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Review of the 2005 Mycology Review of the 2005 Mycology QAP ResultsQAP Results

Royal North Shore Hospital, SydneyRoyal North Shore Hospital, Sydney

Wednesday 28Wednesday 28thth June 2006June 2006

Dispatch 2005:2:7A,B,C Dispatch 2005:2:7A,B,C –– Maria Maria CarinoCarino, SDS Pathology, SDS PathologyDispatch 2005:4:7A,B,C Dispatch 2005:4:7A,B,C –– Kerry Weeks, Kerry Weeks, PaLMSPaLMSDispatch 2005:6:7A,B,C Dispatch 2005:6:7A,B,C –– OkchaOkcha Lee & Catherine Wu, ICPMRLee & Catherine Wu, ICPMRDispatch 2005:8:7A,B,C Dispatch 2005:8:7A,B,C –– MirkaMirka RejRej, , SymbionSymbion HealthHealth

RCPA QUALITY ASSURANCERCPA QUALITY ASSURANCE PROGRAMPROGRAM

DISPATCH 2: 2005 DISPATCH 2: 2005 MYCOLOGYMYCOLOGY

Maria Carino Maria Carino

Case 1Case 1Clinical:Clinical:

Culture A was isolated from a CAPD bag from aCulture A was isolated from a CAPD bag from a30 year old man with peritonitis.30 year old man with peritonitis.

Any ideas?Any ideas?

RhodotorulaRhodotorula mucilaginosamucilaginosa

Rhodotorula mucilaginosaRhodotorula mucilaginosaOn Sabourauds dextrose agar cultures are:• fast growing• smooth, glistering or dull• sometimes roughened or soft • moist to mucoid yeast-like in appearance• cream to pink, coral red, orange in colour.

Microscopic morphology:• spherical to elongate budding yeast-like cells or blastoconidia, 2.5-6.5 x 6.5-14.0 um in size.

India ink preparation:• small capsules present.

Culture on Cornmeal and Tween 80 agar:• budding blastoconidia only. No pseudohyphae areformed.

Physiological tests:• germ tube test is negative• hydrolysis of urea is positive• growth on cycloheximide medium is negative• growth at 37ºC is variable.

Fermentation reactions:• negative for Glucose; Sucrose; Lactose; Galactose;Maltose;Trehalose.

Rhodotorula mucilaginosa is the current name for the species formerly known as Rhodotorula rubra.

Final Results 2005Final Results 200593% of laboratories got it correct to the genus level60% of laboratories got it to correct to the species level

• Morphologically the isolate is very distinctive and a presumptive identification can be made.

• Identification systems. (ID32C) • Keeping up to date with name changes.

Case 2:Case 2:Culture B grew from a scalp scraping taken from Culture B grew from a scalp scraping taken from a 12 year old girl who recently arrived from a 12 year old girl who recently arrived from Sudan.Sudan.


Trichophyton Trichophyton soudanensesoudanense

Trichophyton Trichophyton soudanensesoudanenseOn Sabourauds dextrose agar cultures are:• slow growing• flat to folded, suede-like surface• often there is a broad fringe of submerged growth• surface and reverse pigment are characteristicallya deep apricot-orange in colour.

Microscopy morphology:• hyphae often show reflexive or right-angle branching• pyriform microconidia may occasionally be present• numerous chlamydoconidia are often found in older

cultures

Clinical significance:•T. soudanense is an anthropophilic funguswhich is a frequent cause of tinea capitis inAfrica.

• Invaded hairs show an endothrix infection butdo not fluoresce under Wood’s ultra-violet light.

• Distribution is mainly in Africa with occasional isolates from Europe, Brazil and U.S.A.

Final results 2005Final results 200587% of laboratories got it correct to the genus level57% of laboratories got it correct to the species level

• Follow a schematic key. Process of elimination• Selective media. If available• The absence or the occasional presence of microconidia

• Reflexing hyphae. Bamboo affect• Geographical information

Case 3:Case 3:Culture C was isolated from a subcutaneous Culture C was isolated from a subcutaneous abscess on the forearm in a 29 year old HIV abscess on the forearm in a 29 year old HIV positive patient.positive patient.


CladophialophoraCladophialophora carrioniicarrionii

Cladophialophora carrioniiCladophialophora carrioniiOn Sabourauds dextrose agar cultures are:• slow growing (reaching 3-4cm in dia. after 1 month• compact suede-like to downy surface• colonies are olivaceous-black in colour and have well defined margins

Microscopy morphology:• elongate conidiophores • producing branched acropetal chains of smooth-walled conidia, 1.5-3.0 x 2.0-7.0 um in size

• maximum growth temperature 35-37ºC

Clinical significance:• Cladophialophora carrionii is a recognised agent of chromoblastomycosis and it has been isolated from soils and fence posts made from Eucalyptus sp.

