Laboratory Identification of Dimorphic Fungi

Environment/Routine culture media (SDA) 25-30oC ---Mold form Dimorphic fungi Thermally dimorphic fungi Environment/Routine culture media (SDA) 25-30oC ---Mold form conversion Tissue/Enriched media (BHI) 35 -37oC---Yeast form

Dimorphic Fungi

Dimorphic fungi Histoplasma capsulatum Coccidioides immitis Blastomyces dermatitidis Paracoccidioides brasiliensis Penicillium marneffei

1 2 3 Yeast form (37 0C) 5 4 1=A ; 2=C 3=D ;4=F 5= E Mycelial form (25 0C) A C E F D

Conversion of dimorphic fungi in culture SDA (antibacterial, clycloheximide) 25-300C, incubate take 4-8 weeks or more Mycelial form Inoculate mycelial growth onto BHI agar in a screw-cap tube incubate at 35-370C, preferably in CO2 Yeast form It be necessary several serial transfers to attain complete conversion to yeast phase

Conversion of dimorphic fungi in culture (cont.) Wet preparation (LPCB) of yeastlike area These organisms MUST be handled in a biological safety cabinet

Sporothrix schenckii Sporotrichosis Subcutaneous, pulmonary sporotricosis Scattered worldwide Cutaneous sporotricosis

Sporothrix schenckii Yeast form Round, oval and fusiform budding yeast cells of various sizes 2-3 x 3-10 µm Cigar-bodied 370C on BHI agar

Sporothrix schenckii Mycelial form Hyphae are narrow, septate, with slender conidiophores rising at right angle Conidia formig a “rosette-like”

Sporothrix schenckii Laboratory diagnosis Direct examination Section from a fixed cutaneous lesion showing round positive budding yeast-like cells. (PAS) Cigar-shaped, round form of S.schenckii (GMS-H&E)

Sporothrix schenckii Laboratory diagnosis Culture Conversion to yeast phase

Histoplasma capsulatum Histoplasmosis (systemic mycoses) H.capsulatum var. capsulatum North America, Mexico H.capsulatum var. duboisii Tropical areas of Africa

Histoplasma capsulatum Yeast form Round and oval budding yeast The yeast phase is inhibited by cycloheximide GMS stain of lymph node showing blastoconidia of H.capsulatum

Histoplasma capsulatum Mycelial form Hyaline septate hyphae Pear-shaped microconidia Tuberculate macroconidia Tuberculate macroconidia of H.capsulatum (LPC)

Histoplasma capsulatum Laboratory diagnosis Direct examination KOH wet mount of sputum showing blastoconidia of H.capsulatum Bone marrow aspirate with macrophage containing numerous blastoconidia of H.capsulatum

Histoplasma capsulatum Laboratory diagnosis Culture The organism does not survive well in clinical specimen Conversion to yeast phase BHI +cysteine , at 37C Sepedonium sp., Chrysosporium sp.

Sepedonium species Koloninya mirip Chrysosporium spp, dan tidak dapat dibedakan secara visual (kasat mata ) Mikroskopik : large,spherical,bluntly spiked macroconidia, yang mirip dengan H.capsulatum Ciri-ciri koloni Spedonium sp. : Tumbuh lebih cepat Dihambat oleh cycloheximide Tidak dapat dikonversi jadi yeast phase

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

Histoplasma capsulatum Laboratory diagnosis Serodiagnosis Complement fixation test Immunodiffusion (ID) test with H,M antigen Lines of identify for exoantigens in microimmunodiffusion plate

Coccidioides immitis Coccidioidomycosis (systemic mycoses) Asymptomatic pulmonary infection 60% in normal host Progressive pulmonary infection in immunocompromised patients

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

Coccidioides immitis Hyaline septate hyphae Mycelial form Hyaline septate hyphae Barrel-shaped arthroconidia that alternate with empty cells(disjunctor) C.immitis on PDA, 300C (Lactophenol cotton blue )

Coccidioides immitis Barrel-shaped arthroconidia

Coccidioides immitis Direct examination KOH Calcofluor white Laboratory diagnosis Direct examination KOH Calcofluor white Histophatological stain A typical mature spherule surround by neutrophils is present. (H&E)

Coccidioides immitis Spherules

Coccidioides immitis Culture Laboratory diagnosis Culture The cultures MUST be handled in biological safety cabinet Slide culture should NOT be made

Coccidioides immitis Confirm Specific DNA probe Laboratory diagnosis Confirm Specific DNA probe ID test for exoantigen HS, F, HL Cultivation of spherules in converse medium, 20%CO2, 400C Animal inoculation

Blastomyces dermatitidis Blastomycosis (systemic mycoses) North America, Africa

Blastomyces dermatitidis Yeast form Broad base budding yeast cells The yeast phase is inhibited by cycloheximide

Blastomyces dermatitidis Mycelial form Slow growing Microscopic similar to : Scedosporium apiospermum Chrysosporium B. dermatitidis mycelial form ( lollipop )

Mycelial colony similar to B.dermatitidis S.apiospermum in slide culture Chrysosporium sp.in slide culture

Chrysosporium species Koloninya not distinctive Sesudah pengeraman 2-4 hari, akan tumbuh koloni gray, cottony or wooly mycelium Mikroskopik: subglobose to pyriform conidia are borne singly at the tips of long lateral conidiophores (“lolli-pops’), closely resembling the conidia of B.dermatitidis

The bases of the conidia may appear flattened and scarred Ciri-ciri koloni Chrysosporium spp : Umumnya tumbuh lebih cepat dari B,dermatitidis Tidak tumbuh di medium yang mengandung cycloheximide Mycelial colony tidak dapat di konversi menjadi yeast koloni pada 37C

Untuk membedakan koloni Chry- sosporium spp, dari koloni B Untuk membedakan koloni Chry- sosporium spp, dari koloni B.der-matitidis ialah dengan: uji exoantigen atau nucleic acid probe assay.

