laboratory identification of di hi f idimorphic...

Laboratory Identification of Di hi F iDimorphic Fungi

Di hi f iDimorphic fungi

Thermally dimorphic fungi Thermally dimorphic fungi

Environment/Routine culture media (SDA) o e t/ out e cu tu e ed a (S )25-30oC ---Mold form

conversionconversion

Tissue/Enriched media (BHI) 35 -37oC---Yeast form

Dimorphic Fungi

Di hi f iDimorphic fungi

Histoplasma capsulatumCoccidioides immitisBlastomyces dermatitidisParacoccidioides brasiliensisPenicillium marneffeiu a

1 2 3

Yeast form

(37 0C)4 54 5

1=A ; 2=C

A C

Mycelial form

(25 0C)

3=D ;4=F

5= E

(25 0C)

D E F

Conversion of dimorphic fungi in culture

SDA (antibacterial, clycloheximide) ( , y )25-300C, incubate take 4-8 weeks or more Mycelial formI l t li l th t BHIInoculate mycelial growth onto BHIagar in a screw-cap tube incubate at 35-370C, preferably in CO2 35 37 C, preferably in CO2 Yeast form

It be necessary several serial transfers to attain complete conversion to yeast phase phase

Conversion of dimorphic fungi in culture (cont.)

fWet preparation (LPCB) of yeastlike areah b h dl dThese organisms MUST be handled

in a biological safety cabinet

S th i h kiiSporothrix schenckii

Sporotrichosis Subcutaneous, pulmonary sporotricosisS d ld idScattered worldwide

C t t i iCutaneous sporotricosis

S th i h kiiSporothrix schenckii

Yeast formRound, oval and fusiform budding yeast cells of various sizes 2 3 various sizes 2-3 x 3-10 µmCigar-bodiedCigar-bodied

370C on BHI agar

S th i h kiiSporothrix schenckii

M li l fMycelial formHyphae are narrow septate narrow, septate, with slender conidiophores prising at right angleC idi f i Conidia formig a “rosette-like”

S th i h kiiSporothrix schenckii

Laboratory diagnosisDirect examination

Cigar shaped round form of Section from a fixed cutaneous lesion showing round positive Cigar-shaped, round form of

S.schenckii (GMS-H&E)lesion showing round positive budding yeast-like cells. (PAS)

S th i h kiiSporothrix schenckii

Laboratory diagnosisCultureConversion to yeast phase

Hi t l l tHistoplasma capsulatum

Histoplasmosis (systemic mycoses)H.capsulatum var. capsulatum

North America, Mexico

H.capsulatum var. duboisiiTropical areas of Africa

Hi t l l tHistoplasma capsulatum

Yeast formRound and oval budding yeastThe yeast phase i i hibit d b is inhibited by cycloheximide

GMS stain of lymph node showing blastoconidia of H.capsulatum

Hi t l l tHistoplasma capsulatum

Mycelial formHyaline septate hyphaePear-shaped

i idimicroconidiaTuberculate

idi

Tuberculate macroconidia of H.capsulatum (LPC)

macroconidia

Hi t l l tHistoplasma capsulatumLaboratory diagnosisLaboratory diagnosis

Direct examination

Bone marrow aspirate with macrophage KOH wet mount of sputum o a o a p a a op agcontaining numerous blastoconidia of H.capsulatum

showing blastoconidia of H.capsulatum

Hi t l l tHistoplasma capsulatumLaboratory diagnosisLaboratory diagnosis

CultureTh i d t i ll i The organism does not survive well in clinical specimen

Conversion to yeast phaseConversion to yeast phaseBHI +cysteine , at 37CSepedonium sp., Chrysosporium sp.p p , y p p

Sepedonium speciesSepedonium species

K l i i i Ch i Koloninya mirip Chrysosporium spp, dan tidak dapat dibedakan secara visual (kasat mata )visual (kasat mata )Mikroskopik : large,spherical,bluntly spiked macroconidia, yang mirip p , y g pdengan H.capsulatumCiri-ciri koloni Spedonium sp. :

Tumbuh lebih cepatDihambat oleh cycloheximideTidak dapat dikonversi jadi yeast phaseTidak dapat dikonversi jadi yeast phase

fIn cases of suspected histoplasmosis, nucleic acid probe or exoantigentesting may be performed on testing may be performed on mycelial extracts of young colonies to establish a rapid identificationestablish a rapid identification.

