A guide to diagnosing skin infections in practice

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Special ThanksPfizerAnimalHealthwouldliketothankthefollowingpeopleforkindly

supplyingtheimagesforthisbookletandwithoutwhosesupportthe

visualisationoftheindividualstepswouldnothavebeenpossible:

Albanese,Francesco,ClinicaVeterinarial'Arca,Naples,Italy

Bettenay,Sonya,Deisenhofen,Germany

Leone,Frederico,ClinicaVeterinariaAdriatica,Senigallia,Italy

Nuttall,Tim,UniversityofLiverpool,Liverpool,UK

Peters,Stefanie,TierklinikBirkenfeld,Birkenfeld,Germany

TetonNewMedia,Jackson,USA."DermatologyMadeEasy"series,RalfS.Müller

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IntroductionDuetothefactthat25%ofdogsand18%ofcatspresentedinpracticesufferfromskininfectionsorskindisorders,dermatologyisoneoftheroutinechallengestheveterinarianfaces.*Tofindthecauseofthedisorderandtoachieveasuccessfultherapy,itisimportanttoperformadetaileddiagnosis.

Thereareanumberofdermatologicteststodeterminethecauseofinfection,andnotallrequireaspecialistlaboratory.Someofthesetestscaneasilyandquicklybeperformedinpractice.Theydonotrequirespecialistequipmentandcaninmostcasesdeliveradefiniteresultwhilethepatientisstillinthepractice.

Thisbookletlooksatthesixmostcommonlyuseddermatologicalteststhatcaneasilybeperformedinanypractice.Itdescribes–inaneasy-to-useformat–whenandwhyatestshouldbeperformed,whatitwillshowup,whatutensilsarerequiredandhowitisconducted,withalargeselectionofimagestohelpvisualiseindividualsteps.

ThisbookletisprovidedbyPfizerAnimalHealthtosupporteverydaypracticeandenhancedermatologyservices.

*Bio'satstudy,PfizerAnimalHealth,July2009

Equipmentneededfor“in-practice”dermatologicaltests

Cytology WhendoIdoit?

•Whenbacterialorfungalinfectionissuspected

(inflammatoryalopecia,seborrhoea,scales,

papules,pustules,crusts,erosions,ulcers)

•Inpatientswithnodules/tumours-

docytologyoneverynodule/tumour

•Inpatientswithsuspectedpemphigoid

diseases(erosions,pustules,crusts)

•Ineverypatientwithotitisexterna

WhatcanIfind?

•Cocci(mostlikelyStaphylococcus sp.)

•Rods→cultureandsensitivityadvisable

•Inflammatorycellswithintracellular

bacteria→clinicallyrelevantinfectionthat

requiressystemicantibiotictreatment

•Eosinophils→canpointtoectoparasitesor

allergies

•Macrophages→chronic,sterileand

infectiousprocesses!

•Malassezia spp.→oneormoreMalassezia sp.

perHighPowerField(x1000magnification)

maybeclinicallyrelevant,basalnumbersvary

withtheclimate.IncasesofMalassezia

hypersensitivityamuchlowernumberof

Malassezia(e.g.oneineverytwoorthree

HPFs)cancauseclinicaldisease.Atopical

orsystemictreatmentshouldbeconsidered.

•Neoplasticcells

WhatdoIneed?

•Slides,DiffQuick®orsimilarstain,mineraloil,

adhesivetape,microscope,needleandsyringe

HowdoIdoit?

•Impressionsmear

•Ruborimpressaslideonmoist,exudingor

greasysurfaceofinfectedskin.

•Roleacottonbudontheskinsurfaceor

insertitintheearsandrolecottonbudon

theslide.

•Insertneedle(25-27ga.)intothepustule

holdingtheneedleparalleltotheskinsothat

onlythepustuleispunctured,nodeepercells

orbloodarerequired,topisliftedoffand

slideimpressedontotherupturedpustule.

•Usethestickysurfaceoftheadhesivetape

tocollectcellsandsurfaceorganismsfrom

dryand/orscalyskinandthenplacethis

(stickysidedown)ontoaglassslidewith

adropofDiffQuick®.Thetapeactsasits

owncoverslip.

•Aspirationsmear

•Insertneedleintonodulesorabscesses

andre-insertanumberoftimeswithout

leavingtheskin.Withdrawtheneedle.

