Diagnostic ML b t PLaboratory P

Part Three--OppPart Three Opp

Erik MErik MClinical Mi

Wheaton FranciWheaton FranciWauwatosa

The presenter states no conflict of inteto disclose relevant to the c

Mycology forP f i lProfessionalsortunistic Moldsortunistic Molds

MunsonMunsonicrobiologyscan Laboratoryscan Laboratory, Wisconsin

erest and has no financial relationshipcontent of this presentation. 1

OUTL

I. Introductory statem

A. Review of classB. Important geneB. Important gene

II. Identification of clin

A. Macroscopic mB Microscopic moB. Microscopic moC. Other hints

III. Antifungal suscept

LINE

ments

sificationral criteriaral criteria

nically-significant molds

orphologyorphologyorphology

tibility testing 2

“D#*%it, Jim,I’m not a physician ”I m not a physician.

3

The BBasics

4

SCOPE OAt least 100,000 nam

~1 million to 10 millio1000 to 1500 new sp1000 to 1500 new sp

Fewer than 500 namFewer than 500 namwith animal or human

Less than 50 are pathuman hostshuman hosts

Biol. Rev. 73:

OF FUNGImed fungal species

on unnamed species;pecies per yearpecies per year

ed species associateded species associatedn disease

hogenic in healthy

203-266; 1998 5

PATHOGENICI-- Generally more chronic tha

Generally involves predispo-- Generally involves predispoChemotherapy-induced neutropenia

Organ transplantationOrgan transplantationCorticosteroids

Broad-spectrum antimicrobialsBroad spectrum antimicrobialsParenteral nutrition

DialysisyInvasive medical procedures

-- Certain infections can be “s

ITY OF FUNGIan acuteositionosition

HIVDiabetesDiabetes

AlcoholismIntravenous drug abuseIntravenous drug abuse

Intensive care population (burns, NICU)

Malignancyg yOther immune deficiency

signal diseases”g

6

CLASSIFYING O

Taxonomy

HolomTeleomorph

Sexual reproductionF i f t l i i t tFusion of two nuclei into zygote

Perfect FungiPseudallescheria boydiiy

OPPORTUNISTS

morphAnamorph

Asexual reproductionMit iMitosis

“Fungi Imperfecti”Scedosporium apiospermump p p

7

SEXUAL REPSubphylum Mucoromycotina

Zygopho

Mucoromycotina

Phylum Basidiomycota

Phylum Ascomycotay y

Phylum Deuteromycotay y

PRODUCTIONores meet and fuse (zygosporangium)

Clamp connectionsf ilit t b idifacilitate basidium

Nuclear divisioninside ascus (bag)

NO SEXUAL REPRODUCTION X AL R PR TIOBSERVED 8

CLASSIFYING O

Taxonomy

Cell morphology (coBlastic blaEnlarge, then divide phi

Thallic art“Divide” then enlargeDivide , then enlarge

Mode of entry (implaMode of entry (impla

OPPORTUNISTS

nidiogenesis)astoconidia annelloconidiaialoconidia poroconidia

hroconidia aleurioconidiachlamydoconidiachlamydoconidia

antation; inhalation)antation; inhalation)9

UNIFYING C

Macroscopic observatMacroscopic observat

Microscopic observatMicroscopic observat

GrowthGrowth

CONCEPTS

tion of colonial growthtion of colonial growth

tion of colonial growthtion of colonial growth

h on selective mediumh on selective medium

Rate of growthRate of growth

PigmentationPigmentation10

Wild Card

11

DERMATOInfrequent mortality

Immunocompromise

Tinea (ringworm)

Immunocompromise

Some have niche inSome have niche inGeophilic M

Zoophilic MT.

Anthropophilic M

OPHYTESy

ed host not requireded host not required

n terms of parasitismn terms of parasitismM. gypseum

M. canisT mentagrophytes

Most 12

DERMATOSome have regions oM. audouinii AfricT violaceum MidT. violaceum Mid

NorT concentricum PolyT. concentricum Poly

Poc

DermatophyteMicrosporum spp.

Epidermophyton floccosumTrichophyton spp.

