41. laboratory diagnosis of common fungal diseases
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LABORATORY DIAGNOSIS OF COMMON FUNGAL DISEASES
Prof. Louella A. Dancel, RMT, MSc.
University of Perpetual Help System DALTA
42nd PAMET ANNUAL CONVENTIONManila Hotel – December 2,2006
Clinical specimens submitted for Fungal Isolation 2004-2006*
Clinical specimen 2004 2005 2006 Total
Respiratory 29 35 34 98
Body fluids 37 17 34 88
Tissues 9 0 23 32
Skin 1 0 0 1
Nails 29 31 20 80
Hair 0 1 0 1
CSF 8 5 8 21
Others 5 20 24 49
TOTAL 118 109 153 380
Fungal Isolates 2004-2006*
Isolate 2004 2005 2006
C.albicans 5.1% 13.8% 11.8%
C.tropicalis 1.8% 3.7% 5.2%
C.parapsilosis 5.1% 6.4% 3.9%
C.glabrata 0.91% 0.91% 3.9%
C.famata 1.8% 4.6% 2.0%
Aspergillus spp. 0.91% 0% 0.65%
Fusarium spp. 0% 0% 0.65%
Data from a Five-year review of Fungal Isolates at UPM-CPH
• Clinical specimens (n=545):
–Skin scrapings
–Nail clippings/scrapings
–Hair
–Exudates
–Biopsy materials
Data from a Five-year review of Fungal Isolates at UPM-CPH
Total clinical specimens tested – 545
Results:
10.8%- (+) for both KOH & culture
59.1%- (-) for both KOH &culture
17.1% - (+)culture, (-) KOH
12.8% - (-) culture, (+) KOH
Data from a Five-year review of Fungal Isolates at UPM-CPH
Fungal isolates : Trichophyton mentagrophytes
Trichophyton rubrum
Trichophyton tonsurans
Trichophyton schoenlenii
Trichosporon spp.
Data from a Five-year review of Fungal Isolates at UPM-CPH
• Fungal isolates
Microsporum gypseum
Microsporum canis
Epidermophyton flocossum
Candida albicans
Exophiala werneckii
Data from a Five-year review of Fungal Isolates at UPM-CPH
Fungal isolates from biopsy materials and exudates (31.2% positive):
Fonsecaea compactum
Phialophora verrucosa
Exophiala jeanselmei
Madurella grisea
Laboratory Methods for Diagnosis of Mycoses
I. DIRECT EXAMINATION:
*10-30% KOH
*Calcofluor white stain
*Histological stains- H&E, PAS
*India Ink
*Wet mount
Laboratory Methods for Diagnosis of Mycoses
II. Isolation & Culture
SDA
BHIA/BAP
Media with/without antibiotics
• Macroscopic examination of culture
• Microscopic examination using LPCB
Laboratory Methods for Diagnosis of Mycoses
• III. Biochemical Tests:*Rapid kits for yeasts*Urea test
• IV. Special Tests: *In-vitro hair perforation test
*Germ tube test *Chlamydoconidia formation test
Mycotic Infections
Superficial
Cutaneous
Subcutaneous
Systemic
Opportunistic
*Mycotoxicosis
*Allergies
Superficial mycoses
• superficial cosmetic fungal infections of the skin or hair shaft
• no living tissue is invaded
• no cellular response from the host
• no pathological changes
• patients unaware of infection
Superficial mycoses
Disease
SKIN
• Pityriasis versicolor
• Tinea nigra
Causative organisms
• Malassezia furfur
• Exophiala werneckii
Superficial mycoses
Disease
HAIR
• White piedra
• Black piedra
Causative organisms
• Trichosporon beigelii
• Piedraia hortae
Superficial mycoses
Pityriasis versicolor
• Lesion-An-an”
-Hyperpigmented or hypopigmented macular lesions
www.ethnomed.org
Superficial mycoses
Pityriasis versicolor
• Lesion– scale readily, giving
a chalky branny appearance
– occurs on the trunk, shoulders & arms, face and neck
Modified from www.columbia.edu
Superficial mycoses
Pityriasis versicolor
• Lesion– fluoresce pale greenish under Wood’s
lamp
• Distribution – worldwide– more common in tropical than
temperate climates
Superficial mycoses
Pityriasis versicolor
KOH of skin scrapings
• clusters of budding yeast-like cells & short angular hyphal forms
• “spaghetti and meat balls”
Superficial mycoses
Pityriasis versicolor
PAS of skin scrapings• “spaghetti and meat
balls”
Superficial mycoses
Pityriasis versicolor
• Culture of skin scrapings– Not necessary– diagnostic microscopic
features– SDA overlaid with
peanut oil, olive oil
Superficial mycoses
Pityriasis versicolor
• Etiologic Agent– Malassezia globosa
lipophilic yeast
part of skin normal flora
Superficial mycoses
Pityriasis versicolor
• Treatment– keratinolytic agents applied locally– Mild fungicides– Miconazole– Selenium sulfide (1%) shampoo
Superficial mycoses
Tinea nigra
• Lesion– Gray to black well-
demarcated macular lesions
– most frequently occurring on the palms of the hand
– non-inflammatory & non-scaling lesions
11th.blogspot.com
Superficial mycoses
Tinea nigra
• Distribution– world-wide– more common in tropical regions of