Hi t l l tHistoplasma capsulatum

Laboratory diagnosisSerodiagnosis

Complement fixation testImmunodiffusion (ID) test with H,M antigen

Lines of identify for exoantigens in exoantigens in microimmunodiffusion plate

C idi id i itiCoccidioides immitis

Coccidioidomycosis (systemic mycoses)Asymptomatic pulmonary infection 60% in normal hostProgressive pulmonary infection in immunocompromised patients

C idi id i itiCoccidioides immitis

Yeast formIn tissues or body fluids, C.immitis exists as spherules (10 80 um ) (10-80 um ), which contain endospores (2-5 KOH wet mount of sputum endospores (2 5 µm)

pshowing spherule and endospores of C.immitis

C idi id i itiCoccidioides immitis

Mycelial formHyaline septate hyphaeBarrel-shaped

th idi arthroconidia that alternate with empty with empty cells(disjunctor) C.immitis on PDA, 300C

(Lactophenol cotton blue )

Coccidioides immitisCoccidioides immitis

Barrel-shaped arthroconidia

C idi id i itiCoccidioides immitis

Laboratory diagnosisLaboratory diagnosisDirect examination

KOHCalcofluor whiteHistophatological stain

A typical mature spherule yp psurround by neutrophils is present. (H&E)

Coccidioides immitisCoccidioides immitis

Spherules

C idi id i itiCoccidioides immitis

Laboratory diagnosisLaboratory diagnosisCulture

The cultures MUST be handled in biological safety cabinetSlide culture should NOT be made

C idi id i itiCoccidioides immitis

Laboratory diagnosisLaboratory diagnosisConfirm

Specific DNA probeID test for exoantigen HS, F, HLCultivation of spherules in converse medium, 20%CO2, 400CAnimal inoculation

Bl t d titidiBlastomyces dermatitidis

Blastomycosis (systemic mycoses)North America, Africa

Bl t d titidiBlastomyces dermatitidis

Yeast formBroad base budding yeast cellsTh t h The yeast phase is inhibited by cycloheximidecycloheximide

Bl t d titidiBlastomyces dermatitidis

M li l fMycelial formSlow growingMicroscopic similar to :

S d i Scedosporium apiospermumCh i B dermatitidis mycelial Chrysosporium B. dermatitidis mycelial

form ( lollipop )

Mycelial colony similar to B.dermatitidis

S apiospe m m in slide Chrysosporium sp in slide S.apiospermum in slide culture

Chrysosporium sp.in slide culture

Chrysosporium speciesChrysosporium species

K l i t di ti tiKoloninya not distinctiveSesudah pengeraman 2-4 hari, akan tumbuh koloni gray cottony akan tumbuh koloni gray, cottony or wooly myceliumMikroskopik: subglobose to pyriform Mikroskopik: subglobose to pyriform conidia are borne singly at the tips of long lateral conidiophores (“lolli-

’) l l bli h pops’), closely resembling the conidia of B.dermatitidis

fThe bases of the conidia may appear flattened and scarred

k l hCiri-ciri koloni Chrysosporium spp :Umumnya tumbuh lebih cepat dari B dermatitidisB,dermatitidisTidak tumbuh di medium yang mengandung cycloheximidemengandung cycloheximideMycelial colony tidak dapat di konversi menjadi yeast koloni pada 37C

Untuk membedakan koloni Chry-sosporium spp, dari koloni B.der-matitidis ialah dengan:matitidis ialah dengan:

uji exoantigen atau l i id b nucleic acid probe assay.

Sepedonium speciesSepedonium species

Koloninya mirip Chrysosporium spp, dan tidak dapat dibedakan visually

k k k l h l bl lMikroskopik : large,spherical,bluntly spiked macroconidia, yang mirip dengan H capsulatumdengan H.capsulatumCiri-ciri koloni Spedonium sp. :

T b h l bih tTumbuh lebih cepatDihambat oleh cycloheximideTidak dapat dikonversi jadi yeast phaseTidak dapat dikonversi jadi yeast phase

Bl t d titidiBlastomyces dermatitidis

Laboratory diagnosisDirect examination

B Dermatitidis ( H-E ) B dermatitidis GMSB.Dermatitidis ( H-E ) B.dermatitidis, GMS

Bl t d titidiBlastomyces dermatitidisLaboratory diagnosisLaboratory diagnosis

Culture immediately, organism does not organism does not survive well in specimenConversion to yeast phase on BHIDetection of cell-free antigen A ( exoantigen antigen A ( exoantigen test ).

B d titidi l t SDA B.dermatitidis slant, SDA, 300C, 18 days

P idi id b ili iParacoccidioides brasiliensis

Paracoccidioidomycosis (South American blastomycosis)

d h d lEndemic areas; South and Central AmericaR f h l Rate of growth: very slow; mature within 21 days

P idi id b ili iParacoccidioides brasiliensis

Yeast formMultiple budding yeastShip’s wheel (Mariner’s wheel)

(Pilot wheelP.brasiliensis

Multiple budding yeast (GMS)(GMS)

P idi id b ili iParacoccidioides brasiliensis

li l fMycelial formHyaline septate hyphaehyphaeIntercaraly and terminal chlamydospore

P b ili iP.brasiliensis

P idi id b ili iParacoccidioides brasiliensisLaboratory diagnosisLaboratory diagnosis

Direct examinationKOHKOHCalcofluor white

colourless dye that binds tocolourless dye that binds to chitin (fungal cell walls are made of chitin) and fluorescens when illuminated

