12. Fungal identification methods-New.ppt

class presentation

1. FUNGAL IDENTIFICATION
METHODS
DepartmentofMicrobiologyandImmunology
Presenter:JaphetPeter
Facilitator:Dr.Manyahi
Venue;MicrobiologyTeachingLab.
Date;30/11/2022

2. Learning outcomes
•Describefungalidentificationmethods
•Listanddescribethelaboratoryapproaches
usedfordiagnosisoffungalinfection
•CompareandContrastthedifferentfungal
identificationmethods
•Discussdermatophytesidentification
•DescribeFungalSusceptibilityTesting

3. Outline
•Introduction
•Fungal Identification Methods
•Summary
•References

4. Introduction
•Identificationmethodsoffungiistheprocessof
identifyingcausativeagentofinfectiontohuman
betweennonecausativeagentsofinfectionbyusing
severalapproaches;e.gPhysicalandLaboratory
•Fungiaremembersofthegroupofeukaryotic
organismsthatincludesmicroorganismssuchas
yeasts,moldsandmushrooms
•Differenttypesoffungicancauseinfectionsand
differentmethodscanbeusedtoidentifythecauseof
infectioninmedicalfield.

5. Overview of fungi of Medical importance
Superficialmycoses:
2types:surfaceandcutaneousmycoses
Skin,hair&nails.
Deepmycoses:
2types:subcutaneous&systemicmycoses
Rangefromasymptomaticinfectiontofatal
disease
Opportunisticmycoses;-
mostlysystemicinvolvement

6. Fungal Identification
•Identificationoffungalinfectionisbasedon
morphologicalandphysiologicalpropertiesoffungi,also
clinicalinformationofthepatient
•Laboratoryapproachesusedondiagnosisoffungal
infectionincludes;
Directmicroscopicexamination,Histopathological
methods,cultureoftheorganism,serologictests,
PCRandothermolecularmethods

7. Fungal Identification…
•Theproperdiagnosisoffungidependsonproper
specimencollectionbysteriletechniqueincluding;
–Decontaminationofskinsurfacewith70%ethanol
–Surfacescrapingtoremoveskinscales
–Treatmentofspecimenwith10%potassiumhydroxide
todestroytissueelements

8. Fungal Identification…
•Specimensaresupposedtobedeliveredatclinical
laboratory/histologicallab,inaclearlylabeled,sterile
containersappropriatetothetypeofmaterialbeing
investigated
•Theselectionofappropriatecollectiondevices&
transportcontainers,labelingofthespecimen&
completerequisitionformsareimportant
considerationsinensuringthecorrectdiagnosisof
fungalinfections

9. Fungal specimens for identification
•Specimensinclude;
Sputum-foraspergillosis,histoplasmosis,
blastomycosis
Biopsymaterial-forHistoplasmosis
Skinscrapings-Hairornailclippings,
blood-forsystemicinfections
Vaginalswab-forcandidiasis
BodyfluidsegCSFforCryptococcosis
exudatesfromgranulousorulcerative
lesions

10. Fungal Identification Methods
1.MacroscopicexaminationegWood’slamptest
2.Microscopicexamination
a)Wetpreparation(Mount)-KOH,Indiaink
b)Stainingsmears(Gramstain,giemsastain)
c)Histopathologicalmethods(H&E,PAS)
3.Culturemethods
4.Molecularmethods
5.Biochemicalmethods
6.Immunologicalmethods

11. Microscopy
•ItisRapidmethodandverycrucialinthediagnosis
offungalinfections,(superficialandsubcutaneous)
•Detects;asexualspores,hyphae,oryeastsusing
lightmicroscope.
Examples:-capsuleofCryptococcusneoformans,
Candidaalbicansetc

12. Microscopy
1.Wetmountpreparations-Potassiumhydroxide(KOH)
•SlideMethodprocedure;
–Specimensareplacedonaslidecontainingadropof
10–20%potassiumhydroxide.
–Coverwithcoverslip,examineimmediatelyafter20
minutesusinglight-microscopebyusing10x
objective.

13. Microscopy
•Tube Method
–Mainlypreparedforbiopsyspecimens,whichtake
longertimefordissolution
–Thehomogenizedbiopsytissueisdissolvedin
10%KOHandexaminedafterkeepingfor
overnightincubatedat37
0
C.

