Warrell DA Thwin KT et alAcute Kidney Injury Following Eastern Russellrsquos Viper (Daboia siamensis)Snakebite in Myanmar Kidney Int Rep (2019) 41337ndash41 doi 101016jekir201905017

238 Pinho FMO Zanetta DMT Burdmann EA Acute renal failure after Crotalusdurissus snakebite A prospective survey on 100 patients Kidney Int (2005)67659ndash67 doi 101111j1523-1755200567122x

239 Mise YF Lira-da-Silva R Carvalho FM Time to treatment and severity ofsnake envenoming in Brazil Rev Panam Salud Publica (2018) 42e52doi 1026633RPSP201852

240 Silva A Hlusicka J Siribaddana N Waiddyanatha S Time delays intreatment of snakebite patients in rural Sri Lanka and the need for rapiddiagnostic tests PloS Negl Trop Dis (2020) 14(11)e0008914 doi 101371journalpntd0008914

241 Gutierrez JM Fan HW Silvera CLM Angulo Y Stability distribution anduse of antivenoms for snakebite envenomation in Latin America Report of aworkshop Toxicon (2009) 53625ndash30 doi 101016jtoxicon200901020

242 Gutierrez JM Current challenges for confronting the public health problemof snakebite envenoming in Central America J Venom Anim Toxins InclTrop Dis (2014) 201ndash9 doi 1011861678-9199-20-7

243 Williams HF Vaiyapuri R Gajjeraman P Hutchinson G Gibbins JMBicknell AB et al Challenges in diagnosing and treating snakebites in arural population of Tamil Nadu India The views of clinicians Toxicon(2017) 13044ndash6 doi 101016jtoxicon201702025

244 Harrison RA Gutierrez JM Priority Actions and Progress to Substantiallyand Sustainably Reduce the Mortality Morbidity and Socioeconomic Burdenof Tropical Snakebite Toxins (Basel) (2016) 8351 doi 103390toxins8120351

245 Hifumi T Sakai A Kondo Y Yamamoto A Morine N Ato M et alVenomous snake bites clinical diagnosis and treatment J Intensive Care(2015) 31ndash9 doi 101186s40560-015-0081-8

246 Theakston RDG Laing GD Diagnosis of snakebite and the importance ofimmunological tests in venom research Toxins (Basel) (2014) 61667ndash95doi 103390toxins6051667

247 Laustsen AH Dorrestijn N Integrating Engineering Manufacturing andRegulatory Considerations in the Development of Novel Antivenoms Toxins(Basel) (2018) 10(8)309 doi 103390toxins10080309

248 Williams HF Layfield HJ Vallance T Patel K Bicknell AB Trim SA et alThe urgent need to develop novel strategies for the diagnosis and treatmentof snakebites Toxins (Basel) (2019) 111ndash29 doi 103390toxins11060363

249 Sweet D Lorente JA Valenzuela A Lorente M Villanueva E PCR-basedDNA typing of saliva stains recovered from human skin J Forensic Sci (1997)42447ndash51 doi 101520JFS14146J

250 Puzari U Mukherjee AK Recent developments in diagnostic tools andbioanalytical methods for analysis of snake venom A critical review AnalChim Acta (2020) 1137208ndash24 doi 101016jaca202007054

251 Bawaskar HS Bawaskar PH Bawaskar PH Snake bite in India a neglecteddisease of poverty Lancet (2017) 3901947ndash8 doi 101016S0140-6736(17)32175-X

252 Gutierrez JM Burnouf T Harrison RA Calvete JJ Kuch U Warrell DA Amulticomponent strategy to improve the availability of antivenom fortreating snakebite envenoming Bull World Health Organ (2014) 92526ndash32 doi 102471BLT13132431

253 Neuzil P Zhang C Pipper J Oh S Zhuo L Ultra fast miniaturized real-timePCR 40 cycles in less than six minutes Nucleic Acids Res (2006) 34e77doi 101093nargkl416



254 Vaagt F Haase I Fischer M Loop-mediated isothermal amplification(LAMP)-based method for rapid mushroom species identification J AgricFood Chem (2013) 611833ndash40 doi 101021jf304824b

255 Jiang C Yuan Y Liu L Hou J Jin Y Huang L Homogeneous fluorescentspecific PCR for the authentication of medicinal snakes using cationicconjugated polymers Sci Rep (2015) 51ndash7 doi 101038srep16260