OPHYTESf endemicityca, Haitidle Eastdle East,

rth Africaynesiaynesiackets of C. and S. America

Nails Skin HairNO Yes YesYes Yes NOYes Yes Yes 13

DERMATO

Rev. Inst. Med. trop. S. Paulo45: 259-263; 2003

OPHYTES

Ann. Trop. Med. Pub. Health3: 53-57; 2010 14

Trichophyt~14 days; resistant t

Smooth walled “penc

Diffusible red pigmen

Smooth-walled pencmacroconidia (3-8 ce

i bl i tvariable in amount

Abundant microconidAbundant microconidtear-shaped (“birds o

Urease-negative afte

ton rubrumto cycloheximide

cil”

nt

cilells)

dia;dia;on a wire”)

er 7 days15

Trichophyto~12 days; resistant to

Rare irregular thick

Suede surface with fo

Rare, irregular, thick-macroconidia

Abundant microconid(tears balloons club(tears, balloons, clubsome elongated

Urease-positive after

on tonsuranso cycloheximide; scalp

walled

olds

-walled

diabs);bs);

r 4 days16

Trichophyton m~7-10 days; resistant

Fluffy, white Variable-pigCigar-shape

(Rare macroconidia walled (1-attachme

Small microconidia; Very rountear-shaped (resembling

T. rubrum)clustered

conid

Spiral hyphae

Urease-positive after

mentagrophytest to cycloheximide; foot

gment, granulared, smooth, thin-

)6 cells); narrow ent to hyphaed microconidia; d on branched diophores

r 4 days17

TrichophytoHomogenous suspen

Room temperature; 2p ;

GrowthSelected Trichophyton spp.

Growth Casein

Base + thiaminT. rubrum 4+ 4+

T. tonsurans 1+ 4+T. mentagrophytes 4+ 4+

on AGARSnsion of mycelial growth

2 weeks

in Presence of:in Presence of:Ammonium nitrate

ne Base + histidine3+ 4+1+ 1+4+ 2+

18

Epidermophyt~10 days; resistant to

Starts velvety and khbecomes fluffy white

Smooth, thin- or thickidi d

y

macroconidia; roundesingle or characteristi

No microconidia

Urease-positive after

ton floccosumo cycloheximide; jock

aki;

k-walledd ded ends;

ic clusters

7 days19

Microspor~6-10 days; resistant

Cottony, wooly; lemonclosely-spaced groov

Rough, thick-walled, h d idi

y p g

shaped macroconidiato knob-like ends (6-1

Rare, single microcon

Urease-positive after

rum canist to cycloheximide; kids

n peripheryves

spindle-ta; tapers

15 cells)

nidia

7 days20

Microsporu~6 days; resistant to c

Cinnamon brown to bgranular (sporulates h

Very abundant macrothi ll d ith d

g ( p

thin-walled with round(4-6 cells)

Rare, single microcon

Urease-positive after

um gypseumcycloheximide; kids

buff;heavily)

oconidia;d d ti

y)

ded tips

nidia

7 days21

Pictuures

22

DEMATIACEOUSSoil, plant, moist org

Immunocompromise

Some tropical; som

Immunocompromise

Spectrum of diseasSpectrum of diseasEumycotic mycetomChromoblastomycoChromoblastomycoPhaeohyphomycosChronic sinusitis (pChronic sinusitis (pRare systemic dise

S OPPORTUNISTSganics (some air)

ed host not required

e temperate

ed host not required

eemaosisosissisportal for CNS disease)portal for CNS disease)ase

23

Eumycotic mycetomawith Exophiala etiologywith Exophiala etiology

Chromoblaswith Phialophwith Phialoph

stomycosishora etiologyhora etiology

Phaeohyphomycosiswith Alternaria etiology 24

Fonsecaea spp. Most common worldwchromoblastomycosischromoblastomycosisMaturity in ~14-28 daColony surface darkgreen black or gray;green, black, or gray;reverse is blackC idi ( h ) hilConidia (phores), hilavase-shaped phialidecollarettes, denticles

AND OTHERSwide cause ofssys

Fonsecaea pedrosoi

a,s,

Rhinocladiella spp.

Phialophora spp.25

Fonsecaea spp.

Scan from

Fonsecaea-typeconidiation

Rhinocladiella-typeconidiation

P

AND OTHERS

m Larone

Phialophora-typeconidiation

Cladosporium-typeconidiation

26

Cladosporium spp.,Most common worldwof chromoblastomycoof chromoblastomycoMaturity in ~14-28 daColony surface darkgreen black or gray;green, black, or gray;reverse is blackC idi ( h ) hilConidia (phores), hilavase-shaped phialidecollarettes, denticles

, Cladophialophorawide causeosisosisays

Cladosporium spp

;

Cladosporium spp.

Cladophialophora carrionii; Cladophialophora carrionii(formerly Xylohypha)

a,es,

Cladophialophora bantianaCladophialophora bantiana(formerly Xylohypha)

27

Cladosporium spp.,

ClaCladosporium spp.