Central & South America, Africa, Southeast Asia & Australia
Superficial mycoses
Tinea nigra
• KOH– pigmented brown to dark
olivaceous (dematiaceous) septate hyphal elements & 2-celled yeast cells
Superficial mycoses
Tinea nigra
• Etiologic agent– Exophiala werneckii
saprophyte found in soil, compost, humus &
wood in humid tropical & sub-tropical regions
Superficial mycoses
Tinea nigra
• Culture on SDA– initially mucoid,
yeast-like & shiny black
– with age: aerial mycelia & dark olive color
Superficial mycoses
Tinea nigra
• Lactophenol cotton blue (LPCB) of culture on SDA– 2-celled, pale brown yeast cells– darkly pigmented septa (annelides)– one cell cylindrical, the other cell is
spindle-shaped– occur in aggregated masses
Superficial mycoses
Tinea nigra
• Treatment
– keratinolytic agents (Whitfield’s ointment)
– tincture of iodine, 2% salicylic acid, 3% sulfur
– miconazole nitrate, imidazoles, triazoles
Superficial mycoses
Tinea nigra
• Prevention– avoid exposure to sources,
contaminated material
Superficial mycoses
Piedra
• Fungus infection of the hair shaft
• presence of firm, irregular nodules
• Nodules - fungal elements cemented
together along the hair shaft
• Multiple infections of the same strand
Superficial mycoses
Piedra
Two varieties
–White piedra
–Black piedra
Superficial mycoses
Black piedra
• Lesion– discrete, hard,
gritty, brown to black concretions / nodules
– infection of hair• scalp hair -common• beard, moustache -
less common• axilla & groin hairs -
rare
www.doctorfungus.org
Superficial mycoses
Black piedra - distribution
Superficial mycoses
Black piedra
• Etiologic agent– Piedraia hortae– source of infection
Superficial mycoses
Black piedra - lab diagnosis
• Direct microscopy– specimen - hair with nodules
– 25% NaOH or KOH
– dark septate hyphae
Superficial mycoses
Black piedra - lab diagnosis
• Direct microscopy– round to oval asci;
hyaline, curved to fusiform ascospores
Superficial mycoses
Black piedra - lab diagnosis
• Isolation – medium
– SDA with chloramphenicol
– SDA cycloheximide
Superficial mycoses
Black piedra - lab diagnosis
• Isolation
-growth very slow-dark brown to black
-greenish brown, short aerial mycelium
Heaped center
Flat periphery
Superficial mycoses
White piedra
• Infection of hair shaft– face, axilla, genitals -
common
– scalp, eyebrows, eyelashes - less common
Superficial mycoses
White piedra• Infection of hair
shaft
– less common
scalp
eyebrow
eyelashes
Superficial mycoses
White piedra
Nodule
• Soft, white, yellowish, beige or greenish nodule
• Discrete• more often coalescent,
forming an irregular transparent sheath
Superficial mycoses
White piedra
• Distribution
– common in S. America & Asia
– sporadic in N. America & Europe
• Etiologic agent
– Trichosporon beigelii or T. cutaneum
Superficial mycoses
White piedra
• Ecology
– soil, stagnant water
– decaying fruit
– spoiled food
– sputum & body surfaces
– horses
Superficial mycoses
White piedra - lab diagnosis
• Microscopic direct examination
– specimen - hair with nodules
– 10% KOH or 25% NaOH + 5% glycerin
– hyaline septate hyphae
– oval or rectangular arthroconidia
– occasional blastoconidia
Superficial mycoses
White piedra - lab diagnosisIsolation
• medium - SDA with
chloramphenicol without
cycloheximide
• growth/culture– rapid
– cream-colored, soft
– membranous, wrinkled radial
furrows, irregular folding
Superficial mycoses
White piedra - lab diagnosis
Isolation
• microscopic exam
of culture
– hyaline hyphae
– arthroconidia
– blastoconidia
Superficial mycoses
White piedra - lab diagnosis
• Physiological studies– does not ferment carbohydrates– assimilate dextrose, lactose, D-xylose,
inositol
– negative KNO3 assimilation
– urease positive– arbutin is split
Superficial mycoses
Piedra - Treatment
• Shaving or cutting infected hair
• Topical fungicides
– 1:200 bichloride of mercury
– benzoic acid & salicylic acid combinations
– 3% sulfur ointment
– 2% formalin
Cutaneous mycoses
skin
hair
nails
• No living
tissue
• Host Rxn
to fungus
keratinase
Cutaneous mycosesDisease
• Dermatophytosis
Causative organisms
• Dermatophytes
Microsporum
Trichophyton
Epidermophyton
ringworm
Cutaneous mycoses
Disease
• Candidiasis of skin, mucous membranes & nails
Causative organisms
• Candia albicans & related species
dermatomycosis Soil fungi (Scytalidium, Fusarium, etc.)