256 World Health Organization Guidelines for the management of snakebite 2ndEdition World Health Organization (2016) Available at httpswwwwhointsnakebitesresources9789290225300en

257 Seqirus and Snake Venom Detection Kit Product Leaflet (2017) httpslabelingseqiruscomSVDKAUSnake-Venom-Detection-KitENSnake-Venom-Detection-Kitpdf

258 Isbister GK Maduwage K Shahmy S Mohamed F Abeysinghe C Diagnostic20-min whole blood clotting test in Russellrsquos viper envenoming delaysantivenom administration QJM - Int J Med (2013) 106925ndash32doi 101093qjmedhct102

259 Seifert SA Boyer LV Recurrence phenomena after immunoglobulin therapyfor snake envenomations Part 1 Pharmacokinetics and pharmacodynamicsof immunoglobulin antivenoms and related antibodies Ann Emerg Med(2001) 37189ndash95 doi 101067mem2001113135

260 Ho M Warrell DA Looareesuwan S Phillips RE Chanthavanich PKarbwang J et al Clinical significance of venom antigen levels in patientsenvenomed by the Malayan pit viper (Calloselasma rhodostoma) Am J TropMed Hyg (1986) 35579ndash87 doi 104269ajtmh198635579

261 Faiz MA Ahsan MF Ghose A Rahman MR Amin R Hossain M et al Bitesby the Monocled Cobra Naja kaouthia in Chittagong Division BangladeshEpidemiology Clinical Features of Envenoming and Management of 70Identified Cases Am J Trop Med Hyg (2017) 96876ndash84 doi 104269ajtmh16-0842

262 Rojnuckarin P Banjongkit S Chantawibun W Akkawat B Juntiang JNoiphrom J et al Green pit viper (Trimeresurus albolabris and Tmacrops) venom antigenaemia and kinetics in humans Trop Doct (2007)37207ndash10 doi 101258004947507782332838

263 Sanhajariya S Duffull SB Isbister GK Pharmacokinetics of snake venomToxins (Basel) (2018) 1073 doi 103390toxins10020073

264 Fassett RG Venuthurupalli SK Gobe GC Coombes JS Cooper MA HoyWE Biomarkers in chronic kidney disease A review Kidney Int (2011)80806ndash21 doi 101038ki2011198

265 Wang SQ Sarenac D Chen MH Huang SH Giguel FF Kuritzkes DR et alSimple filter microchip for rapid separation of plasma and viruses fromwhole blood Int J Nanomed (2012) 75019ndash28 doi 102147IJNS32579

266 Tang RH Yang H Choi JR Gong Y Feng SS Pingguan-Murphy B et alAdvances in paper-based sample pretreatment for point-of-care testing CritRev Biotechnol (2017) 37411ndash28 doi 1031090738855120161164664

267 Sajid M Kawde A-N Daud M Designs formats and applications of lateralflow assay A literature review J Saudi Chem Soc (2015) 19689ndash705doi 101016jjscs201409001

268 Deonarain MP Yahioglu G Stamati I Marklew J Emerging formats fornext-generation antibody drug conjugates Expert Opin Drug Discovery(2015) 10463ndash81 doi 1015171746044120151025049

269 Soslashrensen CV Knudsen C Auf Dem Keller U Kalogeropoulos K Gutierrez-Jimenez C Pucca MB et al Do Antibiotics Potentiate Proteases in


Hemotoxic Snake Venoms Toxins (Basel) (2020) 121ndash6 doi 103390toxins12040240

270 Morrison JJ Pearn JH Covacevich J Nixon J Can Australians identify snakesMed J Aust (1983) 266ndash70 doi 105694j1326-53771983tb142105x

271 Viravan C Looareesuwan S Kosakam W Wuthiekanun V McCarthy CJStimson AF et al A national hospital-based survey of snakes responsible forbites in Thailand Trans R Soc Trop Med Hyg (1992) 86100ndash6 doi 1010160035-9203(92)90463-M

272 Ariaratnam CA Rezvi Sheriff MH Arambepola C Theakston RDG WarrellDA Syndromic approach to treatment of snake bite in Sri Lanka based onresults of a prospective national hospital-based survey of patients envenomedby identified snakes Am J Trop Med Hyg (2009) 81725ndash31 doi 104269ajtmh200909-0225