• Cases of chromoblastomycosis caused byC.carrionii are commonly found in Australia, Venezuela, Madagascar and South America.

Final Results 2005Final Results 200588% of laboratories got it correct to the genus level59% of laboratories got it correct to the species level

• Cladosporium carrionii has been transferred to theCladophialophora genus

• Cladophialophora can be distinguished from Cladosporium by the absence or poorly differentiatedconidiophores, by unpigmented conidial scars and by being unable to liquify gelatin.

• Cladosporium species have occasionally been found as opportunists in humans, having a wide clinical spectrum.

• In contrast, each Cladophialophora species provokes a mycoses which is characteristic for that species. Cladophialophora is related to the black yeast

• Keeping up to date with name changes

• Following a schematic key

• Clinical information

AcknowledgementsAcknowledgements

VoulaVoula Henry Henry -- BiomerieuxBiomerieuxDr Pitman Dr Pitman -- SDS PathologistSDS PathologistDr fungus website Dr fungus website -- ImagesImagesQuality Assurance ProgramQuality Assurance Program

Review of Mycology QAP Review of Mycology QAP Results for 2005:4: 7A, B, CResults for 2005:4: 7A, B, C

Kerry WeeksKerry WeeksMycology LaboratoryMycology Laboratory

PaLMS, NSCCAHSPaLMS, NSCCAHS

By

Item 4: 7AItem 4: 7A

Clinical historyClinical history

A 60 y.o. farmer presented with swelling A 60 y.o. farmer presented with swelling & a dark, blood& a dark, blood--tinged discharge from the tinged discharge from the nose. A biopsy taken from the paranasal nose. A biopsy taken from the paranasal sinus grew culture 4: 7Asinus grew culture 4: 7A

4: 7A Conidiobolus coronatus4: 7A Conidiobolus coronatus

This fungus didn’t survive freeze drying This fungus didn’t survive freeze drying by QAP = most labs failed to isolate thisby QAP = most labs failed to isolate thisfungusfungus

ConidiobolusConidiobolus spp.spp.Phylum: Zygomycota, Order: EntomophthoralesPhylum: Zygomycota, Order: Entomophthorales

27 species 27 species Most common sp: Most common sp: C. coronatus,C. coronatus,

C. incongruusC. incongruus & & C. lampraugesC. lamprauges

ConidiobolusConidiobolus spp.spp.Rapid growth of waxy colonies (routine agar), Rapid growth of waxy colonies (routine agar), wide hyphae, coenocytic or irregular septatewide hyphae, coenocytic or irregular septateSimple conidiophores single forcibly Simple conidiophores single forcibly discharged terminal discharged terminal conidium papilla scar remains. conidium papilla scar remains. BallistosporesBallistospores--growth on growth on petripetri dish liddish lidZygospores intercalary, Zygospores intercalary, thick walled, hyaline, w/o thick walled, hyaline, w/o beaksbeaks

Photo from “Mycology Online”

Differentiating between Differentiating between ConidiobolusConidiobolus speciesspecies

NoNo1212--42 um, 42 um, single tapering single tapering basal papillabasal papilla

YesYes< 30 mm< 30 mmC. C. incongruusincongruus

NoNo1313--22 um/22 um/spherical, thin spherical, thin walled, 1walled, 1--many papilla many papilla after liberationafter liberation

NoNo< 30 mm< 30 mmC. C. lampraugeslamprauges

Yes (in older Yes (in older cultures)cultures)

40 um/ 40 um/ prominent prominent papillate basepapillate base

YesYes> 40 mm> 40 mmConidiobolus Conidiobolus coronatuscoronatus

Villose Villose (hair(hair--like) like) conidiaconidia

Primary Primary conidia conidia size/shapesize/shape

Forcibly Forcibly discharged discharged conidia w or conidia w or w/o w/o secondary secondary conidiaconidia

Colony (PDA Colony (PDA 3 days)3 days)

SpeciesSpecies

ConidiobolusConidiobolus coronatuscoronatus

Photo from “Mycology Online”RNSH photo (high magnification)

Primary conidia of C. coronatus

PapillaMultiplicative conidia

C. C. coronatuscoronatusC. coronatus -mature,

spherical villose conidia (hair-like protrusions),

& papillae (site of former attachment to conidiophore)


Papillae

Villose conidia

Differentiating between Differentiating between BasidiobolusBasidiobolus & & ConidiobolusConidiobolus

NoNo-- C. coronatusC. coronatusYesYes-- C. incongruusC. incongruus

C. lampraugesC. lamprauges

YesYesZygosporesZygospores

NoNo(Yes 2 beaks in (Yes 2 beaks in C. lampraugesC. lamprauges))

YesYesZygospores with Zygospores with conjugation beaksconjugation beaks

NoNoYesYesSporangiophores Sporangiophores with swollen with swollen apicesapices

YesYesNoNoEjected Ejected sporangioles with sporangioles with papillapapilla

Conidiobolus spp.Conidiobolus spp.Basidiobolus spp.Basidiobolus spp.Microscopic Microscopic featuresfeatures

BasidiobolusBasidiobolus ranarumranarum

Photos from “Mycology Online”

B. ranarum- beaked zygospores

Discharged globose conidia & conidiophores

RhinoRhino--facial infections caused by facial infections caused by ConidiobolusConidiobolus

Photos from “Mycology Online”

Infections mainly in tropics- eg. Africa, Asia, Central America, India. Infection not evident until swelling/ deformity occurs.