14. Microscopy
Interpretation of the results;-
•Fromskinornailsspecimens;branchinghyphaeor
chainsofarthroconidia(arthrospores)areseeneasily
•Fromhairsspecimen;mostmicrosporumspeciesform
densesheathsofsporesaroundthehair(ectothrix)
TrichophytontonsuransandTricholosporumviolaceum
producingarthroconidiainsidethehairshaft(endothrix)

15. KOH-wet mount
Spores outside a hair (ectothrix) Spores inside a hair (endothrix

16. Microscopy
2.Calcofluorwhitestain;
Thisisnonspecificfungalcellwallfluorescentdyestain
whichisusedforidentificationoffungiintissue
specimensunderfluorescentmicroscope.Fungalelements
inexudatesandsmallskinscalesappears;
Fluorescentbrightgreentoblueandwhiteappearance.
Seeprocedureatnextslide

17. Calcofluor white stain procedure
1.Putthesampleontoacleanglassslide
2.Add1dropofcalcofluorwhitestain
3.Placeacoverslipoverthesample
4.Allowtostandfor1minute
5.Examinetheslideunderuvlightat355nm
wavelength.
Interpretationofresults;
Fungalorparasiticorganismsappear-
fluorescentbrightlightgreentoblue,other--

18. Microscopy

19. Microscopy-----
3. Lactophenol cotton blue stain; is used to
determine the morphology of the conidiogenous
cells and the conidia that identify filamentous
fungus
Procedure;
1.Plae2-3 drops of Lactophenol Cotton blue stain on
the slide.
2.Add fungal culture to the drop of the stain
3.Carefully tease fungal culture using forcepas to
prepare a thin preparation

20. 4.Placeacoversliponthepreparation
5.Allowtostandfor5minutes
6.Observemicroscopicallyusing100xobjective
Interpretationofresults;
-Fungalstructuressuchashyphaeand
sporesarestainedblueincolor.

21. Microscopy…
Aspergillus fumigatusMould

22. Indian ink for C.neoformans
wet mount
. preparation of cerebrospinal fluid (CSF),
highlights Cryptococcus neoformans
capsule (method-insensitive 50% )

23. Microscopy
5.GramStain;detectionof
yeastssuchasCandidaor
Cryptococcus,
FungisuchasAspergillum,
Intercellularyeastformssuch
asHistoplasmacapsulatumin
Wrightstainfor;
peripheralbloodsmear,bone
marrowortissue
•Yeast cells

24. Histopathological methods
•Examinationoftissuesectionsindiagnosisof
subcutaneousanddeepfungalinfections
1.HematoxylinandEosin(H&E)stain;routine
histologicalstainsbutmanyfungistainpoorly
–Asteroidbodyconsistsofacentralbasophilic
yeastcellsurroundedbyradiatingextensionsof
eosinophilicmaterial(depositionsofantigen-
antibodycomplexesandcomplement)

25. H&E stain
Endospores and sporangia
of Rhinosporidium seeberi
Blastomyces dermatitidis

26. Histopathological…
2.Gomorimethenamine-silverstain
(GMS);fungistaineddarkgrayto
black
3.Mayer’smucicarminestain;
specificallyusedtoshowcapsular
materialofCryptococcus,endospores
andsporangiaofRhinosporidium
seeberi
GMS-Stain
Cryptococcus neoformans: Pleomorphic
yeast-like cells and formation of narrow-
based buds are typical. The encapsulated
strains have capsular material detected with
mucin stain.

27. Histopathological…
4. Gridley fungus stain; fungi stained purplish
rose with a yellow background
5. Calcofluor white stain; used on tissue
sections, fungi fluorescent blue white
appearance on a dark background
6.Periodic acid-Schiff (PAS); fungi stained hot
pink to red based on the presence of chitin and
polysaccharide in fungi cell wall

28. Histopathological…
–Detect non-pigmented branching, septate
hyphae is typical of Aspergillus infection
Aspergillus -skin biopsy

29. Histopathological…
7. Direct immunoflourescence
–Immunoperoxidase and immunofluorescent
staining reagents, detects monoclonal and
polyclonal for some fungi
–Immunochemical staining, facilitate the
identification of atypical fungal elements and
assist the diagnosis of mixed infections

30. Histopathological…
8. In situ hybridization
–DNA probes to the nucleic acid of the fungal
agent, directly on the slide (in situ
hybridization) or in a test tube

31. Culture Method
•Specimens; cultured on inhibitory mold agar e.g.,
Sabouraud's dextrose medium (SDA), enriched
media with antibiotics (chloramphenicol ) to
inhibit bacterial growth, and enriched media with
both antibiotics and cycloheximide (which
inhibits many saprophytic fungi). Incubated at
25–35 °C

32. Culture…
•Selective media;-
–Cornmealagar(CMA)suitablefor
sporulation,chlamydosporeformationby
C.albicans.
–Birdseedagar–cryptococcus,formsbrown
colonies
–BrainHeartInfusion(BHI)agar–dimorphic
&otherfastidiousfungi
•Theappearanceofthemyceliumandthenature
oftheasexualsporesarefrequentlysufficientto
identifytheorganism