273 Sulaiman SS Kharusha IK Samara AM Al-Jabi SW Zyoud SH Anassessment of medical studentsrsquo proficiency in the diagnosis andmanagement of snakebites A cross-sectional study from Palestine J OccupMed Toxicol (2020) 151ndash11 doi 101186s12995-020-00254-3

274 White J Clinical toxinology specialty training Toxicon (2013) 69120ndash5doi 101016jtoxicon201303007

275 Gutierrez JM Williams D Fan HW Warrell DA Snakebite envenomingfrom a global perspective Towards an integrated approach Toxicon (2010)561223ndash35 doi 101016jtoxicon200911020

276 Harrison RA Casewell NR Ainsworth SA Lalloo DG The time is now Acall for action to translate recent momentum on tackling tropical snakebiteinto sustained benefit for victims Trans R Soc Trop Med Hyg (2019)113834ndash7 doi 101093trstmhtry134

277 Williams D Gutierrez JM Harrison R Warrell DA White J Winkel KDet al The Global Snake Bite Initiative an antidote for snake bite Lancet(2010) 37589ndash91 doi 101016S0140-6736(09)61159-4

278 Williams DJ Gutierrez JM Calvete JJ Wuumlster W Ratanabanangkoon KPaiva OK et al Ending the drought New strategies for improving the flow ofaffordable effective antivenoms in Asia and Africa J Proteomics (2011)741735ndash67 doi 101016jjprot201105027

Conflict of Interest CK JJ AH RF SD and AL are shareholders in VenomAidDiagnostics ApS CK is enrolled in an industrial PhD programme sponsoredpartially by the company BioPorto Diagnostics AS and is therefore employed bythis company

The remaining authors declare that the research was conducted in the absence ofany commercial or financial relationships that could be construed as a potentialconflict of interest

The handling editor declared a past co-authorship with several of the authors CKJJ and AL

Copyright copy 2021 Knudsen Juumlrgensen Foslashns Haack Friis Dam Bush White andLaustsen This is an open-access article distributed under the terms of the CreativeCommons Attribution License (CC BY) The use distribution or reproduction in otherforums is permitted provided the original author(s) and the copyright owner(s) arecredited and that the original publication in this journal is cited in accordance withaccepted academic practice No use distribution or reproduction is permitted whichdoes not comply with these terms



Introduction

Commonly Adopted Approaches for Diagnosis of Snakebite Envenoming

Snakebite Diagnosis in Australia

Snakebite Diagnosis in Asia

Snakebite Diagnosis in the United States and Canada

Snakebite Diagnosis in Latin America

Snakebite Diagnosis in Africa

Snakebite Diagnosis in Europe

Potential Benefits of Novel Snakebite Diagnostics

Snakebite Diagnostics Reported in the Literature

Design Considerations for Snakebite Diagnostics

Conclusion

Author Contributions

Funding

References

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Frontiers in Immunology | wwwfrontiersin

Edited byManuela Berto Pucca

Federal University of RoraimaBrazil

Reviewed bySakthivel Vaiyapuri

University of ReadingUnited Kingdom

Clara Guerra-DuarteEzequiel Dias Foundation (FUNED)

Brazil

CorrespondenceCecilie KnudsenCecknudtudk

Andreas H LaustsenAholabiodtudk

Specialty sectionThis article was submitted to

Vaccines and Molecular Therapeuticsa section of the journal

Frontiers in Immunology

Received 30 January 2021Accepted 29 March 2021Published 28 April 2021

CitationKnudsen C Juumlrgensen JA Foslashns S

Haack AM Friis RUW Dam SHBush SP White J and Laustsen AH

(2021) Snakebite EnvenomingDiagnosis and DiagnosticsFront Immunol 12661457

doi 103389fimmu2021661457

REVIEWpublished 28 April 2021

doi 103389fimmu2021661457

Snakebite Envenoming Diagnosisand DiagnosticsCecilie Knudsen12 Jonas A Juumlrgensen1 Sofie Foslashns1 Aleksander M Haack1Rasmus U W Friis1 Soslashren H Dam1 Sean P Bush3 Julian White4 and Andreas H Laustsen1

1 Department of Biotechnology and Biomedicine Technical University of Denmark Kongens Lyngby Denmark 2 BioPortoDiagnostics AS Hellerup Denmark 3 Department of Emergency Medicine Yale School of Medicine New HavenCT United States 4 Toxinology Department Womenrsquos and Childrenrsquos Hospital North Adelaide SA Australia