Asyringewiththeplungerpulledbackis

attachedtotheneedleandcontentsis

blownontoaslideandairdried.

•Staintheairdriedslides(e.g.DiffQuick®)

•Puttheslidesunderamicroscope,

condenserup

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Tip

•Incaseofdryskinorintheinterdigitalarea:

•Moistenacottonbudwithsalinesolution

orcarefullyrubtheedgeofaslideonthe

skinandthenrubthematerialontheslide

•Pressclearstickytape(stickysidedown)

ontotheskin.Stainthetapelikeaslide,

letitairdryandpressitontoaslideorput

adropofthebluestainofDiffQuick®ona

slideandpressthetapestickysidedown

onthedrop.Evaluateunderamicroscope.

Superficialpyoderma*

Impressionsmear:slidepressedonskin*

Pyogranulomatousinflammation(deeppyoderma):manyneutrophilsandmacrophages,fewbacteria†

Superficialpyoderma:neutrophilswithintracellularcocci*

Eosinophils,neutrophilsandbacteria*

Usetheadhesivetapetechniqueondryskinorintheinterdigitalarea*

Aspirationsmear:insertneedleintonodule*

Grade1mastcelltumourwitheosinophils*

Malasseziaandbacteria†

4*CourtesyofSonyaBettenay†CourtesyofStefaniePeters

WhendoIdoit?

•Oneverypatientwithpruriticorscalyskin

WhatcanIfind?

•Cheyletiella sp., Scabies sp., Notoedres cati,

Otodectes cynotis→Afindingofoneof

thesemitesortheireggsisdiagnostic

•Dermatophytesporeinfestedhairshafts

WhatdoIneed?

•Slides,coverslips,scalpelblade,mineraloil,

microscope.

HowdoIdoit?

•Gentlycliptheaffectedarea(s)leaving2-3mm

ofhairsothatthescalesandcrustsarenot

dislodged.Putmineraloilonyourscalpel

bladeandseveraldropsdirectlyontotheskin.

•Theoilisgentlyscrapedoffwiththescalpel

bladeandthematerialisputononeormore

slides.Thesemitesliveonorwithinthe

surfacelayers;thereisnoneedtodraw

bloodwithasuperficialskinscrape.

•Examineslidesundermicroscope

withthecondenserdown

Superficial Skin ScrapingTip

•Sampletheareaswherethemitesyouare

lookingforaremostlikelytobefound.

ForSarcoptes spp.thiswouldbeear

margins,elbows,hocksandventralbody.

•Mitesmaybehardtofind.Thebiggerthe

surfaceareascraped,thebetterthechance

ofapositiveresult.Inthecaseofanegative

resultandSarcoptesmitesarestillsuspected

adiagnostictherapeutictrialovera6week

periodisthemostdefinitivenextstep.

•Aserumantibodytestishighlysensitive,

buttheantibodiestake2-5weeks(upto4

months)afterinfestationtorise,andcross

reactionswithdustmitesarepossible.

•Somepeopleprefertouseadhesivetape

tocollectCheyletiellamites.Withthis

technique,thetapeispressedovermultiple

sitesofscalingandisalsodraggedacross

thehairshafts.Thetapeisplaceddirectly

ontoaglassslidewithoutanyoilor

stainingandisagainviewedwiththe

condenserdown.

•WhenDermatophytosisissuspected,use

onlytheminimumofoilnecessaryto“fix”or

“hold”thehairs,surfacescaleanddebris.

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Scalydogskin* ScalydogskincausedbyCheyletiellamites†

Putseveraldropsofmineraloildirectlyontoclippedareas†

Placeoilanddebrisontoaslide.Scrapeseverallargeareasandpreparevariousslidesforevaluation*

Sarcoptes† Cheyletiellamites†

Cheyletiella blakei † Otodectes cynotis†

*CourtesyofSonyaBettenay†CourtesyofFrancescoAlbaneseandFredericoLeone

Deep Skin Scraping WhendoIdoit?

•Incasesofsuspecteddemodicosis

(non-inflammatoryalopecia,comedones,

pustules,crusts,inflammatoryalopecia)

WhatcanIfind?

•Demodexmites→morethanonemiteis

diagnostic

WhatdoIneed?

•Slides,coverslips,scalpelblade,mineraloil,

microscope

HowdoIdoit?