EpidemiologyEpidemiologyFound in soil & decaying debris (found on bananas, Found in soil & decaying debris (found on bananas, rotten wood & a fruit warehouse etc). Ubiquitousrotten wood & a fruit warehouse etc). Ubiquitous--wide range of areaswide range of areas-- AlsaksaAlsaksa, temperate to tropical , temperate to tropical regions (regions (egeg. PNG soil). PNG soil)Insect pathogen (mosquitoes, termites, aphids)Insect pathogen (mosquitoes, termites, aphids)Infects man (80% cases are males) (typically) Infects man (80% cases are males) (typically) nasal mucosa & paranasal mucosa & para--nasal sinus, (sometimes) nasal sinus, (sometimes) subcutaneous, (rarely) pulmonary & pericardial subcutaneous, (rarely) pulmonary & pericardial infections firm, subcutaneous nodules/polypsinfections firm, subcutaneous nodules/polypsImmuneImmune--competent & immunecompetent & immune--suppressedsuppressed-- emerging emerging opportunist. Occur sporadically but rarely.opportunist. Occur sporadically but rarely.

Route: inhalationRoute: inhalationInfects animalsInfects animals-- horses, sheep, deer, dogs & horses, sheep, deer, dogs & chimpanzees.chimpanzees.Hard to treatHard to treat-- high MIC’s high MIC’s –– ?itraconazole, Amp B + ?itraconazole, Amp B + terbinafine, flucon, keto, iodides, plastic surgeryterbinafine, flucon, keto, iodides, plastic surgery

Item 4: 7BItem 4: 7B


This culture was isolated from sputum This culture was isolated from sputum collected from a lung transplant patient collected from a lung transplant patient who presented with a chronic coughwho presented with a chronic cough

4:7B Geotrichum candidum4:7B Geotrichum candidumTeleomorph: Teleomorph: Galactomyces candidumGalactomyces candidumDescription:Description:

Colonies, fast growing (routine agar), white, rough Colonies, fast growing (routine agar), white, rough texture, hairy, creeping, mostly submerged, dry. texture, hairy, creeping, mostly submerged, dry. Fruity odour.Fruity odour.Hyaline hyphae, aerial Hyaline hyphae, aerial hyphaehyphae fragment forming fragment forming arthroconidia (cylindrical, barrel shaped or arthroconidia (cylindrical, barrel shaped or ellipsoidal). ellipsoidal). No blastospores produced. No blastospores produced. No capsules. No capsules. [[TrichosporonTrichosporon spp. produce spp. produce blastosporesblastospores along along pseudohyphaepseudohyphae]]

Differential features of G. candidum:Differential features of G. candidum:Hyphae 12 um wide with dichotomous branching at Hyphae 12 um wide with dichotomous branching at colony margin.colony margin.Assimilation: xylose POS, cellobiose NEGAssimilation: xylose POS, cellobiose NEGNG at 40C.NG at 40C.

Colonial morphologyColonial morphology

G. candidumSabs agar

(28C 4 days)

MicroscopyMicroscopy


Geotrichum candidum- Course true hyphae (no pseudohyphae) that segment into arthoconidia. main & lateral branches with arthroconidia. Dichotomously branching.

Similar species to Similar species to GeotrichumGeotrichumTrichosporonTrichosporon spsp-- blastoconidiablastoconidia & & pseudohyphaepseudohyphae (not seen in (not seen in Geotrichum Geotrichum has has true hyphae)true hyphae)ScytalidiumScytalidium spsp-- dematiacousdematiacous arthroconidiaarthroconidia(hyaline (hyaline arthosarthos in in GeotrichumGeotrichum))ArthrographisArthrographis & & OidiodendronOidiodendron spsp-- have have condiophorescondiophores (which (which GeotrichumGeotrichum lacks)lacks)MalbrancheaMalbranchea spsp-- arthroconidaarthroconida release by release by disjunctordisjunctor cells (cells (GeotrichumGeotrichum produces produces arthrosarthrosby fission)by fission)Coccidioides Coccidioides spsp-- arthrosarthros alternate with alternate with empty cells (empty cells (GeotrichumGeotrichum-- consecutive)consecutive)

Profile number:

3200 3100 13 Xylose assimilation POS Cellobiose “ NEG

Result: Geotrichum candidum

Biomerieux ID 32c result

Differentiating between Differentiating between GeotrichumGeotrichum speciesspecies