Systemic fungi (Histoplasma, etc)
Ecological Groups ofDermatophytes
Geophilic
• inhabit soil where
they decompose
keratinaceous
debris
• Dead animals
Zoophilic
• parasitic on animals
www.saanendoah.com
www.kolumbus.fi
Anthropophilic fungi
• primarily parasitic to man• man as exclusive host• for maintenance & dissemination of
species
Anthropophilic fungi• Anthropophilic
fungi:– Examples:
• M. audonii• T. rubrum• T. schoenleinii• T. tonsurans• T. violaceum
Classification of
Dermatophytes
Microsporum
Macroconidia
Rough walled
Microconidapresent
Trichophyton
Macroconidia
Smooth walled
Microconidia
present
Epidermophyton
ChlamydoconidiaMacroconidia
Smooth walled
Microconidia
none
Clinical Manifestations of Dermatophytes
Tinea capitis
www.emedicinehealth.com
Scalp, eyebrow, eyelashes
Microsporum &
Trichophyton
Tinea capitis Endothrix
Ectothrix
Tinea favosa
• Scutulum
• Mass of mycelia
& epithelial
debris
• Cup shaped
crusts
www.mf.uni-lj.si
Tinea corporis
www.cut.ee/
•Non-hairy skin
•Rings with scaly
centers
•Rxn vs fungus
Tinea corporis
• E. floccosum
• Trichophyton
• Microsporum
Cutaneous
Tinea imbricata
Concentric
rings
Trichophyton
concentricum
Cutaneous
Tinea barbae
• Bearded areas of
face & neck
www.merck.com
www.emedicine.com
Cutaneous
Tinea cruris
www.dermnetnz.org
Jock itch
Moist groin
area
E. floccosum,
T. rubrum
Cutaneous
Tinea pedis
www.doctorfungus.org
dermatologie.free.fr
Athlete’s foot
Toe webs & soles,
even nails
Id reaction,
circulating fungal
antigens
Cutaneous
Tinea manuum
www.dermnetnz.org
• Interdigital
areas & palmar
surfaces
Cutaneous
Tinea unguium
www.dermnetnz.org
Invasion of nail plate by
dermatophytes
Thickened, discolored &
brittle• Onychomycosis- non
dermatophyte
Yeast etc.
Laboratory diagnosis
Wipe with water
www.doctorfungus.org
scalpel
Paper / envelope
active edge
Skin scraping specimen
Direct Examination
• Wet mount
KOH• KOH– 10% to 30%
– with Parker Superquink blue-black ink
– gentle warming
pa
rk
er
Cutaneous
KOH of skin scrapings
Septate hypha
Cutaneous
arthrospores
septate hypha
KOH of skin scrapings
Cutaneous
Ectothrix invasion of hair
• Hair invasion • formation of
arthroconidia on the outside of hair shaft
• cuticle of hair is destroyed
Cutaneous
Ectothrix invasion of hair
• Hair invasion by a dermatophyte– Microsporum canis
– M. gypseum
– Trichophyton equinum
– T. verrucosum
Cutaneous
Ectothrix invasion of hair
• Wood’s UV light
• infected hairs fluoresce
• bright greenish yellow under
Cutaneous
Endothrix invasion of hair
• formation of arthroconidia within hair shaft
• cuticle of hair remains intact
• do not fluoresce under Wood’s UV light
Cutaneous
Endothrix invasion of hair
• ALL AGENTS ARE ANTHROPOPHILIC
• Trichophyton tonsurans,
• T. violaceum
Culture:
• Selective media– SDA with chloramphenicol &
cycloheximide (Mycosel or Mycobiotic agar)
– Dermatophyte test medium
Non-selective medium– Sabouraud’s dextrose agar
Culture:
• IncubationRoom temperature
At least 2 weeks
Identification
• Gross color & texture• Microscopic characteristics• Confirm / compare with
Written descriptionsDrawingsphotographs
Mycology
Cutaneous
Microsporum canis
netti.nic.fi
• Zoophilic– cats and dogs
• Invades– Hair– skin – rarely nails
• distribution– worldwide
www.vet.ohio-state.edu
Cutaneous
golden yellow reverse colony
www2.provlab.ab.ca
Microsporum canislab diagnosis –
culture• white cottony growth