Sepedonium species Koloninya mirip Chrysosporium spp, dan tidak dapat dibedakan visually Mikroskopik : large,spherical,bluntly spiked macroconidia, yang mirip dengan H.capsulatum Ciri-ciri koloni Spedonium sp. : Tumbuh lebih cepat Dihambat oleh cycloheximide Tidak dapat dikonversi jadi yeast phase

Blastomyces dermatitidis Laboratory diagnosis Direct examination B.Dermatitidis ( H-E ) B.dermatitidis, GMS

Blastomyces dermatitidis Laboratory diagnosis Culture immediately, organism does not survive well in specimen Conversion to yeast phase on BHI Detection of cell-free antigen A ( exoantigen test ). B.dermatitidis slant, SDA, 300C, 18 days

Paracoccidioides brasiliensis Paracoccidioidomycosis (South American blastomycosis) Endemic areas; South and Central America Rate of growth: very slow; mature within 21 days

Paracoccidioides brasiliensis Yeast form Multiple budding yeast Ship’s wheel (Mariner’s wheel) (Pilot wheel P.brasiliensis Multiple budding yeast (GMS)

Paracoccidioides brasiliensis Mycelial form Hyaline septate hyphae Intercaraly and terminal chlamydospore

P.brasiliensis

Paracoccidioides brasiliensis Laboratory diagnosis Direct examination KOH Calcofluor white colourless dye that binds to chitin (fungal cell walls are made of chitin) and fluorescens when illuminated with UV light. Calcofluor white wet mount of sputum showing blastoconidia of P.brasiliensis

Paracoccidioides brasiliensis Laboratory diagnosis Direct examination KOH Calcofluor white Mickey Mouse

Paracoccidioides brasiliensis Laboratory diagnosis Culture Conversion to yeast phase on BHI P.brasiliensis, 370C on BHI

Paracoccidioides brasiliensis Laboratory diagnosis Culture Conversion to yeast phase on BHI P.brasiliensis, 370C on BHI

1 2 3 Yeast form 37 0C 5 4 1=A ; 2=C 3=D ;4=F 5 = E Mycelial form 25 0C A C E F D

TERIMA KASIH

Penicillium marneffei Penicilliosis marneffei Southeast Asia Disseminated infection, most commonly in HIV patients

Penicillium marneffei Penicillium marneffei , Segretain : Primary patogen pada manusia & hewan Thermal dimorphism Terbatas di Asia Tenggara & Timur Jauh Reservoir natural :Bamboo rats Rhizomys pruinosus Rhizomys sinensis Rhizomys sumatrensis Cannomys badius

Penicilliosis marneffei : Infeksi diseminata terdapat pada penderita AIDS yang berasal dari Eropah dan Amerika Utara yang melancong ke China Selatan atau Thailand Lesinya mirip dengan lesi oleh Histoplasma capsulatum Ineksi alami ( natural infection) : Asia Tenggara, termasuk Southern China

Penicillium marneffei Yeast form Producing single-celled round to oval as yeastlike cell Reproduce by fission; bud are not produce. A Giemsa stained showing typical septate yeast-like cells of P. marneffei

Penicillium marneffei Mycelial form Colony is flat powdery to velvety, gray-green in the center A deep-reddish soluble pigment diffuses into the medium Culture showing a common green saprophytic Penicillium sp. and the typical reddish yellow with a yellow or white edge colony with distinctive red diffusable pigment of Penicillium marneffei

Penicillium marneffei Laboratory diagnosis Direct examination Wright or Giemsa stain, PAS, H&E Oval yeastlike cells and multiply within histiocytes in tissue (or within monocytes in blood or BM)

Penicillium marneffei Laboratory diagnosis Yeast-form fungi are massively phagocytized by macrophages (H&E) Lymph node biopsy. Recognition of the septa in the yeasts (Grocott's silver)

Penicillium marneffei Laboratory diagnosis Culture

Penicillium marneffei Laboratory diagnosis Conversion to yeast phase on BHI

On Sabouraud's dextrose agar at 25C, colonies are fast growing, suede-like to downy, white with yellowish-green conidial heads. Colonies become greyish-pink to brown with age and produce a diffusible brownish-red to wine red-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 subglobose, 2 to 3 um in diameter, smooth-walled and are produced in basipetal succession from the phialides.

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

Clinical significance: 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. marneffei, especially in AIDS patients have now been reported

TERIMA KASIH

TERIMA KASIH

Laboratory Identification of Dimorphic Fungi Lecturer : Kanya Preechasuth Course: Clinical Diagnostic Microbiology Department of Clinical Microbiology Faculty of Associated Medical Sciences, CMU