PosPosPosPosVVGrowth at 37CGrowth at 37C

PosPosNegNegNegNegArbutinArbutin

NegNegNegNegPosPosVitamin freeVitamin free

PosPosNegNegNegNegSalicinSalicin

NegNegNegNegPosPosDD--xylosexylose

PosPosNegNegNegNegCellobioseCellobiose

G. clavatumG. clavatumG. capitaumG. capitaumG. candidumG. candidum

[Ref: deHoog, Guarro et al. “Atlas of Clinical Fungi” p. 227]

4:7:B QAP results4:7:B QAP results

Geotrichum candidum

Geotrichum sp. G. pennicill- atum

G. captatum G. klebahni Candida kefyr

Unable to ID

82 labs (73.2%)

16 (14.3%)

7 (6.3%)

1 (0.9%)

1 (0.9%)

1 (0.9%)

4 (3.6%)

96% labs correct to genus level73% correct to species level

Results obtained from Microbiology QAP

EpidemiologyEpidemiologyG. G. candidumcandidum found worldwide (tundra, temperate, tropical found worldwide (tundra, temperate, tropical climates) in soil, marine habitats, water, air, sewage, plants,,climates) in soil, marine habitats, water, air, sewage, plants,,freq dairy, cereals (freq dairy, cereals (eg.frozeneg.frozen fruit cake, bread), tomatoes, fruit cake, bread), tomatoes, animals skin & in droppings etc.animals skin & in droppings etc.Found in gas oil in Germany, paper pulp factory in France, Found in gas oil in Germany, paper pulp factory in France, isolated from irradiated soil.isolated from irradiated soil.Normal human floraNormal human flora-- isolated from faeces & sputumisolated from faeces & sputumOpportunistic infections in immune compromised Opportunistic infections in immune compromised (“Geotrichosis”)(“Geotrichosis”)

EndogenousEndogenous-- oral, bronchial, systemic & oral, bronchial, systemic & exogenousexogenous-- skin, allergic skin, allergic & trauma.& trauma.Disseminated infectionDisseminated infection-- poor prognosis.poor prognosis.Route: Ingestion, inhalation or traumaRoute: Ingestion, inhalation or trauma

?Also may cause environmental damage?Also may cause environmental damage-- eg. destroy dataeg. destroy data--storing storing polycarbonate resin found in CDs! polycarbonate resin found in CDs! (disc becomes (disc becomes transparent). Also sig cause of citrus fruit rot.transparent). Also sig cause of citrus fruit rot.

Item 4: 7CItem 4: 7C


This culture was isolated from a corneal This culture was isolated from a corneal scarping collected from a 12 y.o. with an scarping collected from a 12 y.o. with an eye injuryeye injury

4:7:C Candida kefyr4:7:C Candida kefyrSynonym: Synonym: Candida pseudotropicalisCandida pseudotropicalis

Teleomorph: Kluyveromyces marxianusTeleomorph: Kluyveromyces marxianus

ColonyColony-- white, creamwhite, cream-- coloured, smooth texturecoloured, smooth textureMicroscopy (CMA tween 80) shows elongated Microscopy (CMA tween 80) shows elongated blastospores, parallel ”logs in a stream”, curved along blastospores, parallel ”logs in a stream”, curved along pseudohyphaepseudohyphae

Differential features of Differential features of C. kefyrC. kefyr::Raffinose POS, 2Raffinose POS, 2--keto gluconate (2KG) NEG, keto gluconate (2KG) NEG, ethylamine POS, growth on 0.1% ethylamine POS, growth on 0.1% cyclohexcyclohexG at 37C, G w/o niacin.G at 37C, G w/o niacin.

Candida kefyrcolony white, cream coloured, butyrous(buttery) ie. typical yeast morphology.

(Sabs 28C 3 days)

Colonial morphology

Candida kefyr

elongated blastoconidia

Photo from Boekhout et al. CD-ROM “Yeasts of the World”Elongated

blastoconidia

Pseudohyphae

Microscopy

Kluyveromyces marxianis

(Teleomorph of C. kefyr)asciwith ascospores

Photo from Boekhout et al. CD-ROM “Yeasts of the World”ascospore

asci

BiomerieuxBiomerieux ID 32c resultID 32c result

Profile number:

7 6 2 0 2 1 0 0 3 1 Raffinose assimilation POS 2 KG “ Neg

Result: C. kefyr 99.9% T 0.88

4:7:C QAP results4:7:C QAP results

Candidakefyr

Candidasp.