Cutaneous
Microsporum canis
• microscopic:
– spindle shaped,
one end pointed,
other end blunt
– thick walled
verrucose
macroconidia
– 6 to 12 cellswww.doctorfungus.org
Cutaneous
Microsporum gypseum
– geophilic
– usually produces a single inflammatory skin or scalp lesion
• distribution– worldwide
Cutaneous
Microsporum gypseum
lab diagnosis - culture
• flat, spreading suede-like to granular
• cinnamon growth • yellow brown pigment
on reverse of colony
www.ukneqasmicro.org.uk
Cutaneous
Microsporum gypseum
microscopic:
• symmetrical ellipsoidal
• thin walled verrucose
macroconidia
• distal end slightly rounded,
proximal (point of
attachment) is blunt
• 4 to 6 cells
vtpb-www.cvm.tamu.edu
www.medmicro.wisc.edu
Cutaneous
Trichophyton mentagrophytes
– zoophilic: mice, cats, horses, sheep, rabbits
– inflammatory skin or scalp lesions in humans
– ectothrix
• distribution– worldwide
Cutaneous
Trichophyton mentagrophytes
• lab diagnosis - culture
• flat, white to cream color; powdery to granular surface
danival.org
Cutaneous
Trichophyton mentagrophytes
Microscopic
spherical microconidia forming dense clusters,
“en-grappe”vtpb-www.cvm.tamu.edu
Cutaneous
Trichophyton mentagrophytes
• spiral hyphae
smooth thin-walled clavate multiseptate macroconidia
Microscopic
www.vet.ohio-state.edu vtpb-www.cvm.tamu.edu
Cutaneous
Trichophyton mentagrophytes
lab diagnosis
www2.provlab.ab.ca
positive urease production
positive for in-vitro hair perforation
www2.provlab.ab.ca
Cutaneous
Trichophyton rubrum
• anthropophilic– chronic infections of the skin,
nails, rarely scalp
– ectothrix or endothrix hair infection
• distribution– worldwide
Cutaneous
Trichophyton rubrum
lab diagnosis – culture
• white, suede-like to downy
• wine red pigment on reverse side
www.pfizer.ch
www4.medfak.lu.se
Cutaneous
Trichophyton rubrum
www2.provlab.ab.ca • lab diagnosis
– scanty to moderate numbers of slender clavate to pyriform microconidia
– arranged “en-thyrse”
Cutaneous
Trichophyton concentricumAnthropophilic
chronic non-inflammatory tinea corporis
tinea imbricata – concentric scaling of skin
Not invade hair
Cutaneous
Trichophyton concentricum
Distribution
Pacific Islands of Oceania
Southeast Asia
Central and South America
Cutaneous
Trichophyton concentricum
Lab diagnosis
Slow growing deeply folded thallus
Cream to orange brown in color
Reverse buff to brown
Cutaneous
Trichophyton concentricum
Microscopic –
“antler tips” hyphae,
chlamydoconidia
Cutaneous
Trichophyton schoenleiniiAnthropophilic
Cause favus
Chronic scarring form of tinea capitis
Saucer shaped crusted lesions or scutula
Permanent hair loss
Cutaneous
Trichophyton schoenleiniiLab diagnosis
Culture
Waxy or glabrous
Deeply folded honeycomb-like thallus with sub-surface growth
Cutaneous
Trichophyton schoenleiniiLab diagnosis
Microscopic
Favic chandeliers
No macroconidia
No microconidia
Cutaneous
Epidermophyton floccosum
• anthrophophilic
• does not invade hair in vivo
• distribution– worldwide
Cutaneous
Epidermophyton floccosum
Culture• greenish-brown or
“khaki” colored
• suede-like surface
• raised & folded center, with flat periphery
• yellowish brown reverse pigment
Cutaneous
Epidermophyton floccosum
botit.botany.wisc.edu
Microscopic• smooth thin-walled
macroconidia often in clusters growing directly from hyphae
• no microconidia• numerous
chlamydoconidia
www.fns.uniba.sk