Snakebite envenoming is predominantly an occupational disease of the rural tropicscausing death or permanent disability to hundreds of thousands of victims annually Thediagnosis of snakebite envenoming is commonly based on a combination of patienthistory and a syndromic approach However the availability of auxiliary diagnostic tests atthe disposal of the clinicians vary from country to country and the level of experiencewithin snakebite diagnosis and intervention may be quite different for clinicians fromdifferent hospitals As such achieving timely diagnosis and thus treatment is a challengefaced by treating personnel around the globe For years much effort has gone intodeveloping novel diagnostics to support diagnosis of snakebite victims especially in ruralareas of the tropics Gaining access to affordable and rapid diagnostics could potentiallyfacilitate more favorable patient outcomes due to early and appropriate treatment Thisreview aims to highlight regional differences in epidemiology and clinical snakebitemanagement on a global scale including an overview of the past and ongoing researchefforts within snakebite diagnostics Finally the review is rounded off with a discussionon design considerations and potential benefits of novel snakebite diagnostics

Keywords envenoming clinical toxinology diagnosis diagnostics ophidism snakebite managementsyndromic approach

INTRODUCTION

Every year people lose their livelihoods limbs and lives to a disease that is as neglected as it isancient Snakebite envenoming The exact burden of snakebite envenoming is notoriously difficultto assess because data on envenoming prevalence are scarce and the available data points are ofteninaccurate or not representative for broader geographical areas (1ndash4) Nonetheless studies suggestthat mortality due to snakebite envenoming may exceed 125000 deaths per year globally while thenumber of people suffering permanent sequelae may be around 400000 and the toll of associateddisability-adjusted life years might add up to a total of over 6 million (4ndash8) To make matters worsesnakebite envenoming is both a disease mainly affecting the poor and a disease that leads to furtherimpoverishment (7 9ndash12) In spite of the immense burden of snakebite envenoming on victimstheir families and local communities the disease remains largely neglected and has historically onlyreceived few resources and limited efforts have gone into the development of better treatments anddiagnostics (13)

org April 2021 | Volume 12 | Article 6614571











Notes Year Ref

1957 (24)


1968 (26)

Sensitivity 396 (40101) 1974 (27)

1974 (28)

1975 (29)


1977 (30)

1977 (3132)

1978 (33)


1982 (35)



(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614573


samples

Sample matrix

Immuno-assay

Immuno-diffusion


1100000000dilution


Tissue homogenate

Immuno-diffusion



Agglutinationtest

Caprine ampequine


Immuno-diffusion






Serum sample buffer








RIA LeporineIgG



ELISA LeporineIgG


Enzymeimmunoassay

LeporineIgG




Blood urine exudate

ELISA LeporineIgG

A rhodostomaN naja


TABLE 1 | Continued

Notes Year Ref


1984 (38)


1984 (39)

1986 (40)1987 (41)

1987 (42)

1987 (43)

1987 (44)




1988 (47)


1990 (48)



1991 (50)

1991 (51)


1991 (52)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614574

Crosnacon

Nonaja

Kno

Spe


Senand

Spefrom


samples

Sample matrix

ELISA LeporineIgG






MurinemonoclonalIgG





Blood

ELISA LeporineIgG







Blood


Serum

ELISA LeporineIgG


Serum


Serum

Agglutinationtest

LeporineIgG



Serum woundswabs




ELISA LeporineIgG


Serum

TABLE 1 | Continued

Notes Year Ref


1992 (53ndash56)

1992 (57)




1993 (59)

1993 (60)



1993 (62)

1994 (63)

1994 (64)

1994 (65)

1995 (66)

1996 (67)


(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614575


samples

Sample matrix

crossN kaoO haT ery




ELISA LeporineIgG



LeporineIgG

SouthernThailand


Agglutinationtest

LeporineIgG




ELISA LeporineIgG



ELISA LeporineIgG



FluorogenicELISA




ELISA LeporineIgG


ELISA NorthAmerica


Serum


Serum



Yes Plasma urine




Only a

-

ntd

-

e

w

TABLE 1 | Continued

Notes Year Ref


1997 (70)

1999 (71)



2002 (73)


2004 (75)

2006 (76)