•Ifnecessary,clip1cm2oftheareayouwantto

scrapetoremovehairs

•Squeezetheskinpriortoscrapingtopushthe

mitesoutfromthedepthsofthehairfollicles

•Scrapetheskininthedirectionofhairgrowth

withabladecoveredwithmineraloiluntil

capillarybleedingisobserved

•Putthematerialontheslide

•Placetheslideunderamicroscopewith

thecondenserdownandusethe

x400magnificationtolookforadults,

larvae/nymphsandeggs

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Tip

•Feetandfacearehardtoscrape→

Trichogrammaygiveequivalentresults

if1cm2ofhairsaresampled.

•Somebreeds(e.g.SharPei)arehardto

scrapeandmayhavetobebiopsiedfor

diagnosis.

8*CourtesyofSonyaBettenay†CourtesyofFrancescoAlbaneseandFredericoLeone

Dogwithdemodicosis:inflammatoryalopeciaandhyperpigmentation*

Puppy:focalalopecia†

Squeezeskinpriortoscraping* Scapeuntillightcapillarybleedingisobserved*

Excoriationafterdeepskinscraping†

Demodexmites† Demodex canis*

Wood’s Lamp Examination WhendoIdoit?

•IneverypatientwithpossibleMicrosporum

canisinfection(inflammatoryand

non-inflammatoryalopecia)

WhatcanIfind?

•Fluorescinghairshafts

WhatdoIneed?

•Wood’slamp,whichhasbeen“warmedup”

for5minutespriortouse

HowdoIdoit?

•Illuminatetheaffectedareainadarkened

room.In50-60%ofMicrosporum canis

infectionstherewillbegreenishfluorescence

whichrunsalongthehairshafts.

Be careful: A lack of fluorescent areas does

not rule out dermatophytosis as only half of

them show fluorescence.

Inthecaseofnegativeresults→ perform

fungalcultureusingMcKenzietoothbrush

technique(see:FungalCulture,Tip).

•Pluckhairswithfluorescencealonghairshafts

andusethemfortrichoscopyand/orfungal

culture.

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Tip

•Forbetterresultswarmthelampup

for5minutesbeforeusebecausethe

stabilityofthewavelengthandintensity

istemperature-dependent.

•Drugs,soapsandbacteria

(Pseudomonas sp.)oroccasional

individualscalescanfluoresceaswell,

buttheyshouldnotbeassociatedwith

thehairshafts.

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Wood’sLamp†

YorkshireTerrierwithamixedMicrosporum canisandMicrosporum gypseuminfection*

Positivefluorescenceinfelinedermatophytosis†

Microsporum canis:fluorescencerunningalonghairshaft▲

†CourtesyofStefaniePeters*CourtesyofTimNuttall▲CourtesyofTetonNewMedia

Microsporum canis:fluorescencerunning

Fungal Culture WhendoIdoit?

•Ineverypatientwithsuspectedfungalinfection

WhatcanIfind?

•Microsporum canis

•White,woollycolonieswithayellowish

reversepigment

•Abundantthickwalled,spindle-shaped

macroconidiawithknobsattheends

andtypicallymorethansixinternal

compartments

•Microsporum gypseum

•Granular,beigecultureswithyellowish

reversepigmentandthin-walled

macroconidiawithlessthansixinternal

compartments

•Trichophyton mentagrophytes

•Variablecolonieswithveryfew,cigar-shaped

macroconidiaandlargenumbersofsmall,

roundmicroconidia

WhatdoIneed?

•Dermatophytetestmedium(DTM),clear

stickytape,slides,microscope,methylene

blueorDiffQuick®blue

HowdoIdoit?

•Useascalpelwithoutoiltoscrapeand

haemostatstopluck;takehairsandscale

fromtheedgeofalesion(preferablytheones

fluorescingundertheWood’slight)

•ImpresshairsandscalegentlyonDTM;

donotscrewthelidtight

•Incubatetheagarat20-25°C(awarm,dark

spacewithmoisture)

•Checktheagardailyovera3weekperiod

•Colourchange(pHchange)thatoccurswhen

thecolonyisstillsmallandthenspreadsas

thecolonygrows,isindicativeof

dermatophytes

•Whenthecolonyis10-14daysold,impress

clearstickytape(stickysidedown)gentlyon

thesuspiciouscoloniesandlayitdownontoa

dropofmethyleneblueonaslide.

•Evaluatethesampleunderamicroscopewith

thecondenserup.Thestickytapeactsasits

owncoverslip.