Candida(notalbicans)

Candidatropicalis

Geotrichumcandidum

Unable toID

102 labs(90.3%)

6(5.3%)

1(0.9%)

1(0.9%)

1 (0.9%)

2(1.8%)

97% labs correct to genus level90% correct to species level

Results obtained from Microbiology QAP

EpidemiologyEpidemiology

Found worldwide in humans, other Found worldwide in humans, other mammals & dairy foodsmammals & dairy foodsEmerging fungal pathogen.Emerging fungal pathogen.InfectionsInfections-- occasionally superficial occasionally superficial candidiasis. Reported cases of pulmonary candidiasis. Reported cases of pulmonary infections, disseminated infection, infections, disseminated infection, oesophagitisoesophagitis in immune compromised. in immune compromised.

A Review of 2005 Mycology QAP A Review of 2005 Mycology QAP ((despatchdespatch 6)6)

OkchaOkcha Lee Lee Catherine WuCatherine Wu

Mycology Unit, CIDMLS, ICPMR Mycology Unit, CIDMLS, ICPMR Westmead hospitalWestmead hospital

Item 2005:6:7AItem 2005:6:7A

Clinical notes:Clinical notes:

Culture was isolated from fingernail scrapings taken Culture was isolated from fingernail scrapings taken from a 23 yearfrom a 23 year--old African man with suspected old African man with suspected onychomycosis. Direct microscopy showed the onychomycosis. Direct microscopy showed the presence of septate hyphae.presence of septate hyphae.

ColonyColony

Initially whitish, finally becoming dark grayInitially whitish, finally becoming dark graySpreading, fastSpreading, fast


Hyphae: melanized or hyaline, falling intoHyphae: melanized or hyaline, falling intoathroconidiaathroconidia

Chlamydospore: conidia single or in chains, darkChlamydospore: conidia single or in chains, darkbrown, thick walled, swollen up to 7brown, thick walled, swollen up to 7µµm widem wide

IdentificationIdentificationScytalidiumScytalidium sppspp

PathogenicityPathogenicityOnychomycosisOnychomycosisDermatophytosis like infectionDermatophytosis like infection

EcologyEcologySoil fungus or plant pathogen in tropical and subtropical Soil fungus or plant pathogen in tropical and subtropical areasareas

* 60% correct to genus level* 60% correct to genus level

Item 2005:6:7BItem 2005:6:7B


Culture was isolated from a wound swab taken from Culture was isolated from a wound swab taken from a 30 yeara 30 year--old man recovering from severe burns.old man recovering from severe burns.

Colony Colony

Bright yellow with dark yellow reverse.Bright yellow with dark yellow reverse.



Hyphae: sparsely Hyphae: sparsely septatedseptatedSporangiophores:Sporangiophores:Sporangia: <100Sporangia: <100µµmmColumella: sphericalColumella: sphericalSporangiospores: variable in size and shapeSporangiospores: variable in size and shapeChlamydospores: presentChlamydospores: presentRhizoids: presentRhizoids: presentGrowth temperature: growth at 37Growth temperature: growth at 37ººCC

no growth at 42 no growth at 42 ººCC

Comparison between Comparison between Rhizomucor variabilisRhizomucor variabilisand and Mucor hiemalisMucor hiemalis

Ellipsoidal, spherical, up to Ellipsoidal, spherical, up to 38 38 ×× 30 um30 um

Spherical, ellipsoidal to cylindrical Spherical, ellipsoidal to cylindrical in various shape, up to 40in various shape, up to 40µµm m widewide

columellacolumella

Up to 80um diameterUp to 80um diameterSubsphericalSubspherical, up to 100um , up to 100um diameterdiameter

sporangiasporangia

Up to 15mm long, 14um Up to 15mm long, 14um wide, branchedwide, branched

Up to 2mm long, 9Up to 2mm long, 9--23um wide,23um wide,branchedbranched

SporangioSporangio--phoresphores

Whitish to Whitish to ochraceousochraceousGreyishGreyish--ochraceousochraceouscolonycolony

Mucor hiemalisMucor hiemalisRhizomucor variabilisRhizomucor variabilis

Comparison continuedComparison continued

--abundantabundantrhizoidsrhizoids

3030ººCC3838ººCCMaximum Maximum growth Tmgrowth Tm

--abundantabundantchlamydoschlamydosporespores

ellipsoidal, smoothellipsoidal, smooth--walledwalled66--9 9 ×× 33--5 um5 um

Hyaline, Hyaline, smoothwalledsmoothwalled, very , very variable 3variable 3--11 11 ×× 22--7 um7 um

sporangiossporangiosporespores


Molecular Molecular

18S rDNA

(SSU)

ITS 1 5.8S rDNA

ITS 2 28S rDNA

(LSS)

D1 D2

ITS1 ITS3

ITS2 ITS4

LR1

LR16

Schematic diagram of ribosomal gene cluster of fungi

Comparison of sequencing results between Comparison of sequencing results between Rhizomucor variabilisRhizomucor variabilis and and Mucor hiemalisMucor hiemalis

97% 642/658 97% 642/658 bpbp))99% (638/644 99% (638/644 bpbp))LSU D1/D2 region (21LSU D1/D2 region (21--664 664 bpbp))