2007 (77)

2008 (78)


2010 (79)

city 100 2012 (80)



2016 (83)

2017 (84)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614576


samples

Sample matrix

E carinatusN naja



Serum Sensit






Serum plasma




Tissue samples


Only a

Opticalimmunoassay

LeporineIgG


Blood tissuesamples

Crosstested

Agglutinationtest



Opticalimmunoassay

LeporineIgG








ELISA LeporineIgG



Skin blood

Immuno-flourescence


ELISA LeporineIgG


ELISA LeporineIgG


ELISA LeporineIgG




Serum Sensit



Plasma

ELISA LeporineIgG



Sample buffer

-

n

e

fi

e

TABLE 1 | Continued

Notes Year Ref


2018 (85)


2018 (86)


2020 (87)

2020 (88)

sed on (82) 2020 (89)


2001 (90)

2015 (91)

ecificity 100 2016 (92)

2014 (93)

2017 (94)

2018 (95)


Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614577


samples

Sample matrix

Echis sppNaja spp









Serum Quve

LFA Avian ampequine

South-East Asia

Daboia sppNaja spp

10 ngmL (invitro)



Asia ampAfrica



Molecularbiology










Patients 108 Serum







Serum


sa

a










Auxilliary tests

Type Subtype



Serumbiochemistry




Electrocardiagram












































































































Asia ()


April 20

21 | Volume 12


| Article 661457



CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References

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DownsampleGrayImages false GrayImageDownsampleType Bicubic GrayImageResolution 300 GrayImageDepth -1 GrayImageMinDownsampleDepth 2 GrayImageDownsampleThreshold 150000 EncodeGrayImages false GrayImageFilter DCTEncode AutoFilterGrayImages true GrayImageAutoFilterStrategy JPEG GrayACSImageDict ltlt QFactor 040 HSamples [1 1 1 1] VSamples [1 1 1 1] gtgt GrayImageDict ltlt QFactor 015 HSamples [1 1 1 1] VSamples [1 1 1 1] gtgt JPEG2000GrayACSImageDict ltlt TileWidth 256 TileHeight 256 Quality 30 gtgt JPEG2000GrayImageDict ltlt TileWidth 256 TileHeight 256 Quality 30 gtgt AntiAliasMonoImages false CropMonoImages false MonoImageMinResolution 1200 MonoImageMinResolutionPolicy OK DownsampleMonoImages false MonoImageDownsampleType Bicubic MonoImageResolution 300 MonoImageDepth -1 MonoImageDownsampleThreshold 150000 EncodeMonoImages false MonoImageFilter CCITTFaxEncode MonoImageDict ltlt K -1 gtgt AllowPSXObjects false CheckCompliance [ None ] PDFX1aCheck false PDFX3Check false PDFXCompliantPDFOnly false PDFXNoTrimBoxError true PDFXTrimBoxToMediaBoxOffset [ 000000 000000 000000 000000 ] PDFXSetBleedBoxToMediaBox true PDFXBleedBoxToTrimBoxOffset [ 000000 000000 000000 000000 ] PDFXOutputIntentProfile () PDFXOutputConditionIdentifier () PDFXOutputCondition () PDFXRegistryName () PDFXTrapped False Description ltlt ENU (TampF settings for black and white printer PDFs 20081208) gtgt ExportLayers ExportVisibleLayers Namespace [ (Adobe) (Common) (10) ] OtherNamespaces [ ltlt AsReaderSpreads false CropImagesToFrames true ErrorControl WarnAndContinue FlattenerIgnoreSpreadOverrides false IncludeGuidesGrids false IncludeNonPrinting false IncludeSlug false Namespace [ (Adobe) (InDesign) (40) ] OmitPlacedBitmaps false OmitPlacedEPS false OmitPlacedPDF false SimulateOverprint Legacy gtgt ltlt AddBleedMarks false AddColorBars false AddCropMarks false AddPageInfo false AddRegMarks false BleedOffset [ 0 0 0 0 ] ConvertColors NoConversion DestinationProfileName () DestinationProfileSelector DocumentCMYK Downsample16BitImages true FlattenerPreset ltlt ClipComplexRegions true ConvertStrokesToOutlines false ConvertTextToOutlines false GradientResolution 300 LineArtTextResolution 1200 PresetName ([High Resolution]) PresetSelector HighResolution RasterVectorBalance 1 gtgt FormElements false GenerateStructure true IncludeBookmarks true IncludeHyperlinks true IncludeInteractive false IncludeLayers false IncludeProfiles false MarksOffset 6 MarksWeight 0250000 MultimediaHandling UseObjectSettings Namespace [ (Adobe) (CreativeSuite) (20) ] PDFXOutputIntentProfileSelector DocumentCMYK PageMarksFile RomanDefault PreserveEditing true UntaggedCMYKHandling LeaveUntagged UntaggedRGBHandling UseDocumentProfile UseDocumentBleed false gtgt ltlt AllowImageBreaks true AllowTableBreaks true ExpandPage false HonorBaseURL true HonorRolloverEffect false IgnoreHTMLPageBreaks false IncludeHeaderFooter false MarginOffset [ 0 0 0 0 ] MetadataAuthor () MetadataKeywords () MetadataSubject () MetadataTitle () MetricPageSize [ 0 0 ] MetricUnit inch MobileCompatible 0 Namespace [ (Adobe) (GoLive) (80) ] OpenZoomToHTMLFontSize false PageOrientation Portrait RemoveBackground false ShrinkContent true TreatColorsAs MainMonitorColors UseEmbeddedProfiles false UseHTMLTitleAsMetadata true gtgt ]gtgt setdistillerparamsltlt HWResolution [2400 2400] PageSize [612000 792000]gtgt setpagedevice











