1. Practical medical mycology Lab 7 identification of fungi ( part 2)
بسم الله الرحمن الرحيم
Practical medical mycology
Lab 7 identification of fungi ( part 2)
Microsporum
Cryptococcus neoformans
Histoplasma capsulatum
Coccidioides immitis
Blastomyces dermatitidis

2. Microsporum
The genus Microsporum is characterized by fusiform or spindle-shaped macroconidia, often with thick, rough, or spiny walls.
The rough nature of spines may be difficult to demonstrate in some cultures and may be seen only on the tip of the conidium.
Macroconidia and colony morphology are used to identify the genus and species.
Although microconidia may be produced, they do not aid in identification.
spindle مغزل
Rough خشن
tip طرف

3. Most Microsporum species perforate hair in vitro.
Examination of tinea capitis or of cultures derived from it, when illuminated by Wood’s light, demonstrate a bright yellow-green fluorescence with M. canis. M. ferrugineum also has yellow-green fluorescence, while M. Audouinii produces a silver fluorescence.
perforate يخترق

4. Microsporum audouinii • Colony: Growth is slow, with glabrous to velvety dense mycelium, wrinkled, with a radiating edge. • Surface: Color is white-gray, tan, or rust-buff. • Reverse: peach to salmon color. • Microscopic: Macroconidia are rare and poorly developed. When seen they are similar to those of M. canis with terminal spine-like projections, pectinate hyphae, and chlamydospores. M. audouinii is differentiated from other Microsporum species by its poor growth on rice grains.
glabrous امرد
velvety مخملي
Wrinkled متجعد
radiating مشع
Tan اسمر
rust-buff صدأ برتقالي
peach خوخي (قرنفلي)
salmon colorاللون السلموني هو طيف يمتد من البرتقالي الشاحب إلى الزهري الفاتح

5. Spiral لولبي
Pectinate مشطي
Chandelierثريا
Nodule عقدة
Raquetمضرب التنس
Chlamydospore: a thick-walled hyphal cell that functions as a spore
Arthrosporeبوغ مفصلي

7. Microsporum gypseum • Colony: Microsporum gypseum rapidly covers the agar surface with floccose to powdery growth that may become pleomorphic. • Surface: Cinnamon-buff. • Reverse: May be colorless, buff, deep mahogany- brown, or variable. • Microscopic: Abundant clusters of macroconidia are produced: clavate or cylindrical to fusiform-shaped,
floccose عهني
Pleomorphic متغيرة الشكل
Cinnamon-buff. برتقالي قرفي
mahogany- brown بني محمر
Clavate متعجر
Fusiform مغزلي

10. Cryptococcosis
The infections produced by the ubiquitous encapsulated yeast Cryptococcus neoformans which is usually acquired by inhalation.
immunosuppressed patients
Localized in The lung
Primary pulmonary Infection (bronchitis)
cough, low-grade fever and pleuritic pain
Soil, bird droppings

11. Cryptococcosis : Diagnosis
India ink smear or nigrosin stain of the CSF sediments will reveal a capsule, double cell wall.
Fungal culture- mostly done with CSF deposit, sputum and urine.
These are cultured on SDA.
C. neoformans colonies are smooth, convex and yellow or tan on solid media.
It produces brown colonies on bird seed agar (melanin production).

12. It can also be identified by biochemical tests, eg. , API
It can also be identified by biochemical tests, eg ., API . (3 days system)
There are also DNA probes that are available for diagnosis.
Cryptococcal antigen test- this a latex agglutination test that can be performed on CSF and serum that is >90% specific.
Histopathology- Methenamine silver or Periodic acid- Schiff staining show a yeast- like organism with narrow based buds.
Mayer’s mucicarmine stain will stain the capsule rose red.

13. Identification of Crypto. spp
Urease (positive for Crypto sp.)
India ink (positive for Crypto sp.)

14. Grocott’s methenamine silver (GMS) stained tissue section of lung showing typical encapsulated yeast cells of C. neoformans.

15. CRYPTOCOCCOSIS : culture
Bird seed agar plate showing the typical brown colour effect seen with C. neoformans.
Mucoid colonies of C. neoformans

16. Canavanine-glycine-bromothymol blue medium can be used to distinguish C. neoformans var. neoformans (medium remains yellow) from C. neoformans var. gattii (medium turns a deep blue in 2-3 days).

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

18. Histoplasma capsulatum
Yeast form
Round and oval budding yeast
The yeast phase is inhibited by cyclohexamide
GMS stain of lymph node showing blastoconidia of H.capsulatum
Mycelial form
Hyaline septate hyphae
Pear-shaped microconidia
Tuberculate macroconidia
Tuberculate macroconidia of H.capsulatum (LPC)
Gomori Methenamine-Silver (GMS)
Pearاجاص
Tuberculate متدرن

19. 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

20. Culture: Definitive diagnosis is by culture and is done for up to 6 weeks at 300C in brain heart infusion agar, blood, antibiotics and cyclohexamide. Fungus from culture is confirmed using a probe for ribosomal DNA.
Organism detection: This is the detection of polysaccharide antigen in urine and blood.
Serology: (Complement fixation test Immunodiffusion (ID) test)
Histochemical: stains can allow rapid identification of fungus from tissues and body fluids.

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

22. Coccidioides immitis
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(
C.immitis on PDA, 300C (Lactophenol cotton blue )

24. Coccidioides immitis
Spherules

25. Coccidioides immitis Laboratory diagnosis
Culture Isolation of the organism- definitive diagnosis is by culture or the identification of fungal elements within clinical specimens.
There is minimal risk to the medical personnel because infection is not transmitted from primary specimen.
Stains such as silver, periodic acid –Schiff or H&E will reveal spherules.
C. immitis grows on standard microbiological media in aerobic conditions and typically takes the form of a white mold at around 5-7 days. At this point, it is highly infectious.
Unlike the spherule, the mycelial form is not unique, and identification is made by reference laboratories either antigenic detection or by the detection of specific ribosomal RNA.

26. Blastomyces dermatitidis
Yeast form
Broad base budding yeast cells
The yeast phase is inhibited by cycloheximide
Mycelial form
Slow growing
Microscopic similar to :
Scedosporium apiospermum
Chrysosporium
B. dermatitidis mycelial form ( lollipop )

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

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

29. Microscopy of secretions- The characteristic yeast cell may be seen in a wet preparation of sputum or pus.
Body fluids such as urine or pleural fluid should be centrifuged and the sediment examined.
BAL (Bronchio Alveolar Lavage) may be useful in patients who are not producing sputum.
When organisms are low, they may be more easily identified on papanicolaou (PAP) preparations.
Histology- Gmomori’s methenamine silver and PAS stains readily the fungus.

30. Culture- Material should be inoculated onto Sabouraud’s or more-enriched agar and incubated at 300C.
The mycelial form is not diagnostic, and ideally conversion to yeast at 370C should be demonstrated.
This is not always possible and mycelial identification can be achieved by nucleic acid probes for Blastomyces RNA.
Serology-Complement fixation, immunodiffusion and ELISA based tests are available but sensitivity and specificity vary widely.