98% (484/488 98% (484/488 bpbp))

98% (547/557 98% (547/557 bpbp))

99% (487/488 99% (487/488 bpbp))

100% 557/557 100% 557/557 bpbp))

SSU Base pairs (37SSU Base pairs (37--524 524 bpbp))

Base pairs (556Base pairs (556--1112 1112 bpbp))

92% (452/487 92% (452/487 bpbp))not in databasenot in databaseITS 1 and ITS 2 regionITS 1 and ITS 2 region


Relationship between Relationship between Rhizomucor variabilisRhizomucor variabilis and and Mucor hiemalisMucor hiemalis

Rhizomucor variabilisRhizomucor variabilis may concern degenerate cultures of may concern degenerate cultures of Mucor Mucor hiemalishiemalis

Phylogenetically it proved to be very close to Phylogenetically it proved to be very close to Mucor hiemalisMucor hiemalis

IdentificationIdentification

Rhizomucor variabilisRhizomucor variabilis

* 63% answered to * 63% answered to Mucor sppMucor spp and 3.6% to and 3.6% to Rhizomucor variabilisRhizomucor variabilis

Item 2005:6:7CItem 2005:6:7C


Culture was isolated from a lung biopsy taken from a Culture was isolated from a lung biopsy taken from a 15 year15 year--old girl with acute lymphocytic leukaemia.old girl with acute lymphocytic leukaemia.

ColonyColony

Usually green, creamUsually green, cream--buff or honeybuff or honey--yellow where yellow where cleistotheciacleistothecia formform

Reverse may be olive to purpleReverse may be olive to purple--brownbrown


Microscopy Microscopy

Conidiophores: short (< 250um)Conidiophores: short (< 250um)Conidial heads: short columnar (< 80um)Conidial heads: short columnar (< 80um)ConidiogenousConidiogenous cells: cells: biseriatebiseriate, , metulaemetulae and and phialidephialideConidia: globose (3Conidia: globose (3--3.5 um in 3.5 um in diamdiam))Teleomorph: Teleomorph: EmericellaEmericella nidulansnidulans cleistotheciacleistothecia with with reddish ascospores are often surrounded by Hreddish ascospores are often surrounded by Hüüllelle cells cells (up to 25um in (up to 25um in diamdiam))

IdentificationIdentificationAspergillus nidulansAspergillus nidulans

PathogenicityPathogenicityOpportunistic infectionOpportunistic infection

EcologyEcologySoil fungusSoil fungusProminent colonizer of decomposing plant debrisProminent colonizer of decomposing plant debris

* 94% correct to genus level and 74% to species level* 94% correct to genus level and 74% to species level

RCPA Quality Assurance RCPA Quality Assurance Programs Programs

A Review of the 2005 Mycology A Review of the 2005 Mycology QAPQAP

Mycology Items 2005:8:7 A,B,CMycology Items 2005:8:7 A,B,CMirka RejMirka RejMicrobiology DepartmentMicrobiology DepartmentSymbion and Laverty Pathology North RydeSymbion and Laverty Pathology North Ryde

Item 2005:8:7AItem 2005:8:7A

Clinical Notes: Clinical Notes: Culture was isolated from a knee aspirate Culture was isolated from a knee aspirate taken from a ten year old leukemic boy taken from a ten year old leukemic boy with osteomyelitis of the right knee. with osteomyelitis of the right knee.

Identification: Identification: MacromorphologyMacromorphology

•Young colony is cottony or moist (yeasty) and light grey to black.

•Mature colony becomes dark grey to black and may develop white mycelial tufts with age.

•Reverse is grey to black.

Identification: Identification: MicromorphologyMicromorphology

•Septate hyphae

•Unbrachedconidiogenous cells (annellides).

•Swelling of the basal portion of the annellides.

•“Swan necks”

•Conidia are one celled, smooth and ovoid with a slightly narrowed, truncated base.

•Conidigenous cells locally aggregated into small brushes.

•Sympotial conidia

Clinical importance of Clinical importance of ScedosporiumScedosporium prolificansprolificans

osteomyelitisosteomyelitisseptic arthritisseptic arthritisonychomycosisonychomycosisotomycosisotomycosisendophthalmitisendophthalmitissinusitis sinusitis

gangrenous skin gangrenous skin ulcersulcerspulmonary infectionspulmonary infectionsperitonitisperitonitissystemic infections systemic infections (may be isolated (may be isolated from blood cultures)from blood cultures)

Documented as a cause of:

Clinical importance of Clinical importance of ScedosporiumScedosporium prolificansprolificans

ScedosporiumScedosporium prolificansprolificans in nature occurs in soil in nature occurs in soil and is traumatically introduced into humans by and is traumatically introduced into humans by thorns or splinters. thorns or splinters. In the body it has a predilection for cartilage and In the body it has a predilection for cartilage and joint areas. joint areas. In immunocompromised patients or In immunocompromised patients or transplantation patients fatal dissemination may transplantation patients fatal dissemination may occur. occur. A fatal case of endocarditis was reported.A fatal case of endocarditis was reported.