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Tip

•Ifthepatienthasnoclearlycircumscribed

lesionoranasymptomaticcarrieris

suspectedusetheMcKenzie toothbrush

technique.

•Brushthehairwithanewtoothbrush

•Gentlyplacethehairsandscale,using

asterileneedle,ontotheagarorcutthe

bristleswithsterilescissors

•Putallthematerial(bristles,hairs,

scale)ontotheagar

•Agarcolourchangescanalsooccurwith

largesaprophytecolonies.Dailycontrol

ofthecultureisimperativetonoticea

colourchangethataccompaniesagrowing

culture.

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†CourtesyofStefaniePeters*CourtesyofTimNuttall▲

CourtesyofFrancescoAlbanese

DTMfalsepositive:saprophytecolonies†Dogwithlocaliseddermatophytosis(Kerion)†

DTMpositive:Microsporum canis* Microsporum canismacroconidia*

Microsporum gypseummacroconidia▲DTMpositive:Microsporum gypseum▲

DTMpositive:Trichophyton mentagrophytes▲ Trichophyton mentagrophytes macroconidiaandmicroconidia*

Trichogram WhendoIdoit?

•Ineverypatientwithalopecia

•Tolookforbrokenhairtipswhenone

suspectsself-inducedalopecia

•Todetermineifhairsareinanagenortelogen

phase(theinterpretationofratiosoftelogen

hairstoanagenhairsindogsisbreedand

season-dependentandexactratioshavenot

beenestablished)

•Whendermatophytosisissuspected

•Asanalternativetoadeepskinscraping

whenDemodexissuspected

WhatcanIfind?

•Brokenoffhairtips→causedbyself-trauma

•Taperedhairtips→hairlossiscausedby

eventswithinthefolliclee.g.endocrine

disordersorinflammationinvolvingthehair

follicle

•Hairsinanagen(growing)phase→rootsof

anagenhairsarerounded,curled,bentand

oftensmoothandpigmented

•Hairsintelogen(resting)phase→rootsof

telogenhairsarelancet-shapedandlack

pigmentation,althoughthebaseofthehair

mayshowaroughenedorbrush-likeedge

•Thepresenceofnumerousanagenphase

hairsshoulddecreasethesuspicionforan

endocrinopathy

•Inthecaseofdermatophytosis,affectedhairs

arecoveredwithsporesandpenetratedby

hyphae

•Colourdilutionalopecia→melaninis

clumpedinthehairshaft

WhatdoIneed?

•Forceps/haemostatorrubbercoveredclamp,

mineraloil,slide,coverslip,microscope

HowdoIdoit?

•Pluckasmallnumberofhairsinapartially

orcompletelyalopecicareausingforceps/

clampindirectionofhairgrowth;holdthe

forceps/clampclosetotheskinsurfaceand

graspallhairshaftswhichemerge

•Putadropofmineraloilontoaslide,place

thehairsinparallelorderonthemineraloil,

separatethemtoevaluaterootsandtips

adequately

•Coverhairswithacoverslipandevaluate

undermicroscope

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Tip

•Coverthetipsofyourforcepsorclamp

withrubberorsiliconsleevestoavoid

crushingorbreakingthehairshafts

•Youcanalsousethetrichogram

techniquetolookforDemodexmitesin

affectedareasthataredifficulttoscrape

(e.g.closetotheeye,pododermatitis),

ideallya1-2cm2areawillbeplucked,

thesameareaaswithaskinscraping

•YoumightfindDemodexmiteshanging

onthehairsorsometimeshidingbehind

them.Onlypositiveresultsarediagnostic.

•Youcanalsofindlice,Cheyletiellamites

andtheireggs

Samplinghairsforatrichogram*

Trichogramwithpointyhairtips* Trichogramwithbrokenoffhairtips*

Hairbulbsinanagenphase* Hairbulbsintelogenphase*

Demodex canis:twoadultmitesandonelarvaeonahairbulb†

Colourdilutionalopecia-macromelanosomes▲

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*CourtesyofSonyaBettenay†CourtesyofFrancescoAlbaneseandFredericoLeone▲CourtesyofFrancescoAlbanese

Pfizer Animal Health, Walton Oaks, Dorking Road, Walton-on-the-Hill, Tadworth, Surrey KT20 7NS Pfizer Animal Health, 9 Riverwalk, Citywest Business Campus, Dublin 24 AH438/09