Dematiaceous Mold Distinct conidiophores

Hila on conidia

Coclconidiophores conidia le

Cladosporium spp. Yes Yes SC. carrionii Variable Yes MC. bantiana No No L

D. H. Larone, Medically Im

, Cladophialophora

Cladophialophoraadophialophorabantiana

onidial chain

th

Conidial chain

b hi

Gelatin hydrolysis

Growth in 15% N Cl

Max growth °C

carrioniibantiana

ength branching hydrolysis NaCl °CShort Frequent Positive Positive <37edium Moderate Negative Negative 35-37Long Sparse Negative Negative 42-43

mportant Fungi, fourth ed. 28

AlternarTypically contaminanphaeohyphomycosisphaeohyphomycosis,Maturity in ~5 daysColony surface becomgreenish black or brogreenish black or brolight border; reverse iD ti k iDrumstick macroconiwith longitudinal, transeptations; poroconid

ria spp.t; role inallergy, allergy

mesown withown withis blackidiidia

nsversediation (chains)

29

Inhalation

30

ASPERGNasoorbitalEndocardialCutaneousEndocardial

DisseminatedCentral nervous sysCentral nervous sysPulmonaryy

Allergic bronchoAspergilloma (fuAspergilloma (fuInvasive pulmon

GILLOSIS

stem diseasestem disease

pulmonary aspergillosisngus ball)ngus ball)ary aspergillosis 31

24 MEDICAL CEWhat does your (lab) posit

Clin. Infect. Dis. 33

ENTERS; n = 1477tive culture result mean???

3: 1824-1833; 2001 32

UNDERLYING RIS

Clin. Infect. Dis. 33

K AND OUTCOME

3: 1824-1833; 2001 33

ASPERGGILLOSIS

(---)seriatephialides

conidiaphialides

vesicleconidiophore

34

AspergillusMaturity in ~3 days

Conidiophores short

C fColony surface becomdark greenish to grayreverse white to tan

U i i t hi lidUniseriate phialides oupper 2/3 of vesicle; to axis of conidiophor

s fumigatus

& smooth

mesy;

onparallelre

35

AspergillCommonly associate

Conidiophores rough

C fColony surface velveyellow to green or broreverse white to tan

U i i t d bi iUniseriate and biseriaphialides coveringentire vesicle (all dire

lus flavusd with aflatoxins

& spiny

ty,own;

tate

ections)36

AspergilluCommonly considere

Conidiophores short

C fColony surface velvecinnamon brown;reverse white to brow

Bi i t hi lidBiseriate phialides vecompact; can be quitelengthy

us terreused contaminant

& smooth

ty,

wn

erye

37

Emericella (AspeCommonly considereC idi h h tConidiophores short,smooth, brownColony surface typicagreen (yellow in spotsgreen (yellow in spotsreverse purplish redBi i t h t lBiseriate, short, columphialides; cleistothecHülle cells

ergillus) nidulansed contaminant

allys);s);

mnaria,

38

CASE PRES19-year-old male withcongestion and ear pacongestion and ear paPMH of psoriasis in mpdaily ibuprofen for tonPre io s regimens ofPrevious regimens of amoxicillin, otic neomyciprofloxacin-hydrocorPain worsened; ENT c

Courtesy T

Pain worsened; ENT c

SENTATION three-day history ofainain

multiple cutaneous sites;pnsillar hypertrophy

amo icillin cla lanateamoxicillin-clavulanate,ycin-polymyxin, oticrtisoneconsult

T. K. Block

consult39

CASE PRES

Courtesy T

SENTATION

calcofluor white stain;400x total magnificationg

T. K. Block 40

AspergillCan cause disease in

Conidiophores long &

Colony surface startst ll t bl kto yellow, turns blackreverse white to yello

Biseriate phialides;forms a “radiate head

lus nigern debilitated patients

& smooth

s white;

ow

d” 41

Aspergillus niger A. niger at least two tithan A flavus in contethan A. flavus in conte