Isolates are often resistant to antifungal agents.Isolates are often resistant to antifungal agents.

RCPA Quality Assurance RCPA Quality Assurance ProgramsPrograms

84% correct to genus level.

76% correct to species level

0 10 20 30 40 50 60 70 80 90

Scedosporium prolificans

Scedosporium apiospermum

Scedosporuim species

Scedosporium inflatum

Phialophora verrucosa

Phialophora species

Phialophora richardsiae

Fonsecaea species

Exophiala jeanselmei

Wangiella dermatidis

Sporotrichum schenckii

Sporothrix schenckii

Pseudallescheria boydii

Candida guillermondii

Yeast species

Unable to identify

Differences between Differences between S. S. apiospermumapiospermum and S. and S.

prolificansprolificansS. apiospermum S. prolificans

growth on mediacontrainingcycloheximide

+ -

annellides cylindrical flask shaped, swollenbasal portion

annellidic rings difficult to see long thin appendages(swan neck)

formation ofclastothecia

+ (P.boydi) -

synnemata + -cultures white cottony

mycelium later turnsgrey to brown

first moist grey to blacklater dark grey, white

tufts in the centre.growth at 45 degrees - +

•Bottle shaped annellides

•Annellides with parrallel sides may also be seen.

Scedosporium prolificans

Item 2005:8:7BItem 2005:8:7B

Clinical Notes:Clinical Notes:Culture was isolated from the fingernail Culture was isolated from the fingernail scraping taken from a 50 year old woman scraping taken from a 50 year old woman with chronic paronychia. with chronic paronychia.


•White to cream coloured, smooth, glabrous yeast-like colonies.


•Budding yeast like cells.

• cells spherical to broadly ellipsoidal.

•2.0-4.0 x 3.0-6.5 µm.

IdentificationIdentification

•For the identification of germ tube negative yeasts, morphological (Dalmau plate culture) physiological and biochemical tests are essential.

•Reliable commercially available yeast identification kits are the API 20C AUX, ATB32C, Vitek systems.

Microscopy Morphology on Microscopy Morphology on CornmealCornmeal--TweenTween 80 agar80 agar

•Fairly short, fine pseudohyphae.

•Clusters of blastoconidia at septa.

Identification:Identification:

Physiological tests:Physiological tests:

Germ Tube test: Germ Tube test: Hydrolysis of Urea: Hydrolysis of Urea: Growth on Cycloheximide:Growth on Cycloheximide:Growth at 37C:Growth at 37C:

NegativeNegativeNegativeNegativePositivePositivePositivePositive

Identification:Identification:Fermentation reactions:Fermentation reactions:

Glucose:Glucose:Lactose:Lactose:Sucrose:Sucrose:Galactose:Galactose:Trehalose:Trehalose:Maltose:Maltose:

++--++VariableVariable----

Differential DiagnosisDifferential Diagnosis

DD--glucosamineglucosamineDD--arabinosearabinosecellobiosecellobioseinulininulinerythritolerythritolLL--arabinitolarabinitolDD--glucuronateglucuronate

++++++++--++--

The species can be recognised by the following growth characters:

Clinical Importance of Candida Clinical Importance of Candida guilliermondiiguilliermondii

is found from normal skin, in sea water, is found from normal skin, in sea water, faeces of animals, fig wasps, buttermilk, faeces of animals, fig wasps, buttermilk, leather, fish and beer. leather, fish and beer. has been isolated from numerous human has been isolated from numerous human infections. infections.

Clinical ImportanceClinical ImportanceYeasts are considered opportunistic Yeasts are considered opportunistic

pathogens causing disease in patients:pathogens causing disease in patients:with a breakdown in the body’s with a breakdown in the body’s immune system immune system on prolonged treatment with antibioticson prolonged treatment with antibioticson corticosteroids or cytotoxic drugson corticosteroids or cytotoxic drugswith intravascular catheterswith intravascular catheterswith diabetes mellituswith diabetes mellitusknown to be intravenous drug abusersknown to be intravenous drug abusers


0 20 40 60 80 100 120

Candida guilliermondii

Candida species

Candida famata

Candida glabrata

Unable to identify

No growth

96% correct to genus level. 89% correct to species level.

Item 2005:8:7CItem 2005:8:7C

Clinical Notes:Clinical Notes:A 40 year old market gardener presented A 40 year old market gardener presented with a tender subcutaneous nodule on the with a tender subcutaneous nodule on the leg. Fluid aspirated from the nodule grew leg. Fluid aspirated from the nodule grew Culture C. Culture C.


•Initially smooth, black, mucoid and yeast like colonies.