Notes Year Ref

1957 (24)


1968 (26)

Sensitivity 396 (40101) 1974 (27)

1974 (28)

1975 (29)


1977 (30)

1977 (3132)

1978 (33)


1982 (35)



(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614573


samples

Sample matrix

Immuno-assay

Immuno-diffusion


1100000000dilution


Tissue homogenate

Immuno-diffusion



Agglutinationtest

Caprine ampequine


Immuno-diffusion






Serum sample buffer








RIA LeporineIgG



ELISA LeporineIgG


Enzymeimmunoassay

LeporineIgG




Blood urine exudate

ELISA LeporineIgG

A rhodostomaN naja


TABLE 1 | Continued

Notes Year Ref


1984 (38)


1984 (39)

1986 (40)1987 (41)

1987 (42)

1987 (43)

1987 (44)




1988 (47)


1990 (48)



1991 (50)

1991 (51)


1991 (52)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614574

Crosnacon

Nonaja

Kno

Spe


Senand

Spefrom


samples

Sample matrix

ELISA LeporineIgG






MurinemonoclonalIgG





Blood

ELISA LeporineIgG







Blood


Serum

ELISA LeporineIgG


Serum


Serum

Agglutinationtest

LeporineIgG



Serum woundswabs




ELISA LeporineIgG


Serum

TABLE 1 | Continued

Notes Year Ref


1992 (53ndash56)

1992 (57)




1993 (59)

1993 (60)



1993 (62)

1994 (63)

1994 (64)

1994 (65)

1995 (66)

1996 (67)


(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614575


samples

Sample matrix

crossN kaoO haT ery




ELISA LeporineIgG



LeporineIgG

SouthernThailand


Agglutinationtest

LeporineIgG




ELISA LeporineIgG



ELISA LeporineIgG



FluorogenicELISA




ELISA LeporineIgG


ELISA NorthAmerica


Serum


Serum



Yes Plasma urine




Only a

-

ntd

-

e

w

TABLE 1 | Continued

Notes Year Ref


1997 (70)

1999 (71)



2002 (73)


2004 (75)

2006 (76)

2007 (77)

2008 (78)


2010 (79)

city 100 2012 (80)



2016 (83)

2017 (84)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614576


samples

Sample matrix

E carinatusN naja



Serum Sensit






Serum plasma




Tissue samples


Only a

Opticalimmunoassay

LeporineIgG


Blood tissuesamples

Crosstested

Agglutinationtest



Opticalimmunoassay

LeporineIgG








ELISA LeporineIgG



Skin blood

Immuno-flourescence


ELISA LeporineIgG


ELISA LeporineIgG


ELISA LeporineIgG




Serum Sensit



Plasma

ELISA LeporineIgG



Sample buffer

-

n

e

fi

e

TABLE 1 | Continued

Notes Year Ref


2018 (85)


2018 (86)


2020 (87)

2020 (88)

sed on (82) 2020 (89)


2001 (90)