Eur. J. Clin. Microbiol. InfAm. J. Trop. Med. Hy

Superficial infection; imEur J Clin Microbiol Inf

Self manipulation; ma

Eur. J. Clin. Microbiol. Inf

Am. J. Trop. Med. Hy

Self-manipulation; ma

OTOMYCOSISmes more commonext of otomycosisext of otomycosisfect. Dis. 8: 413-437; 1989yg. 29: 620-623; 1980

mmunocompetent hostsfect Dis 8: 413-437; 1989

anipulation by barbers

fect. Dis. 8: 413 437; 1989

yg. 29: 620-623; 1980

anipulation by barbers42

PenicilliTypically contaminanrespiratory cornea erespiratory, cornea, eMaturity in ~4 daysColony surface becompowdery and bluish gpowdery and bluish gwhite border; reverseB h d bBranched or non-branconidiophores; seconbranches known as m

ium spp.t; ear,

endocarditisendocarditis

mesgreen withgreen withe variable

h dnchedndarymetulae 43

PenicilliumEndemic to Southeascompromised and comcompromised and comMold maturity (25°C)Colony surface can bereddish yellow with ligreddish yellow with ligreddish pigment diffusY tlik ll bYeastlike cells observ35-37°C; central croas result of fission (no

m marneffeit Asia;mpetentmpetent) in ~3 daysecome

ght edge;ght edge;sion

d ted atss wall

ot budding)44

FusariuCommon contaminankeratitis disseminatedkeratitis, disseminatedMaturity in ~4 daysCottony surface, deveviolet or pink center wviolet or pink center wlight periphery; reversC h dCanoe-shaped macro± oval 1- to 2-celled in clusters resembling

um spp.nt; mycoticd diseased disease

elopswithwithse light

idioconidiaconidia

g Acremonium45

PseudallescheriaMycetoma; respirator(bone brain eyes m(bone, brain, eyes, mMaturity in ~7 days;mouse-like appearan

Scedosporium apiospermum GrapScedosporium apiospermumasexual

no inhibition by cycloheximide

Grapasex

no inhibition by

HOLOMORPHry/sinus, disseminates

meninges)meninges)

nce

phiumphiumxual

y cycloheximidePseudallescheria boydii

sexualinhibited by cycloheximide

46

ScedosporiumInvasive infection (ostcompetent & compromcompetent & compromMaturity in ~5 days; ginhibited by cyclohexiinhibited by cyclohexiCottony or moist surfabecomes dark gray/blwhite tufts; reverse grgOlive to brown conidiaannellides have swollannellides have swolland elongated neck

m prolificansteomyelitis, arthritis);misedmisedgrowthmidemideace,lack withray/blackya, ovoid;en baseen base

47

MORE M

Chrysosporium spp. Sepedoni

MIMICRY

Malbranchea spp.ium spp.48

MUCORM

Rapid growers

Diabetic susceptibility

Different cellular nom

Ribbon-like hyphae;most are aseptate

www.youtube.com/w

MYCOSIS

y

enclature

watch?v=IK0MtXNKgKI49

MUCORM

Clin. Infect. Dis. 4

MYCOSIS

41: 634-653; 2005 50

RhizopCommon contaminan

Maturity in ~4 days; gcycloheximidey

Cotton candy--white all i h byellowish brown;

reverse white

Rhizoids opposite ofsporangiophoressporangiophores

us spp.nts

growth inhibited by

at first, then gray or

51

Muco

Common contaminan

Maturity in ~4 days; ginhibited by cyclohexiinhibited by cyclohexi

Cottony or moist surfaCottony or moist surfabecomes gray;

hitreverse white

Rhizoids absentRhizoids absent

r spp.

nt

growthmidemide

aceace,

52

LichtheimCommon contaminanM t it i 4 dMaturity in ~4 days; ginhibited by cyclohexiCoarse, wooly-gray seventually covers surfeventually covers surfwith “fluff”; reverse whSporangiophores formconical apophysis jusp p y jcolumella; rhizoids alt

mia spp.nt

thgrowthmideurface--facefacehitemt belowternate 53

OTHER MUCO

Cunninghamellaspp.

Rhizomucorsppspp. spp.

ORMYCETES

A hApophysomycesspp.

Syncephalastrumspp.

54

Antifungal Susceeptibility Testing

55

CLSI DOCUMENT

M38-A2 ReferenceDilution Antifungal of Filamentous FunApproved Standard

M51-A Method foDiffusion SusceptibDiffusion SusceptibNondermatophyte Approved GuidelineApproved Guideline

TS OF INTEREST

e Method for BrothSusceptibility Testingngi, 2nd ed.;g , ;d

or Antifungal Diskbility Testing ofbility Testing ofFilamentous Fungi;ee

56

BROTH MICR“Intended for testing cmoulds, including the demoulds, including the deinvasive and cutaneous A illAspergillus spp.Rhizopus spp.Scedosporium prolificansScedosporium prolificansOpportunistic monilaceous fOpportunistic dematiaceouspp

“Method has not beenyeast or mould form oyeast or mould form o

CLSI M

RODILUTIONcommon filamentous...ermatophytes, which causeermatophytes, which causeinfections, respectively...”