33. Culture…
–Identificationofyeastcultures
•Morphologiccharacteristics(presenceofcapsule,
formationofgermtubesinserum,andmorphology
oncornmealagar)
•Biochemicaltests(urease,nitratereduction,and
carbohydrateassimilationsandfermentations)
•AlsocanbeaccomplishedwithDNAprobes,which
areavailableforCryptococcus

34. Culture…
–Identification of filamentous fungal cultures
•Immunologic criteria; uses an
immunologic method called exoantigen
testing, in which antigens extracted from the
culture to be identified are immuno diffused
against known antisera
-Blood culture should be performed in all cases
of suspected deep fungal infection

35. Fungi on culture plates
•,
Penicillium
Aspergillus
versicolor

36. Dermatophytes Identification
–Species identified based:
•Colonial morphology (growth rate, surface
texture, and any pigmentation)
•Microscopic morphology (macroconidia,
microconidia)
•In some cases, nutritional requirements
Note:dermatophyte-pathogenic fungus that grows on skin, mucous membranes,
hair, nails, feathers, and other body surfaces, causing ringworm and related disease

37. Dermatophytes Identification…
•Microscopicmorphologicalcharacteristic(microconidia
and/ormacroconidia)
–Specimensinoculatedontoinhibitorymoldagaror
Sabouraud'sagarslantscontainingcycloheximide
andchloramphenicoltosuppressmoldandbacterial
growth,incubatedfor1–3weeksatroom
temperature

38. Dermatophytes Identification…
Threegeneraarerecognized:
•Epidermophyton:Smooththin-walledMacroconidia
onlypresent,nomicroconidia,coloniesaregreen-
browntokhakicolour
•Microsporum:Macroconidiawithroughwalls
present,microconidiamayalsobepresent
•Trichophyton:Microconidiapresent,smooth-walled
macroconidiamayormaynotbepresent

40. Dermatophytes Identification…
•Trichophytonspecies;Infectedhair,skin,ornails,
developcylindric,smooth-walledmacroconidia
•Tmentagrophytes-cottonytogranularcolonies,
grape-likeclustersofsphericalmicroconidiaon
terminalbranches.Coiledorspiralhyphaeare
commonlyfoundinprimaryisolates

41. Dermatophytes Identification…
•Trubrum-whitecolonies,cottonysurfaceandadeep
red,nodiffusiblepigmentwhenviewedfromthe
reversesideofthecolony.Themicroconidiaaresmall
andpiriform(pear-shaped)
•Ttonsurans-flat,powderytovelvetycolonyonthe
obversesurfacethatbecomesreddish-brownon
reverse;themicroconidiaaremostlyelongated

42. Dermatophytes Identification…
•Epidermophytonfloccosum;Theonlypathogeninthis
genus,infectstheskinandnails.
-Producesmacroconidia,whicharesmooth-walled,
clavate,two-tofour-celled,andformedingroupsof
twoorthree
-Thecoloniesareusuallyflat,smoothwithatantoolive-
greentinge

43. Dermatophytes Identification…
•Inadditiontogrossandmicroscopicmorphology,a
fewnutritionalorothertests,suchasgrowthat37
°Coratestforinvitrohairperforation,areusefulin
differentiatingcertainspecies.

44. Dermatophytes Identification…
•Microsporumspecies;Infecthairand/orskin,
producedistinctivemulticellularmacroconidiawith
echinulated(smallspines)walls.
•Mcanis-formswhitecottonysurfacecolonies
andadeepyellowcoloronreverse;thethick-
walled,8-to15-celledmacroconidiafrequently
havecurvedorhookedtips

45. Dermatophytes Identification…
•Mgypseum-producesatan,powderycolonyand
abundantthin-walled,four-tosix-celledmacroconidia

46. FUNGAL SUSCEPTIBILITY
TESTING
•Broth micro dilution method is used for
susceptibility testing
•Antifungal resistance is a particular problem
with Candidainfections.
•About 7% of all Candidablood stream isolates
tested at CDC were resistant to fluconazole

47. Biochemical Tests
a.Urease test
–Urease positive e.g.
Cryptococcus neoformans
b.Nitrite reduction
–Fungi that reduce nitrate e.g. Aspergillus spp
c.Carbohydrate fermentation
–C. albicans ferment glucose, galactose and
maltose

48. Molecular Methods
•Directdetectionofnucleicacidamplification
–DNAprobesusedtoidentifyfungicolonies
growingincultureatamuchearlierstage,for
detectionofCoccidioides,Histoplasma,
Blastomyces,andCryptococcus
–Othermethodsincludes,theuseof
conventionalPCRformats,multiplexPCR
(amplifymorethanonetargetsequence-
multiplesetsprimers),pan-fungalPCR,real-
timePCRmethods.