2015 (91)

ecificity 100 2016 (92)

2014 (93)

2017 (94)

2018 (95)


Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614577


samples

Sample matrix

Echis sppNaja spp









Serum Quve

LFA Avian ampequine

South-East Asia

Daboia sppNaja spp

10 ngmL (invitro)



Asia ampAfrica



Molecularbiology










Patients 108 Serum







Serum


sa

a










Auxilliary tests

Type Subtype



Serumbiochemistry




Electrocardiagram












































































































Asia ()


April 20

21 | Volume 12


| Article 661457



CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References


TABLE 1 | Continued

Notes Year Ref


1984 (38)


1984 (39)

1986 (40)1987 (41)

1987 (42)

1987 (43)

1987 (44)




1988 (47)


1990 (48)



1991 (50)

1991 (51)


1991 (52)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614574

Crosnacon

Nonaja

Kno

Spe


Senand

Spefrom


samples

Sample matrix

ELISA LeporineIgG






MurinemonoclonalIgG





Blood

ELISA LeporineIgG







Blood


Serum

ELISA LeporineIgG


Serum


Serum

Agglutinationtest

LeporineIgG



Serum woundswabs




ELISA LeporineIgG


Serum

TABLE 1 | Continued

Notes Year Ref


1992 (53ndash56)

1992 (57)




1993 (59)

1993 (60)



1993 (62)

1994 (63)

1994 (64)

1994 (65)

1995 (66)

1996 (67)


(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614575


samples

Sample matrix

crossN kaoO haT ery




ELISA LeporineIgG



LeporineIgG

SouthernThailand


Agglutinationtest

LeporineIgG




ELISA LeporineIgG



ELISA LeporineIgG



FluorogenicELISA




ELISA LeporineIgG


ELISA NorthAmerica


Serum


Serum



Yes Plasma urine




Only a

-

ntd

-

e

w

TABLE 1 | Continued

Notes Year Ref


1997 (70)

1999 (71)



2002 (73)


2004 (75)

2006 (76)

2007 (77)

2008 (78)


2010 (79)

city 100 2012 (80)



2016 (83)

2017 (84)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614576


samples

Sample matrix

E carinatusN naja



Serum Sensit






Serum plasma




Tissue samples


Only a

Opticalimmunoassay

LeporineIgG


Blood tissuesamples

Crosstested

Agglutinationtest



Opticalimmunoassay

LeporineIgG








ELISA LeporineIgG



Skin blood

Immuno-flourescence


ELISA LeporineIgG


ELISA LeporineIgG


ELISA LeporineIgG




Serum Sensit



Plasma

ELISA LeporineIgG



Sample buffer

-

n

e

fi

e

TABLE 1 | Continued

Notes Year Ref


2018 (85)


2018 (86)


2020 (87)

2020 (88)

sed on (82) 2020 (89)


2001 (90)

2015 (91)

ecificity 100 2016 (92)

2014 (93)

2017 (94)

2018 (95)


Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614577


samples

Sample matrix

Echis sppNaja spp









Serum Quve

LFA Avian ampequine

South-East Asia

Daboia sppNaja spp

10 ngmL (invitro)



Asia ampAfrica



Molecularbiology










Patients 108 Serum







Serum


sa

a










Auxilliary tests

Type Subtype



Serumbiochemistry




Electrocardiagram












































































































Asia ()


April 20

21 | Volume 12


| Article 661457



CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References


TABLE 1 | Continued

Notes Year Ref


1992 (53ndash56)

1992 (57)




1993 (59)

1993 (60)



1993 (62)

1994 (63)

1994 (64)

1994 (65)

1995 (66)

1996 (67)


(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614575


samples

Sample matrix

crossN kaoO haT ery




ELISA LeporineIgG



LeporineIgG

SouthernThailand


Agglutinationtest

LeporineIgG




ELISA LeporineIgG



ELISA LeporineIgG



FluorogenicELISA




ELISA LeporineIgG


ELISA NorthAmerica


Serum


Serum



Yes Plasma urine




Only a

-

ntd

-

e

w

TABLE 1 | Continued

Notes Year Ref


1997 (70)

1999 (71)



2002 (73)


2004 (75)

2006 (76)

2007 (77)

2008 (78)


2010 (79)

city 100 2012 (80)



2016 (83)

