1. Laboratory Identification of Dimorphic Fungi

2. 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

3. Dimorphic Fungi

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

5. 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

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

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

8. Sporothrix schenckii Sporotrichosis
Subcutaneous, pulmonary sporotricosis
Scattered worldwide
Cutaneous sporotricosis

9. 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

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

11. 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)

12. Sporothrix schenckii Laboratory diagnosis Culture
Conversion to yeast phase

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

14. 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

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

16. 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

17. 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.

18. 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

19. 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.

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

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

22. 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

23. 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 )

24. Coccidioides immitis
Barrel-shaped arthroconidia

25. 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)

26. Coccidioides immitis
Spherules

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

28. 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

29. Blastomyces dermatitidis
Blastomycosis (systemic mycoses)
North America, Africa

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

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

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

33. Chrysosporium species
Koloninya not distinctive
Sesudah pengeraman 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

34. 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

35. 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.

36. 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

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

38. 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

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

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

41. Paracoccidioides brasiliensis
Mycelial form
Hyaline septate hyphae
Intercaraly and terminal chlamydospore

42. P.brasiliensis

43. 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

44. Paracoccidioides brasiliensis
Laboratory diagnosis
Direct examination
KOH
Calcofluor white
Mickey Mouse

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

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

47. 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

48. TERIMA KASIH

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

50. 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

51. 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

52. 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

53. 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

54. 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)

55. 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)

56. Penicillium marneffei
Laboratory diagnosis
Culture

57. Penicillium marneffei
Laboratory diagnosis
Conversion to yeast phase on BHI

58. 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.

59. 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

60. 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

61. TERIMA KASIH

62. TERIMA KASIH

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