49. Molecular Methods…
–These techniques permit quantification of the
amounts of fungal nucleic acid that are present
in clinical samples
•Strain typing

50. Immunological Methods
•Testsforthepresenceofantibodies
(histoplasmosis,coccidioidomycosis)and
antigens(cryptococcosis,aspergillus,candidiasis,
histoplasmosis)inthepatient'sserumorspinal
fluidfordiagnosingsystemicmycoses
•DetectionofIgMindicatesrecentexposure
whereasIgGindicateexposuresometimeago

51. Immunological…
•Complementfixationtest,usedinsuspected
casesofcoccidioidomycosis,histoplasmosis,
andblastomycosis
•Latexagglutinationtest,detectedthepresence
ofthepolysaccharidecapsularantigensofC.
neoformansinthespinalfluid(cryptococcal
meningitis)

52. SummaryFungus Microscopic
Morphologic
Features in
Clinical
Specimens
Morphologic Characteristic features in Culture Tests for
Identification
Macroscopic Microscopic
Candida Oval budding
yeasts 2-6 µm in
diameter ( hyphae
& pseudohyphae
may present)
Variable; colonies
usual pasty, white to
tan & opaque (may
have smooth or
wrinkle morphology)
Clusters of blastoconidia,
pseudohyphae and/or
terminal chlamydospores in
some spp
Germ tube,
PNA-FISH
Cryptococuss
neoformans
Spherical budding
yeast of variable
size 2-15µm.
Capsule may
present, no
hyphae or
phseudohypae
Colonies are shiny,
mucoid, dome shaped
and cream to tan in
color
Budding spherical cells of
varying size, capsule
present, no pseudohyphae
Urease (+),
phenoloxidase
(+), EIA test for
polyscharide
Aspergillus Septate,
dichromously
branched hyphae
3-6
Varies of species A.
fumigates; blue-green
to gray A. niger; black
Varies of species
Conidiosphores with
enlarged visceral covered
flask-shaped metulae or
philalides. Hyphae are
hyaline and septate
Base on
microscopic and
colonial
morphology
Gene sequence

53. Summary…Fungus Microscopic
Morphologic
Features in Clinical
Specimens
Morphologic Characteristic features in Culture Tests for
Identification
Macroscopic Microscopic
Histoplasma
capsulatum
Small 2-4 µm
budding yeast within
macrophages
Colonies are slow
growing and white or
buff-brown in
color(25
o
C)
Yeast phase colonies
(37
o
C) are smooth,
white and pasty
Thin septate hyphae that
produce tuberculte
macroconidia and smooth-
walled microcornidia (25

o
C).Small, oval, budding
yeast produced at 37
o
C
Demonstration of
temperature-
regulated
dimorphism by
conversion from
mold to yeast phase
at 37
o
C, acid probe
test allow
identification without
phase conversion
Mucomycetes Broad thin-walled,
pauciseptate hypae, 6-
25 µm with
nonparallel sides and
random branches.
Hyphae stain poorly
with GMS stain and
ofen stain well with
H&E stain
Colonies rapidly
growing, wooly and
gray-brown to gray-
black in color
Broad. Ribbon-like hyphae
with rare septa.
Sporangium or sporangial
produced from
sporangiophore. Rhizoids
present in some species
Identification based
on microscopic and
colonial macroscopic
morphology. Gene
sequencing
Dematiaceous
molds
Pigmented ( brown,
tan or black) hypae,
2-6µm wide, May be
branched or un-
branched
Collonies usually
rapidly growing,
woody and gray,
olive, black or brown
in color
Varies depend on genus
and species. Hyphae are
pigmented. Conidia may be
single or in chains, smooth
or rough and dematiaceous
Identification based
on microscopic and
colonial macroscopic
morphology. Gene
sequencing

54. References
•Johnson,ArthurG.;Ziegler,RichardJ.;Lukasewycz,
OmelanA.;Hawley,LouiseB.inBRSMicrobiologyand
Immunology,4thEdition
•MurrayRosentialPfallerinMedicalMicrobiology,
seventhedition
•http://www.mycology.adelaide.edu.au/virtual/2005/ID2-
Nov05.html
•https://bmcmicrobiol.biomedcentral.com/articles/10.1186/
1471-2180-8-135
•https://www.cdc.gov/fungal/antifungal-resistance.html