Calcofluor white wet t f tfluorescens when illuminated

with UV light.mount of sputum showing blastoconidia of P.brasiliensis

P idi id b ili iParacoccidioides brasiliensisLaboratory Laboratory

diagnosisDirect examinationDirect examination

KOHCalcofluor whiteCalcofluor white

Mickey Mouse

P idi id b ili iParacoccidioides brasiliensis

Laboratory diagnosisCultureConversion to yeast phase on BHI

P.brasiliensis, 370C on BHI,

P idi id b ili iParacoccidioides brasiliensis

Laboratory diagnosisCultureConversion to yeast phase on BHI

P.brasiliensis, 370C on BHI,

1 2 3

Yeast form

37 0C55

4

A C

Mycelial form

25 0C

1=A ; 2=C

3=D ;4=F

5 = E 25 0C5 = E

D E F

P i illi ff iPenicillium marneffei

Penicilliosis marneffeiSoutheast AsiaDisseminated infection, most commonly in HIV patients

Penicillium marneffeiPenicillium marneffei

ffPenicillium marneffei , Segretain :Primary patogen pada manusia & hewanThermal dimorphismThermal dimorphismTerbatas di Asia Tenggara & Timur JauhReservoir natural :Bamboo rats

Rhizomys pruinosusRhizomys sinensisRhizomys sumatrensisRhizomys sumatrensisCannomys badius

P i illi i ff iPenicilliosis marneffei :

fInfeksi diseminata terdapat pada penderita AIDS yang berasal dari Eropah dan Amerika Utara yang Eropah dan Amerika Utara yang melancong ke China Selatan atau ThailandThailandLesinya mirip dengan lesi oleh Histoplasma capsulatumHistoplasma capsulatumIneksi alami ( natural infection) : Asia Tenggara termasuk Asia Tenggara, termasuk Southern China

P i illi ff iPenicillium marneffeiYeast formYeast form

Producing single-celled round to oval as round to oval as yeastlike cellReproduce by fission; Reproduce by fission; bud are not produce.

A Gi t i d h i A Giemsa stained showing typical septate yeast-like cells of P. marneffei

P i illi ff iPenicillium marneffei

M celial fo mMycelial formColony is flat powdery to velvety powdery to velvety, gray-green in the centerA deep-reddish soluble pigment diffuses into the

Culture showing a common green h ti P i illidiffuses into the

mediumsaprophytic Penicillium sp.and the typical reddish yellow with a yellow or white edge colony with distinctive red diffusable pigmentdistinctive red diffusable pigment of Penicillium marneffei

P i illi ff iPenicillium marneffeiLaboratory diagnosisLaboratory diagnosis

Direct examinationW i ht Gi t i PAS H&EWright or Giemsa stain, PAS, H&EOval yeastlike cells and multiply within histiocytes in tissue (or withinwithin histiocytes in tissue (or within monocytes in blood or BM)

P i illi ff iPenicillium marneffei

Laboratory diagnosis

Yeast-form fungi are massively phagocytized by macrophages

Lymph node biopsy. Recognition of the septa in the yeasts (Grocott'sphagocytized by macrophages

(H&E) the septa in the yeasts (Grocott s silver)

P i illi ff iPenicillium marneffei

L b di iLaboratory diagnosisCulture

P i illi ff iPenicillium marneffei

Laboratory diagnosisConversion to yeast phase on BHI

On Sabouraud's dextrose agar at 25C On Sabouraud's dextrose agar at 25C, colonies are fast growing, suede-like to downy, white with yellowish-green conidial heads Colonies become greyish-conidial heads. Colonies become greyish-pink to brown with age and produce a diffusible brownish-red to wine red-pigment. Conidiophores are hyaline, pigment. Conidiophores are hyaline, smooth-walled and bear terminal verticils of 3 to 5 metulae, each bearing 3 to 7 phialides. Conidia are globose to p gsubglobose, 2 to 3 um in diameter, smooth-walled and are produced in basipetal succession from the phialides.

fOn brain heart infusion (BHI) blood agar incubated at 37C, colonies are rough glabrous tan colored and rough, glabrous, tan-colored and yeast-like. Microscopically, yeast-cells are spherical to ellipsoidal 2 to cells are spherical to ellipsoidal, 2 to 6 um in diameter, and divide by fission rather than budding.o a a budd gNumerous short hyphal elements are also present

Clinical significance:

P i illi ff i hibit th l Penicillium marneffei exhibits thermal dimorphism by growing in living tissue or in culture at 37C as a yeast-like fungus or in culture at temperatures below 30C as a mould. This fungus has been isolated from bamboo rats and is endemic in Southeast Asia and the southern region of China. Over 30 cases of hyalohyphomycosis cause by P. ya o yp o ycos s cause bymarneffei, especially in AIDS patients have now been reported

Laboratory Identification of Di hi F iDimorphic Fungi

Lecturer : Kanya PreechasuthCourse: Clinical Diagnostic MicrobiologyDepartment of Clinical MicrobiologyFaculty of Associated Medical Sciences CMUFaculty of Associated Medical Sciences, CMU