•Revers is black

•Grows slower or not at all at 37C


•Colonies become raised and develop tufts of aerial mycelium with age.

•Often dome shaped and suede like in texture.


•Yeast like cells

•Torulose hyphae

Identification: Identification: MicromorphologyMicromorphology•The conidiogenous cells are slender annellides.

•Tips of annellides are narrow, elongated and rocket shaped.

•Conidia gather in clusters at the end, sides of conidiophore and at points along the hyphae.

•Inflated germinating cells.

•Budding yeast like cells.

•Conidia 1-3 x 2-5 µm in size, hyaline, smooth, thin walled and broadly ellipsoidal.

Clinical Importance of Clinical Importance of Exophiala jeanselmeiExophiala jeanselmei

Mainly associated with Mainly associated with phaeohyphomycoticphaeohyphomycotic cysts. cysts. The fungus has also been documented The fungus has also been documented as a cause of blackas a cause of black--grained mycetoma. grained mycetoma. Has been reported from a small number Has been reported from a small number of cases of chromoblastomycosis.of cases of chromoblastomycosis.


76% correct to genus level

52% correct to species level

0 10 20 30 40 50 60 70


Exophiala species

Exophila dermatitidis

Exophila spinifera

Exophila castellanii

Wangiella dermatitidis

Fonsecaea species

Fonsecaea dermatidis

Fonsecaes pedrosoi

Philophora verrucoas

Phialophora species

Phialophora gougerotii

Hortaea werneckii

Homonema deratioides

Sporothrix schenckii

Cladosporium species

Cladosporium carrionii

Yeast species

Unable to identify

Differences Differences betweembetweem Exophiala Exophiala jeanselmei and jeanselmei and WangiellaWangielladermatitidis (Exophiala dermatitidis (Exophiala

dermatitidis)dermatitidis)Exophialajeanselmei

Wangielladermatitidis

growth at 42degrees

- +

conidiogenous cell annellidetapering extensions

to the tip.

phialide.do not showexidence ofextension.


Wangiella dermititidis

ReferencesReferencesBooks:Books:

Ellis, D., Davis, S., Ellis, D., Davis, S., AlexiouAlexiou, H., Pfeiffer, T., , H., Pfeiffer, T., ManatakisManatakis, Z. (1992). ", Z. (1992). "Descriptions of Descriptions of MedicatMedicatQAP fungiQAP fungi". Mycology Unit, Adelaide Children's Hospital. ". Mycology Unit, Adelaide Children's Hospital. Hocking, A., Hocking, A., WoodgyerWoodgyer, A. , A. "Clinically Significant Non"Clinically Significant Non--Dermatophyte Fungi a Practical Dermatophyte Fungi a Practical Guide to IdentificationGuide to Identification". AIMS National Scientific Meeting 2001, Melbourne". AIMS National Scientific Meeting 2001, MelbourneHoogHoog G., G., GuarroGuarro, J. (1995). ", J. (1995). "Atlas of Clinical FungiAtlas of Clinical Fungi" " CentraalbureauCentraalbureau voorvoorSchimmelculturesSchimmelcultures, The Netherlands, and , The Netherlands, and UniversitatUniversitat RoviraRovira i i VirgiliVirgili, Spain], Spain]LaroneLarone, D. (1995). , D. (1995). "Medically Important Fungi" 3rd Edition"Medically Important Fungi" 3rd Edition. American Society for . American Society for Microbiology, Washington. Microbiology, Washington. MurryMurry, P., Baron, E., , P., Baron, E., PfallerPfaller, M., , M., TenoverTenover, F., , F., YolkenYolken, R. (1995). ", R. (1995). "Manual of Clinical Manual of Clinical Microbiology" 6th EditionMicrobiology" 6th Edition. American Society for Microbiology, Washington. . American Society for Microbiology, Washington.

Websites:Websites:www.scielo.brwww.scielo.brmicrobiology.mtsinai.on.ca/mig/defungi/index2.shtmlmicrobiology.mtsinai.on.ca/mig/defungi/index2.shtmlwww.doctorfungus.org/thefungi/Wangiella.htmwww.doctorfungus.org/thefungi/Wangiella.htmwww.mycology.adelaide.edu.au/Fungal_Descriptions/Hyphomycetes_(dwww.mycology.adelaide.edu.au/Fungal_Descriptions/Hyphomycetes_(dematiaceous)/Exoematiaceous)/Exophiala/jeanselmei.htmlphiala/jeanselmei.htmlwww.ukneqasmicro.org.uk/cg.htmlwww.ukneqasmicro.org.uk/cg.htmlhttp://www.reviberoammicol.com/photo_gallery/Candida/guilliermonhttp://www.reviberoammicol.com/photo_gallery/Candida/guilliermondii/dii/onychomycosis.com/images/Table%201.htmonychomycosis.com/images/Table%201.htm

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