2017 (84)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614576


samples

Sample matrix

E carinatusN naja



Serum Sensit






Serum plasma




Tissue samples


Only a

Opticalimmunoassay

LeporineIgG


Blood tissuesamples

Crosstested

Agglutinationtest



Opticalimmunoassay

LeporineIgG








ELISA LeporineIgG



Skin blood

Immuno-flourescence


ELISA LeporineIgG


ELISA LeporineIgG


ELISA LeporineIgG




Serum Sensit



Plasma

ELISA LeporineIgG



Sample buffer

-

n

e

fi

e

TABLE 1 | Continued

Notes Year Ref


2018 (85)


2018 (86)


2020 (87)

2020 (88)

sed on (82) 2020 (89)


2001 (90)

2015 (91)

ecificity 100 2016 (92)

2014 (93)

2017 (94)

2018 (95)


Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614577


samples

Sample matrix

Echis sppNaja spp









Serum Quve

LFA Avian ampequine

South-East Asia

Daboia sppNaja spp

10 ngmL (invitro)



Asia ampAfrica



Molecularbiology










Patients 108 Serum







Serum


sa

a










Auxilliary tests

Type Subtype



Serumbiochemistry




Electrocardiagram












































































































Asia ()


April 20

21 | Volume 12


| Article 661457



CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References


TABLE 1 | Continued

Notes Year Ref


1997 (70)

1999 (71)



2002 (73)


2004 (75)

2006 (76)

2007 (77)

2008 (78)


2010 (79)

city 100 2012 (80)



2016 (83)

2017 (84)

(Continued)

Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614576


samples

Sample matrix

E carinatusN naja



Serum Sensit






Serum plasma




Tissue samples


Only a

Opticalimmunoassay

LeporineIgG


Blood tissuesamples

Crosstested

Agglutinationtest



Opticalimmunoassay

LeporineIgG








ELISA LeporineIgG



Skin blood

Immuno-flourescence


ELISA LeporineIgG


ELISA LeporineIgG


ELISA LeporineIgG




Serum Sensit



Plasma

ELISA LeporineIgG



Sample buffer

-

n

e

fi

e

TABLE 1 | Continued

Notes Year Ref


2018 (85)


2018 (86)


2020 (87)

2020 (88)

sed on (82) 2020 (89)


2001 (90)

2015 (91)

ecificity 100 2016 (92)

2014 (93)

2017 (94)

2018 (95)


Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614577


samples

Sample matrix

Echis sppNaja spp









Serum Quve

LFA Avian ampequine

South-East Asia

Daboia sppNaja spp

10 ngmL (invitro)



Asia ampAfrica



Molecularbiology










Patients 108 Serum







Serum


sa

a










Auxilliary tests

Type Subtype



Serumbiochemistry




Electrocardiagram












































































































Asia ()


April 20

21 | Volume 12


| Article 661457



CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References


TABLE 1 | Continued

Notes Year Ref


2018 (85)


2018 (86)


2020 (87)

2020 (88)

sed on (82) 2020 (89)


2001 (90)

2015 (91)

ecificity 100 2016 (92)

2014 (93)

2017 (94)

2018 (95)


Knudsen

etal

Snakebite

Envenoming

Diagnosis

andDiagnostics

Frontiersin

Immunology

|wwwfrontiersinorg

April2021

|Volum

e12

|Article

6614577


samples

Sample matrix

Echis sppNaja spp









Serum Quve

LFA Avian ampequine

South-East Asia

Daboia sppNaja spp

10 ngmL (invitro)



Asia ampAfrica



Molecularbiology










Patients 108 Serum







Serum


sa

a










Auxilliary tests

Type Subtype



Serumbiochemistry




Electrocardiagram












































































































Asia ()


April 20

21 | Volume 12


| Article 661457



CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References











Auxilliary tests

Type Subtype



Serumbiochemistry




Electrocardiagram












































































































Asia ()


April 20

21 | Volume 12


| Article 661457



CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References












































































































Asia ()


April 20

21 | Volume 12


| Article 661457



CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References




CONCLUSION






FUNDING


REFERENCES




















































































































































































































































































































Introduction











Conclusion


Funding

References




































































































































































































































































































Introduction











Conclusion


Funding

References


Snakebite Envenoming Diagnosis and Diagnostics - DTU Orbit

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Transcript of Snakebite Envenoming Diagnosis and Diagnostics - DTU Orbit