F iFusarium spp.Pseudallescheria boydiiSporothrix schenckii (mould)Sporothrix schenckii (mould)

fungis fungig

n used in studies of theof dimorphic fungi ”of dimorphic fungi.M38-A2 57

BROTH MICRRPMI 1640 broth (MO

7-day filamentous fungpotato dextrose agar sp gFlood with salineWithdraw mixture parWithdraw mixture, parUpper suspension conInoculum (OD530) dep[0 09-0 30]; range of 0[0.09-0.30]; range of 0

CLSI M

RODILUTIONPS buffer, 0.2% dextrose)gus growth;slants

rticles settle 3 5 minrticles settle 3-5 minntains mycotic elementsendent upon fungus

0 6 to 3 0 x 106 CFU/mL0.6 to 3.0 x 10 CFU/mLM38-A2 58

BROTH MICR

Non-dermatophyte

0.03-16 g/mL amphgketocposac

0.125-64 g/mL flucyt

0.015-8 g/mL flucyt

CLSI M

RODILUTION

e filamentous fungi

hotericin B ravuconazoleconazole itraconazoleconazole voriconazole

tosine fluconazole

tosine fluconazole

M38-A2 59

BROTH MICRDermat

0.06-32 g/mL

0 125 64 g/mL0.125-64 g/mL

0.001-0.5 g/m

0 004-8 g/mLCLSI M

0.004-8 g/mL

RODILUTIONophytes

ciclopirox

L i f l iL griseofulvinfluconazole

L itraconazolevoriconazole

posaconazole

terbinafine

M38-A2

posaconazole60

BROTH MICR35°C ambient air

21-26 hours for mucor70-74 hours for Scedo46-50 hours for most o

21-26 hours for echino46-72 hours for Scedo

Amphotericin B: observeAmphotericin B: observe Other agents: observe 5Dermatophytes: observe 8

Echinocandins: lowest cocompact,Mi iMinimum

CLSI M

RODILUTION

mycetesysporium spp.

others

ocandin testingsporium spp./echinocandins

100% inhibition100% inhibition50% inhibition80% inhibition

oncentration resulting in small,, rounded hyphae

Eff ti C t ti (MEC)m Effective Concentration (MEC)

M38-A2 61

Ete

Not FDA-approved for

Etest MIC and broth mbl f t i lcomparable for trizole

than for amphotericin

Etest MIC values highA. flavus, S. prolificanvalues

J. Clin. Microbiol. 39

est

r filamentous fungi

microdilution data more( 90% t)s (>90% agreement)

B (>80% agreement)

her for S. apiospermum,s higher than reference

9: 1360-1367; 2001 62

CLINICAL“Factors related to…..apthan the MIC as predictothan the MIC as predicto

Clin. Infect. Dis. 2

“Very few correlations ofresponse have been rep

Curr. Fungal Infect. R

“ t t tl“…tests are currently moresistance or outliers bai it b k i tin vitro breakpoints or ep

Pfaller et al., Manual of Clin

L UTILITYppear to have more valueors of clinical outcome ”ors of clinical outcome.4: 235-247; 1997

f in vitro results with in vivoported for mold infections.” Rep. 3: 133-141; 2009

t f l f d t tiost useful for detectingsed on either assignedid i l i l t ff ”pidemiological cutoffs.”

nical Microbiology, tenth ed.63

THE Mostly an observationbiochemical may helpbiochemical may helpnote growth distributiA tif l tibilAntifungal susceptibilcontinues to be a worSee you at the Dells

ENDnal science (occasionalp with dermatophytes);p with dermatophytes);on and rate of growthlit t ti f ldlity testing for mouldsrk in progress

64

CRED

mold.phdoctorfungus.comasm.orgmycology.adelaide.edu.auuniprot.orgmikologi.comjbjs.orgels netels.netlabmed.ucsf.edupf.chiba-u.ac.jpgefor 4t comgefor.4t.comcladosporium.netmycota-crcc.mnhn.frhumanpath.compextension.umass.edu

DITS

dehs.umn.edubiotechnologie debiotechnologie.demadsci.orgbotit.botany.wisc.edupfdb netpfdb.netmy wife’s iPhonethunderhouse4-yuri.blogspot.cominfections.consultantlive.comlistal.commycobank.orgen.wikipedia.orgwww.proprofs.comcmpt.capath.umpc.edu

db b f itprgdb.cbm.fvg.itimages.mitrasites.com

65